Topics in Dental Biochemistry

Martin฀Levine

Topics in Dental
Biochemistry
Martin฀Levine
Department฀of฀Biochemistry฀&฀฀
Molecular฀Biology
Colleges฀of฀Medicine฀and฀Dentistry
University฀of฀Oklahoma
Health฀Sciences฀Center
940,฀S.L.฀Young฀Blvd
Oklahoma฀City,฀OK฀73104
USA
martin-levine@ouhsc.edu

ISBN฀978-3-540-88115-5฀ e-ISBN฀978-3-540-88116-2

DOI฀10.1007/978-3-540-88116-2

Springer฀Heidelberg฀Dordrecht฀London฀New฀York

©฀Springer-Verlag฀Berlin฀Heidelberg฀2011

This฀work฀is฀subject฀to฀copyright.฀All฀rights฀are฀reserved,฀whether฀the฀whole฀or฀part฀of฀the฀material฀is฀
฀concerned,฀speciically฀the฀rights฀of฀translation,฀reprinting,฀reuse฀of฀illustrations,฀recitation,฀฀broadcasting,฀
reproduction฀on฀microilm฀or฀in฀any฀other฀way,฀and฀storage฀in฀data฀banks.฀Duplication฀of฀this฀publication฀
or฀parts฀thereof฀is฀permitted฀only฀under฀the฀provisions฀of฀the฀German฀Copyright฀Law฀of฀September฀9,฀
1965,฀in฀its฀current฀version,฀and฀permission฀for฀use฀must฀always฀be฀obtained฀from฀Springer.฀Violations฀
are฀liable฀to฀prosecution฀under฀the฀German฀Copyright฀Law.฀

The฀ use฀ of฀ general฀ descriptive฀ names,฀ registered฀ names,฀ trademarks,฀ etc.฀ in฀ this฀ publication฀ does฀ not฀
imply,฀even฀in฀the฀absence฀of฀a฀speciic฀statement,฀that฀such฀names฀are฀exempt฀from฀the฀relevant฀฀protective฀
laws฀and฀regulations฀and฀therefore฀free฀for฀general฀use.฀

Product฀liability:฀The฀publishers฀cannot฀guarantee฀the฀accuracy฀of฀any฀information฀about฀dosage฀and฀
application฀contained฀in฀this฀book.฀In฀every฀individual฀case฀the฀user฀must฀check฀such฀information฀by฀
consulting฀the฀relevant฀literature.

Cover฀design:฀eStudioCalamar,฀Figueres/Berlin

Printed฀on฀acid-free฀paper

Springer฀is฀part฀of฀Springer฀Science+Business฀Media฀(www.springer.com)
In฀memory฀of฀my฀brother฀Ian฀J฀Levine฀BDS฀1947–2009
 Preface

Over฀the฀last฀30฀years,฀the฀development฀of฀molecular฀biology฀has฀revolutionized฀our฀under-
standing฀of฀the฀biochemistry฀underlying฀biology฀and฀medicine.฀As฀yet,฀there฀is฀no฀intro-
ductory฀text฀that฀relates฀this฀revolution฀to฀topics฀of฀major฀interest฀to฀dentistry.฀Because฀of฀
increasing฀ demands฀ to฀ make฀ biochemistry฀ useful฀ by฀ translating฀ its฀ indings฀ into฀ better฀
treatments฀for฀problems฀in฀medicine,฀the฀dental฀ield฀needs฀a฀similar฀textbook.฀The฀primary฀
aim฀of฀this฀book฀is฀to฀integrate฀general฀biochemistry฀into฀topics฀that฀speciically฀pertain฀to฀
dental฀health฀and฀disease.฀First฀and฀second฀year฀dental฀students฀have฀completed฀a฀general฀
biochemistry฀course,฀but฀have,฀at฀best,฀a฀sketchy฀idea฀of฀how฀the฀material฀in฀that฀course฀
relates฀to฀dentistry.฀In฀a฀traditional฀dental฀curriculum,฀the฀topics฀of฀this฀book฀are฀covered฀in฀
physiology,฀nutrition,฀anatomy,฀histology,฀microbiology฀or฀immunology.฀This฀book฀was฀
written฀to฀enable฀dental฀students฀to฀integrate฀their฀general฀biochemistry฀within฀these฀topics฀
of฀dental฀interest.฀It฀was฀considered฀neither฀desirable฀nor฀practical฀to฀ill฀the฀text฀with฀refer-
ences,฀except฀for฀the฀igures฀and฀tables.
The฀formal฀discipline฀of฀dentistry฀was฀initially฀developed฀in฀the฀late฀19th฀century฀to฀
treat฀ dental฀ caries,฀ but฀ it฀ quickly฀ spread฀ to฀ treat฀ all฀ diseases฀ that฀ affect฀ the฀ oral฀ cavity.฀
Dental฀treatments฀have฀progressed฀enormously฀over฀the฀last฀40฀years,฀as฀have฀treatments฀
for฀many฀other฀diseases.฀The฀most฀powerful฀new฀dental฀treatments฀have฀come฀from฀water฀
luoridation,฀better฀oral฀hygiene฀measures,฀new฀mechanical฀or฀replacement฀materials,฀and฀
the฀adoption฀of฀drugs฀developed฀for฀non-dental฀diseases.฀Nevertheless,฀these฀measures฀are฀
not฀universally฀effective฀and฀improvements฀can฀be฀made฀in฀many฀areas.
The฀most฀widespread฀and฀commonly฀treated฀dental฀diseases,฀dental฀caries฀and฀peri-
odontal฀disease,฀are฀chronic฀conditions฀caused฀by฀interactions฀between฀the฀host฀and฀oral฀
bacteria฀that฀are฀still฀only฀partially฀understood฀in฀detail.฀A฀second฀aim฀of฀this฀book฀is฀
therefore฀to฀point฀out฀the฀current฀knowledge฀for฀a฀future฀generation฀to฀build฀upon.฀While฀
I฀ hope฀ the฀ descriptions฀ of฀ dental฀ caries฀ and฀ luoride฀ are฀ pretty฀ standard,฀ describing฀ a฀
modern฀ and฀ coherent฀ view฀ of฀ periodontal฀ disease฀ was฀ a฀ problem.฀ This฀ is฀ a฀ ield฀ with฀
which฀I฀began฀my฀PhD฀and฀am฀still฀active.฀Unfortunately,฀almost฀every฀researcher฀in฀this฀
ield฀has฀their฀own฀view฀of฀how฀chronic฀periodontitis฀begins฀and฀some฀may฀choose฀to฀
disagree฀strongly฀with฀parts฀of฀Chapters฀13฀and฀14.฀In฀these฀chapters,฀I฀have฀attempted฀to฀
describe฀a฀coherent฀biochemical฀view฀of฀the฀development฀and฀progression฀of฀the฀various฀
chronic฀ and฀ aggressive฀ periodontal฀ diseases.฀ A฀ draft฀ version฀ of฀ these฀ chapters฀ was฀
reviewed฀ by฀ a฀ colleague,฀ Dr.฀ Thomas฀ Van฀ Dyke,฀ newly฀ appointed฀ Vice฀ President฀ of฀
Clinical฀ Research฀ and฀ Chair,฀ Department฀ of฀ Periodontology,฀ at฀ The฀ Forsyth฀ Institute,฀

vii
viii Preface

Boston.฀ Tom฀ gave฀ me฀ valuable฀ insights฀ on฀ how฀ to฀ draft฀ these฀ chapters,฀ but฀ the฀ end฀
฀product฀is฀mine.
I฀am฀indebted฀to฀the฀Oklahoma฀College฀of฀Dentistry฀Faculty,฀Dean฀Steven฀Young฀and฀
Dr.฀Kenneth฀Coy,฀for฀encouraging฀me฀to฀develop฀this฀book,฀which฀is฀based฀on฀my฀lectures฀
to฀irst-year฀dental฀students฀during฀their฀second฀semester.฀I฀very฀much฀thank฀Dr.฀Celeste฀
Wirsig,฀Associate฀Professor,฀Dept฀of฀Cell฀Biology,฀University฀of฀Oklahoma฀Health฀Sciences฀
Center฀(OUHSC),฀for฀reading฀and฀re-reading฀almost฀all฀of฀the฀many฀draft฀chapters,฀and฀for฀
igures฀credited฀to฀her;฀Dr.฀Paul฀DeAngelis,฀professor฀and฀colleague฀in฀the฀Department฀of฀
Biochemistry฀&฀Molecular฀Biology,฀University฀of฀Oklahoma฀Health฀Sciences฀Center,฀who฀
contributed฀ substantially฀ to฀ the฀ chapter฀ on฀ blood฀ clotting;฀ Dr.฀ Chadwick฀ Cox฀ who฀ irst฀
sketched฀the฀igures฀that฀Dr.฀DeAngelis฀provided฀for฀this฀book;฀and฀Ms฀Lindsay฀Collins,฀
my฀technical฀assistant,฀who฀tirelessly฀reformatted฀all฀the฀chapters฀and฀helped฀me฀negotiate฀
copyright฀approval฀for฀igures฀and฀tables฀as฀necessary.
I฀would฀also฀like฀to฀thank฀following฀who฀reviewed฀proofs:฀Dr.฀Sharon฀M.฀Wahl,฀Chief,฀
Oral฀Infection฀and฀Immunity฀Branch฀and฀some฀of฀her฀staff฀at฀NIDCR฀who฀provided฀me฀
with฀ helpful฀ suggestions฀ and฀ comments฀ on฀ many฀ of฀ the฀ chapters;฀ OUHSC฀ Graduate฀
Students฀ in฀ Biochemistry฀ and฀ Dentistry,฀ Mary฀ Tappert฀ (Chapters฀ 1฀ and฀ 2)฀ and฀ John฀ R฀
Lovell฀ (Chapters฀ 12฀ and฀ 16);฀ Dr.฀ Zsolt฀ Lohinai,฀ a฀ colleague฀ at฀ the฀ Department฀ of฀
Conservative฀ Dentistry,฀ Institute฀ of฀ Human฀ Physiology฀ and฀ Clinical฀ Experimental฀
Research,฀Semmelweis฀University,฀Budapest,฀Hungary฀(Chapter฀13);฀Dr.฀Augen฀Pioszak,฀an฀
expert฀in฀calcium฀metabolism฀and฀a฀new฀colleague฀in฀the฀Department฀(Chapters฀19฀and฀10),฀
Dr.฀DeAngelis฀(Chapter฀11)฀and฀Dr.฀Wirsig-Weichmann฀(Chapters฀3฀through฀8).
I฀wish฀to฀thank฀the฀Springer฀Verlag฀Editorial฀Board฀for฀agreeing฀to฀publish฀this฀book,฀
and฀their฀assistants฀who฀asked฀me฀every฀year฀when฀the฀book฀would฀be฀ready฀and฀who฀gave฀
me฀innumerable฀deadlines฀that฀I฀could฀not฀keep.฀I฀hope฀very฀much฀that฀this฀book฀fulils฀
their฀expectations.
Finally,฀I฀dedicate฀this฀book฀to฀my฀wife,฀Laura,฀for฀her฀continuous฀support฀of฀my฀career.฀
I฀ began฀ my฀ career฀ as฀ a฀ BDS฀ degree฀ and฀ was฀ working฀ as฀ a฀ Dentist฀ in฀ the฀ UK฀ National฀
Health฀Service฀alongside฀my฀father฀in฀Glasgow,฀Scotland฀at฀the฀age฀of฀23.฀Laura฀encour-
aged฀me฀to฀follow฀my฀dreams฀and฀undertake฀a฀BSc฀honors฀degree฀in฀Biochemistry,฀fol-
lowed฀by฀a฀PhD฀degree฀from฀the฀University฀of฀Glasgow.฀She฀and฀our฀two฀very฀young฀boys฀
accompanied฀ me฀ for฀ a฀ year฀ in฀ the฀ USA฀ on฀ a฀ Sir฀ Henry฀ Wellcome฀ Fellowship฀ at฀ the฀
University฀of฀Washington,฀Seattle฀in฀1973.฀The฀following฀year,฀I฀was฀invited฀to฀become฀a฀
visiting฀assistant฀professor฀at฀SUNY฀Buffalo,฀where฀I฀started฀to฀teach฀the฀material฀in฀this฀
book.฀In฀1976,฀I฀came฀to฀the฀Dept฀of฀Biochemistry฀and฀Molecular฀Biology฀at฀the฀University฀
of฀Oklahoma฀Health฀Sciences฀Center฀where฀I฀have฀spent฀the฀last฀34฀years.฀

Oklahoma,฀USA฀ Martin฀Levine
 Contents

1฀ Necessary฀Basics:฀Elements,฀Isotopes,฀Ions,฀Chemical฀฀
Reactions,฀Energy฀Metabolism,฀and฀Bacterial฀Structures฀.............................฀ 1
1.1.1.฀ Atomic฀Structure:฀Elements฀and฀Isotopes฀................................................ ฀ 1
1.1.2.฀ ฀Isotopes฀Date฀Paleontology฀Samples฀Such฀as฀Teeth฀................................ ฀ 4
1.1.3.฀ ฀Isotopes฀Indicate฀Ancient฀Life฀Forms฀and฀Climate฀Changes................... ฀ 5
1.1.4.฀ T฀ he฀Elements฀in฀Biology฀......................................................................... ฀ 5
1.1.5.฀ ฀Fluorides฀.................................................................................................. ฀ 6
1.2.1.฀ ฀Chemical฀Bonds฀....................................................................................... ฀ 6
1.2.2.฀ ฀Electrostatic฀Bonds฀(Ions)฀........................................................................ ฀ 6
1.2.3.฀ C฀ ovalent฀Bonds฀........................................................................................ ฀ 7
1.2.4.฀ ฀Polarized฀Covalent฀Bonds฀........................................................................ ฀ 7
1.2.5.฀ ฀Hydrophobic฀Bonds฀................................................................................. ฀ 8
1.3.1.฀ ฀Mechanisms฀of฀Energy฀Production:฀Respiration฀and฀Fermentation฀........ ฀ 9
1.3.2.฀ T฀ he฀Oral฀Microbiota,฀Dental฀Caries,฀and฀Periodontal฀Disease฀................ ฀ 10
1.4.1.฀ ฀Bacterial฀Cell฀Structures฀.......................................................................... ฀ 14
1.4.2.฀ ฀Outer฀Surface฀of฀Bacteria......................................................................... ฀ 14

2฀ Photosynthesis฀and฀Sucrose฀Production฀..........................................................฀ 17
2.1.1.฀ R฀ ole฀of฀Photosynthesis฀in฀Living฀Organisms฀........................................... ฀ 17
2.1.2.฀ ฀The฀Light฀Reaction฀.................................................................................. ฀ 18
2.2.1.฀ ฀The฀Dark฀Reaction฀................................................................................... ฀ 21
2.2.2.฀ Starch฀and฀Sucrose฀Provide฀the฀Carbon฀Skeletons฀฀
of฀All฀Plant฀Compounds฀.......................................................................... ฀ 24
2.2.3.฀ ฀Plants฀Are฀Autotrophs฀.............................................................................. ฀ 24
2.3.1.฀ Sucrose฀Is฀the฀Primary฀Transport฀Sugar฀and฀Plays฀฀
a฀Central฀Role฀in฀Plant฀Growth฀and฀Development฀.................................. ฀ 26

3฀ The฀Connective฀Tissue฀Extracellular฀Matrix฀฀
and฀Its฀Major฀Components฀...............................................................................฀ 29
3.1.1.฀ ฀Major฀Components฀of฀the฀Connective฀Tissue฀(Stromal)฀Matrix.............. ฀ 29
3.1.2.฀ ฀Collagen฀................................................................................................... ฀ 30
3.1.3.฀ ฀Elastic฀Fiber฀System฀................................................................................ ฀ 34

ix
x Contents

3.1.4.฀ G
฀ lycosaminoglycans฀................................................................................ ฀ 36
3.1.5.฀ ฀Teeth,฀Alveolar฀Bone,฀and฀Periodontium฀................................................. ฀ 36
3.2.1.฀ ฀Cell฀Surface฀Binding:฀Integrins,฀Fibronectin,฀and฀Collagen฀.................... ฀ 40
3.2.2.฀ ฀Thrombospondins฀.................................................................................... ฀ 41
3.3.1.฀ ฀Stromal฀Nutrition฀..................................................................................... ฀ 43
3.3.2.฀ ฀Stromal฀Turnover,฀Inlammation,฀and฀Bone฀Loss฀.................................... ฀ 44

4฀ Fibrillar฀and฀Non-ibrillar฀Collagens฀and฀Integrins฀.......................................฀ 45
4.1.1.฀ ฀Fibrillar฀Collagens฀................................................................................... ฀ 45
4.2.1.฀ ฀Collagen฀Fiber฀Formation฀........................................................................ ฀ 50
4.2.2.฀ ฀Fiber฀Cross-Linking:฀Formation฀of฀b-฀and฀g-Tropocollagen฀................... ฀ 52
4.3.1.฀ ฀The฀Collagen฀Superfamily฀....................................................................... ฀ 55
4.3.2.฀ ฀Fiber-Modifying฀Non-ibrillar฀Collagens฀................................................ ฀ 55
4.3.3.฀ ฀General฀Structure฀of฀Non-ibrous฀Collagens............................................ ฀ 56
4.3.4.฀ ฀Beaded฀Collagen฀Filaments฀..................................................................... ฀ 56
4.4.1.฀ ฀Integrin฀Signaling฀.................................................................................... ฀ 58

5฀ Basal฀Laminas฀and฀Epithelia฀............................................................................฀ 65
5.1.1.฀ ฀Basal฀Lamina฀........................................................................................... ฀ 65
5.1.2.฀ ฀Hemidesmosomes฀and฀Intermediate฀Filament฀Proteins฀........................... ฀ 67
5.1.3.฀ B฀ asal฀Lamina฀of฀the฀Dental฀Epithelial฀Attachment฀................................. ฀ 71
5.2.1.฀ G฀ eneral฀Structure฀of฀Skin,฀Oral฀and฀Junctional฀Epithelia฀........................ ฀ 73
5.2.2.฀ P฀ rotein฀Composition฀of฀Desmosomes฀...................................................... ฀ 75
5.2.3.฀ O฀ ral฀and฀Junctional฀Epithelium฀............................................................... ฀ 76

6฀ Elastic฀Fibers฀and฀Proteoglycans฀......................................................................฀ 81
6.1.1.฀ F฀ ibrillin฀.................................................................................................... ฀ 81
6.2.1.฀ E฀ lastin฀...................................................................................................... ฀ 85
6.3.1.฀ G฀ lycosaminoglycans฀................................................................................ ฀ 89
6.4.1.฀ P฀ roteo-Glycosaminoglycan฀Core฀Proteins฀and฀Cartilage฀Collagens฀....... ฀ 95
6.5.1.฀ ฀Major฀Collagen–Glycosaminoglycan฀Interactions฀.................................. ฀ 98

7฀ Collagen฀Synthesis,฀Genetic฀Diseases,฀and฀Scurvy฀..........................................฀ 101
7.1.1.฀ ฀Intracellular฀Collagen฀Synthesis฀.............................................................. ฀ 101
7.2.1.฀ ฀Effects฀of฀Collagen฀Polypeptide฀Mutations฀............................................. ฀ 104
7.2.2.฀ ฀Ehlers-Danlos฀Syndrome฀(EDS)฀.............................................................. ฀ 105
7.3.1.฀ ฀Ascorbate฀and฀Antioxidants฀..................................................................... ฀ 109

8฀ The฀Zincins:฀Collagen฀Fiber฀Processing฀and฀Degradation฀............................฀ 113
8.1.1.฀ ฀The฀Zincin฀Enzyme฀Family฀..................................................................... ฀ 113
8.1.2.฀ ฀Catalytic฀Action฀of฀the฀Metzincin฀Family฀............................................... ฀ 115
8.1.3.฀ ฀Metzincin฀Activation฀............................................................................... ฀ 117
8.2.1.฀ ฀Fibrillar฀Procollagen฀Processing฀.............................................................. ฀ 120
8.3.1.฀ ฀Matrilysins:฀Degradation฀of฀Collagen฀and฀Stromal฀Proteins฀................... ฀ 124
8.3.2.฀ ฀Stromelysins฀............................................................................................. ฀ 126
Contents xi

8.3.3.฀ E฀ namelysin฀............................................................................................... ฀ 126

8.3.4.฀ ฀Collagenases฀and฀Gelatinases฀.................................................................. ฀ 127

9฀ Biological฀Mineralization฀..................................................................................฀ 129
9.1.1.฀ ฀Fundamental฀Properties฀of฀Calcium฀Phosphate฀Precipitation฀.................. ฀ 129
9.1.2.฀ ฀Nature฀of฀the฀Apatite฀Precipitate.............................................................. ฀ 130
9.1.3.฀ Apatite฀Crystal฀Substitutions฀Inluence฀฀
Bone฀Strength฀and฀Solubility฀................................................................... ฀ 131
9.1.4.฀ ฀Nucleation฀................................................................................................ ฀ 131
9.2.1.฀ ฀The฀Structures฀of฀Bone,฀Dentin,฀and฀Cementum฀..................................... ฀ 132
9.2.2.฀ T฀ wo฀Mechanisms฀of฀Mineralization฀........................................................ ฀ 134
9.3.1.฀ S฀ ecretion฀of฀Osteoid฀Matrix฀..................................................................... ฀ 134
9.3.2.฀ Osteoclast฀Transport฀of฀Calcium฀and฀Phosphate฀฀
Ions฀to฀Matrix฀Vesicles฀............................................................................ ฀ 136
9.3.3.฀ Calcium฀and฀Phosphate฀Ions฀Precipitate฀and฀Rupture฀฀
Secreted฀Matrix฀Vesicles฀.......................................................................... ฀ 138
9.3.4.฀ Structure฀of฀the฀Calcium฀Transporter฀Proteins฀฀
in฀Matrix฀Vesicles฀.................................................................................... ฀ 138
9.3.5.฀ The฀Phosphate฀Transporter฀Proteins฀and฀Pyrophosphate฀฀
in฀Matrix฀Vesicles฀.................................................................................... ฀ 140
9.4.1.฀ S฀ IBLING฀Proteins,฀Phex฀Gene฀Function,฀and฀Hypophosphatemia฀......... ฀ 142
9.4.2.฀ O฀ steocalcin฀Is฀Required฀for฀Bone฀Modeling฀........................................... ฀ 143
9.5.1.฀ E฀ namel฀Organ฀and฀Matrix฀Development฀................................................. ฀ 144
9.5.2.฀ P฀ roteins฀Involved฀in฀Enamel฀Synthesis฀................................................... ฀ 146
9.5.3.฀ ฀Proposed฀Mechanism฀of฀Enamel฀Synthesis฀............................................. ฀ 147
9.6.1.฀ ฀Summary฀of฀Ways฀in฀Which฀Enamel฀and฀Bone฀Differ฀............................ ฀ 150
9.6.2.฀ ฀Summary฀of฀the฀Vitamins฀for฀Bone฀and฀Enamel฀Formation.................... ฀ 151

10฀ Bone฀Remodeling฀and฀Calcium฀Metabolism฀...................................................฀ 153

10.1.1.฀฀Bone฀Turnover,฀Osteoclasts,฀and฀Lysosomes฀........................................... ฀ 153
10.1.2.฀฀Proteolysis฀in฀the฀Bone฀Resorbing฀Compartment฀.................................... ฀ 157
10.1.3.฀฀Demineralization฀and฀Remineralization฀.................................................. ฀ 157
10.1.4.฀฀Osteoclast฀Ion฀and฀Proton฀Transport฀........................................................ ฀ 159
10.2.1.฀฀Osteoclast฀Differentiation฀........................................................................ ฀ 160
10.2.2.฀฀Osteoclasts฀and฀Inlammation฀.................................................................. ฀ 161
10.2.3.฀฀Osteoporosis:฀Major฀Causes฀and฀Therapies฀............................................. ฀ 163
10.3.1.฀฀Calcium฀Metabolism,฀Parathyroid฀Hormone,฀and฀Calcitriol฀................... ฀ 165
10.4.1.฀฀Vitamin฀D,฀Calcitriol,฀and฀Calbindins...................................................... ฀ 168
10.4.2.฀฀Rickets฀and฀Osteomalacia฀........................................................................ ฀ 171
10.5.1.฀฀Actions฀of฀Calcitonin฀............................................................................... ฀ 172
10.5.2.฀฀Calcitonin฀and฀PTH฀Therapy฀for฀Osteoporosis฀........................................ ฀ 173

11฀ Blood฀Coagulation฀..............................................................................................฀ 175

11.1.1.฀ T
฀ he฀Vascular฀System฀................................................................................ ฀ 175
11.1.2.฀ B
฀ leeding฀and฀Blood฀Clotting฀Problems฀................................................... ฀ 176
xii Contents

11.2.1.฀ B฀ lood฀Vessel฀Injury,฀von฀Willebrand฀Factor,฀and฀Platelets฀...................... ฀ 178

11.2.2.฀ The฀Gamma-฀(g-)฀Carboxyglutamate฀(gla)฀Domain:฀฀
A฀Calcium฀Ion฀Chelator฀........................................................................... ฀ 180
11.3.1.฀ ฀The฀Extrinsic,฀Intrinsic,฀and฀Common฀Coagulation฀Pathways฀................ ฀ 183
11.3.2.฀ ฀The฀Extrinsic฀Pathway฀............................................................................. ฀ 183
11.3.3.฀ ฀The฀Intrinsic฀Pathway฀.............................................................................. ฀ 185
11.3.4.฀ ฀The฀Common฀Pathway฀............................................................................. ฀ 185
11.3.5.฀ ฀The฀Hemophiliac฀(Excessive฀Bleeding฀Diseases).................................... ฀ 187
11.4.1.฀ ฀The฀Fibrin฀Blood฀Clot:฀Production฀and฀Prevention฀................................. ฀ 188
11.4.2.฀ ฀Removal฀of฀a฀Blood฀Clot฀......................................................................... ฀ 190
11.5.1.฀ ฀Prevention฀of฀Unwanted฀Blood฀Clotting฀................................................. ฀ 192
11.5.2.฀ ฀Protein฀S,฀Factor฀V฀and฀Factor฀VIII฀Mutations฀....................................... ฀ 194
11.6.1.฀ Drugs฀to฀Remove฀a฀Pathogenic฀Thrombus฀฀
or฀Embolus:฀“Clot฀Busters”฀..................................................................... ฀ 195
11.6.2.฀ ฀Drugs฀That฀Inhibit฀Excessive฀Clot฀Formation฀......................................... ฀ 197
11.6.3.฀ D฀ rugs฀That฀Retard฀Clot฀Formation฀.......................................................... ฀ 197
11.6.4.฀ D฀ rugs฀That฀Inhibit฀Platelet฀Activation฀..................................................... ฀ 198
11.6.5.฀ D฀ rugs฀That฀Promote฀Clotting฀................................................................... ฀ 198
11.6.6.฀ Laboratory฀Tests฀to฀Determine฀the฀State฀฀
of฀the฀Blood฀Clotting฀System฀................................................................... ฀ 199

12฀ Saliva฀...................................................................................................................฀ 203

12.1.1.฀฀Cell฀Biology฀of฀Salivary฀Glands฀.............................................................. ฀ 203
12.1.2.฀฀Whole฀Saliva:฀Collection฀and฀Composition฀............................................. ฀ 203
12.1.3.฀฀Functions฀of฀the฀Salivary฀Components฀.................................................... ฀ 205
12.1.4.฀฀Innate฀and฀Acquired฀Immune฀Proteins฀in฀Saliva฀..................................... ฀ 207
12.1.5.฀฀Poor฀Oral฀Hygiene฀Adds฀Bacteria฀and฀Host฀Leukocyte฀Products฀to฀Saliva฀฀ 208
12.2.1.฀฀Physiology฀and฀Biochemistry฀of฀Saliva฀Secretion฀................................... ฀ 209
12.3.1.฀฀Salivary฀Mucin฀Composition฀................................................................... ฀ 211
12.3.2.฀฀Glycan฀Composition฀of฀Salivary฀Mucins฀................................................. ฀ 213
12.4.1.฀฀Mucin฀Glycans,฀ABO฀Antigens,฀and฀Forensic฀Dentistry฀......................... ฀ 216
12.5.1.฀฀Amylase:฀Substrates,฀Products,฀and฀Mode฀of฀Action฀.............................. ฀ 219
12.5.2.฀฀Mechanism฀of฀Action฀of฀Salivary฀Amylase฀............................................. ฀ 221
12.5.3.฀฀Detection฀of฀Salivary฀and฀Pancreatic฀Amylase฀........................................ ฀ 225
12.6.1.฀฀Proline-Rich฀Proteins฀............................................................................... ฀ 227
12.6.2.฀฀Salivary฀Agglutinin฀.................................................................................. ฀ 228

13฀ Chronic฀Periodontitis฀.........................................................................................฀ 231

13.1.1.฀฀Detecting฀Periodontal฀Disease฀................................................................. ฀ 231
13.1.2.฀฀Microbiota฀in฀Gingivitis฀and฀Chronic฀Periodontitis฀................................ ฀ 232
13.1.3.฀฀Drugs฀and฀Tooth฀Cleaning฀to฀Prevent฀Gingivitis฀..................................... ฀ 236
13.2.1.฀฀Mammalian฀Cells฀Recognize฀Prokaryotic฀Molecules฀.............................. ฀ 237
13.2.2.฀PAMPs฀Induce฀PRRs฀to฀Release฀Cytokines฀฀
That฀Attract฀Leukocytes฀........................................................................... ฀ 238
13.2.3.฀฀IL-1฀Induces฀Gingival฀Inlammation฀....................................................... ฀ 241
13.2.4.฀฀Neutrophils฀Function฀in฀Tissue฀Destruction฀............................................ ฀ 241
Contents xiii

13.2.5.฀Gingivitis฀is฀Reversible;฀Antiinlammatory฀฀
Cytokines฀Mediate฀Repair฀....................................................................... ฀ 243
13.3.1.฀Monocytes฀Are฀Converted฀to฀Macrophages฀฀
That฀Over-Activate฀Collagenase฀.............................................................. ฀ 244
13.4.1.฀฀Apoptosis฀in฀Chronic฀Periodontitis฀.......................................................... ฀ 246
13.4.2.฀฀Intracellular฀Induction฀of฀Apoptosis฀........................................................ ฀ 247
13.4.3.฀฀Mechanisms฀of฀Apoptosis฀........................................................................ ฀ 249
13.5.1.฀฀Eicosanoids฀and฀Periodontal฀Repair฀........................................................ ฀ 252
13.5.2.฀฀Eicosanoid฀Structure฀................................................................................ ฀ 252
13.5.3.฀฀Functions฀of฀the฀Proinlammatory฀Eicosanoids฀....................................... ฀ 255
13.5.4.฀฀Lipoxygenase-Mediated฀Resolution฀of฀Inlammation฀............................. ฀ 255
13.5.5.฀฀Antiinlammatory฀Drugs฀.......................................................................... ฀ 257

14฀ Aggressive฀Periodontitis฀....................................................................................฀ 259

14.1.1.฀฀Generalized฀Aggressive฀Periodontitis฀...................................................... ฀ 259
14.2.1.฀฀Localized฀Aggressive฀Periodontitis฀......................................................... ฀ 260
14.2.2.฀฀Aa฀Leukotoxin฀Composition฀and฀Properties฀............................................ ฀ 261
14.2.3.฀฀Mutations฀Enhance฀Aa฀Ltx฀and฀LAP฀Severity฀......................................... ฀ 263
14.2.4.฀฀Cytolethal฀Distending฀Toxin฀(Cdt)........................................................... ฀ 264

15฀ Dental฀Caries฀......................................................................................................฀ 267

15.1.1.฀฀Dental฀Caries:฀Deinition฀and฀Measurement฀............................................ ฀ 267
15.1.2.฀฀Sugar,฀Dental฀Caries,฀and฀the฀Dental฀Profession฀..................................... ฀ 267
15.1.3.฀฀Sucrose฀and฀the฀Appearance฀of฀Acid฀in฀Dental฀Bioilms฀........................ ฀ 269
15.1.4.฀฀Cavities฀in฀Animals฀and฀Streptococcus฀mutans฀....................................... ฀ 270
15.2.1.฀How฀Sucrose฀Connects฀S.฀mutans฀to฀the฀Oral฀Microbiota฀฀
and฀Dental฀Caries฀..................................................................................... ฀ 272
15.2.2.฀฀Sources฀of฀Bacterial฀Lactic฀Acid฀in฀Caries฀.............................................. ฀ 277
15.2.3.฀฀Dentinal฀(Advanced)฀Dental฀Caries฀......................................................... ฀ 277
15.3.1.฀฀Variation฀in฀Individual฀Human฀Caries฀Experience฀.................................. ฀ 278
15.3.2.฀฀Bacterial฀Causes฀of฀the฀Variation฀in฀Caries฀Susceptibility฀...................... ฀ 279
15.3.3.฀฀Saliva฀Causes฀of฀Caries฀Susceptibility฀..................................................... ฀ 281
15.3.4.฀฀Caries฀Immunity฀and฀Susceptibility฀......................................................... ฀ 282

16฀ Fluoride฀...............................................................................................................฀ 285

16.1.1.฀Properties฀of฀Fluorine฀and฀Fluoride฀......................................................... ฀ 285
16.1.2.฀How฀Mottled฀Enamel฀Was฀Related฀to฀Fluoride฀฀
in฀the฀Water฀Supply฀.................................................................................. ฀ 286
16.1.3.฀Mottled฀Enamel฀Is฀Moderate฀to฀Severe฀Enamel฀Fluorosis฀...................... ฀ 287
16.1.4.฀Identiication฀of฀1฀ppm฀Fluoride฀in฀the฀Water฀as฀Optimal฀฀
for฀Cavity฀Protection฀................................................................................ ฀ 289
16.2.1.฀Mechanisms฀of฀Fluoride฀Protection฀from฀Caries฀..................................... ฀ 291
16.2.2.฀How฀Fluoride฀Protects฀from฀Caries฀......................................................... ฀ 292
16.3.1.฀Systemic฀Effects฀of฀Fluoride฀.................................................................... ฀ 294
16.3.2.฀Fluoride฀Toxicity฀...................................................................................... ฀ 295

Index฀..........................................................................................................................฀ 299
 Necessary Basics: Elements, Isotopes,
Ions, Chemical Reactions, Energy 1
Metabolism, and Bacterial Structures

This฀chapter฀describes฀how฀a฀traditional฀discussion฀of฀atomic฀structure฀and฀its฀relationship฀
to฀organic฀chemistry฀ is฀ relevant฀ to฀ dentistry.฀ Although฀ the฀fundamental฀ chemistry฀ and฀
biochemistry฀ described฀ here฀ are฀ common฀ knowledge,฀ the฀ discussion฀ centers฀ on฀ the฀
associations฀ with฀ teeth฀ and฀ dental฀ disease฀ (Sects.฀ 1฀ and฀ 2).฀ In฀ addition,฀ the฀ difference฀
between฀respiration฀and฀fermentation฀and฀the฀different฀types฀of฀fermentations฀involved฀in฀
dental฀caries฀and฀periodontal฀disease฀are฀discussed฀(Sect.฀3).฀The฀chapter฀concludes฀with฀
a฀discussion฀of฀the฀difference฀between฀the฀outer฀cell฀surface฀of฀gram-positive฀and฀gram-
negative฀bacteria฀that฀is฀important฀in฀causing฀these฀diseases฀(Sect.฀4).

1.1.1.
Atomic Structure: Elements and Isotopes

Matter฀ is฀ made฀ up฀ of฀ atoms,฀ which฀ are฀ composed฀ of฀ protons,฀ neutrons,฀ and฀ electrons.฀
Protons฀ are฀ positively฀ charged฀ particles,฀ electrons฀ are฀ negatively฀ charged฀ particles,฀ and฀
neutrons฀are฀uncharged฀particles.฀Neutrons฀glue฀the฀protons฀together฀and฀prevent฀the฀posi-
tively฀ charged฀ protons฀ from฀ repelling฀ each฀ other฀ and฀ destroying฀ the฀ nucleus.฀ The฀ posi-
tively฀charged฀atomic฀nucleus฀is฀electrically฀neutral฀because฀it฀is฀surrounded฀by฀a฀number฀
of฀negatively฀charged฀electrons฀equal฀to฀the฀number฀of฀protons.฀Electrons฀are฀insigniicant฀
with฀ respect฀ to฀ nuclear฀ mass฀ but฀ provide฀ chemical฀ reactivity.฀ The฀ number฀ of฀ protons฀
(atomic฀number)฀deines฀the฀elements฀of฀the฀chemical฀periodic฀table฀(Fig.฀1.1)฀because฀it฀
determines฀the฀number฀of฀electrons.
The฀nucleus฀of฀the฀irst฀element,฀hydrogen,฀consists฀only฀of฀a฀proton.฀The฀second,฀helium,฀
has฀two฀protons฀and฀two฀neutrons.฀The฀third,฀lithium,฀has฀three฀protons฀and฀three฀neutrons,฀
and฀so฀on฀(Fig.฀1.2).฀The฀atomic฀weight,฀measured฀as฀daltons฀(Da),฀is฀the฀sum฀of฀the฀number฀
of฀protons฀and฀neutrons฀in฀an฀element’s฀atomic฀nucleus.฀Hydrogen฀has฀a฀mass฀of฀1฀Da฀(the฀
mass฀of฀a฀proton).฀Helium฀has฀two฀protons฀and฀two฀neutrons฀to฀hold฀the฀protons฀together.฀It฀
therefore฀has฀a฀mass฀of฀4฀Da.฀Lithium฀has฀three฀protons฀and฀three฀neutrons฀and฀therefore฀a฀
mass฀of฀6฀Da฀(Fig.฀1.3).฀Oxygen฀has฀an฀atomic฀number฀of฀8฀and฀atomic฀mass฀of฀16.
Isotopes฀are฀elements฀possessing฀different฀numbers฀of฀neutrons.฀Deuterium,฀a฀hydrogen฀
atom฀ containing฀ a฀ neutron฀ in฀ addition฀ to฀ a฀ proton,฀ has฀ the฀ same฀ chemical฀ properties฀ as฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 1
DOI:฀10.1007/978-3-540-88116-2_1,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
2 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

Fig. 1.1฀ Periodic฀table฀showing฀the฀elements฀in฀the฀major฀groups฀and฀transition฀metals.฀Only฀the฀

elements฀ associated฀ with฀ life฀ are฀ listed.฀ The฀ common฀ elements฀ are฀ in฀ dark฀ green.฀ Light฀
orange/yellow฀or฀pink/white฀backgrounds฀indicate฀the฀metal฀and฀halide฀elements฀that฀occur฀as฀ions.฀
Trace฀elements฀are฀indicated฀in฀red,฀pink,฀or฀light฀green฀(Modiied฀from฀the฀full฀Periodic฀Table฀
found฀in฀Wikipedia฀at฀http://en.wikipedia.org/wiki/Periodic_table)

Fig. 1.2฀ Proton,฀neutron฀and฀electron

1.1.1. Atomic Structure: Elements and Isotopes 3

Fig. 1.3฀ Atomic฀composition฀

of฀hydrogen,฀helium,฀and฀
lithium,฀the฀irst฀three฀
elements฀of฀the฀periodic฀
table.฀(Diagram฀prepared฀by฀
Dr฀Wirsig-Weichmann)

Hydrogen

Deuterium
β Emission

Fig. 1.4฀ Isotopes฀of฀hydrogen.฀Tritium฀is฀unstable.฀฀

It฀changes฀to฀helium฀by฀a฀neutron฀emitting฀a฀ Tritium
b-particle฀and฀becoming฀a฀second฀proton.฀฀
The฀b-particle฀is฀a฀radioactive฀emission.฀(Diagram฀
prepared฀by฀Dr฀Wirsig-Weichmann)฀ Helium

hydrogen฀but฀twice฀the฀atomic฀mass฀(heavy฀hydrogen),฀2฀Da฀instead฀of฀1฀(Fig.฀1.4).฀There฀
is฀also฀a฀rarely฀occurring฀third฀isotope฀of฀hydrogen฀in฀nature,฀tritium.฀Tritium฀is฀important฀
because฀it฀is฀radioactive;฀one฀of฀its฀neutrons฀is฀unstable฀and฀releases฀a฀beta฀(β)฀particle.฀
Beta฀particles฀resemble฀fast-moving,฀free฀electrons฀that฀may฀be฀positively฀charged฀(posi-
trons),฀or฀more฀commonly฀negatively฀charged฀(electrons).฀The฀release฀of฀a฀β-particle฀con-
verts฀one฀of฀the฀two฀neutrons฀of฀tritium฀(Fig.฀1.3)฀to฀a฀proton,฀making฀a฀stable฀isotope฀of฀
helium฀(two฀protons฀and฀one฀neutron,฀Fig.฀1.4).฀Table฀1.1฀lists฀the฀radioactive฀elements฀
important฀in฀biology,฀the฀nature฀of฀the฀emitted฀radioactivity,฀what฀element฀they฀decay฀to,฀
and฀their฀usual฀use฀in฀biology.
By฀the฀third฀decade฀of฀the฀twentieth฀century,฀the฀emitted฀radioactivity฀from฀artiicially฀
synthesized฀tritium฀(3H)฀and฀carbon-14฀(14C)฀was฀used฀to฀follow฀biochemical฀reactions.฀
Glucose,฀amino฀acids,฀and฀other฀chemical฀compounds฀were฀synthesized฀with฀ 3H฀or฀ 14C฀
incorporated฀either฀randomly฀or฀at฀some฀known฀position฀in฀the฀molecule.฀The฀radioac-
tive฀ products฀ derived฀ from฀ radiolabeled฀ glucose฀ or฀ amino฀ acids฀ added฀ to฀ cells,฀ tissue฀
slices,฀or฀a฀whole฀organism฀identiied฀the฀metabolic฀fate฀of฀these฀molecules฀under฀deined฀
4 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

Table 1.1฀ Radioactive฀isotopes฀important฀in฀biology

Element Symbol Isotope Symbol Decays฀to Symbol Primary฀Use

Hydrogen 1
H a
Tritium 3
H Heliumb 3
He Metabolic฀pathways
Carbon 12
C a
Carbon-14 14
C Nitrogen 14
N Metabolic฀pathways
Phosphorus 31
P c
Phosphorus-32 32
P Sulfur 32
S Phosphorylations
Calcium 40
Ca a
Calcium-41 41
C Potassium 41
K Bone฀metabolism
Iodine 127
I a
Iodine-131 131
I Xenon 131
Xe Thyroid฀cancer฀
therapy
Iodine 127
I d
Iodine-125 125
I Telurium- 125
Te Protein฀labeling
125d
Notes:
a
Weak b-emission
b
Rare isotope
c
Strong b-emission
d 125
Emits weak gamma rays giving an unstable isotope of Te that stabilizes by capturing a photon and
emitting an Xray

conditions.฀ These฀ studies฀ led฀ to฀ the฀ establishment฀ of฀ the฀ metabolic฀ pathways฀ that฀฀
we฀ now฀ take฀ for฀ granted฀ such฀ as฀ protein,฀ fatty฀ acid,฀ amino฀ acid,฀ and฀ nucleic฀ acid฀
metabolism.

1.1.2.
Isotopes Date Paleontology Samples Such as Teeth

Another฀use฀of฀radioactive฀carbon-14฀is฀to฀date฀prehistoric฀bones฀and฀teeth,฀the฀longest-
surviving฀parts฀of฀the฀body.฀Carbon-14฀(14C;฀six฀protons฀and฀eight฀neutrons)฀is฀continually฀
formed฀in฀the฀upper฀atmosphere฀by฀cosmic฀rays฀acting฀on฀nitrogen฀(14N;฀seven฀protons฀and฀
seven฀neutrons).฀The฀energy฀of฀the฀cosmic฀rays฀causes฀a฀proton฀and฀electron฀in฀atoms฀of฀14N฀
to฀fuse฀into฀a฀neutron.฀The฀newly฀formed฀14C฀is฀rapidly฀oxidized฀to฀14CO2,฀which฀enters฀the฀
earth’s฀plant฀and฀animal฀life฀through฀photosynthesis฀and฀the฀food฀chain.฀The฀ratio฀of฀14C฀to฀
nonradioactive฀carbon฀is฀approximately฀constant฀over฀time.฀When฀plants฀or฀animals฀die,฀
carbon฀uptake฀ceases,฀14C฀is฀not฀replenished,฀and฀it฀slowly฀decays฀with฀a฀half-life฀of฀close฀to฀
5,700฀years.฀The฀radioactivity฀of฀a฀sample฀whose฀age฀is฀not฀known฀can฀be฀used฀to฀indicate฀
the฀amount฀of฀ 14C฀remaining,฀provided฀it฀is฀not฀more฀than฀40,000฀years฀of฀age.฀After฀that฀
time,฀so฀much฀14C฀has฀decayed฀that฀what฀is฀left฀is฀not฀measurable.฀From฀the฀ratio฀of฀radioac-
tive฀carbon฀to฀total฀carbon฀content,฀it฀is฀possible฀to฀determine฀how฀much฀was฀lost฀relative฀to฀
the฀current฀ratio฀(unchanged฀over฀billions฀of฀years)฀and฀therefore฀how฀long฀ago฀the฀bone฀or฀
tooth฀was฀part฀of฀a฀living฀organism.
1.1.4. The Elements in Biology 5

1.1.3.
Isotopes Indicate Ancient Life Forms and Climate Changes

Not฀ all฀ isotopes฀ are฀ radioactive,฀ and฀ even฀ some฀ stable฀ isotopes฀ can฀ be฀ useful.฀ A฀ stable฀
isotope฀of฀carbon,฀carbon-13฀(13C;฀6฀protons฀and฀7฀neutrons)฀has฀accounts฀for฀about฀1%฀of฀
carbon฀atoms฀on฀earth.฀Because฀it฀is฀chemically฀more฀stable฀than฀ 12C,฀living฀organisms฀
preferentially฀utilize฀12C฀for฀chemical฀reactions฀(metabolism).฀Therefore,฀rocks฀containing฀
a฀greater฀than฀usual฀12C/13C฀ratio฀are฀potentially฀‘chemical฀ingerprints’฀of฀life.฀Minute฀resi-
dues฀ in฀ some฀ of฀ the฀ oldest฀ rocks฀ on฀ Earth,฀ from฀ Akilia฀ Island฀ near฀ Greenland,฀ have฀ a฀
chemical฀ingerprint฀that฀may฀have฀come฀from฀living฀organisms.฀Analysis฀of฀selected฀tiny฀
samples฀ using฀ an฀ ion฀ microprobe฀ revealed฀ ratios฀ of฀ carbon-13฀ to฀ carbon-12฀ that฀ were฀
2–5%฀less฀than฀expected.฀Some฀prebiotic฀process฀may฀have฀enriched฀the฀rock฀with฀car-
bon-12฀atoms,฀and฀it฀lay฀apparently฀undisturbed฀(based฀on฀the฀surrounding฀crystal฀struc-
ture)฀for฀over฀3,700฀million฀years฀(3.7฀Giga-years,฀Gyr).฀The฀age฀of฀ancient฀rock฀samples฀
is฀independently฀determined฀from฀the฀rate฀of฀decay฀of฀another฀isotope,฀87Rubidium฀(87Rb)฀
to฀87Strontium฀(87Sr).฀The฀half-life฀of฀this฀decay฀is฀about฀1.0฀Gyr.฀Amounts฀of฀87Sr฀and฀87Rb฀
are฀measured,฀and฀a฀ratio฀is฀derived฀and฀compared฀with฀the฀ratio฀of฀87Sr฀to฀stable฀strontium฀
(86Sr)฀in฀the฀rock฀sample.฀The฀age฀of฀the฀rock฀sample฀is฀then฀derived฀mathematically฀from฀
these฀ratios,฀given฀the฀half-life฀of฀87Rb.
The฀common฀element฀of฀oxygen฀has฀eight฀protons฀and฀eight฀neutrons.฀A฀stable฀isotope฀
with฀ two฀ additional฀ neutrons฀ is฀ also฀ relatively฀ common.฀ Because฀ water-containing฀ 18O฀
evaporates฀more฀slowly฀and฀condenses฀more฀rapidly,฀the฀ratio฀of฀ 18O/16O฀in฀Antarctic฀ice฀
cores฀indicates฀major฀climate฀shifts฀since฀early฀in฀geologic฀time.฀Increased฀18O฀indicates฀a฀
sudden฀warming,฀and฀increased฀16O฀indicates฀a฀sudden฀cooling.

1.1.4.
The Elements in Biology

The฀electrons฀of฀the฀elements฀are฀arranged฀in฀shells฀surrounding฀the฀nucleus.฀The฀irst฀shell฀
consists฀of฀two฀electrons,฀the฀second฀and฀third฀each฀of฀eight฀electrons,฀and฀the฀fourth฀and฀
ifth฀each฀of฀18฀electrons.฀Elements฀take฀part฀in฀a฀chemical฀reaction฀by฀gaining฀or฀sharing฀
electrons฀ to฀ complete฀ their฀ outer฀ shell฀ except฀ for฀ the฀ noble฀ gases฀ (helium,฀ neon,฀ argon,฀
krypton฀and฀xenon,฀far฀right-hand฀column฀of฀the฀periodic฀table).฀These฀elements฀already฀
have฀a฀complete฀outermost฀electron฀shell.฀They฀therefore฀have฀no฀chemical฀reactivity฀and฀
are฀incompatible฀with฀life.
Figure฀ 1.1฀ shows฀ the฀ elements฀ important฀ for฀ all฀ of฀ life.฀ The฀ common฀ elements฀ are฀
depicted฀in฀dark฀green฀boxes:฀carbon,฀hydrogen,฀nitrogen,฀oxygen,฀phosphorous,฀and฀sulfur.฀
Elements฀depicted฀in฀light฀orange/yellow฀or฀pink/white฀boxes฀make฀up฀the฀major฀cations฀
and฀anions฀of฀living฀organisms:฀sodium,฀potassium,฀magnesium,฀calcium,฀manganese,฀iron,฀
cobalt,฀nickel,฀copper,฀zinc,฀chlorine,฀and฀iodine.฀Trace฀elements฀(light฀green,฀pink,฀or฀red฀
boxes)฀occur฀as฀occasional฀enzyme฀cofactors:฀boron,฀luorine,฀silicon,฀arsenic,฀selenium฀and฀
bromine,฀aluminum,฀gallium,฀chromium,฀vanadium,฀molybdenum,฀tungsten,฀and฀cadmium.
6 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

1.1.5.
Fluorides

Fluorine฀is฀present฀on฀earth฀only฀as฀luoride,฀a฀negatively฀charged฀anion฀that฀is฀especially฀
important฀ in฀ dentistry฀ because฀ of฀ its฀ ability฀ to฀ mediate฀ protection฀ from฀ dental฀ caries฀
(Chapter฀16,฀section฀16.2.1.).฀Although฀metabolites฀of฀chlorine฀and฀iodine฀are฀ubiquitous,฀
few฀ biological฀ products฀ contain฀ luoride฀ because฀ of฀ its฀ tight฀ hydration฀ shell.฀ The฀ few฀
enzymes฀that฀utilize฀luoride฀as฀a฀cofactor฀must฀overcome฀an฀exceptionally฀high฀desolva-
tion฀energy฀barrier.฀
The฀luoride฀content฀of฀a฀solution฀is฀measured฀with฀an฀electrode฀made฀from฀a฀mixture฀
of฀lanthanum฀and฀europium฀luorides.฀Lanthanum฀follows฀barium฀in฀the฀periodic฀table฀and฀
has฀an฀atomic฀number฀of฀57.฀Lanthanum฀is฀also฀the฀irst฀of฀a฀series฀of฀15฀heavy฀metals฀
before฀hafnium฀called฀lanthanides.฀Europium฀is฀a฀lanthanide฀and฀its฀atomic฀number฀is฀63.฀
The฀mode฀of฀action฀of฀the฀luoride฀electrode฀is฀described฀in฀Chapter฀16,฀sect.฀16.1.1.

Matter฀consists฀of฀atoms฀that฀are฀made฀up฀of฀protons฀(electropositive),฀electrons฀(electro-
negative),฀and฀neutrons฀(electrically฀neutral).฀Because฀the฀number฀of฀electrons฀and฀pro-
tons฀is฀equal,฀elements,฀atoms฀with฀different฀numbers฀of฀protons,฀have฀different฀numbers฀
of฀electrons.฀The฀chemical฀properties฀of฀an฀element฀depend฀on฀the฀number฀of฀electrons,฀
but฀because฀the฀electrons฀have฀almost฀no฀mass,฀the฀atomic฀weight฀of฀an฀element฀is฀its฀
number฀of฀protons฀and฀neutrons.฀Neutrons฀are฀needed฀to฀hold฀the฀protons฀together฀in฀the฀
nucleus.฀ Isotopes฀ are฀ elements฀ with฀ different฀ numbers฀ of฀ neutrons.฀ Isotopes฀ with฀ too฀
many฀neutrons฀are฀unstable฀and฀emit฀radioactivity.฀Radioactive฀and฀nonradioactive฀iso-
topes฀are฀used฀to฀follow฀biochemical฀reactions฀in฀health฀and฀disease,฀to฀date฀paleontology฀
specimens,฀usually฀bones฀and฀teeth,฀and฀detect฀traces฀of฀life฀in฀ancient฀rocks.

1.2.1.
Chemical Bonds

Four฀ chemical฀ bonds฀ are฀ important฀ in฀ living฀ organisms:฀ electrostatic฀ bonds,฀ covalent฀
bonds,฀polarized฀covalent฀bonds,฀and฀hydrophobic฀bonds.

1.2.2.
Electrostatic Bonds

Electrostatic฀ bonds฀ usually฀ form฀ ions;฀ sodium฀ chloride฀ and฀ potassium฀ chloride฀ are฀
examples฀of฀ionic฀compounds.฀Sodium฀(or฀potassium)฀has฀a฀single฀electron฀in฀its฀outer฀
1.2.4. Polarized Covalent Bonds 7

shell,฀whereas฀chlorine฀is฀missing฀an฀electron฀in฀its฀outer฀shell.฀The฀salt,฀sodium฀chlo-
ride,฀is฀an฀electrostatic฀compound฀consisting฀of฀sodium฀ions,฀sodium฀atoms฀that฀have฀
donated฀an฀electron฀and฀become฀positively฀charged฀(cations),฀and฀chloridions,฀chlorine฀
atoms฀that฀have฀received฀an฀electron฀and฀become฀negatively฀charged฀(anions).
Most฀ electrostatically฀ bonded฀ solids฀ form฀ crystals.฀ In฀ sodium฀ chloride,฀ the฀ sodium฀
cations฀ and฀ chloride฀ anions฀ forms฀ an฀ electrically฀ neutral฀ square฀ –฀ a฀ crystal฀ cell.฀ This฀
simple฀shape฀causes฀sodium฀chloride฀crystals฀to฀be฀granular.฀By฀contrast,฀calcium฀phos-
phate฀ is฀ a฀ more฀ complex฀ ion฀ pair,฀ and฀ it฀ has฀ a฀ correspondingly฀ more฀ complex฀ crystal฀
structure฀(apatite).฀Apatite฀forms฀the฀mineralized฀structures฀of฀bones฀and฀teeth฀–฀a฀hard,฀
smooth฀agranular฀surface.฀Calcium฀forms฀cations฀by฀losing฀two฀electrons.฀Phosphate฀is฀
not฀ an฀ elemental฀ anion;฀ it฀ contains฀ four฀ oxygen฀ atoms฀ that฀ are฀ covalently฀ bound฀ to฀ a฀
central฀phosphorus฀atom.฀Phosphates฀have฀one,฀two,฀or฀three฀negative฀charges฀(monova-
lent,฀divalent,฀or฀trivalent)฀depending฀on฀the฀pH฀of฀the฀surrounding฀solution.฀As฀the฀pH฀
of฀ the฀ solution฀ increases,฀ so฀ also฀ does฀ the฀ net฀ charge฀ of฀ the฀ phosphate฀ ion,฀ causing฀ a฀
calcium฀ phosphate฀ precipitate฀ to฀ undergo฀ intramolecular฀ rearrangements฀ that฀ produce฀
apatite฀and฀decrease฀solubility฀(see฀Chapter฀9:฀Sect.฀9.1.2).
Amorphous฀ solids฀ including฀ proteins฀ have฀ no฀ crystal฀ cell,฀ but฀ may฀ crystallize฀ and฀
precipitate฀ under฀ appropriate฀ conditions.฀ The฀ repeating฀ crystal฀ cell฀ provides฀ an฀ x-ray฀
diffraction฀ pattern฀ that฀ can฀ provide฀ a฀ detailed฀ 3-dimensional฀ protein฀ structure.฀ To฀ be฀
crystallized,฀a฀protein฀must฀be฀pure฀and฀in฀solution.฀It฀is฀then฀tested฀for฀crystal฀formation฀
by฀very฀slow฀evaporation฀under฀a฀variety฀of฀pH฀and฀ionic฀strength฀conditions.

1.2.3.
Covalent Bonds

Carbon฀has฀4฀electrons฀in฀its฀outermost฀shell.฀Removing฀all฀4฀would฀make฀the฀atom฀too฀
positively฀charged฀to฀be฀stable.฀Conversely,฀adding฀4฀electrons฀would฀make฀the฀atom฀
too฀negatively฀charged฀to฀be฀stable.฀Because฀the฀formation฀of฀carbon฀ions฀is฀not฀ener-
getically฀feasible,฀carbon฀shares฀its฀electrons฀by฀forming฀covalently฀bonded฀molecules.฀
Each฀electron฀points฀in฀a฀different฀geometrical฀direction฀so฀that฀each฀bond฀of฀a฀carbon฀
atom฀points฀in฀a฀different฀direction.฀Although฀many฀different฀elements฀can฀share฀elec-
trons฀ with฀ carbon,฀ the฀ ones฀ depicted฀ in฀ green฀ in฀ Fig.฀ 1.1฀ are฀ preferred฀ by฀ biological฀
systems.฀

1.2.4.
Polarized Covalent Bonds

Polarized฀covalent฀bonding,฀is฀evident฀in฀water.฀Because฀oxygen฀has฀six฀electrons฀in฀its฀outer฀
shell,฀the฀electron฀donated฀by฀each฀hydrogen฀atom฀is฀pulled฀towards฀the฀oxygen฀atom฀to฀try฀
and฀complete฀the฀latter’s฀outer฀shell฀of฀eight฀electrons.฀The฀electrons฀are฀therefore฀unevenly฀
8 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

distributed฀around฀each฀water฀molecule.฀They฀are฀polarized฀to฀the฀oxygen฀atom,฀giving฀it฀a฀
slight฀ negative฀ charge,฀ and฀ the฀ hydrogen฀ atoms฀ a฀ slight฀ positive฀ charge.฀ Water฀ is฀ a฀ luid฀
because฀the฀polarized฀molecules฀attract฀each฀other฀without฀forming฀a฀solid฀at฀atmospheric฀
temperatures฀above฀0ºC฀and฀condense฀back฀from฀a฀gas฀to฀the฀liquid฀form฀at฀temperatures฀
below฀100ºC.฀The฀polarization฀also฀attracts฀the฀ions฀in฀solids.฀The฀anions฀and฀cations฀to฀dis-
sociate฀in฀the฀liquid฀and฀the฀solid฀dissolves฀forming฀a฀solution.
Carbon/oxygen฀ bonds฀ resemble฀ hydrogen/oxygen฀ bonds.฀ In฀ carbon฀ dioxide,฀ the฀ two฀
oxygen฀ atoms฀ are฀ negatively฀ charged฀ and฀ the฀ carbon฀ atom฀ is฀ positively฀ charged.฀ The฀
arrangement฀resembles฀water,฀except฀that฀the฀positive฀charge฀is฀on฀the฀central฀carbon฀atom฀
and฀the฀negative฀charge฀on฀the฀two฀oxygen฀atoms฀is฀on฀the฀outside.฀Carbon฀dioxide฀is฀less฀
polarized฀than฀water฀and฀is฀therefore฀a฀gas,฀although฀it฀liqueies฀easily฀under฀pressure.฀The฀
bonding฀of฀carbon฀to฀a฀single฀oxygen฀atom,฀as฀in฀an฀alcohol,฀ketone,฀aldehyde,฀or฀carboxy-
lic฀acid,฀is฀more฀electrically฀withdrawing฀(polarizing),฀which฀provides฀greater฀water฀solu-
bility฀and฀a฀point฀of฀attack฀for฀metabolic฀reactions.
Nitrogen฀and฀sulfur฀bonding฀also฀withdraw฀electrons฀from฀a฀carbon฀atom,฀causing฀it฀to฀
become฀ polarized฀ like฀ oxygen/carbon฀ bonds฀ and฀ provide฀ additional฀ sites฀ for฀ metabolic฀
reactions.฀Nitrogen฀withdraws฀electrons฀less฀strongly฀than฀oxygen,฀whereas฀carbon-halide฀
bonds฀ are฀ more฀ electron฀ withdrawing,฀ but฀ do฀ not฀ form฀ biologically.฀ As฀ elements฀ move฀
from฀ the฀ 5th฀ to฀ the฀ 6th฀ and฀ 7th฀ column฀ of฀ the฀ periodic฀ table฀ (Fig.฀ 1.1),฀ their฀ electron-
withdrawing฀power฀increases฀when฀bonded฀to฀carbon.฀

1.2.5.
Hydrophobic Bonds

The฀electrons฀in฀hydrogen฀–฀carbon฀bonds฀are฀evenly฀shared,฀leaving฀a฀molecule฀com-
pletely฀uncharged฀(apolar)฀and฀insoluble฀in฀water.฀In฀large฀molecules฀composed฀mostly฀
of฀carbon-hydrogen฀bonds฀but฀with฀a฀small฀polarized฀region฀such฀as฀a฀carboxylic฀acid฀at฀
one฀end฀as฀in฀a฀fatty฀acid,฀the฀hydrocarbon฀regions฀clump฀together.฀If฀the฀fatty฀acids฀are฀
part฀ of฀ a฀ phospholipid,฀ the฀ hydrocarbon฀ regions฀ form฀ the฀ interior฀ of฀ membranes฀ that฀
delineate฀ cells฀ from฀ their฀ environment.฀ In฀ proteins,฀ regions฀ of฀ hydrocarbon฀ promote฀
hydrophobic฀regions฀that฀attract฀each฀other.฀Together฀with฀charged฀and฀polarized฀regions฀
caused฀by฀carbon฀bound฀to฀oxygen,฀nitrogen฀and฀sulfur฀atoms,฀the฀hydrophobic฀attrac-
tion฀promotes฀folding฀and฀protein-protein฀and/or฀enzyme-substrate฀interactions.

Molecules฀are฀composed฀of฀atoms฀that฀are฀attached฀to฀each฀other฀by฀sharing฀electrons฀to฀
complete฀ their฀ respective฀ electron฀ shells.฀ Electrostatic฀ bonds฀ arise฀ when฀ electrons฀ are฀
added฀or฀subtracted฀from฀elements,฀giving฀rise฀to฀positive฀and฀negatively฀charged฀particles฀
called฀ions.฀Covalent฀bonds฀do฀not฀give฀rise฀to฀ions฀but฀may฀be฀polarized฀as฀a฀consequence฀
of฀ the฀ electron-withdrawing฀ properties฀ of฀ adjacent฀ atoms฀ in฀ the฀ molecule,฀ permitting฀
interactions฀with฀water,฀the฀solvent฀for฀biochemical฀reactions.฀Polar฀bonds฀are฀water฀solu-
ble,฀whereas฀apolar฀bonds฀(mostly฀carbon–hydrogen)฀are฀water฀insoluble฀(hydrophobic).
1.3.1. Mechanisms of Energy Production: Respiration and Fermentation 9

1.3.1.
Mechanisms of Energy Production: Respiration and Fermentation

All฀living฀organisms฀get฀their฀chemical฀energy฀from฀ATP฀and฀a฀hydride:฀a฀reduced฀form฀of฀
nicotinamide฀adenosine฀nucleotide฀diphosphate,฀NADH฀+฀H+,฀or฀its฀phosphorylated฀analog,฀
NADPH฀+฀H+฀(Fig.฀1.5).

Organisms฀obtain฀ATP฀and฀NADH฀in฀one฀of฀three฀ways:
1.฀ Respiration฀(conversion฀of฀ingested฀foods฀to฀CO2฀and฀H2O)
2.฀ Fermentation฀(partial฀oxidation฀of฀carbon฀compounds)
3.฀ Photosynthesis฀(action฀of฀sunlight฀on฀water฀and฀CO2฀in฀chloroplasts,฀Chap.฀2)

In฀respiration,฀substrate฀organic฀molecules฀containing฀carbon–hydrogen฀bonds฀(food)฀and฀
oxygen฀ are฀ absorbed฀ by฀ prokaryotic฀ cells฀ or฀ by฀ the฀ mitochondria฀ of฀ eukaryotic฀ cells.฀฀
The฀oxygen฀reacts฀with฀electrons฀that฀are฀derived฀from฀metabolic฀changes฀to฀the฀carbon–
hydrogen฀bonds฀of฀the฀substrates.฀The฀inal฀steps฀of฀substrate฀metabolism,฀the฀Krebs฀cycle,฀

+ 2e−
NAD + NADH + H +
+ 2H+
Oxidixed form Reduced form

H H H
H H
O O
Nicotinamide
H H

N+ NH2 N NH2
O O O H
H
O Ribose
P
NH2
O O
HO N +
OH
H
O P H+
N
O N
O O
N
Ribose H
Adenine
OH
HO O
H in NAD+ P O in NADP+

OH

Fig. 1.5฀ Nicotinamide฀adenine฀dinucleotide฀(NAD+)฀and฀its฀phosphorylated฀analog฀(NADP+).฀The฀

difference฀is฀indicated฀in฀purple.฀NAD+฀and฀NADP+฀undergo฀reduction฀to฀NADH฀and฀NADPH฀by฀
accepting฀ a฀ hydride฀ ion฀ and฀ two฀ electrons฀ and฀ releasing฀ a฀ proton฀ from฀ an฀ oxidized฀ substrate.฀
(Adapted฀from฀Fig.฀14-13฀in฀Berg฀JM,฀Tymoczko฀JL฀and฀Stryer฀L.฀Biochemistry,฀5th฀Ed.฀2002.฀
W.H.฀Freeman฀&฀Co.,฀New฀York)
10 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

Fig. 1.6฀ Respiration.฀Energy฀is฀obtained฀by฀

converting฀foodstuffs฀(organic฀material฀composed฀
mainly฀of฀carbohydrate,฀fat,฀and฀protein)฀฀
to฀oxygen฀and฀water.฀These฀organisms฀obtain฀฀
the฀food฀from฀many฀sources฀and฀are฀called฀
heterotrophs

produce฀carbon฀dioxide,฀NADH,฀and฀protons.฀The฀electrons฀are฀transferred฀from฀NADH฀to฀
oxygen฀ along฀ with฀ protons,฀ forming฀ water฀ and฀ ATP.฀ The฀ water฀ and฀ carbon฀ dioxide฀ are฀
excreted,฀and฀the฀ATP฀is฀used฀intracellularly฀with฀some฀of฀the฀NADH฀for฀cell฀maintenance฀
and฀ growth.฀ In฀ vertebrates,฀ the฀ oxygen฀ and฀ carbon฀ dioxide฀ are฀ respectively฀ absorbed฀฀
from฀and฀excreted฀to฀the฀environment฀through฀the฀circulation฀of฀hemoglobin฀between฀the฀
lungs฀and฀tissues฀(Fig.฀1.6).
In฀fermentation,฀energy฀is฀obtained฀by฀shufling฀organic฀molecules฀so฀that฀NADH฀pro-
duced฀in฀one฀step฀is฀oxidized฀by฀a฀subsequent฀product฀or฀products,฀which฀are฀reduced฀and฀
excreted.฀Due฀to฀the฀small฀amount฀of฀energy฀obtained,฀only฀microbes฀can฀rely฀on฀fermenta-
tion฀as฀their฀sole฀energy฀source.฀More฀complex฀organisms฀utilize฀respiration฀alone฀or฀in฀
combination฀with฀sunlight.
Dental฀caries฀results฀from฀microbial฀fermentations฀that฀produce฀lactate฀by฀glycolysis฀
from฀ monosaccharides฀ following฀ the฀ hydrolysis฀ of฀ disaccharide฀ and฀ polysaccharides฀
(Sect.฀15.2.2).฀These฀bacteria฀are฀commonly฀referred฀to฀as฀saccharolytic฀(sugar฀metaboliz-
ing)฀ to฀ distinguish฀ them฀ from฀ asaccharolytic฀ (nonsugar฀ metabolizing)฀ bacteria,฀ most฀ of฀
which฀hydrolyze฀proteins฀and฀utilize฀the฀amino฀acids฀for฀energy.฀A฀third฀type฀of฀fermenta-
tion฀is฀the฀use฀of฀an฀inorganic฀molecule฀such฀as฀nitrate฀as฀electron฀acceptor.฀The฀nitrate฀is฀
reduced฀to฀nitrite฀by฀electron฀transport฀in฀a฀manner฀similar฀to฀the฀reduction฀of฀oxygen฀in฀
respiration,฀but฀only฀1฀mol฀of฀ATP฀is฀produced฀per฀mol฀of฀nitrate.฀Reducing฀oxygen฀pro-
duces฀2–3฀mol฀of฀ATP฀per฀mol฀of฀water฀(Fig.฀1.7).

1.3.2.
The Oral Microbiota, Dental Caries, and Periodontal Disease

Teeth฀adherent฀bacterial฀bioilms,฀commonly฀called฀plaque฀or฀plaques,฀are฀responsible฀for฀
the฀common฀forms฀of฀periodontal฀disease฀(Chap.฀13)฀and฀dental฀caries฀(Chap.฀15).฀In฀chil-
dren฀or฀adults฀who฀keep฀their฀teeth฀clean฀and฀have฀no฀periodontal฀disease,฀the฀bacteria฀in฀a฀
bioilm฀(the฀microbiota)฀is฀mostly฀gram฀positive฀and฀resembles฀that฀in฀saliva฀or฀adhering฀to฀
the฀oral฀mucosa.฀The฀microbiota฀is฀predominantly฀saccharolytic฀and฀the฀major฀fermentation฀
1.3.2. The Oral Microbiota, Dental Caries, and Periodontal Disease 11

Saccharolytic fermentation
produces acid
Carbohydrates Hexose
(HexK) C6
-1ATP

(PFK)
-1ATP

Triose phosphate Triose phosphate

C3 C3

NAD+
(G3PDH)
+2ATP
(Enolase) NADH
+2ATP + H+

Pyruvate Lactate
C3 C3
Net gain of 2ATP/mol lactate excreted
NOTE: The mass of a molecule is identified by the sum of its atomic weights, the molecular
weight equivalent (mole). The standard symbol for mole is 'mol'

Fig. 1.7฀ Saccharolytic฀ fermentation฀ produces฀ acid.฀ The฀ conversion฀ of฀ glucose฀ to฀ lactic฀ acid฀
(glycolysis)฀is฀an฀example฀of฀fermentation.฀Adenosine฀nucleoside฀triphosphate฀(ATP)฀is฀synthe-
sized฀and฀NADH฀is฀oxidized฀from฀carbon฀substrates฀without฀any฀need฀for฀oxygen.฀For฀example,฀
bacteria฀adherent฀to฀teeth฀in฀the฀oral฀cavity฀may฀obtain฀energy฀from฀ingested฀carbohydrates฀by฀
glycolysis฀(Chapter฀15).฀Two฀ATP฀are฀used฀up฀to฀convert฀monosaccharide฀hexose฀to฀2฀molecules฀
of฀ 3-phosphoglyceraldehyde.฀ One฀ ATP฀ is฀ used฀ by฀ hexokinase฀ (HexK)฀ to฀ make฀ the฀ hexose฀
฀6-phosphate฀and฀one฀by฀phosphofructokinase฀(PFK)฀to฀make฀fructose฀bisphosphate.฀Two฀triose฀
phosphate฀molecules฀are฀made฀from฀fructose฀bisphosphate฀and฀both฀are฀converted฀to฀glyceralde-
hyde฀ 3-phosphate฀ (G3P).฀ The฀ G3P฀ is฀ acted฀ on฀ by฀ its฀ dehydrogenase฀ (G3PDH)฀ to฀ produce฀ to฀
2฀molecules฀of฀3-phosphoglycerate,฀2฀ATP฀and฀2฀NADH.฀The฀3-phosphoglycerate฀molecules฀are฀
both฀converted฀to฀phosphoenolpyruvate฀(PEP),฀which฀provides฀2฀ATP฀when฀the฀two฀PEP฀mole-
cules฀ are฀ converted฀ to฀ pyruvate฀ by฀ enolase.฀ There฀ are฀ therefore฀ a฀ total฀ of฀ 4฀ ATP฀ produced฀ of฀
which฀2฀are฀used฀up฀in฀converting฀hexose฀to฀triose,฀leaving฀a฀net฀gain฀of฀2฀ATP฀per฀hexose฀mol-
ecule฀for฀the฀organism.฀The฀2฀molecules฀of฀NADH฀are฀reoxidized฀to฀NAD+฀when฀the฀2฀molecules฀
of฀pyruvate฀are฀converted฀to฀lactate.฀The฀lactate฀is฀extruded฀from฀the฀cell฀as฀lactic฀acid฀which฀
decreases฀the฀pH฀and฀causes฀dental฀caries฀at฀the฀tooth฀surface฀(Chapter฀15).฀(Figure฀is฀adapted฀
from฀Fig.฀1,฀Chapter฀15,฀in฀Harper’s฀Review฀of฀Biochemistry.฀฀D.฀W.฀Martin฀et฀al.,฀20th฀edition,฀
1985,฀Lange฀Medical฀Publications,฀Los฀Altos,฀CA)

end฀product฀is฀lactic฀acid.฀Whole฀saliva฀contains฀mucins,฀proteins฀covered฀with฀numerous฀
saccharide฀(glycan)฀residues฀that฀are฀more฀accessible฀to฀enzymes฀than฀the฀polypeptide.฀In฀
addition,฀the฀repeated฀intake฀of฀dietary฀carbohydrate฀predisposes฀to฀a฀saccharolytic฀micro-
biota฀(Sect.฀15.1.4).฀These฀bacteria฀are฀predominantly฀gram฀positive฀and฀possess฀a฀thick฀
12 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

outer฀wall฀(Sect.฀1.4.1).฀The฀thick฀cell฀walls฀enable฀some฀of฀these฀bacteria฀to฀tolerate฀the฀low฀
pH฀caused฀by฀their฀production฀of฀large฀amounts฀of฀lactate,฀which฀causes฀caries฀by฀dissolving฀
tooth฀enamel฀and฀dentin฀(Sects.฀15.1.4฀and฀15.2.3).
By฀contrast,฀beneath฀a฀healthy฀gingival฀sulcus,฀there฀is฀an฀intermittent฀low฀of฀proteins฀
from฀serum,฀blood฀plasma฀proteins฀in฀which฀clotting฀has฀been฀inactivated฀(Sect.฀11.4.1).฀
This฀exudate฀of฀serum฀proteins,฀the฀gingival฀crevicular฀luid฀(GCF),฀provides฀a฀sulcus฀that฀
is฀richer฀in฀proteins฀than฀saliva฀and฀an฀environment฀that฀is฀more฀suited฀for฀an฀asaccharo-
lytic฀ microbiota฀ (Sect.฀ 13.1.2).฀ Asaccharolytic฀ bacteria฀ secrete฀ proteases฀ that฀ digest฀฀
proteins฀to฀small฀peptides,฀which฀they฀digest฀(ferment)฀in฀the฀cytosol.
In฀asaccharolytic฀fermentations,฀amino฀acids฀are฀deaminated฀to฀ammonia฀in฀a฀reaction฀that฀
converts฀NAD+฀to฀NADH:฀for฀example,฀the฀deamidation฀of฀glutamate฀to฀a-ketoglutarate฀and฀
ammonia฀(Fig.฀1.8).฀a-Ketoglutarate฀may฀then฀be฀decarboxylated฀and฀reduced฀to฀butyrate.฀
Other฀amino฀acids฀are฀manipulated฀to฀produce฀short฀chain฀fatty฀acids฀such฀as฀formate,฀acetate,฀
and฀propionate,฀which฀are฀excreted฀with฀a฀net฀gain฀of฀ATP฀to฀the฀organism.฀These฀reactions฀
are฀bacteria฀speciic฀and฀therefore฀extremely฀varied,฀but฀the฀common฀factor฀is฀ammonia฀pro-
duction฀and฀short฀chain฀fatty฀acids฀whose฀products฀are฀toxic฀to฀mammalian฀cells฀(Sect.฀13.4.1).฀
Figure฀1.8฀illustrates฀relationships฀of฀glycine฀to฀acetate;฀of฀cysteine,฀alanine฀and฀aspartate฀to฀
propionate;฀and฀of฀threonine฀and฀glutamate฀to฀butyrate.฀Despite฀the฀short฀chain฀fatty฀acids,฀
ammonia฀accumulates฀enough฀to฀make฀the฀gingival฀sulcus฀alkaline.฀The฀high฀pH฀of฀the฀sulcus฀
prevents฀caries฀from฀developing฀beneath฀the฀gingival฀sulcus฀(Sect.฀15.3.2).
The฀alkaline฀environment฀also฀precipitates฀calcium฀and฀phosphate฀ions฀from฀the฀GCF,฀
causing฀ dental฀ calculus.฀ Dental฀ calculus฀ interferes฀ with฀ self-administered฀ oral฀ hygiene฀
(Sect.฀ 13.1.2)฀ and฀ asaccharolytic฀ metabolism฀ intensiies.฀ When฀ sulfur-containing฀ amino฀
acids฀(cysteine฀and฀methionine)฀are฀metabolized,฀they฀release฀hydrogen฀sulide฀along฀with฀
the฀ammonia฀and฀short฀chain฀fatty฀acids.฀Hydrogen฀sulide฀is฀a฀major฀contributor฀of฀oral฀
malodor฀that฀often฀accompanies฀moderate฀to฀severe฀periodontal฀disease.
Within฀the฀bioilms฀or฀plaques,฀different฀bacteria฀utilize฀each฀other’s฀products฀in฀order฀
to฀grow฀more฀eficiently฀(a฀mechanism฀called฀symbiosis).฀For฀example,฀a฀bacterium฀that฀
metabolizes฀glucose฀by฀reducing฀it฀to฀lactate฀may฀enable฀another฀bacterium฀to฀grow฀by฀
reducing฀the฀lactate฀to฀propionate.฀The฀formation฀of฀bioilms฀is฀driven฀by฀three฀factors:฀(1)฀
the฀presence฀of฀substrate฀in฀saliva฀or฀gingival฀serum฀exudate฀for฀one฀or฀more฀of฀the฀bacteria฀
in฀a฀symbiotic฀group;฀(2)฀the฀production฀of฀a฀metabolite฀such฀as฀lactate฀that฀can฀be฀utilized฀
by฀other฀bacteria฀in฀the฀group,฀and฀(3)฀proteins฀and฀other฀components฀that฀permit฀the฀bac-
teria฀in฀the฀group฀to฀attach฀to฀each฀another.

Bacteria฀ obtain฀ energy฀ by฀ fermentation,฀ in฀ which฀ shufling฀ carbon฀ compounds฀฀
produces฀ATP฀without฀a฀need฀for฀oxygen.฀NADH฀is฀produced,฀but฀reoxidized฀by฀the฀
product฀of฀the฀shufling,฀which฀is฀excreted.฀Bacteria฀ferment฀sugars฀and฀excrete฀lac-
tate฀(saccharolytic),฀or฀ferment฀amino฀acids฀and฀excrete฀ammonia,฀sulides,฀and฀short฀
chain฀ fatty฀ acids฀ (asaccharolytic).฀ Fermentations฀ may฀ alternatively฀ reduce฀ an฀ inor-
ganic฀molecule฀such฀as฀nitrate฀to฀reoxidize฀their฀NADH.฀Nitrate฀is฀secreted฀by฀the฀
salivary฀glands฀and฀some฀of฀the฀bacteria฀that฀normally฀infect฀saliva฀reduce฀the฀nitrate฀
to฀nitrite฀by฀electron฀transport฀similar฀to฀the฀reduction฀of฀oxygen฀to฀water฀in฀respira-
tion.฀ This฀ reduction฀ provides฀ only฀ enough฀ energy฀ to฀ produce฀ 1฀ mol฀ of฀ ATP/mol฀
nitrate,฀whereas฀the฀reduction฀of฀oxygen฀to฀water฀produces฀3฀moles.฀Saccharolytic฀
1.3.2. The Oral Microbiota, Dental Caries, and Periodontal Disease 13

bacteria฀are฀associated฀with฀lactic฀acid฀production฀and฀dental฀caries,฀whereas฀asac-
charolytic฀bacteria฀are฀associated฀with฀periodontal฀disease.฀The฀latter฀fermentations฀
result฀in฀oral฀odor฀from฀sulides฀and฀an฀alkaline฀environment฀from฀ammonia฀that฀is฀
especially฀important฀in฀causing฀calculus฀to฀precipitate฀around฀the฀teeth฀as฀periodontal฀
disease฀develops.฀The฀bacteria฀adhere฀to฀teeth฀as฀bioilms฀or฀plaques฀in฀which฀differ-
ent฀bacteria฀utilize฀each฀other’s฀products฀and฀grow฀better฀(symbiosis).

Asaccharolytic fermentation produces

ammonia and short chain fatty acids

CH3 − COO- Acetate

NH2 − CH2 − COO- Glycine

CH3 − CH2 − COO- Propionate

CH2 − CH − COO- Cysteine

SH NH2

CH3 − CH − COO- Alanine

NH2
-OOC − CH − CH − COO- Aspartate
2

NH2

CH3 − CH2 − CH2 − COO- Butyrate

CH3 − CH2 − CH − COO- Threonine

OH NH2
-OOC − CH − CH −CH − COO-
2 2 Glutamate
NH2

Fig. 1.8฀ Asaccharolytic฀fermentation฀produces฀ammonia฀and฀short-chain฀fatty฀acids.฀This฀group฀

of฀fermentations฀by฀oral฀bacteria฀utilizes฀proteins,฀which฀are฀converted฀to฀peptides฀and฀amino฀
acids.฀The฀free฀amino฀acids฀are฀then฀deaminated฀to฀ammonia฀in฀a฀reaction฀that฀converts฀nicotin-
amide฀adenine฀dinucleotide฀(NAD)฀to฀NADH.฀For฀example,฀alanine฀is฀converted฀to฀pyruvate฀and฀
ammonia.฀The฀pyruvate฀is฀reduced฀to฀lactate,฀and฀ammonium฀lactate฀is฀excreted฀into฀the฀envi-
ronment.฀ Unlike฀ lactate฀ from฀ glucose,฀ ammonium฀ lactate฀ is฀ a฀ neutral฀ salt.฀ The฀ common฀ end฀
products฀in฀from฀plaque฀are฀ammonium฀acetate,฀ammonium฀propionate,฀and฀ammonium฀butyrate,฀
ammonium฀salts฀of฀short฀chain฀fatty฀acids.฀For฀example,฀glycine฀is฀reduced฀to฀acetate฀and฀ammo-
nia.฀Cysteine฀is฀reduced฀to฀propionate,฀hydrogen฀sulide,฀and฀ammonia;฀alanine฀to฀propionate,฀
water,฀and฀ammonia;฀and฀aspartate฀to฀propionate,฀carbon฀dioxide,฀and฀ammonia.฀Threonine฀is฀
reduced฀to฀butyrate,฀water,฀and฀ammonia฀and฀glutamate฀is฀reduced฀to฀butyrate,฀carbon฀dioxide,฀
and฀ammonia.฀Other฀amino฀acids฀are฀involved฀in฀more฀complicated฀metabolic฀reactions฀that฀give฀
rise฀to฀these฀short-chain฀amino฀acids,฀sometimes฀with฀succinate,฀another฀common฀end฀product฀
in฀plaque.
14 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

1.4.1.
Bacterial Cell Structures

The฀cell฀wall฀protects฀bacterial฀cells฀from฀the฀environment,฀just฀as฀skin฀or฀fur฀protects฀
mammals.฀ Its฀ thickness฀ distinguishes฀ two฀ major฀ classes฀ of฀ bacteria:฀ gram-positive฀
staining฀ (thick฀ walled)฀ and฀ gram-negative฀ staining฀ (thin฀ walled).฀ The฀ gram฀ negative฀
cell฀wall฀(blue฀in฀Fig.฀1.9a฀but฀red฀when฀gram-stained)฀is฀composed฀of฀short฀peptides฀of฀
d-฀and฀l-amino฀acids฀that฀are฀cross-linked฀by฀short฀glycan฀chains฀to฀form฀a฀peptidogly-
can฀network฀containing฀other฀substances.฀The฀cell฀wall฀covers฀the฀outer฀surface฀of฀the฀
plasma฀membrane฀from฀which฀imbriae฀(singular:฀imbria)฀and฀lagella฀(singular:฀lagel-
lum)฀extrude฀(Fig.฀1.9a,฀upper฀left฀side).฀A฀bacterium฀has฀many฀imbriae,฀but฀only฀one฀
or฀two฀lagella.฀Penicillin฀and฀related฀antibiotics฀inhibit฀an฀enzyme฀involved฀in฀synthe-
sizing฀ the฀ cell฀ wall฀ peptidoglycan฀ network.฀ In฀ addition,฀ lysozyme,฀ an฀ enzyme฀ in฀ the฀
acid-activated฀lysosomal฀vesicles฀of฀most฀mammalian฀cells฀(Sect.฀10.1.1),฀hydrolyzes฀a฀
repeating฀bond฀in฀the฀peptidoglycan฀polymer,฀breaking฀it฀up฀into฀small฀fragments.฀Both฀
penicillin฀and฀lysozyme฀lyse฀a฀sensitive฀bacterial฀cell.
In฀periodontitis,฀bacterial฀cells฀that฀no฀longer฀survive฀in฀the฀gingival฀sulcus฀leave฀pepti-
doglycan฀fragments฀that฀may฀be฀absorbed฀into฀the฀cytosol฀of฀surrounding฀host฀cells.฀Within฀
host฀cells,฀peptidoglycan฀fragments฀may฀either฀be฀toxic฀or฀cause฀a฀release฀of฀inlammatory฀
mediators฀(Sect.฀13.4.1).

1.4.2.
The Bacterial Outer Surface

Fimbriae,฀ known฀ also฀ as฀ pili฀ (singular:฀ pilus),฀ are฀ outer฀ surface฀ protein฀ appendages฀ that฀
mediate฀adhesion,฀whereas฀lagella฀are฀a฀separate฀group฀of฀proteins฀responsible฀for฀motility.฀
Fimbriae฀are฀very฀common.฀They฀are฀synthesized฀in฀the฀cytosol฀and฀assemble฀attached฀to฀
the฀cell฀inner฀membrane.฀Many฀imbrial฀proteins฀require฀secretion฀through฀the฀cell฀mem-
brane,฀and฀they฀do฀so฀with฀the฀aid฀of฀an฀N-terminal฀peptide฀signal฀sequence฀and฀signal฀rec-
ognition฀particles฀containing฀small฀RNA฀resembling฀the฀small฀cytosolic฀RNA฀of฀eukaryotes฀
(Type฀II฀or฀Type฀IV฀secretion).฀By฀contrast,฀lagellar฀proteins฀are฀synthesized฀in฀the฀cytosol฀
and฀then฀interact฀with฀other฀proteins฀to฀translocate฀their฀extracellular฀components฀through฀
the฀membrane.฀Flagella฀are฀assembled฀intra-฀and฀extracellularly฀around฀the฀cell฀membrane฀
(Type฀III฀secretion).฀Bacterial฀toxins฀use฀these,฀or฀a฀fourth฀mechanism฀(Type฀I฀secretion฀
described฀in฀Sect.฀14.2.2),฀in฀order฀to฀reach฀the฀extracellular฀luid฀and฀attack฀a฀target฀cell.
In฀ many฀ bacteria,฀ the฀ cell฀ wall฀ is฀ surrounded฀ by฀ a฀ polysaccharide฀ (glycan)฀ capsule฀
through฀which฀the฀imbriae฀and฀lagellum฀protrude.฀One฀end฀of฀the฀glycan฀may฀be฀cova-
lently฀attached฀to฀fatty฀acids฀in฀the฀plasma฀membrane,฀or฀they฀may฀adhere฀by฀non-covalent฀
bonds฀to฀the฀glycan-synthesizing฀enzyme฀within฀the฀plasma฀membrane.฀Capsules฀vary฀in฀
content,฀ composition,฀ size฀ and฀ thickness,฀ especially฀ among฀ gram-positive฀ bacteria฀ in฀
which฀a฀capsule฀may฀be฀absent.฀A฀capsule฀made฀from฀dietary฀sucrose฀by฀the฀gram-positive฀
bacterium,฀Streptococcus฀mutans,฀likely฀mediates฀dental฀caries.
1.4.2. The Bacterial Outer Surface 15

Fig. 1.9฀ Bacterial฀ structure.฀ (a)฀ Bacterial฀ cell.฀ Bacteria฀ have฀ a฀ simple฀ internal฀ structure฀ with฀ few฀
organelles.฀They฀have฀no฀nucleus฀internally,฀but฀a฀nucleoid฀region฀where฀DNA฀is฀found.฀Plasmids฀
are฀ independent฀ pieces฀ of฀ DNA฀ that฀ can฀ be฀ exchanged฀ between฀ cells฀ and฀ often฀ carry฀ genes฀ that฀
promote฀survival,฀for฀example฀genes฀encoding฀enzymes฀that฀remove฀antibiotics.฀The฀interior฀of฀the฀
bacterial฀cell฀is฀full฀of฀ribosomes฀because฀the฀half-life฀of฀bacterial฀proteins฀is฀so฀short฀that฀they฀are฀
constantly฀resynthesized.฀Bacterial฀proteins฀turn฀over฀constantly฀whereas฀many฀mammalian฀proteins฀
turn฀over฀slowly.฀The฀exterior฀of฀the฀cell฀lies฀outside฀the฀plasma฀membrane.฀It฀consists฀of฀a฀cell฀wall฀
and฀capsule.฀Fimbria฀and฀lagella฀extend฀from฀the฀plasma฀membrane฀and฀pass฀through฀the฀cell฀wall฀
and฀capsule.฀In฀gram-negative฀bacteria,฀much฀of฀the฀cell฀wall฀is฀thinner฀than฀in฀gram-positive฀bacte-
ria.฀Instead฀there฀is฀a฀second,฀outer฀membrane฀with฀an฀attached฀polysaccharide฀capsule.฀The฀space฀
between฀the฀inner฀(plasma)฀and฀outer฀membranes฀is฀called฀the฀periplasmic฀space.฀In฀gram-positive฀
cells,฀the฀periplasmic฀space฀is฀small฀and฀lies฀between฀the฀thick฀outer฀cell฀wall฀and฀the฀plasma฀mem-
brane.฀Inclusion฀bodies฀are฀aggregates฀of฀viral฀proteins฀that฀may฀represent฀sites฀of฀multiplication฀or฀
attempted฀multiplication฀of฀a฀bacteriophage฀(virus฀that฀infects฀bacteria).฀Inclusion฀bodies฀may฀occur฀
in฀any฀bacterium,฀gram฀positive฀as฀well฀as฀gram฀negative.฀(b)฀Structures฀of฀the฀double฀membrane,฀
cell฀wall,฀and฀capsule฀of฀gram-negative฀bacteria.฀Each฀membrane฀is฀a฀phospholipid฀bilayer.฀The฀
inner฀ membrane฀ is฀ the฀ plasma฀ membrane฀ and฀ separates฀ the฀ cytosol฀ from฀ the฀ periplasmic฀ space฀
between฀the฀two฀bilayers.฀The฀major฀components฀of฀the฀periplasmic฀space฀are฀cell฀wall฀peptidogly-
can,฀peptidoglycan-associated฀lipoprotein฀(lipoprotein),฀and฀outer฀membrane฀protein฀A฀(omp฀A).฀A฀
similar฀double฀membrane฀structure฀is฀present฀in฀mitochondria฀but฀the฀protein฀composition฀of฀the฀
intermembrane฀space฀is฀very฀different.฀Lipopolysaccharide฀(LPS)฀consists฀of฀an฀outer฀polysaccha-
ride฀chain฀attached฀by฀a฀core฀polysaccharide฀to฀a฀lipid,฀which฀is฀part฀of฀the฀outer฀membrane฀(lipid฀
A),฀shown฀as฀darker฀regions฀of฀the฀outer฀membrane.฀The฀precise฀composition฀of฀the฀outer฀and฀core฀
polysaccharides฀and฀lipid฀A฀varies,฀with฀species฀and฀strain฀of฀the฀bacterium฀(Figure฀is฀modiied฀from฀
Wikipedia฀public฀domain:฀http://en.wikipedia.org/wiki/Bacterial_outer_membrane)

To฀make฀up฀for฀their฀thin฀peptidoglycan,฀gram-negative฀bacteria฀possess฀a฀second฀(outer)฀
membrane฀to฀which฀is฀attached฀a฀glycan฀capsule฀called฀lipopolysaccharide฀(LPS).฀Figure฀1.9a฀
illustrates฀ the฀ inner฀ and฀ outer฀ membranes฀ of฀ these฀ bacteria.฀ Figure฀ 1.9b฀ illustrates฀ the฀
detailed฀structure฀of฀the฀double฀membrane฀with฀the฀attached฀LPS.฀As฀its฀name฀suggests,฀LPS฀
is฀composed฀of฀a฀polysaccharide฀that฀is฀covalently฀attached฀to฀a฀large฀complex฀lipid฀(lipid฀
A)฀in฀the฀outer฀membrane,฀unlike฀the฀attachment฀of฀imbriae฀or฀lagella,฀which฀is฀to฀the฀inner฀
(plasma)฀ membrane฀ as฀ in฀ gram-positive฀ bacteria.฀ The฀ polysaccharide฀ portion฀ of฀ LPS฀ is฀
16 1 Necessary Basics: Elements, Isotopes, Ions, Chemical Reactions, Energy Metabolism, and Bacterial Structures

฀composed฀ of฀ a฀ core฀ with฀ side฀ chains฀ containing฀ a฀ variety฀ of฀ monosaccharides.฀ The฀ lipid฀
A฀moiety฀usually฀contains฀unusual฀fatty฀acids.
As฀gram฀negative฀bacteria฀invade฀and฀grow฀within฀the฀dental฀bioilm,฀they฀shed฀LPS฀
into฀ the฀ environment.฀ The฀ LPS฀ penetrates฀ the฀ surrounding฀ tissues฀ and฀ is฀ recognized฀ as฀
foreign฀by฀mammalian฀cell฀surface฀receptors.฀This฀recognition฀may฀be฀important฀for฀acti-
vating฀gingival฀inlammation฀(gingivitis฀Sect.฀13.2.1).฀LPS฀receptors฀are฀promiscuous฀in฀
that฀ they฀ recognize฀ almost฀ all฀ the฀ various฀ saccharides฀ and฀ lipids฀ in฀ LPS฀ from฀ different฀
gram฀negative฀bacteria.฀Inhibiting฀gingival฀inlammation฀by฀inhibiting฀LPS฀receptor฀acti-
vation฀therefore฀seems฀impracticable.฀Although฀mechanically฀removing฀the฀dental฀bioilm฀
by฀oral฀hygiene฀is฀the฀established฀method฀of฀controlling฀periodontal฀disease,฀it฀only฀works฀
well฀in฀about฀80%฀of฀patients.฀Chemical฀methods฀of฀inhibiting฀the฀colonization฀of฀dental฀
bioilm฀by฀gram฀negative฀bacteria฀are฀discussed฀in฀Sect.฀13.1.3.

Bacteria฀are฀simple฀unicellular฀organisms฀that฀constantly฀grow.฀They฀have฀a฀membrane฀
and฀cell฀wall.฀Fimbriae฀are฀especially฀important฀for฀bacterial฀adhesion,฀a฀critical฀factor฀
in฀dental฀disease฀development.฀Lipopolysaccharide฀is฀a฀covalent฀lipid฀and฀polysaccha-
ride฀structure฀that฀contains฀unusual฀saccharides฀and฀fatty฀acids.฀The฀lipid฀is฀at฀one฀end฀
and฀inserts฀it฀into฀the฀plasma฀membrane.฀LPS฀is฀invariably฀recognized฀as฀foreign฀by฀
receptors฀on฀mammalian฀cells฀that฀recognize฀the฀unique฀structure฀and฀activate฀inlam-
mation฀such฀as฀gingivitis.
 Photosynthesis and Sucrose Production
2

Starch฀and฀sucrose,฀key฀substrates฀for฀the฀development฀of฀dental฀caries,฀are฀exclusively฀
synthesized฀by฀plants.฀They฀are฀made฀in฀plant฀leaves฀by฀a฀process฀called฀photosynthe-
sis,฀which฀utilizes฀sunlight฀as฀the฀energy฀source.฀This฀chapter฀outlines฀the฀light฀and฀dark฀
reactions฀of฀photosynthesis฀and฀compares฀the฀light฀reaction฀with฀mitochondrial฀electron฀
transport฀(Sect.฀1).฀The฀key฀dark฀reaction,฀the฀production฀of฀phosphoglycerate฀by฀the฀
enzyme฀ribulose฀bisphosphate฀carboxylase฀(rubisco),฀is฀described฀along฀with฀the฀pro-
duction฀of฀fructose,฀sucrose,฀and฀starch฀(Sect.฀2).฀The฀chapter฀concludes฀with฀a฀detailed฀
discussion฀of฀the฀roles฀of฀starch฀and฀sucrose฀in฀plant฀metabolism฀(Sect.฀3).

2.1.1.
Role of Photosynthesis in Living Organisms

As฀discussed฀in฀Sect.฀1.3.1,฀insects,฀animals,฀and฀bacteria฀respire฀by฀consuming฀oxygen฀
gas฀and฀a฀complex฀mixture฀of฀organic฀material฀containing฀carbon–hydrogen฀bonds฀(pro-
teins,฀fats,฀and฀carbohydrates)฀for฀energy,฀growth,฀and฀maintenance.฀By฀contrast,฀the฀pho-
tosynthetic฀ organisms,฀ plants฀ and฀ algae,฀ consume฀ carbon฀ dioxide฀ gas฀ from฀ which฀ they฀
make฀all฀their฀molecular฀carbon.฀Nitrogen-containing฀molecules฀(amino฀acids,฀nucleic฀acid฀
bases,฀and฀various฀other฀compounds)฀require฀ammonia,฀which฀comes฀from฀decaying฀organ-
isms฀in฀the฀soil,฀or฀from฀root-associated฀bacteria฀that฀produce฀it฀from฀nitrogen฀gas.
Photosynthesis฀splits฀water฀into฀hydrogen฀and฀oxygen฀atoms฀in฀a฀reaction฀that฀requires฀
sunlight฀(light฀reaction).฀Oxygen฀is฀passed฀into฀the฀atmosphere฀and฀the฀hydrogen฀is฀used฀to฀
assimilate฀carbon฀dioxide฀in฀a฀dark฀(non-photosynthetic)฀reaction฀that฀forms฀starch,฀sucrose,฀
and฀another฀disaccharide฀called฀maltose.฀Metabolites฀such฀as฀a-ketoglutarate฀are฀derived฀
from฀starch฀and฀sucrose฀and฀incorporate฀(ix)฀the฀ammonia฀absorbed฀from฀the฀soil฀into฀the฀
nitrogen-containing฀compounds.฀The฀cycle฀is฀summarized฀in฀Fig.฀2.1.

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 17
DOI:฀10.1007/978-3-540-88116-2_2,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
18 2 Photosynthesis and Sucrose Production

Fig. 2.1฀ Photosynthesis฀ and฀ respiration.฀ Left฀ side฀ is฀ Fig.฀ 1.6.฀ Right฀ side฀ shows฀ photosynthesis฀ in฀
which฀sunlight฀and฀water฀in฀the฀atmosphere฀are฀absorbed฀by฀plants฀and฀algae฀to฀generate฀ATP฀and฀
NADPH,฀which฀make฀carbohydrates฀and฀other฀organic฀carbon฀products฀from฀carbon฀dioxide,฀which฀
is฀ absorbed฀ from฀ the฀ atmosphere฀ separately.฀ All฀ of฀ the฀ carbon฀ in฀ plants฀ and฀ algae฀ is฀ ultimately฀
derived฀from฀a฀single฀source,฀carbon฀dioxide,฀and฀they฀are฀called฀autotrophs.฀Nitrogen฀is฀obtained฀
mostly฀as฀ammonia฀from฀bacterial฀metabolism฀of฀proteins฀from฀dead฀organism฀in฀the฀soil

2.1.2.
The Light Reaction

Photosynthesis฀is฀the฀sunlight-mediated฀splitting฀of฀water฀into฀oxygen฀and฀energy.฀It฀occurs฀
within฀a฀special฀membrane,฀the฀thylakoid฀membrane,฀which฀contains฀chlorophyll฀and฀sur-
rounds฀a฀lumen.฀This฀membrane฀resembles฀the฀thick฀inner฀membrane฀of฀mitochondria฀or฀
oxygen-utilizing฀bacteria.฀The฀thylakoid฀membrane฀lies฀within฀an฀organelle฀called฀a฀chlo-
roplast,฀which฀is฀exclusive฀to฀leaf฀cells฀and฀algae฀where฀photosynthesis฀occurs.฀Central฀to฀
the฀process฀of฀photosynthesis฀is฀the฀light-mediated฀loss฀of฀two฀electrons฀and฀two฀hydrogen฀
atoms฀from฀a฀molecule฀of฀water฀(Fig.฀2.2).฀Both฀electrons฀and฀one฀of฀the฀two฀hydrogen฀
atoms฀pass฀to฀NADPH,฀the฀equivalent฀of฀NADH฀in฀mitochondria฀or฀bacteria;฀the฀other฀
hydrogen฀atom฀becomes฀a฀hydrogen฀ion฀(H+),฀a฀proton.฀When฀two฀molecules฀of฀water฀are฀
split฀by฀chlorophyll,฀a฀molecule฀of฀oxygen฀(O2)฀is฀released.฀For฀each฀molecule฀of฀oxygen,฀
two฀molecules฀of฀NADPH฀and฀two฀protons฀(H+)฀are฀made฀(Fig.฀2.3).
Figure฀2.4฀illustrates฀electron฀and฀proton฀transport฀processes.฀Electrons฀are฀initially฀ener-
gized฀by฀sunlight฀hitting฀photosystem฀II฀(PSII;฀see฀igure฀legend)฀and฀transported฀to฀photo-
system฀I฀(PSI).฀In฀PSI,฀sunlight฀energy฀is฀again฀imparted฀and฀the฀electrons฀are฀transferred฀by฀
ferredoxin,฀another฀electron฀carrier,฀to฀NADPH.฀Electron฀transport฀from฀PSII฀to฀PSI฀is฀via฀
plastoquinone฀(PQ),฀cytochrome฀b6/cytochrome฀f฀complex,฀and฀plastocyanin฀(blue฀arrows฀
in฀Fig.฀2.4).฀During฀electron฀transport,฀protons฀are฀taken฀up฀by฀plastoquinone฀(similar฀to฀
2.1.2. The Light Reaction 19

Fig. 2.2฀ In฀plants,฀light฀mediates฀the฀loss฀of฀two฀electrons฀

and฀two฀hydrogen฀atoms฀from฀a฀molecule฀of฀water.฀See฀ O O
text฀for฀discussion฀of฀this฀process +
H H H H

2H2O

Photosynthesis

O H:H
+
O H:H

− +
O2 + 2H + 2H (2H2)

NADP+ NADPH + H+
Sunlight
H O O
H H
C C +
NH2 NH2 + H
+
N H O฀฀ H N̈
R R

O
O

H O H O฀฀ H O
H H
C C +
NH2 NH2 + H
+
Fig. 2.3฀ The฀electrons฀are฀passed฀from฀water฀to฀ N N̈
NADPH.฀See฀text R R

ubiquinone฀in฀mitochondria)฀and฀released฀into฀the฀lumen฀(see฀legend฀to฀Fig.฀2.4).฀As฀they฀
accumulate,฀the฀protons฀start฀diffusing฀across฀the฀thylakoid฀membrane฀through฀an฀ATP฀syn-
thase฀F0/F1฀complex฀identical฀to฀that฀of฀mitochondria฀(red฀arrows฀in฀Fig.฀2.4).฀Electrons฀that฀
reach฀PSI฀are฀re-energized฀by฀sunlight฀and฀reduce฀NADP+฀by฀transport฀through฀ferredoxin฀
(blue฀arrows฀in฀Fig.฀2.4).฀The฀ATP,฀NADPH,฀and฀protons฀accumulate฀in฀the฀stroma฀of฀the฀
chloroplast฀where฀they฀assist฀in฀the฀synthesis฀of฀triose฀phosphate฀and฀starch.
Figure฀2.5฀compares฀the฀orientation฀of฀the฀ATP฀synthase฀F0/F1฀complex฀in฀mitochondria฀
with฀that฀in฀chloroplasts.฀The฀lumen฀enclosed฀by฀the฀thylakoid฀membrane฀is฀slightly฀acidic;฀
it฀ corresponds฀ to฀ the฀ mitochondrial฀ intermembrane฀ space฀ where฀ electron฀ transport฀ irst฀
pumps฀protons฀(H+).฀In฀chloroplasts,฀ATP฀is฀made฀as฀protons฀diffuse฀from฀the฀thylakoid฀
lumen฀through฀the฀membrane฀to฀the฀chloroplast฀stroma฀(Fig.฀2.4).฀In฀mitochondria,฀ATP฀is฀
made฀as฀protons฀diffuse฀from฀the฀mitochondrial฀intermembrane฀space฀through฀the฀inner฀
mitochondrial฀membrane฀to฀the฀mitochondrial฀“lumen”฀or฀matrix.
20 2 Photosynthesis and Sucrose Production

Fig. 2.4฀ Phosphorylation฀and฀electron฀transport฀in฀chloroplasts.฀Electron฀low฀is฀shown฀in฀blue฀and฀

the฀proton฀low฀in฀red.฀The฀thylakoid฀membrane฀is฀colored฀yellow฀and฀the฀lumen฀is฀colored฀green.฀
Light฀activates฀photosynthesis฀site฀II฀(photosystem฀II฀[PSII])฀within฀chlorophyll฀whose฀manga-
nese฀ions฀bind฀water฀molecules.฀Light฀rays฀attack฀a฀bound฀water฀molecule,฀breaking฀it฀up฀into฀
oxygen฀atoms฀that฀combine฀to฀form฀oxygen฀gas,฀and฀hydrogen฀atoms฀that฀split฀into฀protons฀and฀
electrons.฀Each฀two฀molecules฀of฀water฀give฀two฀pairs฀of฀electrons฀that฀are฀transported฀along฀with฀
four฀protons฀to฀plastoquinone฀(2PQH2).฀The฀protons฀are฀released฀into฀the฀thylakoid฀lumen฀(red฀
arrows)฀as฀PQH2฀is฀reoxidized฀to฀PQ฀by฀the฀cytochrome฀complex฀(blue฀arrows).฀Protons฀accumu-
late฀in฀the฀lumen฀(P฀side฀of฀thylakoid฀membrane)฀and฀diffuse฀into฀the฀chloroplast฀stroma฀through฀
carriers฀that฀synthesize฀ATP฀(CF0฀and฀CF1;฀purple฀at฀the฀foot฀of฀igure).฀ATP฀is฀made฀from฀ADP฀
and฀phosphate฀(Pi)฀in฀the฀chloroplast฀stroma฀(N฀side฀of฀thylakoid฀membrane).฀As฀detailed฀in฀the฀
text,฀electrons฀are฀transported฀from฀PSII฀to฀photosystem฀I฀([PSI],฀pink)฀where฀they฀are฀activated฀
by฀light฀to฀reduce฀NADP+฀(top฀right฀of฀igure).฀Extra฀protons฀are฀taken฀back฀into฀the฀thylakoid฀
lumen฀by฀plastoquinone.฀The฀plant฀partitions฀its฀electron฀low฀and฀protons฀so฀that฀the฀ratio฀of฀ATP฀
to฀NADPH฀matches฀its฀need฀for฀assimilation฀of฀carbon฀dioxide฀into฀carbohydrates,฀the฀primary฀
product฀of฀photosynthesis฀(Adapted฀from฀Fig.฀ 19-57฀in฀Lehninger฀Principles฀of฀Biochemistry.฀
D.L.฀Nelson฀&฀M.M.฀Cox,฀4th฀Ed.฀2005.฀W.H.฀Freeman฀&฀Co.,฀New฀York)
2.2.1. The Dark Reaction 21

Fig. 2.5฀ ATPase฀orientation฀in฀

mitochondria฀and฀
฀chloroplasts.฀Blue฀dots฀
indicate฀the฀ATP฀synthetase฀in฀
mitochondria฀(top฀left)฀and฀
green฀dots฀in฀chloroplasts฀(top฀
right).฀See฀text฀for฀further฀
description฀(Adapted฀from฀
Fig.฀19-58฀in฀Lehninger฀
Principles฀of฀Biochemistry.฀
D.L.฀Nelson฀&฀M.M.฀Cox,฀4th฀
Ed.฀2005.฀W.H.฀Freeman฀&฀
Co.,฀New฀York)

Animals฀and฀bacteria฀are฀heterotrophs;฀they฀obtain฀carbon฀in฀various฀forms฀as฀food฀and฀
metabolize฀many฀forms฀of฀it฀to฀provide฀energy฀and฀body฀structure.฀Plants฀are฀autotro-
phs;฀ all฀ their฀ carbon฀ comes฀ from฀ CO2฀ powered฀ by฀ photosynthesis.฀ Photosynthesis฀
occurs฀within฀the฀thylakoid฀membranes฀of฀chloroplasts฀in฀plant฀leaves,฀and฀it฀is฀medi-
ated฀by฀chlorophyll.฀The฀light฀reaction฀splits฀water฀into฀O2,฀electrons,฀and฀protons฀(H+).฀
NADPH฀ is฀ produced฀ by฀ electron฀ transport฀ and฀ ATP฀ synthesis฀ by฀ associated฀ proton฀
transport.

2.2.1.
The Dark Reaction

The฀utilization฀of฀carbon฀dioxide฀by฀ATP฀and฀NADPH฀occurs฀in฀the฀chloroplast฀matrix,฀
(outside฀the฀thylakoid฀lumen).฀A฀series฀of฀reactions฀assimilates฀carbon฀dioxide฀(Fig.฀2.6),฀
the฀Calvin฀cycle฀or฀dark฀reaction,฀and฀generates฀fructose฀6-phosphate.฀Fructose฀6-phosphate฀
is฀ the฀ immediate฀ precursor฀ of฀ glucose฀ 6-phosphate฀ for฀ the฀ synthesis฀ of฀ starch฀ in฀ the฀
22 2 Photosynthesis and Sucrose Production

Fig. 2.6฀ Overall฀design฀of฀photosynthesis.฀The฀light฀

reactions฀make฀ATP฀and฀NADPH฀to฀be฀used฀in฀the฀
carbon฀dioxide฀assimilation฀reactions฀that฀form฀fructose฀
and฀glucose฀(Modiied฀from฀Fig.฀19-37฀in฀Lehninger฀
Principles฀of฀Biochemistry.฀D.L.฀Nelson฀&฀M.M.฀Cox,฀
4th฀Ed.฀2005.฀W.H.฀Freeman฀&฀Co.,฀New฀York)

H2O O2

Light
reactions

NADP+ NADPH
ADP+ Pi ATP

Carbon-assimilation
reactions

Carbohydrate CO2

CH2OPO32− CH2OPO32−

C O CO2 HO C COO− H2O CH2OPO32−

H C OH C O 2 HO C H

H C OH H C OH CO2−
3-Phosphoglycerate
CH2OPO32− CH2OPO32−
Ribulose Unstable
1,5-bisphosphate intermediate

Fig. 2.7฀ Assimilation฀of฀carbon฀dioxide.฀The฀key฀reaction฀of฀the฀dark฀reaction฀is฀the฀assimilation฀฀

of฀ carbon฀ dioxide฀ by฀ ribulose฀ 1:5-bisphosphate฀ carboxylase฀ (Simpliied฀ from฀ Fig.฀ 26-31฀ in฀
Biochemistry.฀L.฀Stryer,฀4th฀Ed.฀1995.฀W.H.฀Freeman฀&฀Co.,฀New฀York)

c฀ hloroplast฀matrix฀and฀sucrose฀in฀the฀leaf฀cell฀cytosol.฀The฀substrate฀for฀carbon฀dioxide฀is฀
ribulose฀1:5-bisphosphate,฀and฀the฀reaction฀is฀mediated฀by฀a฀substrate-speciic฀carboxy-
lase,฀rubisco฀(Fig.฀2.7).฀The฀products฀are฀two฀molecules฀of฀3-phosphoglycerate,฀each฀of฀
2.2.1. The Dark Reaction 23

Stage 1:
CH2O P Fixation
ADP C O
ATP (3) CO2
Stage 3: CHOH
Regeneration (3) (3)
CHOH
of acceptor
CH2O P rubisco
Energy Ribulose 1,5-
(5)
production bisphosphate
via glycolysis; (3)
starch or CHO COO–
sugar (1) CHOH CHOH
synthesis
CH2O P CH2O P
3-Phosphoglycerate
Glyceraldehyde 3-phosphate
(6)
(GA3P) (6)

Stage 2:
Reduction
Pi
(6) ATP
NADP+
ADP (6)
(6) NADPH + H+
(6)
(6)

Fig. 2.8฀ Summary฀of฀the฀Calvin฀cycle.฀The฀cycle฀consists฀of฀three฀stages,฀culminating฀in฀the฀regen-

eration฀of฀ribulose฀1:5-bisphosphate฀and฀a฀net฀increase฀in฀glyceraldehyde฀3-phosphate.฀Numbers฀
in฀ parenthesis฀ reveal฀ the฀ fate฀ of฀ carbon฀ atoms฀ entering฀ and฀ leaving฀ the฀ cycle฀ (Modiied฀ from฀
Fig.฀ 20-04฀ in฀ Lehninger฀ Principles฀ of฀ Biochemistry.฀ D.L.฀ Nelson฀ &฀ M.M.฀ Cox,฀ 4th฀ Ed.฀ 2005.฀
W.H.฀Freeman฀&฀Co.,฀New฀York)

which฀ reacts฀ with฀ an฀ ATP฀ molecule฀ to฀ make฀ 1:3-bisphosphoglycerate,฀ and฀ then฀ with฀
NADPH฀to฀be฀reduced฀to฀glyceraldehyde฀3-phosphate.฀For฀every฀six฀molecules฀of฀glyc-
eraldehyde฀3-phosphate฀obtained฀from฀three฀molecules฀of฀ribulose฀1:3-bisphosphate,฀one฀
is฀used฀for฀energy฀via฀glycolysis,฀or฀to฀synthesize฀monosaccharides฀or฀starch฀(Fig.฀2.8).฀
The฀other฀ive฀interact฀to฀make฀three฀molecules฀of฀ribulose฀5-phosphate,฀each฀of฀which฀
utilizes฀three฀ATP฀molecules฀to฀regenerate฀three฀ribulose฀1:5-bisphosphate฀molecules.
The฀enzymes฀that฀regenerate฀ribulose฀5-phosphate฀from฀glyceraldehyde฀3-phosphate฀
are฀the฀same฀as฀those฀of฀the฀pentose฀phosphate฀path฀in฀non-photosynthetic฀organisms.฀
The฀three฀stages฀of฀the฀Calvin฀cycle฀are฀(1)฀ixation฀of฀carbon฀dioxide฀by฀rubisco,฀which฀
utilizes฀ ATP;฀ (2)฀ reduction฀ of฀ the฀ 1:3-bisphosphoglycerate฀ to฀ 3-bisglyceraldehyde,฀
which฀ utilizes฀ ATP฀ and฀ NADPH;฀ and,฀ inally,฀ the฀ formation฀ of฀ ribulose฀ 5-phosphate,฀
which฀reacts฀with฀ATP฀to฀regenerate฀the฀rubisco฀substrate,฀ribulose฀1:3-bisphosphate.
24 2 Photosynthesis and Sucrose Production

2.2.2.
Starch and Sucrose Provide the Carbon Skeletons of All Plant Compounds

During฀ photosynthesis,฀ starch฀ is฀ synthesized฀ and฀ stored฀ in฀ the฀ chloroplast฀ matrix฀ and฀
sucrose฀is฀synthesized฀in฀the฀leaf฀cytosol฀from฀which฀it฀diffuses฀to฀the฀rest฀of฀the฀plant.฀
Starch฀resembles฀glycogen,฀but฀it฀has฀few฀or฀no฀a-1:6฀glycosidic฀linkages,฀the฀glucose฀resi-
dues฀are฀little฀branched฀(amylopectin)฀or฀not฀branched฀(amylose).฀Amylose฀and฀amylopec-
tin฀are฀substrates฀for฀salivary฀amylase,฀and฀their฀structures฀are฀illustrated฀in฀Fig.฀12.10.฀The฀
substrate฀ for฀ starch฀ synthase฀ is฀ ADP-glucose,฀ whereas฀ the฀ substrate฀ for฀ glycogen฀ and฀
sucrose฀synthases฀is฀UDP-glucose.
Sucrose฀is฀a฀highly฀soluble฀disaccharide฀that฀provides฀a฀mobile฀energy฀source฀for฀all฀the฀
plant฀cells.฀Sugar฀cane฀stores฀large฀amounts฀of฀sucrose฀in฀its฀leaves฀and฀stalk,฀whereas฀sugar฀
beet฀stores฀it฀in฀roots.฀All฀plants฀make฀sucrose฀from฀two฀molecules฀of฀fructose฀6-phosphate.฀One฀
molecule฀is฀activated฀with฀UDP฀and฀isomerized฀to฀UDP-glucose.฀Sucrose฀6-phosphate฀syn-
thase฀reacts฀with฀UDP-glucose฀and฀fructose฀6-phosphate฀to฀make฀sucrose฀6-phosphate.฀The฀
latter฀then฀reacts฀with฀a฀phosphatase฀to฀produce฀sucrose฀(Fig.฀2.9).
The฀amount฀of฀sucrose฀is฀regulated฀by฀a฀kinase฀that฀inhibits฀sucrose฀synthase฀by฀phos-
phorylating฀a฀serine฀residue฀on฀its฀polypeptide฀and฀a฀phosphatase฀that฀activates฀the฀synthase฀
by฀dephosphorylating฀the฀serine฀residue.฀This฀mode฀of฀regulation฀resembles฀that฀of฀glyco-
gen฀synthase฀in฀the฀mammalian฀liver.฀Both฀enzymes฀make฀enzyme฀energy฀storage฀com-
pounds:฀sucrose฀for฀the฀plant฀as฀a฀whole฀and฀glycogen฀speciically฀for฀liver฀or฀muscles.

2.2.3.
Plants Are Autotrophs

The฀light-powered฀incorporation฀of฀carbon฀dioxide฀into฀ribulose฀bisphosphate฀by฀the฀Calvin฀
cycle฀enzymes฀synthesizes฀starch,฀which฀remains฀in฀the฀chloroplast฀stroma.฀Once฀the฀space฀
for฀starch฀in฀the฀chloroplast฀stroma฀is฀exhausted,฀the฀3-phosphoglycerate฀intermediate฀is฀
converted฀by฀triosephosphate฀isomerase฀to฀dihydroxyacetone฀phosphate฀(DHAP).฀DHAP฀is฀
the฀only฀metabolite฀allowed฀to฀leave฀the฀chloroplast,฀and฀it฀does฀so฀in฀exchange฀for฀phos-
phate฀(Pi)฀from฀the฀cytosol฀(Fig.฀2.10).฀The฀incoming฀Pi฀reacts฀with฀ADP฀to฀form฀ATP฀in฀
the฀ chloroplast,฀ and฀ it฀ eventually฀ ends฀ up฀ in฀ a฀ new฀ molecule฀ of฀ triosephosphate.฀ The฀
exchange฀of฀DHAP฀for฀Pi฀is฀mediated฀by฀a฀protein฀transporter฀called฀an฀antiport฀that฀allows฀
the฀two฀metabolites฀to฀pass฀in฀opposite฀directions฀through฀the฀membrane฀and฀down฀their฀
respective฀ concentration฀ gradients.฀ Similar฀ antiport฀ transport฀ gradients฀ are฀ required฀ for฀
bone฀metabolism฀(Sects.฀9.3.5,฀and฀10.1.4).฀Once฀in฀the฀leaf฀cytosol,฀the฀DHAP฀is฀converted฀
back฀ to฀ 3-phosphoglycerate,฀ which฀ is฀ metabolized฀ to฀ various฀ compounds,฀ especially฀
sucrose.
At฀ night,฀ there฀ is฀ no฀ photosynthesis฀ and฀ respiratory฀ energy฀ becomes฀ as฀ important฀ in฀
plants฀as฀in฀other฀organisms.฀The฀starch฀that฀accumulated฀during฀the฀day฀is฀metabolized฀by฀
the฀activation฀of฀phosphorylase฀and฀some฀glycolytic฀enzymes฀to฀triose฀phosphate฀(Fig.฀1.7),฀
2.2.3. Plants Are Autotrophs 25

Fructose 6-phosphate UDP-glucose

O
CH2OH
HOH2C O
O HN
HO + OH
HO O O O
HO CH2OPO32− P P O
OH O
OH
O O O O

Sucrose
6-phosphate
OH OH
synthase

CH2OH HN
O HOH2C O
OH HO + O O O
P P O
HO O 2− O
CH2OPO3
OH O O O O
OH
Sucrose 6-phosphate
UDP OH OH

Sucrose
6-phosphatase

CH2OH
O HOH2C O
OH HO + HPO42−
HO O
CH2OH
OH
OH
Sucrose

Fig. 2.9฀ Synthesis฀of฀sucrose.฀See฀text฀(Modiied฀from฀Fig.฀20-13฀in฀Berg฀JM,฀Tymoczko฀JL฀and฀

Stryer฀L.฀Biochemistry,฀5th฀Ed.฀2002.฀W.H.฀Freeman฀&฀Co.,฀New฀York฀to฀also฀show฀the฀phosphatase฀
step).฀Note:฀The฀diagram฀of฀fructose฀in฀this฀igure฀(red)฀is฀rotated฀so฀that฀its฀anomeric฀carbon฀atom฀
(C2)฀lies฀to฀the฀right฀of฀the฀ring฀structure฀instead฀of฀the฀left฀as฀in฀a฀conventional฀diagram.฀Because฀of฀
the฀rotation,฀it฀is฀not฀obvious฀that฀the฀fructose฀bond฀in฀sucrose฀is฀in฀the฀b-anomeric฀coniguration.฀
Only฀the฀glucose฀bond฀is฀in฀the฀a-anomeric฀coniguration.฀The฀conventional฀diagrams฀of฀glucose฀
and฀fructose฀alone฀and฀in฀sucrose฀are฀illustrated฀in฀Fig.฀15.6.

which฀passes฀through฀the฀antiport฀as฀DHAP฀in฀exchange฀for฀Pi฀(Fig.฀2.10).฀In฀the฀cytosol,฀
DHAP฀moves฀to฀the฀mitochondria฀as฀a฀source฀of฀ATP.฀The฀Pi฀enters฀the฀chloroplast฀where฀
it฀is฀a฀co-substrate฀for฀phosphorylase฀for฀starch฀breakdown.฀
26 2 Photosynthesis and Sucrose Production

a b
Day (photosynthesis) Night (respiration)

Starch
Triose Triose
ATP Phosphorylase phosphate
Chloroplast phosphate
stroma

Cytosol Pi Pi
To cell mass
Sucrose Mitochondrial respiration

Fig. 2.10฀ Phosphoglycerate฀utilization.฀(a)฀During฀the฀day.฀Photosynthesis฀in฀the฀chloroplast฀makes฀

starch฀until฀there฀is฀no฀more฀room.฀The฀Calvin฀cycle฀continues฀to฀make฀triose฀phosphate,฀which฀฀
exits฀the฀chloroplast฀in฀exchange฀for฀organic฀phosphate฀(Pi)฀entering฀the฀chloroplast฀and฀convert-
ing฀ADP฀to฀ATP.฀In฀the฀cytosol,฀the฀triose฀phosphate฀is฀mostly฀converted฀to฀sucrose฀but฀also฀to฀
small฀ amounts฀ of฀ other฀ compounds฀ such฀ as฀ amino฀ acids฀ for฀ transport฀ throughout฀ the฀ plant.฀ (b)฀
During฀the฀night.฀Phosphorylase฀is฀activated฀and฀it฀breaks฀up฀the฀starch฀to฀glucose฀6-phosphate฀
from฀which฀triose฀phosphate฀is฀made.฀The฀triose฀phosphate฀is฀exchanged฀for฀Pi.฀The฀Pi฀is฀a฀sub-
strate฀for฀phosphorylase฀and฀keeps฀it฀active.฀Once฀in฀the฀cytosol,฀the฀triose฀phosphate฀is฀transferred฀
mostly฀to฀mitochondria฀for฀respiration

The฀dark฀reaction฀(Calvin฀cycle)฀uses฀the฀NADPH฀and฀ATP฀to฀make฀glyceraldehyde฀
3-phosphate฀(triose฀phosphate),฀which฀is฀metabolized฀initially฀to฀starch,฀sucrose,฀and฀
cellulose.฀Starch฀and฀sucrose฀are฀the฀major฀plant฀storage฀products.฀Starch฀is฀synthesized฀
from฀ ADP-glucose฀ in฀ the฀ chloroplast,฀ sucrose฀ from฀ fructose฀ 6-phosphate฀ and฀ UDP-
glucose฀in฀the฀leaf฀cytosol.

2.3.1.
Sucrose Is the Primary Transport Sugar and Plays a Central
Role in Plant Growth and Development

The฀availability฀of฀metabolites฀for฀sucrose฀synthesis฀and฀the฀need฀for฀products฀of฀sucrose฀
degradation฀regulate฀gene฀expression.฀For฀respiration,฀sucrose฀is฀hydrolyzed฀by฀invertase฀
to฀free฀glucose฀and฀fructose,฀which฀are฀phosphorylated฀and฀undergo฀glycolysis฀to฀pyruvate.฀
The฀pyruvate฀is฀then฀either฀metabolized฀by฀mitochondrial฀electron฀transport฀to฀ATP฀and฀
NADH฀(respiration),฀or฀metabolized฀to฀provide฀starting฀products฀for฀amino฀acid,฀lipid,฀and฀
nucleotide฀syntheses.
2.3.1. Sucrose Is the Primary Transport Sugar and Plays a Central Role in Plant Growth and Development 27

Cellulose฀helps฀protect฀plants฀from฀their฀environment฀and฀to฀access฀sunlight฀by฀pro-
viding฀a฀irm฀structure.฀Cellulose฀is฀b1→4฀glucose,฀whereas฀starch฀is฀a1→4฀glucose.฀
Both฀ are฀ derived฀ from฀ sucrose฀ outside฀ of฀ chloroplasts.฀ The฀ sucrose฀ synthase฀ reaction฀
(Fig.฀2.9)฀is฀reversed:฀sucrose฀is฀phosphorylated฀with฀ATP฀to฀make฀sucrose฀6-phosphate,฀
an฀excess฀of฀which฀reverses฀the฀reaction.฀The฀products฀are฀UDP-glucose฀and฀fructose฀
6-phosphate.฀ Cellulose฀ is฀ synthesized฀ from฀ UDP-glucose฀ by฀ cellulose฀ synthase.฀
Additional฀UDP-glucose฀reacts฀with฀ATP฀to฀form฀ADP-glucose,฀the฀precursor฀of฀starch.฀
The฀ fructose฀ 6-phosphate฀ is฀ metabolized฀ to฀ provide฀ the฀ necessary฀ energy฀ (ATP฀ and฀
NADPH)฀by฀respiration.

Sucrose฀ plays฀ a฀ central฀ role฀ in฀ plant฀ growth฀ and฀ development.฀ Invertase-catalyzed฀
hydrolysis฀ of฀ sucrose฀ is฀ associated฀ with฀ the฀ respiration฀ required฀ for฀ plant฀ growth,฀
whereas฀sucrose฀synthase-catalyzed฀hydrolysis฀is฀linked฀to฀cell฀wall฀or฀other฀storage฀
product฀biosynthesis.฀Cellulose฀is฀synthesized฀from฀UDP-glucose฀using฀cellulose฀syn-
thase฀in฀membranes.฀Cellulose฀protects฀plant฀cells฀from฀the฀environment฀and฀provides฀
them฀with฀a฀irm฀structure฀to฀access฀sunlight.
 The Connective Tissue Extracellular
Matrix and Its Major Components 3

Dentists฀are฀concerned฀with฀the฀structures฀of฀the฀oral฀cavity,฀the฀teeth,฀the฀oral฀mucosa,฀
the฀underlying฀dermis฀and฀the฀alveolar฀bone.฀The฀dermis฀beneath฀the฀oral฀mucosa฀and฀
surrounding฀the฀teeth฀is฀similar฀to฀the฀dermis฀beneath฀the฀skin.฀Both฀are฀primarily฀com-
posed฀of฀collagen฀ibers฀within฀a฀connective฀tissue฀or฀stromal฀matrix.฀Variants฀of฀this฀
matrix฀are฀present฀in฀the฀soft฀and฀hard฀tissues฀of฀the฀body,฀including฀the฀teeth,฀gingiva,฀
periodontium,฀and฀alveolar฀bone.฀The฀Sects.฀1฀and฀2฀of฀this฀introductory฀chapter฀describe฀
the฀gingival฀stromal฀matrix฀and฀its฀major฀components.฀Sect.฀3฀describes฀how฀the฀cells฀
and฀stroma฀interact.฀Subsequent฀chapters฀describe฀in฀detail฀the฀structures฀of฀the฀various฀
types฀of฀collagen,฀elastin,฀and฀other฀proteins฀and฀proteoglycans฀that฀contribute฀to฀the฀
matrix฀(Chap.฀6),฀the฀synthesis฀and฀breakdown฀of฀collagen฀(Chaps.฀7฀and฀8),฀the฀struc-
ture฀and฀synthesis฀of฀bone฀and฀tooth฀enamel฀(Chap.฀9),฀and฀bone฀dissolution฀and฀turn-
over฀related฀to฀overall฀calcium฀metabolism฀(Chap.฀10).

3.1.1.
Major Components of the Connective Tissue (Stromal) Matrix

The฀stroma฀or฀internal฀solid฀mass฀of฀an฀organism฀comprises฀its฀cells฀and฀embedded฀matrix.฀
The฀major฀components฀of฀the฀stromal฀matrix฀of฀vertebrates฀are฀collagen฀ibers฀embedded฀
within฀a฀polysaccharide฀ground฀substance฀of฀hyaluronan฀and฀various฀proteo-glycosamino-
glycans฀(Fig.฀3.1).฀The฀stromal฀cells฀are฀derived฀from฀mesodermal฀lineage฀cells฀that฀have฀
differentiated฀into฀ibroblasts,฀chrondroblasts,฀and฀osteoblasts฀that฀make,฀respectively,฀con-
nective฀tissue,฀cartilage฀and฀bone.฀The฀stroma฀and฀bone฀are฀permeated฀with฀low฀molecular฀
weight฀nutrients฀(glucose,฀amino฀acids,฀vitamins,฀etc.)฀and฀oxygen.฀Within฀the฀blood,฀some฀
white฀cells฀become฀attracted฀to฀the฀stroma฀and฀differentiate,฀depending฀on฀the฀tissue฀and฀its฀
environment.฀The฀stromal฀matrix฀is฀covered฀by฀an฀epithelium฀derived฀from฀ectoderm฀(e.g.,฀
epidermis฀or฀oral฀mucosa),฀or฀endoderm฀(e.g.,฀capillary,฀lung฀alveoli,฀kidney฀tubules,฀or฀gut฀
lining).฀The฀stroma฀beneath฀an฀epidermis฀is฀referred฀to฀as฀the฀dermis.
Where฀a฀stromal฀surface฀meets฀the฀epithelium,฀the฀ibroblasts฀interact฀with฀epithelial฀
cells฀to฀form฀a฀basal฀lamina.฀This฀thin,฀lexible฀layer฀of฀specialized฀extracellular฀matrix฀
(40฀ to฀ 120฀ nm฀ thick)฀ underlies฀ all฀ epithelial฀ cell฀ sheets฀ or฀ tubes฀ and฀ is฀ detected฀ by฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 29
DOI:฀10.1007/978-3-540-88116-2_3,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
30 3 The Connective Tissue Extracellular Matrix and Its Major Components

Epithelium
Basal lamina

Collagen fiber

Connective tissue
Macrophage

matrix
Capillary

Elastic fiber
Fibroblast
Mast cell

Hyaluronan/glycos-
aminoglycan matrix

50 µm

Fig. 3.1฀ Composition฀of฀the฀connective฀tissue฀extracellular฀matrix.฀Collagen฀ibers,฀maroon:฀Elastic฀

ibers,฀green:฀Hyaluronan-proteoglycan฀matrix,฀gray.฀Fibroblasts,฀a฀macrophage,฀a฀mast฀cell,฀and฀a฀
capillary฀containing฀a฀red฀blood฀cell฀are฀also฀shown฀(Modiied฀from฀Fig.฀19–34฀in฀The฀Molecular฀
Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀Taylor฀&฀Francis฀Group,฀NY)

visualizing฀the฀tissue฀under฀an฀electron฀microscope.฀Figure฀3.2฀shows฀three฀alternative฀
o฀ rganizations฀of฀a฀basal฀lamina:฀surrounding฀skeletal฀muscles,฀underlying฀epithelia,฀and฀
interposed฀between฀two฀cell฀sheets฀as฀in฀the฀kidney฀glomeruli.฀A฀basal฀lamina฀acts฀as฀a฀
semipermeable฀membrane,฀to฀separate฀specialized฀cells฀such฀as฀epithelial฀or฀muscle฀cells฀
from฀soluble฀stromal฀proteins,฀or฀in฀the฀case฀of฀the฀kidneys,฀to฀act฀as฀a฀membrane฀through฀
which฀metabolic฀end฀products฀from฀the฀body฀can฀pass.฀The฀rich฀capillary฀network฀imme-
diately฀beneath฀an฀epithelium฀ensures฀that฀essential฀nutrients฀and฀low฀molecular฀weight฀
cell-modifying฀agents฀(chemokines)฀have฀access฀to฀the฀overlying฀cells.฀Basal฀laminas฀
prevent฀soluble฀plasma฀proteins฀from฀passing฀into฀stromal฀luid฀or฀soluble฀stromal฀pro-
teins฀from฀passing฀into฀epithelial฀luid.

3.1.2.
Collagen

Collagen฀ is฀ the฀ major฀ protein฀ of฀ the฀ stroma.฀ There฀ are฀ two฀ major฀ groups฀ of฀ collagens฀
encoded฀in฀the฀genome:฀ibrillar฀and฀non-ibrillar฀(Table฀3.1).฀Collagen฀ibers฀are฀the฀most฀
abundant฀group฀and฀visible฀to฀the฀naked฀eye.฀Collagen฀ibrils฀are฀visible฀only฀through฀a฀
3.1.2. Collagen 31

Muscle Epithelium
Basal lamina Connective tissue Kidney glomerulus
Lumen or
external surface
Blood Endothelial cell

Urine
Muscle cell plasma membrane Connective tissue Basal lamina Epithelial cell Basal lamina

Fig. 3.2฀ Relationship฀of฀the฀connective฀tissue฀stroma฀to฀an฀epithelium.฀Fibroblasts฀secrete฀unique฀

types฀of฀laminin฀proteins฀that฀interact฀with฀each฀other฀and฀type฀IV฀collagen,฀to฀form฀a฀basal฀lamina฀
(yellow)฀that฀is฀tightly฀attached฀to฀the฀connective฀tissue฀(stromal)฀cells฀and฀also฀to฀the฀associated฀
muscle฀ or฀ epithelial฀ cell.฀ The฀ kidney฀ glomerulus฀ is฀ a฀ specialized฀ tissue฀ in฀ which฀ the฀ stroma฀ is฀
absent฀and฀endothelial฀cells฀and฀epithelial฀cells฀are฀separated฀by฀the฀basal฀lamina฀which฀ilters฀the฀
blood฀as฀the฀irst฀step฀in฀urine฀collection.฀The฀attachment฀of฀an฀epithelium฀to฀stromal฀collagen฀is฀
discussed฀in฀detail฀in฀Chap.฀5฀(Fig.฀19-55฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀
4th฀Ed.฀2002.฀Garland฀Science,฀Taylor฀&฀Francis฀Group,฀NY)

Table 3.1฀ Major฀functions,฀genetic,฀and฀polypeptide฀composition฀of฀some฀common฀types฀of฀collagen

Collagen฀functions฀and฀types Gene฀name Polypeptide฀composition
FIBRILLAR฀collagens฀฀ COL1A1,฀COL1A2 [a1(I)]2[a2(I)]฀฀
(Types฀I,฀II,฀III,฀V,฀&฀XI) (Most฀common)
COL2A1 [a1(II)]3
Dermal฀reticular฀ibers฀฀ COL3A1 [a1(III)]3
(Type฀III฀with฀V) COL5A1,฀COL5A2 [a1(V)]2[a2(V)]
COL11A1,฀COL11A2 [a1(XI)]2[a2(XI)]
NETWORK฀(ine฀bone฀marrow/ COL8A1,฀COL8A2 [a1(VIII)]2[a2(IV)]
epiphyseal฀reticular฀ibers)฀฀ COL10A1 [a1(X)]3
(Type฀III฀with฀VIII฀&฀X)
BASEMENT฀MEMBRANE฀ COL4A1,฀COL4A2,฀ [a1(IV)]2[a2(IV)]฀฀
collagens฀(Type฀IV) COL4A3 (In฀all฀tissues)
COL4A4,฀COL4A5,฀ [a3(IV)][a4(IV)][a5(IV)]฀
COL4A6 (Glomerulus)
[a5(IV)]2[a6(IV)]฀
(Restricted)
MICROFIBRILLAR฀collagens฀฀ COL6A1,฀COL6A2,฀ [a1(VI)][a2(VI)][a3(VI)]
(Type฀VI) COL6A3
ANCHORING฀ibrils฀with฀฀ COL7A1,฀COL17A1 [a1(VII)]3฀[a1(XVII)]3
interrupted฀triple฀helix;฀long฀฀
chain฀collagen฀(Type฀VII฀&฀XVII)
FACIT฀Fibril-associated฀collagens฀ COL9A1,฀COL9A2,฀ [a1(IX)][a2(IX)][a3(IX)]
with฀interrupted฀triple฀helices฀฀ COL9A3
(Types฀IX฀&฀XII) COL12A1 [a1(XII)]3
Modiied฀from฀public฀domain฀data฀at฀http://en.wikipedia.org/wiki/Collagen
32 3 The Connective Tissue Extracellular Matrix and Its Major Components

light฀ ฀microscope฀ and฀ collagen฀ microibrils฀ or฀ ilaments฀ only฀ though฀ an฀ electron฀ micro-
scope.฀In฀addition฀to฀forming฀and฀maintaining฀tissue฀integrity฀and฀stability,฀collagens฀also฀
form฀a฀bioactive฀surface฀that฀regulates฀cell฀differentiation,฀morphogenesis,฀and฀migration,฀
as฀well฀as,฀wound฀repair,฀inlammation,฀and฀thrombosis.฀In฀addition,฀alterations฀in฀colla-
gen฀ metabolism฀ lead฀ to฀ a฀ large฀ spectrum฀ of฀ diseases,฀ including฀ common฀ disorders฀ like฀
osteoarthritis฀and฀atherosclerosis.฀An฀equal฀number฀of฀other฀genes฀encode฀collagen-like฀
domains฀ in฀ various฀ other฀ proteins฀ such฀ as฀ the฀ complement฀ proteins฀ of฀ blood฀ plasma฀
(see฀Sect.฀3.3.2.).
The฀ibrillar฀collagens฀are฀made฀from฀types฀I,฀II,฀III,฀V,฀and฀XI฀polypeptides฀(Table฀3.1).฀
The฀most฀predominant฀collagen฀is฀type฀I,฀but฀mixtures฀with฀other฀collagen฀types฀affects฀the฀
iber฀structure.฀Type฀I฀ibers฀are฀often฀found฀as฀a฀complex฀with฀type฀V฀ibers฀for฀various฀
reasons:฀to฀facilitate฀corneal฀transparency;฀to฀limit฀iber฀thickness฀during฀tissue฀repair฀and฀to฀
help฀form฀the฀architecture฀of฀various฀collagen-containing฀tissues฀such฀as฀tendons฀or฀the฀pla-
centa.฀Type฀II฀ibers,฀which฀are฀unique฀to฀cartilage,฀form฀a฀complex฀with฀type฀XI฀collagen฀to฀
limit฀thickness฀and฀enhance฀binding฀to฀proteo-glycosaminoglycans.฀Reticular฀ibers฀are฀deli-
cate฀ibers฀composed฀mainly฀of฀type฀III฀collagen฀and฀extensively฀covered฀with฀glycosamino-
glycans฀and฀glycoproteins.฀In฀the฀dermis฀of฀the฀skin฀and฀gingiva,฀the฀reticular฀ibers฀extend฀
out฀from฀type฀I฀collagen฀ibers฀that฀have฀already฀associated฀with฀type฀V฀polypeptides.฀Within฀
the฀bone฀marrow฀and฀other฀less฀ibrous฀tissues,฀the฀type฀III฀collagen฀of฀reticular฀ibers฀associ-
ates฀with฀a฀mixture฀of฀non-ibrillar฀type฀VIII฀and฀type฀X฀polypeptides.
All฀collagen฀ibers,฀ibrils฀and฀microibrils฀have฀an฀alternating฀light฀and฀dark฀staining฀
pattern฀ (striated฀ or฀ banding฀ pattern)฀ that฀ has฀ a฀ characteristic฀ appearance฀ (upper฀ half฀ of฀
Fig.฀3.3).฀This฀pattern฀is฀due฀to฀the฀staggered฀array฀of฀tropocollagen฀triple฀helices฀(lower฀
half฀of฀Fig.฀3.3,฀also฀discussed฀in฀Sects.฀4.2.1,฀8.2.1฀and฀8.3.4).฀The฀ibrillar฀collagens฀are฀
synthesized฀ as฀ a฀ precursor,฀ procollagen.฀ On฀ secretion,฀ the฀ N-terminal฀ and฀ C-terminal฀
domains฀are฀removed.฀Sequences฀identical฀or฀similar฀(homologous)฀to฀the฀N-terminal฀pro-
collagen฀domain฀of฀the฀commonly฀found฀ibrillar฀collagens฀are฀expressed฀on฀other฀proteins฀
and฀are฀referred฀to฀as฀procollagen฀domains.

Fig. 3.3฀ The฀ striated฀ appearance฀ of฀ collagen฀ ibers.฀ Collagen฀ ibrils฀ are฀ made฀ up฀ of฀ tropocollagen฀
molecules฀aligned฀in฀a฀staggered฀fashion฀and฀cross-linked฀for฀strength.฀(a)฀Electron฀micrograph฀show-
ing฀cross-striations฀from฀native฀rat฀tail฀collagen.฀Alternate,฀broad,฀mainly฀light฀and฀mainly฀dark฀bands฀
with฀a฀period฀of฀approximately฀64฀nm฀are฀prominent฀(From฀Fig.฀1฀of฀Cox฀RW,฀Grant฀RA฀and฀Kent฀
CM฀(1972)฀“The฀interpretation฀of฀electron฀micrographs฀of฀negatively฀stained฀native฀collage.”฀Journal฀
of฀ Cell฀ Science฀ 10:547–554).฀ (b)฀ Less฀ magniied฀ view฀ of฀ collagen฀ ibers.฀ (c)฀ Self-aggregation฀ of฀
tropocollagen.฀The฀N-฀and฀C-terminal฀ends฀of฀tropocollagen฀are฀called฀telopeptide฀domains฀and฀they฀
interact฀ with฀ adjacent฀ tropocollagen฀ molecules฀ (yellow฀ lines).฀ (d)฀ Tropocollagen฀ self-aggregating฀
unit.฀A฀tropocollagen฀molecule฀is฀a฀self-aggregating฀unit฀of฀three฀long฀polypeptides฀that฀are฀inter-
twined฀in฀a฀triple฀helix.฀It฀is฀cut฀out฀from฀a฀genetically฀encoded฀polypeptide฀called฀procollagen฀as฀
described฀in฀Sect.฀4.2.1฀(Figs.฀(b)–(d)฀modiied฀from฀Fig.฀4–13฀in฀Lehninger฀Principles฀of฀Biochemistry.฀
D.L.฀Nelson฀and฀M.M.฀Cox,฀4th฀Ed.฀2005.฀W.H.฀Freeman฀&฀Co.,฀NY)
3.1.2. Collagen 33

Cross-striations 640 Å (64 nm)

250
nm

Cross-striations
Tropocollagen 640 Å (64 nm)
c
N C

Section of
collagen
molecule

N C
34 3 The Connective Tissue Extracellular Matrix and Its Major Components

The฀kidney฀contains฀small฀amounts฀of฀ibrillar฀collagen,฀but฀its฀major฀collagen฀is฀a฀non-
ibrillar฀ network฀ collagen฀ (type฀ IV),฀ a฀ part฀ of฀ basal฀ laminas฀ called฀ the฀ lamina฀ densa฀
(Sect.฀5.1.1).฀Basal฀laminas฀are฀especially฀well฀developed฀in฀the฀kidney฀as฀exempliied฀in฀
the฀glomerulus฀(Fig.฀3.2).฀The฀collagen฀contents฀of฀various฀tissues฀are฀indicated฀in฀Table฀3.2฀
and฀their฀iber฀arrangements฀in฀Table฀3.3.

3.1.3.
Elastic Fiber System

Elastic฀ibers฀endow฀a฀stroma฀with฀recoil฀after฀stretching.฀They฀are฀composed฀of฀ibrillin฀
with฀or฀without฀a฀central฀portion฀of฀elastin฀(Table฀3.4).฀These฀two฀proteins฀are฀described฀in฀
detail฀in฀Sect.฀6.1.1฀and฀6.2.1).฀Elastic฀ibers฀are฀especially฀prominent฀in฀ligaments฀and฀

Table 3.2฀ Collagen฀and฀elastin฀content฀of฀some฀tissues฀(g/100฀g฀of฀dry฀weight)

Tissue Collagen Elastin
Ligament 17 50
Aorta 12–24 30–57
Tendon฀(achilles) 86 4
Dermis฀of฀skin 72 2–5
Liver 3.9 0
Cartilage 46–63 0
Cornea 68.1 0
Bone,฀mineral฀free 88 0
Collagen฀data฀is฀from฀Table฀2.9฀in฀R.฀Montgomery฀et฀al.฀Biochemistry,฀A฀case฀oriented฀approach,฀
4th฀Ed.,฀The฀CV฀Mosby฀Co.,฀St฀Louis฀MO฀1983.฀Elastin฀data฀is฀from฀S.฀Mithieux฀and฀A.S.฀Weiss,฀
Adv.฀Prot.฀Chem.฀79:437–461,฀2005

Table 3.3฀ Collagen฀iber฀arrangements฀in฀some฀tissues

Tissue Arrangement฀of฀ibrils฀or฀microibrils1
Tendon/Periodontium Parallel฀iber฀bundles
Cartilage No฀distinct฀arrangement
Dermis฀of฀skin Planar฀sheets฀of฀microibrils฀฀
layered฀at฀many฀angles
Cornea Planar฀sheets฀stacked฀crossways฀for฀strength
฀ ibers฀seen฀by฀naked฀eye,฀ibrils฀only฀by฀light฀microscope,฀microibrils฀only฀by฀electron฀micro-
F
1

scope
Data฀is฀from฀Table฀2.10฀in฀R.฀Montgomery฀et฀al.฀Biochemistry,฀A฀case฀oriented฀approach,฀4th฀Ed.,฀
The฀CV฀Mosby฀Co.,฀St฀Louis,฀MO,฀1983
3.1.3. Elastic Fiber System 35

major฀arteries฀(Table฀3.2).฀Thin฀elastic฀ibers฀consist฀of฀ibrillin฀bundles฀around฀very฀little฀
elastin฀(elaunin฀ibers).฀Within฀the฀dermis฀of฀the฀skin฀there฀is฀a฀core฀of฀thick฀elastin฀ibers฀
within฀which฀a฀deeply฀embedded฀elaunin฀plexus฀radiates฀thin฀ibers฀towards฀the฀supericial฀
layers฀beneath฀the฀epidermis.฀Oxytalan฀ibers฀are฀ibrillin฀bundles฀with฀no฀elastin.฀They฀are฀
important฀at฀four฀major฀sites฀throughout฀the฀body฀(Fig.฀3.4):฀(A)฀Between฀the฀iber฀bundles฀
of฀the฀periodontium฀(where฀they฀were฀irst฀found฀and฀described฀below,฀Sect.฀3.1.5);฀(B)฀
beneath฀the฀endothelial฀cell฀lining฀of฀blood฀vessels,฀also฀called฀the฀intima฀(Sect.฀11.1.1);฀
(C)฀ in฀ the฀ dermis฀ perpendicular฀ to฀ the฀ dermo-epidermal฀ junction;฀ and฀ (D)฀ beneath฀ the฀
epithelial฀ covering฀ of฀ organs฀ or฀ tissues฀ (adventitia)฀ such฀ as฀ surrounding฀ lung฀ alveoli฀
(Tables฀3.2฀and฀3.4).
Unlike฀ ligaments฀ which฀ attach฀ bone฀ to฀ other฀ bones฀ or฀ teeth,฀ tendons฀ attach฀ bone฀ to฀
muscle฀and฀have฀few฀elastic฀ibers฀(Table฀3.2).฀Ultraviolet฀radiation฀and฀aging฀disorganize฀
the฀elastic฀iber฀system฀of฀the฀skin฀by฀activating฀proteases฀that฀degrade฀elastin฀and฀ibrillin.฀

Table 3.4฀ Composition฀of฀three฀major฀types฀of฀elastic฀ibers

Fiber฀type Arrangement Tissuea
Elastic Elastin฀surrounded฀by฀ibrillin Ligaments฀&฀large฀arteries
Elaunin Fibrillin฀with฀a฀little฀elastin Deeper฀layer฀of฀skin฀dermis
Oxytalan Fibrillin฀only Throughout฀body
a
More฀details฀in฀text

Fig. 3.4฀ An฀oxytalan฀iber฀

tract.฀(a)฀Cementum;฀฀
(b)฀Principal฀oxytalan฀฀
iber;฀(c)฀Oxytalan฀tract;฀฀
(d)฀Periodontal฀vessel฀(From฀
c
Fig.฀2.4,฀Oxytalan฀ibers฀
forming฀a฀network฀that฀
attaches฀blood฀vessels฀to฀the฀
a
cementum.฀In฀article฀titled,฀
“Force฀Generation฀and฀
Reaction฀within฀the฀ d
Periodontium.”฀Caputo฀AA฀
and฀Wylie฀RS:฀UCLA฀School฀
of฀Dentistry฀website;฀http://
www.dent.ucla.edu/pic/
b
members/force/index.html)฀
Figure฀modiied฀slightly฀and฀
redrawn฀by฀Dr฀Wirsig-
Weichmann.฀Permission฀
granted฀by฀the฀Authors
36 3 The Connective Tissue Extracellular Matrix and Its Major Components

Elastin฀ is฀ eficiently฀ synthesized฀ only฀ in฀ the฀ fetus฀ and฀ children฀ because฀ it฀ is฀ normally฀
extremely฀ stable฀ (half฀ life฀ ~70฀ years).฀ By฀ contrast฀ ibrillar฀ collagens฀ including฀ those฀ in฀
bone฀have฀a฀turnover฀half-life฀of฀about฀6฀months.฀The฀failure฀to฀replace฀elastin฀causes฀the฀
skin฀to฀become฀less฀elastic,฀more฀wrinkled,฀and฀thinner฀with฀age.

3.1.4.
Glycosaminoglycans

Glycosaminoglycans฀are฀carbohydrates฀important฀for฀the฀development฀and฀repair฀of฀the฀
stroma,฀and฀also฀for฀the฀formation฀of฀cartilage.฀During฀an฀organism’s฀development,฀or฀fol-
lowing฀the฀removal฀of฀damaged฀tissue฀from฀an฀infection฀or฀injury,฀ibroblasts฀are฀activated฀
to฀ proliferate฀ and฀ secrete฀ hyaluronan,฀ a฀ large,฀ repeating฀ dimer฀ of฀ glucuronate฀ linked฀ to฀
N-acetylglucosamine.฀The฀ibroblasts฀then฀invade฀the฀hyaluronan฀(ground฀substance)฀along฀
with฀proliferating฀endothelial฀cells฀that฀form฀new฀capillaries.฀Once฀within฀hyaluronan,฀the฀
ibroblasts฀secrete฀collagen฀ibers฀that฀increase฀stromal฀density.฀Proteo-glycosaminoglycans฀
are฀ proteins฀ that฀ have฀ covalently฀ bound฀ glycosaminoglycans.฀ They฀ are฀ synthesized฀ by฀
ibroblasts,฀chrondroblasts,฀and฀osteoblasts฀along฀with฀collagen.฀These฀negatively฀charged฀
polymers฀impart฀resilience฀(pliability)฀to฀the฀stroma,฀and฀are฀especially฀important฀in฀carti-
lage.฀The฀stroma฀is฀both฀resilient฀and฀ibrous.฀Long฀hyaluronan฀molecules฀are฀especially฀
important฀in฀maintaining฀the฀viscosity฀of฀joint฀luid฀around฀the฀cartilage฀between฀bones,฀
and฀ within฀ the฀ eyeballs.฀ Fibroblast฀ production฀ of฀ shorter฀ length฀ hyaluronans฀ stimulates฀
angiogenesis฀(new฀capillary฀formation).

3.1.5.
Alveolar Bone, Teeth, and Periodontium

Bone฀is฀synthesized฀by฀osteoblasts฀which฀transport฀calcium฀ions฀from฀blood฀into฀a฀secreted,฀
uncalciied฀ osteoid฀ matrix฀ composed฀ mostly฀ of฀ type฀ I฀ collagen.฀ During฀ calciication,฀
monocyte-like฀cells฀are฀attracted฀out฀of฀the฀adjacent฀blood฀capillaries฀and฀adhere฀to฀irregu-
larities฀ in฀ the฀ calcifying฀ bone฀ surface฀ and฀ eventually฀ become฀ osteoclasts.฀ These฀ cells,฀
which฀resorb฀the฀bone,฀develop฀according฀to฀genetic฀and฀environmental฀stimuli฀that฀deter-
mine฀bone฀shape฀and฀response฀to฀stress.
A฀tooth฀and฀its฀surrounding฀tissue฀(the฀periodontium)฀are฀diagrammed฀in฀Fig.฀3.5.฀The฀
enamel฀is฀made฀by฀ameloblasts,฀cells฀of฀ectodermal฀origin฀unrelated฀to฀osteoclasts฀which฀
are฀of฀mesodermal฀origin.฀Enamel฀is฀the฀only฀calciied฀tissue฀that฀does฀not฀contain฀colla-
gen฀in฀the฀vertebrate฀body.฀It฀forms฀a฀tight,฀impenetrable฀seal฀around฀the฀dentin,฀much฀like฀
a฀skin.฀The฀dentin฀and฀cementum฀are฀calciied฀over฀type฀I฀collagen฀ibers฀like฀bone.฀They฀
are฀respectively฀made฀by฀odontoblasts฀and฀cementoblasts,฀both฀closely฀related฀to฀osteo-
blasts.฀Once฀dentin฀has฀been฀synthesized,฀odontoblasts฀remain฀viable฀on฀its฀inner฀surface฀
3.1.5. Alveolar Bone, Teeth, and Periodontium 37

Fig. 3.5฀ The฀periodontium.฀

The฀periodontium฀consists฀
of฀the฀gingiva฀and฀
underlying฀periodontal฀
membrane.฀Both฀are฀is฀
composed฀of฀large฀bundles฀
of฀collagen฀mixed฀with฀
large฀and฀small฀oxytalan฀
ibers฀that฀allow฀the฀
periodontium฀to฀respond฀to฀
violent฀movements฀of฀the฀
tooth฀within฀the฀jaws฀during฀
mastication

within฀the฀pulp฀cavity,฀a฀region฀of฀uncalciied฀connective฀tissue฀rich฀in฀blood฀vessels฀and฀
nerves฀in฀the฀center฀of฀the฀teeth฀(Fig.฀3.5).฀On฀the฀outer฀surface฀of฀dentin฀apical฀to฀enamel,฀
cementoblasts฀ differentiate฀ from฀ ibroblasts฀ and฀ form฀ cementum.฀ The฀ synthesis฀ of฀ the฀
calciied฀tissues฀is฀discussed฀in฀detail฀in฀Chap.฀9.
The฀ periodontium฀ comprises฀ the฀ gingiva,฀ cementum,฀ periodontal฀ ligament,฀ and฀ sur-
rounding฀alveolar฀bone.฀The฀gingiva฀and฀periodontal฀attachment฀are฀largely฀composed฀of฀
large฀collagen฀iber฀bundles฀to฀absorb฀masticatory฀forces.฀The฀periodontal฀collagen฀ibers฀
are฀called฀ periodontal฀ligaments฀because฀both฀ends฀are฀ calciied฀like฀the฀ligaments฀that฀
attach฀bones.฀Instead฀of฀elastin,฀the฀ligaments฀and฀blood฀vessels฀are฀surrounded฀by฀oxyta-
lan฀ibers฀composed฀of฀ibrillin฀which฀provide฀the฀lesser฀elasticity฀required฀for฀tooth฀move-
ment฀ as฀ compared฀ with฀ bone฀ movement.฀ The฀ gingiva฀ lies฀ coronal฀ to฀ the฀ periodontal฀
ligaments฀and฀is฀composed฀of฀free฀and฀attached฀segments.฀The฀free฀gingiva฀is฀the฀soft฀tis-
sue฀wall฀adjacent฀to฀the฀gingival฀sulcus.฀Its฀coronal฀end฀is฀the฀gingival฀margin฀and฀its฀apical฀
end฀is฀continuous฀with฀the฀attached฀gingiva฀on฀the฀oral฀side฀and฀with฀a฀junctional฀epithelial฀
attachment฀on฀the฀dental฀side฀(Fig.฀3.6).฀The฀free฀gingiva฀is฀covered฀by฀keratinized฀epithe-
lium฀ on฀ its฀ oral฀ cavity฀ surface฀ and฀ non-keratinized฀ epithelium฀ in฀ the฀ sulcus฀ against฀ the฀
tooth฀surface฀(Sects.฀5.2.2฀and฀5.2.3).
38 3 The Connective Tissue Extracellular Matrix and Its Major Components

Fig. 3.6฀ Diagrams฀showing฀the฀attached฀gingiva฀(cross-hatched)฀and฀free฀gingiva฀(dark฀line฀against฀

the฀teeth):฀(a).฀is฀a฀clinical฀view฀and฀(b).฀enlarged฀horizontal฀and฀cross-sectional฀views.฀The฀coronal฀
end฀of฀the฀attached฀gingiva฀is฀the฀free฀gingiva฀which฀lies฀beneath฀the฀gingival฀margin฀and฀extends฀
apically฀for฀1฀–฀2฀mm฀to฀the฀gingival฀groove฀where฀collagen฀ibers฀attach฀the฀gingival฀epithelium฀
to฀alveolar฀bone.฀The฀free฀gingiva฀forms฀the฀outer฀wall฀of฀the฀gingival฀sulcus.฀The฀free฀gingiva฀is฀
shorter฀and฀the฀attached฀gingiva฀longer฀than฀in฀this฀diagram฀which฀is฀not฀drawn฀to฀scale.฀In฀perio-
dontitis฀(Chap.฀13),฀the฀gingival฀margin฀migrates฀apically฀to฀beneath฀the฀cementoenamel฀junction,฀
causing฀the฀attached฀gingiva฀to฀shorten฀in฀height฀and฀the฀cementum฀of฀the฀teeth฀to฀appear฀in฀the฀oral฀
cavity.฀(From฀Fig.฀1–1,฀Contemporary฀Periodontics,฀edited฀by฀RJ฀Genco,฀HM฀Goldman฀and฀DW.฀
Cohen.฀Chapter฀1:฀The฀Gingiva,฀Structure฀and฀Function฀by฀H฀Loe.,฀MA฀Listgarten฀&฀VP฀Terranova.฀
Publisher฀was฀The฀CV฀Mosby฀Co.,฀St฀Louis,฀MO,฀1990;฀Copyright฀Elsevier,฀2008).฀Figure฀was฀
modiied฀slightly฀and฀redrawn฀by฀Dr฀Wirsig-Weichmann

The฀ gingiva฀ and฀ periodontium฀ both฀ possess฀ ive฀ groups฀ of฀ collagen฀ ibers฀ or฀ liga-
ments,฀which฀are฀categorized฀by฀orientation฀and฀function.฀฀The฀gingival฀ibers฀are:฀(A)฀
dentogingival;฀ (B)฀ alveologingival;฀ (C)฀ circumferential;฀ (D)฀ transseptal;฀ and฀ (E)฀
periosteal฀ (Fig.฀ 3.7).฀ The฀ irst฀ two฀ are฀ free฀ gingival฀ ibers,฀ which฀ are฀ calciied฀ into฀
cementum฀or฀bone฀at฀one฀end฀and฀free฀at฀the฀other.฀They฀hold฀the฀free฀gingiva฀tightly฀
against฀the฀tooth฀surface.฀The฀gingival฀circumferential฀ibers฀anchor฀in฀the฀cementum฀
and฀ partially฀ encircle฀ a฀ tooth฀ beneath฀ the฀ free฀ gingiva.฀ The฀ transseptal฀ ibers฀ are฀
3.1.5. Alveolar Bone, Teeth, and Periodontium 39

ligament-like฀ibers฀attached฀to฀cementum฀at฀each฀end.฀They฀maintain฀teeth฀alignment฀
by฀connecting฀the฀teeth฀interdentally฀(not฀shown฀in฀Fig.฀3.7).฀Periosteal฀ibers฀hold฀the฀
attached฀gingiva฀tightly฀to฀the฀outer฀surface฀of฀alveolar฀bone฀(Fig.฀3.7).฀The฀periodontal฀
ligament฀ ibers฀ are฀ also฀ called฀ the฀ principal฀ iber฀ bundles฀ based฀ also฀ on฀ location฀ and฀
orientation:฀(A)฀alveolar฀crest;฀(B)฀horizontal;฀(C)฀oblique;฀(D)฀periapical;฀and฀(E)฀inter-
radicular฀in฀multi-rooted฀teeth.฀The฀portions฀of฀these฀ibers฀anchored฀into฀cementum฀or฀
alveolar฀bone฀are฀called฀Sharpey’s฀ibers.
The฀stroma฀within฀which฀the฀principal฀periodontal฀ibers฀are฀embedded฀is฀composed฀of฀
proteoglycans฀within฀which฀are฀embedded฀oxytalan฀ibers฀(Sect.฀3.1.3)฀whose฀elasticity฀is฀
important฀(Sect.฀3.1.3).฀Mini-oxytalan฀ibers฀surround฀blood฀vessels฀and฀are฀attached฀to฀

Fig. 3.7฀ Gingival฀iber฀groups.฀Two฀of฀the฀four฀groups฀of฀gingival฀collagen฀ibers฀are฀indicated:฀

dentogingival฀and฀alveologingival.฀The฀third฀group,฀the฀circumferential฀ibers,฀is฀shown฀as฀green฀
circles฀in฀vertical฀cross฀section.฀The฀fourth฀group,฀the฀transseptal฀ibers,฀is฀not฀shown.฀These฀ibers฀
run฀between฀the฀teeth฀and฀are฀obscured฀by฀the฀teeth฀in฀the฀plane฀of฀the฀igure.฀The฀dentoalveolar฀
ibers฀ lie฀ immediately฀ above฀ the฀ periodontal฀ membrane฀ ibers.฀ The฀ periosteal฀ ibers฀ hold฀ the฀
attached฀gingiva฀tightly฀to฀the฀alveolar฀bone฀and฀are฀not฀part฀of฀the฀periodontium.฀The฀ibers฀are฀
briely฀described฀in฀the฀text฀(Modiied฀from฀a฀PowerPoint฀slide฀entitled฀“Dental,฀Gingival฀Fiber฀
Groups”฀ accompanying฀ an฀ Histology฀ Full-Text:฀ William฀ A฀ Beresford:฀ West฀ Virginia฀ Univer-
sity.฀ http://wberesford.hsc.wvu.edu/histolch24.htm).฀ Figure฀ modiied฀ slightly฀ and฀ redrawn฀ by฀
Dr฀Wirsig-Weichmann.฀Permission฀given฀by฀author
40 3 The Connective Tissue Extracellular Matrix and Its Major Components

cementum฀by฀regular฀oxytalan฀ibers฀(Fig.฀3.4).฀These฀ibers฀may฀support฀the฀blood฀vessels฀
within฀the฀periodontium฀during฀chewing.฀Biting฀causes฀the฀teeth฀to฀compress฀the฀blood฀
vessels฀in฀the฀periodontium.฀Oxytalan฀ibers฀allow฀the฀capillaries฀to฀recoil฀so฀that฀the฀blood฀
supply฀ with฀ its฀ oxygen฀ and฀ other฀ essential฀ nutrients฀ is฀ immediately฀ replaced฀ when฀ the฀
pressure฀is฀released฀in฀the฀human฀periodontium.

The฀connective฀tissue฀extracellular฀matrix฀along฀with฀the฀associated฀cells฀comprises฀the฀
stroma฀or฀internal฀solid฀mass฀of฀an฀organism.฀Collagen฀is฀the฀major฀stromal฀protein฀in฀
vertebrates.฀It฀may฀be฀ibrillar฀or฀non-ibrillar,฀depending฀on฀the฀types฀of฀collagen฀poly-
peptides฀expressed.฀Fibrous฀collagen฀forms฀ilaments,฀microibrils฀and฀ibers฀in฀ascend-
ing฀order฀of฀size฀and฀molecular฀complexity.฀Other฀ibers฀in฀the฀stroma฀are฀made฀from฀
ibrillin฀and฀elastin.฀The฀various฀stromal฀ibers฀provide฀tensile฀strength,฀iltration฀barri-
ers,฀and฀shape฀to฀the฀tissue฀or฀organ.฀The฀stroma฀is฀separated฀from฀the฀overlying฀epithe-
lial฀cells฀by฀a฀basal฀lamina.฀The฀basal฀lamina฀acts฀as฀a฀ilter฀to฀control฀the฀movement฀of฀
proteins฀and฀other฀molecules฀between฀luid฀compartments,฀e.g.,฀between฀the฀blood฀and฀
stroma฀or฀between฀the฀stroma฀and฀epithelium.฀Collagen฀interacts฀with฀other฀proteins,฀
glycans฀and฀proteoglycans฀to฀build฀calciied฀and฀uncalciied฀tissues฀and฀organs.฀Bone,฀
dentin,฀ and฀ cementum฀ are฀ formed฀ from฀ collagen฀ ibers฀ synthesized฀ by฀ osteoblasts,฀
odontoblasts,฀ and฀ cementoblasts,฀ respectively,฀ and฀ around฀ which฀ calcium฀ phosphate฀
has฀precipitated.฀Uncalciied฀collagen฀iber฀bundles,฀and฀collagen฀calciied฀in฀cemen-
tum฀and฀bone฀are฀the฀major฀components฀of฀the฀gingiva฀and฀periodontium.฀The฀ends฀of฀
the฀collagen฀ibers฀of฀the฀periodontal฀ligament฀are฀embedded฀in฀calciied฀cementum฀and฀
bone฀to฀anchor฀the฀tooth฀into฀the฀bony฀socket.฀Collagen฀ibers฀in฀the฀gingiva฀provide฀
structural฀ support฀ to฀ the฀ gingival฀ tissue฀ and฀ maintain฀ the฀ alignment฀ of฀ the฀ teeth.฀
Oxytalan฀ibers฀are฀made฀of฀ibrillin฀and฀elastin.฀They฀support฀the฀blood฀vessels฀within฀
the฀periodontium฀during฀chewing.

3.2.1.
Cell Surface Binding: Integrins, Fibronectin, and Collagen

The฀stromal฀ibers฀generally฀remain฀tightly฀attached฀to฀ibroblast,฀chondroblast,฀or฀osteoblast฀
cells฀that฀produce฀them.฀The฀ibroblast฀cell฀surface฀contains฀integrins,฀a฀family฀of฀proteins฀that฀
attach฀laminin,฀ibronectin฀and฀many฀other฀stromal฀proteins฀by฀an฀Arg-Gly-Asp฀motif฀(RGD฀
in฀the฀one-letter฀amino฀acid฀code;฀see฀also฀Sects.฀4.4.1,฀5.1.2,฀8.2.1,฀10.1.1,฀and฀11.2.1).฀An฀
important฀insight฀into฀the฀importance฀of฀the฀RGD฀motif฀of฀ibronectin฀and฀other฀proteins฀in฀
mediating฀ stromal฀ cohesion฀ came฀ from฀ snake฀ bites.฀ A฀ family฀ of฀ polypeptides฀ (45–84฀
amino฀ acids)฀ in฀ snake฀ venoms,฀ the฀ modiied฀ saliva฀ of฀ many฀ snakes,฀ contains฀ the฀ RGD฀
sequence.฀These฀polypeptides฀are฀known฀as฀disintegrins.฀They฀break฀up฀or฀disintegrate฀the฀
stroma฀by฀competing฀out฀normal฀integrin–ligand฀interactions฀including฀platelet฀aggregations฀
3.2.2. Thrombospondins and Transforming Growth Factor-β 41

that฀normally฀initiate฀blood฀clots฀(Sect.฀11.2.1)฀at฀the฀site฀of฀a฀snake฀bite.฀The฀disintegrin฀activ-
ity฀permits฀other฀components฀of฀snake฀venom฀such฀as฀nerve฀inhibitor฀enzymes฀to฀reach฀the฀
systemic฀circulation.
Collagen฀binds฀integrins฀by฀an฀unrelated฀motif,฀a฀short,฀proline-poor฀peptide฀which฀is฀
lexible฀within฀the฀triple฀helix฀backbone฀as฀discussed฀in฀Sect.฀4.4.1.฀Fibronectin฀is฀a฀com-
plex฀multidomain฀protein฀containing฀a฀motif฀that฀recognizes฀integrins฀(FN-1฀motif)฀and฀
collagen฀ibers฀(FN-2).฀A฀third฀ibronectin฀motif฀(FN-3)฀recognizes฀heparin฀sulfate,฀a฀cell฀
surface฀glycosaminoglycan฀(Sect.฀6.3.1).฀Fibronectin฀usually฀forms฀a฀dimer฀that฀exposes฀
the฀integrin-binding฀RGD฀motif฀in฀one฀region฀and฀the฀collagen฀binding฀motif฀in฀another.฀
Thus,฀ibronectin฀attaches฀collagen฀ibers฀in฀the฀stroma฀to฀cell฀surface฀integrins฀indepen-
dently฀of฀collagen-integrin฀binding.฀This฀separation฀allows฀collagen฀alone฀to฀control฀the฀
cell฀cycle฀and฀its฀own฀synthesis฀(Sect.฀4.4.1).฀The฀segment฀of฀the฀triple฀helical฀region฀of฀
collagen฀ that฀ binds฀ to฀ the฀ ibronectin฀ FN2฀ motif฀ is฀ rich฀ in฀ proline฀ and฀ entirely฀ separate฀
from฀the฀collagen฀integrin-binding฀motif.

3.2.2.
Thrombospondins and Transforming Growth Factor-β

Thrombospondins฀are฀ive฀independently฀encoded฀proteins฀unique฀to฀vertebrates.฀The฀orig-
inal฀ThromboSpondin฀(TSP-1)฀was฀irst฀identiied฀as฀a฀product฀of฀thrombin-activated฀plate-
lets฀during฀blood฀clotting฀(Sect.฀11.3.4),฀and฀was฀designated฀a฀“thrombin-sensitive฀protein.”฀
The฀TSP฀protein฀family฀is฀divided฀into฀two฀groups฀that฀differ฀in฀structure฀and฀biological฀
role฀(Fig.฀3.8).฀Group฀A฀consists฀of฀TSP1฀and฀TSP2,฀which฀are฀homotrimers฀of฀a฀145฀kDa฀
polypeptide.฀Human฀thrombospondin-1฀is฀secreted฀as฀a฀1152฀amino฀acid฀chain฀following฀
loss฀of฀an฀18฀amino฀acid฀secretion฀signal.฀Group฀B฀(TSP3,฀TSP4฀and฀TSP5)฀are฀homopen-
tamers,฀but฀their฀subunit฀polypeptide฀is฀smaller฀(~110฀kDa฀corresponding฀to฀934฀amino฀
acids฀after฀removal฀of฀a฀22฀amino฀acid฀N-terminal฀signal฀sequence).฀The฀group฀B฀family฀
lacks฀a฀procollagen฀domain฀(see฀legend฀to฀Fig.฀3.8)฀and฀repeats฀of฀the฀type฀I฀proteins฀(TSB฀
type฀I฀repeats),฀but฀it฀contain฀four฀rather฀than฀three฀type฀II฀TSB฀repeats฀which฀are฀related฀
to฀epidermal฀growth฀factor฀protein.
TSP-1฀ functions฀ by฀ activating฀ transforming฀ growth฀ factor-b฀ (TGF-b),฀ a฀ 112฀ amino฀
acid฀protein฀whose฀three฀variants฀(b1,฀b2,฀and฀b3)฀control฀proliferation,฀differentiation,฀
apoptosis฀ (Sect.฀ 13.4.1),฀ and฀ various฀ other฀ functions฀ in฀ many฀ different฀ cell฀ types.฀ All฀
three฀ forms฀ of฀ TGF-b฀ have฀ three฀ domains:฀ (1)฀ an฀ N-terminal฀ signal฀ peptide฀ of฀ 20–30฀
amino฀acids฀that฀is฀required฀for฀secretion฀and฀removed฀in฀the฀endoplasmic฀reticulum;฀(2)฀
an฀ intermediate฀ 280฀ amino฀ acid฀ pro-region฀ (called฀ the฀ latency฀ associated฀ peptide฀ or฀
LAP);฀and฀(3)฀a฀112–114฀amino฀acid฀C-terminal฀region฀that฀becomes฀the฀mature฀TGF-b฀
molecule.฀TSP-1฀binds฀to฀LAP,฀and฀this฀exposes฀the฀C-terminal฀domain฀to฀cleavage฀by฀
stromal฀proteases฀and฀forming฀the฀active฀TGF-b.
The฀active฀portion฀of฀TGF-b฀(the฀C-terminal฀region)฀is฀composed฀of฀a฀cysteine฀knot,฀
nine฀conserved฀cysteine฀residues,฀of฀which฀eight฀form฀disulide฀bonds฀within฀the฀molecule฀
42 3 The Connective Tissue Extracellular Matrix and Its Major Components

Pro-collagen C-ter
homology globular

N-ter Type-1 Type-2 Type-3

globular repeats repeats repeats

TSP-1

TSP-2

TSP-3

B TSP-4

TSP-5

Fig. 3.8฀ Domain฀organization฀of฀the฀thrombospondin฀gene฀family.฀Shapes฀and฀colors฀illustrate฀the฀

different฀ domains฀ discussed฀ in฀ the฀ text.฀ The฀ procollagen฀ domain฀ is฀ homologous฀ to฀ C-฀ and฀
N-terminal฀ domains฀ that฀ are฀ excised฀ from฀ collagen฀ polypeptides฀ when฀ a฀ iber฀ forms฀ (Modiied฀
from฀Fig.฀1฀in฀Adams฀JC,฀Tucker฀RP฀(2000฀Jun)฀“The฀thrombospondin฀type฀1฀repeat฀(TSR)฀super-
family:฀diverse฀proteins฀with฀related฀roles฀in฀neuronal฀development.”฀Developmental฀Dynamics,฀
218(2):280–299)

to฀ create฀ a฀ TGF-b฀ superfamily฀ structure,฀ while฀ the฀ ninth฀ cysteine฀ connects฀ to฀ the฀ ninth฀
cysteine฀of฀another฀TGF-b฀molecule฀to฀produce฀the฀active฀form,฀a฀dimer.฀The฀free฀cysteine฀
residues฀of฀an฀almost฀identical฀cysteine฀knot฀sequence฀at฀the฀C-terminus฀of฀von฀Willebrand฀
factor฀(Sect.฀11.2.1)฀similarly฀causes฀dimer฀formation.฀A฀virtually฀identical฀structure฀at฀the฀
C-terminus฀of฀salivary฀Mucin฀Glycoprotein-1฀(MG-1;฀Sect.฀12.3.1)฀is฀partially฀responsible฀
for฀its฀multi-monomeric฀structure.
Many฀cells฀secrete฀at฀least฀one฀of฀the฀three฀immature฀forms฀of฀TGF-b,฀and฀essentially฀
all฀cells฀have฀receptors฀that฀respond฀to฀the฀presence฀of฀mature฀TGF-b฀in฀the฀stroma.฀In฀the฀
periodontium,฀TGF-b฀stimulates฀ibroblast฀and฀osteoblast฀proliferation฀during฀connective฀
tissue฀or฀bone฀remodeling฀(Sect.฀10.1.3),฀and฀maintains฀the฀proliferation฀of฀dentally฀attached฀
epithelial฀cells฀(Sect.฀5.2.3).฀The฀linker฀domains฀that฀connect฀calcium฀binding฀domains฀in฀
ibrillin฀are฀identical฀to฀the฀sequence฀of฀protein฀receptors฀that฀bind฀to฀TGF-b฀(Sect.฀6.1.1).
Thrombospondin-2฀is฀the฀major฀expressed฀thrombospondin฀in฀the฀human฀body.฀Mice฀
deicient฀in฀TSP2฀exhibit฀excessive฀collagen฀degradation.฀Secretion฀of฀TSP2฀by฀capillary฀
endothelium฀inhibits฀the฀collagen฀degradation฀required฀for฀remodeling฀and฀causes฀dense฀
ibers฀to฀form฀during฀development฀or฀wound฀healing฀by฀inhibiting฀matrix฀metalloprotei-
nase฀(gelatinase)฀secretion฀(Sect.฀8.3.3).฀Many฀synthetic฀implants฀directly฀promote฀TSP2฀
3.3.1. Stromal Nutrition 43

secretion฀and฀the฀formation฀of฀thick฀collagen฀ibers฀which฀have฀an฀avascular,฀poorly฀per-
meable฀ capsule฀ (Foreign฀ Body฀ Reaction).฀ Delivering฀ RNA฀ complimentary฀ to฀ the฀ pro-
cessed฀mRNA.
In฀mice,฀TSP-3฀slows฀(normalizes)฀the฀rate฀of฀vascular฀invasion฀and฀post-natal฀ossiica-
tion฀of฀the฀cartilage฀during฀long฀bone฀development฀(Sect.฀9.2.2)฀without฀affecting฀prenatal฀
skeletal฀patterning.฀TSP-4฀is฀an฀adhesive฀glycoprotein฀that฀mediates฀cell-to-cell฀and฀cell-
to-matrix฀interactions฀like฀ibronectin.฀TSP-5฀contributes฀to฀the฀structures฀of฀cartilage฀and฀
tendons฀by฀zinc฀or฀nickel฀ion-mediated฀binding฀to฀collagen.

I฀ ntegrins฀determine฀the฀attachment฀of฀collagen฀ibers฀directly฀or฀through฀ibronectin,฀a฀
multi-domain฀protein฀that฀also฀attaches฀collagen.฀Collagen฀binds฀to฀integrin฀by฀a฀differ-
ent฀amino฀acid฀motif฀from฀that฀attaching฀to฀ibronectin.฀Direct฀collagen฀binding฀to฀inte-
grin฀ causes฀ changes฀ in฀ ibroblast฀ metabolism.฀ Thrombospondins฀ (TSP)฀ are฀ ive฀
independently฀encoded฀proteins฀unique฀to฀vertebrates.฀The฀family-1฀thrombospondin-1฀
and฀ -2฀ function฀ differently.฀ Thrombospondin-2฀ is฀ expressed฀ throughout฀ the฀ stroma฀
where฀it฀mediates฀the฀development฀of฀a฀thick฀collagen฀ibrous฀network฀that฀walls฀off฀a฀
foreign฀body฀such฀as฀a฀dental฀implant฀(foreign฀body฀reaction).฀Thrombospondin-1฀acti-
vates฀transforming฀growth฀factor-b฀which฀stimulates฀ibroblast฀and฀osteoblast฀prolifera-
tion฀ during฀ connective฀ tissue฀ or฀ bone฀ remodeling฀ and฀ maintains฀ the฀ proliferation฀ of฀
dentally฀ attached฀ epithelial฀ cells.฀ The฀ family-2฀ thrombospondins-3,฀ and฀ -5฀ promote฀
cartilage฀ development฀ and฀ its฀ ossiication฀ to฀ bone.฀ The฀ family-2฀ thrombospondin-4฀
mediates฀cell฀and฀stromal฀protein฀adhesion.

3.3.1.
Stromal Nutrition

The฀ibroblasts฀and฀other฀cells฀of฀the฀stroma฀are฀surrounded฀by฀a฀dense฀layer฀of฀secreted฀
materials฀through฀which฀nutrients฀must฀reach฀the฀cells฀and฀waste฀must฀be฀excreted.฀The฀
arteriolar฀ends฀of฀blood฀capillaries฀have฀tiny฀junctions฀between฀the฀endothelial฀cells฀so฀that฀
small฀molecules฀leak฀out฀under฀hydrostatic฀pressure.฀This฀luid,฀interstitial฀luid,฀feeds฀the฀
stroma฀ and฀ then฀ drains฀ back฀ into฀ the฀ venous฀ end฀ of฀ capillaries฀ under฀ the฀ inluence฀ of฀
increased฀capillary฀osmotic฀pressure฀and฀reduced฀hydrostatic฀pressure.฀It฀contains฀glucose,฀
amino฀ acids,฀ some฀ metabolites฀ such฀ as฀ citrate,฀ pyrophosphate,฀ and฀ extracellular฀ ATP฀
(Sect.฀9.1.4)฀as฀well฀as฀vitamins฀and฀inorganic฀ions.฀It฀is฀free฀of฀the฀proteins฀and฀other฀large฀
molecules฀present฀in฀blood฀plasma,฀but฀it฀receives฀soluble฀proteins฀that฀are฀secreted฀into฀it฀
by฀matrix฀cells฀such฀as฀ibroblasts.
The฀ basal฀ cells฀ of฀ a฀ layered฀ epithelium฀ such฀ as฀ skin฀ or฀ gingiva฀ (Sect.฀ 5.2.1),฀ or฀ the฀
periosteal฀cells฀covering฀bone,฀have฀such฀high฀nutrient฀demands฀that฀they฀need฀to฀be฀close฀to฀
a฀rich฀capillary฀bed.฀Thus,฀although฀capillaries฀permeate฀the฀stroma,฀they฀are฀especially฀dense฀
beneath฀ the฀ basal฀ lamina฀ of฀ a฀ layered฀ epithelium฀ (Sect.฀ 13.3.1)฀ or฀ within฀ a฀ periosteum฀
(Sect.฀9.2.1).฀The฀gingival-tooth฀interface฀is฀especially฀susceptible฀to฀bacterial฀agents฀that฀
44 3 The Connective Tissue Extracellular Matrix and Its Major Components

affect฀the฀subepithelial฀capillary฀plexus.฀The฀capillaries฀become฀leaky฀and฀exude฀traces฀of฀
blood฀plasma฀and฀leukocytes฀into฀the฀gingival฀sulcus฀as฀an฀inlammatory฀exudate฀which฀฀can-
not฀clot฀because฀of฀plasmin฀activation฀(Sect.฀13.1.2฀and฀Fig.฀13.5a).

3.3.2.
Stromal Turnover, Inflammation, and Bone Loss

Hormones,฀infections฀and฀physical฀stresses฀promote฀degradation฀of฀the฀stroma฀by฀inducing฀
affected฀cells฀to฀release฀cytokines.฀This฀set฀of฀small฀proteins฀mediates฀cell-to-cell฀interac-
tions,฀proliferation,฀differentiation,฀the฀activation฀of฀cell-speciic฀organelles฀and฀diverse฀
molecular฀ pathways.฀ Some฀ cytokines,฀ called฀ interleukins,฀ are฀ proinlammatory;฀ they฀
increase฀capillary฀permeability฀so฀that฀white฀blood฀cells฀pass฀into฀the฀stroma฀where฀their฀
lysosomes฀are฀activated฀to฀secrete฀tissue฀destructive฀enzymes฀(Sect.฀13.2.2).฀Lysosomes฀are฀
membrane฀bound฀vesicles฀containing฀various฀acid-activated฀digestive฀enzymes.฀They฀are฀
present฀ in฀ all฀ cells,฀ but฀ are฀ only฀ activated฀ if฀ the฀ cell฀ has฀ been฀ damaged฀ by฀ physical฀ or฀
chemical฀injury฀directly,฀or฀by฀an฀infecting฀organism.฀The฀cytokines฀cause฀leukocytes฀to฀be฀
extruded฀from฀nearby฀capillaries฀and฀activated฀to฀enhance฀lysosomal฀enzyme฀content฀at฀the฀
site฀of฀damage฀or฀infection฀(Sect.฀13.3.1).฀As฀the฀affected฀tissues฀are฀digested฀and฀removed,฀
the฀surrounding฀and฀invading฀cells฀secrete฀anti-inlammatory฀interleukins฀and฀other฀sub-
stances฀ that฀ promote฀ acquired฀ immunity฀ and฀ tissue฀ repair.฀ A฀ physiological฀ example฀ of฀
cytokine฀action฀(Sect.฀10.2.1)฀is฀the฀response฀to฀stress-induced฀micro-cracks฀that฀continu-
ally฀develop฀on฀bone฀surfaces.
Acquired฀immunity,฀for฀example฀antibodies฀that฀react฀to฀an฀antigen฀on฀the฀surface฀of฀
an฀ infecting฀ bacterium,฀ activate฀ proteins฀ called฀ complement฀ in฀ the฀ blood฀ plasma.฀
Complement฀proteins฀attract฀white฀blood฀cells฀(leukocytes)฀and฀activate฀them฀to฀digest฀
(phagocytose)฀ antigen-antibody฀ complexes฀ (Sect.฀ 13.3.1).฀ Complement฀ proteins฀ were฀
named฀because฀they฀were฀initially฀determined฀to฀complement฀or฀complete฀the฀action฀of฀
antigen-antibody฀complexes฀in฀the฀body.฀The฀interactions฀of฀antigen,฀antibody,฀and฀com-
plement฀protect฀the฀body฀from฀speciic฀bacterial฀infection฀and฀tissue฀damage.฀

฀A฀stromal฀matrix฀is฀permeated฀by฀interstitial฀luid฀from฀capillaries.฀This฀luid฀drains฀
back฀into฀the฀capillaries฀except฀where฀the฀tissue฀is฀damaged,฀in฀which฀case฀the฀capillar-
ies฀become฀leaky฀and฀proteins฀enter฀with฀the฀luid,฀causing฀edema.฀Infections฀and฀phys-
ical฀stresses฀promote฀stromal฀degradation฀by฀inducing฀the฀cells฀to฀release฀proinlammatory฀
cytokines฀ that฀ attract฀ and฀ activate฀ blood฀ granulocytes฀ and฀ macrophages.฀ These฀ cells฀
secrete฀lysosomal,฀acid-activated,฀tissue฀destructive฀enzymes.฀As฀the฀infection฀or฀tissue฀
damage฀is฀removed,฀the฀surrounding฀cells฀invade฀and฀start฀secreting฀anti-inlammatory฀
cytokines฀that฀stimulate฀immunity฀and฀tissue฀regeneration.
 Fibrillar and Non-fibrillar Collagens
and Integrins 4

This฀chapter฀describes฀the฀isolation฀and฀amino฀acid฀composition฀of฀the฀common฀type฀of฀
collagen฀ iber฀ polypeptides฀ and฀ how฀ they฀ associate฀ into฀ collagen฀ ibers฀ (Sect.฀ 1).฀฀
A฀ description฀ of฀ how฀ collagen฀ ibers฀ are฀ stabilized฀ by฀ crosslinking฀ then฀ follows฀
(Sect.฀2).฀The฀non-ibrillar฀class฀of฀collagen฀is฀described,฀along฀with฀its฀role฀in฀modify-
ing฀ ibers฀ and฀ in฀ forming฀ ilaments฀ (Sect.฀ 3).฀ The฀ chapter฀ concludes฀ with฀ a฀ detailed฀
discussion฀of฀integrin฀proteins,฀with฀special฀emphasis฀on฀how฀integrins฀control฀ibro-
blast฀cell฀attachment฀to฀collagen฀ibers฀and฀collagen฀synthesis฀(Sect.฀4).

4.1.1.
Fibrillar Collagens

Collagen฀ibers฀are฀readily฀obtained฀from฀vertebrate฀bones,฀teeth,฀cartilage,฀ligaments,฀and฀
dermis฀of฀the฀skin.฀Periodontal฀ibers฀from฀washed,฀extracted฀teeth฀may฀be฀cut฀out฀and฀the฀
collagen฀ibers฀examined฀under฀a฀dissecting฀microscope.฀Collagen฀ibers฀in฀bones฀or฀dentin฀
may฀be฀freed฀from฀the฀mineral฀by฀boiling฀them฀in฀disodium฀ethylene฀diamine฀until฀soft.฀
Puriied฀ collagen฀ ibers฀ possess฀ a฀ unique฀ amino฀ acid฀ composition:฀ a฀ third฀ of฀ the฀ amino฀
acids฀are฀glycine฀and฀another฀23%฀are฀proline,฀of฀which฀almost฀half฀are฀hydroxyproline.฀
Lysine฀comprises฀another฀2฀–฀4%฀of฀residues,฀of฀which฀many฀are฀hydroxylysine.฀Short-
chain฀amino฀acids฀such฀as฀alanine฀and฀serine฀total฀another฀13%฀of฀residues฀(Table฀4.1).฀Of฀
the฀20฀encoded฀amino฀acids,฀tryptophan฀and฀cysteine฀are฀absent฀and฀the฀remaining฀13฀are฀
present฀in฀small฀amounts.฀Because฀tryptophan฀is฀nutritionally฀essential,฀a฀diet฀consisting฀
only฀of฀collagen฀cannot฀support฀life฀in฀humans฀or฀other฀mammals;฀nutritionally,฀collagen฀
is฀said฀to฀have฀a฀biological฀value฀of฀zero.
Collagen฀ibrils,฀ibers฀and฀iber฀bundles฀may฀be฀isolated฀from฀calf฀skin฀dermis฀by฀dis-
aggregation฀ with฀ a฀ neutral฀ salt฀ buffer฀ followed฀ by฀ differential฀ centrifugation.฀ The฀ pure฀
ibers฀฀are฀boiled฀in฀sodium฀dodecyl฀sulfate฀(SDS)฀and฀subjected฀to฀polyacrylamide฀gel฀
electrophoresis฀(SDS-PAGE).฀In฀this฀modern฀system,฀the฀collagen฀polypeptides฀are฀sepa-
rated฀by฀molecular฀weight.฀Figure฀4.1฀shows฀that฀collagen฀ibers฀are฀composed฀of฀a1-,฀a2-,฀
b-,฀and฀g-฀polypeptides฀called฀tropocollagen.฀Marker฀proteins฀of฀known฀molecular฀weight฀
indicate฀that฀the฀smallest฀polypeptides฀a1-฀and฀a2-tropocollagens฀are฀both฀approximately฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 45
DOI:฀10.1007/978-3-540-88116-2_4,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
46 4 Fibrillar and Non-fibrillar Collagens and Integrins

Table 4.1฀ Amino฀acid฀composition฀of฀alpha-1฀chain฀of฀tropocollagen

Important฀amino฀acid %฀of฀total฀฀ Function
amino฀acids
Glycine 33 Enhances฀van฀der฀Waals฀forces฀and฀hydrogen฀
bonds฀that฀hold฀three฀helical฀polypeptides฀
together.
Proline 14 Responsible฀for฀the฀extended฀helix.
Hydroxyproline 9 Hydroxyl฀group฀stabilizes฀the฀extended฀฀
helix฀at฀higher฀temperatures.
Alanine 12 Small฀side฀chain฀allows฀polypeptides฀฀
to฀lie฀alongside฀each฀other฀and฀form฀ibers.
Serine 4 Small฀side฀chain฀(same฀effect฀as฀alanine).
Lysine 3 Responsible฀for฀covalent฀cross-linking.
Hydroxylysine 1 Attaches฀carbohydrate฀and฀is฀involved฀฀
with฀lysine฀in฀covalent฀cross-linking.
Percentage฀of฀total฀amino฀acids฀from฀Table฀38.1,฀p฀1134฀of฀Principles฀of฀Biochemistry,฀White,฀A.฀
et฀al.,฀6th฀Ed.฀1978.฀McGraw฀Hill,฀New฀York

100฀kDa;฀the฀a1-polypeptide฀being฀slightly฀larger฀because฀it฀migrates฀less.฀The฀origins฀of฀
the฀b-฀andg-฀chains฀are฀discussed฀below฀(Sect.฀4.2.2).
The฀a1฀and฀a2-polypeptides฀may฀be฀cut฀out฀of฀the฀gel,฀each฀partially฀digested฀with฀a฀
suitable฀protease฀and฀the฀fragments฀separated,฀blotted฀onto฀a฀membrane฀and฀the฀irst฀18–23฀
N-terminal฀residues฀of฀each฀peptide฀sequenced.฀After฀sorting฀for฀overlaps,฀large฀segments฀
of฀the฀sequence฀may฀be฀obtained.฀The฀results฀consistently฀indicate฀that฀glycine฀is฀present฀
at฀every฀third฀residue฀(Gly-X-Y฀repeating฀motif)฀in฀both฀a-polypeptides฀and฀that฀proline฀is฀
often฀present฀at฀the฀X฀or฀Y฀position.฀Other฀studies฀indicate฀that฀artiicial฀polypeptides฀con-
taining฀ a฀ Gly-X-Pro฀ repeating฀ sequence฀ have฀ an฀ extended฀ (unfolded)฀ chain฀ due฀ to฀ the฀
conformation฀of฀the฀proline฀peptide฀bond฀(Fig.฀4.2),฀a฀left-handed฀extended฀helix฀called฀the฀
collagen฀helix฀(Fig.฀4.3)฀because฀of฀its฀presence฀in฀all฀types฀of฀collagen.
This฀left-handed฀secondary฀structure฀of฀collagen฀polypeptides฀differs฀from฀the฀a-helix,฀
whose฀ tightly฀ coiled,฀ right-handed฀ helix฀ is฀ disrupted฀ by฀ proline.฀ It฀ also฀ differs฀ from฀ a฀
b-sheet,฀a฀series฀of฀six฀to฀ten฀amino฀acids฀in฀extended฀chain฀coniguration฀in฀which฀proline฀
residues฀create฀sharp฀turns,฀allowing฀the฀chains฀to฀lie฀alongside฀each฀other.฀The฀secondary฀
structure฀of฀a฀ibrous฀collagen฀a-polypeptide฀is฀an฀extended฀helical฀rod฀(Fig.฀4.4a฀and฀b).฀
The฀glycine฀residues฀permit฀strong฀associations฀between฀the฀peptides฀(quaternary฀struc-
ture)฀due฀to฀hydrogen฀bonding฀between฀the฀hydrogen฀atom฀residue฀of฀glycine฀and฀carbonyl฀
and฀amide฀groups฀of฀nearby฀peptide฀bonds.฀Tertiary฀structure฀is฀absent.฀The฀result฀is฀an฀
extended฀triple฀helix,฀tropocollagen฀(Fig.฀4.4c),฀which฀forms฀the฀monomeric฀unit฀of฀all฀
ibrous฀collagens.฀Figure฀4.5฀is฀a฀cut-through฀view฀of฀the฀chains฀in฀Fig.฀4.4.฀It฀shows฀a฀
central฀cavity,฀across฀which฀the฀glycine฀hydrogen฀atom฀(glycine฀side-chain)฀holds฀the฀three฀
a-polypeptides฀together฀by฀hydrogen฀bonding฀between฀alternate฀pairs฀of฀the฀chains.
Recently,฀ luoroproline,฀ made฀ by฀ artiicially฀ replacing฀ the฀ hydroxyproline฀ hydroxyl฀
group฀with฀a฀luoride฀atom,฀was฀found฀to฀form฀a฀collagen฀helix฀that฀was฀more฀stable฀to฀heat฀
denaturation฀than฀hydroxyproline฀or฀proline.฀These฀studies฀also฀indicated฀that฀luoropro-
line฀and฀hydroxyproline฀stabilize฀the฀collagen฀helix฀to฀heating฀by฀promoting฀an฀extended฀
4.1.1. Fibrillar Collagens 47

Origin

g
(300 kDa)
b1, 2 b1 1
(200 kDa)

a1
a2 (100 kDa)

Fig. 4.1฀ The฀ polypeptide฀ composition฀ of฀ collagen฀ ibers.฀ Periodontal฀ membrane฀ ibers฀ were฀ dis-
sected,฀boiled฀with฀sodium฀dodecyl฀sulfate฀(SDS),฀in฀the฀presence฀of฀mercaptoethanol฀to฀provide฀a฀
reducing฀environment,฀and฀separated฀by฀polyacrylamide฀gel฀electrophoresis฀(PAGE).฀Polypeptides฀
were฀visualized฀by฀staining฀with฀a฀dye฀after฀electrophoresis.฀The฀smallest฀polypeptides฀migrate฀most฀
from฀the฀origin.฀The฀a-tropocollagen฀chains฀are฀about฀100฀kDa฀mol฀wt,฀the฀a2-chain฀being฀slightly฀
smaller฀(nearer฀the฀foot฀of฀the฀gel)฀than฀the฀a1-chain.฀The฀b-bands฀have฀a฀molecular฀weight฀of฀about฀
200฀kDa.฀The฀b1,฀2-band฀consists฀of฀an฀a1-chain฀covalently฀cross-linked฀to฀an฀a2-chain;฀the฀b1,฀
1-band฀consists฀of฀two฀cross-linked฀a1-chains.฀The฀g-tropocollagen฀chain฀has฀a฀molecular฀weight฀of฀
300,000฀and฀consists฀of฀three฀covalently฀cross-linked฀a฀chains฀(Adapted฀from฀Fig.฀2.7฀published฀in:฀
Biochemistry,฀A฀Case-Oriented฀Approach,฀4th฀Edition.฀R.฀Montgomery,฀R.L.฀Dryer,฀R.C.฀Conway,฀
and฀A.฀Spector,฀C.V.฀Mosby฀Co.,฀St฀Louis,฀MO฀1983;฀Copyright฀Elsevier,฀2008)

OH
4 3
HC CH2
Side-chain CH2 CH COO−
5 2
attached to N+
alpha-amino H H
group 4-Hydroxyproline
(HOpro)

1
OH COO−
6 5 4 3 2
+H N
3 CH2 CH2 CH2 CH2 CH
Regular alpha-amino H N+
group
H H
Fig. 4.2฀ Structures฀of฀hydroxyproline฀and฀ 5-Hydroxylysine
hydroxylysine (HOlys)

(trans)฀form฀of฀the฀peptide฀bond.฀Other฀studies฀indicate฀that฀a฀proline฀helix฀becomes฀unsta-
ble฀above฀10°C,฀but฀that฀replacing฀about฀half฀of฀the฀proline฀residues฀with฀hydroxyproline฀
as฀in฀mammals฀(Table฀4.1)฀stabilizes฀the฀helix฀at฀37°C.฀In฀cold-blooded฀vertebrates฀such฀as฀
ish,฀the฀tropocollagen฀must฀dissociate฀at฀20°C฀or฀lower,฀not฀at฀37°C.฀Less฀hydroxyproline฀
is฀ present฀ in฀ cold-water฀ ish฀ collagens.฀ These฀ studies฀ suggest฀ that฀ it฀ is฀ the฀ secondary฀
48 4 Fibrillar and Non-fibrillar Collagens and Integrins

Pro
Pro

Gly

Gly
Pro Pro

Fig. 4.3฀ Collagen฀helix.฀This฀secondary฀structure฀is฀created฀by฀peptide฀bond฀conformation฀around฀

the฀proline฀and฀hydroxyproline฀residues฀(From฀Fig.฀2-39฀in฀Biochemistry.฀L.฀Stryer,฀4th฀Ed.฀1995.฀฀
W.H.฀Freeman฀&฀Co.,฀New฀York)

Fig. 4.4฀ Collagen฀triple฀฀

helix฀formation.฀฀
(a)฀A฀repeating฀tripeptide฀
sequence฀Gly-X-Pro฀or฀
Gly-X-4-Hyp฀adopts฀a฀
left-handed฀helical฀structure฀
with฀three฀residues฀per฀turn.฀
The฀repeating฀sequence฀
used฀to฀generate฀this฀model฀
is฀Gly-Pro-4-Hyp.฀฀
(b)฀Space-illing฀model฀of฀
the฀same฀chain.฀(c)฀Three฀of฀
these฀chains฀(gray,฀blue,฀
and฀purple)฀wrap฀around฀
one฀another฀with฀a฀
right-handed฀twist฀
(Figure฀4.12a,฀b฀and฀c฀in฀
Lehninger฀Principles฀of฀
Biochemistry.฀D.L.฀฀
Nelson฀and฀M.M.฀Cox,฀4th฀
Ed.฀2005.฀W.H.฀Freeman฀&฀
Co.,฀New฀York)
4.1.1. Fibrillar Collagens 49

Fig. 4.5฀ Glycine฀residues฀

make฀up฀the฀interior฀of฀a฀
tropocollagen฀triple฀helix.฀
The฀same฀three-stranded฀
collagen฀super-helix฀is฀
depicted฀as฀in฀Fig.฀4.4,฀but฀
looking฀down฀the฀center฀of฀a฀
ball-and-stick฀representation.฀
Glycine฀residues฀(−H)฀are฀
shown฀in฀red.฀Because฀of฀its฀
small฀size,฀glycine฀is฀
required฀where฀the฀three฀
chains฀contact.฀The฀balls฀in฀
this฀illustration฀do฀not฀
represent฀the฀van฀der฀Waals฀
radii฀of฀the฀individual฀atoms฀
(Figure฀4-12d฀in฀Lehninger฀
Principles฀of฀Biochemistry.฀
D.L.฀Nelson฀and฀M.M.฀Cox,฀
4th฀Ed.฀2005.฀W.H.฀Freeman฀
&฀Co.,฀New฀York)

structure฀(the฀collagen฀helix)฀that฀stabilizes฀the฀triple฀helix฀to฀heat฀denaturation.฀Quaternary฀
structure฀hydrogen฀bonds฀involving฀the฀OH฀group฀of฀hydroxyproline฀or฀the฀side฀chain฀of฀
glycine฀to฀carbonyl฀groups฀of฀the฀peptide฀bond฀seem฀less฀important.
After฀its฀expression฀from฀ibroblasts,฀the฀a-polypeptides฀of฀tropocollagen฀form฀ibers฀
spontaneously฀(without฀an฀enzyme).฀The฀standard฀model฀of฀tropocollagen฀is฀derived฀from฀
peptides฀such฀as฀(pro-pro-gly)n฀that฀suggest฀a฀helical฀repeat฀length฀of฀30฀amino฀acid฀resi-
dues.฀Unfortunately,฀this฀inding฀is฀inconsistent฀with฀X-ray฀diffraction฀patterns฀from฀native฀
collagens,฀which฀suggest฀a฀repeat฀length฀of฀only฀21฀amino฀acids.฀Indeed,฀recent฀analysis฀of฀
nonpolar฀residues฀(Val,฀Leu,฀Ile,฀Met,฀and฀Phe)฀in฀the฀a1฀tropocollagen฀sequence฀of฀the฀
most฀ common฀ form฀ of฀ collagen฀ iber฀ reveals฀ them฀ to฀ predominate฀ near฀ the฀ centers฀ of฀
21-residue฀ segments.฀ The฀ spontaneity฀ of฀ collagen฀ ibril฀ assembly฀ may฀ be฀ initiated฀ by฀
hydrophobic฀ interactions฀ between฀ tropocollagen฀ molecules฀ at฀ these฀ centers฀ within฀ the฀
extended฀chains฀and฀that฀this฀guides฀the฀subsequent,฀stabilizing฀formation฀of฀the฀glycine-
bonded฀triple฀helix.

The฀alpha฀chains฀of฀ibrous฀collagens฀(tropocollagen)฀have฀many฀proline฀(and฀hydroxy-
proline)฀ residues฀ responsible฀ for฀ the฀ collagen฀ helix,฀ an฀ extended฀ chain,฀ left-handed฀
helix฀ (secondary฀ structure)฀ different฀ from฀ the฀ a-helix฀ and฀ b-sheet฀ in฀ other฀ proteins.฀
Glycine฀and฀hydroxyproline฀are฀responsible฀for฀the฀association฀of฀the฀polypeptides฀into฀
a฀ triple฀helix.฀The฀ glycine฀ side฀ chains฀ (hydrogen฀ atoms)฀ hydrogen฀ bond฀ to฀ carbonyl฀
groups฀of฀a฀nearby฀peptide฀bond฀in฀the฀helical฀backbone฀and฀hydroxyproline฀OH฀groups฀
50 4 Fibrillar and Non-fibrillar Collagens and Integrins

to฀ amide฀ groups฀ of฀ other฀ nearby฀ peptide฀ bonds.฀ The฀ hydroxyproline฀ bonds฀ enhance฀
triple฀helix฀formation฀at฀37°C.฀Cold-blooded฀vertebrates฀have฀less฀hydroxyproline฀in฀
their฀ collagens฀ (non-ibrillar฀ as฀ well฀ as฀ ibrillar).฀ Serine฀ and฀ alanine฀ have฀ short฀ side฀
chains฀ that฀ allow฀ the฀ three฀ chains฀ to฀ come฀ together฀ more฀ easily฀ than฀ long฀ residues.฀
Recent฀X-ray฀diffraction฀studies฀of฀native฀collagen฀suggest฀that฀a฀21฀amino฀acid฀repeat-
ing฀unit฀whose฀central฀region฀contains฀hydrophobic฀amino฀acid฀residues฀initiates฀triple฀
helix฀formation.

4.2.1.
Collagen Fiber Formation

The฀a1-฀and฀a2-฀tropocollagen฀polypeptides฀are฀each฀a฀long,฀central฀portion฀(domain)฀of฀
two฀ larger฀ polypeptides฀ (procollagen)฀ encoded฀ by฀ genomic฀ DNA฀ (genes฀ COL1A1฀ and฀
COL1A2).฀The฀sequences฀of฀the฀respective฀genes฀are฀similar฀(homologous).฀And฀assem-
ble฀so฀that฀two฀a1-฀and฀one฀a2-tropocollagen฀polypeptides฀interact฀by฀their฀hydrophobic฀
domains฀ and฀ initiate฀ triple฀ helix฀ formation.฀ The฀ tropocollagen฀ polypeptides฀ are฀ then฀
cleaved฀out฀as฀the฀ibril-forming฀monomeric฀unit฀(Fig.฀4.6).฀The฀removed฀portions฀are฀
called฀ the฀ collagen฀ propeptides.฀ The฀ N-฀ and฀ C-terminal฀ ends฀ of฀ the฀ excised฀ central฀
domains฀ are฀ called฀ the฀ tropocollagen฀ telopeptide฀ domains.฀ These฀ telopeptide฀ domains฀
interact฀with฀adjacent฀tropocollagen฀molecules฀(yellow฀lines฀in฀Fig.฀3.3)฀so฀that฀a฀stag-

Fig. 4.6฀ Procollagen฀is฀the฀precursor฀of฀tropocollagen.฀Tertiary฀structures฀are฀absent฀from฀tropocol-

lagen,฀but฀present฀on฀procollagen฀whose฀N-฀and฀C-termini฀fold฀like฀a฀regular฀protein.฀These฀globu-
lar฀non-collagenous฀(NC)฀domains฀at฀each฀end฀of฀the฀procollagen฀molecule฀(green)฀form฀soluble,฀
tertiary฀structures฀that฀keep฀the฀central,฀glycine/proline-rich฀(collagenous)฀domain฀in฀solution฀until฀
secreted฀from฀the฀cell.฀These฀domains฀are฀called฀propeptides.฀The฀C-terminal฀NC฀domain฀is฀usu-
ally฀referred฀to฀as฀NC1฀and฀the฀N-terminal฀domain฀as฀NC2.฀In฀non-ibrillar฀collagens,฀and฀in฀ibril-
lar฀ collagens฀ with฀ interrupted฀ collagenous฀ domains฀ (see฀ Table฀ 3.1),฀ there฀ are฀ additional฀ NC฀
domains฀which฀are฀numbered฀so฀that฀the฀C-terminus฀propeptides฀remains฀NC1.฀For฀example,฀the฀
N-terminal฀domain฀of฀type฀IX฀collagen฀in฀cartilage฀is฀NC4฀(see฀Fig.฀6.14a).฀The฀propeptides฀are฀
removed฀ from฀ ibrillar฀ procollagens฀ after฀ secretion฀ to฀ form฀ a-tropocollagen฀ (red,฀ right฀ side฀ of฀
igure),฀but฀remain฀uncleaved฀in฀non-ibrillar฀collagens.฀The฀N-฀and฀C-terminal฀ends฀of฀a-tropocol-
lagen฀are฀also฀non-helical฀and฀referred฀to฀as฀the฀telopeptides฀(N฀and฀C฀ends฀of฀red฀portion฀on฀right฀
side฀of฀igure).฀Telopeptides฀are฀involved฀in฀cross-linking฀the฀ibers฀(see฀Fig.฀4.9)฀(Modiied฀from฀
Fig.19-47฀in฀The฀Molecular฀Biology฀of฀the฀Cell,฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀
Taylor฀&฀Francis฀Group,฀New฀York)
4.2.1. Collagen Fiber Formation 51

gered฀ array฀ forms฀ with฀ gaps฀ (bottom฀ half฀ of฀ Fig.฀ 3.3).฀ The฀ one-quarter฀ overlap฀ of฀ the฀
arrayed฀molecules฀causes฀a฀striated฀appearance,฀the฀dark฀and฀light฀banding฀pattern฀at฀the฀
top฀of฀Fig.฀3.3.฀The฀gaps฀also฀control฀how฀the฀ibers฀stain฀with฀dyes,฀cross-link,฀calcify฀
and฀degrade฀(Table฀4.2).฀Figure฀4.7฀shows฀how฀the฀quarter-staggered฀arrays฀aggregate฀
into฀ibrils฀(thin,฀small฀ibers฀<30฀nm฀diameter)฀and฀ibers฀(>300฀nm฀diameter).฀Filaments฀
and฀shorter฀length฀microibrils฀are฀thin,฀extended฀aggregates฀(>10฀nm฀diameter).
The฀extended฀triple-helical฀structure,฀repeated฀over฀and฀over฀in฀the฀quarter-staggered฀
array,฀gives฀the฀collagen฀iber฀its฀stiffness฀strength฀and฀insolubility.฀The฀helical฀wrapping฀
of฀three฀polypeptides฀in฀tropocollagen฀provides฀greater฀strength฀than฀steel฀wire฀and฀the฀
staggered฀array฀provides฀resistance฀to฀external฀forces.฀Heating฀to฀more฀than฀90°C฀disrupts฀
the฀hydrogen฀bonding฀between฀the฀polypeptides฀in฀a฀collagen฀iber,฀and฀is฀analogous฀to฀
the฀disruption฀of฀double-stranded฀DNA.฀The฀a-tropocollagen฀denatures฀and฀individual฀
polypeptides฀are฀released฀from฀the฀iber.฀The฀a-tropocollagen฀telopeptide฀domains฀cannot฀
spontaneously฀reassemble฀into฀a฀triple฀helix฀because฀the฀excised฀propeptides฀are฀critical฀
for฀ helix฀ formation.฀ When฀ cooled,฀ the฀ tropocollagen฀ polypeptides฀ lose฀ their฀ solubility฀
and฀aggregate฀to฀form฀a฀clear฀gel.฀The฀denatured฀collagen฀(gelatin)฀is฀the฀basis฀of฀many฀
manufactured฀ food฀ products฀ and฀ also฀ an฀ effective฀ surface฀ for฀ in฀ vitro฀ cell฀ and฀ organ฀
culture.

Table 4.2฀ Functions฀of฀gaps฀in฀the฀ibrillar฀collagen฀array

1. Dye฀staining฀gives฀the฀microibril฀a฀striated฀appearance฀
under฀the฀electron฀microscope.
2. Lysine฀residues฀predominate฀around฀the฀gaps,฀causing฀glycan฀
attachment฀and฀polypeptide฀cross-linking.
3. Phosphorylation฀nucleates฀calcium฀as฀bone฀forms.
4. Site฀of฀initial฀collagenase฀degradation.
5. Site฀of฀iber฀interactions฀with฀non-ibrous฀collagens

Collagen fiber
Tropocollagen
0.5-3 mm

10-300
nm

Collagen fibril

Fig. 4.7฀ Super-arrays฀of฀tropocollagen฀form฀collagen฀ibrils฀or฀ibers.฀Each฀ibril฀(left)฀is฀composed฀

of฀a฀10฀×฀300฀nm฀bundle฀of฀a-tropocollagen฀monomeric฀units฀that฀have฀aggregated฀in฀quarter-
staggered฀array.฀A฀single฀tropocollagen฀polypeptide฀is฀indicated฀as฀a฀red฀arrow฀and฀the฀ends฀of฀the฀
others฀in฀the฀ibril฀are฀seen฀as฀black฀dots฀(far฀left).฀Collagen฀ibers฀in฀the฀periodontium฀or฀a฀tendon฀
are฀made฀up฀from฀bundles฀of฀ibrils฀that฀are฀50฀times฀thicker฀and฀300฀times฀longer฀(right)฀(Adapted฀
from฀Fig.19-47฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀ et฀ al.,฀ 4th฀ Ed.฀ 2002.฀ Garland฀
Science,฀Taylor฀&฀Francis฀Group,฀New฀York)
52 4 Fibrillar and Non-fibrillar Collagens and Integrins

4.2.2.
Fiber Cross-Linking: Formation of b- and g-Tropocollagen

The฀tropocollagen฀b-฀and฀g-chains฀in฀Fig.฀4.1฀are฀respectively฀twice฀and฀three-times฀the฀
size฀ of฀ the฀ a-chain.฀ Indeed,฀ their฀ amino฀ acid฀ composition฀ is฀ virtually฀ identical฀ to฀ the฀
a-chain฀in฀Fig.฀4.1,฀except฀for฀the฀appearance฀of฀derivatives฀of฀lysine฀and฀hydroxylysine,฀
mainly฀lysyl฀hydroxyl-norleucine฀and฀pyridinoline.฀They฀increase฀in฀amount฀with฀age฀of฀
the฀organism.
Along฀with฀procollagen,฀ibroblasts฀secrete฀an฀enzyme,฀lysyl฀oxidase,฀which฀binds฀to฀
and฀activates฀oxygen฀gas฀with฀copper฀ions฀at฀its฀catalytic฀site.฀Lysyl฀oxidase฀attaches฀to฀gaps฀
in฀the฀ibrils฀where฀it฀converts฀side-chain฀terminal฀amino฀group฀of฀lysine฀or฀hydroxylysine฀
residues฀(e-amino฀group)฀within฀the฀tropocollagen฀telopeptide฀domains฀into฀an฀aldehyde,฀
allysine,฀ (a-aminoadipic-d-semialdehyde)฀ or฀ hydroxyl-allysine฀ (hydroxy-a-aminoadipic-
d-semialdehyde).฀ Subsequent฀ cross-linking฀ reactions฀ are฀ all฀ spontaneous฀ and฀ due฀ to฀ the฀
reactivity฀of฀the฀generated฀aldehyde฀group.฀The฀allysine฀or฀the฀hydroxyallysine฀aldehyde฀
group฀reacts฀with฀the฀e-amino฀group฀of฀lysine฀or฀hydroxylysine฀in฀an฀adjacent฀polypep-
tide฀to฀cross-link฀two฀polypeptides,฀forming฀dehydro-hydroxylysinorleucine฀or฀dehydro-
lysinorleucine฀ cross-links฀ (Fig.฀ 4.8).฀ The฀ illustrated฀ reaction฀ between฀ allysine฀ and฀
hydroxylysine฀is฀more฀frequent฀than฀between฀allysine฀and฀lysine,฀but฀either฀gives฀rise฀to฀
b-tropocollagen฀in฀which฀the฀lysine-derived฀cross-link฀is฀stable.฀Some฀of฀the฀dehydro-
hydroxylysinorleucine฀ or฀ dehydro-lysinorleucine฀ molecules฀ are฀ reduced฀ in฀ vivo฀ to฀
hydroxylysinorleucine฀or฀lysinorleucine.
g-Tropocollagen฀forms฀when฀an฀α-polypeptide฀becomes฀linked฀to฀two฀others฀by฀sepa-
rate฀dehydro-hydroxylysinorleucine฀bonds,฀or฀by฀dehydro-hydroxylysinorleucine฀being฀
transformed฀into฀pyridinoline฀through฀adding฀a฀third฀allysine฀residue฀from฀another฀α-chain฀
(Fig.฀4.9).฀Pyridinoline฀cross-links฀appear฀mostly฀in฀the฀telopeptide฀region฀of฀tropocolla-
gen฀(Sect.฀4.2.1),฀but฀few฀are฀present฀and฀the฀g-chain฀band฀in฀Fig฀4.1฀is฀faint.฀The฀older฀
the฀ organism,฀ the฀ greater฀ is฀ the฀ extent฀ of฀ collagen฀ cross-linking.฀ Collagen฀ ibers฀ from฀
young฀animals฀contain฀few฀b-฀and฀almost฀no฀g-polypeptides,฀whereas฀the฀ibers฀from฀old฀
animals฀ have฀ substantial฀ amounts฀ of฀ b-polypeptides฀ and฀ some฀ clearly฀ detectable฀
g-polypeptides.
Cross-linking฀contributes฀to฀tissue฀strength฀and฀limits฀the฀need฀for฀iber฀replacement,฀
but฀it฀also฀inhibits฀repair฀following฀a฀mechanical฀injury฀or฀infection฀(Sect.฀8.1.3.).฀Lysyl฀
oxidase฀catalysis฀is฀self-limiting฀to฀avoid฀excessive฀cross-linking.฀The฀oxidation฀rate฀of฀
lysine฀amine฀residues฀is฀limited฀to฀approximately฀100฀catalytic฀turnovers฀per฀enzyme฀mol-
ecule฀because฀ammonia฀and฀other฀reaction฀by-products฀inactivate฀it฀irreversibly.
Collagen฀ibers฀must฀be฀stable฀for฀some฀years฀for฀measurable฀amounts฀of฀pyridinoline฀to฀
form.฀Because฀bone฀is฀the฀major฀source฀of฀long-term฀stable฀collagen฀ibers,฀an฀increase฀in฀
peptides฀containing฀pyridinoline฀in฀the฀blood฀or฀urine฀is฀a฀good฀marker฀of฀increased฀bone฀
remodeling฀and฀resorption.฀Normally,฀amounts฀of฀these฀peptides฀are฀low฀except฀at฀adoles-
cence฀and฀in฀women฀shortly฀after฀menopause฀(Fig.฀4.9).฀Large฀luctuations฀in฀collagen฀turn-
over฀outside฀of฀bone฀make฀measurements฀of฀hydroxyproline฀in฀the฀blood฀or฀urine฀an฀unreliable฀
measure฀of฀the฀extent฀of฀collagen฀degradation.฀Most฀hydroxyproline฀is฀metabolized฀in฀the฀
4.2.2 Fiber Cross-Linking: Formation of β-฀and γ-฀Tropocollagen 53

Polypeptide Polypeptide
chain-1 chain-2
Adjacent lysine residues
H
H N N H
CH CH2 CH2 CH2 CH N CH2 CH CH2 CH2 CH
O C C O
OH
H2N H

+ O2
Lysy1
oxidase − NH3
(Cu ion)

H
H N N H
CH CH2 CH2 CH2 CH N CH2 CH CH2 CH2 CH
O C Allysine C O
O OH
H

−H2 O
H N N H
CH CH2 CH2 CH2 CH N CH2 CH CH2 CH2 CH
O C C O
OH
Lys residue
Polypeptide minus e-amino HyLys Polypeptide
chain group (norleucine) residue chain
Dehydrohydroxylysinorleucine
crosslink

Fig. 4.8฀ Formation฀of฀b-polypeptides฀from฀tropocollagen.฀The฀e-amino฀group฀of฀a฀lysine฀residue฀within฀

a฀tropocollagen฀polypeptide฀(chain฀1,฀colored฀black)฀is฀oxidized฀to฀an฀aldehyde฀by฀lysyl฀oxidase,฀an฀
enzyme฀secreted฀by฀ibroblasts.฀This฀enzyme฀adheres฀to฀the฀gap฀regions฀of฀the฀quarter-staggered฀arrays฀
and฀uses฀molecular฀oxygen฀(blue)฀which฀reacts฀with฀cupric฀ions฀at฀the฀catalytic฀center฀of฀the฀oxidase.฀
One฀oxygen฀atom฀oxidizes฀the฀e-amino฀group฀of฀an฀adjacent฀lysine฀residue,฀spontaneously฀driving฀out฀
ammonia฀(NH3).฀The฀aldehyde฀reacts฀spontaneously฀with฀a฀nearby฀hydroxylysine฀or฀lysine฀residue฀on฀
another฀ chain฀ (chain฀ 2,฀ red)฀ to฀ form฀ a฀ dehydro-hydroxylysinorleucine฀ (or฀ dehydro-lysinorleucine)฀
crosslink.฀Because,฀the฀peptide฀is฀reduced฀on฀hydrolysis฀with฀6.0฀M฀HCl฀to฀detect฀these฀cross-links,฀
the฀products฀found฀are฀always฀hydroxylysinorleucine฀or฀lysinorleucine฀(not฀the฀dehydro฀forms).฀It฀is฀
likely฀that฀some฀cross-links฀remain฀in฀the฀dehydro-฀form฀in฀vivo฀and฀that฀others฀are฀reduced฀(Adapted฀
from฀the฀diagram฀at฀the฀foot฀of฀p.128฀in฀Lehninger฀Principles฀of฀Biochemistry.฀D.L.฀Nelson฀and฀M.M.฀
Cox,฀4th฀Ed.฀2005.฀W.H.฀Freeman฀&฀Co.,฀New฀York)

liver฀to฀4-hydroxy฀2-ketoglutarate฀and฀ultimately฀to฀glycollate฀and฀glycine฀(or฀glyoxylate).฀
By฀ contrast,฀ dehydro-hydroxylysinorleucine฀ and฀ dehydro-lysinorleucine฀ are฀ reduced฀ to฀
hydroxylysinorleucine฀and฀lysinorleucine฀and฀returned฀to฀blood฀plasma฀where฀they฀are฀reli-
able฀measures฀of฀pathology฀(See฀also,฀Sect.฀10.1.3).
54 4 Fibrillar and Non-fibrillar Collagens and Integrins

NH2 COOH
COOH
CH
CH

NH2 CH2 CH2

CH2 OH

a1
...YDEKSTGG..
a2
N QYDGKGYG...

CH2

CH−OH

CH2
K
N-telopeptide
CH2

CH

NH2 COOH
Pyridinoline

C Gap N
a1
a2
a1
N C
Quarter-staggered array

Fig. 4.9฀ Formation฀of฀pyridinoline฀from฀tropocollagen.฀Dehydro-hydroxylysinorleucine฀forms฀at฀the฀

N-฀(or฀C)฀telopeptide฀region฀near฀a฀gap฀region.฀Pyridinoline฀(top฀left)฀is฀formed฀when฀a฀lysine฀residue฀
from฀a฀third฀polypeptide฀at฀this฀site฀is฀oxidized฀by฀lysine฀oxidase฀and฀becomes฀covalently฀bonded฀to฀
the฀dehydro-hydroxylysinorleucine.฀The฀orientation฀of฀the฀three฀polypeptides฀that฀form฀pyridinoline฀
is฀shown฀in฀the฀quarter-staggered฀array฀at฀the฀foot฀of฀the฀igure.฀When฀collagen฀ibers฀are฀degraded฀
in฀vivo,฀two฀telopeptide฀sequences฀are฀incompletely฀degraded฀(single฀letter฀sequences),฀but฀the฀third฀
polypeptide฀is฀removed฀except฀for฀its฀attached฀lysine฀residue.฀(top,฀right)฀(Slightly฀modiied฀from฀
Figs.฀1฀and฀3,฀D.A.฀Hanson,฀“A฀Speciic฀Immunoassay฀for฀Monitoring฀Human฀Bone฀Resorption:฀
Quantitation฀of฀Type฀I฀Collagen฀Cross-Linked฀N-telopeptides฀in฀Urine.”฀J.฀Bone฀Miner.฀Res.฀7(11):฀
1251–1258,฀1992.฀Also฀Fig.฀3,฀Ureña,฀Pablo฀and฀Marie-Christine฀de฀Vernejoul.฀‘Circulating฀bio-
chemical฀ markers฀ of฀ bone฀ remodeling฀ in฀ uremic฀ patients.”฀ Kidney฀ International.฀ 55:2141–2156฀
(1999))

Tropocollagen฀alpha฀chains฀are฀encoded฀as฀parts฀of฀larger฀polypeptides฀from฀which฀
the฀ N-฀ and฀ C-terminal฀ regions฀ (propeptides)฀ are฀ cleaved.฀ The฀ wrapping฀ of฀ three฀
a-polypeptides฀into฀a฀triple฀helix,฀repeated฀over฀and฀over,฀gives฀the฀collagen฀ibers฀
their฀stiffness,฀strength฀and฀insolubility.฀The฀triple฀helical฀monomeric฀tropocollagen฀
molecule฀polymerizes฀into฀a฀quarter-staggered฀array,฀forming฀gaps฀that฀are฀critical฀
for฀ staining,฀ cross-linking,฀ calciication฀ and฀ degradation.฀ Covalent฀ cross-links฀
between฀a-polypeptides฀strengthen฀the฀iber฀and฀are฀caused฀by฀the฀co-secretion฀of฀
lysyl฀oxidase,฀near฀gaps฀in฀the฀collagen฀array.฀Lysyl฀oxidase฀uses฀molecular฀oxygen฀
and฀copper฀ions฀to฀oxidize฀the฀terminal฀e-amino฀groups฀of฀a฀lysine฀residue฀near฀the฀
4.3.2. Fiber-Modifying Non-fibrillar Collagens 55

N-฀or฀C-terminal฀end฀of฀an฀a-polypeptide฀(telopeptide฀region).฀The฀lysyl฀aldehydes฀
(allysines)฀spontaneously฀but฀slowly฀interact฀with฀nearby฀e-amino฀lysine฀resides฀on฀
the฀same฀or฀different฀polypeptides.฀The฀result฀is฀covalent฀cross-linking฀between฀two฀
or฀ three฀ a-polypeptides฀ (b-฀ or฀ g-chains)฀ that฀ increases฀ whose฀ amounts฀ in฀ collagen฀
ibers฀ increase฀ with฀ age.฀ The฀ covalent฀ links฀ are฀ dehydro-hydroxylysyl-norleucine฀
(b-฀or฀g-chains),฀or฀pyridinoline฀(g-chains).฀Pyridinoline฀mostly฀forms฀in฀bone฀whose฀
collagen฀is฀least฀remodeled.฀The฀blood฀plasma฀content฀of฀peptides฀containing฀pyridi-
noline฀and฀hydroxylysinorleucine฀are฀good฀indicators฀of฀collagen฀degradation.

4.3.1.
The Collagen Superfamily

Although฀by฀far฀the฀most฀abundant,฀ibrillar฀collagens฀comprise฀only฀ive฀of฀the฀27฀฀collagen฀
types฀in฀the฀body,฀the฀remainder฀being฀non-ibrillar.฀All฀collagens฀are฀made฀from฀procol-
lagen฀monomeric฀units฀composed฀of฀three฀polypeptides฀in฀a฀triple฀helix฀(Fig.฀4.6).฀Proteins฀
that฀contain฀a฀short฀tropocollagen-like฀sequence฀are฀not฀collagens.฀Each฀type฀of฀collagen฀
is฀made฀up฀from฀identical฀or฀different฀polypeptides฀encoded฀in฀the฀genome.฀As฀noted฀also฀
in฀Table฀3.1,฀the฀different฀types฀of฀collagen฀polypeptides฀are฀numbered฀in฀Latin฀characters฀
(I,฀II,฀etc.)฀and฀correspond฀to฀different฀gene฀products.฀A฀triple฀helix฀may฀be฀composed฀of฀a฀
single฀polypeptide฀as฀in฀type฀II฀procollagen฀[a(II)1]3฀and฀encoded฀as฀a฀single฀gene฀(COL2A1).฀
Alternatively,฀a฀triple฀helix฀may฀be฀composed฀of฀two฀polypeptides฀as฀in฀type฀I฀procollagen฀
[a(I)1]2,a(I)2,฀ which฀ is฀ encoded฀ as฀ 2฀ genes฀ (COL1A1฀ +฀ COL1A2),฀ or฀ of฀ three฀ different฀
polypeptides฀ as฀ in฀ some฀ type฀ IV฀ procollagens,฀ a(IV)3,a(IV)4,a(IV)5฀ encoded฀ as฀ 3฀ genes฀
(COL4A3฀+฀COL4A4฀+฀COL4A5).฀The฀different฀polypeptide฀sequences฀of฀the฀different฀
collagen฀types฀therefore฀give฀rise฀to฀different฀structures.

4.3.2.
Fiber-Modifying Non-fibrillar Collagens

Only฀ive฀procollagens:฀types฀1,฀II,฀III,฀V฀and฀XI,฀are฀processed฀to฀tropocollagen.฀Type฀II฀
ibers฀are฀unique฀to฀cartilage฀and฀are฀limited฀in฀thickness฀by฀complexing฀with฀types฀XI฀and฀
type฀IX฀collagens฀(Table฀3.1).฀Other฀collagen฀types฀are฀expressed฀in฀small฀amounts฀and฀they฀
inluence฀the฀iber฀thickness฀and฀shape฀of฀type฀I฀collagen฀,฀or฀anchor฀a฀group฀of฀ibers฀to฀each฀
other฀and฀the฀surrounding฀tissues.
Type฀III฀ibers฀(fetal,฀reticular฀and฀vascular฀collagen)฀are฀delicate฀compared฀with฀type฀I฀
ibers.฀In฀the฀fetus,฀type฀III฀collagen฀is฀incorporated฀within฀the฀type฀I฀collagen฀to฀impart฀the฀
greater฀lexibility฀critical฀for฀fetal฀development.฀After฀birth,฀the฀delicate฀type฀III฀collagen฀
ibers฀contribute฀to฀reticular฀ibers฀and฀also฀type฀I฀collagen฀ibers฀that฀are฀present฀in฀cardio-
vascular฀and฀lymphoid฀tissues฀and฀also฀beneath฀epithelial฀basal฀cell฀layers,฀muscles,฀and฀
nervous฀tissue฀Schwann฀cells.
56 4 Fibrillar and Non-fibrillar Collagens and Integrins

In฀the฀cornea฀and฀lens฀of฀the฀eye,฀the฀presence฀of฀small฀amounts฀of฀type฀V฀collagen฀
around฀type฀I฀ibrils฀adjusts฀their฀orientation฀so฀that฀they฀are฀translucent.฀In฀developing฀
long฀ bones,฀ a฀ network฀ collagen,฀ type฀ X,฀ is฀ induced฀ to฀ attract฀ blood฀ vessels฀ that฀ invade฀
cartilage฀prior฀to฀its฀transformation฀into฀bone฀(Sect.฀9.2.2.).฀The฀blood฀vessels฀bring฀osteo-
blasts,฀which฀replace฀the฀resorbed฀type฀II฀collagen฀of฀the฀cartilage฀with฀type฀I฀collagen.฀In฀
children฀and฀adolescents,฀the฀site฀of฀this฀replacement฀and฀growth,฀the฀epiphyseal฀growth฀
plate,฀is฀almost฀exclusively฀composed฀of฀type฀X฀collagen.

4.3.3.
General Structure of Non-fibrous Collagens

Non-ibrillar฀collagens฀are฀composed฀of฀polypeptides฀that฀have฀sequences฀of฀glycine,฀proline฀
and฀hydroxyproline฀residues฀as฀in฀ibrous฀collagens฀and฀also฀form฀a฀triple฀helical฀monomeric฀
unit.฀However,฀the฀helical฀region฀is฀shorter฀or฀interrupted฀and฀the฀C-฀and฀N-terminal฀propep-
tides,฀except฀for฀the฀short฀N-terminal฀secretory฀signal฀sequence฀(pre-propeptide฀sequence),฀are฀
not฀removed.฀As฀in฀ibrous฀collagens,฀the฀propeptides฀possess฀a฀non-collagenous฀(NC)฀struc-
ture฀and,฀in฀addition฀to฀controlling฀triple฀helix฀formation,฀they฀control฀how฀the฀triple฀helical฀
units฀ (protomers)฀ aggregate.฀ The฀ non-ibrillar฀ collagens฀ form฀ basal฀ laminas฀ (i.e.฀ basement฀
membranes),฀anchors,฀and฀microibrils฀(Table฀3.1).฀The฀N฀and฀C-terminal฀regions฀of฀the฀triple฀
helices฀are฀not฀cleaved฀off฀like฀ibrous฀collagens,฀nor฀are฀they฀฀฀cross-linked฀by฀oxidized฀lysine฀
residues.฀Indeed,฀the฀major฀cross-links฀found฀in฀types฀IV,฀VII฀and฀XVII฀collagens฀are฀mediated฀
by฀cysteine-disulide฀bonding,฀mostly฀within฀or฀close฀to฀the฀C-terminal฀propeptide฀regions.฀
Some฀relevant฀non-ibrous฀collagen฀structures฀are฀discussed฀in฀Chapter฀5.

4.3.4.
Beaded Collagen Filaments

Microibrillar฀collagen฀(type฀VI)฀and฀elastic฀microibrils฀called฀oxytalan฀ibers฀(described฀
in฀Sect.฀3.1.3)฀are฀beaded฀ilaments฀(sometimes฀called฀microibers).฀These฀two฀types฀of฀
beaded฀ilaments฀in฀the฀connective฀tissue฀stroma฀are฀collagen฀type฀VI,฀and฀ibrillin฀with฀or฀
without฀elastin.฀Collagen฀type฀VI฀ilaments฀are฀non-stretchable,฀whereas฀ibrillin฀ilaments฀
are฀stretchable.฀Type฀VI฀collagen฀forms฀small฀diameter฀beaded฀microilaments฀that฀inter-
weave฀among฀large฀collagen฀ibers฀by฀crossing฀the฀gap฀regions฀of฀the฀type฀I฀and฀type฀III฀
ibers฀ (Table฀ 4.2).฀ These฀ ilaments฀ strengthen฀ collagen฀ iber฀ resistance฀ to฀ mechanical฀
forces฀beneath฀the฀dermis฀and฀also฀around฀arteries฀and฀capillaries฀(Sects.฀3.1.3฀and฀11.1.1).฀
The฀collagen฀฀ilaments฀are฀composed฀of฀three฀separately฀encoded฀a-polypeptides฀with฀
similar฀length฀triple฀helical฀domains฀but฀different฀sizes฀of฀N-฀and฀C-terminal฀non-helical฀
domains฀(illustrated฀at฀the฀top฀of฀Fig.฀4.10).฀It฀is฀commonly฀observed฀as฀a฀microibril฀with฀
a฀ double-beaded฀ period฀ of฀ about฀ 100฀ nm฀ due฀ to฀ aggregated฀ non-collagenous฀ C-฀ and฀
N-terminal฀domains.฀The฀formation฀of฀a฀typical฀collagenous฀beaded฀ilament฀is฀illustrated฀
in฀Fig.฀4.10฀and฀its฀legend.
4.3.4. Beaded Collagen Filaments 57

Polypeptides
a1 N1 C1 C2

a2 N1 C1 C2

a3 N10 N9 N8 N7 N6 N5 N4 N3 N2 N1 C1 C2 C3 C4 C5

‘A’ domains Proline-rich Fibronectin Kunitz protease

N1 - N10; C1 and C2 repeat (C3) type III repeat inhibitor domain
(C4) (C5)

Assembly

N-ter Monomer
C-ter
Antiparallel
Dimer
Triple helical region
(monomer is a polypeptide trimer.)

Tetramer

Beaded miocrofibril filament

Fig. 4.10฀ Beaded฀ilaments฀of฀type฀VI฀collagen.฀Top:฀Diagram฀of฀the฀3฀a-polypeptides฀with฀their฀

central฀ collagenous฀ domain฀ (wavy฀ line)฀ connecting฀ non-collagenous฀ N-฀ and฀ C-terminal฀ regions฀
squares฀indicating฀the฀various฀domains฀in฀the฀N-฀and฀C-฀terminal฀non-collagenous฀domain.฀The฀
non-collagenous฀N-฀and฀C-terminal฀domains฀(C1฀and฀C2)฀are฀Von฀Willebrand฀Factor฀(VWF)฀“A”฀
domains฀ (Fig.฀ 11.2).฀ Domains฀ C3,฀ C4,฀ and฀ C5฀ are฀ unrelated฀ domains฀ described฀ in฀ the฀ igure.฀
Bottom:฀Assembly฀from฀a฀monomeric฀protomer,฀a฀collagen฀triple฀helix฀with฀a฀massive฀non-collag-
enous฀domain฀at฀each฀end฀(bar-bell฀model).฀The฀helical฀region฀is฀interrupted,฀allowing฀the฀triple฀
helical฀domain฀to฀twist฀into฀a฀segmented฀super-coil฀(not฀shown).฀The฀N-฀and฀C-terminal฀ends฀adhere฀
to฀each฀other฀to฀form฀an฀anti-parallel฀dimer.฀Two฀dimers฀adhere฀similarly฀to฀form฀a฀tetramer฀that฀
extends฀laterally฀such฀that฀only฀N-terminal฀ends฀adhere฀to฀C-terminal฀ends฀(bottom฀left).฀The฀mas-
sive฀ non-collagenous฀ domains฀ form฀ the฀ beaded฀ ilaments฀ and฀ are฀ stabilized฀ by฀ intra-and฀ inter-
molecular฀disulide฀bonds฀(From฀Fig.฀2฀in฀Knupp฀C฀and฀Squire฀JM฀(2005)฀“Molecular฀Packing฀in฀
Network-Forming฀Collagens.”฀Adv.฀Prot.฀Chem.฀70:375–403.฀Assembly฀is฀from฀Fig.฀3฀in฀Fauvel-
Lafève฀ F฀ (1999)฀ “Microibrils฀ from฀ the฀ arterial฀ subendothelium.”฀ Int.฀ Rev.฀ Cytol.฀ 188:1–40).฀
Figure฀was฀modiied฀by฀Dr฀Wirsig-Weichmann
58 4 Fibrillar and Non-fibrillar Collagens and Integrins

The฀ collagen฀ superfamily฀ has฀ two฀ major฀ classes,฀ ibrillar฀ and฀ non-ibrillar,฀ based฀ on฀
their฀gene฀sequences฀and฀how฀the฀polypeptides฀are฀processed฀and฀assembled.฀There฀are฀
more฀than฀27฀collagen฀genes,฀of฀which฀only฀ive฀are฀ibrillar.฀The฀ibrillar฀collagens,฀
especially฀type฀I฀are฀the฀most฀common.฀Although฀each฀monomeric฀unit฀is฀composed฀of฀
a฀ speciic฀ tropocollagen฀ type,฀ a฀ mixture฀ of฀ different฀ types฀ inluences฀ assembly฀ in฀ a฀
given฀tissue.฀The฀non-helical฀N-฀and฀C-terminal฀regions฀are฀removed฀from฀all฀ibrillar฀
collagens,฀but฀remain฀intact฀or฀mostly฀intact฀in฀all฀non-ibrillar฀collagens.฀Microibrillar฀
collagen฀(type฀VI)฀and฀elastic฀microibrils฀called฀oxytalan฀ibers฀are฀beaded฀ilaments฀
or฀microibers.฀Beaded฀collagen฀ibers฀composed฀of฀type฀VI฀collagen฀strengthen฀larger฀
type฀I฀and฀type฀III฀collagen฀ibers฀in฀the฀dermis฀and฀around฀arteries฀and฀capillaries฀by฀
binding฀at฀the฀gap฀regions฀of฀the฀quarter-staggered฀array฀of฀the฀latter฀collagens.฀The฀
large฀non-collagenous฀N-฀and฀C-terminal฀regions฀of฀type฀VI฀collagen฀interact฀with฀the฀
triple฀helices฀and฀aggregate฀into฀antiparallel฀tetramers.฀The฀tetramers฀form฀beaded฀ila-
ments฀by฀end-to-end฀aggregation.

4.4.1.
Integrins and Regulation of Stromal Composition

Integrins฀are฀a฀family฀of฀transmembrane฀proteins฀that฀control฀when฀a฀cell฀expresses฀connec-
tive฀tissue฀matrix฀components,฀or฀responds฀to฀environmental฀stresses.฀Integrins฀are฀heterodi-
meric฀receptors฀composed฀of฀one฀a-฀and฀one฀b-polypeptide.฀Each฀heterodimer฀is฀a฀single฀
molecule฀with฀two฀sites:฀an฀extracellular฀receptor฀site฀and฀an฀intracellular฀signaling฀site.฀The฀
former฀is฀the฀N-terminal฀head฀region฀which฀binds฀to฀extracellular฀proteins฀such฀as฀collagen,฀
ibronectin฀or฀laminin.฀The฀latter฀is฀the฀C-terminal฀tail฀region฀which฀remains฀on฀the฀cytoso-
lic฀ side฀ of฀ the฀ membrane฀ and฀ interacts฀ with฀ cytoplasmic฀ effectors.฀ Integrin฀ signaling฀ is฀
passed฀to฀the฀cell฀by฀extracellular฀ligands฀(outside-in฀signal),฀or฀from฀the฀cell฀by฀intracel-
lular฀ligands฀(inside-out฀signal).฀Human฀integrins฀are฀selected฀from฀one฀of฀18฀a-polypep-
tides฀and฀one฀of฀8฀b-polypeptides,฀although฀only฀24฀of฀the฀144฀combinations฀exist฀(Fig.฀4.11).฀
The฀b-polypeptides฀(~90฀kDa)฀are฀smaller฀than฀the฀a-polypeptides฀(~130฀kDa).
Flexible฀domains฀inluence฀whether฀an฀extracellular฀ligand฀will฀bind฀to฀an฀integrin.฀The฀
domains฀are฀listed฀and฀illustrated฀in฀Fig.฀4.12a.฀Integrins฀exist฀in฀three฀major฀confor฀mations฀
(Fig.฀ 4.12b):฀ ฀ i)฀ low-afinity฀ (inactive);฀ ii)฀ ligand-bound฀ (activated)฀ or฀ iii)฀ high-afinity฀
(primed).฀The฀change฀from฀inactive฀to฀activated฀conformation฀is฀mediated฀by฀a฀large฀global฀
rearrangement฀ whereby฀ the฀ integrin฀ subunits฀ extend฀ with฀ a฀ sharp,฀ ‘switchblade’-
like฀motion.฀A฀‘b-propeller’฀domain฀(Fig.฀12a)฀is฀N-terminal฀in฀about฀half฀of฀the฀a-poly-
peptides,฀but฀an฀additional฀domain฀extends฀from฀two฀blades฀of฀the฀propeller,฀the฀inserted฀
domain฀ (I-domain)฀ in฀ the฀ remainder.฀ The฀ I-domain฀ is฀ homologous฀ to฀ the฀ N-terminal฀
domain฀ of฀ the฀ b-polypeptide,฀ which฀ is฀ called฀ the฀ I-like฀ domain.฀ Four฀ of฀ the฀ 9฀ human฀
a-polypeptides฀possessing฀an฀I-domain฀(a1,฀a2,฀a10฀and฀a11)฀bind฀collagens฀when฀partnered฀
with฀a฀b1-polypeptide.
4.4.1. Integrins and Regulation of Stromal Composition 59

a1*
a10*
aL* aIIb
a11*
aE* b7
aM* a2*
b3
b2 a3
aX* b5
a4 b1 aV
b6
aD* a5
b8
b4 a6
a7
a8
a9

Fig. 4.11฀ Integrin฀subunit฀combinations.฀Integrin฀a-฀and฀b-subunits฀form฀24฀heterodimers฀that฀rec-

ognize฀distinct฀but฀overlapping฀ligands.฀Half฀of฀the฀a-subunits฀contain฀I-domains฀(asterisks).฀The฀
b2฀integrin฀subunit฀is฀also฀called฀CD18฀(Cell฀Differentiation฀antigen฀18)฀and฀its฀various฀a-subunits฀
are฀called฀CD11a,฀b,฀etc.฀The฀a-฀and฀b2-subunit฀and฀its฀partners฀are฀important฀in฀lymphocyte฀adhe-
sion฀and฀activation,฀as฀discussed฀in฀Sect.฀13.฀2.4.฀(Reprinted฀from฀Advances฀in฀Protein฀Chemistry,฀
Volume฀68,฀Springer,฀TA฀and฀Wang,฀J-H.,฀The฀three-dimensional฀structure฀of฀integrins฀and฀their฀
ligands,฀and฀conformational฀regulation฀of฀cell฀adhesion,฀pp฀29–63,฀2004,฀with฀permission฀from฀
Elsevier)

In฀integrins฀whose฀a-polypeptides฀do฀not฀possess฀an฀I-domain,฀the฀b-propeller฀domain฀
contacts฀the฀I-like฀domain฀of฀the฀b-subunit฀when฀the฀conformation฀is฀extended฀(Fig.฀4.13a).฀
Ligands฀ such฀ as฀ laminin฀ (Sect.฀ 5.1.1)฀ are฀ attracted฀ to฀ the฀ extracellular฀ integrin฀ surface฀
where฀the฀RGD฀aspartic฀acid฀residue฀in฀the฀consensus฀motif฀(Sect.฀3.2.1),฀or฀a฀glutamic฀
acid฀ within฀ other฀ integrin-binding฀ motifs,฀ open฀ a฀ metal฀ ion฀ dependent฀ adhesion฀ site฀
(MIDAS),฀exposing฀a฀divalent฀cation฀(usually฀Ca+2)฀within฀the฀I-like฀domain.฀Exposing฀the฀
cation฀moves฀the฀C-terminal฀helix฀of฀the฀I-like฀domain฀down฀(white฀arrow฀in฀Fig.฀4.13aii)฀
which฀stabilizes฀the฀integrin-ligand฀complex฀by฀causing฀an฀outward฀swing฀of฀the฀hybrid฀
domain฀of฀the฀b-polypeptide฀(curved฀black฀arrow฀in฀Fig.฀4.13aii).
In฀a-polypeptides฀possessing฀an฀I-domain,฀the฀mechanism฀is฀more฀complex฀because฀the฀
I-domain฀irst฀interacts฀with฀the฀I-like฀domain฀to฀stabilize฀a฀binding฀site฀that฀is฀not฀RGD-
dependent.฀Contact฀with฀collagen฀(or฀another฀ligand)฀activates฀a฀glutamic฀acid฀residue฀in฀a฀
consensus฀motif฀on฀the฀ligand฀to฀expose฀the฀I-domain’s฀MIDAS฀site฀containing฀Mg2+฀or฀
Mn2+.฀ The฀ C-terminal฀ helix฀ of฀ the฀ I-domain฀ is฀ pushed฀ down฀ (upper฀ white฀ arrow฀ in฀
Fig.฀3.13bii),฀exposing฀an฀acidic฀amino฀acid฀that฀binds฀to฀a฀Ca+2฀ion฀in฀the฀MIDAS฀site฀of฀
the฀I-like฀domain฀(lower฀white฀arrow฀in฀Fig.฀4.13bii).฀The฀b-polypeptide’s฀hybrid฀domain฀
then฀swings฀out฀as฀in฀integrins฀without฀an฀I-domain฀(curved฀black฀arrow).
Cytoplasmic฀effectors฀can฀bring฀the฀cytoplasmic฀ends฀closer,฀closing฀the฀metal฀binding฀
site฀of฀the฀I-฀or฀I-like฀domain฀and฀releasing฀the฀ligand.฀If฀effectors฀bring฀the฀cytoplasmic฀
domain฀ still฀ closer,฀ they฀ cause฀ the฀ integrin฀ to฀ collapse฀ to฀ the฀ bent฀ state฀ (Fig.฀ 4.12bi).฀
Conversely,฀ other฀ cytoplasmic฀ effectors฀ can฀ promote฀ integrin฀ binding฀ to฀ extracellular฀
ligands฀by฀causing฀the฀cytoplasmic฀ends฀to฀move฀apart.
60 4 Fibrillar and Non-fibrillar Collagens and Integrins

Fig. 4.12฀ Domain฀structure฀and฀integrin฀conformations.฀(a)฀The฀a-฀and฀b-polypeptide฀domains.฀In฀

a-polypeptides,฀the฀N-terminus฀is฀a฀b-propeller฀domain฀(red)฀made฀up฀from฀seven฀b-sheets฀inter-
woven฀into฀a฀seven฀bladed฀propeller,฀or฀an฀inserted฀domain฀(I-domain;฀brown)฀that฀extends฀out฀
from฀the฀N-terminal฀β-propeller฀(see฀text).฀Beneath฀the฀propeller฀are฀the฀thigh฀domain฀(maroon),฀
and฀two฀leg฀domains,฀calf฀1฀(orange)฀and฀calf฀2฀(yellow).฀The฀region฀between฀the฀thigh฀and฀calf฀1฀
domains฀is฀lexible฀and฀the฀thigh฀and฀propeller฀domains฀(with฀or฀without฀an฀I-domain)฀comprise฀
the฀a-polypeptide฀head฀region.฀In฀β-polypeptides,฀the฀N-terminal฀domain฀is฀homologous฀to฀the฀
I-domain฀(I-like฀domain;฀purple).฀The฀I-like฀domain฀is฀attached฀to฀a฀hybrid฀fold฀related฀to฀a฀fold฀in฀
immunoglobulins฀(dark฀blue),฀followed฀by฀4฀cysteine฀rich฀EGF฀domains฀(shades฀of฀blue)฀similar฀
to฀those฀in฀ibrillin฀(Sect.฀6.1.1)฀but฀not฀calcium-ion฀binding.฀The฀EGF฀domains฀are฀followed฀by฀a฀
b฀tail฀domain,฀and฀a฀single฀transmembrane฀helix฀that฀anchors฀each฀polypeptide฀within฀the฀lipid฀
membrane฀bilayer.฀Between฀the฀hybrid฀and฀irst฀EGF฀domain฀of฀the฀b-polypeptide฀is฀a฀lexible฀
region,฀ the฀ plexin-semaphorin-integrin฀ (PSI)฀ region,฀ named฀ for฀ common฀ sequence฀ homologies.฀
The฀I-like฀and฀hybrid฀domains฀form฀the฀b-polypeptide฀head฀region.฀The฀respective฀domain฀struc-
tures฀are฀stabilized฀by฀intra-chain฀disulide฀bonding.฀Both฀polypeptides฀possess฀a฀C-terminal฀intra-
cellular฀ region฀ that฀ interacts฀ with฀ cytoplasmic฀ signaling฀ components.฀ (b)฀ Conformations฀ of฀ the฀
alpha฀ (a)฀ and฀ beta฀ (b)฀ subunits.฀ Integrins฀ possess฀ bent,฀ partially฀ extended฀ and฀ fully฀ extended฀
conformations฀analogous฀to฀those฀of฀an฀almost-closed,฀partially฀open,฀and฀fully฀open฀switchblade฀
4.4.1. Integrins and Regulation of Stromal Composition 61

The฀collagen฀binding฀site฀for฀integrins฀is฀a฀short฀lexible฀region฀(Gly-X-X-Gly-Glu-Arg฀
where฀X฀is฀any฀amino฀acid).฀This฀motif฀is฀conserved฀in฀all฀ibrous฀and฀some฀non-ibrous฀
collagens,฀especially฀types฀IV฀and฀VI.฀Different฀amino฀acid฀sequences฀(at฀the฀N-terminus฀
of฀each฀integrin฀α-polypeptide)฀determine฀which฀type฀of฀collagen฀will฀bind.฀For฀instance,฀
the฀I-domain฀of฀a2-฀or฀a10-polypeptides฀attaches฀ibrillar฀collagens,฀whereas฀the฀I-domain฀
of฀a1-฀or฀a11-polypeptides฀attaches฀non-ibrillar฀collagens.฀Each฀of฀these฀integrin฀α฀sub-
units฀have฀different฀cytoplasmic฀partners฀that฀result฀in฀different฀cell฀responses฀when฀the฀
appropriate฀ligand฀binds.฀Fibroblasts฀secreting฀collagen฀are฀long฀and฀thin฀and฀do฀not฀divide฀
whereas฀dividing฀ibroblasts฀are฀rounded฀up฀and฀release฀any฀integrin-bound฀stromal฀pro-
teins.฀ This฀ difference฀ is฀ mediated฀ by฀ growth฀ factors฀ or฀ by฀ small฀ cell-signaling฀ proteins฀
called฀cytokines฀that฀produce฀ligands฀that฀determine฀whether฀the฀cytosolic฀ends฀of฀each฀
integrin฀polypeptide฀can฀associate.฀Collagen฀ibers฀are฀synthesized฀only฀when฀the฀cell฀can-
not฀divide.
A฀ few฀ integrin฀ a-฀ and฀ β-polypeptides฀ bind฀ multiple฀ ligands.฀ For฀ instance฀ the฀ aMb2฀
integrin฀(Mac-1฀receptor฀of฀neutrophils฀and฀macrophages฀(Sect.฀13.2.4)฀has฀a฀ligand฀bind-
ing฀region฀(Lys245฀–฀Arg261)฀that฀can฀attach฀more฀than฀30฀different฀proteins฀including฀
laminin,฀collagen฀ibers฀and฀the฀ibrin฀of฀clotted฀blood฀(Sect.฀11.3.1).฀Mice฀made฀deicient฀
in฀integrin฀α-polypeptides฀that฀bind฀laminin฀and฀some฀other฀connective฀tissue฀components฀
do฀not฀survive.฀However,฀mice฀deicient฀in฀a1฀or฀a2฀subunits฀appear฀normal฀and฀only฀exhibit฀
a฀defective฀ibroblast฀response฀to฀injury.฀Table฀4.3฀lists฀the฀integrins฀that฀bind฀to฀collagen.฀

Table 4.3฀ Functions฀of฀฀collagen฀binding฀integrins

Integrin Function
α1β1 induces฀ibroblast฀proliferation฀and฀decreases฀collagen฀synthesis฀in฀
response฀to฀cytokines฀in฀angiogenesis,฀ibrosis,฀chronic฀inlammation฀
and฀bone฀healing
α2β1 stromal฀protease฀activation฀(remodeling)฀in฀response฀to฀stress฀or฀injury฀
with฀de฀novo฀synthesis฀of฀both฀collagenase฀and฀collagen
α10β1 expressed฀on฀chondrocytes฀and฀inluences฀cartilage฀maturation
α11β1 skeletal฀development฀around฀vertebral฀cartilages฀and฀ibers

knife.฀i.฀ Bent฀(low฀afinity).฀The฀region฀between฀the฀calf฀1฀and฀thigh฀region฀of฀the฀α-polypeptide฀
(arrows)฀bends฀along฀with฀the฀PSI฀region฀of฀the฀b-polypeptide.฀Both฀polypeptide฀head฀regions฀lose฀
ligand฀binding฀and฀face฀the฀calf฀2฀domain.฀The฀intracellular฀(C-terminal)฀regions฀of฀the฀a-฀and฀
b-polypeptides฀overlap.฀ii.฀Partially฀extended฀(closed฀head;฀intermediate฀afinity).฀The฀two฀intrac-
ellular฀regions฀partially฀separate,฀the฀a-polypeptide฀calf฀1฀to฀thigh฀connection฀(arrows)฀straightens฀
up฀and฀the฀b-propeller฀of฀the฀a-polypeptide฀(red)฀pulls฀up฀the฀I-like฀domain฀of฀the฀b-polypeptide฀
(purple)฀ straightening฀ the฀ PSI฀ domain.฀ ฀ iii.฀ Fully฀ extended฀ (open฀ head;฀ high฀ afinity).฀ The฀ PSI฀
region฀of฀the฀b-polypeptide฀rotates฀around฀the฀around฀the฀a-polypeptide฀calf฀domains.฀The฀move-
ment฀opens฀the฀binding฀site฀and฀also฀completely฀separates฀the฀two฀intracellular฀regions฀(Fig.฀4.13).฀
Fig฀is฀based฀on฀Fig.฀1฀in฀Carman฀CV,฀Springer฀TA฀(2003฀Oct)฀“Integrin฀avidity฀regulation:฀are฀
changes฀in฀afinity฀and฀conformation฀underemphasized?”฀Current฀Opinion฀in฀Cell฀Biology,฀15(5):฀
547฀–฀556.฀(Modiications฀were฀by฀Dr฀Wirsig-Weichmann)
62 4 Fibrillar and Non-fibrillar Collagens and Integrins

Fig. 4.13฀ Ligand-integrin฀receptor฀interactions.฀Only฀the฀head฀regions฀are฀shown.฀The฀presence฀or฀

absence฀ of฀ an฀ I-domain฀ in฀ the฀ a-polypeptide฀ affects฀ the฀ mode฀ of฀ receptor฀ activation.฀ (a)฀ I-like฀
domain฀ receptor.฀ (i)฀ The฀ propeller฀ domain฀ of฀ the฀ a-polypeptide฀ (red)฀ interacts฀ with฀ the฀ I-like฀
domain฀of฀the฀b฀polypeptide฀(purple)฀and฀the฀MIDAS฀cation฀making฀up฀the฀ligand฀receptor฀site฀
(see฀text),฀but฀the฀site฀remains฀closed.฀(ii)฀An฀acidic฀amino฀acid฀in฀the฀ligand฀causes฀a฀downward฀
displacement฀ of฀ the฀ seventh฀ and฀ most฀ C-terminal฀ a-helix฀ within฀ the฀ I-like฀ domain฀ (a-7฀ helix),฀
opening฀the฀ligand฀binding฀site฀and฀stabilizing฀it฀with฀an฀outward฀movement฀of฀the฀hybrid฀domain฀
of฀the฀β-polypeptide฀(curved฀black฀arrow).฀(b)฀I-domain฀receptor.฀(ii)฀A฀MIDAS฀site฀cation฀in฀the฀
I-domain฀interacts฀with฀the฀I-like฀domain฀to฀open฀and฀stabilize฀the฀ligand฀binding฀site.฀In฀these฀
integrins,฀the฀I-domain฀MIDAS฀possesses฀Mg+2฀or฀Mn+2,฀and฀the฀I-like฀domain฀MIDAS฀possesses฀
Ca+2฀(see฀text).฀(ii)฀The฀ligand-binding,฀high-afinity฀conformation฀of฀the฀I-domain฀is฀opened฀by฀
ligand฀binding฀(irst฀downward-pointing฀white฀arrow),฀and฀stabilized฀by฀a฀downward฀displacement฀
of฀the฀C-terminal฀a-7฀helix฀containing฀an฀acidic฀residue฀that฀binds฀to฀the฀Ca+2฀ion฀in฀the฀I-like฀
domain฀(second฀downward-pointing฀white฀arrow).฀This฀movement฀stabilizes฀the฀I-like฀domain฀by฀
attaching฀it฀to฀the฀propeller฀domain฀along฀with฀an฀outward฀movement฀of฀the฀hybrid฀domain฀of฀the฀
β-polypeptide฀(curved฀arrow฀as฀in฀a).฀Fig฀is฀based฀on฀Fig.฀1฀in฀Carman฀CV,฀Springer฀TA฀(2003฀Oct)฀
“Integrin฀avidity฀regulation:฀are฀changes฀in฀afinity฀and฀conformation฀underemphasized?”฀Current฀
Opinion฀in฀Cell฀Biology,฀15(5):฀547–556.฀(Modiications฀were฀by฀Dr฀Wirsig-Weichmann)
4.4.1. Integrins and Regulation of Stromal Composition 63

Despite฀all฀possessing฀the฀b1฀subunit฀and฀a฀limited฀range฀of฀a฀subunits,฀the฀effects฀on฀col-
lagen฀synthesis฀and฀degradation฀vary฀greatly.฀Integrin฀a11฀is฀also฀strongly฀expressed฀with฀
β-subunits฀other฀than฀b1฀in฀mature฀tendon฀and฀ligament฀cells,฀but฀the฀signaling฀properties฀
of฀this฀integrin฀combination฀are฀as฀yet฀uncertain.

Integrins฀are฀composed฀of฀transmembrane฀heterodimers฀with฀an฀N-terminal,฀extracel-
lular฀binding฀site฀for฀a฀ligand฀such฀as฀laminin฀or฀collagen,฀and฀a฀C-terminal฀intracellular฀
binding฀site฀for฀a฀cytosolic฀activating฀or฀deactivating฀proteins.฀The฀ligand฀binding฀site฀
is฀ activated฀ by฀ a฀ “switch-blade”฀ change฀ in฀ conformation,฀ from฀ bent฀ to฀ activated,฀ to฀
primed.฀Presentation฀of฀the฀binding฀site฀is฀determined฀by฀stromal฀cytokines฀and฀growth฀
factors฀that฀inluence฀cytosolic฀factors,฀or฀by฀intracellularly฀induced฀cytosolic฀factors.฀
Binding฀to฀ligand:฀(1)฀strengthens฀cellular฀attachment฀to฀the฀extracellular฀matrix;฀(2)฀
induces฀ speciic฀ changes฀ in฀ ibroblast฀ metabolism;฀ and฀ (3)฀ affects฀ growth฀ factor฀ and฀
cytokine฀ signaling฀ involved฀ in฀ mesenchymal฀ tissue฀ development,฀ maintenance,฀ and฀
repair.฀Changes฀in฀the฀cell฀environment฀mediate฀changes฀in฀the฀approximation฀of฀the฀
cytosolic฀ends฀of฀the฀heterodimers฀that฀close฀or฀open฀the฀“switchblade.”
 Basal Laminas and Epithelia
5

This฀chapter฀describes฀the฀organization฀of฀the฀major฀proteins฀that฀form฀a฀basal฀lamina฀
which฀connects฀an฀epithelium฀to฀its฀underlying฀stroma฀(Sect.฀1).฀The฀organization฀and฀
major฀protein฀composition฀of฀oral฀and฀gingival฀epithelium฀and฀the฀junctional฀epithelial฀
dental฀attachment฀at฀the฀base฀of฀a฀gingival฀sulcus฀are฀described฀(Sect.฀2).

5.1.1.
Basal Lamina and its Stromal Attachment

An฀epithelium฀such฀as฀the฀skin฀or฀oral฀mucosa฀is฀separated฀from฀the฀underlying฀stroma฀by฀฀
a฀basal฀lamina.฀The฀basal฀lamina฀is฀derived฀from฀both฀epithelium฀and฀connective฀tissue,฀but฀
its฀major฀components฀are฀induced฀from฀connective฀tissue฀ibroblasts฀by฀the฀overlying฀epi-
thelial฀cells.฀All฀basal฀laminas฀consist฀of฀two฀parts฀(Fig.฀5.1):฀a฀lightly฀staining฀layer฀con-
taining฀laminin,฀the฀lamina฀lucida฀which฀contacts฀basal฀epithelial฀cells;฀and฀an฀intensely฀
staining฀layer฀beneath฀that฀contains฀type฀IV฀collagen฀(lamina฀densa)฀and฀contacts฀the฀col-
lagens฀and฀other฀proteins฀of฀the฀stroma.
Laminins฀ are฀ composed฀ of฀ three฀ polypeptides,฀ a,฀ b,฀ and฀ g฀ that฀ are฀ homologous฀ and฀
encoded฀by฀separate฀genes฀in฀the฀mammalian฀genome.฀What฀genes฀are฀expressed฀is฀deter-
mined฀by฀tissue฀type.฀There฀are฀ive฀different฀฀types฀of฀a฀chain,฀three฀types฀of฀b฀chain,฀and฀
three฀types฀of฀g฀chain,฀but฀fewer฀laminins฀than฀integrins฀(Sect.฀4.4.1).฀The฀major฀laminin฀in฀
the฀lamina฀lucida฀is฀laminin-1฀(1a,1b,1g;฀Fig.฀5.2)฀which฀is฀secreted฀by฀ibroblasts,฀but฀its฀
minor฀ laminin,฀ laminin-5฀ (5a1b1g)฀ is฀ secreted฀ by฀ epithelial฀ cells.฀ The฀ nomenclature฀ for฀
laminins฀ was฀ recently฀ changed฀ to฀ indicate฀ which฀ of฀ the฀ alpha,฀ beta฀ and฀ gamma฀ chains฀
are฀used฀in฀a฀particular฀laminin.฀Thus,฀the฀two฀laminins฀referred฀to฀above฀are฀now฀written฀
laminin-111฀ and฀ laminin-511.฀ The฀ older฀ laminin฀ ฀ nomenclature฀ is฀ used฀ in฀ this฀ book.฀
Laminin-1฀ assembles฀ into฀ a฀ clear,฀ web-like฀ polymer฀ (Fig.฀ 5.2)฀ above฀ the฀ lamina฀ densa,฀
whereas฀laminin-5฀forms฀long,฀thin฀strands฀that฀pass฀through฀the฀lamina฀lucida฀and฀bind฀to฀
type฀IV฀collagen฀in฀the฀lamina฀densa.฀A฀few฀laminin-5฀strands฀pass฀right฀through฀lamina฀
lucida฀and฀lamina฀densa฀and฀become฀attached฀to฀the฀head฀region฀of฀type฀VII฀collagen฀anchor-
ing฀ibrils฀(see฀end฀of฀Sect.฀5.1.2).
Type฀ VII฀ collagen฀ is฀ a฀ non-ibrous,฀ anchoring฀ ibril฀ that฀ binds฀ large฀ type฀ I฀ collagen฀
ibers฀in฀the฀stroma฀to฀the฀lamina฀densa฀section฀of฀the฀basal฀lamina.฀Type฀VII฀procollagen฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 65
DOI:฀10.1007/978-3-540-88116-2_5,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
66 5 Basal Laminas and Epithelia

Fig. 5.1฀ Diagram฀of฀a฀basal฀lamina.฀The฀various฀components฀are฀shown฀with฀reference฀to฀histo-

logical฀markers฀(bold)฀and฀their฀major฀biochemical฀components฀(small฀print).฀The฀lamina฀lucida฀
and฀lamina฀densa฀are฀the฀light฀blue฀and฀dark฀green฀regions฀beneath฀a฀basal฀cell฀(epithelial฀or฀
epidermal฀ cell).฀ The฀ lamina฀ lucida฀ is฀ usually฀ composed฀ of฀ a฀ laminin-1฀ network฀ (light฀ blue)฀
within฀ilaments฀of฀laminin-5฀(white)฀(From฀Fig.฀4฀in฀Giehl฀KA,฀Ferguson,฀DJP,฀Dean฀D,฀Chuang,฀
YH,฀Allen฀J,฀De฀Berker,฀DAR,฀Tosti,฀A.,฀Dawber฀RPR฀and฀F.Wojnarowska,฀F.฀Alterations฀in฀the฀
basement฀membrane฀zone฀in฀pili฀annulati฀hair฀follicles฀as฀demonstrated฀by฀electron฀microscopy฀
and฀ immunohistochemistry.฀ British฀ Journal฀ of฀ Dermatology,฀ 150:722–727,฀ 2004.฀ With฀ copy-
right฀ permission฀ from฀ Wiley-Blackwell,฀ PO฀ Box฀ 805,฀ 9600฀ Garsington฀ Road,฀ Oxford฀ OX4฀
2DQ,฀UK,฀as฀modiied฀by฀Dr฀Wirsig-Wiechmann.)

has฀a฀145-kDa฀N-terminal฀non-collagenous฀(NC1)฀domain,฀an฀extended฀central,฀triple-
helical฀domain,฀and฀a฀short฀34-kDa฀non-collagenous฀C-terminal฀(NC2)฀domain฀(Fig.฀5.3a).฀
The฀contribution฀of฀type฀VII฀procollagen฀฀to฀the฀mechanical฀stability฀of฀the฀dermal–epi-
dermal฀junction฀depends฀on฀the฀ability฀of฀single฀molecules฀to฀self-assemble฀into฀highly฀
ordered฀ anchoring฀ ibrils.฀ On฀ secretion,฀ some฀ of฀ the฀ C-terminal฀ NC2฀ domain฀ is฀ prote-
olytically฀removed฀and฀cysteine฀cross-links฀form฀triple฀helical฀reverse฀dimers฀(Fig.฀5.3b).฀
At฀either฀end฀of฀a฀dimer,฀the฀large฀NC1฀domain฀can฀interact฀with฀type฀IV฀collagen,฀type฀I฀
collagen,฀or฀laminin-5.฀The฀interaction฀with฀type฀I฀collagen฀ibers฀may฀promote฀both฀type฀
VII฀dimerization฀and฀ibril฀formation.฀As฀type฀VII฀ibrils฀form,฀the฀stromal฀ends฀bind฀to฀
type฀IV฀collagen฀anchoring฀plaques฀deep฀within฀the฀stroma฀(Fig.฀5.1).
Type฀ IV฀ collagen฀ is฀ responsible฀ for฀ the฀ dense฀ network฀ that฀ characterizes฀ the฀ lamina฀
densa฀and฀anchoring฀plaques฀in฀all฀mammals.฀It฀is฀made฀up฀from฀two฀or฀three฀of฀six฀homol-
ogous฀ a-procollagen฀ type฀ IV฀ polypeptide฀ chains฀ (Fig.฀ 5.4a)฀ that฀ assemble฀ at฀ their฀
C-terminal฀procollagen฀domain฀into฀one฀of฀three฀heterotrimers฀called฀protomers฀(Fig.฀5.4b):฀
[a1(IV)]2[a2(IV)]฀ beneath฀ an฀ epidermis฀ such฀ as฀ the฀ gingival฀ mucosa฀ or฀ skin;฀ [a3(IV)]฀
[a4(IV)][a5(IV)]฀in฀the฀glomerulus฀of฀the฀kidney฀and฀lungs;฀and฀[a5(IV)]2[a6(IV)]฀around฀
smooth฀muscle฀cells.฀Each฀individual฀polypeptide฀has฀a฀long,฀collagenous฀domain฀฀(gly-X-Y฀
repeats)฀but฀฀frequent฀interruptions฀reduce฀rigidity.฀The฀C-terminal฀non-collagenous฀region฀
(NC1฀ region)฀ of฀ the฀ three฀ polypeptides฀ making฀ up฀ each฀ protomer฀ (Fig.฀ 5.4C)฀ links฀ the฀
C-terminal฀end฀of฀each฀pair฀of฀protomers฀(Fig.฀5.4D).
5.1.2. Hemidesmosomal Proteins 67

100 nm

α-Chain β-Chain γ-Chain

COOH

COOH

Globular Coiled-coil α-helical domain

domains

20 nm

Fig. 5.2฀ Structure฀of฀laminin.฀Laminin฀is฀composed฀of฀three฀homologous฀polypeptides฀(a,฀b,฀and฀g),฀

each฀more฀than฀1,500฀amino฀acids฀long.฀They฀contain฀many฀domains฀that฀come฀together฀in฀a฀central,฀
disulide-bonded,฀triple฀helical฀coiled-coil฀with฀globular฀ends.฀Each฀trimer฀spontaneously฀interacts฀
with฀ others฀ to฀ form฀ a฀ web-like฀ polymer฀ illustrated฀ in฀ the฀ electron฀ microscope฀ photomicrograph฀
(upper฀right).฀Laminin-1฀(a1,฀b1,฀g1)฀forms฀an฀asymmetric฀cross-linked฀structure฀shown฀on฀the฀
electron฀micrographs฀of฀laminin฀shadowed฀with฀platinum,฀whereas฀laminin-5฀(a5,฀b1,฀g1)฀forms฀a฀
ilament฀resembling฀a฀thin,฀barbell฀rod฀shown฀in฀Fig.฀5.6฀(Upper฀igure;฀Reprinted฀from฀J.฀Mol.฀Biol.฀
Vol.฀150,฀Shapes,฀domain฀organizations฀and฀lexibility฀of฀laminin฀and฀ibronectin,฀two฀multifunc-
tional฀proteins฀of฀the฀extracellular฀matrix.฀Engel฀J,฀Odermatt฀E,฀Engel฀A,฀Madri฀JA,฀Furthmayr฀H,฀
Rohde฀H,฀and฀Timpl฀R.฀Pages฀No.฀97–120,฀1981:฀with฀permission฀from฀Elsevier.฀Lower฀igure฀is฀
adapted฀ from฀ Fig.19-57฀ in฀ The฀ Molecular฀ Biology฀ of฀ the฀ Cell.฀ B.฀ Alberts฀ et฀ al.,฀ 4th฀ Ed.฀ 2002.฀
Garland฀Science,฀Taylor฀&฀Francis฀Group,฀New฀York)

5.1.2.
Hemidesmosomal Proteins

Hemidesmosomes฀(HDs)฀are฀membrane-associated฀adhesive฀junctions฀linked฀to฀the฀ila-
mentous฀networks฀of฀the฀epithelial฀cell฀cytoskeleton฀and฀the฀lamina฀lucida฀(฀light฀green/
dark฀blue฀region฀in฀Fig.฀5.1).฀The฀cytoskeleton฀of฀all฀mammalian฀cells฀is฀composed฀of฀three฀
kinds฀of฀ilaments:฀microilaments,฀intermediate฀ilaments฀and฀microtubules.฀Microilaments฀
68 5 Basal Laminas and Epithelia

Fig. 5.3฀ Dimeric฀structure฀of฀ a Procollagen VII triple helix

collagen฀type฀VII฀anchoring฀
C2634
ibrils.฀(a)฀Type฀VII฀ 1
collagen฀triple฀helix.฀The฀ NC1 Fibrillar triple helical region NC2
non-collagenous฀(NC)฀
N-฀and฀C-terminal฀ 2
procollagen฀domains฀฀ 3
N-terminal end C-terminal end
(NC1฀and฀NC2฀regions)฀are฀
Noncollagenous 1 Noncollagenous 2
respectively฀colored฀red฀and฀
green.฀(b)฀Fibril฀formation.฀
Antiparallel฀binding฀of฀two฀ Alignment of procollagen VII into cysteine
triple฀helices฀may฀be฀ b cross-linked hexamers (triple helical dimers)
orientated฀by฀each฀helix฀ C2634
binding฀to฀a฀type฀I฀collagen฀
iber฀followed฀by฀prote-
olytic฀cleavage฀within฀the฀ NC2
C-terminal฀non-collagenous฀
region฀(NC2฀region).฀The฀ NC2
cysteine฀at฀residue฀2634฀
aligns฀with฀a฀cysteine฀
residue฀near฀the฀cleaved฀ Cleavage of the NC2 domains
C-terminus฀so฀that฀two฀ C2634
triple฀helical฀regions฀are฀
correctly฀cross-linked฀to฀ 6
form฀a฀hexamer฀from฀which฀
a฀triple฀helix฀extends฀in฀
either฀direction฀(Modiied฀
from฀Figs.฀1฀and฀2฀in฀฀
R.฀Brittingham฀et฀al.฀฀
(2005฀Jan฀7)฀“Single฀amino฀ Formation of disulfide bonds
acid฀substitutions฀in฀ C2634
procollagen฀VII฀affect฀early฀
stages฀of฀assembly฀of฀
anchoring฀ibrils.”฀J.฀Biol.฀
Chem.฀280(1):191–198)

are฀thin฀and฀made฀of฀actin,฀whereas฀microtubules฀are฀thick฀and฀made฀of฀tubulin.฀Intermediate฀
ilaments฀are฀of฀intermediate฀size฀(~10฀nm฀thick)฀and฀they฀include฀keratins฀in฀epithelial฀
cells฀(which฀also฀secrete฀them฀to฀form฀hair฀and฀nails);฀nuclear฀lamins฀(which฀form฀a฀net-
work฀that฀stabilizes฀the฀inner฀membrane฀of฀the฀nuclear฀envelope);฀neuroilaments฀(which฀
strengthen฀the฀long฀axons฀of฀neurons);฀and฀vimentins฀(which฀provide฀mechanical฀strength฀
to฀ muscle฀ and฀ other฀ cells).฀ All฀ intermediate฀ ilaments฀ possess฀ a฀ pronounced฀ a-helix.฀
Keratins฀are฀described฀in฀detail฀in฀Sect.฀5.2.2).
Hemidesmosomes฀are฀made฀up฀of฀four฀proteins:฀two฀bullous฀pemphigoid฀(BP)฀antigens,฀
(BP180,฀an฀anchoring฀ibril฀identiied฀as฀type฀XVII฀collagen,฀and฀BP230,฀a฀plakin฀protein),฀
and฀two฀other฀proteins;฀plectin฀(a฀second฀plakin฀protein),฀and฀integrin฀a6b4.฀BP฀antigens฀
are฀proteins฀that฀function฀abnormally฀in฀the฀disease,฀epidermolysis฀bullosa฀(Sect.฀5.1.3.).฀
5.1.2. Hemidesmosomal Proteins 69

Fig. 5.4฀ Schematic฀illustration฀of฀type฀IV฀collagen฀supramolecular฀network฀assembly.฀(a)฀Type฀IV฀col-

lagen฀chains.฀There฀are฀6฀a-chains฀(a1-6)฀encoded฀in฀the฀mammalian฀genome,฀each฀characterized฀by฀a฀
long฀central฀collagen฀triple฀helix,฀a฀7S฀domain฀at฀the฀N฀terminus,฀and฀a฀globular,฀non-collagenous฀(NC)฀
trimer฀at฀the฀C฀terminus฀(NC1).฀(b)฀Association฀into฀protomers.฀The฀NCl฀domain฀at฀the฀C-terminus฀
(Type฀IV฀collagen฀has฀only฀one฀non-collagenous฀domain)฀induces฀triple฀helix฀formation฀from฀C-฀to฀
N-terminus฀in฀a฀protomer.฀The฀formation฀of฀protomer฀[a3,a4,a5]฀is฀illustrated.฀(c)฀Only฀a฀few฀protomers฀
form.฀The฀ability฀of฀only฀certain฀7S฀or฀NCl฀domains฀to฀associate฀[(a1)2฀a2],฀[a3,a4,a5]฀and฀[(a5)2฀a5]฀
explains฀the฀limited฀triple-helical฀associations฀of฀the฀polypeptides.฀(d)฀Network฀formation฀and฀cross-
linking.฀The฀supramolecular฀network฀is฀assembled฀by฀four฀protomers฀associating฀at฀the฀non-collage-
nous฀7S฀domain,฀followed฀by฀dimerization฀and฀disulide฀cross-linking฀at฀the฀C-terminus.฀(b฀–฀Slightly฀
modiied฀from฀Fig.฀1B฀from฀Borza฀DB,฀Hudson฀BG฀(2003฀May)฀“Molecular฀characterization฀of฀the฀
target฀ antigens฀ of฀ anti-glomerular฀ basement฀ membrane฀ antibody฀ disease.”฀ Springer฀ Semin฀
Immunopathol.฀24(4):345–361);฀a,฀c,฀d฀–฀Slightly฀modiied฀from฀Hudson฀BG,฀Tryggvason฀K฀et.฀al.฀
(2003฀Jun฀19)฀“Alport’s฀Syndrome,฀Goodpasture฀Syndrome,฀and฀Type฀IV฀Collagen.”฀N.฀Engl.฀J.฀Med.฀
348(25):2543–2556)

Plakin฀ proteins฀ link฀ intracellular฀ keratin฀ intermediate฀ ilaments฀ to฀ hemidesmosomes฀ of฀
basal฀ epidermal฀ cells,฀ or฀ to฀ desmosomes฀ on฀ suprabasal฀ epidermal฀ cells฀ (Sect.฀ 5.2.1),฀
whereas฀type฀XVII฀collagen฀(the฀only฀collagen฀secreted฀by฀epithelial฀cells)฀and฀integrins฀
attach฀hemidesmosomes฀to฀a฀basal฀lamina.฀Three฀b1฀integrins฀(with฀a2,฀a3฀or฀a5฀partners)฀
70 5 Basal Laminas and Epithelia

Fig. 5.5฀ Model฀of฀the฀hemidesmosome฀(HD)–basal฀lamina฀junction.฀This฀is฀an฀enlarged฀region฀of฀

one฀of฀the฀hemidesmosomes฀pictured฀in฀Fig.฀5.1.฀There฀are฀4฀components:฀integrin฀alpha฀and฀beta฀
polypeptides฀ (dark฀ brown/yellow฀ typescript),฀ BP180฀ (type฀ XVII฀ collagen,฀ dark฀ blue฀ tadpole-
shaped),฀BP230฀(light฀brown)฀and฀plectin฀(dark฀green).฀They฀all฀cluster฀together฀around฀the฀intracel-
lular฀ side฀ of฀ the฀ integrin฀ beta฀ polypeptide฀ within฀ a฀ hemidesmosome฀ (HD,฀ blue฀ oval).฀ The฀ head฀
portion฀of฀type฀XVII฀collagen฀(BP180)฀is฀attached฀intracellularly฀along฀with฀BP230฀to฀the฀b-integrin฀
subunit.฀The฀tail฀portion฀extends฀through฀the฀cell฀membrane฀alongside฀the฀β-integrin฀subunit฀and฀
together฀they฀enfold฀a฀laminin-5฀head฀(dark฀bluish฀gray).฀The฀b1฀integrins฀which฀are฀present฀in฀
addition฀to฀integrin฀a6b4฀bind฀to฀many฀other฀lamina฀lucida฀components฀besides฀laminin-5,฀and฀initi-
ate฀keratinocyte฀differentiation฀intracellularly฀(see฀text).฀BP230฀and฀plectin฀are฀each฀connected฀to฀
keratin฀5/14฀dimers฀intracellularly฀and฀therefore฀attach฀the฀hemidesmosome฀to฀keratin฀intermediate฀
ilaments฀that฀hold฀an฀epithelium฀together฀with฀desmosomes฀(Sect.฀5.2).฀(Modiied฀from฀Fig.฀8฀in฀
Colognato฀H,฀Yurchenko฀PD฀(2000฀Jun)฀“Form฀and฀Function:฀the฀Laminin฀Family฀of฀Heterotrimers.”฀
Dev.฀Dyn.฀218(2):213–234)

together฀with฀integrin฀a6b4฀can฀attach฀any฀of฀the฀laminins฀in฀the฀lamina฀lucida,฀not฀just฀
laminin-5.฀The฀b1฀integrins฀attach฀laminin-5฀by฀various฀laminin฀motifs,฀not฀exclusively฀the฀
arg-gly-asp฀(RGD)฀motif฀described฀in฀Sect.฀3.2.1.
Type฀ XVII฀ collagen฀ is฀ composed฀ of฀ three฀ identical฀ procollagen฀ polypeptides,฀ each฀
about฀1,500฀amino฀acids฀in฀length.฀It฀has฀a฀tadpole-like฀shape฀under฀physiological฀condi-
tions฀ (deduced฀ from฀ rotary฀ shadowing฀ electron฀ microscopy฀ of฀ bovine฀ cell฀ lines฀ or฀ the฀
pure฀protein).฀The฀protein฀is฀one฀of฀very฀few฀proteins฀whose฀N-฀and฀C-termini฀are฀inverted฀
with฀respect฀to฀the฀membrane.฀In฀nearly฀all฀transmembrane฀proteins,฀for฀example฀integ-
rins,฀the฀C-terminus฀is฀cytosolic฀and฀the฀N-terminus฀is฀extracellular.฀In฀type฀XVII฀colla-
gen,฀the฀cytosolic฀N-terminal฀domain฀comprises฀about฀a฀third฀of฀the฀amino฀acid฀residues฀
5.1.3. Basal Lamina of the Dental Epithelial Attachment 71

Fig. 5.6฀ Basal฀lamina฀anchoring฀to฀the฀dermis.฀The฀long฀ilament฀of฀laminin-5฀(blue฀gray)฀attached฀

to฀integrin฀and฀type฀XVII฀collagen฀beneath฀the฀hemidesmosome฀(HD)฀(top฀right)฀protrudes฀through฀
the฀type฀IV฀collagen฀network฀(brown฀ibers)฀to฀attach฀type฀VII฀anchoring฀collagen฀(dark฀orange฀
ibers฀ with฀ three฀ heads฀ corresponding฀ to฀ each฀ constituent฀ a1-procollagen฀ polypeptides;฀ bottom฀
right).฀Type฀VII฀collagen฀forms฀dimers฀(Fig.฀5.3)฀that฀loop฀around฀type฀I฀collagen฀ibers฀to฀the฀
anchoring฀plaques฀(Modiied฀from฀Fig.฀8฀in฀Colognato฀H,฀Yurchenko฀PD฀(2000฀Jun)฀“Form฀and฀
Function:฀the฀Laminin฀Family฀of฀Heterotrimers.”฀Dev.฀Dyn.฀218(2):213–234)

and฀ends฀in฀a฀short,฀trans-membrane฀hydrophobic฀sequence.฀The฀C-terminal฀extracellular฀
domain฀is฀a฀long,฀interrupted฀Gly-Pro-X฀sequence฀which฀forms฀a฀lexible฀triple฀helix฀like฀
type฀IV฀collagen฀and฀anchors฀itself฀to฀the฀head฀region฀of฀a฀laminin-5฀ilament฀(Fig.฀5.6),฀
supplementing฀the฀integrin฀connection฀and฀strengthening฀the฀basal฀cell฀attachment฀to฀the฀
lamina฀lucida฀by฀hemidesmosomes.฀At฀their฀stromal฀end,฀the฀laminin-5฀ibrils฀are฀anchored฀
to฀type฀VII฀anchoring฀collagen฀ibrils฀(see฀Sect.฀5.1.1).฀The฀epidermis฀may฀be฀pulled฀away฀
from฀the฀dermis฀at฀the฀lamina฀lucida,฀which฀does฀not฀have฀the฀strength฀of฀collagen.฀This฀
weakness฀may฀also฀allow฀the฀epidermis฀to฀repair฀or฀grow฀along฀with฀changes฀to฀the฀dermis฀
as฀dictated฀by฀development฀or฀the฀environment.

5.1.3.
Basal Lamina of the Dental Epithelial Attachment

At฀the฀base฀of฀a฀gingival฀sulcus,฀the฀junctional฀epithelium฀mediates฀the฀epithelial฀attach-
ment.฀This฀epithelium฀possesses฀two฀basal฀laminas,฀an฀outer฀one฀that฀is฀continuous฀with฀the฀
sulcular฀epithelium฀around฀the฀free฀gingiva฀and฀an฀inner฀one฀which฀mediates฀the฀actual฀
72 5 Basal Laminas and Epithelia

dental฀epithelial฀attachment฀(Fig.฀5.7a).฀The฀inner฀basal฀lamina฀contains฀only฀keratinocyte-
secreted฀products:฀type฀XVII฀collagen,฀laminin-5,฀and฀integrins.฀Laminin-1฀and฀type฀IV฀
collagen,฀the฀ibroblast-secreted฀components,฀are฀absent฀(Fig.฀5.7b).฀Teeth฀movements฀con-
tinuously฀ alter฀ the฀ junctional฀ epithelial฀ cell฀ environment,฀ causing฀ the฀ integrins฀ and฀ type฀
XVII฀collagen฀to฀release฀and฀reattach฀laminin-5฀to฀the฀tooth฀surface฀(Fig.฀13.1).
Epidermolysis฀bullosa฀(EB)฀is฀caused฀by฀mutations฀that฀affect฀epidermal฀basal฀cells,฀
the฀basal฀lamina฀that฀they฀synthesize,฀or฀ibroblast฀cell฀products฀with฀which฀they฀interact.฀
A฀mutation฀that฀causes฀EB฀may฀affect฀any฀one฀of฀the฀following:฀epithelial฀basal฀cell฀kera-
tins฀(Sect.฀5.2.2),฀plectin,฀integrin฀a6b4,฀type฀XVII฀collagen,฀laminin-5,฀or฀type฀VII฀colla-
gen.฀ The฀ disease฀ manifests฀ as฀ a฀ blistering฀ if฀ the฀ skin฀ and฀ mucous฀ membranes฀ at฀ the฀
dermal-epidermal฀junction.฀A฀similar฀aberrant฀interaction฀prevents฀enamel-forming฀cells฀
(ameloblasts)฀from฀aligning฀correctly฀against฀calciied฀collagen฀ibrils฀of฀dentin,฀the฀tem-
plate฀for฀enamel฀matrix฀calciication฀(Sect.฀9.5.1).฀The฀enamel฀does฀not฀properly฀calcify฀
(hypoplastic฀enamel),฀and฀the฀affected฀individual฀becomes฀prone฀to฀severe฀dental฀caries.฀
These฀latter฀individuals฀usually฀present฀with฀a฀form฀of฀EB฀that฀is฀classiied฀as฀junctional฀
epithelial฀bullosa฀in฀which฀the฀blistering฀is฀due฀to฀mutations฀of฀a฀structural฀component:฀
integrin฀a6b4,฀type฀XVII฀collagen฀or฀laminin-5฀(Table฀7.1).
In฀addition฀to฀integrin฀a6b4,฀epidermal฀basal฀cells,฀which฀include฀junctional฀epithelial฀
cells,฀possess฀two฀β1฀integrins฀that฀affect฀subsequent฀basal฀cell฀differentiation฀(Sect.฀5.2.2).฀
The฀ β1฀ integrins฀ transmit฀ inside-out฀ signaling฀ via฀ kindlin-1,฀ a฀ protein฀ that฀ binds฀ to฀ the฀

a b
Gingival Nucleus
epithelium
Gingival Basal
Keratin 5/14
fibres epithelial
Alveolar (BP230) cell
Marginal bone Plakin
Plectin
gingiva crest
EBL
of JE XVII Coll β4
Periodontal α6
Gingival membrane
sulcus fibers DAT Cell membrane
Laminin-5
Enamel Cementum
IBL
of JE
Enamel

Fig. 5.7฀ Composition฀ of฀ the฀ gingival฀ epithelial฀ attachment’s฀ basal฀ lamina.฀ (a)฀ Gingival฀ region฀
rotated฀anti-clockwise฀–฀See฀Fig.฀12.1฀for฀an฀un-rotated฀view.฀(b)฀Components฀of฀the฀internal฀basal฀
lamina฀of฀junctional฀epithelium฀(IBL฀of฀JE)฀Dentally฀attached฀(DAT)฀cells฀make฀hemidesmosomes฀
which฀form฀the฀internal฀basal฀lamina฀of฀junctional฀epithelium฀(IBL฀of฀JE).฀The฀DAT฀cell฀intracel-
lular฀hemidesmosome฀connections฀are฀as฀shown฀in฀Fig.฀5.5,฀but฀its฀internal฀basal฀lamina฀(IBL)฀is฀
composed฀only฀of฀a฀thin฀layer฀of฀laminin-5฀polymers฀attached฀to฀type฀XVII฀collagen฀and฀integrin฀
a6b4฀at฀one฀end,฀and฀to฀tooth฀enamel฀at฀the฀other.฀Laminin-1฀and฀type฀IV฀collagen฀are฀absent฀from฀
the฀IBL฀because฀there฀are฀no฀ibroblasts฀to฀make฀them.฀The฀external฀basal฀lamina฀of฀junctional฀
epithelium฀(EBL฀of฀JE)฀contacts฀the฀gingival฀connective฀tissue฀stroma฀as฀shown฀for฀the฀epithelial-
connective฀tissue฀basal฀lamina฀in฀Figs฀5.1,฀5.5,฀and฀5.6฀(Slightly฀modiied฀and฀updated฀version฀of฀
Fig.฀5฀in฀Pollanen฀MT,฀Salonen฀JI,฀and฀Uitto฀V-J.฀Structure฀and฀function฀of฀the฀tooth–epithelial฀
interface฀in฀health฀and฀disease,฀Periodontology฀2000,฀31:12–31,฀2002.฀With฀copyright฀permission฀
from฀Wiley-Blackwell,฀PO฀Box฀805,฀9600฀Garsington฀Road,฀Oxford฀OX4฀2DQ,฀UK)
5.2.1. General Structure of Skin, Oral and Junctional Epithelia 73

integrin฀cytoplasmic฀domain.฀Mutations฀of฀kindlin-1฀upset฀the฀differentiation฀of฀epidermal฀
basal฀cells฀and฀give฀rise฀to฀a฀form฀of฀EB,฀Kindler฀syndrome฀accompanied฀by฀aggressive฀
periodontitis฀(Sect.฀14.1.1).฀In฀other฀forms฀of฀EB,฀the฀absence฀of฀a฀ibroblast฀interaction฀may฀
protect฀the฀junctional฀epithelial฀attachment฀from฀developing฀aggressive฀periodontitis.

Basal฀laminas฀are฀made฀up฀of฀type฀IV฀collagen฀and฀laminin,฀each฀a฀trimer฀of฀separately฀
encoded฀polypeptides.฀The฀outer฀epithelial฀side฀(lamina฀lucida),฀is฀composed฀of฀lami-
nin-1฀ilaments฀and฀laminin-5฀ilaments.฀Laminin-5฀is฀attached฀to฀hemidesmosomes฀by฀
integrin฀ a6b4฀ and฀ type฀ XVII฀ anchoring฀ collagen฀ (bullous฀ pemphigoid฀ antigen-180,฀
BP180).฀The฀latter฀is฀a฀transmembrane฀protein฀whose฀non-collagenous฀N-terminal฀head฀
lies฀in฀the฀cytosol.฀The฀head฀is฀linked฀to฀intermediate฀ilaments฀(keratins)฀by฀the฀cyto-
plasmic฀domain฀of฀integrin฀b4฀via฀two฀proteins฀(plectin฀and฀BP230).฀The฀ibroblasts฀on฀
the฀dermal฀(stromal)฀side฀secrete฀a฀lamina฀densa,฀mostly฀laminin-1฀and฀type฀IV฀collagen.฀
The฀latter฀is฀tightly฀attached฀to฀the฀dermis฀by฀anchoring฀collagen฀ibrils฀(type฀VII)฀which฀
attach฀type฀IV฀collagen฀to฀type฀I฀collagen฀ibers฀and฀other฀stromal฀proteins.฀Epidermolysis฀
bullosa฀(EB)฀is฀caused฀by฀a฀mutation฀that฀alters฀laminar฀structure,฀or฀dermal฀or฀hemides-
mosomal฀ attachments.฀ It฀ is฀ manifest฀ as฀ blistering฀ within฀ the฀ basal฀ lamina.฀ A฀ variant,฀
junctional฀EB,฀is฀due฀to฀mutations฀of฀type฀XVII฀collagen฀or฀laminin-5฀which฀additionally฀
interfere฀with฀ameloblast/odontoblast฀interactions.฀Another฀variant,฀Kindler฀syndrome,฀
is฀the฀only฀form฀of฀EB฀accompanied฀by฀aggressive฀periodontitis.฀This฀EB฀variant฀is฀due฀
to฀a฀mutation฀in฀kindlin-1,฀an฀intracellular฀protein฀that฀provides฀an฀inside-out฀signal฀for฀
β1฀integrin-mediated฀control฀of฀epidermal฀cell฀differentiation.

5.2.1.
General Structure of Skin, Oral and Junctional Epithelia

An฀ epithelium฀ (plural:฀ epithelia)฀ is฀ classiied฀ as฀ simple,฀ stratiied฀ or฀ transitional฀ (Fig.฀
5.8a).฀A฀simple฀epithelium฀consists฀of฀a฀single฀layer฀of฀continually-dividing฀cells฀that฀medi-
ate฀ an฀ exchange฀ of฀ metabolites฀ between฀ compartments.฀ Capillary,฀ kidney฀ tubular,฀ and฀
intestinal฀epithelia฀are฀examples฀of฀simple฀epithelia.฀They฀respectively฀exchange฀metabo-
lites฀ in฀ interstitial฀ luid฀ (Chapter฀ 3,฀ section฀ 3.3.1.),฀ between฀ the฀ glomerular฀ iltrate฀ and฀
blood฀plasma฀(Chapter฀10,฀section฀10.3.1.),฀or฀pass฀mono-฀and฀di-saccharides,฀amino฀acids฀
and฀fatty฀acids฀from฀digested฀food฀to฀blood฀plasma.฀By฀contrast,฀a฀stratiied฀epithelium฀
such฀as฀that฀of฀the฀skin฀is฀layered,฀and฀designed฀to฀prevent฀luid฀diffusion฀and฀metabolite฀
exchange.฀A฀transitional฀epithelium฀is฀a฀layered฀epithelium฀that฀becomes฀simple฀when฀the฀
tissue฀is฀stretched,฀for฀example฀the฀bladder฀epithelium.
A฀ basal฀ lamina฀ is฀ permeable฀ to฀ interstitial฀ luid฀ which฀ provides฀ the฀ nutrients฀ for฀ the฀
basal฀layer฀of฀cells฀to฀proliferate.฀As฀discussed฀in฀Sect.฀5.1,฀basal฀cells฀adhere฀to฀the฀basal฀
lamina฀using฀hemidesmosomes.฀In฀simple฀epithelia,฀basal฀cells฀either฀divide฀into฀identical฀
daughter฀cells฀or฀undergo฀apoptosis฀(programmed฀cell฀death,฀Sect.฀13.4.1).฀In฀a฀stratiied฀
74 5 Basal Laminas and Epithelia

Fig. 5.8฀ Skin฀ epithelium.฀ (a)฀ Types฀ of฀ epithelium.฀ Epithelia฀ are฀ primarily฀ classiied฀ as฀ simple฀ or฀
stratiied฀and฀secondarily฀as฀squamous,฀cuboidal,฀or฀columnar.฀A฀transitional฀epithelium฀is฀neither฀
simple฀nor฀stratiied.฀(b)฀The฀four฀epithelial฀cell฀layers฀of฀a฀fully฀stratiied฀epithelium.฀Names฀of฀the฀
layers฀ are฀ indicated฀ on฀ the฀ right.฀ The฀ basal฀ cell฀ layer฀ consists฀ of฀ undifferentiated฀ (stem)฀ cells฀
between฀the฀rete฀pegs฀and฀committed,฀actively฀dividing฀(transit-amplifying)฀cells฀along฀the฀length฀
of฀the฀rete฀pegs.฀The฀transit-amplifying฀cells฀move฀down,฀toward฀the฀tip฀of฀a฀rete฀peg฀where฀they฀
start฀to฀differentiate.฀Each฀stromal฀papilla฀surrounding฀the฀basal฀and฀transit-amplifying฀cells฀is฀rich฀
in฀collagen฀and฀capillaries฀(Slightly฀modiied฀from฀Fig.฀22-6฀in฀The฀Molecular฀Biology฀of฀the฀Cell,฀
B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀Taylor฀&฀Francis฀Group,฀New฀York)

epithelium,฀the฀basal฀cells฀are฀a฀mixture฀of฀undifferentiated฀stem฀cells฀and฀transit-amplifying฀
cells.฀The฀former฀divide฀slowly฀and฀remain฀strongly฀attached฀to฀the฀basal฀lamina฀(Fig.฀5.8b),฀
whereas฀the฀latter฀are฀stem฀cell฀progeny฀that฀differentiate฀into฀a฀layer฀next฀to฀the฀basal฀layer฀
(Fig.฀5.8b).฀Until฀they฀move฀out฀of฀the฀basal฀layer,฀they฀divide฀faster฀than฀stem฀cells,฀creat-
ing฀inger-like฀projections฀(rete฀pegs)฀that฀bulge฀into฀the฀stroma.
Differentiation฀ starts฀ when฀ the฀ transit-amplifying฀ cells฀ auto-digest฀ their฀ hemidesmo-
somes฀ and฀ synthesize฀ additional฀ desmosomes,฀ which฀ appear฀ as฀ thick฀ tufts฀ resembling฀
‘microscopic฀prickles’฀on฀their฀outer฀surface.฀As฀they฀are฀pushed฀further฀away฀by฀the฀con-
tinuing฀basal฀cell฀dividion,฀the฀prickle฀cells฀synthesize฀different฀keratins฀(Sect.฀5.2.2)฀whose฀
strong฀desmosomal฀attachment฀causes฀the฀cells฀to฀latten฀and฀elongate.฀Eventually,฀a฀thin,฀
dark-staining฀region฀develops,฀the฀granular฀layer,฀in฀which฀cells฀contain฀brown฀granules฀of฀
keratin฀precursors฀called฀hyalokeratin.฀Desmosomes฀mediate฀mechanical฀cohesion฀and฀luid฀
permeability฀in฀the฀basal฀and฀prickle฀layers,฀but฀the฀granular฀layer฀is฀luid-impermeable.฀
The฀lattened฀cells฀of฀the฀granular฀layer฀undergo฀apoptosis฀(Chapter฀13,฀section฀13.4.1.),฀
5.2.2. Composition of Desmosomes and Keratins 75

losing฀their฀internal฀cytosolic฀structures฀and฀becoming฀scales฀or฀squames,฀i.e.฀cell฀mem-
branes฀illed฀only฀with฀keratin.฀Unlike฀skin,฀the฀cells฀of฀the฀outer฀surface฀of฀the฀hard฀palate฀
and฀gingival฀mucosa฀retain฀their฀nuclei฀and฀are฀said฀to฀be฀parakeratinized,฀not฀fully฀kerati-
nized฀(orthokeratinized)฀like฀the฀skin.

5.2.2.
Composition of Desmosomes and Keratins

Desmosomes฀(Fig.฀5.9)฀are฀punctate฀adhesions฀between฀adjacent฀keratinocyte฀cell฀mem-
branes.฀These฀membranes฀are฀unusually฀thick฀and฀are฀referred฀to฀as฀plaques,฀thick฀mem-
brane฀discs฀connected฀by฀desmosomal฀proteins฀that฀anchor฀adjacent฀keratinocytes฀and฀they฀
provide฀ strong฀ intercellular฀ cohesion.฀ The฀ desmosomal฀ plaques฀ should฀ not฀ be฀ confused฀
with฀bacterial฀plaques฀or฀bioilms฀(Chapter฀1,฀section฀1.3.2.).฀The฀desmosomal฀proteins฀
anchor฀adjacent฀cells฀and฀provide฀proximal฀cohesion.฀Desmosomes฀are฀composed฀mostly฀
of฀two฀different฀types฀of฀proteins:฀cadherins฀and฀plakins.฀The฀cadherins,฀mostly฀desmog-
lein฀and฀desmocollin,฀are฀transmembrane฀proteins฀that฀provide฀intermembrane฀attachments฀
by฀forming฀heterodimers.฀On฀the฀cytosolic฀side฀of฀the฀membrane,฀cadherins฀are฀attached฀to฀
plakin฀proteins฀that฀link฀desmosomes฀to฀the฀intracellular฀keratin฀ilaments฀just฀as฀they฀link฀
hemidesmosomes฀ in฀ the฀ basal฀ cell฀ layer.฀ In฀ the฀ outer฀ layers฀ of฀ the฀ epithelium฀ where฀
mechanical฀stress฀is฀important,฀the฀types฀of฀desmogleins฀and฀desmocollins฀change฀and฀the฀
content฀ of฀ desmoplakin฀ increases.฀ There฀ are฀ other,฀ less฀ prominent฀ types฀ of฀ junctions฀
between฀keratinocytes,฀primarily฀the฀adherens฀and฀tight฀junctions.฀Small฀numbers฀of฀these฀
other฀junctions฀are฀critical฀for฀maintaining฀epidermal฀health฀and฀the฀luid฀barrier.
The฀keratin฀family฀of฀proteins฀comprises฀two฀of฀the฀six฀classes฀of฀intermediate฀ilament฀
proteins฀found฀in฀the฀cytosol฀of฀eukaryotic฀cells.฀They฀provide฀the฀intracellular฀structural฀
stability฀that฀complements฀the฀intercellular฀mechanical฀cohesion฀provided฀by฀desmosomes.฀
Keratin฀ilaments฀cross฀the฀entire฀cytosol฀of฀keratinocytes฀and฀their฀ends฀are฀tightly฀attached฀
to฀desmosomes฀by฀desmoplakin฀(Fig.฀5.9)฀or,฀in฀basal฀cells,฀to฀the฀related฀plakin฀(BP230)฀
and฀plectin฀proteins฀in฀hemidesmosomes.

Note:฀Keratin฀is฀an฀intracellular฀ilamentous฀protein฀whereas฀keratan฀is฀a฀sulfated฀poly-
saccharide฀found฀extracellularly฀in฀cartilage฀and฀dermal฀(stromal)฀connective฀tissue.

Keratin฀polypeptides฀are฀encoded฀as฀two฀classes:฀type฀I,฀acidic฀(K9฀through฀K19)฀and฀
type฀II,฀basic฀(K1฀through฀K8).฀Both฀classes฀form฀extended฀a-helical฀domains.฀Keratin฀
ilaments฀are฀composed฀of฀heterodimers:฀type฀I฀keratin฀a-helices฀supercoiled฀around฀type฀
II฀ keratin฀ a-helices฀ in฀ a฀ (left-handed)฀ coiled-coil฀ (Fig.฀ 5.10).฀ Monomeric฀ heterodimers฀
become฀cross-linked฀to฀each฀other฀by฀disulide฀bonds฀and฀then฀assemble฀rapidly฀and฀spon-
taneously฀ into฀ 100฀ Ǻ฀ diameter฀ ilaments฀ in฀ staggered฀ arrays฀ as฀ in฀ ibrillar฀ collagen฀
(Sect.฀4.1.1).฀The฀basal฀cells฀express฀K5฀and฀K14,฀which฀change฀to฀K1฀and฀K10฀as฀they฀
differentiate฀into฀prickle฀cells.฀The฀K1–K10฀heterodimer฀provides฀structural฀stability฀and฀
rigidity฀for฀the฀skin,฀hard฀palate,฀and฀buccal฀surface฀of฀the฀free฀and฀attached฀gingiva.฀The฀
76 5 Basal Laminas and Epithelia

Fig. 5.9฀ Molecular฀composition฀of฀a฀desmosome.฀The฀upper฀portion฀of฀the฀igure฀shows฀the฀general฀

outline฀of฀a฀desmosome฀crossing฀the฀intercellular฀space฀between฀keratinocytes฀and฀its฀attachment฀
plaque฀consisting฀of฀plakin฀proteins฀attached฀to฀the฀respective฀inner฀cell฀membranes.฀The฀lower฀
half฀is฀a฀blow-up฀of฀part฀of฀that฀region฀to฀show฀the฀protein฀structures฀and฀interactions.฀Transmembrane฀
cadherin฀proteins,฀desmocollin฀(blue)฀and฀desmoglein฀(green)฀form฀an฀extended฀chain฀heterodimer฀
that฀links฀the฀plakin฀proteins฀on฀adjacent฀cells.฀On฀the฀inner฀phospholipid฀layer,฀desmocollin฀is฀
attached฀to฀plakophilin฀and฀desmoglein฀to฀plakoglobin.฀In฀turn฀plakoglobin฀and฀plakophilin฀are฀
attached฀to฀one฀end฀of฀desmoplakin฀whose฀other฀end฀is฀attached฀to฀the฀keratin฀intermediate฀ila-
ment.฀ Desmoplakin฀ therefore฀ mediates฀ the฀ attachment฀ of฀ the฀ cytoskeleton฀ to฀ the฀ desmosomes฀
which฀increases฀as฀the฀cells฀differentiate฀(see฀text)฀(Original฀igure฀by฀Dr฀Wirsig-Wiechmann)

gingiva฀also฀contains฀the฀K6/K16฀pair฀(Fig.฀5.11a).฀Besides฀K5฀and฀K14,฀the฀basal฀layer฀of฀
all฀squamous฀epithelia฀expresses฀three฀integrins฀(a2b1,฀a3b1,฀and฀a6b4)฀on฀their฀outer฀
cell฀surface.฀The฀two฀β1฀integrins฀interact฀with฀kindlin-1฀to฀control฀the฀differentiation฀of฀
basal฀cells฀to฀prickle฀cells฀(Sect.฀5.1.3).

5.2.3.
Oral and Junctional Epithelium

The฀nonkeratinized฀regions฀of฀the฀oral฀mucosa,฀the฀mobile฀mucosa฀of฀the฀cheeks,฀lips,฀
ventral฀surface฀of฀the฀tongue,฀soft฀palate฀and฀the฀oral฀sulcular฀and฀junctional฀epithelia฀are฀
permeable฀ to฀ luids฀ and฀ small฀ molecules฀ (Sect.฀ 5.2.1).฀ The฀ prickle฀ cell฀ layer฀ of฀ these฀
epithelia฀expresses฀mostly฀K4฀and฀K13฀instead฀of฀K1฀and฀K10.
5.2.3. Oral and Junctional Epithelium 77

Fig. 5.10฀ Keratin฀ structure.฀ The฀ protein฀ is฀ almost฀ entirely฀ composed฀ of฀ a-helical฀ rod฀ domains฀
(orange,฀red,฀and฀green฀boxes)฀linked฀by฀non-helical฀linker฀regions.฀The฀latter฀are฀indicated฀as฀two฀
internal฀lines฀and฀a฀thin฀yellow฀square.฀The฀N-terminal฀domain฀(head)฀forms฀a฀b-sheet฀and฀the฀
C-terminal฀domain฀(tail)฀has฀a฀complex฀tertiary฀structure.฀A฀typical฀keratin฀a-helix฀domain฀(1B)฀is฀
indicated฀along฀with฀its฀position฀with฀other฀helical฀domains฀and฀the฀N-฀and฀C-terminal฀domains฀in฀
a฀ dimer.฀ Two฀ dimers฀ assemble฀ alongside฀ each฀ other฀ staggered฀ in฀ opposite฀ directions฀ to฀ form฀ a฀
tetramer฀ protoilament.฀ Eight฀ tetramers฀ then฀ assemble฀ to฀ form฀ a฀ ilament฀ (bottom)฀ (Figure฀ is฀ a฀
pastiche.฀ Top฀ two฀ rows฀ are฀ from฀ Fig.฀ 1A฀ and฀ B฀ from฀ Kirfel฀ J,฀ et฀ al.฀ (2003฀ Jan)฀ “Keratins:฀฀
A฀Structural฀Scaffold฀with฀Emerging฀Functions.”฀Cell.฀Mol.฀Life฀Sci.฀60(1):56–71.฀These฀are฀inter-
spersed฀with฀the฀top฀two฀lines฀of฀Fig.฀4-11฀from฀Lehninger฀Principles฀of฀Biochemistry.฀D.L.฀Nelson฀
and฀M.M.฀Cox,฀4th฀Ed.฀2005.฀W.H.฀Freeman฀&฀Co.,฀New฀York;฀the฀remainder฀is฀from฀Fig.฀16-16฀฀
(C฀ through฀ E)฀ in฀ The฀ Molecular฀ Biology฀ of฀ the฀ Cell.฀ B.฀ Alberts฀ et฀ al.,฀ 4th฀ Ed.฀ 2002.฀ Garland฀
Science,฀Taylor฀&฀Francis฀Group,฀New฀York)
78 5 Basal Laminas and Epithelia

FGM
JE
OSE Free gingival
K4,K13
K16 epithelium

Enamel K1,K10

DAT cells
K6,K16
Attached
To B. K5,K14 gingival
K8,K18 epithelium
K19
Dentin
K5,K14
K19

Alveolar
Bone

Alveolar
K4,K13
Epithelium

Fig. 5.11฀ Keratin฀ composition฀ of฀ junctional฀ and฀ gingival฀ epithelia.฀ The฀ free,฀ attached฀ and฀ alveolar฀
epithelia฀are฀not฀drawn฀to฀scale.฀The฀thicker฀surface฀and฀internal฀broken฀lines฀indicate฀a฀corniied฀kera-
tinized฀layer฀that฀characterizes฀the฀epithelium฀of฀the฀free฀and฀attached฀gingiva.฀In฀the฀junctional฀epi-
thelium฀(JE)฀and฀oral฀sulcular฀epithelium฀(OSE)฀to฀just฀past฀the฀free฀gingival฀margin฀(FGM),฀and฀also฀
in฀the฀alveolar฀epithelium,฀the฀prickle฀cells฀possess฀K1฀and฀K10฀instead฀of฀K1฀and฀K10.฀These฀epithe-
lia฀are฀non-keratinized;฀the฀granular฀and฀corniied฀layers฀are฀absent.฀K16฀is฀present฀in฀the฀oral฀sulcular฀
epithelium฀without฀a฀partner฀and฀its฀function฀there฀is฀not฀known.฀K19฀pairs฀with฀any฀type฀II฀keratin.฀฀
(Slightly฀modiied฀by฀Dr.฀Wirsig-Weichmann฀from฀Fig.฀5฀in฀Pollanen฀MT,฀Salonen฀JI฀and฀Uitto฀V-J,฀
Structure฀and฀function฀of฀the฀tooth-epithelial฀interface฀in฀health฀and฀disease.฀Periodontology฀2000,฀
31:12–31,฀2003)

DNA฀for฀genetic฀analysis฀is฀often฀obtained฀from฀the฀nuclei฀of฀nonkeratinized฀mucosal฀
cells฀ from฀ inside฀ the฀ cheek.฀ Collecting฀ these฀ cells฀ is฀ less฀ invasive฀ and฀ simpler฀ than฀
obtaining฀a฀sample฀of฀blood฀cells.

Junctional฀epithelium฀seals฀the฀periodontium฀from฀the฀oral฀cavity฀at฀the฀base฀of฀a฀gingival฀
sulcus฀and฀it฀is฀often฀referred฀to฀as฀the฀junctional฀epithelial฀attachment฀or฀just฀the฀epithelial฀
attachment฀(Fig.฀5.12a).฀Hubert฀Schroeder฀and฀Max฀Listgarten฀published฀the฀irst฀compre-
hensive฀description฀of฀this฀epithelium฀in฀a฀monograph฀in฀1973.฀The฀junctional฀epithelium฀
develops฀at฀the฀base฀of฀a฀sulcus฀where฀the฀reduced฀enamel฀epithelium฀(Sect.฀9.5.1)฀merges฀
with฀the฀apical฀end฀of฀the฀oral฀sulcular฀epithelium฀(OSE)฀(Fig.฀5.12b).฀It฀may฀also฀develop฀
from฀regenerating฀oral฀sulcular฀epithelium฀if฀the฀sulcus฀is฀surgically฀excised฀by฀a฀procedure฀
called฀ gingivectomy.฀ The฀ external฀ (outer)฀ epithelial฀ basal฀ layer฀ forms฀ a฀ basal฀ lamina฀ that฀
contacts฀the฀gingival฀stroma฀and฀is฀continuous฀with฀the฀basal฀layer฀and฀lamina฀of฀the฀oral฀
sulcular฀epithelium.฀At฀its฀apical฀extremity฀(only฀a฀few฀cells฀thick),฀the฀external฀basal฀cell฀
layer฀and฀lamina฀unite฀with฀an฀internal฀(inner)฀basal฀layer฀of฀cells฀whose฀lamina฀is฀dentally฀
attached฀and฀extends฀coronally฀to฀slightly฀above฀the฀base฀of฀the฀gingival฀sulcus฀(Fig.฀5.12b).
5.2.3. Oral and Junctional Epithelium 79

The฀biochemistry฀and฀metabolism฀of฀junctional฀epithelium฀has฀been฀studied฀by฀many฀
investigators,฀but฀most฀thoroughly฀by฀Ian฀Mackenzie฀and฀Jukka฀Salonen.฀Both฀junctional฀
basal฀layers฀are฀rapidly฀proliferating,฀transit-amplifying฀cells.฀They฀are฀derived฀from฀stem฀
cells฀located฀where฀the฀external฀basal฀layer฀meets฀the฀basal฀layer฀of฀oral฀sulcular฀epithelium฀
(Fig.฀5.12a).฀Because฀basal฀cells฀must฀proliferate฀to฀produce฀a฀basal฀lamina฀and฀remain฀
attached฀ to฀ an฀ underlying฀ surface,฀ interstitial฀ luid฀ must฀ penetrate฀ the฀ whole฀ junctional฀
epithelium฀to฀the฀tooth฀surface฀to฀maintain฀internal฀basal฀cell฀proliferation฀and฀attachment.฀
Junctional฀epithelium฀is฀therefore฀permeable฀to฀stromal฀luid฀and฀in฀this฀respect฀it฀resem-
bles฀a฀simple฀epithelium.฀Interstitial฀luid฀transudes฀throughout฀the฀junctional฀epithelium฀

Fig. 5.12฀ Coronal฀extremity฀of฀the฀periodontium฀showing฀the฀gingiva.฀(a)฀The฀gingival฀region.฀The฀

oral฀sulcular฀epithelium฀and฀oral฀epithelium฀below฀the฀free฀gingival฀margin฀(FGM)฀comprise฀the฀
free฀gingiva฀which฀is฀tightly฀held฀against฀the฀tooth฀by฀free฀collagen฀ibers.฀The฀attached฀gingiva฀
(length฀slightly฀exaggerated)฀is฀attached฀to฀the฀alveolar฀bone฀by฀collagen฀ibers.฀The฀junctional฀
epithelial฀ attachment฀ has฀ outer฀ and฀ inner฀ basal฀ cells฀ (thick฀ black฀ line)฀ that฀ merge฀ apically.฀ The฀
outer฀basal฀cells฀merge฀coronally฀with฀the฀basal฀cells฀at฀the฀apical฀end฀of฀the฀oral฀sulcular฀epithe-
lium฀and฀the฀site฀of฀junctional฀epithelial฀stem฀cells฀(b)฀The฀gingival฀sulcus.฀The฀gingival฀sulcus฀
(Sulcus)฀ is฀ bounded฀ by฀ the฀ enamel฀ cuticle฀ (Cuticle)฀ and฀ oral฀ sulcular฀ epithelium฀ (OSE)฀ which฀
terminates฀in฀the฀free฀gingival฀margin฀(FGM)฀coronally.฀Immediately฀beneath฀the฀sulcus,฀intersti-
tial฀luid฀(ISF)฀leaks฀from฀capillary฀blood฀vessels฀(BV)฀in฀the฀stroma฀and฀transudes฀through฀the฀
junctional฀epithelium฀to฀provide฀nutrients฀for฀the฀proliferative฀dentally฀attached฀cells฀(DAT฀cells),฀
at฀the฀base฀of฀the฀sulcus฀(long฀arrow)฀and฀apically฀(two฀short฀arrows).฀The฀DAT฀cells฀lie฀against฀
the฀inner฀basal฀lamina฀(IBL)฀and฀the฀similarly฀proliferative฀basal฀cells฀continuous฀with฀those฀of฀
oral฀sulcular฀epithelium฀lie฀against฀the฀external฀basal฀lamina฀(EBL).฀This฀area฀is฀enlarged฀from฀the฀
box฀ around฀ the฀ gingival฀ sulcus฀ in฀ a.฀ Opposite฀ (Modiied฀ from฀ Fig.฀ 1-2฀ in฀ Contemporary฀
Periodontics,฀ edited฀ by฀ RJ฀ Genco,฀ HM฀ Goldman฀ and฀ DW.฀ Cohen.฀ Chapter฀ 1:฀ The฀ Gingiva,฀
Structure฀and฀Function฀by฀H฀Loe.,฀MA฀Listgarten฀&฀VP฀Terranova.฀Pub.,฀The฀CV฀Mosby฀Co.,฀St฀
Louis฀MO.)฀b฀–฀Modiied฀from฀Fig.฀36A฀in฀Schroeder,฀H.E.฀&฀Listgarten,฀M.A.฀(1977)฀Monographs฀
in฀ Developmental฀ Biology,฀ Vol.฀ 2,฀ Fine฀ Structure฀ of฀ the฀ Developing฀ Epithelial฀ Attachment฀ of฀
Human฀Teeth.฀Series฀editor,฀A.฀Wolsky,฀Vol฀2,฀2nd฀Ed.฀Pub.,฀S.฀Karger,฀Basel,฀Switzerland)
80 5 Basal Laminas and Epithelia

from฀capillaries฀beneath฀the฀external฀basal฀lamina฀(EBL).฀Traces฀of฀this฀luid฀reach฀the฀
most฀coronal฀region฀of฀dentally฀attached฀cells฀by฀passing฀through฀the฀base฀of฀the฀gingival฀
sulcus฀(Fig.฀5.12a).
Basal฀cells฀that฀stop฀dividing฀also฀stop฀secreting฀a฀basal฀lamina฀and฀lose฀their฀attachment.฀
They฀are฀squeezed฀by฀adjacent,฀dividing฀basal฀cells฀into฀the฀body฀of฀the฀junctional฀epithe-
lium฀and฀are฀expelled฀into฀the฀base฀of฀a฀sulcus.฀The฀few฀tight฀junctions฀in฀the฀body฀of฀the฀
junctional฀epithelium฀are฀consistent฀with฀its฀permeability฀to฀interstitial฀luid.฀The฀rapidly฀
dividing฀cells฀of฀both฀internal฀and฀external฀basal฀layers฀express฀K5฀and฀K14฀like฀all฀stratiied฀
epithelial฀basal฀cells,฀but฀the฀interior฀cells฀express฀K4฀and฀K13,฀not฀K1฀and฀K10฀(Fig.฀5.11).฀
The฀composition฀of฀the฀internal฀basal฀lamina฀is฀described฀in฀Fig.฀5.7b.
The฀junctional฀epithelium฀is฀continually฀regenerating฀itself฀from฀stem฀cells฀at฀the฀union฀
of฀internal฀and฀external฀basal฀lamina.฀It฀expresses฀a฀fourth฀integrin,฀αvβ6฀in฀addition฀to฀the฀
two฀β1฀integrins฀and฀α4β6฀integrin.฀Integrin฀αvβ6฀is฀a฀marker฀for฀newly฀developing฀or฀regen-
erating฀epithelia฀and฀it฀binds฀to฀the฀RGD฀sequence฀of฀laminin-5฀(Sect.฀5.1.2).฀A฀regulatory฀
protein,฀TGF-β1฀(Section฀3.2.2.)฀is฀continually฀secreted฀by฀junctional฀epithelium฀and฀it฀binds฀
to฀integrin฀αvβ6.฀The฀binding฀exposes฀TGF-β1฀to฀proteases฀that฀hydrolyze฀off฀a฀latent฀acti-
vation฀peptide฀(LAP),฀providing฀mature฀(processed)฀TGF-β1฀in฀the฀inner฀and฀outer฀basal฀
laminae.฀Among฀its฀many฀functions,฀mature฀TGF-β1฀inhibits฀inlammatory฀responses.฀Thus,฀
the฀activation฀of฀TGF-β1฀by฀integrin฀αvβ6฀in฀the฀junctional฀epithelium฀inhibits฀a฀potential฀
inlammatory฀response฀to฀masticatory฀tooth฀movements฀that,฀uncontrolled,฀could฀result฀in฀
spontaneous฀periodontitis.฀The฀junctional฀epithelium฀is฀the฀initial฀target฀of฀bacteria฀that฀initi-
ate฀gingivitis฀and฀is฀eventually฀destroyed฀(Chapter฀13,฀section฀13.3.1.).

Epithelial฀ cells฀ contain฀ keratins:฀ cytosolic,฀ intermediate-sized฀ ilaments.฀ Each฀ acidic฀

keratin฀(type฀I)฀is฀partnered฀with฀a฀basic฀keratin฀(type฀II)฀to฀form฀extended,฀intertwined฀
alpha฀helices.฀These฀heterodimers฀extend฀longitudinally฀and฀laterally฀in฀staggered฀and/
or฀end-to-end฀arrays฀to฀form฀cytosolic฀intermediate฀ilaments.฀Different฀keratin฀types฀
account฀for฀the฀properties฀of฀different฀types฀of฀epithelia.฀A฀stratiied฀epithelium฀provides฀
a฀barrier฀consisting฀of฀multiple฀layers฀of฀cells฀in฀which฀the฀basal฀layer฀expresses฀K5–K14฀
but฀the฀supra-basal฀layers฀express฀K1–K10.฀Junctional฀epithelium฀has฀inner฀and฀outer฀
basal฀layers฀that฀contain฀K5–K14.฀Its฀supra-basal฀layers฀possess฀K4฀and฀K13,฀not฀K1฀
and฀K10฀and฀it฀therefore฀has฀no฀granular฀layer.฀Desmosomes฀permit฀water฀and฀metabo-
lite฀ exchange฀ beneath฀ the฀ granular฀ layer,฀ but฀ they฀ are฀ mostly฀ absent฀ from฀ junctional฀
epithelium฀which฀is฀luid-permeable฀like฀a฀simple฀epithelium.฀The฀cells฀of฀both฀basal฀
layers฀of฀junctional฀epithelium฀proliferate฀and฀shed฀their฀progeny฀into฀the฀base฀of฀the฀
sulcus.฀ Junctional฀ epithelium฀ also฀ secretes฀ and฀ activates฀ TGF-b1,฀ which฀ prevents฀ an฀
inlammatory฀response฀to฀masticatory฀trauma.
 Elastic Fibers and Proteoglycans
6

Extracellular฀matrix฀(stroma)฀contains฀elastic฀ibers฀and฀glycosaminoglycans฀(GAGs)฀
that฀have฀long฀been฀identiied฀histologically.฀Fibrillin฀is฀a฀major฀component฀of฀microi-
brils฀that฀surround฀elastin฀in฀elastic฀tissues฀and,฀along฀with฀collagen,฀is฀an฀important฀
component฀of฀the฀periodontal฀ligament.฀Section฀1฀describes฀how฀ibrillin฀was฀isolated,฀
major฀ features฀ of฀ its฀ structure,฀ and฀ mutations฀ that฀ affect฀ the฀ face฀ and฀ oral฀ cavity.฀
Section฀ 2฀ describes฀ how฀ elastin฀ was฀ isolated,฀ the฀ major฀ features฀ of฀ its฀ structure,฀ the฀
importance฀of฀copper฀in฀elastin฀processing฀and฀its฀contribution฀to฀stromal฀lexibility.฀
Section฀3฀describes฀stromal฀glycosaminoglycan฀composition฀and฀synthesis.฀Section฀4฀
describes฀the฀glycosaminoglycan฀proteins฀and฀their฀association฀with฀type฀II฀collagen฀in฀
cartilage.฀The฀chapter฀concludes฀with฀a฀discussion฀of฀the฀structure฀and฀function฀of฀all฀
collagen-glycosaminoglycan-associated฀proteins฀(Sect.฀5).

6.1.1.
Fibrillin

When฀tissues฀containing฀elastic฀ibers฀are฀extracted฀with฀6.0฀M฀guanidine฀buffers,฀a฀large฀
molecular฀weight฀aggregate฀appears฀with฀a฀characteristic฀beads-on-a-string฀structure.฀The฀
major฀component฀of฀this฀structure฀is฀a฀~350฀kDa฀glycoprotein฀protein฀identiied฀as฀ibrillin.฀
Using฀ gold-conjugated฀ antibodies,฀ ibrillin฀ was฀ located฀ to฀ microibrils฀ with฀ or฀ without฀
elastin.฀ Fibrillin฀ is฀ extremely฀ long,฀ 2,871฀ amino฀ acid฀ residues฀ in฀ humans.฀ The฀ beaded฀
regions฀bind฀calcium฀ions฀and฀have฀a฀periodicity฀of฀56฀nm,฀whereas฀the฀periodicity฀of฀col-
lagen฀ibrils฀and฀microibrils฀(types฀I฀and฀VI฀collagen)฀is฀64฀and฀3฀nm,฀respectively.
Fibrillin-1฀is฀made฀up฀of฀numerous฀calcium-binding฀(cb)฀domains,฀all฀homologous฀to฀
the฀sequence฀of฀epidermal฀growth฀factor฀(cbEGF฀domains,฀Fig.฀6.1).฀Each฀cbEGF฀domain฀
is฀a฀beta-sheet฀held฀together฀by฀cysteine฀disulide฀bonds.฀In฀addition,฀calcium฀ions฀bind฀to฀
aspartate฀and฀glutamate฀residues฀at฀the฀N-terminus฀of฀each฀cbEFG฀domain฀(Fig.฀6.1a).฀EGF฀
domains฀occur฀in฀diverse฀membrane-bound฀or฀secreted฀animal฀proteins,฀but฀usually฀with-
out฀the฀calcium-binding฀sub-domain฀which฀is฀a฀separate฀N-terminal฀domain฀in฀EGF฀itself.฀
The฀entire฀ibrillin-1฀molecule฀consists฀of฀43฀cbEGF฀domains฀(shown฀in฀blue฀in฀Fig.฀6.1b).฀
Short฀ linker฀ domains฀ between฀ the฀ cbEGF฀ (green฀ ovals฀ in฀ Fig.฀ 6.1b)฀ are฀ homologous฀ to฀
domains฀in฀proteins฀that฀bind฀transforming฀growth฀factor-b1฀(TB฀domains).฀As฀discussed฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 81
DOI:฀10.1007/978-3-540-88116-2_6,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
82 6 Elastic Fibers and Proteoglycans

Fig. 6.1฀ Fibrillin฀individual฀domains.฀(a)฀Detailed฀structure฀of฀the฀ibrillin฀cbEGF13–14฀pair.฀The฀

amino฀acids฀are฀numbered฀from฀N-฀to฀C-terminus.฀The฀bound฀calcium฀ions฀(one฀per฀cbEFG฀domain)฀
are฀ indicated฀ as฀ black฀ circles฀ close฀ to฀ the฀ N-terminus฀ of฀ each฀ domain.฀ The฀ cysteine฀ cross-links฀
(three฀per฀cbEFG฀domain)฀are฀indicated฀by฀zigzag฀lines.฀(b)฀Fibrillin฀domain฀structure.฀The฀43฀
cbEGF฀domains฀(blue)฀are฀numbered.฀There฀are฀four฀additional฀more฀complete฀epidermal฀growth฀
factor฀(EGF)฀domains฀at฀the฀N-terminal฀region฀(gray).฀There฀are฀nine฀TB฀domains,฀but฀only฀seven฀
are฀linker฀domains฀(green฀ovals).฀The฀TB1฀domain฀is฀linked฀to฀a฀separate฀proline-rich฀domain฀(dark฀
blue)฀and฀TB3฀(linked฀to฀cbEGF10)฀is฀modiied฀(green฀diamond).฀The฀TB฀linker฀domains฀are฀a฀
potential฀source฀of฀lexibility,฀through฀their฀interaction฀with฀their฀lanking฀cbEGF฀domains.฀The฀
interbead฀ segment,฀ TB4-6฀ and฀ intervening฀ cbEGF฀ domains฀ (orange฀ segment฀ at฀ the฀ foot฀ of฀
Fig.฀4.17b)฀are฀the฀compressed,฀normal฀form฀that฀expands฀under฀tension฀by฀releasing฀Ca2+฀to฀give฀
the฀70-nm฀beaded฀form฀(a฀–฀From฀Fig.฀3฀in฀Whiteman฀P.฀et฀al.฀(1998฀Apr฀3)฀“A฀Gly฀–>฀Ser฀change฀
causes฀defective฀folding฀in฀vitro฀of฀calcium-binding฀epidermal฀growth฀factor-like฀domains฀from฀
factor฀IX฀and฀ibrillin-1.”฀J.฀Biol.฀Chem.฀273(14):7807–7813.฀b฀–฀Reprinted฀with฀minor฀changes฀
from฀ Arnaout฀ MA฀ (2004)฀ “The฀ structural฀ basis฀ of฀ elasticity฀ in฀ ibrillin-based฀ microibrils.”฀
Structure฀12(4):734–736;฀Copyright฀2004,฀with฀permission฀from฀Elsevier)

previously฀ (Sect.฀ 3.2.2),฀ latent฀ transforming฀ growth฀ factor-b฀ (latent฀ TGF-β)฀ binds฀ to฀
t฀hrombospondin-1฀ (TSP-1)฀ by฀ its฀ latent฀ activated฀ peptide฀ (LAP)฀ domain,฀ exposing฀ the฀
potentially฀active฀C-terminal฀domain฀to฀be฀released฀by฀stromal฀protease฀cleavage.฀An฀alter-
native฀activation฀mechanism฀exists฀in฀which฀the฀latent฀TGF-b฀binds฀instead฀to฀ibrillin฀at฀
TB-cbEGF฀hybrid฀domains฀near฀the฀N฀terminus฀(Narrow฀green฀ovals฀on฀the฀N-terminal฀side฀
of฀cbEGF฀domains฀1฀and฀10฀in฀Fig.฀6.1b).฀Fibrillin฀also฀binds฀bone฀morphogenic฀proteins฀
(BMPs)฀at฀this฀site.฀BMPs฀other฀than฀BMP-1฀which฀is฀a฀zincin฀protease฀(Sect.฀8.2.1)฀are฀
6.1.1. Fibrillin 83

N CN C
Pro-rich TB3 TB7 Pro-rich TB3 TB7

150 nm

Pro-rich TB3 TB7

C/N Pro-rich
100 nm TB7
Pro-rich

TB3 C/N

70 nm
TB7

TB3
56 nm

Fig. 6.2฀ The฀hinged฀model฀of฀ibrillin฀elasticity.฀Fibrillin฀forms฀dimers฀by฀covalent฀N-฀and฀C-terminal฀

binding฀and฀this฀permits฀folding฀initiation฀to฀the฀150-nm฀periodicity.฀As฀folding฀then฀proceeds฀to฀
the฀100-nm฀periodicity,฀glutaminase฀attaches฀and฀forms฀glutamine-lysine฀intra-molecular฀cross-
links฀between฀the฀folded฀segments฀(blue).฀As฀folding฀proceeds฀further,฀the฀ibrillin฀polypeptide฀
dimer฀bends฀at฀the฀TB3฀and฀TB7฀regions฀and฀additional฀proteins฀attach฀to฀this฀region.฀The฀N฀and฀
C฀termini฀are฀in฀yellow฀and฀red,฀respectively.฀The฀interbead฀region฀between฀TB4฀and฀TB6฀is฀elastic.฀
Its฀b-sheet฀structure฀loses฀calcium฀ions฀under฀tension,฀allowing฀disulide-bonded฀cysteine฀residues฀
to฀move฀by฀rotating฀away฀from฀each฀other฀(shown฀in฀orange).฀Numbers฀on฀the฀left฀indicate฀the฀
periodicity.฀ Details฀ of฀ folding฀ are฀ described฀ in฀ the฀ text฀ (Reprinted฀ with฀ minor฀ changes฀ from฀
Structure,฀Vol฀12฀(4),฀Arnaout,฀MA,฀The฀structural฀basis฀of฀elasticity฀in฀ibrillin-based฀microibrils,฀
pp.฀734–736;฀Copyright฀2004,฀with฀permission฀from฀Elsevier)

homologous฀to฀TGF-b.฀A฀ibrillin฀microibril฀that฀binds฀a฀BMP฀(not฀BMP-1)฀along฀with฀
latent฀TGF-β฀causes฀the฀BMP฀prodomain฀to฀promote฀TGF-β฀activation,฀presumably฀by฀a฀
stromal฀protease฀like฀TSP-1฀activation.฀The฀active฀disulide฀peptide฀dimer฀is฀released฀and฀
induces฀osteoblast฀differentiation฀and฀activation฀for฀bone฀development.
Fibrillin฀forms฀head-to-tail฀polymers฀that฀progressively฀fold฀at฀deined฀sites฀between฀
฀successive฀ molecules฀ (Fig.฀ 6.2).฀ Initial฀ internal฀ folding฀ at฀ the฀ head-tail฀ junction฀ (red/฀
yellow)฀฀produces฀a฀150-nm฀periodicity.฀Further฀folding฀at฀the฀proline-rich฀region฀(dark฀
green)฀produces฀an฀approximately฀100-nm฀bead฀periodicity฀and฀creates฀cross-links฀(not฀
shown)฀between฀the฀folded฀regions.฀These฀cross-links฀are฀mediated฀by฀the฀enzyme฀trans-
glutaminase,฀which฀replaces฀the฀terminal฀amide฀of฀glutamine฀with฀the฀e-amino฀group฀of฀an฀
84 6 Elastic Fibers and Proteoglycans

Fig. 6.3฀ The฀transglutaminase฀ COO− COO−

reaction.฀Folding฀brings฀the฀ COO− COO−
+ +
+ +
H3N H H3N C H
glutamine฀and฀lysine฀residues฀ H3N C H H3N C H C
into฀close฀proximity฀where฀ CH2 CH2
CH2 CH2
bound฀glutaminase฀replaces฀฀
CH2 CH2
the฀glutamine฀amide฀group฀by฀ CH2 CH2
linking฀it฀to฀the฀e-amino฀group฀ C CH2
C CH2
of฀lysine.฀The฀amide฀is฀฀ O
H2N O CH2
given฀off฀as฀ammonia
CH2 +
+
Glutamine +
NH3 NH
NH3
Bond between glutamine and
Lysine
lysine catalysed by glutaminase
ammonia is given off

adjacent฀ lysine฀ residue,฀ creating฀ an฀ isopepide,฀ intermolecular฀ cross-link฀ (Fig.฀ 6.3).฀
Transglutaminase฀also฀acts฀on฀various฀other฀proteins,฀most฀notably฀ibrinogen฀(Sect.฀11.3.4).฀
Transglutaminase฀ requires฀ calcium฀ ions฀ for฀ activity฀ and฀ it฀ attaches฀ to฀ folded฀ (beaded)฀
regions฀(light฀blue,฀egg-shaped฀region฀in฀Fig.฀6.2)฀along฀with฀other฀proteins.
The฀extended฀central฀regions฀of฀the฀ibrillin฀polymer฀(orange฀in฀Fig.฀6.2)฀remain฀free฀
of฀ associated฀ proteins.฀ Further฀ intramolecular฀ folding฀ at฀ the฀ TB7-฀ and฀ TB3-cbEGF฀
linker฀regions฀result฀in฀a฀~70-nm฀bead฀periodicity฀which฀corresponds฀to฀a฀“stretched”฀
form.฀The฀interbead฀segment฀(cbEGF฀domains฀from฀TB4฀to฀TB-6;฀orange฀in฀Fig.฀6.2)฀
spontaneously฀bind฀calcium฀ions,฀which฀compresses฀them฀further฀to฀give฀the฀observed฀
56-nm฀beaded฀periodicity,฀“relaxed”฀form.฀Stretching฀occurs฀within฀this฀segment฀and฀is฀
reversible.฀ On฀ stretching,฀ ibrillin฀ periodicity฀ increases฀ from฀ 56฀ to฀ 70฀ nm฀ due฀ to฀ the฀
interbead฀ segment฀ dissociating฀ bound฀ calcium฀ ions.฀ When฀ the฀ stretching฀ force฀ is฀
released,฀ calcium฀ ions฀ can฀ bind฀ again฀ and฀ this฀ pulls฀ the฀ segment฀ back฀ to฀ the฀ 56-nm฀
“relaxed”฀form.฀The฀folded฀beaded฀region฀normally฀stays฀intact,฀but฀severe฀stretching฀
that฀ partially฀ unfolds฀ the฀ beaded฀ regions฀ to฀ above฀ 100฀ nm฀ prevents฀ the฀ beads฀ from฀
returning฀to฀the฀“relaxed”฀form฀(overstretched).฀Along฀with฀elastin฀(Sect.฀6.2.1),฀ibril-
lin฀is฀an฀important฀component฀of฀ligaments฀(Sect.฀3.1.3).฀If฀a฀ligament฀is฀overstretched฀
it฀is฀the฀ibrillin฀microibrils฀that฀are฀damaged฀and฀take฀a฀long฀time฀to฀be฀repaired.฀
Fibrillin-2฀has฀an฀amino฀acid฀sequence฀that฀is฀68%฀identical฀to฀ibrillin-1฀and฀is฀coex-
pressed฀with฀ibrillin-1฀in฀many฀tissues฀early฀in฀mammalian฀development.฀It฀forms฀head-
to-tail฀ibrillin1/2฀alternating฀heterodimers฀that฀resemble฀ibrillin-1฀homodimers฀shown฀at฀
the฀top฀of฀Fig.฀6.3.฀During฀mammalian฀development,฀some฀tissues฀express฀ibrillin-2฀with-
out฀ ibrillin-1฀ and฀ ibrillin-2฀ homodimers฀ may฀ assemble฀ by฀ a฀ mechanism฀ that฀ does฀ not฀
involve฀ibrillin-1฀but฀perhaps฀utilizing฀ibrillin-3,฀a฀third฀member฀of฀the฀ibrillin฀family.฀
Fibrillin-2฀binds฀to฀the฀precursor฀of฀elastin฀during฀development฀and฀forms฀stronger฀elastic฀
ibers฀than฀ibrillin-1.฀Fibrillin-3฀is฀a฀minor฀component฀whose฀functions฀are฀uncertain.
Mutations฀of฀ibrillin-1฀and฀-2฀disrupt฀elastic฀tissue฀scaffolding,฀particularly฀in฀the฀aorta,฀
eyes,฀and฀skin.฀An฀ especially฀ obvious฀ effect฀ of฀ certain฀ ibrillin-1฀ and฀ -2฀ mutations฀ is฀ an฀
overgrowth฀of฀the฀long฀bones฀of฀the฀body,฀resulting฀in฀long฀limbs฀and฀a฀tall฀stature฀(Marfan’s฀
syndrome).฀In฀addition,฀there฀are฀major฀changes฀to฀the฀face,฀oral฀cavity,฀and฀teeth,฀most฀
notably,฀a฀highly฀arched฀palate,฀crowded฀incompletely฀developed฀(hypoplastic)฀teeth฀and฀
deformities฀ of฀ the฀ roots.฀ These฀ changes฀ may฀ all฀ stem฀ from฀ those฀ ibrillin฀ mutations฀ that฀
6.2.1. Elastin 85

Fibrillin฀ is฀ the฀ major฀ component฀ of฀ beaded฀ microilaments฀ possessing฀ elasticity.฀ It฀ is฀
secreted฀ as฀ a฀ 150฀ nm฀ polypeptide฀ possessing฀ almost฀ 50฀ calcium-binding฀ domains฀ sur-
rounded฀by฀lexible฀domains฀that฀allow฀folding.฀The฀calcium-binding฀domains฀are฀homolo-
gous฀to฀epidermal฀growth฀factor฀and฀the฀lexible฀domains฀to฀proteins฀that฀bind฀transforming฀
growth฀factor-b.฀Fibrillin฀is฀secreted฀as฀covalently฀connected,฀head-to-tail฀dimers.฀The฀N-฀
to฀ C-terminal฀ regions฀ are฀ central฀ to฀ the฀ folding฀ that฀ provides฀ the฀ beaded฀ appearance.฀
Folding฀is฀stabilized฀by฀transglutaminase฀cross-linking฀between฀glutamine฀and฀lysine฀resi-
dues฀and฀various฀small฀proteins฀that฀bind฀to฀the฀folded฀(beaded)฀ibrillin.฀The฀central฀region฀
of฀each฀molecule฀is฀free฀of฀associated฀proteins฀and฀consists฀of฀partially฀folded฀calcium-
binding฀domains฀that฀stretch,฀expanding฀the฀bead฀periodicity฀from฀56฀nm฀to฀70฀nm.฀There฀
are฀three฀homologous฀ibrillin฀molecules฀(ibrillin-1฀through฀3).฀Disruption฀of฀elastic฀tissue฀
scaffolding฀due฀to฀mutations฀in฀ibrillin-1฀and฀-2฀affect฀the฀aorta,฀eyes,฀and฀skin.฀฀Some฀
ibrillin฀mutations฀also฀cause฀abnormal฀bone฀growth฀(Marfan’s฀syndrome)฀perhaps฀due฀to฀
an฀uncontrolled฀activation฀of฀latent฀TGF-β฀attached฀to฀microibrils฀together฀with฀any฀bone฀
morphogenic฀protein฀family฀member฀except฀BMP-1.

interfere฀ with฀ a฀ normal฀ interaction฀ between฀ TGF-β฀ and฀ BMP฀ on฀ the฀ ibrillin฀ surface.฀
Mutations฀of฀stromal฀proteins฀that฀affect฀the฀teeth฀are฀listed฀in฀Table฀7.1.฀It฀should฀be฀clear฀
that฀ibrillin฀is฀absent฀from฀teeth฀and฀bones,฀which฀are฀essentially฀calciied฀type฀I฀collagen.

6.2.1.
Elastin

Animals฀placed฀on฀a฀copper-deicient฀diet฀exhibit฀a฀decreased฀content฀of฀elastic฀ibers฀and฀
suffer฀aneurysms฀of฀the฀aorta.฀This฀observation฀suggested฀an฀impaired฀cross-linking฀of฀
elastin,฀and฀led฀to฀the฀isolation฀of฀a฀soluble฀precursor,฀tropoelastin฀(~72,000฀kDa),฀from฀
the฀aortas฀of฀the฀copper-deicient฀animals.฀Elastin฀is฀encoded฀by฀a฀single฀gene฀with฀an฀
exceptionally฀high฀intron/exon฀ratio.฀It฀is฀mainly฀expressed฀by฀ibroblasts฀and฀chondro-
blasts.฀ Sequencing฀ the฀ tropoelastin฀ polypeptide฀ identiied฀ alternating฀ short฀ hydrophilic฀
and฀hydrophobic฀domains.฀The฀hydrophilic฀domains฀are฀rich฀in฀lysine฀(K)฀with฀adjacent฀
alanine฀(A)฀or฀proline฀(P)฀residues฀that฀become฀cross-linked฀(KA฀and฀KP฀domains).฀The฀
hydrophobic฀domains฀are฀rich฀in฀valine฀(V),฀proline฀(P),฀and฀glycine฀(G),฀often฀as฀VPGVG฀
or฀ VGGVG฀ repeats฀ with฀ or฀ without฀ alanine฀ (A)฀ and฀ are฀ ultimately฀ responsible฀ for฀ the฀
elasticity฀(Fig.฀6.4a).
After฀tropoelastin฀is฀synthesized฀into฀the฀rough฀endoplasmic฀reticulum,฀its฀signal฀pep-
tide฀is฀removed฀by฀a฀protease.฀The฀hydrophobic฀domains฀in฀the฀molecule฀bind฀to฀a฀67-kDa฀
chaperone,฀preventing฀self-aggregation฀and฀additional฀proteolysis.฀[Chaperones฀are฀pro-
teins฀that฀mediate฀the฀folding฀of฀proteins฀and฀sometimes฀stabilize฀conformations฀that฀pro-
mote฀binding฀to฀other฀proteins.]฀The฀hydrophilic,฀C-terminal฀domain฀is฀a฀KP฀domain฀which฀
does฀ not฀ bind฀ the฀ chaperone.฀ This฀ KP฀ domain฀ mediates฀ the฀ attachment฀ of฀ tropoelastin,฀
mostly฀ to฀ ibrillin-2฀ microibrils฀ with฀ bound฀ microibril-associated฀ glycoprotein-1,฀
86 6 Elastic Fibers and Proteoglycans

a
2 4 6 8 10 12 14 16 18 20 22 24 26 26A 28 30 32 36

Signal peptide KP domains KA domains Hydrophobic domains

*Alternatively spliced domains Domain 26A Domain 36

b
Compressed
Fibrillin
aggregate
MAGP-1
with other
proteins
Tropoelastin molecules that have lost their chaperone
protein by associating with MAGP-1 and fibrillin

Fig. 6.4฀ Human฀tropoelastin฀domains.฀Each฀domain฀corresponds฀to฀an฀exon,฀which฀is฀irregular฀in฀

size;฀the฀gene฀has฀a฀ratio฀of฀intron฀to฀exon฀DNA฀of฀about฀20:1,฀one฀of฀the฀largest฀known.฀This฀ratio฀
is฀only฀about฀8:1฀in฀ibrillar฀collagens.฀The฀signal฀peptide฀is฀cleaved฀in฀the฀endoplasmic฀reticulum,฀
leaving฀tropoelastin.฀(a)฀Elastin฀has฀hydrophilic฀and฀hydrophobic฀domains.฀The฀lysine-rich฀KA฀
and฀KP฀domains฀(colored)฀are฀hydrophilic฀and฀involved฀in฀cross-linking.฀They฀are฀interspersed฀
within฀mostly฀longer฀hydrophobic฀regions฀(white).฀Asterisks฀indicate฀domains฀that฀may฀be฀included฀
or฀spliced฀out฀(alternative฀splicing),฀permitting฀minor฀differences฀in฀elastin฀structure฀for฀different฀
tissues.฀The฀C-terminal฀domain฀(domain฀36)฀is฀essential฀for฀the฀interaction฀of฀tropoelastin฀with฀
ibrillin฀ to฀ form฀ elastic฀ ibers฀ (illustrated฀ in฀ b).฀ Domain฀ 26฀ is฀ critical฀ for฀ the฀ succeeding฀ step,฀
coacervation,฀whereby฀the฀ibrillin–tropoelastin฀aggregates฀form฀ilamentous฀structures฀that฀then฀
cross-link฀and฀develop฀into฀elastic฀ibers;฀recombinant฀tropoelastin฀lacking฀in฀domain฀26฀neither฀
coacervates฀ nor฀ cross-links.฀ Domain฀ 26A฀ is฀ an฀ exceptionally฀ hydrophilic฀ region฀ that฀ is฀ usually฀
spliced฀ out.฀ (b)฀ Association฀ of฀ ibrillin฀ with฀ elastin.฀ Fibrillin-associated฀ protein฀ MAGP-1฀ (light฀
blue฀ovals)฀binds฀to฀elastin฀(green)฀by฀displacing฀the฀chaperone฀(a฀–฀Reprinted฀from฀Mithieux฀S,฀
Weiss฀ AS.฀ (2005)฀ “Elastin.”฀ Adv.฀ Prot.฀ Chem.฀ 70:437–461;฀ with฀ permission฀ from฀ Elsevier.฀ b–
Original฀igure฀derived฀by฀adding฀a฀representation฀of฀elastin฀to฀Fig.฀6.2)

(MAGP-1;฀Fig.฀6.4b).฀The฀positively฀charged฀lysine฀residues฀of฀tropoelastin฀interact฀with฀
the฀ negatively฀ charged฀ ibrillin,฀ forcing฀ release฀ of฀ the฀ chaperone฀ and฀ exposing฀ the฀ tro-
poelastin฀hydrophobic฀domains฀to฀the฀aqueous฀environment.฀Exposure฀of฀one฀such฀domain฀
two-thirds฀ of฀ the฀ way฀ to฀ the฀ tropoelastin฀ C-terminus฀ causes฀ the฀ microibril-bound฀ tro-
poelastin฀molecules฀to฀repel฀water฀by฀coaggregating฀(coacervation).฀Coacervation฀causes฀
the฀ attached฀ microibrils฀ to฀ enclose฀ the฀ tropoelastin฀ molecules,฀ limiting฀ their฀ aqueous฀
exposure.฀Coacervation฀is฀essential฀for฀the฀tropoelastin฀molecules฀to฀align฀for฀cross-link-
ing.฀Later฀in฀life,฀the฀lack฀of฀expression฀of฀ibrillin-2฀makes฀repaired฀elastic฀ibers฀weaker฀
than฀those฀laid฀down฀during฀development.
Cross-linking฀of฀the฀tropoelastin฀molecules฀within฀the฀tropoelastin–ibrillin฀aggregates฀
is฀ mediated฀ by฀ lysyl฀ oxidase,฀ the฀ same฀ enzyme฀ responsible฀ for฀ cross-linking฀ collagen฀
ibers.฀In฀the฀KA฀domains,฀lysine฀residues฀are฀typically฀found฀in฀clusters฀of฀two฀or฀three฀
amino฀acids,฀separated฀by฀two฀or฀three฀alanine฀residues.฀These฀regions฀are฀proposed฀to฀be฀
a-helical฀with฀3.6฀residues฀per฀turn฀of฀helix,฀which฀has฀the฀effect฀of฀positioning฀two฀lysine฀
6.2.1. Elastin 87

side-chains฀on฀the฀same฀side฀of฀the฀helix,฀facilitating฀the฀formation฀of฀desmosine฀cross-
links฀ (Fig.฀ 6.5).฀ In฀ the฀ KP฀ domains,฀ the฀ lysine฀ pairs฀ are฀ lanked฀ by฀ prolines฀ and฀ bulky฀
hydrophobic฀amino฀acids.฀Desmosine฀and฀isodesmosines฀have฀not฀been฀found฀in฀associa-
tion฀with฀KP฀domains,฀probably฀due฀to฀the฀steric฀constraints฀imposed฀by฀the฀prolines.฀The฀
loss฀of฀many฀positively฀charged฀lysine฀residues฀following฀cross-linking฀makes฀elastin฀ibers฀
among฀the฀most฀insoluble฀proteins฀in฀the฀body฀and฀much฀less฀soluble฀than฀tropoelastin.฀It฀
is฀not฀clear฀exactly฀how฀lysyl฀oxidase฀accesses฀the฀KA฀and฀KP฀domains฀at฀the฀center฀of฀the฀
tropoelastin–ibrillin฀aggregates.
The฀hydrophobic฀domain฀of฀elastin฀is฀a฀compact,฀dynamic฀structure฀which฀forms฀short-
lived฀interconverting฀structures:฀distorted฀b฀strands,฀luctuating฀b฀turns,฀and฀buried฀hydro-
phobic฀residues.฀Nevertheless,฀the฀numerous฀amide฀groups฀in฀the฀peptide฀bonds฀can฀still฀
hydrogen฀ bond฀ with฀ water.฀ The฀ overall฀ structure฀ is฀ therefore฀ a฀ compact฀ amorphous฀

a K K c C
K H H O
K A
1 1 N
4 4 5 C C
2 2
A A
3 3 D (CH2)2
B
H N N H
CH2
A A
H C CH2 CH2 CH2 CH2 C H
b O C
C +
C O
N
CH2 CH2 D B A
CH CH CH2
+
C-CHO N N (CH2)3
C C N
A
O H H

Fig. 6.5฀ Elastin฀cross-linking.฀(a)฀Lysine฀residue฀relationships.฀In฀any฀a-helical฀domain,฀each฀resi-

due฀is฀related฀to฀the฀next฀one฀by฀a฀translation฀of฀1.5Å฀along฀the฀helical฀axis฀and฀a฀rotation฀of฀100°,฀
forming฀a฀rod-like฀main฀chain฀structure฀with฀the฀side฀chains฀extended฀outward฀in฀a฀helical฀array฀as฀
indicated฀in฀the฀igure.฀For฀elastin฀KA฀sequences฀(...Lys-Ala-Ala-Lys…฀and฀...Lys-Ala-Ala-Ala-
Lys...)฀the฀140°฀rotation฀makes฀the฀fourth฀lysine฀side฀chain฀close฀to฀the฀irst฀lysine.฀The฀lysine฀in฀
position-5฀is฀equally฀close,฀but฀on฀the฀opposite฀site.฀Absent฀from฀KA฀sequences฀are฀lysine฀residues฀
in฀position-3฀or฀position-6,฀which฀would฀lie฀on฀the฀opposite฀side฀of฀the฀helix฀from฀lysine฀1฀and฀be฀
unable฀to฀participate฀in฀desmosine฀formation฀(From฀Rosenbloom฀J,฀et฀al.฀(1993฀Oct)฀“Extracellular฀
Matrix฀4:฀The฀Elastic฀Fiber.”฀FASEB฀J.฀7(13):1208–1218).฀(b)฀Tropoelastin฀lysyl฀oxidase฀action.฀
Oxidation฀of฀one฀of฀these฀lysine฀residues฀by฀lysyl฀oxidase฀results฀in฀a฀“within฀chain”฀cross-link฀
precursor฀(dehydro-lysinorleucine,฀Fig.฀4.8).฀The฀latter฀spontaneously฀interacts฀with฀allysine฀resi-
dues฀from฀a฀similar฀pair฀of฀lysine฀residues฀in฀an฀aligned,฀adjacent฀tropoelastin฀molecule฀to฀give฀
desmosine.฀As฀with฀collagen,฀the฀cross-linking฀is฀due฀to฀spontaneous฀reactivity฀of฀the฀lysyl฀oxi-
dase-generated฀ allysine฀ aldehyde฀ (Adapted฀ by฀ permission฀ from฀ Macmillan฀ Publishers฀ Ltd.฀
Rosenbloom฀J.฀(1984฀Dec)฀“Elastin:฀relation฀of฀protein฀and฀gene฀structure฀to฀disease.”฀Laboratory฀
Investigation฀51(6):605–623).฀(c)฀Structure฀of฀desmosine฀showing฀the฀lysine฀residue฀attachment฀
sites.฀Isodesmosine฀is฀similar฀except฀that฀attachment฀C฀is฀at฀carbon฀5,฀between฀A฀and฀B฀instead฀of฀
between฀D฀and฀B.฀KP฀domain฀lysine฀residues฀also฀participate฀in฀cross-links,฀corresponding฀to฀those฀
not฀ involving฀ hydroxylysine฀ in฀ collagen:฀ dehydro-lysinorleucine฀ (double)฀ or฀ dehydromerodes-
mosine฀(triple)฀cross-links.฀Elastin฀has฀an฀interchain฀lysine-derived฀cross-link฀at฀about฀every฀68฀
residues:฀i.e.,฀involving฀all฀but฀~5฀of฀the฀34฀lysine฀residues฀of฀tropoelastin฀(From฀Figure฀11.26฀in฀
Biochemistry,฀L.฀Stryer,฀3rd฀Ed.฀1988.฀W.H.฀Freeman฀&฀Co.,฀New฀York)
88 6 Elastic Fibers and Proteoglycans

structure฀less฀densely฀packed฀than฀in฀soluble฀proteins฀such฀as฀albumin฀or฀hemoglobin.฀The฀
elasticity฀is฀due฀to฀the฀increase฀in฀entropy฀from฀hydrophobic฀residues฀exposed฀to฀the฀aque-
ous฀environment฀after฀stretching฀and฀favors฀collapse฀when฀the฀stretching฀force฀is฀removed฀
(Fig.฀6.6).฀Elastin฀becomes฀brittle฀if฀dried฀and฀in฀vivo฀it฀remains฀in฀a฀dynamic฀relaxed฀state,฀
not฀a฀conformationally฀rigid฀state฀like฀ibrillin.
Elastin฀is฀synthesized฀and฀secreted฀mostly฀during฀early฀development฀and฀it฀has฀a฀half-life฀
of฀~70฀years.฀Otherwise,฀it฀is฀only฀made฀after฀an฀injury฀or฀infection,฀which฀induces฀white฀
blood฀cells,฀especially฀neutrophilic฀granulocytes฀(neutrophils)฀to฀the฀site฀(Sect.฀13.2.3).฀One฀
of฀the฀many฀proteolytic฀products฀of฀neutrophils฀is฀elastase฀(Sect.฀8.3.1),฀which฀hydrolyzes฀
elastin฀at฀sites฀between฀the฀cross-linked฀region.฀Cleavage฀is฀most฀common฀on฀the฀C-terminal฀
side฀of฀valine฀bonded฀to฀alanine฀(i.e.,฀between฀val–ala฀residues).

Fig. 6.6฀ Elastic฀iber฀ Elastic fiber

structure.฀Elastin฀is฀
cross-linked฀at฀the฀KA฀and฀
KP฀domains.฀The฀remainder฀
of฀the฀molecule฀is฀hydro-
phobic.฀When฀stretched฀
these฀hydrophobic฀regions฀
come฀into฀excessive฀contact฀
with฀the฀water฀and฀return฀to฀
a฀more฀globular฀structure฀on฀
relaxation฀(Adapted฀from฀ Relax
Fig.19-52฀in฀The฀Molecular฀
Biology฀of฀the฀Cell.฀B.฀
Stretch
Alberts฀et฀al.,฀4th฀Ed.฀2002,฀
Garland฀Science,฀Taylor฀&฀ Single elastin molecule
Francis฀Group,฀New฀York)
Cross link

Elastin฀ is฀ encoded฀ by฀ a฀ single฀ gene,฀ tropoelastin,฀ which฀ has฀ an฀ exceptionally฀ high฀
intron/exon฀ratio.฀It฀is฀expressed฀by฀ibroblasts฀and฀chondroblasts฀along฀with฀microi-
brillar฀ components.฀ In฀ the฀ endoplasmic฀ reticulum,฀ tropoelastin฀ remains฀ soluble฀ by฀
binding฀ to฀ a฀ chaperone.฀ On฀ secretion,฀ the฀ chaperone-free฀ C-terminal฀ region฀ of฀ tro-
poelastin฀and฀its฀many,฀positively฀charged฀lysine฀residues฀are฀attracted฀to฀negatively฀
charged฀ ibrillin-2฀ and฀ an฀ associated฀ glycoprotein฀ on฀ co-secreted฀ microibrils.฀ The฀
bound฀tropoelastin฀now฀has฀its฀many฀hydrophobic฀domains฀exposed฀to฀water.฀One฀such฀
hydrophobic฀domain฀causes฀the฀tropoelastin฀to฀coacervate฀so฀that฀the฀tropoelastin฀mol-
ecules฀come฀together฀at฀the฀center฀of฀the฀microibrils฀instead฀of฀outside.฀The฀lysine฀resi-
dues฀ can฀ then฀ react฀ with฀ lysine฀ oxidase฀ and฀ cross-link฀ into฀ a฀ large฀ elastin฀ aggregate฀
within฀the฀microibers.฀Stretching฀exposes฀the฀many฀central,฀disorganized฀hydrophobic฀
regions฀to฀water฀so฀that฀it฀collapses฀when฀the฀force฀is฀removed.฀Elastin฀has฀a฀dynamic฀
relaxed฀state,฀not฀a฀conformationally฀rigid฀state฀like฀ibrillin.
6.3.1. Glycosaminoglycans 89

6.3.1.
Glycosaminoglycans

The฀most฀abundant฀glycans฀ in฀ connective฀ tissue฀ ground฀ substance฀ and฀cartilage฀ are฀ the฀

glycosaminoglycans฀ (GAGs):฀ hyaluronan,฀ and฀ protein-bound฀ glycosaminoglycans฀
฀(proteo-GAGs)฀called฀chondroitin,฀keratan฀and฀heparin฀sulfates.฀Hyaluronan฀is฀a฀polymer฀
of฀glucuronate,฀the฀salt฀of฀glucuronic฀acid฀at฀physiological฀pH,฀and฀N-acetyl฀glucosamine฀
(Fig.฀6.7).฀Hyaluronan฀is฀involved฀with฀other฀glycosaminoglycans฀in฀diverse฀physiological฀
functions:฀matrix฀structure,฀development,฀and฀ovulation.฀Various฀glycosaminoglycans฀are฀
involved฀in฀blood฀coagulation฀and฀pathological฀conditions.฀Table฀6.1฀lists฀the฀various฀pro-
teo-glycosaminoglycans฀and฀their฀functions.฀Heparin฀and฀dermatan฀sulfate฀are฀especially฀
important฀glycosaminoglycans฀on฀the฀luminal฀surface฀of฀intact฀endothelial฀cells.฀At฀this฀

− CH2OH
6 COO
O O
H 5 O H O
H H
4 1β H 1β
OH H
H HO H
3 2
H OH H NHCOCH3
Glucuronic acid N-Acetylglucosamine
OH −
CH2 C O COO O
HO O OH 1 1 O
HO NH 1
1 O O 3 O 4 OH
O HO
3 NH 4 COO O
O HO CH2
C O −
OH
CH3 Hyaluronan (hyaluronate)

Fig. 6.7฀ Hyaluronan฀ structure.฀ Hyaluronan฀ is฀ composed฀ of฀ a฀ repeating฀ dimer฀ of฀ glucuronate฀
attached฀to฀N-acetylglucosamine.฀Top฀half฀shows฀the฀ring฀conigurations฀of฀the฀glycans฀and฀the฀
bottom฀half฀shows฀the฀boat฀conigurations.฀The฀carbon฀atoms฀of฀glucuronate฀(top฀left)฀are฀num-
bered฀from฀the฀anomeric฀OH฀group฀carbon฀(C1).฀The฀OH฀group฀attached฀to฀C1฀may฀be฀conigured฀
a฀(down)฀or฀b฀(up)฀in฀the฀D-series฀sugars฀(determined฀by฀the฀coniguration฀of฀H฀and฀OH฀groups฀
around฀C5฀of฀a฀hexose,฀i.e.,฀glucose฀and฀fructose),฀and฀it฀freely฀rotates฀between฀these฀two฀con-
igurations฀through฀a฀straight฀chain฀aldehyde฀coniguration฀in฀which฀the฀ring฀is฀broken.฀This฀end฀
of฀ the฀ free฀ monosaccharide฀ and฀ of฀ hyaluronan฀ is฀ also฀ known฀ as฀ the฀ reducing฀ end฀ because฀ it฀
reduces฀cupric฀to฀cuprous฀salts฀(see฀Fig.฀15.08฀for฀a฀diagram฀of฀the฀monosaccharide฀ring฀and฀
straight฀chain฀forms).฀In฀hyaluronan,฀the฀C1฀OH฀group฀is฀always฀connected฀to฀the฀adjoining฀resi-
due฀in฀the฀b฀coniguration฀as฀shown.฀Glucuronate฀is฀glucose฀in฀which฀the฀CH2OH฀group฀attached฀
to฀C6฀is฀oxidized฀to฀a฀carboxyl฀group฀(COO−).฀N-acetylglucosamine฀(top฀right)฀is฀an฀aminogly-
can,฀glucose฀with฀its฀C2฀the฀OH฀group฀replaced฀by฀an฀amine฀(forming฀glucosamine)฀and฀then฀one฀
of฀the฀two฀hydrogen฀atoms฀of฀the฀amino฀group฀replaced฀by฀an฀acetyl฀group.฀In฀hyaluronan,฀each฀
glucuronate฀residue฀is฀attached฀b1฀→฀3฀to฀N-acetylglucosamine฀and฀each฀N-acetylglucosamine฀
is฀connected฀b1฀→฀4฀to฀the฀adjacent฀glucuronate฀(bottom฀half)฀(Upper฀half฀is฀from฀Fig.฀18-15฀in฀
Biochemistry,฀ L.฀ Stryer,฀ 4th฀ Ed.฀ 1995.฀ W.H.฀ Freeman฀ &฀ Co.,฀ New฀ York฀ and฀ lower฀ half฀ is฀
Fig.฀19-38฀in฀The฀Molecular฀Biology฀of฀the฀Cell,฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002,฀Garland฀Science,฀
Taylor฀&฀Francis฀Group,฀New฀York)
90 6 Elastic Fibers and Proteoglycans

Table 6.1฀ Some฀common฀proteo-glycosaminoglycans฀(proteo-GAGs)

Proteoglycan Core฀protein฀฀ Type฀of฀GAG฀ No.฀of฀ Location Functions
mol฀wt chains chains
Aggrecan 210,000 Chondroitin฀฀ ~130 Cartilage Mechanical฀support;฀
and฀keratan฀ large฀aggregates฀with฀
sulfate hyaluronan
Betaglycan ฀ 36,000 Chondroitin฀฀ 1 Cell฀surface฀฀ Binds฀TGF-beta
or฀dermatan฀ and฀matrix
sulfate
Decorin ฀ 40,000 Chondroitin฀฀ 1 All฀ Binds฀type฀I฀collagen฀
or฀dermatan฀ connective฀ ibrils฀and฀TGF-beta
sulfate tissues
Perlecan 600,000 Heparan฀฀ 2–15 Basal฀฀ Structural฀and฀iltering฀
sulfate laminae function฀in฀basal฀lamina
Syndecan ฀ 32,000 Chondroitin฀฀ 1–3 Epithelial฀฀ Cell฀adhesion;฀binds฀
and฀heparan฀ cell฀surface ibroblast฀and฀other฀
sulfate growth฀factors
Adapted฀from฀Table.19-4฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀
Garland฀Science,฀Taylor฀&฀Francis฀Group,฀New฀York

site,฀the฀inner฀surface฀of฀blood฀vessels,฀they฀inactivate฀blood฀clotting฀outside฀sites฀of฀injury฀
(Sect.฀ 11.5.1).฀ In฀ addition,฀ heparin฀ is฀ stored฀ in฀ mast฀ cells฀ within฀ the฀ blood฀ and฀ may฀ be฀
released฀to฀inhibit฀excessive฀blood฀clotting฀after฀injury฀or฀infection.฀Heparin฀on฀the฀cell฀
surface฀is฀also฀part฀of฀the฀ibroblast฀growth฀factor฀receptor฀associated฀with฀tissue฀develop-
ment฀and฀repair฀(Sect.฀13.2.5).
Glycosaminoglycans฀ are฀ solubilized฀ from฀ stromal฀ or฀ other฀ tissues฀ by฀ extracting฀ the฀
source฀tissue฀with฀dilute฀acid฀or฀alkali.฀Hyaluronan฀is฀electrostatically฀bound฀to฀speciic฀
proteins฀ called฀ hyaladherins,฀ which฀ possess฀ a฀ structural฀ domain฀ of฀ ~100฀ amino฀ acids฀
termed฀a฀link฀module.฀Other฀glycosaminoglycans฀are฀O-linked฀to฀serine฀and฀threonine฀resi-
dues฀of฀polypeptides฀and฀these฀bonds฀hydrolyze฀before฀the฀rest฀of฀the฀polysaccharide.฀The฀
protein฀moiety฀precipitates฀when฀trichloroacetic฀acid฀or฀ammonium฀sulfate฀is฀added฀to฀the฀
cooled฀mixture.฀The฀composition฀of฀the฀GAGs฀(including฀hyaluronan)฀was฀identiied฀by฀
chromatographic฀separation฀of฀the฀puriied฀polysaccharides,฀followed฀by฀their฀hydrolysis฀
in฀ boiling฀ 1.0฀ M฀ HCl฀ for฀ 2–4฀ h฀ and฀ identiication฀ of฀ the฀ individual฀ monosaccharide฀
components.
Hyaluronan฀is฀synthesized฀by฀a฀transmembrane฀enzyme฀(hyaluronan฀synthetase)฀from฀
ibroblasts,฀chondroblasts,฀and฀osteoblasts฀and฀is฀degraded฀by฀liver฀endothelial฀cells.฀The฀
inner฀(cytosolic)฀face฀of฀hyaluronan฀synthetase฀binds฀to฀uridine฀diphosphate฀(UDP)฀acti-
vated฀precursors,฀UDP-glucuronate฀and฀UDP-N-acetylglucosamine฀(Fig.฀6.8a).฀There฀are฀
two฀binding฀domains,฀one฀for฀glucuronate฀and฀one฀for฀N-acetyl฀glucosamine฀that฀the฀syn-
thetase฀alternates,฀so฀that฀the฀correct฀order฀of฀insertion฀into฀the฀polymer฀is฀maintained.฀The฀
UDP฀attached฀to฀the฀monosaccharide฀added฀to฀the฀chain฀is฀not฀hydrolyzed฀until฀the฀next฀
UDP-monosaccharide฀ is฀ added฀ (Fig.฀ 6.8b).฀ The฀ chain฀ therefore฀ grows฀ by฀ the฀ incoming฀
UDP-monosaccharide฀hydrolyzing฀the฀UDP฀attached฀to฀the฀hyaluronan’s฀reducing฀end.฀By฀
6.3.1. Glycosaminoglycans 91

Fig. 6.8฀ Hyaluronan฀biosynthesis.฀Hyaluronan฀is฀synthesized฀by฀extending฀the฀reducing฀end฀where฀

uridine฀diphosphate฀(UDP)฀is฀attached฀to฀the฀glycan฀residues.฀(a)฀Structure฀of฀UDP-glucuronate฀
(A-UDP).฀ (b)฀ Synthesis฀ of฀ hyaluronan.฀ Top:฀ The฀ chain฀ begins฀ when฀ the฀ UDP-glucuronate฀ adds฀
UDP-aminoglycan฀(N),฀which฀retains฀its฀UDP฀(red฀UDP฀terminus).฀Middle:฀The฀next฀glycan฀must฀
be฀the฀UDP-glucuronate฀(A),฀which฀displaces฀the฀terminal฀UDP฀(green฀UDP฀terminus).฀Bottom:฀
The฀third฀glycan฀must฀be฀another฀UDP-aminoglycan฀(N)฀molecule,฀which฀displaces฀the฀terminal฀
UDP฀of฀UDP-glucuronate฀(A)฀at฀the฀end฀of฀the฀chain฀when฀it฀adds฀(red฀UDP฀terminus).฀This฀syn-
thesis฀ is฀ the฀ reverse฀ of฀ the฀ synthesis฀ of฀ most฀ other฀ polysaccharides฀ (e.g.,฀ glycogen,฀ starch,฀ and฀
other฀glycosaminoglycans),฀which฀elongate฀by฀adding฀to฀the฀nonreducing฀end.฀The฀UDP฀remains฀
attached฀to฀the฀irst฀residue฀of฀the฀chain฀and฀each฀donor฀loses฀its฀UDP฀when฀it฀adds฀to฀the฀chain฀(a฀฀
–฀Slightly฀modiied฀from:฀Lehninger฀Principles฀of฀Biochemistry,฀Nelson,฀D.L.฀and฀Cox,฀M.M.,฀4th฀
Ed.฀2005.฀W.H.฀Freeman฀&฀Co.,฀New฀York;฀b฀–฀Adapted฀from฀Tlapak-Simmons,฀V.฀et฀al.฀(2005฀
Apr฀ 1)฀ “Hyaluronan฀ biosynthesis฀ by฀ class฀ I฀ streptococcal฀ hyaluronan฀ synthases฀ occurs฀ at฀ the฀
reducing฀end.”฀J.฀Biol.฀Chem.฀280(13):13012–13018)
92 6 Elastic Fibers and Proteoglycans

contrast,฀glycogen฀or฀starch฀are฀extended฀from฀the฀non-reducing฀end;฀the฀incoming฀NDP฀is฀
hydrolyzed,฀not฀the฀NDP฀at฀the฀end฀of฀the฀polymer.฀
The฀length฀of฀the฀polymer฀is฀probably฀controlled฀by฀the฀intracellular฀supply฀of฀the฀two฀
UDP-monosaccharides.฀If฀one฀monomer฀becomes฀depleted,฀chain฀lengthening฀stops฀and฀
the฀terminal฀UDP฀is฀hydrolyzed.฀The฀reducing฀end฀of฀the฀polymer฀in฀the฀cytosol฀is฀now฀
free฀to฀pass฀through฀the฀membrane฀but฀it฀may฀remain฀bound฀to฀the฀cell฀surface.฀Hyaluronan฀
is฀degraded฀into฀large฀fragments฀in฀the฀stroma฀by฀hyaluronidase฀from฀the฀same฀cells฀that฀
make฀hyaluronan.฀Hyaluronan฀fragments฀diffuse฀into฀the฀lymphoid฀circulation฀and฀eventu-
ally฀the฀blood.฀In฀the฀liver,฀endothelial฀cells฀possessing฀hyaluronan฀receptors฀bind฀to฀and฀
endocytose฀the฀fragments,฀passing฀them฀to฀lysosomal฀vesicles฀where฀they฀are฀degraded฀to฀
glucuronate฀and฀N-acetylglucosamine.
The฀numerous฀negative฀charges฀on฀the฀hyaluronan฀molecule฀cause฀an฀extended฀chain฀
conformation,฀allowing฀hydrogen฀bonding฀to฀salts฀and฀water฀in฀the฀extracellular฀luid.฀This฀
interaction฀expands฀the฀extracellular฀space฀and฀provides฀a฀long,฀water-absorbent฀region฀to฀
which฀various฀proteins฀bind,฀forming฀a฀gel-like฀ground฀substance.฀The฀ibrous฀proteins฀of฀
the฀ extracellular฀ matrix฀ (collagen฀ etc.)฀ lie฀ within฀ and฀ strengthen฀ the฀ gel.฀ Hyaluronan฀ is฀
secreted฀irst฀during฀organ฀formation,฀or฀when฀damaged฀tissues฀are฀repaired,฀and฀is฀replaced฀
as฀the฀tissue฀develops฀(Sect.฀13.2.5).฀The฀large฀area฀that฀a฀hyaluronan฀molecule฀occupies฀
compared฀with฀tropocollagen฀or฀albumin฀is฀illustrated฀in฀Fig.฀6.9.
Proteo-GlycosAminoGlycans฀(Proteo-GAGs)฀contain฀chondroitin-,฀dermatan-,฀keratan-,฀
or฀ heparin-sulfate฀ and฀ have฀ the฀ properties฀ of฀ proteins฀ called฀ mucins฀ (Sect.฀ 12.3.1).฀ The฀
names฀refer฀to฀the฀tissues฀where฀they฀were฀irst฀identiied฀and฀are฀usually฀most฀prominent.฀
Dermatan฀and฀keratan฀sulfate฀are฀named฀for฀the฀dermis฀of฀the฀skin฀and฀gingiva,฀chondroitin฀
for฀cartilage฀and฀heparin฀for฀liver.
Figure฀ 6.10฀ compares฀ the฀ composition฀ of฀ various฀ proteo-glycosaminoglycans฀ with฀
hyaluronan.฀UDP-glucuronate฀is฀epimerized฀to฀UDP-iduronate฀in฀dermatan฀and฀heparin฀
sulfate฀(bottom฀row).฀UDP-galactose฀is฀incorporated฀instead฀of฀UDP-glucuronate฀in฀kera-
tan฀sulfate฀(top,฀right).฀UDP-N-acetylglucosamine฀is฀incorporated฀into฀keratan฀and฀hepa-
rin,฀but฀UDP-N-acetylgalactosamine฀is฀incorporated฀into฀chondroitin฀and฀dermatan.฀Once฀

Fig. 6.9฀ Hyaluronan฀and฀other฀molecules.฀The฀volume฀of฀a฀single,฀hydrated฀hyaluronan฀molecule฀is฀

compared฀with฀those฀of฀monomeric฀tropocollagen฀(triple฀helix),฀glycogen฀and฀albumin฀(Adapted฀
from฀Fig.฀19-37฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002,฀Garland฀
Science,฀Taylor฀&฀Francis฀Group,฀New฀York)
6.3.1. Glycosaminoglycans 93

the฀polymer฀is฀made,฀the฀aminoglycan฀of฀chondroitin฀and฀keratan฀is฀sulfated฀at฀the฀C-6฀
position,฀ whereas฀ that฀ of฀ dermatan฀ is฀ sulfated฀ at฀ the฀ C-4฀ position.฀ Heparin฀ sulfate฀ is฀ a฀
unique฀dimer.฀The฀repeating฀link฀in฀the฀dimer฀is฀b1,4฀(not฀b1,3฀as฀in฀the฀other฀glycosamino-
glycans),฀and฀each฀dimer฀is฀linked฀a1,4.฀Heparin฀is฀additionally฀unique฀because฀its฀idu-
ronate฀ residues฀ are฀ sulfated฀ (in฀ the฀ 3¢-OH฀ position)฀ and฀ its฀ N-acetyl฀ group฀ on฀ the฀
glucosamine฀residues฀is฀replaced฀with฀an฀N-sulfate฀group.
Glycosaminoglycan฀synthesis฀occurs฀in฀the฀Golgi,฀which฀contains฀enzymes฀that฀cata-
lyze฀the฀sequential฀addition฀of฀uronate฀and฀aminoglycans.฀Serine฀hydroxyl฀groups฀in฀the฀

Glycosaminoglycan (GAG) Repeating Disaccharide Unit (A and B)

Hyaluronan Glucuronate and N-acetyl glucosamine
Chrondroitin sulfate Glucuronate and N-acetyl galactosamine
Keratan sulfate Galactose and N-acetyl galactosamine
Dermatan sulfate Iduraonate and N-acetyl galactosamine
Heparin sulfate Idurinate and N-acetyl glucosamine

COO− CH2OSO3− CH2OH CH2OSO3−

O O O O
H O HO HO H O
O H H
H H O
H H OH H
OH H H H
H H H H

H NHCOCH3 H OH H NHCOCH3
H OH
Chondroitin 6-sulfate Keratan sulfate
Glucuronate N-Acetyl-galactosamine Galactose N-Acetyl-glucosamine
−O CH2OH
H 3S H CH2OSO3−
O O O
H O O O O
H H H
COO− H
COO− H
H O
OH H OH H OH H O
H H H
H
H NHCOCH3
H OH H OSO3− H NHSO3−
Dermaten sulfate Heparin sulfate
Iduronate N-Acetyl-galactosamine Iduronate N-Acetyl-glucosamine

Fig. 6.10฀ Other฀glycosaminoglycan฀structures.฀Structures฀of฀chondroitin,฀keratan,฀dermatan,฀and฀heparin฀

sulfate฀ (The฀ hyaluronan฀ structure฀ is฀ shown฀ in฀ Fig.฀ 6.7).฀ In฀ proteo-glycosaminoglycans฀ containing฀
N-acetylgalacto฀samine฀ (chondroitin฀ and฀ dermatan),฀ the฀ uronate฀ is฀ connected฀ b1฀ →฀ 3฀ to฀
N-acetylgalactosamine฀and฀b1฀→฀4฀to฀uronate฀in฀the฀succeeding฀dimer฀(left฀half฀of฀igure).฀If฀the฀amino-
glycan฀is฀a฀glucosamine฀derivative,฀the฀galactose฀is฀connected฀b1฀→฀4฀to฀N-acetylglucosamine฀and฀b1฀
→฀3฀to฀the฀succeeding฀uronate฀for฀keratan฀sulfate฀(top฀right฀of฀igure),฀but฀iduronate฀is฀connected฀b1฀→฀
4฀and฀a1฀→฀4฀in฀heparin฀sulfate฀(bottom฀right฀of฀igure).฀(One฀way฀to฀remember฀the฀different฀sugars฀is฀
to฀ think฀ of฀ hyaluronan฀ having฀ only฀ glucose฀ derivatives,฀ keratan฀ as฀ having฀ N-acetyl฀ galactosamine฀
instead฀of฀N-acetyl฀glucosamine,฀chondroitin฀sulfate฀as฀containing฀glucuronate฀and฀galactosamine฀and฀
dermatan฀as฀containing฀iduronate฀in฀which฀the฀uronic฀sugar฀points฀down฀compared฀with฀glucuronate)฀
(Adapted฀from฀Fig.฀18-15฀in฀Biochemistry,฀L.฀Stryer,฀4th฀Ed.฀1995.฀W.H.฀Freeman฀&฀Co.,฀New฀York)
94 6 Elastic Fibers and Proteoglycans

core฀polypeptide฀have฀a฀surrounding฀domain฀that฀activates฀an฀enzyme฀to฀add฀UDP-xylose.฀
Additional฀enzymes฀add฀other฀monosaccharides,฀forming฀a฀chondroitin฀sulfate฀linker฀gly-
can฀(Fig.฀6.11)฀that฀may฀be฀a฀recognition฀signal฀for฀activating฀the฀appropriate฀synthetase฀to฀
attach฀the฀irst฀GAG฀monosaccharide.฀Keratan฀sulfate฀linkers฀฀resemble฀those฀of฀salivary฀
glycoproteins฀(Sect.฀12.2.1)฀and฀mucins฀(Sect.฀12.3.1).฀A฀class฀of฀keratan฀sulfate฀originally฀
found฀in฀the฀cornea฀of฀the฀eye฀(KS-I)฀is฀N-linked฀to฀asparagine฀by฀N-acetylglucosamine฀
and฀mannose฀residues.฀The฀attachment฀is฀irst฀synthesized฀on฀dolichol฀phosphate,฀trans-
ferred฀to฀the฀core฀protein฀and฀reduced฀in฀size฀to฀contain฀only฀the฀attachment฀glycans฀(Fig.฀
12.3).฀The฀other฀two฀keratan฀sulfate฀types฀are฀O-linked฀to฀serine฀or฀threonine฀by฀N-acetyl฀
galactosamine฀as฀described฀for฀salivary฀mucins.฀
Synthetases฀ in฀ the฀ Golgi฀ add฀ each฀ UDP-activated฀ monosaccharide฀ and฀ aminoglycan฀
alternately฀to฀the฀core฀protein฀linker฀monosaccharide.฀The฀synthetases฀are฀homologous฀to฀
hyaluronan฀synthetase,฀but฀their฀mechanism฀of฀addition฀is฀to฀the฀non-reducing฀end฀like฀
glycogen฀ or฀ starch฀ synthesis.฀ Each฀ incoming฀ monosaccharide฀ releases฀ its฀ UDP฀ as฀ it฀ is฀
added฀to฀the฀C3฀or฀C4฀−OH฀group฀of฀the฀growing฀glycosaminoglycan฀chain.฀During฀gly-
osaminoglycan฀synthesis,฀the฀GAGs฀are฀sulfated฀by฀enzymatic฀transfer฀of฀an฀acidic฀sul-
phate฀ residue฀ from฀ 3’-phosphoadenylyl-phosphosulfate.฀ The฀ completed฀ polymers฀ are฀
much฀smaller฀than฀hyaluronan,฀only฀70–200฀residues฀in฀length฀14–40฀kDa).

The฀glycosaminoglycans฀are฀beta-linked฀polysaccharides.฀Hyaluronan฀is฀unique฀in฀that฀
it฀is฀not฀O-linked฀to฀a฀protein฀in฀the฀Golgi,฀not฀synthesized฀at฀the฀nonreducing฀end฀and฀
not฀ sulfated.฀ Hyaluronan฀ is฀ also฀ much฀ longer฀ than฀ other฀ glycosaminoglycans฀ and฀
entirely฀ composed฀ of฀ glucose฀ derivatives.฀ Hyaluronan฀ is฀ synthesized฀ by฀ a฀ incoming฀
UDP-monosaccharide฀displacing฀the฀UDP฀at฀the฀reducing฀end฀of฀the฀polymer,฀whereas฀
the฀others฀are฀synthesized฀by฀loss฀of฀their฀UDP฀residue฀when฀it฀is฀added฀to฀the฀non-
reducing฀end฀like฀glycogen฀or฀starch.฀The฀protein-linked฀glycosaminoglycans฀are฀chon-
droitin฀sulfate,฀dermatan฀sulfate,฀keratan฀sulfate,฀and฀heparin฀sulfate.฀Except฀for฀some฀฀
short฀chains฀of฀heparin฀sulfate฀at฀cell฀surfaces,฀all฀four฀sulfated฀glycosaminoglycans฀are฀
found฀covalently฀linked฀to฀a฀protein.

Serine Attaching
C O
Glucuronic
H C CH2 O Xylose Galactose Galactose A B
acid
H N n
Link tetrasaccharide
Chondroitin sulfate
Core protein

Fig. 6.11฀ Linker฀ glycan฀ for฀ chondroitin฀ sulfate฀ synthesis.฀ In฀ the฀ Golgi,฀ the฀ serine฀ residue฀ –OH฀
group฀on฀a฀protein฀activates฀a฀speciic฀synthetase฀in฀the฀Golgi฀membrane฀to฀transfer฀UDP-xylose.฀
UDP฀is฀lost฀in฀making฀the฀attachment฀shown.฀UDP-activated฀galactose฀and฀glucuronate฀are฀then฀
added฀by฀other฀synthetases฀before฀chondroitin฀sulfate฀synthetase฀is฀activated;฀each฀donor฀loses฀its฀
UDP฀unlike฀hyaluronan฀synthesis.฀Keratan฀sulfate฀is฀added฀different฀linker฀glycans฀(Adapted฀from฀
Fig.฀19-39฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀
Taylor฀&฀Francis฀Group,฀New฀York)
6.4.1. Proteo-Glycosaminoglycan Core Proteins and Cartilage Collagens 95

6.4.1.
Proteo-Glycosaminoglycan Core Proteins and Cartilage Collagens

Aggrecan฀is฀a฀large฀keratan฀sulfate/chondroitin฀sulfate-linked฀proteoglycan฀(2,500฀kDa฀
mass)฀ which฀ is฀ prominent฀ in฀ cartilage,฀ but฀ also฀ present฀ in฀ brain,฀ aorta,฀ and฀ tendon.฀
Aggrecan฀ provides฀ a฀ hydrated,฀ space-illing฀ gel฀ caused฀ by฀ more฀ than฀ 100฀ polyanionic฀
(negatively฀charged)฀glycosaminoglycan฀chains฀attached฀to฀a฀polypeptide฀core.฀The฀mass฀
of฀this฀proteo-GAG฀varies฀slightly฀with฀age฀in฀humans฀(210–250฀kDa).฀Aggrecan฀฀provides฀
a฀ground฀substance฀that฀holds฀together฀the฀ibrous฀component฀of฀cartilage฀(type฀II฀colla-
gen)฀ and฀ provides฀ it฀ with฀ resilience.฀ Similar฀ amounts฀ ar฀ e฀ present฀ in฀ brain,฀ aorta,฀ and฀
tendon,฀but฀little฀is฀present฀elsewhere฀in฀the฀body.฀Versican฀is฀a฀more฀widely฀expressed,฀
related฀proteoglycan฀with฀an฀apparent฀molecular฀mass฀of฀about฀1,000฀kDa.฀It฀has฀many฀
fewer฀attached฀glycosaminoglycans฀than฀aggrecan฀and฀so฀provides฀a฀less฀viscous฀stromal฀
ground฀substance฀than฀does฀cartilage.
Together฀with฀neurocan฀and฀brevican,฀which฀are฀only฀found฀in฀brain,฀they฀form฀the฀aggre-
can฀family,฀also฀called฀the฀lectican฀or฀hyalectan฀family.The฀aggrecan฀family฀of฀proteins฀pos-
sesses฀ a฀ multi-domain฀ structure.฀ Their฀ N-terminal฀ domain฀ binds฀ to฀ hyaluronan฀ in฀ an฀
interaction฀stabilized฀by฀pair฀of฀link฀protein฀hyaladherins.฀The฀central฀domain฀of฀aggrecan฀
contains฀ many฀ serine-glycine฀ repeats฀ that฀ mostly฀ attach฀ chondroitin฀ sulfate,฀ and฀ their฀
C-terminal฀domain฀binds฀to฀oligosaccharides฀in฀the฀extracellular฀matrix฀or฀cell฀surface฀in฀an฀
interaction฀ requiring฀ calcium฀ ions.฀ The฀ C-terminal฀ domain฀ is฀ made฀ up฀ of฀ three฀ smaller฀
domains฀(modules);฀one฀or฀two฀EGF-like฀repeats,฀a฀lectin฀(glycan฀binding)-like฀module,฀and฀
a฀complement฀regulatory฀protein-like฀module฀that฀binds฀oligosaccharides฀in฀a฀reaction฀stabi-
lized฀by฀calcium฀ions฀(Fig.฀6.12).฀This฀last฀module฀is฀a฀single฀copy฀of฀a฀short฀repeat฀sequence฀
found฀in฀receptors฀for฀complement,฀a฀group฀of฀proteins฀from฀blood฀plasma฀(Sect.฀3.3.2).

Fig. 6.12฀ Peptide฀ domains฀ and฀ attached฀ glycosaminoglycans฀ in฀ aggrecan฀ and฀ versican.฀ HABR:฀
Hyaluronic฀ Acid-Binding฀ Region;฀ GAG:฀ Glycosaminoglycan-Attachment฀ domain;฀ EGF:฀
Epidermal฀Growth฀Factor-like฀repeat;฀LEC:฀C-type฀Lectin-like฀module;฀C฀(yellow):฀Complement฀
regulatory฀ protein-like฀ module฀ (see฀ text)฀ (From฀ GlycoWord฀ website:฀ http://www.glycoforum.
gr.jp/science/word/proteoglycan/PGA03E.html฀(contributed฀by฀Toshikazu฀Yada))
96 6 Elastic Fibers and Proteoglycans

a 1 µm Aggrecan aggregate

Aggrecan
molecules

b
Link proteins

Hyaluronan
molecule

Keratan
sulfate Chondroitin sulfate

Fig. 6.13฀ Aggrecan฀ and฀ aggrecan฀ aggregate฀ from฀ fetal฀ bovine฀ cartilage.฀ (a)฀ Comprehensive฀ dia-
gram฀of฀the฀hyaluronan/aggrecan฀aggregate.฀Hyaluronan฀=฀blue;฀aggrecan฀=฀green.฀(b)฀Details฀of฀
hyaluronan/aggrecan฀aggregate.฀Link฀proteins฀attach฀the฀N-terminal฀(head)฀region฀of฀numerous฀
aggrecan฀ molecules฀ to฀ the฀ hyaluronan฀ polymer฀ (blue).฀ The฀ aggrecan-attached฀ chondroitin฀ and฀
keratan฀ chains฀ (red)฀ are฀ shown฀ spreading฀ out฀ from฀ the฀ aggrecan฀ polypeptide฀ (green)฀ (From฀฀
Fig.฀19-41฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀
Taylor฀&฀Francis฀Group,฀New฀York)

Figure฀6.13a฀shows฀the฀noncovalent฀aggrecan/hyaluronan฀complex,฀which฀comprises฀
the฀ground฀substance฀(central฀mass)฀of฀cartilage฀interspersed฀between฀the฀type฀II฀collagen฀
ibers.฀The฀central฀extended฀chain฀of฀hyaluronan฀(blue)฀has฀one฀end฀still฀protruding฀into฀the฀
chondrocyte฀(not฀shown).฀The฀link฀proteins฀(hyaluronan-binding)฀attach฀the฀double฀globu-
lar฀head฀of฀aggrecan฀to฀the฀hyaluronan.฀The฀negatively฀charged฀glycosaminoglycans฀are฀
partly฀ neutralized฀ by฀ the฀ sodium฀ ions฀ in฀ the฀ extracellular฀ luid.฀ The฀ glycan฀ residues฀ all฀
stain฀strongly฀with฀uranyl฀acetate฀causing฀the฀aggrecan฀aggregate฀structure฀diagrammed฀in฀
Fig.฀6.13b฀to฀be฀clearly฀visible฀in฀an฀electron฀micrograph฀(not฀shown).
Type฀II฀collagen฀ibers฀are฀smaller฀in฀diameter฀than฀type฀I฀and฀more฀randomly฀oriented฀
within฀ the฀ proteoglycan฀ matrix.฀ These฀ ibers฀ impart฀ strength฀ and฀ compressibility฀ to฀ the฀
cartilage฀matrix,฀so฀that฀it฀resists฀large฀deformations฀in฀shape฀as฀the฀joints฀absorb฀physio-
logical฀shocks฀during฀function.฀Type฀II฀ibers฀are฀noncovalently฀cross-linked฀to฀proteogly-
cans฀by฀type฀IX฀collagen,฀a฀ibril-associated฀collagen.฀The฀triple฀helix฀of฀type฀IX฀collagen฀
is฀composed฀of฀three฀separately฀encoded฀polypeptides฀(a1IX,฀a2IX,฀a3IX)฀that฀form฀three฀
triple฀helical฀regions฀and฀two฀lexible฀noncollagenous฀domains฀(NC2฀and฀NC3)฀in฀addition฀
to฀the฀N-฀and฀C-฀terminal฀noncollagenous฀domains,฀NC1฀and฀NC4฀(Fig.฀6.14a).฀The฀lexi-
ble฀NC3฀domain฀of฀the฀a2(IX)฀chain฀has฀a฀serine฀residue฀which฀is฀covalently฀attached฀to฀a฀
chondroitin฀ or฀ dermatan฀ sulfate฀ molecule฀ and฀ is฀ ive฀ amino฀ acids฀ longer฀ than฀ the฀ same฀
6.4.1. Proteo-Glycosaminoglycan Core Proteins and Cartilage Collagens 97

Fig. 6.14฀ Structures฀of฀cartilage฀collagens.฀The฀large฀gray฀cylinders฀represent฀the฀type฀II฀colla-

gen฀triple฀helix฀with฀polypeptides฀running฀N-฀to฀C-terminal.฀Type฀II฀collagen฀is฀the฀major฀col-
lagen฀of฀cartilage.฀Wavy฀lines฀represent฀the฀triple฀helix฀of฀type฀IX฀collagen,฀a฀ibril-associated฀
collagen฀with฀interrupted฀triple฀helices฀(Table฀3.1).฀(a)฀Type฀IX฀collagen:฀NC1฀through฀NC4฀
indicate฀the฀four฀noncollagenous฀(NC)฀domains฀(see฀text)฀and฀COL1฀through฀COL3฀the฀three฀
collagenous฀ (COL)฀ domains.฀ The฀ domains฀ are฀ numbered฀ C-terminal฀ to฀ N-terminal.฀ Type฀ IX฀
collagen฀it฀around฀the฀triple฀helical฀region฀of฀type฀II฀collagen฀such฀that฀the฀COL3฀and฀NC4฀
ends฀protrude.฀A฀glycosaminoglycan฀(GAG)฀is฀covalently฀attached฀to฀a฀serine฀residue฀at฀NC3฀
and฀ also฀ protrudes฀ from฀ the฀ iber.฀ The฀double฀ arrow฀ points฀ to฀ two฀ pyridinoline฀ crosslinks฀ at฀
each฀ end฀ of฀ the฀ ibrillar฀ gap฀ regions.฀ The฀ right฀ arrow฀ cuts฀ through฀ a฀ pyridinoline฀ cross-link฀
between฀the฀COL2฀and฀the฀NC1฀region฀of฀type฀IX฀collagen,฀and,฀immediately฀below฀it,฀also฀a฀
pyridinoline฀between฀the฀same฀region฀and฀the฀other฀end฀of฀the฀COL฀2฀region.฀The฀NC1฀region฀
is฀ also฀ disulide฀ bonded.฀ (b)฀ Diagram฀ of฀ interaction฀ between฀ types฀ IX฀ and฀ type฀ II฀ collagens฀
derived฀from฀a฀study฀of฀pyridinoline฀cross-linked฀polypeptides.฀A฀collagen฀XI฀iber฀with฀two฀
collagenous฀domains฀separated฀by฀a฀central฀noncollagenous฀domain฀is฀shown฀at฀the฀bottom฀left฀
of฀the฀igure.฀Both฀Collagen฀type฀IX฀and฀XI฀limit฀the฀thickness฀of฀the฀ibers฀of฀type฀II฀collagen.฀
Type฀ IX฀ collagen฀ also฀ re-orientates฀ the฀ ibers฀ (Adapted฀ from฀ Fig.฀ 2฀ in฀ Eyre฀ DR,฀ Wu฀ J-J,฀
Fernandes฀RJ,฀Pietka฀TA฀and฀Weis฀MA.฀Recent฀developments฀in฀cartilage฀research:฀matrix฀biol-
ogy฀of฀the฀collagen฀II/IX/XI฀heteroibril฀network.฀Biochemical฀Society฀Transactions.฀(30):฀part฀
6฀894–899,฀2002:฀Reproduced฀with฀฀permission฀from฀Portland฀Press)
98 6 Elastic Fibers and Proteoglycans

Aggrecan฀ is฀ a฀ large฀ keratan฀ sulfate/chondroitin฀ sulfate-linked฀ proteoglycan฀ (2,500฀

kDa฀mass)฀which฀is฀prominent฀in฀cartilage,฀but฀also฀present฀in฀brain,฀aorta,฀and฀tendon.฀
It฀provides฀a฀ground฀substance฀for฀type฀II฀collagen,฀the฀ibrous฀component฀of฀cartilage,฀
which฀it฀provides฀with฀resilience.฀Its฀N-terminal฀domain฀binds฀to฀hyaluronan฀an฀inter-
action฀ stabilized฀ by฀ link฀ protein.฀ Its฀ central฀ domain฀ contains฀ many฀ serine-glycine฀
repeats฀that฀attach฀glycosaminoglycan,฀and฀its฀C-terminal฀domain฀binds฀to฀oligosac-
charides฀in฀the฀extracellular฀matrix฀or฀attached฀to฀the฀cell฀surface.฀Type฀II฀collagen฀is฀
associated฀with฀types฀IX฀and฀XI฀collagen฀which฀control฀the฀iber฀thickness฀and฀orienta-
tion.฀Type฀IX฀collagen฀is฀especially฀important฀for฀interaction฀with฀chondroitin฀sulfate฀
in฀the฀aggrecan–glycosaminoglycan฀aggregate.฀Within฀the฀glycosaminoglycan฀aggre-
gate,฀the฀reducing฀end฀of฀the฀hyaluronan฀remains฀attached฀to฀the฀outer฀surface฀of฀the฀
chondroblast฀and฀the฀rest฀of฀the฀molecule,฀to฀which฀the฀aggrecan฀glycosaminoglycan฀
unit฀is฀attached,฀protrudes฀into฀the฀stroma.฀The฀ibers฀impart฀strength฀and฀compress-
ibility฀to฀a฀viscous฀matrix฀enabling฀it฀to฀resist฀large฀deformations฀in฀shape฀as฀the฀joints฀
absorb฀physiological฀shocks฀during฀function.

globular฀ domains฀ in฀ the฀ a1(IX)฀ a3(IX)฀ chains.฀ The฀ NC4฀ domain฀ also฀ protrudes,฀ and฀
together฀with฀the฀glycosaminoglycan-attached฀NC3฀domain,฀anchors฀the฀cartilage฀collagen฀
ibril฀to฀proteoglycans฀and฀other฀components฀of฀the฀matrix.฀The฀lexible฀NC2฀domain฀of฀
type฀IX฀collagen฀interacts฀with฀type฀XI฀collagen฀whose฀collagen฀domain฀has฀a฀rigid฀bend฀
that฀limits฀the฀thickness฀of฀the฀type฀II฀cartilage฀collagen฀ibers฀(Fig.฀6.14b).

6.5.1.
Major Collagen–Glycosaminoglycan Interactions

Figure฀6.15฀summarizes฀all฀the฀major฀proteins฀and฀glycosaminoglycans฀involved฀in฀three฀
types฀of฀connective฀tissues:฀cartilage,฀basal฀membrane,฀and฀dermis.฀Each฀of฀these฀tissues฀
has฀a฀distinctive฀glycosaminoglycan฀and฀collagen.฀Cartilage฀contains฀type฀II฀collagen฀and฀
aggrecan฀as฀discussed฀above.฀An฀epithelial฀lamina฀densa฀contains฀contain฀type฀IV฀collagen฀
(Sect.฀5.1.1)฀and฀also฀perlecan,฀a฀proteo-GAG฀containing฀two฀to฀15฀heparin฀sulfate฀chains.฀
The฀dermis฀beneath฀the฀basal฀lamina฀contains฀type฀I฀collagen฀and฀decorin,฀a฀small฀mole-
cule฀with฀a฀single฀long฀chondroitin฀or฀dermatan฀sulfate฀chain.
Together฀with฀type฀I฀collagen,฀decorin฀is฀widespread฀within฀the฀connective฀tissue฀stroma฀
where฀it฀ensures฀proper฀formation฀and฀stability฀of฀collagen฀ibrils.฀It฀is฀composed฀of฀an฀
N-terminal฀region฀to฀which฀the฀single฀chondroitin/dermatan฀sulfate฀side฀chain฀is฀attached,฀
and฀a฀central฀region฀composed฀of฀ten฀leucine-rich฀repeats฀(LX2LXLX2NXL).฀The฀leucine฀
repeats฀are฀surrounded฀by฀a฀distinct฀pattern฀of฀Cys฀residues฀(CX3CXCX6C)฀which฀separate฀
the฀leucine฀repeats฀from฀the฀N-terminus฀and฀also฀make฀up฀the฀C-terminus฀of฀the฀protein.฀
The฀ leucine-rich฀ region฀ is฀ the฀ site฀ of฀ interaction฀ with฀ other฀ proteins฀ including฀ collagen฀
ibrils,฀to฀which฀decorin฀binds฀(“decorates”)฀with฀high฀afinity.฀Decorin฀binds฀to฀type฀I฀col-
lagen฀triple฀helices฀at฀the฀major฀intermolecular฀cross-link฀site฀near฀the฀C-terminus.฀Biglycan฀
6.5.1. Major Collagen–Glycosaminoglycan Interactions 99

Fig. 6.15฀ Major฀extracellular฀

matrix฀proteins฀and฀
glycosaminoglycans.฀
Protein฀is฀shown฀in฀green฀
and฀glycosaminoglycans฀in฀
red.฀The฀length฀of฀the฀
polypeptides฀and฀glycan฀
chains฀are฀approximately฀
proportional฀to฀their฀sizes.฀
The฀list฀is฀limited฀to฀major฀
structural฀proteins฀discussed฀
in฀this฀and฀the฀previous฀฀
two฀chapters.฀(a)฀Cartilage.฀฀
(b)฀Basal฀lamina.฀(c)฀Dermis฀
(Modiied฀from฀Fig.฀19-59฀
in:฀The฀Molecular฀Biology฀
of฀the฀Cell,฀B.฀Alberts฀et฀al.,฀
4th฀Ed.฀2002,฀Garland฀
Science,฀Taylor฀&฀Francis฀
Group,฀New฀York)

(not฀ shown฀ in฀ Fig.฀ 6.15)฀ is฀ homologous฀ to฀ decorin,฀ but฀ it฀ contains฀ two฀ attached฀ GAGs฀
instead฀of฀only฀one,฀chondroitin฀sulfate฀in฀addition฀to฀dermatan฀sulfate.฀In฀tendons,฀carti-
lage฀ and฀ periodontal฀ ligament,฀ changes฀ in฀ biglycan฀ binding฀ to฀ collagen฀ or฀ proteo-GAG฀
aggregates฀activate฀TGF-β฀after฀tissue฀damage,฀whereas฀the฀ibrillin-mediated฀activation฀
(Sect฀6.1.1)฀may฀associate฀more฀with฀developmental฀changes.

Proteo-glycosaminoglycans฀ determine฀ the฀ structure฀ of฀ cartilage,฀ the฀ permeability฀ of฀

basement฀ membranes฀ to฀ small฀ molecules,฀ the฀ stability฀ of฀ collagen฀ ibrils,฀ and฀ the฀
response฀of฀tendons,฀cartilage฀and฀periodontal฀ligaments฀to฀injury.
 Collagen Synthesis, Genetic
Diseases, and Scurvy 7

Collagen฀synthesis฀and฀degradation฀are฀central฀to฀the฀well-being฀of฀the฀teeth฀and฀perio-
dontium.฀ Section฀ 1฀ is฀ an฀ overview฀ of฀ collagen฀ synthesis฀ and฀ secretion฀ with฀ special฀
emphasis฀on฀the฀ibrillar฀collagens.฀Section฀2฀describes฀mutations฀of฀ibrillar฀collagen,฀
the฀associated฀diseases,฀and฀the฀effects฀of฀these฀and฀other฀stromal฀protein฀mutations฀on฀
tooth฀structure.฀Section฀3฀describes฀the฀enzymes฀and฀their฀cofactors฀that฀process฀procol-
lagen฀in฀the฀endoplasmic฀reticulum.฀The฀chapter฀concludes฀with฀a฀discussion฀of฀the฀role฀
of฀ascorbate฀in฀collagen฀synthesis,฀the฀changes฀associated฀with฀its฀deiciency฀(scurvy),฀
and฀its฀function฀as฀a฀reducing฀agent฀(antioxidant)฀in฀plants฀and฀animals฀(Sect.฀4).

7.1.1.
Intracellular Collagen Synthesis

Fibrillar฀collagen฀is฀synthesized฀by฀ibroblasts,฀chrondroblasts,฀osteoblasts,฀odontoblasts฀
and฀cementoblasts.฀As฀each฀collagen฀a-polypeptide฀is฀synthesized,฀an฀N-terminal฀translo-
cation฀signal฀moves฀it฀into฀the฀ER฀lumen฀where฀the฀signal฀peptide฀is฀removed฀by฀a฀protease.฀
The฀resulting฀product฀is฀procollagen,฀a฀tropocollagen฀extended฀domain฀with฀small฀folded฀
domains฀at฀its฀N-฀and฀C-terminus฀(Sect.฀4.2.1).฀The฀tropocollagen฀domains฀are฀hydroxy-
lated฀at฀selected฀proline฀and฀lysine฀residues,฀glycosylated฀at฀some฀hydroxylysine฀residues,฀
and฀form฀trimers฀at฀the฀C-terminal฀propeptide฀regions฀within฀the฀ER฀lumen฀(Fig.฀7.1).
Trimer฀formation฀begins฀when฀a฀domain฀within฀the฀C-terminal฀a1-propeptide฀sponta-
neously฀binds฀to฀an฀acceptor฀domain฀in฀the฀C-terminal฀a2-propeptide.฀The฀heterodimer฀
then฀attaches฀a฀second฀a1฀C-terminal฀propeptide฀to฀form฀the฀trimer,฀2฀a1฀chains฀and฀one฀
a2฀chain.฀The฀heterotrimer฀is฀stabilized฀by฀cystine฀cross-links฀catalyzed฀by฀protein฀disul-
ide฀isomerase,฀β-subunits฀of฀proline฀hydroxylase฀whose฀a-subunits฀hydroxylate฀the฀tropo-
collagen฀ proline฀ residues.฀ Lysine฀ is฀ hydroxylated฀ by฀ lysine฀ hydroxylase฀ which฀ is฀
homologous฀to฀the฀proline฀hydroxylase฀a-subunits฀only฀(Sect.฀7.3.1).฀After฀hydroxylation,฀
glycosylation฀and฀cross-linking฀are฀completed,฀a฀soluble฀chaperone฀called฀hsp47฀replaces฀
proline฀hydroxylase฀and฀guides฀triple฀helix฀formation฀across฀the฀tropocollagen฀domains฀
(Fig.฀7.1b).฀This฀last฀process฀is฀reminiscent฀of฀the฀chaperone฀which฀causes฀newly฀synthe-
sized฀elastin฀polypeptides฀to฀associate฀with฀ibrillin฀(Sect.฀6.2.1).

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 101
DOI:฀10.1007/978-3-540-88116-2_7,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
102 7 Collagen Synthesis, Genetic Diseases, and Scurvy

a b
Association of chains with
1. Synthesis of PH / PDI and Hsp47 chaperone
pro-a chain
2. Hydroxylation of PH / PDI
selected prolines
and lysines Ribosomes
OH OH
OH at ER N-ter
s C-ter
3. Glycosylation of H2N s disufide
selected hydroxy- OH OH Newly-synthesized OH bonds
procollagen chains
lysines ss
H2N COOH
OH
N-ter propeptide OH OH
H 4. HSP47-Guided assembly Hsp47
O OH of 3 pro-α chains
HO (from disulfide-bonded
3 pro-a C-ter propeptide OH OH
chains OH OH associations) N C
OH
OH OH

ER ER
Cytosol

Cytosol
OH OH
Transport vesicle
Transport
vesicle OH OH
OH O O OH
H H N C
OH OH
to cis-Golgi stack (cisterna)

Fig. 7.1฀ Collagen฀synthesis฀and฀processing฀in฀the฀endoplasmic฀reticulum.฀(a)฀Synthesis฀and฀process-

ing฀of฀procollagen.฀Steps฀1฀through฀4฀are฀explained฀in฀the฀text.฀The฀N-terminal฀propeptide฀(green฀on฀
left)฀contains฀a฀short฀triple฀helical฀region฀that฀is฀removed฀with฀the฀propeptide.฀The฀C-terminal฀pro-
peptide฀ (green)฀ is฀ larger฀ than฀ the฀ N-terminal฀ propeptide.฀ The฀ tropocollagen฀ region฀ (red)฀ is฀ much฀
larger฀ than฀ illustrated.฀ Blue฀ hexagons฀ indicate฀ position฀ of฀ the฀ hydroxylated฀ lysine฀ residues฀ with฀
attached฀glycan.฀(b)฀Procollagen฀triple฀helix฀formation.฀Proline฀hydroxylase฀(PH,฀dark฀green฀oval)฀
is฀part฀of฀the฀inner฀wall฀of฀the฀endoplasmic฀reticulum.฀It฀binds฀to฀individual฀polypeptides฀and฀cata-
lyzes฀ proline฀ hydroxylation.฀ Following฀ hydroxylation,฀ the฀ β฀ subunit฀ of฀ PH,฀ disulide฀ isomerase฀
(PDI),฀catalyzes฀the฀formation฀of฀trople฀helical฀tropocollagen฀domain฀by฀disugide฀bonding฀between฀
cysteine฀residues฀on฀the฀C-terminal฀propeptides.฀Hsp47฀(red฀oval)฀is฀a฀chaperone฀that฀binds฀to฀the฀
disulide฀bonded฀tropocollagen฀and฀completes฀the฀triple฀helix฀formation.฀Procollagen฀α-polypeptides฀
fail฀to฀form฀a฀triple฀helix฀and฀aggregate฀in฀the฀endoplasmic฀reticular฀lumen฀if฀Hsp47฀is฀absent.฀Once฀
the฀triple฀helix฀has฀formed,฀the฀procollagen฀is฀moved฀into฀a฀transport฀vesicle฀and฀transferred฀to฀the฀
Golgi.฀The฀N-฀and฀C-terminal฀propeptide฀regions฀are฀indicated฀as฀green฀circles฀and฀green฀ovals.฀(a)฀
Revised฀and฀partially฀updated฀from฀Fig.19-47฀in฀The฀Molecular฀Biology฀of฀the฀Cell,฀Alberts฀et฀al.,฀
4th฀Ed.฀2002.฀Garland฀Science,฀Taylor฀&฀Francis฀Group,฀New฀York.฀(b)฀Slightly฀modiied฀from฀
Fig.฀6฀in฀Ishida,฀Y.,฀et฀al.,฀Molecular฀Biology฀of฀the฀Cell,฀Vol.฀17,฀2347–2355,฀May฀2006;฀Reprinted฀
with฀permission฀from฀the฀American฀Society฀for฀Cell฀Biology;฀PDF฀access฀http://www.molbiolcell.
org/cgi/reprint/17/5/2346)

The฀ procollagen฀ trimers฀ are฀ taken฀ into฀ transport฀ vesicles฀ which฀ pass฀ them฀ to฀ cis-
Golgi฀cisternae฀(Fig.฀7.2).฀There,฀they:฀(a)฀aggregate฀into฀dense฀material฀(procollagen฀
bundles฀seen฀in฀electron฀micrographs);฀(b)฀lose฀their฀N-terminal฀propeptide,฀which฀re-
enters฀the฀cytosol฀and฀inhibits฀the฀translation฀of฀additional฀collagen;฀and฀(c)฀are฀is฀sorted฀
into฀secretory฀vesicles฀in฀the฀trans-Golgi.฀Secretion฀strongly฀activates฀propeptide฀removal฀
extracellularly฀(Sect.฀8.2.1),฀generating฀spontaneous฀tropocollagen฀iber฀formation฀and฀
cross-linking฀฀(Sect.฀4.2.2.).฀The฀synthesis฀and฀processing฀of฀non-ibrillar฀collagens฀are฀
similar,฀except฀that฀their฀N-฀and฀C-terminal฀propeptides฀remain฀attached฀for฀use฀in฀the฀
polymeric฀assemblages฀(Sect.฀5.1.2).
7.1.1. Intracellular Collagen Synthesis 103

Fig. 7.2฀ Intracellular฀organelle฀path฀

taken฀by฀collagen฀polypeptides฀
before฀secretion.฀Diagram฀shows฀
the฀rough฀and฀smooth฀endoplasmic฀
reticulum,฀the฀budding฀of฀primary฀
transport฀vesicles฀from฀the฀smooth฀
endoplasmic฀reticulum฀to฀the฀
cis-Golgi,฀the฀Golgi฀cisternae,฀and฀
the฀budding฀of฀secretory฀or฀
secondary฀transport฀granules฀
(vesicles)฀from฀the฀trans-Golgi฀for฀
secretion,฀delivery฀to฀lysosomes,฀or฀
insertion฀into฀cell฀membranes฀฀
from฀the฀trans-Golgi฀to฀form฀
secretory฀granules฀or฀lysosomes฀
(Slightly฀adapted฀from฀Fig.฀27-35฀
in฀Lehninger฀Principles฀of฀
Biochemistry.฀D.L.฀Nelson฀฀
&฀M.M.฀Cox,฀4th฀Ed.฀2005.฀฀
W.H.฀Freeman฀&฀Co.,฀New฀York)

Collagen฀is฀synthesized฀in฀the฀endoplasmic฀reticular฀lumen.฀Each฀α-polypeptide฀pos-
sesses฀a฀signal฀sequence฀that฀guides฀the฀N-terminus฀into฀the฀ER฀where฀it฀is฀removed,฀
leaving฀a฀long฀extended฀tropocollagen฀domain฀with฀small,฀folded฀N-฀and฀C-terminal฀
propeptide฀extensions฀(procollagen).฀Proline฀hydroyxlase฀has฀two฀α฀subunits฀and฀two฀
β฀ subunits.฀ The฀ latter฀ is฀ called฀ protein฀ disulide฀ isomerase฀ and฀ it฀ disulide฀ bonds฀
C-terminal฀propeptides฀that฀have฀self-associated฀into฀trimers฀of฀two฀α1-chains฀and฀one฀
α2-chain.฀ Lysine฀ is฀ hydroxylated฀ by฀ an฀ enzyme฀ loosely฀ homologous฀ to฀ the฀ proline฀
hydroxylase฀ α-subunits.฀ After฀ selected฀ proline฀ and฀ lysine฀ residues฀ are฀ hydroxylated฀
and฀ hydroxylysine฀ glycosylated,฀ chaperone฀ hsp47฀ replaces฀ proline฀ hydroxylase฀ and฀
guides฀ triple฀ helix฀ formation฀ of฀ the฀ tropcollagen฀ domain.฀ Procollagen฀ then฀ passes฀
through฀the฀Golgi฀to฀secretory฀vesicles฀where฀it฀forms฀large฀procollagen฀bundles.฀The฀
N-terminal฀propeptides฀are฀hydrolyzed฀and฀re-enter฀the฀cytosol฀where฀they฀inhibit฀col-
lagen฀α-chain฀synthesis.฀Synthesis฀and฀processing฀of฀non-ibrillar฀collagens฀is฀similar,฀
except฀that฀their฀N-฀and฀C-terminal฀propeptides฀are฀not฀removed.
104 7 Collagen Synthesis, Genetic Diseases, and Scurvy

7.2.1.
Effects of Collagen Polypeptide Mutations

The฀ ibrous฀ collagens฀ of฀ all฀ vertebrates฀ and฀ invertebrates฀ possess฀ about฀ 340฀ gly-X-Y฀
sequences฀ encoded฀ as฀ groups฀ of฀ approximately฀ 18฀ amino฀ acids฀ in฀ their฀ tropocollagen฀
domain.฀The฀41฀to฀42฀exons฀are฀all฀multiples฀of฀9฀bases.฀Most฀are฀54฀bases฀in฀size,฀but฀some฀
are฀also฀multiples฀of฀54฀bases,฀or฀combinations฀of฀45-฀and฀54-bases.฀The฀sequences฀of฀three฀
such฀exons฀(Fig.฀7.3),฀which฀suggest฀that฀the฀a-procollagen฀polypeptide฀evolved฀from฀a฀
precursor฀of฀the฀N-฀and฀C-terminal฀domains฀by฀duplication฀of฀a฀54฀base฀exon฀that฀origi-
nally฀encoded฀18฀amino฀acids฀in฀the฀central฀portion฀of฀an฀ancient฀protein฀precursor฀of฀the฀
procollagen฀N-฀and฀C-terminal฀domains.฀At฀about฀the฀same฀time,฀a฀small฀insertion฀within฀
the฀C-terminal฀region฀enabled฀different฀a-polypeptide฀mixtures฀to฀associate.฀The฀resultant฀฀
“molecular฀incestuous”฀mixing฀resulted฀in฀allowed฀the฀formation฀of฀collagen฀heterotrimers฀
compatible฀ with฀ bone฀ formation฀ and฀ the฀ appearance฀ of฀ vertebrates฀ from฀ invertebrates.฀
Invertebrate฀collagens฀are฀homotrimers฀and฀cannot฀calcify.
Mutations฀that฀interfere฀with฀collagen฀iber฀formation฀mostly฀cause฀lethal฀or฀nonlethal฀
osteogenesis฀imperfecta,฀also฀known฀as฀brittle฀bone฀disease.฀The฀bones฀break฀easily฀and฀
apparently฀spontaneously.฀The฀disorder฀occurs฀in฀about฀one฀in฀50,000฀live฀births฀in฀the฀US.฀
Osteogenesis฀imperfecta฀is฀clinically฀divided฀by฀whether฀the฀teeth฀are฀also฀affected.฀They฀
may฀appear฀opalescent฀blue-gray฀or฀yellow-brown฀because฀of฀abnormal฀dentin฀฀calciication.฀

13
-Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg-
22
-Gly-Leu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp-
Intron
31
-Gly-Pro-Gln-Gly-Phe-Gln-Gly-Pro-Hyp-
40
-Gly-Glu-Hyp-Gly-Glu-Hyp-Gly-Ala-Ser-
Intron
49
-Gly-Pro-Met-Gly-Pro-Arg-Gly-Pro-Hyp-
58
-Gly-Pro-Hyp-Gly-Lys-Asn-Gly-Asp-Asp-
Fig. 7.3฀ Translated฀ sequence฀ of฀ a฀ tropocollagen฀ exon.฀ The18฀ amino฀ acid฀ residues฀ (3x6฀ gly-X-Y฀
sequence)฀encoded฀as฀a฀54฀bp฀exon.฀Sequence฀homologies฀between฀amino฀acids฀1–18฀in฀exon฀1฀of฀
the฀tropocollagen฀domain฀of฀the฀COL1A1฀gene฀compared฀with฀the฀sequence฀of฀18฀amino฀acid฀resi-
dues฀in฀exons฀2฀and฀3฀respectively.฀Numbering฀is฀according฀to฀den฀Dunnen฀JT฀and฀Antonarakis฀SE฀
(2000).฀Human฀mutation,฀15:7–12.฀Underlines฀show฀identity฀between฀exon฀1฀and฀exon฀2฀sequences.฀
Highlights฀show฀identity฀between฀exon฀1฀and฀exon฀3฀sequences฀.฀The฀collagen฀genes฀are฀listed฀in฀
Table฀3.1.฀The฀COL1A1฀gene฀encodes฀the฀type฀I฀α1฀procollagen฀polypeptide฀and฀the฀COL1A2฀
gene฀encodes฀the฀type฀I฀α2฀procollagen฀polypeptide.
7.2.2. Ehlers-Danlos syndrome (EDS) 105

Table 7.1฀ Stromal฀protein฀mutations฀affecting฀teeth

Mutationa Disease Symptom
Type฀I฀collagen฀mutations฀associated฀ Dentinogenesis฀ Opalescent฀blue-gray฀฀
with฀osteogenesis฀imperfecta imperfecta฀type฀I฀฀ or฀yellow-brown฀teeth฀
(see฀also฀Chap.฀9) because฀of฀abnormal฀dentin฀
calciication
Kindlin-1฀(Chap.฀5) Kindler฀syndrome
Unknown฀genes฀on฀chromosome฀12 Ehlers–Danlos฀฀ Aggressive฀฀
syndrome฀–฀type฀VIII periodontal฀disease
Laminin-5฀(Chap.฀5) Junctional฀ Generalized฀enamel฀
epidermolysis฀bullosa hypoplasia;฀฀
increased฀caries
Fibrillin-1฀and฀-2฀(Chap.฀6) Marfan฀syndrome Crowded฀incompletely฀
developed฀(hypoplastic)฀฀
teeth฀and฀deformities฀of฀the฀
roots
Dentin฀sialophosphoprotein฀(DSPP),฀a฀ Dentinogenesis฀฀ Changes฀resemble฀฀
glycoprotein฀of฀the฀connective฀tissue฀ imperfecta฀type฀II type฀I฀dentinogenesis฀
stroma฀(Chap.฀9) imperfecta฀฀
(Chap.฀9)
Mutations฀of฀amelogenin฀formation฀ Amelogenesis฀฀ Brittle฀or฀absent฀฀
and฀processing฀(Chap.฀9) imperfecta enamel
I฀ talic฀type฀indicates฀the฀various฀collagen฀polypeptides฀and฀an฀enzyme฀mutation฀described฀in฀this฀
a

chapter

Occasionally,฀some฀teeth฀may฀be฀missing.฀Table฀7.1฀is฀a฀list฀of฀stromal฀protein฀diseases฀that฀
affect฀tooth฀development.
There฀are฀two฀major฀types฀of฀spontaneous฀genetic฀mutations:฀a฀change฀in฀a฀nucleotide฀
base฀ in฀ the฀ organism’s฀ DNA฀ (point฀ mutation),฀ or฀ a฀ base฀ deletion.฀ Either฀ mutation฀ can฀
sometimes฀alter฀intron/exon฀splicing฀boundaries,฀causing฀a฀more฀extensive฀mutation฀and฀
serious฀or฀fatal฀disease.฀Osteogenesis฀imperfecta฀is฀usually฀caused฀by฀a฀point฀mutation฀of฀
the฀codon฀for฀glycine,฀which฀is฀always฀encoded฀as฀GGT฀within฀the฀COL1A1฀and฀COL1A2฀
genes฀(Fig.฀7.3).฀Since฀the฀triple฀helix฀develops฀from฀C-฀to฀N-terminus฀(Fig.฀7.2),฀a฀muta-
tion฀near฀the฀C-terminus฀interrupts฀helix฀formation฀more฀completely฀than฀the฀same฀muta-
tion฀near฀the฀N-terminus฀and฀is฀therefore฀more฀likely฀to฀be฀lethal.

7.2.2.
Ehlers-Danlos syndrome (EDS)

Other฀mutations฀of฀ibrillar฀collagen,฀or฀mutations฀that฀affect฀collagen-processing,฀cause฀
Ehlers-Danlos฀syndrome฀(EDS),฀a฀group฀of฀heritable฀connective฀tissue฀disorders฀causing:฀
skin฀hyperextensibility,฀articular฀hypermobility,฀and฀tissue฀fragility.฀The฀3฀major฀types฀are฀
classic฀(EDS-I฀and฀EDS-II),฀hypermobility฀(EDS-III)฀and฀vascular฀(EDS-IV).
106 7 Collagen Synthesis, Genetic Diseases, and Scurvy

Classical฀EDS฀is฀usually฀due฀to฀type฀V฀collagen฀mutations฀that฀unduly฀limit฀iber฀thick-
ness.฀Mice฀made฀haploid฀for฀type฀V฀collagen฀express฀only฀half฀the฀amount฀in฀normal฀mice฀
and฀exhibit฀classical฀EDS฀symptoms.฀Occasionally,฀a฀non-glycine฀residue฀is฀mutated฀to฀
cysteine฀in฀type฀I฀collagen฀and฀the฀resulting฀disulide฀cross-linked฀polypeptides฀also฀limit฀
iber฀thickness.฀The฀corresponding฀mutation฀is฀more฀common฀in฀type฀II฀collagen,฀where฀
instead฀of฀classical฀EDS,฀it฀causes฀a฀chondrodysplasia฀such฀as฀dwarism.฀The฀genetic฀muta-
tions฀involved฀in฀joint฀hypermobility฀(EDS-III)฀are฀mostly฀unknown.
Vascular฀EDS,฀(EDS-IV;฀fragile฀blood฀vessels)฀is฀exclusively฀associated฀with฀mutations฀
of฀COL3A1,฀the฀gene฀encoding฀type฀III฀collagen฀(Table฀3.1).฀Most฀mutations฀(66%)฀are฀
substitutions฀of฀glycine฀for฀another฀amino฀acid฀in฀one฀of฀the฀gly-X-Y฀triplets฀and฀nearly฀all฀
the฀rest฀are฀the฀result฀of฀exon฀skipping.
EDS-V฀is฀an฀X-linked฀form฀of฀EDS-I฀in฀which฀skin฀fragility฀is฀prominent.฀EDS฀VI฀is฀a฀
severe฀form฀of฀EDS-I฀associated฀with฀corneal฀degeneration.฀The฀genetic฀mutations฀of฀both฀
are฀unknown,฀but฀EDS-VI฀has฀reduced฀lysine฀hydroxylase฀activity฀despite฀no฀detectable฀
mutation฀of฀any฀of฀its฀three฀genes฀(Sect.฀7.3.1)฀or฀of฀the฀genes฀involved฀in฀ascorbate฀metab-
olism฀(Sect.฀7.4.1).
EDS฀VII฀is฀a฀combination฀of฀classical฀and฀vascular฀EDS฀caused฀mostly฀by฀in-frame฀
deletions฀of฀18฀or฀24฀amino฀acid฀residues฀encoding฀exon-6฀of฀COL1A1฀or฀COL1A2,฀the฀
downstream฀splice฀site฀for฀procollagen฀N-peptidase.฀Mutations฀of฀procollagen฀N-peptidase฀
(Sect.฀8.2.1,฀Fig.฀8.5)฀also฀cause฀this฀form฀of฀EDS.
EDS฀ VIII฀ is฀ associated฀ with฀ aggressive฀ periodontal฀ disease.฀ It฀ may฀ be฀ caused฀ by฀ a฀
mutation฀in฀one฀or฀more฀genes฀other฀than฀the฀collagen฀gene฀(COL2A1)฀on฀chromosome฀12.฀
Individuals฀resemble฀Marfan’s฀syndrome฀(Table฀6.1),฀but฀have฀normal฀teeth.฀They฀display฀
excessive฀bleeding฀around฀the฀knees฀and฀fragile฀skin,฀hallmarks฀of฀vascular฀EDS.

The฀similarity฀of฀collagen฀structure฀in฀all฀species฀suggests฀a฀common฀origin฀in฀which฀
the฀central฀segment฀of฀a฀precursor฀globular฀protein฀resembling฀the฀N-฀and฀C-propeptides฀
was฀ repeatedly฀ duplicated.฀ Vertebrates฀ contain฀ collagen฀ heterotrimers฀ and฀ bone,฀ but฀
invertebrates฀contain฀a฀simpler฀procollagen฀C-terminal฀domain฀and฀homotrimers฀which฀
cannot฀calcify.฀Mutations฀of฀glycine฀residues฀at฀one฀of฀the฀~340฀gly-X-Y฀sequences฀in฀
a฀ collagen฀ gene฀ inhibits฀ adequate฀ triple฀ helix฀ formation฀ and฀ results฀ in฀ fragile฀ bones฀
(osteogenesis฀imperfecta),฀sometimes฀accompanied฀by฀opalescent฀or฀completely฀miss-
ing฀teeth฀(dentinogenesis฀imperfecta).฀Ehlers-Danlos฀syndrome฀(EDS),฀skin฀hyperex-
tensibility,฀ articular฀ hypermobility,฀ and฀ tissue฀ fragility,฀ is฀ caused฀ by฀ mutations฀ that฀
affect฀collagen฀iber฀assembly.฀The฀three฀major฀types฀of฀EDS฀are฀classical,฀hypermobil-
ity,฀and฀vascular.฀Classical฀EDS฀is฀mostly฀due฀to฀mutations฀of฀type฀V฀or฀type฀I฀collagen฀
that฀limit฀iber฀thickness.฀Vascular฀EDS฀is฀exclusively฀due฀to฀mutations฀of฀type฀III฀col-
lagen฀that฀promote฀vascular฀fragility.฀An฀unusual฀form฀of฀EDS,฀in฀which฀a฀gene฀other฀
than฀ the฀ collagen฀ gene฀ encoded฀ on฀ chromosome฀ 12฀ is฀ mutated,฀ is฀ associated฀ with฀
aggressive฀periodontal฀disease.
7.2.2. Ehlers-Danlos syndrome (EDS) 107

Proline฀hydroxylase฀(PH)฀and฀lysine฀hydroxylase฀(LH)฀bind฀to฀substrate฀motifs฀on฀procol-
lagen฀ in฀ the฀ endoplasmic฀ reticulum.฀ The฀ proline฀ substrate฀ motif฀ has฀ the฀ sequence฀ X-Y-Gly฀
where฀X฀is฀any฀amino฀acid฀and฀Y฀is฀the฀proline฀residue฀to฀be฀hydroxylated.฀The฀lysine฀substrate฀
motif฀has฀an฀analogous฀sequence฀in฀which฀Y฀is฀a฀lysine฀residue.฀The฀amino฀acid฀sequences฀sur-
rounding฀the฀tripeptide฀substrate฀motif฀for฀lysine฀are฀more฀variable฀than฀those฀surrounding฀the฀
proline฀motif.฀This฀variability฀facilitates฀or฀inhibits฀lysine฀hydroxylase฀binding฀and฀catalysis,฀
explaining฀variations฀in฀the฀hydroxylysine฀content฀of฀different฀collagen฀types.฀The฀two฀motifs฀
are฀not฀unique฀to฀collagen;฀other฀proteins฀contain฀these฀motifs฀and฀are฀hydroxylated฀similarly.
PH฀and฀LH฀are฀mixed฀function฀oxidases.฀Molecular฀oxygen฀and฀α-ketoglutarate฀are฀co-
substrates฀along฀with฀procollagen.฀One฀atom฀of฀molecular฀oxygen฀is฀reduced฀by฀α-ketoglutarate฀
and฀the฀other฀by฀an฀electron฀from฀a฀ferrous฀ion฀within฀the฀hydroxylase.฀The฀oxidized฀products฀
are฀a฀coordinated฀ferric฀ion฀hydroxide฀at฀the฀catalytic฀center฀of฀the฀enzyme,฀plus฀succinate฀and฀
carbon฀dioxide฀from฀the฀oxidation฀of฀α-ketoglutarate.฀The฀ferric฀ion฀is฀reduced฀to฀ferrous฀ion฀
by฀a฀cofactor฀(ascorbate)฀releasing฀the฀hydroxide฀as฀a฀nucleophile฀to฀hydroxylate฀a฀proline฀or฀
lysine฀residue฀in฀procollagen฀(Fig.฀7.4).฀Ascorbate฀is฀oxidized฀to฀dehydroascorbate฀and฀regen-
erated฀by฀glutathione฀(Sect฀7.4.1).฀Some฀of฀the฀electrons฀that฀restore฀ascorbate฀come฀from฀
procollagen฀C-terminal฀cysteine฀residues฀oxidized฀by฀PDI฀to฀stabilize฀the฀procollagen฀trimers฀
(Sect.฀7.1.1),฀but฀the฀numerous฀proline฀residues฀to฀be฀hydroxylated฀require฀many฀additional฀
electrons฀which฀are฀provided฀by฀glutathione฀(Sect.฀7.4.1).฀PDI฀is฀also฀found฀as฀a฀single฀dimer฀
in฀the฀ER฀where฀it฀catalyzes฀an฀exchange฀of฀disulide฀bonds฀in฀many฀proteins.

Prolyl residue
-pro-pro-gly- _
O
COO
H
N C C Molecular
oxygen gas CH2

Fig. 7.4฀ Catalytic฀action฀of฀ H2C CH2 + O2 + CH2

proline฀hydroxylase.฀One฀ C
atom฀of฀oxygen฀gas฀(red)฀ (Fe2+)
C O
oxidizes฀ketoglutarate฀and฀ H H Prolyl hydroxylase
appears฀in฀succinate฀along฀ _
Dehydro- COO
with฀CO2.฀The฀other฀oxygen฀ ascorbate
atom฀forms฀a฀Fe3+–OH฀ a-Ketoglutarate
complex฀attached฀to฀the฀ Ascorbate
enzyme.฀When฀Fe3+฀is฀
(Fe3+)
reduced฀by฀ascorbate,฀H+฀ Red/Ox Prolyl hydroxylase
dissociates฀and฀the฀O−1฀ Glutathione _
species฀(red)฀is฀released฀to฀a฀ O COO
proline฀residue฀in฀the฀ H
polypeptide,฀forming฀a฀ N C C CH2
hydroxyproline฀residue.฀The฀
H2C CH2 + CH2 +
enzymes฀recognize฀prolyl-฀or฀ CO2
lysyl฀residue฀sequence฀motifs฀ C
C O
described฀in฀the฀text฀(From฀ H O H
Fig.฀11.3฀in฀Biochemistry,฀L.฀ _
O
Stryer,฀฀ 4-Hydroxyprolyl
3rd฀Ed.฀1988.฀W.H.฀Freeman฀ residue Succinate
&฀Co.,฀New฀York) ..pro-HOpro-gly..
108 7 Collagen Synthesis, Genetic Diseases, and Scurvy

+
NH2
CH2OH
CH2
HO O
O C H
Galactose H Hydroxylysine
OH H CH2
H H
CH2 O
H
N C C
Glucose CH2OH
H H
H O H O
H Peptide backbone
OH H
HO

H OH

Fig. 7.5฀ Structure฀of฀the฀diglycan฀attached฀to฀hydroxylysine.฀The฀LH3฀isoform฀of฀lysine฀hydroxylase฀

attaches฀UDP-฀galactose฀to฀hydroxylysine฀in฀a฀b-linkage,฀and฀then฀a-links฀UDP฀glucose฀to฀the฀C2฀
atom฀of฀the฀galactose.฀This฀reaction฀occurs฀in฀the฀endoplasmic฀reticulum฀and฀in฀the฀connective฀tissue฀
matrix฀(From฀Fig.฀11.4฀in฀Biochemistry,฀L.฀Stryer,฀3rd฀Ed.฀1988.฀W.H.฀Freeman฀&฀Co.,฀New฀York)

There฀ are฀ also฀ three฀ encoded฀ lysine฀ hydroxylases฀ in฀ the฀ genome.฀ LH1฀ and฀ LH2฀ are฀
more฀ closely฀ related฀ (homologous)฀ to฀ each฀ other฀ and฀ the฀ PH฀ α-subunits฀ than฀ LH3.฀ All฀
three฀LH฀isoforms฀are฀homodimers฀that฀adhere฀to฀the฀ER฀luminal฀membrane฀by฀a฀hydro-
phobic฀fold฀in฀their฀iron-binding฀domain฀and฀are฀unattached฀to฀PDI.฀Some฀LH3฀detaches฀
from฀ the฀ inner฀ wall฀ of฀ the฀ endoplasmic฀ reticulum฀ along฀ with฀ individual฀ α-procollagen฀
polypeptides฀in฀which฀all฀available฀proline฀and฀lysine฀residues฀have฀been฀hydroxylated.฀
Once฀detached,฀LH3฀has฀a฀second,฀unrelated฀enzymatic฀activity.฀It฀adds฀galactose฀and฀then฀
glucose฀to฀certain฀hydroxylysine฀residues฀in฀each฀procollagen฀polypeptide฀(Fig.฀7.5).฀The฀
sugars฀are฀activated฀in฀the฀cytosol฀by฀UDP-sugar฀transferase฀and฀transported฀into฀the฀endo-
plasmic฀reticular฀lumen.฀Once฀glycosylated,฀the฀processed฀tropocollagen฀domain฀binds฀to฀
chaperone฀hsp47฀to฀guide฀triple฀helix฀formation฀(Sect.฀7.1.1).฀The฀fate฀of฀LH3฀is฀uncertain฀
but฀it฀likely฀reattaches฀to฀the฀ER฀lumen฀wall.

Summary:฀Proline฀and฀lysine฀hydroxylase฀(PH฀and฀LH)฀molecules฀are฀homodimers฀
containing฀ an฀ iron฀ atom.฀ They฀ are฀ attached฀ to฀ the฀ luminal฀ wall฀ of฀ the฀ endoplasmic฀
reticulum.฀PH฀(α-subunit)฀is฀held฀loosely฀by฀protein฀disulide฀isomerase฀(PDI,฀β-subunit),฀
whereas฀LH฀is฀directly฀attached.฀Hydroxylation฀is฀by฀a฀mixed฀function฀oxidase฀reac-
tion.฀One฀atom฀of฀an฀oxygen฀molecule฀oxidizes฀the฀ferrous฀ion฀to฀a฀ferric฀ion฀hydroxide฀
and฀the฀other฀oxidizes฀α-ketoglutarate,฀producing฀succinate฀and฀carbon฀dioxide.฀When฀
ferric฀hydroxide฀is฀reduced฀by฀a฀cofactor,฀ascorbate,฀the฀second฀oxygen฀atom฀is฀released฀
and฀hydroxylates฀the฀substrate.฀Ascorbate฀is฀reduced฀to฀dehydroascorbate฀and฀reoxi-
dized฀ by฀ glutathione฀ which฀ receives฀ electrons฀ from฀ various฀ sources฀ including฀ PDI฀
which฀oxidizes฀cysteine฀to฀stabilize฀procollagen฀trimers.฀Three฀homologous฀PH฀poly-
peptides฀are฀encoded฀by฀different฀genes฀and฀expressed฀in฀different฀tissues.฀There฀are฀
also฀3฀homologous฀LH฀polypeptides,฀of฀which฀one,฀the฀LH3฀polypeptide,฀glycosylates฀
some฀of฀the฀hydroxylysine฀residues฀with฀galactose฀and฀glucose.
7.3.1. Ascorbate and Antioxidants 109

7.3.1.
Ascorbate and Antioxidants

Ascorbate฀ (ascorbic฀ acid฀ or฀ vitamin฀ C)฀ is฀ an฀ antioxidant฀ (reducing฀ agent).฀ Besides฀ its฀
importance฀as฀a฀cofactor฀for฀proline฀and฀lysine฀hydroxylases฀in฀vertebrates,฀it฀ascorbate฀
protects฀macromolecules฀from฀oxidative฀damage฀by฀neutralizing฀reactive฀oxygen฀species฀
(ROS),฀by-products฀of฀respiration.฀Cells฀possess฀large฀amounts฀of฀catalase,฀peroxidase฀and฀
superoxide฀ dismutase฀ enzymes฀ that฀ rapidly฀ neutralize฀ these฀ harmful฀ agents฀ within฀ the฀
cytosol฀(Sect.฀16.3.2.).฀The฀anti-oxidant฀property฀of฀ascorbate฀is฀more฀important฀extracel-
lularly฀where฀it฀neutralizes฀the฀ROS฀from฀leukocytes฀during฀inlammation฀(Sect.฀13.3.1).
Dehydroascorbate฀forms฀directly฀when฀two฀electrons฀are฀removed฀from฀ascorbate฀(Fig.฀7.6a,฀
lower฀ left),฀ for฀ example,฀ after฀ reacting฀ with฀ proline฀ or฀ lysine฀ hydroxylase฀ (Fig.฀ 7.4a).฀
Dehydroascorbate฀forms฀indirectly฀if฀ascorbate฀loses฀a฀single฀electron฀(for฀example,฀to฀an฀
electron-deicient฀ radical฀ group)฀ that฀ irst฀ produces฀ mono-dehydroascorbate฀ (Fig.฀ 7.6a,฀
top).฀The฀two฀parts฀of฀the฀indirect฀reaction฀occur฀spontaneously฀(without฀an฀enzyme)฀in฀
aqueous฀phase.฀Both฀ascorbate฀and฀dehydroascorbate฀are฀transported฀in฀and฀out฀of฀cells.฀
Ascorbate฀ enters฀ through฀ sodium-dependent฀ transporters,฀ and฀ dehydroascorbate฀ through฀
glucose฀ transporters-1฀ and฀ -3฀ (GLUT1฀ and฀ GLUT3).฀ Dehydroascorbate฀ is฀ immediately฀
reduced฀to฀ascorbate฀inside฀cells,฀but฀it฀predominates฀extracellularly฀where฀it฀spontaneously฀
hydrolyzes฀ to฀ 2,3-diketogulonate฀ (Fig.฀ 7.6b,฀ lower฀ right)฀ with฀ a฀ half-life฀ of฀ 2–15฀ min.฀

Fig. 7.6฀ Structures฀of฀ascorbate,฀ascorbyl฀radical฀and฀dehydroascorbate.฀(a)฀Ring฀structures.฀Red฀

numbers฀indicate฀the฀conventional฀carbon฀atom฀numbering.฀The฀COO−฀group฀of฀ascorbate฀(esteri-
ied฀to฀C4)฀is฀C1.฀The฀green฀and฀blue฀hydrogen฀atoms฀indicate฀sites฀of฀electron฀loss฀leading฀to฀mono-
dehydroascorbate฀(ascorbyl฀radical)฀and฀then฀to฀dehydroascorbate฀(upper฀half฀of฀Fig.฀7.6a).฀The฀
mono-dehydroascorbate฀is฀stabilized฀by฀having฀its฀free฀electron฀shared฀among฀the฀attached฀oxygen฀
atoms.฀The฀loss฀of฀a฀second฀electron฀and฀proton฀(blue),฀or฀of฀both฀protons฀and฀electrons฀directly฀as฀
in฀the฀proline฀or฀lysine฀hydroxylase฀reaction฀(bottom฀half),฀gives฀rise฀to฀dehydroascorbate฀(Original฀
igure).฀(b)฀Straight฀chain฀structures.฀Ascorbate฀is฀in฀the฀l-form฀because฀of฀the฀orientation฀around฀
C5.฀ Mono-dehydroascorbate฀ is฀ not฀ shown.฀ Instead,฀ the฀ degradation฀ form฀ of฀ dehydroascorbate,฀
l-diketogulonate฀is฀diagrammed฀at฀the฀far฀right฀(Adapted฀from฀drawings฀on฀p.฀1359,฀Chap.฀50฀in฀
Principles฀of฀Biochemistry,฀White,฀A.,฀et฀al.฀6th฀Ed.฀1978.฀McGraw฀Hill฀Inc.,฀New฀York)
110 7 Collagen Synthesis, Genetic Diseases, and Scurvy

Diketogulonate฀ cannot฀ be฀ reduced฀ back฀ to฀ ascorbate.฀ Most฀ animals฀ synthesize฀ ascorbate฀
using฀the฀path฀shown฀in฀Fig.฀7.7,฀but฀primates฀including฀humans฀have฀accumulated฀muta-
tions฀ in฀ the฀ gene฀ for฀ l-gulonolactone฀ oxidase฀ and฀ cannot฀ synthesize฀ ascorbate฀ de฀ novo.฀
They฀ require฀ a฀ dietary฀ source฀ of฀ vitamin฀ C฀ to฀ compensate฀ for฀ the฀ gradual฀ loss฀ of฀ dehy-
droascorbate.฀The฀current฀Recommended฀Dietary฀Allowance฀(RDA)฀from฀the฀US฀Food฀and฀฀
Drug฀Administration฀is฀60–95฀mg/day.
Depletion฀of฀ascorbate฀causes฀scurvy,฀which฀was฀irst฀identiied฀in฀sailors฀during฀long฀voy-
ages฀between฀the฀ifteenth฀and฀seventeenth฀centuries.฀It฀was฀a฀common฀cause฀of฀illness฀and฀
death฀if฀fresh฀fruits฀and฀meat฀were฀not฀eaten฀within฀6฀weeks.฀Citrus฀fruits,฀especially฀lemon฀and฀
lime฀in฀which฀the฀reduced฀form฀of฀ascorbate฀stores฀well,฀were฀found฀to฀prevent฀or฀cure฀the฀
disease.฀An฀early฀symptom฀of฀ascorbate฀deiciency฀is฀the฀loss฀of฀gingival฀and฀periodontal฀mem-
brane฀ibers฀accompanied฀by฀loosening฀of฀the฀teeth.฀The฀reason฀is฀that฀anchoring฀ibrils฀and฀
ibers฀of฀the฀gingival฀cuff฀and฀upper฀periodontium฀(Chap.฀3)฀turn฀over฀every฀24฀h฀due฀to฀tooth฀
movements฀that฀stimulate฀ibroblasts฀to฀replace฀collagen฀and฀renew฀the฀attachment฀continu-
ously.฀Movement฀of฀blood฀causes฀a฀similar฀instability฀of฀collagen฀in฀blood฀vessel฀walls.฀Without฀

HC=O CH2OH COO−

HCOH HCOH HOCH

HOCH NADPH + H+ HOCH Same HOCH -H O

2
as
HCOH D-glucuronate HCOH HCOH L-gulonolacton
dehydrogenase synthetase
HCOH HCOH HOCH

COO− COO− CH OH
2
D-glucuronate D-gulonate
O=C
O=C O=C
HOC
HOCH HOCH O
O O Ene-diol
-2H HOC
HOCH O=C formation
L-gulonolactone
(spontaneous) HC
HC oxidase HC
HOCH
HOCH HOCH
CH2OH
CH2OH CH2OH
L-Gulonolactone 3-Keto-L-gulonolactone D-ascorbate

Fig. 7.7฀ Synthesis฀ of฀ ascorbate.฀ UDP-d-glucuronate฀ is฀ the฀ precursor฀ in฀ mammals.฀ The฀ precursor฀ of฀
endogenous฀ ascorbate฀ in฀ animals฀ is฀ D-glucuronate,฀ which฀ is฀ derived฀ from฀ D-glucose฀ by฀ glucose฀
6-phosphate฀dehydrogenase.฀The฀straight฀chain฀forms฀of฀D-glucuronate฀and฀L-gulonate,฀the฀next฀inter-
mediate,฀are฀shown฀along฀with฀the฀enzymes฀that฀catalyze฀the฀changes฀(top฀row).฀The฀L-gulonate฀inter-
mediate฀is฀acted฀on฀by฀a฀synthetase฀to฀lose฀water,฀forming฀L-gulonolactone฀which฀is฀oxidized฀to฀form฀
3-keto-L-gulonolactone.฀ The฀ latter฀ spontaneously฀ isomerizes฀ to฀ L-ascorbate.฀ L-gulono-1,4-lactone฀
oxidase฀is฀inactive฀in฀humans฀and฀most฀other฀primates฀because฀of฀a฀mutation฀not฀present฀in฀other฀ani-
mals.฀Plants฀make฀large฀amounts฀of฀ascorbate฀by฀a฀different฀path.฀(Adapted฀from฀drawings฀on฀p.฀1359,฀
Chap.฀50฀in฀Principles฀of฀Biochemistry,฀White,฀A.,฀et฀al.฀6th฀Ed.฀1978.฀McGraw฀Hill฀Inc.,฀New฀York)
7.3.1. Ascorbate and Antioxidants 111

ascorbate,฀ibers฀are฀removed฀but฀not฀replaced;฀fresh฀collagen฀cannot฀be฀secreted.฀The฀collagen฀
in฀bone฀turns฀over฀more฀slowly,฀but฀if฀scurvy฀becomes฀advanced,฀cessation฀of฀this฀turnover฀
causes฀deep,฀intense฀bone฀pain.฀Late฀in฀the฀disease,฀a฀lack฀of฀type฀IV฀and฀other฀collagens฀sur-
rounding฀blood฀vessels฀cause฀their฀degeneration,฀leading฀to฀potentially฀fatal฀aneurisms.
In฀addition฀to฀collagen฀metabolism฀and฀scavenging฀ROS฀species฀to฀limit฀inlammation฀
as฀noted฀above,฀ascorbate฀is฀required฀for฀the฀synthesis฀of฀norepinephrine฀from฀tyrosine,฀of฀
carnitine฀from฀g-butyrobetaine฀whose฀immediate฀precursor฀is฀made฀by฀trimethylating฀lysine,฀
for฀folinic฀acid฀production฀from฀folic฀acid.฀In฀the฀absence฀of฀ascorbate,฀the฀reduced฀activity฀
of฀these฀processes฀slows฀nerve,฀energy฀and฀cardiac฀output,฀causingthe฀affected฀person฀to฀
become฀exhausted฀and฀irritable.฀฀Scurvy฀is฀the฀old฀English฀word฀for฀ill-tempered.
A฀minority฀of฀scientists,฀most฀notably฀Linus฀Pauling,฀believed฀that฀the฀failure฀of฀humans฀
and฀a฀few฀other฀animals฀to฀synthesize฀vitamin฀C฀is฀a฀genetic฀defect฀that฀should฀be฀overcome฀
by฀ supplementing฀ the฀ diet฀ with฀ large฀ amounts฀ of฀ vitamin฀ C฀ (megadoses฀ of฀ vitamin฀ C).฀
Viruses฀and฀bacteria฀stimulate฀white฀blood฀cells฀to฀release฀large฀amounts฀of฀hydrogen฀perox-
ide฀and฀oxygen-free฀radicals,฀an฀oxidative฀burst฀that฀persists฀after฀the฀causative฀agents฀are฀
gone.฀Megadoses฀of฀vitamin฀C฀were฀proposed฀to฀prevent฀chronic฀damage฀by฀reducing฀the฀
extent฀of฀this฀burst฀if฀an฀infection฀occurs.฀Current฀studies฀suggest฀that฀regularly฀taking฀larger฀
doses฀of฀vitamin฀C฀(0.5–3.0฀g฀up฀to฀18฀g/day)฀may฀inhibit฀some฀cancer฀cells฀and฀viruses฀from฀
growing฀in฀vitro.฀There฀is฀less฀evidence฀that฀it฀prevents฀cancers฀or฀viral฀infections฀in฀vivo.฀
Some฀better-established฀uses฀of฀ascorbate฀are฀as฀an฀antioxidant฀in฀foods,฀an฀antiwrinkle฀agent฀
for฀skin,฀and฀as฀an฀antidote฀to฀ascorbate฀depletion฀associated฀with฀nickel฀or฀lead฀poisoning.
Ascorbate฀ is฀ the฀ major฀ antioxidant฀ in฀ chloroplasts฀ and฀ large฀ amounts฀ are฀ synthesized฀
from฀glucose฀in฀the฀cytosol฀of฀leaves฀by฀the฀path฀shown฀in฀Fig.฀7.7.฀However,฀in฀animals฀
glutathione฀(GSH)฀is฀the฀major฀antioxidant,฀not฀ascorbate฀(Fig.฀7.8).฀Glutathione฀(GSH)฀is฀a฀
tripeptide฀(Glu-Cys-Gly),฀in฀which฀the฀amino฀group฀of฀cysteine฀is฀attached฀to฀the฀g-carboxyl฀
group฀of฀glutamate.฀It฀is฀synthesized฀and฀degraded฀by฀speciic฀enzymes฀in฀the฀cytosol.฀Two฀
reduced฀glutathione฀molecules฀lose฀two฀electrons฀and฀form฀a฀disulphide฀bond฀(GS−SG).฀In฀
primates,฀GSH฀transfers฀its฀electrons฀to฀dehydroascorbate฀or฀peroxidase฀and฀GSSG฀accu-
mulates฀(Fig.฀7.8,฀red฀arrows).฀The฀peroxidase฀action฀is฀described฀in฀connection฀with฀its฀
inhibition฀by฀luoride฀(Sect.16.3.2;฀Fig.฀16.8b).฀In฀collagen฀synthesizing฀cells,฀protein฀dis-
ulide฀isomerase฀or฀GSH-dehydroascorbate฀reductase฀can฀transfer฀their฀electrons฀to฀dehy-
droascorbate฀(Fig.฀7.8,฀green฀or฀red฀arrows฀on฀right).฀GSSG฀reductase฀obtains฀its฀electrons฀
from฀NADPH฀from฀the฀pentose฀phosphate฀path฀(Fig.฀7.8,฀blue฀arrow).฀Note฀that฀the฀use฀of฀
protein฀disulide฀isomerase฀(PDI)฀to฀oxidize฀cysteine฀residues฀(Sect.฀7.3.1)฀appears฀limited฀
to฀its฀association฀with฀proline฀hydroxylase.฀As฀its฀name฀implies,฀PDI฀primarily฀exchanges฀
disulide฀bonds฀to฀stabilize฀protein฀structures.

Ascorbate฀ is฀ a฀ cofactor฀ for฀ proline฀ and฀ lysine฀ hydroxylation.฀ The฀ oxidized฀ form,฀ dehy-
droascorbate,฀predominates฀extracellularly฀but฀degrades฀spontaneously.฀Most฀vertebrates฀
synthesize฀ascorbate฀de฀novo฀from฀glucose,฀but฀the฀last฀enzyme฀of฀the฀path,฀l-gulonolactone฀
oxidase,฀is฀inactive฀in฀primates฀and฀is฀therefore฀nutritionally฀essential.฀Ascorbate฀(vitamin฀
C)฀is฀present฀in฀fresh฀citrus฀fruits฀and฀meat,฀but฀it฀is฀gradually฀lost฀because฀its฀oxidation฀
product,฀dehydroascorbate,฀is฀unstable.฀Scurvy฀develops฀in฀the฀absence฀of฀vitamin฀C,฀pre-
venting฀ collagen฀ re-synthesis฀ in฀ response฀ to฀ stress฀ in฀ tissues฀ such฀ as฀ the฀ gingiva,฀ blood฀
vessels฀and฀bone.฀Ascorbate฀also฀functions฀as฀a฀nonenzymatic฀scavenger฀of฀water-soluble฀
oxidizing฀ agents.฀ It฀ reduces฀ peroxides฀ and฀ oxygen฀ free฀ radicals฀ and฀ is฀ regenerated฀ by฀
112 7 Collagen Synthesis, Genetic Diseases, and Scurvy

reductases,฀the฀protein฀disulide฀isomerase฀subunit฀of฀proline฀hydroxylase฀or฀glutathione.฀
Oxidized฀glutathione฀is฀reduced฀by฀electrons฀which฀are฀transferred฀from฀NADPH฀by฀gluta-
thione฀reductase.฀The฀NADP+฀is฀in฀turn฀reduced฀by฀the฀oxidation฀of฀glucose฀in฀the฀pentose฀
phosphate฀path.฀฀Glutathione฀is฀a฀cofactor฀for฀peroxidases฀that฀maintain฀normal฀cell฀function฀
by฀reducing฀peroxides฀made฀by฀reactive฀oxygen฀species฀on฀membranes฀and฀the฀cytosol.

Fig. 7.8฀ Top฀half฀of฀the฀igure฀depicts฀the฀spontaneous฀oxidation฀of฀ascorbate฀by฀oxygen-free฀radicals,฀

peroxides฀and฀proline฀hydroxylase฀(black฀arrow)฀and฀the฀reduction฀of฀dehydroascorbate฀to฀ascorbate฀
by฀dehydroascorbate฀via฀PDI,฀or฀providing฀reducing฀equivalents฀as฀a฀cofactor฀for฀peroxidases฀and฀
other฀reductases฀(upper฀and฀lower฀red฀arrows).฀The฀oxidized฀form฀(GSSG)฀is฀reduced฀by฀NADPH฀
(straight฀ blue฀ arrow).฀ (Slightly฀ modiied฀ from฀ Meister฀ A.,฀ “Glutathione–ascorbic฀ acid฀ antioxidant฀
system฀in฀animals.”฀J.฀Biol.฀Chem.,฀269(13):9397–9400,฀1994)
 The Zincins: Collagen Fiber
Processing and Degradation 8

This฀ chapter฀ discusses฀ the฀ zinc-containing฀ metalloendoproteinases.฀ These฀ enzymes฀

remodel฀the฀stroma฀during฀development฀and฀around฀tissues฀that฀have฀been฀injured฀or฀
stressed.฀Section฀1฀describes฀the฀three฀major฀classes฀of฀metalloproteases,฀their฀metal฀ion฀
cofactors,฀their฀functions฀in฀biology,฀and฀the฀metzincin฀catalytic฀mechanism฀in฀procol-
lagen฀and฀stromal฀protein฀processing.฀Section฀2฀describes฀the฀astacin฀and฀adamalysin฀
metzincin฀ subclasses฀ and฀ how฀ they฀ process฀ procollagen฀ to฀ tropocollagen.฀ Section฀ 3฀
describes฀the฀matrilysin฀metzincin฀subclass฀and฀their฀roles฀in฀collagen฀and฀stromal฀tis-
sue฀degradation฀and฀in฀enamel฀synthesis.

8.1.1.
The Zincin Enzyme Family

The฀zincin฀proteases฀remodel฀the฀stroma฀during฀development฀and฀around฀tissues฀that฀have฀
been฀ injured฀ or฀ stressed฀ (Sects.฀ 13.2.4).฀ All฀ proteases฀ possess฀ one฀ of฀ the฀ three฀ types฀ of฀
activities:฀(a)฀(Sects.฀13.2.5)฀that฀cut฀within฀a฀polypeptide;฀(b)฀(Sects.฀13.3.1)฀that฀cut฀at฀the฀
C-฀or฀N-terminus;฀and฀(c)฀peptidases฀that฀cut฀small฀polypeptides.฀The฀ive฀structural฀classes฀
of฀proteases฀are฀listed฀in฀Table฀8.1฀and฀the฀enzymatic฀activities฀of฀the฀various฀metallopro-
tease฀classes฀and฀subclasses฀are฀listed฀in฀Table฀8.2.
Most฀metalloproteinases฀contain฀a฀catalytic฀zinc฀ion฀bound฀to฀two฀histidines฀within฀a฀
conserved฀motif,฀usually฀HEXXH฀in฀the฀one฀letter฀amino฀acid฀code฀where฀X฀stands฀for฀any฀
amino฀acid฀(Fig.฀8.1).฀These฀enzymes฀are฀known฀as฀zincins,฀and฀they฀comprise฀by฀far฀the฀
largest฀clan฀of฀metalloendoprotease฀families.฀The฀zincin฀clan฀assignments฀depend฀on฀the฀
nature฀of฀a฀third฀zinc-binding฀residue:฀glutamate฀(E)฀in฀gluzincins,฀aspartate฀(D)฀in฀aspz-
incins,฀and฀histidine฀or฀aspartate฀(H/D)฀in฀metzincins.฀Aspzincins฀are฀absent฀from฀the฀human฀
genome฀(Fig.฀8.1)฀and฀gluzincins฀encode฀proteases฀other฀than฀endoproteases฀(Table฀8.2).
All฀human฀metalloendoproteinases฀are฀metzincins,฀named฀for฀a฀downstream฀methion-
ine฀ residue฀ involved฀ in฀ regulating฀ catalysis฀ by฀ mediating฀ a฀ critical฀ turn฀ that฀ brings฀ an฀
adjacent฀tyrosine฀or฀proline฀residue฀close฀to฀the฀catalytic฀zinc฀ion.฀Matrilysins฀(also฀called฀
matrix฀metalloendoproteinases,฀MMPs)฀are฀the฀major฀class฀of฀metzincin฀endopeptidases฀
involved฀ in฀ collagen฀ and฀ stromal฀ degradation.฀ The฀ other฀ two฀ classes,฀ adamalysins฀ and฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 113
DOI:฀10.1007/978-3-540-88116-2_8,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
114 8 The Zincins: Collagen Fiber Processing and Degradation

Table 8.1฀ All฀the฀protease฀families

Catalytic฀sitea Genesb Representativec Catalytic฀mechanism
Serine 175 Trypsin/chymotrypsin/ Residue฀forms฀−OH฀฀
caspasesd from฀bound฀H2O
Threonine 28 Proteasome฀enzymese Residue฀forms฀−OH฀฀
from฀bound฀H2O
Aspartic฀acid 21 Pepsin/HIV฀retropepsin Aspartyl฀residue
Cysteine 150 Cathepsins Cysteinyl฀residue
Metal฀ion฀(Zn2+) 187 Collagenase Zn2+฀ion-bound฀฀
water฀moleculef
a
฀ rotease฀structure฀is฀classiied฀by฀its฀catalytic฀action.฀A฀subclass฀of฀the฀serine฀proteases฀requires,฀
P
in฀addition,฀one฀or฀two฀histidine฀residues฀for฀catalytic฀activity
b
Number฀of฀protease฀genes฀in฀the฀human฀genome
c
Representative฀enzymes
d
Subgroup฀requiring฀calcium฀ions฀for฀activity฀are฀called฀calpains
e
Mostly฀exopeptidases
f
Rarely,฀the฀catalytic฀ion฀is฀Ni2+฀or฀Co2+฀(not฀in฀humans)
(Adapted฀from฀Table฀1฀in฀Puente฀et฀al.฀“Human฀and฀mouse฀proteases:฀a฀comparative฀genom-
ic฀ ฀approach.”฀ Nat.฀ Rev.฀ Genet.฀ 4(7):544–558,฀ 2003฀ and฀ updated฀ according฀ to฀ Puente฀ et฀ al.,฀฀
“A฀genomic฀view฀of฀the฀complexity฀of฀mammalian฀proteolytic฀systems.”฀Biochem฀Soc฀Trans.฀33฀
(Part฀2):331–334,฀2005)

Table 8.2฀ Types฀of฀zincin฀metalloproteases฀in฀humans

Type฀of฀enzyme Action Typical฀enzyme Structural฀class
Exoproteinase Removes฀N-terminal฀ Alanyl฀Aminopeptidase a
Metzincin
amino฀acid
Exoproteinase Removes฀C-terminal฀฀ Carboxypeptidase฀Ab Gluzincin
amino฀acid
Peptidase Removes฀a฀C-terminal฀ Angiotensin-converting฀ Gluzincin
dipeptide enzymec
Peptidase Cuts฀a฀peptide฀฀ Neurolysind Gluzincin
(<13฀aa)฀internally
Endoproteinase Cuts฀a฀large฀polypeptide฀ Matrilysins,฀adamalysins฀ Metzincin
internally and฀astacinse
a
฀ minopeptidase฀N฀(APN฀or฀CD13)฀is฀expressed฀in฀many฀cells,฀tissues,฀and฀species.฀It฀cleaves฀the฀
A
N-terminal฀amino฀acids฀from฀bioactive฀peptides,฀leading฀to฀their฀inactivation฀or฀degradation.฀It฀
has฀putative฀involvement฀in฀antigen฀processing฀and฀presentation,฀cell฀adhesion,฀tumor฀invasion฀
and฀metastasis,฀neurotransmitter฀degradation,฀and฀as฀a฀coronavirus฀receptor
b
฀Carboxypeptidase฀is฀a฀digestive฀enzyme.฀It฀is฀synthesized฀in฀the฀pancreas,฀secreted฀into฀the฀small฀
intestine,฀and฀hydrolyzes฀the฀C-terminal฀end฀of฀proteins฀and฀peptides
c
฀Angiotensin-converting฀enzyme฀(ACE)฀cleaves฀dipeptides฀from฀the฀C-terminus฀of฀oligopeptides,฀
notably฀converting฀angiotensin฀I฀(eight฀amino฀acids)฀to฀angiotensin฀II฀(six฀amino฀acids);฀angiotensin฀
converting฀enzyme฀(ACE).฀The฀product฀of฀enzyme฀activity฀causes฀an฀increase฀in฀blood฀pressure
d
฀Neurolysin฀acts฀only฀on฀substrates฀of฀less฀than฀about฀19฀amino฀acid฀residues฀(oligopeptides),฀with฀
a฀ particular฀ preference฀ for฀ cleaving฀ near฀ the฀ C-terminus.฀ Neurolysin฀ is฀ known฀ by฀ many฀ other฀
names,฀including฀oligopeptidase฀M฀and฀soluble฀angiotensin฀II-binding฀protein
e
Major฀metalloprotease฀families฀in฀humans฀(see฀text)
8.1.2. Catalytic Action of the Metzincin Family 115

Metalloendopeptidase

Inverzincins
Zincins HEXXH
HXXEH

Metzincins
Gluzincins Aspzincins
HEXXHXXGXXH/D
HEXXH + E HEXXH + D
Met turn
Pitrilysin
(Insulin processing)
Neurolysin
Astacins
Mitochondrial Neprilysin G. frondosa Adamalysins
processing Thermolysins metalloprotease Matrilysins (MMPs)
peptidase B. Anthracis
A. oryzae Serralysins
lethal factor
deuterolysin Snapalysin
C. botulinum
Leishmanolysin
neurotoxin

Fig. 8.1฀ Classiication฀of฀metallopeptidases฀by฀zinc-binding฀motifs.฀The฀major฀amino฀acid฀motif฀in฀

zincins฀has฀two฀histidine฀residues฀that฀coordinate฀with฀the฀metal฀ion฀and฀a฀glutamate฀residue฀(E)฀
for฀catalysis.฀A฀third฀residue฀that฀coordinates฀with฀the฀metal฀may฀be฀glutamate,฀aspartate฀(D),฀or฀
histidine.฀In฀metzincins,฀the฀third฀coordinating฀residue฀is฀histidine฀or฀aspartate฀(H/D),฀but฀the฀name฀
is฀taken฀from฀the฀presence฀of฀a฀downstream฀invariant฀methionine฀residue฀(see฀Fig.฀8.2฀and฀text).฀
The฀red฀type฀indicates฀enzymes฀or฀enzyme฀subfamilies฀encoded฀in฀the฀human฀genome฀(Slightly฀
modiied฀from฀Fig.฀1A฀of฀F.X.฀Gomis-Ruth,฀“Structural฀aspects฀of฀the฀metzincin฀clan฀of฀metal-
loendopeptidases.”฀Mol.฀Biotechnol.฀24(2):157–202,฀2003)

astacins฀(Fig.฀8.1),฀are฀involved฀in฀procollagen฀processing.฀Metzincin฀classes฀not฀encoded฀
in฀the฀human฀genome฀are฀mostly฀bacterial฀virulence฀factors฀such฀as฀anthrax฀lethal฀toxin,฀a฀
serralysin.฀Additional฀metzincins฀have฀recently฀been฀identiied฀in฀plants฀and฀bacteria.

8.1.2.
Catalytic Action of the Metzincin Family

The฀metzincin฀catalytic฀domain฀consists฀of฀a฀lat฀surface฀within฀a฀small฀cleft฀within฀which฀
peptide฀substrates฀bind฀and฀are฀hydrolyzed฀(Fig.฀8.2a).฀In฀astacins,฀the฀catalytic฀domain฀
is฀stable,฀but฀adamalysins฀require฀a฀calcium฀ion฀to฀stabilize฀the฀lat฀surface฀of฀the฀domain฀
above฀the฀cleft.฀Matrilysins฀require฀two฀calcium฀ions฀and฀a฀second,฀noncatalytic฀zinc฀ion฀
to฀stabilize฀this฀domain฀(Fig.฀8.3).฀Table฀8.3฀reviews฀the฀roles฀of฀speciic฀metal฀ions฀that฀
participate฀ in฀ the฀ various฀ stages฀ of฀ collagen฀ processing฀ discussed฀ in฀ this฀ chapter฀ and฀
Chap.฀7.
116 8 The Zincins: Collagen Fiber Processing and Degradation

a b
ENZYME
S2
S1’ S3’
P2
P1’ P3’
N Substrate Polypeptide C

P1 2+
P3 Zn P2’
S3 S1 S2’

c
Rn CH
CH Rn+1 O
O Rn CH
Glu C NH C CH Rn+1
C H
O − HO C NH
O− O
O 1 H+
H O−
++
Zn++ Zn

His His His

2

H2N CH

Rn CH + Rn+1 HN CH
O H
O Rn CH + Rn+1
C OH
C
C H C OH
O O−
O− 3 O
O
H
Zn++
Zn++

Fig. 8.2฀ Catalytic฀metzincin฀domain.฀(a)฀Catalytic฀endopeptidase฀subunit฀showing฀the฀peptide฀bind-

ing฀cleft฀and฀surrounding฀domains.฀The฀site฀binds฀six฀amino฀acids฀on฀either฀side฀of฀the฀bond฀to฀be฀
hydrolyzed.฀Substrate฀is฀in฀an฀extended฀conformation฀and฀the฀bound฀amino฀acids฀are฀numbered฀
(N-฀to฀C-terminus):฀…S3–S2–S1–S1¢–S2¢–S3¢…฀The฀products฀are฀two฀peptides:฀…P3–P2–P1–COOH฀
and฀NH2–P1¢–P2¢–P3¢…฀(Slightly฀modiied฀from฀Fig.฀1B฀of฀F.X.฀Gomis-Ruth,฀“Structural฀aspects฀of฀
the฀metzincin฀clan฀of฀metalloendopeptidases.”฀Mol.฀Biotechnol.฀24(2):157-202,฀2003).฀(b)฀Active฀
site฀crevice฀of฀the฀metzincin฀catalytic฀domain฀from฀above.฀The฀conserved฀zinc-binding฀motif฀has฀
the฀ structure:฀ His176-Glu177-Xaa-Xaa-His180-Xaa-Xaa-Gly-Xaa-Xaa-His186.฀ The฀ numbering฀ is฀ for฀
serralysin,฀ an฀ astacin฀ family฀ protein,฀ but฀ the฀ relative฀ positions฀ of฀ these฀ amino฀ acids฀ within฀ the฀
motif฀are฀identical฀in฀all฀metzincins.฀The฀catalytic฀zinc฀ion฀(large฀sphere)฀has฀a฀coordinated฀water฀
molecule฀(small฀sphere)฀whose฀hydrogen฀atoms฀are฀H-bonded฀to฀Glu177฀(bond฀shown฀in฀Fig.฀8.2c).฀
About฀20฀residues,฀downstream฀lies฀a฀tyrosine฀phenolic฀side฀chain฀that฀points฀back฀from฀beneath฀
the฀active฀site฀as฀a฀result฀of฀a฀turn฀induced฀by฀the฀invariant฀methionine฀residue.฀The฀ligands฀involv-
ing฀tyrosine฀form฀a฀distorted฀trigonal฀bipyramid฀in฀which฀His176,฀His186,฀and฀the฀water฀molecule฀are฀
equatorial.฀His180฀and฀Tyr216,฀respectively,฀form฀the฀upper฀and฀lower฀axial฀ends฀(peaks฀of฀each฀pyra-
mid).฀The฀Zn2+฀ion฀is฀approximately฀2.2฀Å฀from฀the฀histidine฀ligands,฀2฀Å฀from฀the฀water฀molecule,฀
and฀ 2.8฀ Å฀ from฀ the฀ phenolic฀ O฀ atom฀ of฀ Tyr216.฀ Adamalysins฀ and฀ matrilysins฀ (and฀ some฀ other฀
8.1.3. Metzincin Activation 117

The฀glutamate฀(E)฀residue฀in฀the฀common฀zincin฀motif฀(HEXXH)฀participates฀in฀cataly-
sis฀ by฀ attaching฀ a฀ water฀ molecule฀ that฀ also฀ coordinately฀ bonds฀ to฀ the฀ catalytic฀ zinc฀ ion฀
(Fig.฀ 8.2b).฀ A฀ likely฀ catalytic฀ mechanism฀ involves฀ the฀ loss฀ of฀ a฀ proton฀ from฀ the฀ water฀
molecule,฀ followed฀ by฀ nucleophilic฀ attack฀ of฀ OH−1฀ on฀ the฀ peptide฀ bond฀ (illustrated฀ in฀
Fig.฀8.2c).฀During฀this฀process฀in฀astacins฀such฀as฀serralysin,฀a฀tyrosine฀residue฀lips฀back฀
and฀forth฀during฀substrate฀anchoring,฀cleavage,฀and฀product฀release,฀in฀a฀motion฀referred฀to฀
as฀a฀“tyrosine฀switch.”฀The฀phenol฀group฀of฀this฀tyrosine฀coordinates฀with฀the฀catalytic฀zinc฀
ion฀and฀displaces฀the฀water฀molecule,฀thus฀inhibiting฀catalysis.฀Interactions฀of฀the฀enzyme฀
away฀from฀the฀catalytic฀site฀are฀transmitted฀to฀the฀tyrosine฀residue฀and฀weaken฀or฀strengthen฀
the฀ coordination฀ of฀ its฀ phenolic฀ group฀ to฀ the฀ catalytic฀ zinc฀ ion.฀ In฀ matrilysins฀ and฀ ada-
malysins,฀ the฀ “tyrosine-switch”฀ is฀ replaced฀ by฀ a฀ “cysteine-switch.”฀ The฀ thiol฀ group฀ of฀
cysteine฀coordinates฀to฀the฀catalytic฀zinc฀ion฀instead฀of฀tyrosine,฀similarly฀displacing฀the฀
water฀ molecule฀ required฀ for฀ catalysis.฀ Unlike฀ the฀ tyrosine฀ switch,฀ this฀ “off”฀ position฀ is฀
virtually฀permanent฀unless฀the฀N-terminal฀propeptide฀that฀contains฀this฀cysteine฀residue฀is฀
removed,฀allowing฀the฀thiol฀group฀to฀be฀replaced฀on฀the฀zinc฀ion฀by฀the฀water฀molecule฀that฀
participates฀in฀catalysis฀as฀in฀astacins฀(Fig.฀8.2).

8.1.3.
Metzincin Activation

All฀human฀metzincins฀are฀secreted฀as฀proenzymes.฀Astacins฀and฀adamalysins฀are฀mostly฀
activated฀by฀calcium-ion-dependent฀serine฀proteases฀(pro-protein฀convertases)฀that฀meet฀
up฀ with฀ their฀ substrates฀ in฀ trans-Golgi฀ and฀ secretory฀ vacuoles.฀ These฀ proenzymes฀ are฀
known฀as฀furin-like฀convertases฀because฀of฀their฀homology฀to฀a฀serine฀protease฀called฀furin฀
and฀a฀bacterial฀endoprotease฀called฀subtilisin.฀The฀furin-like฀enzymes฀require฀calcium฀ions฀
to฀maintain฀structural฀stability฀whereas฀other฀serine฀proteases,฀represented฀by฀trypsin฀and฀
chymotrypsin,฀ do฀ not.฀ The฀ furin-like฀ pro-protein฀ convertases฀ autocleave฀ their฀ own฀
N-terminal฀ domain฀ propeptide฀ (self-activate)฀ during฀ secretion฀ and฀ then฀ convert฀ the฀
N-terminal฀domains฀of฀co-secreted฀metzincins.฀Activation฀cascades฀also฀occur฀among฀the฀

฀ etzincins)฀have฀proline฀instead฀of฀tyrosine฀at฀the฀turn.฀In฀these฀enzymes,฀a฀cysteine฀residue฀within฀
m
the฀pro-domain฀blocks฀the฀cleft฀by฀binding฀to฀the฀catalytic฀zinc฀ion฀and฀excluding฀water฀molecule฀
binding฀(see฀text)฀(Modiied฀from฀Park,฀H.I.฀and฀Ming,฀L.J.฀“Mechanistic฀studies฀of฀the฀astacin-like฀
Serratia฀metalloendopeptidase฀serralysin:฀highly฀active฀(>2,000%)฀Co(II)฀and฀Cu(II)฀derivatives฀
for฀ further฀ corroboration฀ of฀ a฀ “metallotriad”฀ mechanism.”฀ J.฀ Biol.฀ Inorg.฀ Chem.฀ 7(6):600–610.฀
2002).฀(c)฀Mechanism฀of฀catalysis.฀(1)฀The฀tyrosine฀residue฀moves฀away฀from฀the฀zinc฀ion฀and฀the฀
water฀molecule฀(green)฀attacks฀the฀substrate฀polypeptide฀(red)฀under฀the฀inluence฀of฀the฀deproto-
nated฀ glutamate฀ residue.฀ (2)฀ The฀ C-terminal฀ peptide฀ amino฀ group฀ picks฀ up฀ the฀ proton฀ and฀ is฀
released.฀(3)฀The฀remaining฀hydroxide฀anion฀attacks฀the฀carboxyl฀group,฀which฀remains฀held฀by฀the฀
zinc฀ion฀but฀it฀is฀quickly฀replaced฀by฀another฀water฀molecule฀(Modiied฀from฀University฀of฀Tours,฀
France฀Web฀site:฀http://delphi.phys.univ-tours.fr/Prolysis/introprotease.html)
118 8 The Zincins: Collagen Fiber Processing and Degradation

II II II

A A A
C C
I I I

III III III

Ca
V V VZn
Ca
IV IV IV
B B B
Glu Glu Glu
His Active His Active His Active
His His
site His
site Gly
site
Gly Zn Gly Zn
Zn Met
His Met N Met His
Tyr His
C N
Glu
Met- Met- Asp Met-
N turn
turn turn C
C C

Matrix
Astacin Adamalysin
metalloproteinase
(ADAMTS-2)
(MMP8)

Fig. 8.3฀ Architecture฀of฀astacins,฀adamalysin฀type-2,฀and฀matrix฀metalloproteases.฀Topology฀scheme฀

of฀astacins,฀adamalysin฀type-2,฀and฀matrix฀metalloproteinases.฀The฀a-helices฀are฀shown฀as฀rods,฀
b-sheet฀strands฀as฀arrows,฀and฀unstructured฀regions฀as฀thin฀lines.฀Key฀amino฀acids฀in฀the฀catalytic,฀
zinc-binding฀motif฀are฀shown฀(white฀in฀black฀ellipse).฀Distinguishing฀features฀of฀each฀structure฀are฀
shown฀as฀lighter฀gray฀for฀amino฀acids฀(black฀letter),฀and฀as฀unlabeled,฀secondary-structure฀ele-
ments.฀In฀addition฀to฀the฀catalytic฀zinc฀ion฀in฀all฀three฀structures,฀the฀calcium฀ion฀in฀adamalysin฀
type-2,฀and฀the฀two฀calcium฀ions฀and฀second฀zinc฀ion฀in฀the฀matrix฀metalloproteinases฀(Abbreviated฀
from฀Fig.฀3฀in฀F.X.฀Gomis-Ruth,฀“Structural฀aspects฀of฀the฀metzincin฀clan฀of฀metalloendopepti-
dases.”฀Mol.฀Biotechnol.฀24(2):157–202,฀2003)

Table 8.3฀ Metal฀ions฀required฀for฀collagen฀processing

Metal฀Ion/s Enzyme/s
Fe 2+
Proline/lysine฀hydroxylase
Cu1+ Lysyl฀oxidase
Mg2+/Mn2+ Integrin฀attachment฀of฀collagen฀to฀cells
Zn /Ca2+ 2+
Metalloproteases

zincins฀due฀to฀self-฀or฀hetero-catalysis.฀For฀example,฀an฀activated฀adamalysin฀can฀remove฀
the฀propeptide฀from฀another฀adamalysin,฀a฀matrilysin฀or฀an฀astacin,฀all฀independently฀of฀
the฀initiating฀furin-like฀activation.
A฀similar฀cascade฀is฀associated฀with฀inlammation฀following฀an฀injury,฀infection,฀or฀
environmental฀stress฀(Fig.฀8.4).฀It฀involves฀chymotrypsinogen-like฀serine฀proteases฀called฀
8.1.3. Metzincin Activation 119

Plasminogen

t-PA Fibrin
u-PA
PAI-1
PAI-2

Plasmin
α2-Antiplasmin
α2-Macroglobulin

Fibrinolysis Matrilysin Stromal

activation breakdown

Fig. 8.4฀ Matrilysin฀activation.฀Pro-matrilysins฀are฀activated฀by฀plasmin.฀This฀serine฀protease฀does฀not฀

require฀Ca2+฀for฀activation,฀unlike฀furin,฀which฀activates฀the฀procollagen฀peptidases฀(Fig.฀8.5).฀Plasmin฀
is฀obtained฀from฀a฀precursor,฀plasminogen,฀by฀plasminogen฀activators.฀Plasminogen฀is฀made฀in฀the฀
liver฀and฀secreted฀into฀the฀blood฀plasma.฀Plasminogen฀activators฀are฀secreted฀in฀the฀tissues฀as฀uroki-
nase฀related,฀u-PA,฀or฀as฀a฀tissue฀type,฀t-PA.฀tPA฀is฀made฀by฀ibroblasts฀and฀basal฀epithelial฀cells.฀It฀is฀
present฀in฀small฀amounts฀in฀the฀stroma฀where฀it฀activates฀plasminogen฀released฀from฀injured฀capillar-
ies฀and฀causes฀ibrinolysis฀(Sect.฀11.4.2)฀(Copy฀of฀Fig.฀1฀from฀B.฀Kinnby,฀“The฀plasminogen฀activat-
ing฀ system฀ in฀ periodontal฀ health฀ and฀ disease”.฀ Biol.฀ Chem.฀ 383:85–92,฀ January฀ 2002;฀ copyright฀
permission฀given฀by฀Walter฀de฀Gruyter,฀Berlin,฀New฀York฀and฀the฀author)

tissue฀ or฀ urokinase฀ plasminogen฀ activators฀ (plasminogen฀ pro-protein฀ convertases).฀

Plasminogen฀is฀made฀in฀the฀liver,฀secreted฀into฀the฀blood฀plasma,฀and฀activated฀its฀single฀
polypeptide฀chain฀being฀cut฀into฀two฀disulide฀linked฀chains,฀like฀the฀cutting฀of฀prothrom-
bin฀by฀factor฀Xa฀(Sect.฀11.3.4.).฀If฀plasminogen฀escapes฀from฀capillaries,฀it฀is฀converted฀
by฀ a฀ plasminogen฀ activator฀ enzyme฀ in฀ the฀ tissues฀ (usually฀ t-PA,฀ Fig.฀ 8.4)฀ to฀ plasmin.฀
Plasmin฀ degrades฀ ibrin฀ blood฀ clots฀ (Chap.฀ 11).฀ In฀ the฀ stroma,฀ plasmin฀ also฀ cleaves฀
ibronectin,฀ thrombospondin฀ (TS),฀ laminin,฀ and฀ the฀ matrilysins,฀ especially฀ procollage-
nase.฀These฀activities฀lyse฀a฀damaged฀stroma฀so฀that฀it฀can฀later฀be฀replaced฀with฀healthy฀
tissue.
Excessive฀activation฀of฀matrilysin฀by฀plasmin฀is฀prevented฀by฀tissue฀inhibitors฀of฀matri-
lysin฀proteases฀(TIMPs)฀in฀a฀healthy฀stroma,฀or฀during฀the฀repair฀phase฀of฀inlammation฀
(Sect.฀13.2.5),฀or฀by฀thrombospondin-2.฀(Sect.฀3.2.2),฀or฀during฀the฀repair฀phase฀of฀inlam-
mation฀(Sect.฀13.2.5).฀The฀N-terminal฀domains฀of฀one฀of฀four฀homologous฀TIMPs฀bind฀
tightly฀and฀irreversibly฀to฀the฀catalytic฀site฀of฀activated฀matrilysins฀and฀adamalysins.฀Tissue฀
damage฀must฀be฀maintained฀at฀suficiently฀high฀levels฀for฀enough฀plasmin฀to฀have฀been฀
activated฀ before฀ these฀ inhibitors฀ are฀ signiicantly฀ depleted฀ along฀ with฀ the฀ matrix.฀
Uncontrolled฀ matrilysin฀ activation฀ characterizes฀ many฀ chronic฀ diseases฀ including฀ peri-
odontal฀disease฀(Sect.฀13.3.1).
120 8 The Zincins: Collagen Fiber Processing and Degradation

Collagen฀processing฀and฀degradation฀are฀accomplished฀by฀zinc-containing฀metalloen-
doproteinases฀(zincins)฀that฀cleave฀polypeptides฀into฀large฀fragments.฀The฀catalytic฀
zinc฀ion฀is฀coordinated฀to฀two฀histidine฀residues฀in฀a฀motif฀(HEXXH)฀and฀subdivided฀
by฀a฀third฀zinc-coordinated฀residue:฀glutamate฀(gluzincins),฀aspartate฀(aspzincins),฀or฀
histidine฀or฀aspartate฀associated฀with฀a฀downstream฀methionine-mediated฀fold฀(metz-
incins).฀Human฀metzincin฀endoproteinases฀are฀astacins,฀adamalysins฀and฀matrilysins.฀
Catalysis฀involves฀a฀zinc-bound฀water฀molecule,฀the฀glutamate฀residue฀of฀the฀zincin฀
motif,฀and฀an฀enzyme-speciic฀recognition฀site.฀The฀coordinated฀water฀molecule฀may฀
be฀ displaced฀ by฀ a฀ downstream฀ phenol฀ (tyrosine)฀ in฀ astacins,฀ or฀ thiol฀ (cysteine)฀ in฀
adamalysins฀and฀matrilysins.฀The฀cysteine฀is฀part฀of฀the฀methionine-mediated฀fold,฀
which฀is฀disconnected฀by฀activation฀when฀a฀large฀N-terminal฀peptide฀is฀removed฀by฀
serine฀proteases:฀furin-like฀during฀development฀or฀plasminogen฀activators฀induced฀by฀
stress.฀ Stromal฀ activation฀ is฀ resisted฀ by฀ tissue฀ inhibitors฀ of฀ matrilysin฀ proteases฀
(TIMPs).

8.2.1.
Fibrillar Procollagen Processing

Once฀the฀procollagen฀triple฀helix฀has฀assembled฀in฀the฀lumen฀of฀the฀endoplasmic฀reticulum฀
(Chap.฀6),฀it฀moves฀to฀the฀cis-Golgi฀cisternae฀in฀transport฀vesicles฀and฀then฀through฀the฀
Golgi฀to฀the฀trans-Golgi,฀where฀it฀forms฀bundles฀before฀being฀released฀to฀secretory฀vacu-
oles฀(Fig.฀7.1).฀The฀bundles฀develop฀as฀procollagen฀is฀cleaved฀to฀tropocollagen.
Collagen฀types฀I,฀II,฀III,฀and฀a2V,฀all฀use฀one฀of฀the฀three฀adamalysins฀as฀procollagen฀
N-proteases฀(PNPs)฀and฀one฀of฀the฀three฀astacins฀as฀procollagen฀C-peptidases฀(PCPs).฀The฀
adamalysin฀consensus฀sequence฀is฀pro-gln฀with฀the฀hydrolytic฀cleavage฀C-terminal฀to฀the฀
proline฀residue฀as฀indicated฀by฀the฀down฀pointing฀arrow฀(P↓Q)฀in฀type฀I฀collagen,฀ala-gln฀
(A↓Q)฀in฀types฀II฀and฀III฀collagens,฀and฀pro-ala฀(P↓A)฀in฀a2V฀collagen.฀The฀amino฀acid฀that฀
follows฀the฀down-pointing฀arrow฀is฀the฀N฀terminal฀amino฀acid฀of฀the฀tropocollagen฀that฀is฀
cleaved฀ out฀ by฀ PNP.฀ The฀ a1V,฀ a1XI,฀ and฀ a2XI฀ collagens฀ use฀ an฀ astacin฀ to฀ remove฀ the฀
N-propeptide฀ and฀ a฀ furin-like฀ pro-protein฀ convertase฀ to฀ remove฀ the฀ C-propeptide.฀
Figure฀8.5฀indicates฀the฀polypeptide฀motifs฀of฀the฀nonadamalysin฀enzymes฀that฀cleave฀the฀
various฀ibrillar฀collagens.
Furin-like฀ pro-protein฀ convertases฀ activate฀ all฀ the฀ procollagen฀ processing฀ enzymes฀
(Fig.฀8.6).฀Figure฀8.7a฀shows฀the฀domain฀structure฀of฀the฀astacin฀family.฀The฀three฀common฀
ones฀(grouped฀at฀the฀top)฀are฀bone฀morphogenetic฀protein฀1฀(BMP-1),฀mammalian฀tolloid฀
(mTld),฀and฀tolloid฀like฀1฀protein฀(TLL-1).฀Protein฀mTld฀is฀the฀preferred฀PCP,฀but฀it฀cleaves฀
slowly฀in฀secretory฀vesicles,฀preventing฀the฀bundles฀from฀growing฀too฀rapidly.฀Indeed,฀a฀
separately฀secreted฀enhancer฀protein฀upregulates฀mTld฀activity฀after฀secretion฀at฀the฀cell฀
surface,฀where฀a฀rapid฀self-assembly฀of฀collagen฀ibers฀occurs.฀All฀three฀astacins฀also฀pro-
cess฀pro-lysyl฀oxidase฀(Sect.฀4.2.2),฀the฀g2฀and฀a3฀chains฀of฀laminin-5,฀and฀pro-biglycan,฀a฀
glycosaminoglycan฀ similar฀ to฀ decorin฀ (Sect.฀ 6.5.1)฀ but฀ possessing฀ two฀ attached฀ gly-
cosaminoglycans฀residues฀instead฀of฀one.
8.2.1. Fibrillar Procollagen Processing 121

a BMP Procollagen C peptidase

Pro-α1I Tyr Tyr Arg Ala Asp Asp Ala
Y Y R A D D A
Pro-α2I Phe Tyr Arg Ala Asp GIn Pro
F Y R A D Q P
Pro-αII Y M R A D Q A
Pro-αIII P Y Y G D E P
Pro-α2V E F T E D Q A

b BMP Procollagen N peptidase

Pro-α1V T P Q S Q D P
Pro-α1XI A A Q A Q E P
Pro-α2XI R P Q N Q Q P

c Furin Procollagen C peptidase

Pro-α1V R T R R N I D
Pro-α1XI K T R R H T E
Pro-α2XI K T R R S V D

Fig. 8.5฀ Procollagen฀amino฀acid฀cleavage฀motifs฀other฀than฀adamalysins.฀See฀text฀for฀adamalysin฀

cleavage฀sequences.฀All฀sequences฀are฀from฀human฀procollagens.฀The฀sequences฀around฀the฀cleav-
age฀site฀for฀Pro-α1I฀and฀Pro-α2I฀are฀given฀in฀both฀three-letter฀and฀(beneath)฀one-letter฀amino฀acid฀
abbreviations.฀The฀sequences฀for฀the฀other฀procollagen฀chains฀are฀only฀the฀one-letter฀abbreviations.฀
(a)฀Astacin฀procollagen฀C฀peptidase฀(PCP).฀Amino฀acids฀motifs฀cleaved฀in฀common฀ibrillar฀colla-
gens฀ by฀ PCP,฀ the฀ astacin฀ bone฀ morphogenetic฀ protein฀ 1฀ (BMP1)฀ and฀ the฀ homologous฀ enzymes฀
(Fig.฀8.6).฀Bold฀indicates฀a฀motif,฀in฀this฀case:฀fXX↓DX(A/P)฀where฀f฀=฀hydrophobic฀residue;฀pep-
tide฀bond฀cut฀=฀↓฀and฀X฀=฀any฀amino฀acid฀residue.฀(b)฀Astacin฀procollagen฀N฀peptidase.฀Amino฀acids฀
motifs฀cleaved฀in฀type฀V฀and฀XI฀ibrillar฀collagens฀by฀procollagen฀N-protease฀(PNP)฀where฀PNP฀is฀
BMP1฀instead฀of฀adamalysin฀PNP฀(motif฀is฀QX↓QXP).฀(c)฀Furin฀procollagen฀C฀peptidase.฀Amino฀
acids฀motifs฀cleaved฀in฀above฀ibrillar฀collagens฀where฀PCP฀is฀a฀furin-like฀enzyme฀instead฀of฀BMP1฀
(motif฀is฀BTRR↓XXX฀where฀B฀is฀a฀basic฀residue฀other฀than฀R).฀This฀motif฀is฀very฀similar฀to฀RXRR↓,฀
one฀of฀two฀common฀furin฀consensus฀sequences฀(Sequences฀of฀all฀human฀procollagen฀polypeptides฀
are฀public฀and฀can฀be฀downloaded฀from฀the฀Swiss-Protein฀Database.฀For฀example,฀Type฀I฀collagen฀
alpha1฀ and฀ alpha2฀ procollagen฀ polypeptides,฀ COL1A1฀ and฀ COL1A2,฀ are฀ respectively฀ at฀ http://
www.uniprot.org/uniprot/P02452฀and฀http://www.uniprot.org/uniprot/P08123).฀Data฀for฀type฀pro-
aII,฀pro-aIII,฀and฀pro-a2V฀are฀from฀C.฀Unsold฀et฀al.,฀J.฀Biol.฀Chem.฀277(7):596–5602.฀Data฀for฀
pro-a1V,฀pro-a1XI฀and฀pro-a2XI฀are฀from฀Imamura฀et฀al.,฀J.฀Biol.฀Chem.฀273(42):27511–27517)

Note:฀Astacins฀were฀named฀from฀two฀unrelated฀sources.฀Bone฀morphogenetic฀protein฀1฀(BMP-
1)฀was฀originally฀identiied฀as฀a฀zinc฀metalloprotease฀in฀extracts฀of฀demineralized฀bovine฀bone฀
together฀ with฀ TGFa-like฀ growth฀ factors฀ (described฀ in฀ Chap.฀ 3฀ and฀ 8)฀ termed฀ BMP-2A฀ and฀
BMP-3.฀Amino฀acid฀sequencing฀and฀cDNA฀cloning฀demonstrated฀that฀mouse฀PCP-1฀was฀iden-
tical฀to฀BMP-1฀and฀that฀chicken฀PCP-2฀was฀identical฀to฀a฀protein฀named฀mammalian฀tolloid฀
(mTld)฀after฀a฀homologous฀Drosophila฀proteinase฀tolloid฀(TLD).฀BMP-1฀and฀mTld฀are฀two฀of฀
six฀splice฀variants฀of฀the฀bmp1฀gene.฀Two฀related฀genes฀encoding฀proteases฀similar฀to฀mTld฀
have฀been฀identiied฀in฀bmp1฀null฀mice:฀mammalian฀tolloid฀like-1฀and฀-2฀(mTLL-1฀and฀mTLL-
2).฀The฀bmp1฀null฀mice฀make฀abnormal฀collagen฀ibrils฀in฀the฀skin฀and฀fail฀to฀close฀the฀ventral฀
body฀wall.฀Homozygous฀loss฀of฀bmp1฀is฀lethal฀in฀utero,฀but฀a฀skeleton฀develops฀because฀mTll-1฀
has฀PCP฀activity฀that฀partially฀compensates฀for฀BMP1.
122 8 The Zincins: Collagen Fiber Processing and Degradation

Subtilisin-like P- and cystein- Furin or related

Pro
catalytic domain rich domains proprotein convertases

(RTKR|DVYQ)

RSRR|AATS

Pro Astacin CUB,EGF & specific domains PCP (BMP-1,

catalytic domain alternatively spliced mTLD or TLL-1)
RARR|HAAD

Adamalysin Disintegrin, TSP-1 cysteine- PNP (ADAM

Pro catalytic domain rich and C-terminal domains
TS-2, 3, 14)

Procollagen
N C

Tropocollagen

Fig. 8.6฀ Activation฀of฀procollagen฀N-฀and฀C-proteinases.฀The฀signal฀sequence฀for฀secretory฀targeting฀

is฀represented฀as฀a฀black฀rectangle฀and฀is฀removed฀in฀the฀endoplasmic฀reticulum฀prior฀to฀secretion.฀
Each฀of฀the฀C-terminal฀domains฀is฀labeled฀in฀the฀igure.฀The฀activator฀pro-protein฀convertases฀furin,฀
procollagen฀C-peptidase฀(PCP)฀and฀procollagen฀N-protease฀(PNP)฀each฀contain฀a฀pro-domain฀(Pro;฀
purple)฀that฀ensures฀proper฀folding.฀The฀pro-domain฀of฀furin฀is฀auto-catalytically฀removed,฀but฀the฀
pro-domains฀of฀PNP฀and฀PCP฀are฀not.฀The฀irst฀domain฀of฀furin฀after฀its฀signal-฀and฀pro-domains฀is฀
the฀protease฀domain,฀related฀to฀the฀subtilase฀superfamily฀of฀Ca2+-dependent฀proteases.฀Similarly,฀
the฀ irst฀ domain฀ of฀ PCP฀ and฀ PNP฀ after฀ their฀ signal฀ and฀ pro-domains฀ is฀ a฀ metalloendoprotease.฀
Following฀the฀catalytic฀domain฀of฀each฀protein฀is฀a฀mixture฀of฀C-terminal฀domains.฀In฀furin฀and฀
other฀ pro-protein฀ convertases,฀ the฀ so-called฀ P-domain฀ is฀ essential฀ for฀ activity฀ C-terminal฀ exten-
sions฀ for฀ correct฀ localization.฀ The฀ C-terminal฀ domains฀ of฀ the฀ PCPs฀ modulate฀ activity,฀ whereas฀
those฀of฀PNP฀specify฀collagen-type฀substrate฀(Modiied฀slightly฀from฀Fig.฀4฀in฀E.C.฀Canty฀and฀K.E.฀
Kadler.฀“Procollagen฀Traficking,฀Processing,฀and฀Fibrillogenesis.”฀J.฀Cell฀Sci.฀Vol.฀118(7):1341–
1353,฀2005;฀Reproduced฀with฀permission฀of฀the฀Company฀of฀Biologists)

Removal฀of฀the฀less฀bulky฀N-propeptide฀of฀procollagen฀proceeds฀similarly.฀The฀released฀
collagen฀N-terminal฀propeptide฀is฀reabsorbed฀back฀into฀the฀cytosol฀where฀it฀inhibits฀collagen฀
translation฀and฀prevents฀excessive฀iber฀formation฀(feedback฀inhibition).฀The฀most฀common฀
PNP฀is฀an฀ada฀malysin฀of฀the฀thrombospondin฀type-2฀class฀(ADAMTS-2),฀which฀is฀structur-
ally฀large฀and฀complex,฀containing฀nine฀domains฀(Fig.฀8.7b).฀The฀pro-domain฀is฀essential฀for฀
correct฀folding฀during฀polypeptide฀synthesis.฀The฀thrombospondin฀(TS)฀domains฀through฀the฀
C-terminus฀ of฀ the฀ protein฀ specify฀ the฀ proper฀ orientation฀ of฀ enzyme฀ binding฀ for฀ catalysis.฀
Replacing฀the฀three฀C-terminal฀TS฀repeats฀with฀those฀in฀a฀closely฀related฀protein,฀ADAMTS-14฀
(PNP฀for฀type฀a2V฀collagen),฀prevents฀all฀enzymatic฀activity฀toward฀type฀I฀collagen,฀whereas฀
removing฀only฀the฀C-terminal฀domain฀enhances฀the฀type฀I฀collagen฀activity.
Snake฀ venoms฀ cause฀ a฀ rapid฀ disintegration฀ of฀ the฀ stroma฀ (disintegrin)฀ due฀ to฀ short฀฀
peptides฀each฀containing฀an฀RGD฀integrin-binding฀sequence.฀The฀RGD฀sequence฀displaces฀
8.2.1. Fibrillar Procollagen Processing 123

a PCP and astacin family domains

BMP-1
mTId
mTLL-1

mTLL-2

TLD
Pro MMP C1 C2 E1 C3 E2 C4 C5
Signal

b PNP and ADAMTS-2 domains

MP cataytic Central
Furin cleavage site domain
sites

N-ter 1 2 3 4
Pro- Metallo- TS-1 Spacer TS-1 C-terminal
peptide proteinase repeat repeats
Signal Dis- Cys-
peptide integrin rich

Fig. 8.7฀ Domain฀organization฀of฀the฀two฀procollagen฀peptidases.฀(a)฀Domain฀organization฀of฀pro-

collagen฀C-peptidase฀(PCP)฀and฀related฀astacins.฀Signal฀peptides฀are฀dark฀purple,฀prodomains฀are฀
green,฀proteinase฀domains฀are฀yellow,฀CUB฀domains฀are฀light฀blue,฀EGF-like฀domains฀are฀light฀
purple,฀and฀domains฀unique฀to฀each฀protein฀are฀shown฀in฀pink.฀The฀CUB฀domains฀are฀labeled฀C1฀
through฀C5฀and฀the฀EGF฀domains฀are฀labeled฀E1฀and฀E2.฀CUB฀domains฀are฀present฀in฀functionally฀
diverse,฀mostly฀developmentally฀regulated฀proteins฀and฀also฀in฀peptidases฀belonging฀to฀astacin฀and฀
chymotrypsin฀ families.฀ It฀ is฀ an฀ extracellular฀ domain฀ of฀ approximately฀ 110฀ amino฀ acid฀ residues฀
containing฀ four฀ conserved฀ cysteines฀ that฀ form฀ adjacent฀ disulide฀ bridges.฀ The฀ CUB฀ domain฀ is฀
predicted฀to฀have฀a฀beta-barrel฀structure฀similar฀to฀that฀of฀immunoglobulins฀(antibody฀molecules).฀
The฀EGF฀domain฀does฀not฀include฀the฀N-terminal฀Ca2+-binding฀subdomain฀discussed฀in฀relation฀to฀
ibrillin฀(Sect.฀6.1.1)฀(Slightly฀modiied฀from฀Fig.฀1฀in฀G.฀Ge.฀and฀D.S.฀Greenspan,฀Developmental฀
Roles฀of฀the฀BMP1/TLD฀Metalloproteinases.฀Birth฀Defects฀Research฀(Part฀C),฀78:47–68,฀2006).
(b)฀Domain฀organization฀of฀procollagen฀N-peptidase,฀ADAMTS-2฀adamalysin.฀฀The฀four฀thrombo-
spondin฀type฀1฀(TS-1)฀repeats฀(circles)฀are฀numbered฀as฀indicated฀Thrombospondins฀are฀discussed฀
in฀Sect.฀3.2.2).฀ADAMTS-3฀and฀ADAMTS-14฀proteins฀are฀homologous฀(Slightly฀modiied฀from฀
Fig.฀ 1฀ in฀ A.฀ Colige฀ et฀ al.฀ “Domains฀ and฀ maturation฀ processes฀ that฀ regulate฀ the฀ activity฀ of฀
ADAMTS-2,฀a฀metalloproteinase฀cleaving฀the฀aminopropeptide฀of฀ibrillar฀procollagens฀types฀I-III฀
and฀V.”฀J.฀Biol.฀Chem.฀280(41):34397–34408,฀2005)

adamalysins฀from฀integrins฀on฀the฀cell฀surface฀(Sect.฀3.2.1).฀The฀released฀adamalysins฀loat฀
freely฀in฀the฀stroma฀and฀behave฀like฀activated฀nonspeciic฀matrilysins.฀Integrin-bound฀met-
alloproteases฀are฀critical฀for฀ovum฀fertilization,฀and฀so฀the฀name฀of฀this฀group฀of฀proteins฀
was฀ cleverly฀ transformed฀ into฀ a฀ biochemical฀ name:฀ A฀ Disintegrin฀ And฀ Metalloprotease฀
Domain฀ (ADAM),฀ or฀ ADAMalysin.฀ The฀ ADAMTS-2฀ proteins฀ comprise฀ a฀ subfamily฀ of฀
adamalysins฀possessing฀thrombospondin฀domains.฀The฀ADAM฀family฀proper฀consists฀of฀
over฀40฀proteins,฀one฀of฀which฀is฀described฀in฀Chap.฀13฀(Sect.฀13.2.2).฀These฀latter฀proteins฀
possess฀ a฀ canonical฀ disintegrin฀ domain฀ that฀ keeps฀ them฀ integrin-bound฀ at฀ the฀ outer฀ cell฀
surface.฀ A฀ different฀ disintegrin฀ domain฀ in฀ the฀ ADAMTS฀ family฀ enables฀ their฀ secretion฀
instead฀of฀remaining฀cell฀surface฀bound.
124 8 The Zincins: Collagen Fiber Processing and Degradation

Procollagen฀N-terminal฀processing฀is฀performed฀mostly฀by฀adamalysins฀and฀C-terminal฀
processing฀mostly฀by฀astacins.฀Processing฀begins฀with฀procollagen฀bundle฀formation.฀
Some฀ cleavage฀ of฀ all฀ three฀ polypeptides฀ at฀ mostly฀ the฀ N-terminus฀ occurs฀ within฀ the฀
secretory฀vacuole,฀but฀secretion฀activates฀rapid฀C-terminal฀cleavage฀and฀spontaneous฀
iber฀formation.฀The฀adamalysin฀responsible฀for฀procollagen฀N-terminal฀processing฀is฀
held฀to฀integrins฀at฀the฀cell฀surface฀following฀secretion฀and฀this฀also฀promotes฀faster฀
extracellular฀procollagen฀processing฀to฀tropocollagen.

8.3.1.
Matrilysins (MMPs) Hydrolyze Collagen and Stromal Proteins

Matrilysins฀(MMPs)฀are฀required฀for฀stromal฀remodeling฀during฀development,฀pregnancy,฀
and฀ growth,฀ and฀ also฀ following฀ trauma฀ or฀ infection.฀ Different฀ classes฀ degrade฀ different฀
extracellular฀matrix฀protein฀components:฀ibers,฀anchoring฀and฀basement฀membrane฀col-
lagens,฀proteoglycans,฀laminin,฀ibronectin,฀and฀other฀stromal฀proteins.฀Many฀also฀partici-
pate฀in฀proteolytic฀events฀required฀to฀control฀diverse฀physiological฀processes:฀cell฀surface฀
release฀ of฀ growth฀ factors,฀ activation฀ of฀ cytokines฀ and฀ receptors,฀ and฀ the฀ inactivation฀ of฀
proteinase฀and฀angiogenesis฀inhibitors.
As฀noted฀in฀the฀previous฀section,฀matrilysin฀catalysis฀is฀held฀in฀check฀by฀endogenous฀
tissue฀ inhibitors฀ of฀ metalloproteinases฀ (TIMPs),฀ which฀ irreversibly฀ bind฀ to฀ the฀ active฀
site.฀ Different฀ TIMPs฀ irst฀ bind฀ to฀ hemopexin-like฀ domains฀ on฀ almost฀ all฀ matrilysins฀
before฀they฀can฀bind฀to฀the฀active฀site,฀thus฀providing฀some฀TIMP฀speciicity.฀Hemopexin฀
is฀a฀plasma฀protein฀that฀binds฀to฀heme฀and฀transports฀it฀to฀the฀liver฀for฀conversion฀to฀bile.฀
It฀is฀formed฀by฀the฀repetition฀of฀a฀variable฀length฀unit฀of฀35฀to฀45฀residues,฀the฀hemopexin-
like฀domain.฀TIMPs฀are฀the฀ligands฀for฀a฀modiied,฀homologous฀hemopexin-like฀domain฀
on฀ matrilysins.฀ Figure฀ 8.8฀ diagrams฀ the฀ structures฀ of฀ matrilysins฀ most฀ relevant฀ to฀ the฀
topics฀in฀this฀book.
There฀are฀at฀least฀28฀matrilysins฀that฀participate฀in฀connective฀tissue฀degradation฀as฀col-
lagenases,฀gelatinases,฀elastases,฀and฀stromelysins.฀All฀28฀matrilysin฀enzymes฀are฀listed฀in฀
Table฀8.4฀along฀with฀their฀matrilysin฀(MMP)฀number฀and฀cell฀expression.฀The฀molecular฀
weights฀of฀the฀most฀relevant฀pro-฀and฀activated฀enzymes,฀and฀their฀substrate฀speciicities,฀
are฀listed฀in฀Table฀8.5.฀Collagen฀is฀primarily฀degraded฀by฀MMP-1฀and฀-8฀(ibroblast฀and฀
neutrophilic฀ granulocyte฀ collagenase)฀ and฀ MMP-2฀ and฀ -9฀ (ibroblast฀ and฀ neutrophilic฀
granulocyte฀gelatinase).฀Fibroblast฀gelatinase฀(MMP-2)฀secretion฀is฀inhibited฀by฀thrombo-
spondin-2,฀causing฀excessive฀collagen฀synthesis฀(ibrosis),฀which฀not฀only฀limits฀the฀spread฀
of฀an฀infection,฀but฀also฀destroys฀tissue฀architecture฀and฀causes฀implants฀rejection฀(foreign฀
body฀reaction;฀Sect.฀3.2.2).
8.3.1. Matrilysins (MMPs) Hydrolyze Collagen and Stromal Proteins 125

MMP-1, -8
MMP-3
MMP-20 MMP-2, -9

II II
I III I
IV III
IV
Catalytic domain with
active site cleft (orange),
catalytic zinc (red) and 2 3 Fibronectin
structural calcium ions type II repeats

Activation Domain linker

cleavage site
II Hemopexin-like domain
Prodomain I III with four-fold blade-like
IV
symmetry

Fig. 8.8฀ The฀ domain฀ arrangement฀ of฀ the฀ matrilysins.฀ White฀ dots฀ represent฀ two฀ calcium฀ ions฀ that฀
contribute฀to฀the฀structural฀integrity฀of฀the฀zincin฀catalytic฀domain.฀The฀red฀dot฀represents฀the฀zinc฀
ion,฀the฀yellow฀dot฀represents฀the฀active฀site฀cleft฀with฀substrate฀binding฀sites฀represented฀by฀the฀
‘smile’.฀The฀thick฀light-blue฀arrow฀฀represents฀the฀prodomain฀in฀the฀binding฀cleft฀and฀the฀scissors฀
represent฀where฀the฀prodomain฀is฀removed฀by฀plasmin.฀Figure฀is฀composed฀of฀the฀right฀top฀two฀parts฀
of฀Fig.฀1฀in฀W.฀Bode฀and฀K.฀Maskos,฀Structural฀basis฀of฀the฀matrix฀metalloproteinases฀and฀their฀
physiological฀inhibitors,฀the฀tissue฀inhibitors฀of฀metalloproteinases.฀Biol.฀Chem.฀384฀(June):863–
872,฀2003;฀Copyright฀permission฀given฀by฀Walter฀de฀Gruyter,฀Berlin,฀New฀York฀and฀both฀authors

Table 8.4฀ Cellular฀sources฀of฀matrix฀metalloproteases

Protease฀class MMP฀number Keratinocyte/leukocyte฀
expression
Collagenases MMP-1a,฀8฀and฀13 Keratinocyteb:฀MMP-1,฀3,฀฀
9,฀10,฀and฀28
Gelatinases MMP-2a฀and฀9 Granulocytesc:฀MMP-8,฀9,฀and฀25
Stromelysins MMP-3฀and฀10 B฀Cellsd:฀MMP-11,฀26,฀and฀27
Membrane-type฀MMPs MT-MMPs฀14a฀–฀17;฀฀ T฀cellse:฀MMP-15,16,฀24,฀and฀28
24,฀and฀25
Others MMP-7,฀11,฀12,฀19,฀20,฀฀ Monocytesf:฀MMP-1,฀2,฀3,฀฀
21,฀23,฀26,฀27,฀and฀28 9,฀10,฀14,฀17,฀19,฀and฀25
a
Major฀MMPs฀expressed฀by฀ibroblasts
b฀
Expression฀enhanced฀in฀proliferative/migratory฀basal฀keratinocytes฀at฀wound฀edge.฀Around฀ul-
cerations฀of฀mucosal฀tissues,฀such฀as฀lung฀and฀intestine,฀MMP-28฀(epilysin)฀is฀absent฀but฀MMP-7฀
(matrilysin฀ -฀ from฀ which฀ all฀ this฀ group฀ of฀ MMPs฀ takes฀ its฀ name.)฀ is฀ present฀ instead฀ ฀ MMP-7฀
degrades฀most฀major฀non-collagen฀proteins฀in฀a฀stromal฀matrix
c
Mostly฀in฀neutrophilic฀granulocytes฀attracted฀to฀a฀region฀of฀stromal฀injury
d
Antibody-producing฀lymphocytes฀that฀become฀attracted฀to฀a฀site฀of฀infection
e
Non-antibody฀producing฀lymphocytes฀also฀attracted฀to฀a฀site฀of฀infection
f
฀Macrophage฀precursors฀that฀develop฀from฀the฀white฀blood฀cell฀iniltrates฀at฀sites฀of฀stromal฀injury฀
or฀infection
126 8 The Zincins: Collagen Fiber Processing and Degradation

Table 8.5฀ Matrilysin฀connective฀tissue฀degrading฀enzyme฀speciicities

MMP# Enzyme฀name Molecular฀฀ Molecular฀฀ Substrates
mass,฀latent mass,฀active
(kDa) (kDa)
MMP-1 Collagenase-1฀ 55 45 Fibrillar฀collagens,฀฀
(ibroblasts) gelatin,฀proteoglycans
MMP-8 Collagenase-2฀ 75 58 Fibrillar฀collagens
(neutrophils)
MMP-13 Collagenase-3฀฀ 65 55 Collagen฀type฀II
(many฀cells)
MMP-2 Gelatinase฀A฀ 72 66 Gelatin,฀collagen฀฀
(ibroblasts) type฀IV,฀elastin,฀ibronectin
MMP-9 Gelatinase฀B฀ 92 86 Gelatin,฀collagen฀฀
(neutrophils) type฀IV,฀elastin
MMP-7 Matrilysin 28 19 Matrix฀components฀except฀
ibrillar฀collagens
MMP-3 Stromelysin-1 57 45 All฀matrix฀components฀except฀
elastin฀and฀ibrillar฀collagen
MMP-10 Stromelysin-2 57 44 Matrilysin฀without฀elastase฀฀
or฀laminin฀activity
MMP-11 Stromelysin-3 59 44 Laminin
MMP-12 Metalloelastase 53 45/22 Elastin,฀ibronectin,฀฀
collagen฀type฀lV
MMP-20 Enamelysin 54 43 Amelogenin

8.3.2.
Stromelysins

Stromelysins-1,฀-2,฀and฀-3฀(MMP-3,฀MMP10,฀and฀MMP-11)฀degrade฀stromal฀components฀
other฀than฀collagen.฀Skin฀ibroblasts฀constitutively฀express฀progelatinase฀(MMP-2),฀and฀
activate฀it฀by฀co-secreting฀a฀membrane-adherent฀matrilysin฀on฀their฀cell฀surface,฀especially฀
MMP-14.฀In฀contrast,฀following฀exogenous฀stresses฀or฀exposure฀to฀cytokines฀and฀ultravio-
let฀irradiation,฀ibroblasts฀secrete฀procollagenase฀(MMP-1)฀and฀neutrophils฀secrete฀proge-
latinase฀ (MMP-9).฀ These฀ enzymes฀ are฀ activated฀ by฀ plasmin฀ from฀ stress-activated฀
plasminogen฀pro-protein฀convertases฀(see฀Sect.฀8.1.3).

8.3.3.
Enamelysin

Enamelysin฀(MMP-20)฀has฀major฀domains฀and฀an฀overall฀structure฀identical฀to฀ibroblast฀
collagenase,฀ gelatinase,฀ and฀ some฀ stromelysins,฀ but฀ it฀ lacks฀ conserved฀ residues฀ that฀
8.3.4. Collagenases and Gelatinases 127

determine฀collagenase฀or฀stromelysin฀speciicity.฀A฀shorter฀amino฀acid฀sequence฀of฀the฀
catalytic฀domain’s฀C-terminal฀region฀further฀distinguishes฀it฀from฀collagenase฀and฀gelati-
nase.฀Enamelysin฀cleaves฀amelogenin,฀a฀major฀protein฀that฀determines฀enamel฀crystalli-
zation฀ (Chap.฀ 9).฀ Except฀ for฀ collagenases฀ (Sect.฀ 8.3.4),฀ the฀ roles฀ of฀ the฀ remaining฀
matrilysins฀are฀not฀yet฀known.

8.3.4.
Collagenases and Gelatinases

Collagenases฀ act฀ on฀ collagen฀ ibers฀ at฀ neutral฀ pH.฀ They฀ recognize฀ a฀ three-dimensional฀
structure฀that฀recurs฀at฀the฀gaps฀in฀the฀quarter-staggered฀array฀of฀tropocollagen฀molecules฀
and฀cleave฀all฀three฀polypeptides฀at฀that฀point.฀This฀cut฀(Fig.฀8.9a)฀causes฀the฀tropocollagen฀
triple฀helix฀to฀spontaneously฀unwind,฀exposing฀individual฀one-quarter฀and฀three-quarter฀

a
Collagenase Gap of 35 nm
N-ter C-ter
300 nm
1
2
3
4
5
6

Collagen triple helix 67 nm

b
N-ter Collagenase action site C-ter
Fig. 8.9฀ Modes฀of฀action฀of฀
neutral฀collagenase฀and฀
gelatinase฀on฀collagen฀iber. Collagen triple helix
(a)฀Initial฀step:฀Collagen฀
degradation฀begins฀with฀
Gelatinase cuts between leu-pro sequences
neutral฀collagenase฀cutting฀a฀ of each tropocollagen polypeptide fragment
triple฀helix฀into฀N-ter฀¾฀and฀
C-ter฀¼฀fragments.฀(b)฀Final฀
step:฀The฀¾฀and฀¼-length฀
1
tropocollagen฀a-chain฀
fragments฀unwind,฀exposing฀ 2
its฀leu-pro฀bonds฀to฀ 3
gelatinase฀and฀it฀is฀degraded฀ 3/4 Length fragment 1/4 Length
to฀small฀peptides฀ fragment
128 8 The Zincins: Collagen Fiber Processing and Degradation

length฀polypeptides฀to฀gelatinase.฀The฀gelatinase฀cleaves฀exposed฀leucine–proline฀bonds,฀
which฀are฀common฀in฀the฀tropocollagen฀sequence฀(Fig.฀8.9b).฀The฀resultant฀small฀peptides฀
are฀taken฀up฀by฀local฀cells฀and฀degraded฀to฀free฀amino฀acids฀in฀their฀lysosomal฀vesicles.฀
Excessive฀collagen฀iber฀cross-linking฀slows฀the฀unwinding฀of฀the฀three฀chains฀and฀their฀
rate฀of฀degradation฀is฀slowed฀considerably.฀Collagen฀ibers฀are฀therefore฀dificult฀to฀turn฀
over฀in฀old฀age฀because฀of฀extensive฀cross-linking.฀(Sect.฀4.2.2),฀resulting฀in฀tissue฀mal-
functions฀associated฀with฀senescence.
Collagenase฀and฀gelatinase฀are฀produced฀by฀ibroblasts฀and฀neutrophils.฀Although฀cata-
lytically฀identical,฀the฀respective฀cells฀utilize฀different฀genes฀with฀homologous฀but฀non-
identical฀amino฀acid฀sequences฀(Table฀8.5).฀The฀ibroblast฀enzymes฀are฀larger฀and฀produced฀
in฀ a฀ different฀ environment฀ from฀ the฀ neutrophil฀ enzyme.฀ Fibroblast฀ gelatinase฀ cleaves฀
monocyte฀ chemoattractant฀ protein-3฀ (MCP-3),฀ which฀ prevents฀ leukocyte฀ iniltration฀ of฀
developing฀ or฀ remodeling฀ tissues.฀ The฀ MCP-3฀ cleavage฀ products฀ bind฀ to฀ and฀ inhibit฀ a฀
monocyte฀receptor฀that฀intact฀MCP-3฀activates฀on฀monocytes฀and฀neutrophils.฀MCP3฀is฀
cleaved฀because฀it฀฀binds฀to฀the฀hemopexin฀domain฀of฀ibroblast฀gelatinase฀(MMP-2),฀but฀
not฀to฀the฀corresponding฀domain฀of฀neutrophil฀gelatinase฀(MMP-9).฀Neutrophilic฀granulo-
cytes฀are฀absent฀during฀development,฀but฀present฀in฀large฀numbers฀following฀tissue฀dam-
age฀or฀infection฀when฀intact฀MCP3฀actively฀recruits฀granulocytes฀to฀the฀affected฀region฀
(Sect.฀13.3.1).

There฀are฀28฀matrilysins฀that฀degrade฀various฀stromal฀proteins,฀most฀importantly฀ibrous฀
collagen:฀MMP-1฀and฀-8฀(collagenases)฀and฀MMP-2฀and฀-9฀(gelatinases).฀Collagenases฀
cleave฀all฀three฀tropocollagen฀polypeptides฀into฀large฀N-terminal฀and฀small฀C-terminal฀
fragments฀ that฀ spontaneously฀ unwind,฀ exposing฀ leu-pro฀ bonds.฀ Gelatinase฀ cleaves฀
these฀bonds฀to฀short฀peptides฀that฀are฀endocytosed฀and฀digested฀to฀amino฀acids฀in฀lyso-
somal฀vesicles.฀The฀collagenases฀and฀gelatinases฀are฀expressed฀by฀ibroblasts฀and฀neu-
trophils,฀respectively.฀They฀are฀separately฀encoded:฀ibroblast฀gelatinase฀will฀hydrolyze฀
monocyte฀chemoattractant฀protein-3฀(MCP-3),฀preventing฀inlammation฀during฀devel-
opment฀when฀neutrophils฀are฀absent.฀Neutrophil฀gelatinase฀cannot฀cleave฀MCP-3.
 Biological Mineralization
9

Mineralization฀is฀the฀precipitation฀of฀calcium฀phosphate,฀but฀biochemical฀mediation฀of฀
this฀process฀is฀not฀fully฀understood.฀In฀this฀chapter,฀the฀chemistry฀underlying฀mineral-
ization฀(Sect.฀1)฀and฀the฀structures฀of฀bones฀and฀teeth฀(Sect.฀2)฀are฀described.฀Osteoblasts฀
secrete฀osteoid฀matrix฀and฀matrix฀vesicles฀that฀transport฀type฀I฀collagen฀and฀calcium฀
phosphate,฀respectively,฀to฀the฀matrix฀where฀they฀will฀mineralize.฀Secreted฀matrix฀ves-
icles฀take฀up฀calcium฀and฀phosphate฀until฀they฀burst฀and฀release฀the฀calcium฀phosphate,฀
which฀ then฀ redissolves฀ and฀ remineralizes฀ around฀ the฀ type฀ I฀ collagen฀ (Sect.฀ 3).฀
Glycoproteins฀involved฀in฀correctly฀modeling฀bone฀and฀dentin,฀and฀the฀role฀of฀osteocal-
cin฀ in฀ limiting฀ excessive฀ bone฀ growth฀ is฀ then฀ discussed฀ (Sect.฀ 4).฀ There฀ follows฀ a฀
detailed฀description฀of฀enamel฀(E)฀mineralization฀and฀of฀the฀major฀proteins฀involved฀
(Sect.฀5)฀followed฀by฀two฀summaries:฀the฀difference฀between฀enamel฀and฀bone฀miner-
alization,฀and฀the฀vitamins฀required฀for฀mineralization฀(Sect.฀6).

9.1.1.
Fundamental Properties of Calcium Phosphate Precipitation

Calcium฀ ions฀ precipitate฀ with฀ phosphate฀ ions฀ where฀ their฀ dissolved฀ free฀ ion฀ activities฀
(concentrations฀at฀low฀ionic฀strength)฀exceed฀their฀solubility฀product฀–฀the฀product฀of฀the฀
molar฀concentrations฀of฀each฀ion฀powered฀to฀its฀respective฀charge.฀Calcium฀is฀invariably฀
present฀as฀a฀divalent฀ion฀(Ca2+),฀but฀phosphate฀ions฀assume฀one฀of฀three,฀pH-dependent฀
forms฀ (Fig.฀ 9.1a,฀ left):฀ dihydrogen฀ phosphate฀ (H2PO41−),฀ monohydrogen฀ phosphate฀
(HPO42−),฀and฀phosphate฀(PO43−).฀In฀solution฀above฀pH฀6.2,฀a฀predominance฀of฀calcium฀
dihydrogen฀ phosphate฀ transitions฀ to฀ a฀ predominance฀ of฀ calcium฀ monohydrogen฀ phos-
phate.฀ Calcium฀ monohydrogen฀ phosphate฀ (solubility฀ product฀ ~1฀ ×฀ 10−6)฀ is฀ about฀ 100฀
times฀ less฀ soluble฀ than฀ calcium฀ dihydrogen฀ phosphate฀ (solubility฀ product฀ ~1฀ ×฀ 10-4).฀
Precipitated฀calcium฀phosphate฀is฀commonly฀referred฀to฀as฀apatite.

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 129
DOI:฀10.1007/978-3-540-88116-2_9,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
130 9 Biological Mineralization

Fig. 9.1฀ Calcium฀phosphate฀precipitation฀and฀structures฀of฀orthophosphate฀(Pi)฀and฀pyrophosphate฀

(PPi)฀ions.฀(a)฀Calcium฀phosphate฀precipitation฀and฀solubility฀at฀different฀pH.฀Left฀side฀of฀equation฀
summarizes฀that,฀at฀pH฀6.2,฀the฀fraction฀of฀monohydrogen฀and฀dihydrogen฀phosphate฀ions฀is฀about฀
equal฀and฀that฀calcium฀dihydrogen฀phosphate฀is฀100฀times฀less฀soluble฀than฀calcium฀monohydro-
gen฀phosphate.฀Right฀side฀of฀the฀igure฀shows฀that฀phosphate฀ions฀exist฀mostly฀as฀monohydrogen฀
phosphate฀ at฀ pH฀ 7.฀ Calcium฀ monohydrogen฀ phosphate฀ precipitates฀ as฀ an฀ amorphous฀ solid฀ that฀
spontaneously฀rearranges฀to฀form฀hydroxyapatite฀crystals.฀(b)฀Structures฀of฀Pi฀and฀PPi.฀Structures฀
are฀the฀ionized฀forms฀at฀physiological฀pH฀(~7.2).฀(Original฀igures)

9.1.2.
Nature of the Apatite Precipitate

The฀apatite฀that฀initially฀precipitates฀is฀crumbly฀and฀brittle฀due฀to฀its฀amorphous฀(noncrystal-
line)฀structure.฀If฀the฀surrounding฀luid฀remains฀above฀pH฀7,฀this฀apatite฀undergoes฀a฀series฀
of฀spontaneous,฀solid-state฀rearrangements฀whose฀major฀end฀product฀is฀hydroxyapatite,฀a฀
crystal฀containing฀ten฀calcium฀ions,฀six฀phosphate฀(PO43−)฀ions,฀and฀two฀hydroxide฀(OH1−)฀
ions.฀These฀changes฀are฀due฀to฀a฀spontaneous฀alkalinization฀of฀the฀apatite,฀in฀which฀mono-
hydrogen฀ phosphate฀ (HPO42−)฀ ions฀ lose฀ a฀ proton฀ and฀ hydroxide฀ ions฀ appear฀ from฀ water฀
molecules฀trapped฀in฀the฀initial฀amorphous฀precipitate฀(Fig.฀9.1a,฀right).฀Hydroxyapatite฀is฀a฀
long,฀thin,฀lat฀crystal฀that฀forms฀a฀thick,฀hard,฀lat฀surface฀that฀is฀primarily฀responsible฀for฀
the฀strength฀of฀bones฀and฀teeth.฀The฀long฀axis฀is฀referred฀to฀as฀the฀“c”฀axis.฀The฀width฀is฀the฀
“b”฀axis฀and฀the฀thickness฀is฀the฀“a”฀axis.
9.1.4. Nucleation 131

During฀bone฀formation,฀the฀crystals฀form฀with฀their฀“c”฀axis฀parallel฀to฀the฀collagen฀iber฀
and฀thicken฀by฀accretion฀at฀their฀“a”฀and฀“b”฀axes.฀During฀enamel฀formation,฀apatite฀crystals฀
join฀end-to-end฀at฀their฀“c”฀axis,฀forming฀thin฀ribbons฀that฀become฀enamel฀rods.

9.1.3.
Apatite Crystal Substitutions Influence Bone Strength and Solubility

The฀hydroxyapatite฀crystals฀in฀bone฀and฀teeth฀are฀imperfect฀due฀to฀other฀anions฀and฀cations,฀
especially฀ magnesium,฀ chloride,฀ carbonate,฀ and฀ luoride฀ ions.฀ Carbonate฀ (CO32−)฀ is฀ the฀
most฀important.฀At฀low฀carbonate฀contents฀(<4%฀by฀weight),฀a฀carbonate฀ion฀replaces฀a฀
phosphate฀ion฀in฀the฀crystal฀(“A”฀site฀substitution),฀but฀at฀higher฀contents฀(>4%฀by฀weight)฀
it฀replaces฀a฀hydroxide฀ion฀(“B”฀site฀substitution).฀Either฀substitution฀slightly฀shortens฀and฀
fattens฀the฀crystal฀(“c”฀or฀“a”฀axes฀increase)฀and฀increases฀solubility.฀In฀contrast,฀if฀hydrox-
ide฀ions฀are฀present,฀they฀can฀be฀replaced฀by฀luoride,฀which฀decreases฀apatite฀solubility฀
(Sect.฀ 16.2.1).฀ Crystallographic฀ analyses฀ indicate฀ that,฀ in฀ bone฀ and฀ dentin,฀ phosphate฀ is฀
often฀ replaced฀ by฀ carbonate,฀ whereas฀ in฀ enamel฀ it฀ is฀ more฀ often฀ replaced฀ with฀ chloride฀
(Cl1−).฀Carbonated฀hydroxyapatite฀is฀critical฀for฀enamel฀development฀(see฀Sect.฀9.5.3).
Enamel฀mineral฀has฀many฀large฀hydroxyapatite฀crystals,฀whereas฀bone฀has฀many฀small฀
ones฀with฀numerous฀vacancies฀and฀substitutions.฀These฀differences฀increase฀the฀elasticity฀
of฀bone฀compared฀with฀enamel฀and฀promote฀its฀interactions฀with฀the฀surrounding฀collagen.฀
Recently,฀a฀tightly฀bound฀“hydration฀shell”฀that฀ills฀a฀porous฀collagen-apatite฀junction฀was฀
discovered฀around฀normal฀bone฀crystals.฀The฀water-illed฀pores฀are฀normally฀immobile,฀but฀
repeated฀stresses฀cause฀the฀water฀to฀leak฀out฀from฀between฀the฀mineral฀and฀collagen.฀The฀
drying฀increases฀mineralization฀and฀crystal฀formation,฀which฀may฀explain฀the฀decreased฀
elasticity฀of฀bones฀with฀age.

9.1.4.
Nucleation

Dissolved฀calcium฀and฀phosphate฀ions฀may฀remain฀soluble฀despite฀their฀concentrations฀exceed-
ing฀the฀solubility฀product฀in฀blood฀plasma฀and฀stromal฀extracellular฀(interstitial)฀luid฀where฀the฀
pH฀is฀just฀above฀7฀(Sect.฀3.3.1).฀In฀blood฀plasma,฀mineralization฀is฀prevented฀by฀polyanions,฀
especially฀albumin,฀citrate,฀and฀pyrophosphate฀(PPi),฀which฀chelate฀calcium฀ions฀and฀prevent฀
their฀ precipitation฀ with฀ monohydrogen฀ phosphate฀ ions฀ (orthophosphate,฀ Pi,฀ or฀ HPO42−).฀
Pyrophosphate฀(PPi)฀inhibits฀the฀premature฀aggregation฀of฀calcium฀with฀monohydrogen฀phos-
phate฀ions฀in฀mineralizing฀tissues฀and฀interstitial฀luid฀throughout฀the฀body฀(Fig.฀9.1b).
For฀mineralization,฀the฀normal,฀metastable฀state฀is฀adjusted฀by฀nucleation,฀measured฀by฀
the฀seed฀and฀solubility฀tests.฀The฀seed฀test฀measures฀amount฀of฀solid฀apatite฀required฀to฀
precipitate฀ Ca2+฀ and฀ HPO42−฀ ion฀ concentrations฀ exceeding฀ their฀ solubility฀ product.฀ The฀
solubility฀test฀measures฀the฀minimal฀concentrations฀of฀Ca2+฀and฀HPO42−฀necessary฀to฀induce฀
precipitation.฀Type฀I฀collagen฀ibers฀nucleate฀bone฀formation฀as฀the฀concentrations฀of฀Ca2+฀
132 9 Biological Mineralization

and฀ HPO42−฀ ions฀ increase.฀ Premature฀ nucleation฀ is฀ prevented฀ by฀ pyrophosphate฀ (PPi;฀
Fig.฀9.1b),฀small฀amounts฀of฀which฀strongly฀inhibit฀nucleation.
PPi฀ is฀ made฀ in฀ three฀ ways:฀ (1)฀ in฀ the฀ nucleus฀ as฀ a฀ by-product฀ of฀ RNA฀ synthesis฀
(nNTP฀→฀NMPn฀+฀nPPi);฀(2)฀in฀the฀cytosol฀as฀a฀by-product฀of฀amino฀acid฀activation฀for฀
protein฀synthesis฀(aa฀+฀ATP฀→฀aaAMP฀+฀PPi);฀and฀(3)฀by฀acetyl฀CoA฀synthetase฀on฀the฀
outer฀mitochondrial฀membrane฀prior฀to฀its฀degradation฀for฀ATP฀production฀(R–COOH฀+฀
ATP฀ +฀ HS–CoA฀ →฀ R–Co–SCoA฀ +฀ AMP฀ +฀ PPi).฀ Amino฀ acid฀ activation฀ is฀ the฀ major฀
source฀of฀cytosolic฀PPi,฀which฀is฀transported฀by฀the฀ANK฀protein฀to฀the฀osteoid฀matrix฀
to฀inhibit฀premature฀mineralization฀(see฀Sect.฀9.3.5).

Mineralization฀is฀the฀precipitation฀of฀calcium฀and฀phosphate฀ions฀above฀pH฀7.฀Initial฀pre-
cipitates฀are฀soft฀and฀noncrystalline฀(amorphous).฀If฀left฀alone,฀a฀solid-state฀rearrangement฀
slowly฀and฀spontaneously฀forms฀hydroxyapatite,฀whose฀crystals฀each฀contain฀ten฀calcium฀
(Ca2+),฀ six฀ phosphate฀ (PO43−),฀ and฀ two฀ hydroxide฀ (OH1−)฀ ions.฀ The฀ resulting฀ hard,฀ lat฀
surface฀is฀primarily฀responsible฀for฀the฀strength฀of฀bones฀and฀teeth.฀Substituting฀hydroxide฀
or฀phosphate฀ions฀with฀carbonate฀ions฀increases฀crystal฀solubility,฀whereas฀substituting฀
hydroxide฀ ions฀ with฀ luoride฀ ions฀ decreases฀ crystal฀ solubility.฀ In฀ bone,฀ hydroxyapatite฀
crystals฀have฀many฀spaces฀and฀substitutions,฀permitting฀a฀water฀layer฀between฀apatite฀and฀
collagen฀that฀dries฀up฀and฀decreases฀bone’s฀elasticity฀with฀age.฀Biological฀luids฀are฀super-
saturated฀ with฀ calcium฀ and฀ phosphate,฀ but฀ contain฀ pyrophosphate฀ and฀ polyanions฀ that฀
inhibit฀ spontaneous฀ precipitation.฀ Pyrophosphate฀ interferes฀ with฀ calcium฀ phosphate฀
aggregation฀and฀polyanions฀(citrate,฀albumin,฀and฀other฀negatively฀charged฀proteins)฀che-
late฀calcium฀ions฀and฀prevent฀them฀from฀being฀free฀in฀solution฀to฀precipitate.

9.2.1.
The Structures of Bone, Dentin, and Cementum

There฀are฀two฀types฀of฀bone฀tissue:฀dense฀(compact฀or฀cortical฀)฀and฀spongy฀(cancellous฀or฀
trabecular).฀The฀difference฀lies฀in฀how฀tightly฀the฀tissue฀is฀packed฀together.฀Bone฀matrix฀is฀
predominantly฀a฀mixture฀of฀type฀I฀collagen฀ibrils,฀that฀resist฀pulling฀forces,฀and฀calcium฀phos-
phate฀mineral฀(apatite฀crystals)฀that฀resist฀compression.฀The฀volumes฀of฀collagen฀and฀mineral฀
in฀bone฀are฀about฀equal,฀but฀the฀collagen฀accounts฀for฀only฀~20%฀of฀the฀bone฀weight.
Compact฀bone฀consists฀of฀closely฀packed฀osteons฀(Haversian฀systems).฀In฀these฀osteons,฀
central฀canals฀called฀the฀osteonic฀(Haversian)฀canals฀are฀surrounded฀by฀concentric฀rings฀
(lamellae)฀of฀calciied฀matrix฀(Fig.฀9.2).฀Bone฀cells฀(osteocytes)฀lie฀between฀the฀calciied฀
rings,฀in฀spaces฀called฀lacunae.฀Small฀channels฀(canaliculi฀containing฀osteocyte฀processes)฀
radiate฀from฀the฀lacunae฀to฀an฀osteonic฀(Haversian)฀canal฀to฀provide฀passageways฀for฀nutri-
ents฀and฀excreted฀products.฀Each฀osteonic฀canal฀contains฀a฀central฀large฀blood฀capillary฀
vessel฀that฀parallels฀the฀long฀axis฀of฀the฀bone.฀The฀capillaries฀are฀connected฀to฀each฀other฀
and฀to฀larger฀blood฀vessels฀within฀a฀thin฀ibroblast-rich฀stroma฀on฀the฀surface฀of฀the฀bone฀
(the฀periosteum)฀by฀mineral-perforating฀(Volkmann’s)฀canals.
Cancellous฀bone฀is฀less฀dense.฀It฀consists฀of฀thin฀plates฀and฀bars฀of฀bone฀(trabeculae)฀
adjacent฀ to฀ small,฀ irregular฀ cavities฀ (bone฀ marrow)฀ containing฀ a฀ connective฀ tissue฀ from฀
9.2.1. The Structures of Bone, Dentin, and Cementum 133

Osteon of
Lacunae containing osteocytes compact bone

Lamellae
Trabecula of
Canaliculi cancellous bone

Osteon Osteonic (Haversian)

(Haversian canal
System)

Periosteum

Volkmann’s canal

Fig. 9.2฀ Bone฀structure.฀Diagram฀of฀a฀long฀bone฀indicating฀the฀compact฀and฀cancellous฀structures฀

(see฀ text)฀ (http://training.seer.cancer.gov/module_anatomy/unit3_2_bone_tissue.html;฀ funded฀ by฀
the฀U.S.฀National฀Cancer฀Institute’s฀Surveillance,฀Epidemiology,฀and฀End฀Results฀(SEER)฀Program,฀
via฀ contract฀ number฀ N01-CN-67006,฀ with฀ Emory฀ University,฀ Atlanta฀ SEER฀ Cancer฀ Registry,฀
Atlanta,฀Georgia,฀U.S.A.)

which฀the฀various฀cells฀that฀form฀the฀red฀and฀white฀blood฀cells฀and฀platelets฀differentiate฀
(Sect.฀11.1.1).฀The฀cavities฀are฀the฀equivalent฀of฀osteonic฀canals฀and฀are฀surrounded฀by฀
trabeculae฀containing฀lacunae฀and฀canaliculi฀in฀which฀the฀osteocyte฀cell฀bodies฀and฀pro-
cesses฀are฀respectively฀situated฀(Fig.฀9.2).฀Although฀the฀trabeculae฀appear฀haphazard,฀they฀
are฀in฀fact฀organized฀to฀provide฀maximum฀strength,฀like฀braces฀that฀support฀a฀building.฀The฀
trabeculae฀of฀cancellous฀bone฀follow฀stress฀lines.฀Stress฀creates฀boney฀microcracks฀that฀
activate฀osteoclasts฀and฀osteoblasts฀(Sect.฀10.2.1),฀leading฀to฀appropriate฀remodeling฀and฀
realignment.฀Bone฀cells฀develop฀as฀osteoblasts฀in฀the฀periosteum฀or฀on฀the฀surface฀of฀tra-
beculae฀and฀become฀osteocytes฀in฀lacunae฀following฀matrix฀mineralization.฀Osteoblasts฀
and฀osteocytes฀account฀for฀about฀15%฀of฀the฀bone฀mass.
Dentin฀is฀secreted฀unmineralized฀like฀bone.฀The฀predentin฀matrix฀consists฀of฀collagen,฀
glycoproteins฀and฀proteoglycans฀like฀the฀osteoid฀matrix฀described฀below฀(Sect฀9.3.1).฀The฀
collagen฀ibers฀aggregate฀with฀their฀long฀axes฀parallel฀to฀long฀thin฀odontoblast฀processes฀
which฀extend฀through฀the฀predentin฀and฀remain฀in฀the฀mineralized฀tissue฀as฀the฀center฀of฀
dentinal฀tubules.฀To฀mineralize฀the฀dentin฀around฀the฀tubules,฀Ca2+฀ions฀are฀transported฀to฀
the฀mineralization฀front฀from฀underlying฀blood฀vessels฀in฀the฀developing฀pulp฀cavity.฀The฀
process฀of฀mineralization฀is฀likely฀mediated฀by฀matrix฀vesicles฀as฀described฀for฀bone฀(Sect.฀
9.3.1).฀ The฀ innermost฀ lining฀ of฀ dentinal฀ tubules฀ is฀ mineralized฀ last฀ and฀ becomes฀ more฀
dense฀than฀the฀inter-tubular฀dentin.฀The฀dentinal฀tubules฀have฀lateral฀branches฀that฀permit฀
the฀odontoblasts฀to฀communicate฀with฀each฀other฀like฀osteocytes.฀These฀lateral฀branches฀
are฀much฀more฀numerous฀in฀root฀dentin฀than฀in฀coronal฀dentin.฀Unlike฀bone,฀dentin฀con-
tains฀no฀blood฀vessels.฀Cementum฀is฀deposited฀in฀layers฀above฀the฀dentin฀on฀the฀external฀
surface฀of฀the฀root฀as฀a฀calciied฀matrix฀for฀the฀insertion฀of฀Sharpey’s฀periodontal฀ligament฀
ibers฀(Sect.฀3.1.5).฀Cementum฀is฀less฀mineralized฀than฀compact฀bone฀or฀dentin.
134 9 Biological Mineralization

9.2.2.
Two Mechanisms of Mineralization

Intramembranous฀ ossiication฀ is฀ responsible฀ for฀ most฀ of฀ the฀ mineralization฀ of฀ the฀ skull,฀
including฀the฀maxilla฀and฀mandible.฀It฀begins฀with฀the฀differentiation฀and฀activation฀of฀osteo-
blasts฀from฀ibroblast-related฀precursors฀within฀a฀region฀of฀connective฀tissue฀that฀demarcates฀
where฀the฀bone฀will฀develop.฀The฀osteoblasts฀secrete฀a฀nonmineralized฀protein-rich฀(osteoid)฀
matrix฀and,฀as฀they฀move฀away,฀the฀matrix฀mineralizes฀(Fig.฀9.3a).฀The฀periosteum฀remains฀
uncalciied฀ and฀ contains฀ latent฀ and฀ undifferentiated฀ osteoblasts฀ for฀ bone฀ remodeling.฀
Odontoblasts฀ (Ob)฀ and฀ cementoblasts฀ secrete฀ an฀ osteoid-like฀ matrix฀ similar฀ to฀ that฀ of฀
intramembraneous฀ossiication.
Endochondral฀ ossiication฀ is฀ responsible฀ for฀ the฀ mineralization฀ of฀ long฀ bones฀ and฀ it฀
begins฀after฀chondroblasts฀have฀formed฀a฀three-dimensional฀cartilaginous฀template฀of฀the฀
future฀bone฀(Fig.฀9.3b).฀Blood฀vessels฀grow฀into฀the฀center฀of฀the฀cartilage฀and฀osteoblasts฀
develop฀alongside฀invading฀endothelial฀cells฀at฀the฀growth฀plate฀where฀type฀II฀collagen฀is฀
already฀ present฀ and฀ type฀ X฀ collagen฀ will฀ develop฀ (Sect.฀ 4.3.2).฀ The฀ invading฀ osteoblasts฀
replace฀the฀type฀II฀and฀type฀X฀collagen฀of฀the฀endochondral฀growth฀plate฀with฀type฀I฀colla-
gen.฀As฀the฀osteoid฀matrix฀is฀laid฀down,฀the฀chondrocytes฀proliferate฀and฀then฀undergo฀apop-
tosis฀(Fig.฀9.3b).฀Apoptosis฀is฀described฀in฀Sect.฀13.4.1.฀Traces฀of฀type฀II฀and฀type฀X฀collagen฀
and฀ proteo-glycosaminoglycans฀ may฀ remain฀ from฀ the฀ cartilage฀ and฀ become฀ ossiied.฀ A฀
periosteum฀forms฀around฀the฀compact฀outer฀surface.

Bone฀ is฀ synthesized฀ by฀ osteoblasts฀ that฀ differentiate฀ from฀ assembled,฀ mesenchymal,฀
ibroblast-like฀ precursors฀ (intramembranous฀ ossiication),฀ or฀ from฀ precursors฀ that฀
migrate฀ into฀ cartilage฀ (endochondral฀ ossiication).฀ Bone฀ mineralizes฀ over฀ an฀ osteoid฀
matrix฀ composed฀ of฀ type฀ I฀ collagen฀ ibers,฀ which฀ nucleate฀ (initiate)฀ and฀ control฀ the฀
process.฀Outer฀surfaces฀of฀bone฀are฀hard฀(compact฀bone)฀but฀the฀insides฀form฀a฀cavity฀
that฀is฀poorly฀mineralized฀(cancellous฀bone).฀The฀dentin฀and฀cementum฀of฀teeth฀resem-
ble฀compact฀bone.฀The฀outer฀surfaces฀of฀bones฀are฀covered฀by฀an฀organic฀periosteum฀
containing฀capillaries฀and฀an฀uncalciied฀cell-rich฀stroma.฀The฀central฀cavities฀also฀con-
tain฀capillaries฀and฀a฀different฀ibroblast-like฀stroma฀within฀which฀blood฀cells฀develop.฀
As฀they฀form,฀they฀enter฀the฀circulation฀where฀they฀replenish฀the฀red฀and฀white฀cells฀and฀
platelets฀that฀mediate฀oxygen฀transport,฀immunity,฀and฀blood฀clotting.

9.3.1.
Secretion of Osteoid Matrix

Skeletal฀tissue฀mineralization฀(bone฀formation)฀is฀initiated฀by฀osteoblasts,฀which฀secrete฀the฀
osteoid฀matrix฀(Fig.฀9.4).฀They฀express฀type฀I฀procollagen฀in฀secretory฀vesicles฀together฀with฀
matrix฀vesicles฀that฀pinch฀off฀from฀the฀membrane.฀The฀matrix฀vesicles฀are฀pushed฀away฀
from฀the฀cell฀surface,฀possibly฀by฀the฀low฀of฀luid฀containing฀calcium฀and฀phosphate฀ions฀
that฀are฀also฀transported฀through฀the฀cell฀from฀the฀extracellular฀luid฀on฀the฀outer฀surface.฀
Collagen฀ibers฀develop฀further฀away฀from฀the฀cell฀surface฀than฀from฀ibroblasts.
9.3.1. Secretion of Osteoid Matrix 135

Fig. 9.3฀ Bone฀synthesis.฀(a)฀Membrane฀ossiication฀is฀typiied฀by฀cranial฀bone฀fusion.฀Osteoblasts฀

differentiate฀ from฀ loose฀ mesenchymal฀ cells฀ resembling฀ primitive฀ ibroblasts.฀ The฀ center฀ of฀
cranial฀bones฀becomes฀cancellous฀as฀in฀long฀bones.฀(b)฀Endochondral฀ossiication฀is฀typiied฀by฀
long฀bone฀development฀and฀growth.฀In฀this฀more฀complex฀process,฀mesenchymal฀cells฀aggre-
gate฀(1)฀and฀differentiate฀into฀cartilage฀in฀the฀shape฀of฀the฀bone฀(2).฀The฀central฀region฀becomes฀
hypertrophic฀and฀encapsulated฀(3).฀The฀lack฀of฀nutrients฀causes฀apoptosis฀(cell฀death),฀which฀
attracts฀blood฀vessels฀and฀other฀mesenchymal฀cells฀from฀which฀osteoblasts฀differentiate฀and฀
grow฀so฀that฀bone฀and฀periosteum฀replace฀the฀central฀cartilage฀(4).฀As฀osteoclasts฀resorb฀the฀
enlarged฀central฀cavity฀(5),฀chondroblasts฀proliferate฀at฀each฀end฀(6).฀The฀periosteum฀induces฀
appositional฀ growth฀ resembling฀ membrane฀ bone฀ and฀ the฀ central฀ cavity฀ (bone฀ marrow,฀ red)฀
forms฀cancellous฀bone,฀see฀text฀(7).฀At฀each฀end฀of฀the฀bone,฀the฀chondrocytes฀become฀apop-
totic,฀and฀attract฀blood฀vessels฀so฀that฀secondary฀ossiication฀centers฀develop฀above฀and฀below฀
the฀epiphyseal฀cartilage฀and฀bone฀growth฀ceases฀(8)฀(Adapted฀from฀Developmental฀Biology,฀
Ed.฀S.F.฀Gilbert,฀Sinauer฀Assoc.฀Inc.,฀1997฀and฀copied฀from฀Web฀site฀http://classes.aces.uiuc.
edu/AnSci312/Bone/Bonelect.htm)
136 9 Biological Mineralization

Osteogenic cell
(osteoblast precursor)
Osteoblast

Type I collagen secretion

Osteoid (uncalcified
bone) matrix
Osteoblast process
Calcified bone matrix

Cell process
in canaliculus
Osteocyte
(end-stage
osteoblast)

Fig. 9.4฀ Osteoblast฀secretion฀and฀matrix฀vesicle฀formation.฀The฀outer฀surface฀of฀all฀bones฀is฀covered฀

by฀ibroblast-like฀cells฀that฀differentiate฀into฀pre-osteoblasts฀that฀secrete฀osteoid฀matrix฀to฀remodel฀
the฀surface฀as฀necessary.฀The฀surface฀osteoblasts฀extend฀into฀the฀osteoid฀tissue฀by฀long฀processes฀
that฀attach฀to฀osteocytes฀(fully฀differentiated,฀nondividing฀osteoblasts)฀within฀the฀bone.฀Changes฀฀
in฀the฀environment฀may฀be฀sensed฀by฀the฀osteocytes,฀which฀transmit฀them฀as฀remodeling฀signals฀to฀
the฀osteoblasts.฀The฀osteoid฀matrix฀is฀illed฀with฀many฀small฀membrane-covered฀matrix฀vesicles฀
containing฀various฀amounts฀of฀precipitated฀basic฀calcium฀phosphate฀(white฀circles)฀(Modiied฀from฀
Fig.฀22-52฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀
Taylor฀&฀Francis฀Group,฀New฀York)

Matrix฀vesicles฀are฀dificult฀to฀isolate฀from฀developing฀membrane฀bone฀and฀the฀only฀
well-characterized฀matrix฀vesicles฀available฀are฀from฀chondroblasts฀about฀to฀be฀replaced฀by฀
osteoblasts฀during฀endochondral฀ossiication฀(Fig.฀9.3).฀The฀chondroblast฀matrix฀vesicles฀
shown฀in฀Fig.฀9.4฀are฀surrounded฀by฀cartilage฀collagens฀(type฀II฀and฀type฀X)฀and฀aggrecan฀
(Sect.฀6.5.1).฀As฀osteoblasts฀invade฀and฀secrete฀their฀own฀matrix฀vesicles฀and฀type฀I฀colla-
gen,฀the฀cartilage฀collagen฀and฀proteoglycans฀are฀almost฀entirely฀removed,฀presumably฀by฀
matrilysins฀expressed฀by฀dying฀chrondroblasts฀or฀invading฀osteoblasts฀ahead฀of฀type฀I฀col-
lagen฀expression.฀It฀is฀not฀clear฀how฀cartilage฀derived฀matrix฀vesicles฀shown฀in฀Fig.฀9.5฀are฀
related฀to฀osteoblast-derived฀matrix฀vesicles฀but฀they฀are฀assumed฀to฀be฀similar.

9.3.2.
Osteoblast Transport of Calcium and Phosphate Ions to Matrix Vesicles

Osteoblasts฀take฀up฀Ca2+฀ions฀from฀the฀periosteal฀extracellular฀luid฀using฀Na+/Ca2+-exchangers฀
NCX1฀ and฀ NCX3.฀ Once฀ in฀ the฀ cytosol,฀ the฀ Ca2+฀ ions฀ must฀ be฀ transported฀ to฀ the฀ osteoid฀
matrix฀side฀(basal฀side)฀by฀calbindins,฀which฀require฀the฀active฀form฀of฀vitamin฀D฀(calcitriol)฀
for฀synthesis฀and฀expression.฀The฀Ca2+฀ions฀are฀passed฀out฀to฀the฀osteoid฀matrix฀through฀an฀
ATP-dependent฀ plasma฀ membrane฀ Ca2+-ATPase฀ 1b฀ (PMCA1b).฀ The฀ orientations฀ of฀ the฀
cells,฀the฀transporters,฀and฀the฀calbindins฀are฀described฀in฀detail฀in฀Sect.฀10.4.1.฀Once฀in฀the฀
osteoid฀matrix,฀the฀matrix฀vesicles฀take฀up฀the฀Ca2+฀ions฀via฀an฀annexin฀transporter.
9.3.2. Osteoblast Transport of Calcium and Phosphate Ions to Matrix Vesicles 137

a Ca2+
Annexin V Annexin II
Pi
Type X Ca2+
Collagen
Annexin VI
Ca2+
P
Type II Initiation of type I
Mineral
Collagen Pi collagen mineralization

Phospholipid
bilayer

b Hyaluronan binding
TNAP and NTP-PPi
head of proteoglycan
hydrolases

Lactate dehydrogenase
Calbindin and proteinase

Annexins
Carbonic anhydrase
Type II
Actin
collagen
microfibril
Apatite
Type II crystals
collagen
microfibril
Type X
collagen

Fig. 9.5฀ Matrix฀vesicle฀composition.฀(a)฀Transporters.฀The฀major฀calcium฀transport฀channel฀is฀made฀

from฀annexin฀V฀(thick฀blue฀arrow).฀Lesser฀amounts฀of฀annexins฀II฀and฀VI฀are฀also฀present฀(thin฀
blue฀arrows)฀but฀their฀role฀in฀Ca2+฀ion฀transport฀is฀uncertain.฀Calcium฀ion฀transport฀is฀enhanced฀by฀
the฀ presence฀ of฀ type฀ II฀ and฀ type฀ X฀ collagen฀ ibers฀ during฀ endochondral฀ ossiication.฀ Phosphate฀
transporters฀ (PiT)฀ are฀ shown฀ in฀ yellow฀ with฀ a฀ red฀ arrow฀ indicating฀ the฀ direction฀ of฀ phosphate฀
transport.฀As฀each฀HPO42−฀ion฀is฀transported฀into฀the฀vesicle,฀a฀sodium฀ion฀is฀transported฀out฀(not฀
shown)฀(Modiied฀from฀Fig.฀5฀of฀T.฀Kirsch,฀Annexins฀and฀tissue฀mineralization:฀matrix฀vesicle,฀ion฀
channel฀ activity฀ of฀ annexins฀ and฀ annexin฀ V/collagen฀ interactions,฀ published฀ in฀ Annexins:฀
Biological฀importance฀and฀annexin-related฀pathologies฀(2003).฀Edited฀by฀J.฀Bandorowicz-Pialuka,฀
Kluwer/Plenum฀Publishers,฀233฀Spring฀St.,฀New฀York,฀NY฀10013).฀(b)฀Matrix฀vesicle-associated฀
enzymes฀and฀proteins.฀Collagen฀Type฀X,฀the฀proteoglycan฀link฀protein฀and฀the฀hyaluronan฀binding฀
region฀of฀proteoglycans฀may฀attach฀cartilage฀collagen฀(type฀II)฀to฀the฀outer฀surface฀of฀matrix฀vesi-
cles.฀Nucleoside฀triphosphate฀pyrophosphohydrolase฀(NTAP)฀and฀NTP-PPi฀hydrolase฀are฀anchored฀
in฀the฀matrix฀vesicle฀membrane.฀Annexin฀V฀and฀carbonic฀anhydrase฀are฀concentrated฀just฀below฀
the฀ matrix฀ vesicle฀ membrane.฀ Lactate฀ dehydrogenase฀ (LDH),฀ calbindin฀ D9K,฀ and฀ proteases฀ are฀
soluble฀in฀the฀center฀of฀the฀matrix฀vesicle฀(Modiied฀to฀refer฀to฀osteoblast฀matrix฀vesicles฀from฀
Anderson฀HC฀(1992)฀“Conference฀introduction฀and฀summary.”฀Bone฀and฀Mineral฀17:110)

Osteoblasts฀also฀take฀up฀orthophosphate฀(Pi)฀from฀the฀periosteal฀extracellular฀luid฀using฀
a฀type฀I฀sodium/Pi฀co-transporter.฀Pi฀consists฀of฀about฀60%฀monohydrogen฀phosphate฀and฀
40%฀dihydrogen฀phosphate฀at฀pH฀7.0.฀The฀Pi฀diffuses฀freely฀through฀the฀cytosol฀and฀exits฀
138 9 Biological Mineralization

into฀the฀osteoid฀matrix฀by฀an฀unknown฀mechanism฀independently฀of฀matrix฀vesicle฀secre-
tion.฀Pi฀then฀enters฀the฀matrix฀vesicles฀within฀the฀osteoid฀matrix฀through฀a฀type฀III฀sodium/
Pi฀co-transporter฀(Fig.฀9.5a)฀whose฀interior฀is฀made฀more฀alkaline฀by฀carbonic฀anhydrase฀
(Fig.฀9.5b)฀removing฀protons฀from฀dihydrogen฀phosphate฀by฀reaction฀with฀sodium฀bicar-
bonate.฀ The฀ carbonic฀ acid฀ is฀ unstable฀ and฀ breaks฀ down฀ into฀ water฀ and฀ carbon฀ dioxide,฀
which฀bubbles฀off฀and฀the฀sodium฀ions฀are฀exchanged฀to฀the฀osteoid฀matrix฀for฀incoming฀
Pi.฀Lactic฀dehydrogenase฀is฀also฀present฀and฀it฀may฀function฀to฀prevent฀the฀pH฀from฀becom-
ing฀too฀alkaline,฀keeping฀the฀hydroxyapatite฀crystals฀small฀and฀poorly฀formed.

9.3.3.
Calcium and Phosphate Ions Precipitate and Rupture Secreted Matrix Vesicles

Nucleation฀of฀calcium฀phosphate฀precipitation฀within฀the฀matrix฀vesicles฀is฀mediated฀by฀
phosphatidylserine,฀which฀comprises฀about฀8%฀of฀the฀phospholipids฀of฀the฀inner฀cytosolic฀
membrane฀surface฀(Fig.฀9.5a).฀Calbindin฀in฀the฀vesicle฀(Fig.฀ 9.5b)฀may฀also฀contribute.฀
Rapid฀mineral฀growth฀within฀the฀vesicle฀keeps฀the฀concentration฀of฀dissolved฀calcium฀and฀
inorganic฀phosphate฀ions฀so฀low฀that฀additional฀Ca2+฀and฀Pi฀ions฀spontaneously฀enter฀from฀
the฀extracellular฀luid฀via฀their฀respective฀transporters.฀Attached฀type฀II฀and฀type฀X฀colla-
gens฀ from฀ cartilage฀ in฀ the฀ growth฀ plate฀ enhance฀ calcium฀ ion฀ transport฀ and฀ calciication฀
during฀endochondral฀ossiication฀(Fig.฀9.5b).
Once฀ the฀ solid฀ calcium฀ phosphate฀ reaches฀ a฀ certain฀ size,฀ the฀ vesicle฀ ruptures.฀ The฀
exposed฀mineral฀partially฀redissolves฀and฀nucleates฀the฀extracellular฀luid.฀Type฀I฀collagen฀
ibers฀propagate฀crystal฀growth฀within฀the฀gaps฀of฀the฀quarter-staggered฀array฀of฀ibers฀and฀
also฀between฀the฀ibers.฀Within฀the฀gaps,฀serine฀residues฀become฀spontaneously฀phospho-
rylated฀and฀further฀nucleate฀mineralization฀such฀that฀the฀crystals฀align฀parallel฀to฀the฀ibers.฀
The฀nucleation฀of฀collagen฀ibers฀is฀further฀controlled฀by฀alkaline฀phosphatase฀removing฀
pyrophosphate,฀which฀also฀accumulates฀in฀the฀osteoid฀matrix฀(Fig.฀9.5b).

9.3.4.
Structure of the Calcium Transporter Proteins in Matrix Vesicles

The฀ annexins฀ bind฀ to฀ phospholipids฀ in฀ a฀ reversible฀ Ca2+-dependent฀ manner.฀ They฀ are฀
implicated฀ in฀ membrane฀ fusion,฀ vesicular฀ traficking,฀ and฀ ion-channel฀ formation.฀ X-ray฀
crystal฀structures฀of฀various฀soluble฀annexins฀all฀reveal฀a฀common฀backbone฀fold฀in฀which฀
each฀of฀four฀repeats฀in฀the฀core฀domain฀contains฀ive฀a-helices฀connected฀by฀short฀loops฀
(Fig.฀9.6a).฀Two฀of฀these฀loops฀come฀together฀to฀form฀Ca2+-binding฀sites฀that฀coordinate฀
with฀the฀negatively฀charged฀head-group฀of฀phosphatidylserine฀on฀the฀cytosolic฀surface฀of฀
membranes.฀Unfortunately,฀this฀association฀does฀not฀explain฀its฀ion-channel฀activity.฀That฀
9.3.4. Structure of the Calcium Transporter Proteins in Matrix Vesicles 139

Inner membrane (Ca2+ binds to

b phosphatidyl serine side)

2+
Ca
H+
Trimer binds
internal surface
Monomer with seven
transmembrane helices

H+

Membrane’s extracellular surface

Fig. 9.6฀ Crystal฀structure฀and฀membrane฀insertion฀model฀of฀human฀annexin.฀(a)฀Annexin฀structure.฀

Annexins฀all฀possess฀four฀amino฀acid฀sequence฀repeats฀in฀the฀core฀domain.฀X-ray฀crystallography฀
indicates฀that฀each฀repeat฀has฀ive฀right-turn฀helices฀connected฀by฀short฀loops,฀two฀of฀which฀come฀
together฀to฀form฀a฀very฀tight฀Ca2+-binding฀site฀that฀coordinates฀with฀phosphatidylserine฀residues฀on฀
the฀cytosolic฀side฀of฀a฀membrane.฀Different฀colors฀highlight฀the฀four฀annexin฀repeat฀sequences฀I฀
through฀IV:฀green,฀blue,฀red,฀and฀violet฀(or฀cyan).฀High฀and฀low฀Ca2+฀binding฀forms฀are฀due฀to฀a฀
conformational฀change฀in฀repeat฀III฀to฀expose฀Trp-187฀for฀insertion฀into฀the฀membrane฀(high฀bind-
ing฀form฀is฀in฀cyan).฀The฀bound฀Ca2+฀ions฀are฀depicted฀as฀yellow฀spheres฀(Image฀is฀Fig.฀2฀from฀
Gerke฀V฀and฀Moss฀SE฀(April฀2002)฀“Annexins:฀from฀structure฀to฀function.”฀Physiological฀Reviews฀
82(2):331–371.฀With฀permission฀from฀the฀American฀Physiological฀Society).฀(b)฀Ca2+฀binding฀and฀
membrane฀insertion฀modiications.฀How฀annexin฀V฀inserts฀into฀bilayers฀to฀create฀a฀calcium฀chan-
nel฀is฀poorly฀understood.฀One฀possibility฀is฀that฀when฀annexin฀interacts฀with฀the฀phosphate฀resi-
dues฀of฀the฀membrane,฀the฀acidic฀environment฀protonates฀its฀four฀loops,฀causing฀a฀rearrangement฀
into฀a฀seven฀helix฀transmembrane฀form฀that฀can฀potentially฀transport฀Ca2+.฀Annexins฀V฀and฀XII฀are฀
each฀reversibly฀converted฀between฀three฀states:฀surface฀trimer,฀phosphatidylserine฀binding฀mono-
mer,฀and฀transmembrane฀monomer.฀Equilibrium฀is฀regulated฀by฀the฀concentration฀of฀H+฀and฀Ca2+.฀
It฀has฀not฀yet฀been฀determined฀whether฀the฀transmembrane฀form฀can฀be฀directly฀converted฀to฀the฀
surface฀trimer฀when฀pH฀and฀Ca2+฀concentrations฀are฀raised฀(Modiied฀from฀Fig.฀7฀in฀Isas฀JM฀et฀al.฀
(2000)฀ “Annexins฀ V฀ and฀ XII฀ insert฀ into฀ bilayers฀ at฀ mildly฀ acidic฀ pH฀ and฀ form฀ ion฀ channels.”฀
Biochemistry฀39(11):3015–3022)
140 9 Biological Mineralization

action฀may฀be฀mediated฀by฀a฀form฀of฀the฀protein฀that฀inserts฀into฀the฀hydrophobic฀core฀of฀
the฀vesicle฀lipid฀bilayer.
Annexins฀isolated฀from฀chondroblast฀matrix฀vesicles฀may฀be฀reconstituted฀with฀phos-
pholipids฀ to฀ form฀ calcium฀ ion฀ channels฀ in฀ the฀ complete฀ absence฀ of฀ Ca2+฀ ions.฀ Indeed,฀
annexin฀V฀has฀domains฀that฀directly฀bind฀calcium฀ions;฀glutamate฀and฀aspartate฀residues฀
provide฀the฀ion฀binding฀site฀(EF-hand฀domains).฀Figure฀9.6b฀illustrates฀putative฀annexin฀V฀
channels฀that฀mediate฀an฀inlux฀of฀Ca2+฀ions฀into฀artiicial฀bilayers฀and฀liposomes฀(detect-
able฀with฀a฀calcium-sensitive฀luorescent฀dye).฀These฀in฀vitro฀annexin฀Ca2+฀channels,฀and฀
also฀the฀Ca2+฀inlux฀into฀matrix฀vesicles฀in฀cell฀culture฀and฀in฀vivo,฀are฀blocked฀by฀Zn2+฀
ions,฀or฀a฀derivative฀of฀1,4-benzothiazepine฀(inhibitor฀K201).

9.3.5.
The Phosphate Transporter Proteins and Pyrophosphate in Matrix Vesicles

The฀type฀III฀sodium-dependent฀phosphate฀(Na/Pi)฀transporters฀involved฀in฀mineralization฀
are฀members฀of฀the฀inorganic฀phosphate฀transporter฀(PiT)฀family,฀which฀is฀conserved฀in฀all฀
biology.฀In฀osteoclasts,฀a฀proton฀(H+)฀gradient฀instead฀of฀a฀sodium฀(Na+)฀gradient฀transports฀
Pi.฀These฀transporters฀are฀antiports:฀the฀Na+฀or฀H+฀is฀transported฀out฀as฀the฀Pi฀is฀transported฀
in฀(Sects.฀2.2.3฀and฀10.1.4).฀All฀these฀transporters฀are฀composed฀of฀repeating฀alpha฀helices฀
that฀weave฀in฀and฀out฀of฀a฀membrane฀with฀intra-฀and฀extracellular฀turns฀(Fig.฀9.7).฀A฀similar฀
kind฀of฀structure฀is฀proposed฀for฀the฀annexins฀that฀mediate฀calcium฀ion฀transport.
A฀physiologic฀phosphate฀concentration฀is฀required฀for฀bone฀mineralization.฀Lowering฀the฀
concentration฀prevents฀mineralization,฀but฀raising฀it฀does฀not฀ensure฀precipitation฀because฀
pyrophosphate฀is฀present฀to฀inhibit฀precipitation.฀The฀concentration฀of฀PPi฀in฀cartilage฀and฀
bone฀is฀controlled฀by฀three฀enzymes,฀two฀on฀the฀outer฀surface฀of฀matrix฀vesicles฀(Fig.฀9.5b).฀
One฀is฀tissue-nonspeciic฀alkaline฀phosphatase฀(TNAP),฀which฀decreases฀stromal฀pyrophos-
phate฀and฀the฀other฀is฀NTP-PPi฀hydrolase฀(also฀called฀plasma฀cell฀membrane฀glycoprotein-1),฀
which฀increases฀it.฀The฀progressive฀ankylosis฀gene฀product฀(ANK฀protein)฀is฀expressed฀by฀
osteoblasts฀to฀add฀to฀the฀pyrophosphate฀of฀the฀osteoid฀matrix฀from฀osteoblast฀cytosol.
Figure฀9.8฀outlines฀how฀matrix฀vesicles฀increase฀and฀decrease฀the฀concentration฀of฀pyro-
phosphate.฀NTP-PPi฀hydrolase฀synthesizes฀pyrophosphate฀from฀stromal฀luid฀nucleotides,฀
mostly฀ATP฀(ATP฀→฀AMP฀+฀PPi).฀Many฀cells฀secrete฀ATP฀into฀the฀extracellular฀luid฀and฀
it฀passes฀into฀the฀blood฀plasma฀where฀it฀affects฀a฀variety฀of฀cells฀independently฀of฀its฀func-
tion฀in฀intracellular฀energy฀metabolism.฀In฀mice,฀a฀nonfunctional฀ANK฀protein฀or฀a฀deletion฀
of฀NTP-PPi฀hydrolase฀decreases฀the฀extracellular฀pyrophosphate฀concentration฀and฀the฀phe-
notype฀exhibits฀extensive฀mineralization.฀Thus,฀the฀hydrolysis฀of฀pyrophosphate฀appears฀to฀
be฀a฀major฀function฀of฀alkaline฀phosphatase฀(TNAP)฀after฀the฀calcium฀phosphate฀precipitate฀
has฀ruptured฀the฀matrix฀vesicles.฀Rapid฀mineralization฀of฀collagen฀and฀the฀rest฀of฀the฀osteoid฀
matrix฀ensue฀without฀a฀need฀to฀transport฀any฀more฀Ca2+฀or฀Pi฀to฀the฀region.
Mineralization฀ therefore฀ occurs฀ in฀ bone฀ because฀ of฀ the฀ exclusive฀ co-expression฀ in฀
osteoblasts฀of฀type฀I฀collagen฀and฀tissue-nonspeciic฀alkaline฀phosphatase฀(TNAP).฀The฀
abnormal฀appearance฀of฀TNAP฀in฀any฀cell฀that฀also฀produces฀ibrillar฀collagen฀(ectopic฀
TNAP฀expression)฀gives฀rise฀to฀pathological฀(nonbacterial)฀mineralization,฀which฀is฀out-
side฀the฀scope฀of฀this฀text.
9.3.5. The Phosphate Transporter Proteins and Pyrophosphate in Matrix Vesicles 141

Extracellular side
D506 in the C-terminal
E55 N81
signature sequence S593

D28 in the N-terminal S113 E575

signature sequence

Intracellular (cytosol)
side

Fig. 9.7฀ Model฀of฀the฀PiT-2฀transporter.฀There฀are฀two฀homologous฀transporters฀in฀humans,฀PiT-1฀

and฀PiT-2.฀Diagram฀illustrates฀the฀membrane฀topology฀model฀of฀PiT=2฀derived฀mostly฀from฀trans-
membrane฀ (TM)฀ predictions,฀ tagging฀ N-฀ and฀ C-terminal฀ sequences,฀ and฀ glycosylated฀ residues.฀
Duplicated฀sequences฀are฀assumed฀to฀exhibit฀similar฀membrane฀topologies.฀The฀strongest฀indica-
tion฀for฀overall฀orientation฀of฀the฀protein฀in฀the฀membrane฀is฀that฀N81฀is฀glycosylated฀in฀vitro฀and฀
therefore฀ extracellular.฀ Dotted฀ curves฀ indicate฀ no฀ experimental฀ evidence฀ for฀ the฀ location฀ of฀ the฀
loop฀between฀membrane฀insertions.฀Amino฀acids฀that฀are฀important฀for฀PiT=2฀transport฀of฀Pi฀and฀
Na฀are฀indicated฀with฀arrows:฀D28฀and฀D506,฀E55฀and฀E575,฀S113฀and฀S593.฀(Note:฀Na฀is฀in฀high฀
concentration฀in฀all฀extracellular฀luids฀including฀the฀luid฀around฀matrix฀vesicles฀and฀that฀it฀spon-
taneously฀diffuses฀through฀the฀membrane.฀In฀cells,฀Na฀is฀actively฀extruded฀by฀the฀Na/K฀exchanger,฀
which฀is฀absent฀from฀matrix฀vesicles.)฀A฀consensus฀amino฀acid฀sequence฀common฀to฀all฀members฀
of฀this฀transporter฀family฀appears฀intracellularly฀before฀membrane฀insertion฀2,฀and฀extracellularly฀
before฀membrane฀insertion฀9.฀In฀addition,฀all฀phosphate฀transporters฀possess฀a฀large฀cytosolic฀loop฀
of฀variable฀sequences฀and฀lengths฀between฀membrane฀insertions฀7฀and฀8฀(Image฀is฀Fig.฀7A฀from฀
Bøttger฀P฀and฀Pedersen฀L฀(2005)฀“Evolutionary฀and฀experimental฀analyses฀of฀inorganic฀phosphate฀
transporter฀PiT฀family฀reveals฀two฀related฀signature฀sequences฀harboring฀highly฀conserved฀aspartic฀
acids฀critical฀for฀sodium-dependent฀phosphate฀transport฀function฀of฀human฀PiT2.”฀FEBS฀Journal฀
272:3060–3074.฀With฀copyright฀permission฀from฀Wiley-Blackwell,฀PO฀Box฀805,฀9600฀Garsington฀
Road,฀Oxford,฀OX4฀2DQ,฀UK)

Osteoblasts฀ secrete฀ osteoid,฀ a฀ matrix฀ rich฀ in฀ type฀ I฀ collagen฀ ibers฀ and฀ vesicles.฀
Precipitation฀of฀calcium฀phosphate฀is฀inhibited฀by฀a฀high฀concentration฀of฀pyrophos-
phate฀in฀stromal฀interstitial฀luids,฀and฀a฀high฀concentration฀also฀of฀albumin฀and฀citrate฀
in฀blood฀plasma.฀Pyrophosphate฀is฀derived฀from:฀(1)฀transport฀out฀of฀the฀cytosol,฀and฀
(2)฀synthesis฀from฀nucleoside฀triphosphates฀in฀the฀stromal฀interstitial฀luid฀that฀perme-
ates฀the฀osteoid฀matrix.฀Precipitation฀occurs฀only฀when฀calcium฀and฀phosphate฀ions฀
are฀taken฀up฀into฀vesicles฀whose฀inner฀membrane฀is฀composed฀of฀phosphatidylserine.฀
The฀high฀concentration฀of฀calcium฀and฀phosphate฀ions฀in฀the฀vesicle฀is฀mediated฀by฀
annexin฀and฀type฀III฀Pi฀Na-dependent฀transporters.฀This฀overwhelms฀the฀pyrophos-
phate฀ and฀ nucleation฀ occurs.฀ As฀ the฀ precipitate฀ grows฀ and฀ ruptures฀ the฀ membrane,฀
tissue-nonspeciic฀ alkaline฀ phosphatase฀ is฀ activated฀ to฀ remove฀ pyrophosphate฀ from฀
the฀osteoid฀matrix฀luid฀so฀that฀calcium฀phosphate฀precipitates฀around฀phosphorylated฀
serine฀residues฀within฀the฀collagen฀ibers.
142 9 Biological Mineralization

PPi TNAP Pi Ppte

ANK
PC-1 TNAP
Pi and Ca
Intra from diet
cellular NTPS
PPi

Fig. 9.8฀ Removal฀of฀pyrophosphate฀is฀necessary฀for฀precipitation.฀Pyrophosphate฀(PPi)฀inhibits฀the฀

precipitation฀of฀calcium฀phosphate.฀In฀the฀bone฀matrix,฀PC-1฀(red)฀is฀the฀major฀producer฀of฀PPi฀
from฀nucleotide฀triphosphates฀(NTPs,฀thick฀arrow฀on฀left)฀and฀ANK฀is฀a฀minor฀producer฀by฀trans-
porting฀it฀from฀the฀cytosol฀of฀osteoblasts.฀TNAP฀(green)฀causes฀mineralization฀by฀its฀phosphatase฀
activity฀converting฀PPi฀to฀two฀molecules฀of฀Pi.฀TNAP฀also฀generates฀Pi฀directly฀from฀NTPs฀and฀
PPi,฀but฀most฀Pi฀and฀most฀Ca2+฀are฀derived฀directly฀from฀the฀diet฀(thick฀arrow฀on฀right)฀(Slightly฀
modiied฀from฀Fig.฀4฀in฀Hessle฀L฀et฀al.฀(2002)฀“Tissue-nonspeciic฀alkaline฀phosphatase฀and฀plasma฀
cell฀ membrane฀ glycoprotein-1฀ are฀ central฀ antagonistic฀ regulators฀ of฀ bone฀ mineralization.”฀
Proceedings฀ of฀ the฀ National฀ Academy฀ of฀ Sciences฀ 99:9445–9449.฀ Copyright฀ (2002)฀ National฀
Academy฀of฀Sciences,฀U.S.A)

9.4.1.
Non-collagenous Bone Proteins in Bone and Dentin

There฀are฀a฀number฀of฀non-collagenous฀proteins฀in฀bone฀and฀dentin.฀Most฀are฀proteins฀of฀
the฀SIBLING฀(Small฀Integrin-Binding฀Ligand,฀N-linked฀Glycoprotein)฀family.฀Members฀
of฀this฀family฀are฀osteopontin,฀matrix฀extracellular฀phosphoglycoprotein฀(MEPE),฀bone฀
sialoprotein฀ (BSP),฀ dentin฀ matrix฀ protein-1฀ (DMP-1)฀ and฀ dentin฀ sialophosphoprotein฀
(DSPP).฀These฀proteins฀are฀all฀calcium฀binding฀and฀found฀mostly฀in฀the฀non-mineralized฀
or฀mineralized฀osteoid฀or฀dentinal฀matrix.฀The฀SIBLING฀proteins฀are฀encoded฀together฀at฀
the฀same฀locus฀on฀human฀chromosome฀4฀and฀have฀a฀similar฀gene฀organization฀in฀which฀the฀
downstream฀encoded฀exons฀are฀the฀longest฀and฀encode฀an฀RGD฀motif฀that฀binds฀integrins฀
(Sect฀4.4.1).฀They฀also฀undergo฀similar฀post-translational฀modiications:฀phosphorylation,฀
glycosylation,฀ proteolytic฀ processing,฀ sulphation,฀ and฀ transglutaminase฀ cross-linking.฀
Extent฀of฀expression฀differs฀between฀bone฀and฀dentin.
Osteopontin฀is฀discussed฀in฀Sect.฀10.1.1.฀Little฀is฀known฀about฀MEPE฀except฀that฀it฀is฀
required฀for฀skeletal฀development.฀BSP฀is฀hydrolyzed฀at฀a฀single฀site฀and฀DMP-1฀at฀two฀
sites.฀The฀fragments฀bind฀integrins฀on฀the฀surface฀of฀the฀long฀processes฀of฀osteocytes฀or฀
odontoblasts,฀ where฀ they฀ likely฀ control฀ apatite฀ growth฀ on฀ collagen฀ so฀ that฀ canaliculi฀
develop฀in฀bone฀or฀tubules฀in฀dentin.฀DMP1฀fragments฀may฀also฀participate฀in฀activating฀
ostecalcin฀expression฀from฀osteoblasts฀(Sect.฀9.4.2).
Mice฀and฀humans฀possess฀a฀gene฀that฀encodes฀a฀membrane-bound฀gluzincin฀metalloen-
dopeptidase฀(Chapter฀8,฀Table฀8.2).฀This฀enzyme฀hydrolyzes฀small฀peptides฀(<3฀kDa)฀on฀
the฀amino-terminal฀side฀of฀aspartate฀residues฀on฀various฀proteins.฀If฀this฀gluzincin฀loses฀its฀
proteolytic฀activity฀due฀to฀mutation,฀BSP฀and฀DMP-1฀fragments฀are฀absent฀and฀the฀osteoid฀
matrix฀ fails฀ to฀ mineralize.฀ There฀ is฀ also฀ an฀ excessive฀ excretion฀ of฀ phosphate฀ into฀ urine฀
(phosphaturia)฀and฀a฀low฀content฀of฀phosphate฀in฀the฀blood฀(hypophosphatemia).฀Rickets฀
9.4.2. Osteocalcin Is Required for Bone Modeling 143

develops฀ as฀ in฀ vitamin฀ D฀ deiciency฀ (Sect.฀ 10.4).฀ The฀ normal฀ (non-mutated)฀ gluzincin฀
prevents฀phosphate฀excretion฀(PHEX)฀and฀hypophosphatemia฀by฀hydrolyzing฀an฀unknown฀
protein฀in฀the฀kidney฀where฀BSP฀and฀DMP-1฀are฀normally฀absent.฀Hypophosphatemia฀is฀
alternatively฀ caused฀ by฀ a฀ non-functional฀ (mutated)฀ ANK฀ protein฀ or฀ a฀ deleted฀ NTP-PPi฀
hydrolase฀ (Sect.฀ 9.3.5),฀ but฀ then฀ BSP฀ and฀ DMP-1฀ fragments฀ are฀ present฀ in฀ the฀ osteoid฀
matrix.
Type฀I฀collagen฀is฀essential฀for฀normal฀bone฀formation.฀Improper฀collagen฀iber฀forma-
tion฀due฀to฀mutations฀in฀one฀of฀the฀collagen฀genes,฀or฀in฀the฀associated฀processing฀enzymes,฀
cause฀skeletal฀abnormalities฀called฀osteogenesis฀imperfecta฀(Sect.฀7.2.1.),฀often฀accompa-
nied฀ by฀ improper฀ dentin฀ development,฀ dentinogenesis฀ imperfecta฀ type฀ I฀ (DGI-I).฀ Other฀
causes฀of฀abnormal฀dentin฀are฀due฀to฀mutations฀of฀DSPP฀(Table฀7.1),฀dentinogenesis฀imper-
fecta฀types฀II฀or฀III฀(DGI-II฀or฀III),฀or฀dentin฀dysplasia฀type฀II฀(DD-II).฀In฀all฀forms฀of฀DGI฀
irrespective฀of฀cause,฀the฀teeth฀are฀discoloured฀and฀show฀structural฀defects฀such฀as฀bulbous฀
crowns฀and฀small฀pulp฀chambers.฀Dentin฀dysplasia฀type฀I฀(DD-1)฀is฀unrelated฀and฀charac-
terized฀by฀rootless฀teeth฀of฀unknown฀origin.
One฀of฀the฀irst฀DSPP฀mutations฀to฀be฀identiied฀was฀the฀mutation฀of฀a฀tyrosine฀residue฀
(encoded฀as฀TAT)฀to฀an฀aspartic฀acid฀(GAT)฀at฀amino฀acid฀6฀from฀the฀N-terminus.฀This฀฀
T฀→฀G฀mutation฀changes฀a฀neutral฀amino฀acid฀to฀an฀acidic฀one฀that฀inhibits฀the฀binding฀of฀
the฀N-terminal฀secretion฀signal฀to฀the฀signal฀recognition฀particle.฀The฀mutated฀DSPP฀can-
not฀translocate฀into฀the฀endoplasmic฀reticulum฀and฀is฀destroyed฀in฀the฀cytosol.฀Many฀other฀
mutations฀ of฀ DSPP฀ have฀ since฀ been฀ found,฀ some฀ of฀ which฀ also฀ affect฀ hearing.฀ DSPP฀
appears฀important฀for฀the฀development฀of฀inner฀ear฀bones,฀and฀it฀is฀also฀expressed฀by฀osteo-
blasts.฀DSPP฀mutations฀are฀autosomal฀dominant;฀a฀mutation฀in฀only฀one฀of฀the฀two฀genes฀
results฀in฀DGI-II฀or฀III,฀or฀DD-II.฀Like฀other฀SIBLING฀proteins,฀normal฀DSPP฀is฀cleaved.฀
The฀major฀products฀are฀dentin฀sialoprotein฀(DSP,฀N-terminal฀end)฀and฀dentinphosphopro-
tein฀(DPP,฀C-terminal฀end).฀The฀function฀of฀the฀N-terminal฀DSP฀fragment฀is฀not฀known,฀
but฀the฀C-terminal฀DPP฀fragment฀is฀an฀important฀initiator฀and฀modulator฀of฀apatite฀crystal฀
formation฀and฀its฀content฀in฀dentin฀or฀bone฀is฀exceeded฀only฀by฀osteocalcin฀(Sect.฀9.4.2).

9.4.2.
Osteocalcin Is Required for Bone Modeling

The฀non-collagenous฀proteins฀in฀bone฀and฀dentin฀appear฀primarily฀involved฀in฀postminer-
alization฀bone฀modeling.฀Osteocalcin฀is฀the฀major฀noncollagenous฀protein฀of฀bone฀matrix.฀
It฀contains฀49฀amino฀acids,฀including฀three฀residues฀of฀gamma-carboxyglutamic฀acid฀(gla),฀฀
a฀posttranslational฀modiication฀of฀glutamate฀residues฀that฀cause฀the฀protein฀to฀bind฀tightly฀
to฀calcium฀ions฀on฀the฀hydroxyapatite฀surface.฀Osteocalcin,฀also฀known฀as฀the฀bone฀gla฀
protein฀(BGP),฀is฀induced฀by฀calcitriol฀from฀vitamin฀D฀(Sect.฀10.4.1).฀Vitamin฀A฀(retinoic฀
acid)฀is฀also฀involved฀in฀regulating฀osteocalcin฀synthesis.฀Finally,฀vitamin฀K฀is฀required฀to฀
make฀ osteocalcin’s฀ gamma-carboxyglutamic฀ acid฀ residues฀ by฀ the฀ same฀ mechanism฀
described฀for฀the฀gla-containing฀blood-clotting฀proteins฀(Sect.฀11.2.2).฀Besides฀osteocalcin฀
and฀blood-clotting฀proteins,฀some฀kidney฀and฀spleen฀proteins฀are฀also฀gla-containing.
Warfarin฀ is฀ a฀ poison฀ that฀ inhibits฀ the฀ action฀ of฀ vitamin฀ K฀ by฀ preventing฀ gla฀ residue฀
synthesis฀(Sect.฀11.2.2)฀and฀therefore฀causes฀an฀osteocalcin฀deiciency.฀Mice฀in฀which฀the฀
144 9 Biological Mineralization

gene฀for฀osteocalcin฀was฀deleted฀or฀given฀warfarin฀developed฀larger฀bones฀of฀improved฀
functional฀quality.฀The฀absence฀of฀osteocalcin฀therefore฀causes฀a฀histologically฀detectable฀
increase฀in฀bone฀formation฀without฀impairing฀bone฀resorption.฀Human฀studies฀indicate฀that฀
osteocalcin฀in฀blood฀plasma฀is฀increased฀in฀diseases฀in฀which฀bone฀turnover฀is฀high,฀but฀
decreased฀in฀diseases฀in฀which฀bone฀turnover฀is฀low.฀The฀association฀of฀enhanced฀osteocal-
cin฀production฀with฀faster฀osteoblast฀turnover,฀but฀less฀bone฀formation฀than฀in฀osteocalcin฀
deiciency฀ seems฀ contradictory.฀ One฀ explanation฀ is฀ that฀ osteocalcin฀ prevents฀ excessive฀
bone฀growth฀due฀to฀its฀gla฀residues฀binding฀tightly฀to฀hydroxyapatite฀and฀inhibiting฀unlim-
ited฀osteoid฀matrix฀formation฀during฀synthesis.

The฀osteoblast฀noncollagenous฀proteins,฀bone฀sialoprotein฀(BSP),฀osteopontin฀(OPN),฀
dentin฀ sialophosphoprotein฀ (DSPP),฀ dentin฀ matrix฀ protein-1฀ (DMP1),฀ and฀ matrix฀
extracellular฀phosphoglycoprotein฀(MEPE)฀are฀SIBLING฀proteins฀(Small฀Integrin-
Binding฀ Ligand,฀ N-linked฀ Glycoproteins).฀ They฀ bind฀ strongly฀ to฀ Ca2+฀ ions฀ or฀
hydroxyapatite฀ and฀ are฀ involved฀ with฀ osteocalcin฀ (not฀ a฀ sibling฀ protein)฀ in฀ bone฀
remodeling.฀ DSPP฀ is฀ important฀ in฀ dentin฀ formation฀ and฀ mutations฀ cause฀ various฀
forms฀of฀abnormal฀dentin.฀Two฀of฀the฀SIBLING฀proteins,฀BSP฀and฀DMP1,฀are฀hydro-
lyzed฀by฀a฀gluzincin฀(the฀PHEX฀protein)฀in฀normal฀osteoid฀matrix.฀Absence฀of฀the฀
PHEX฀protein฀also฀results฀in฀hypophosphatemia,฀which฀may฀prevent฀skeletal฀devel-
opment฀irrespective฀of฀the฀lack฀of฀BSP฀and฀DMP1฀cleavage฀in฀the฀osteoid฀matrix.฀
Osteocalcin฀is฀the฀major฀noncollagenous฀protein฀of฀bone฀matrix฀and฀its฀gene฀requires฀
vitamins฀ D฀ and฀ A฀ to฀ activate฀ transcription฀ and฀ its฀ subsequent฀ processing฀ requires฀
vitamin฀ K.฀ A฀ deiciency฀ of฀ osteocalcin฀ appears฀ to฀ enhance฀ skeletal฀ mass,฀ whereas฀
increased฀levels฀indicate฀greater฀osteoblast฀turnover.฀This฀is฀useful฀for฀categorizing฀
osteoporosis฀(bone฀loss)฀in฀the฀elderly.฀Osteocalcin฀likely฀prevents฀excessive฀bone฀
mineralization฀by฀its฀gamma-carboxyglutamic฀acid฀(gla)฀residues฀binding฀tightly฀to฀
hydroxyapatite฀ and฀ inhibiting฀ osteoid฀ matrix฀ formation฀ during฀ remodeling.฀ Proper฀
synthesis฀of฀bone฀requires฀vitamins฀A,฀C,฀D,฀and฀K.

9.5.1.
Enamel Organ and Matrix Development

Teeth฀develop฀from฀tooth฀buds,฀an฀aggregation฀of฀cells฀derived฀from฀the฀ectoderm฀of฀the฀
irst฀branchial฀arch฀and฀ectomesenchyme฀of฀the฀neural฀crest.฀The฀tooth฀bud฀is฀divided฀into฀
enamel฀organ฀(EO),฀dental฀papilla฀(DP),฀and฀dental฀follicle.฀An฀enamel฀organ฀has฀four฀lay-
ers:฀outer฀and฀inner฀enamel฀epithelium,฀stratum฀intermedium฀(SI),฀and฀stellate฀reticulum฀
(SR).฀ The฀ inner฀ enamel฀ epithelium฀ induces฀ the฀ development฀ of฀ odontoblasts฀ from฀ the฀
opposing฀mesenchymal฀cells฀of฀the฀dental฀papilla.฀As฀dentin฀forms,฀the฀inner฀enamel฀epi-
thelium฀becomes฀converted฀to฀ameloblasts฀(Fig.฀9.9).
The฀ameloblasts฀develop฀a฀tall฀columnar฀shape฀in฀which฀the฀nuclei฀move฀toward฀the฀
stratum฀intermedium.฀On฀the฀dentinal฀side,฀a฀Tome’s฀process฀appears฀and฀enamel฀matrix฀is฀
9.5.1. Enamel Organ and Matrix Development 145

Fig. 9.9฀ Light฀micrograph฀of฀a฀portion฀฀

of฀a฀molar฀tooth฀germ฀from฀a฀3-day-old฀
rat.฀The฀enamel฀organ฀(EO)฀shows฀the฀
outer฀epithelium฀(OE),฀stellate฀
reticulum฀(SR),฀stratum฀intermedium฀
(SI),฀and฀the฀ameloblast฀layer฀(AM).฀฀
A฀portion฀of฀the฀dental฀papilla฀(DP)฀
shows฀odontoblasts฀(Ob)฀adjacent฀to฀
early฀dentine฀(D).฀E฀enamel,฀DF฀dental฀
follicle฀outside฀the฀outer฀epithelium,฀AB฀
alveolar฀bone.฀Scale฀bar฀=฀70฀mm฀(From฀
Fig.฀1฀in฀Cerri฀PS,฀de฀Faria฀FP,฀Villa฀RG,฀
et฀al.฀(2004)฀“Light฀microscopy฀and฀
computer฀three-dimensional฀reconstruc-
tion฀of฀the฀blood฀capillaries฀of฀the฀
enamel฀organ฀of฀rat฀molar฀tooth฀germs.”฀
Journal฀of฀Anatomy฀204:฀191–195.฀
With฀copyright฀permission฀from฀
Wiley-Blackwell,฀PO฀Box฀805,฀9600฀
Garsington฀Road,฀Oxford฀฀
OX4฀2DQ,฀UK)

secreted฀into฀a฀mildly฀acidic฀pH฀that฀prevents฀mineralization฀(secretory฀stage;฀Fig.฀9.10).฀
As฀the฀matrix฀increases฀in฀volume,฀the฀ameloblast฀layer฀(AM)฀is฀pushed฀away฀from฀the฀
amelo-dentinal฀boundary฀and฀the฀Tome’s฀process฀starts฀to฀disappear.฀A฀rufled฀membrane฀
develops฀on฀either฀side฀of฀the฀resorbing฀Tome’s฀process฀and฀it฀starts฀transporting฀calcium,฀
phosphate,฀and฀bicarbonate฀ions฀slowly฀at฀irst฀and฀faster฀as฀its฀area฀increases.฀The฀matrix฀
and฀slow฀rate฀of฀pH฀increase฀and฀mineralization฀cause฀carbonated฀hydroxyapatite฀crystals฀
to฀develop฀and฀attach฀end-to-end,฀forming฀long฀enamel฀ribbons฀(Fig.฀9.10).฀The฀direction฀
of฀the฀ribbons฀is฀determined฀by฀the฀alignment฀of฀hydroxyapatite฀crystals฀in฀the฀adjacent฀
dentinal฀ collagen.฀ As฀ the฀ ribbons฀ mature฀ and฀ mineral฀ ion฀ transport฀ increases,฀ proteases฀
secreted฀along฀with฀the฀matrix฀begin฀to฀hydrolyze฀it.฀This฀allows฀the฀ribbons฀to฀thicken฀and฀
become฀transformed฀to฀rods฀of฀hydroxyapatite.฀The฀rufled฀border฀functions฀as฀an฀endocy-
totic฀membrane฀and฀absorbs฀the฀matrix฀protein฀fragments฀(maturation฀stage).
Finally,฀the฀ameloblasts฀secrete฀an฀unusual฀basal฀lamina,฀which฀partially฀mineralizes฀
into฀the฀surface฀enamel฀crystals,฀the฀enamel฀cuticle.฀By฀this฀time,฀the฀enamel฀organ฀has฀
shrunk฀ so฀ that฀ its฀ outer฀ layers฀ have฀ merged฀ with฀ the฀ ameloblast฀ layer฀ at฀ the฀ completed฀
enamel฀surface฀(postmaturation฀stage).฀This฀reduced฀enamel฀epithelium฀is฀rubbed฀off฀as฀
the฀teeth฀erupt,฀but฀the฀enamel฀cuticle฀is฀abraded฀more฀slowly฀and฀gradually฀replaced฀by฀
proteins฀from฀saliva,฀the฀acquired฀pellicle฀(Sect.฀12.1.3).
146 9 Biological Mineralization

Fig. 9.10฀ Ameloblasts฀in฀developing฀enamel.฀This฀igure฀is฀not฀to฀scale฀and฀oriented฀upside฀down฀

with฀respect฀to฀Fig.฀9.9;฀i.e.,฀dentin฀is฀above฀enamel฀in฀this฀igure฀but฀below฀the฀enamel฀in฀Fig.฀9.9.฀
Left:฀Amelogenin฀is฀secreted฀from฀the฀rod฀growth฀site฀(RGS)฀of฀each฀ameloblast฀Tomes’฀process฀
(shown฀in฀detail฀on฀the฀right).฀The฀amelogenin฀aggregates฀into฀nanospheres฀that฀bind฀linearly฀onto฀
ribbons฀of฀carbonated฀hydroxyapatite.฀In฀dentin฀above฀the฀enamel,฀plate-like฀apatite฀crystals฀grow฀
within฀collagen฀ibril฀gaps฀(dark฀regions฀center)฀before฀enamel฀mineralization฀begins.฀The฀long฀
axis฀of฀the฀amelogenin฀rod฀hydroxyapatite฀(“c”฀axis)฀parallels฀the฀long฀axis฀of฀the฀collagen฀ibers฀
that฀have฀penetrated฀between฀the฀amelogenin฀rod฀ribbons฀at฀the฀enamel–cemental฀junction฀(From฀
igure฀in฀Veis฀A฀(2005)฀“A฀window฀on฀biomineralization.”฀Science฀(307):1419–1420,฀March฀4,฀
2005.฀Reprinted฀with฀permission฀from฀AAAS).฀Right:฀Enamel฀matrix฀proteins฀are฀secreted฀from฀
Tomes’฀processes฀from฀which฀the฀enamel฀rods฀extend฀(rod฀growth฀site,฀RGS).฀Amelogenin฀( )฀is฀
present฀in฀the฀enamel฀rods฀but฀minor฀proteins฀such฀as฀intact฀ameloblastin฀(*)฀are฀more฀evident฀than฀
amelogenin฀( )฀within฀a฀Tomes’฀process฀beneath฀the฀rod.฀In฀addition,฀the฀N-terminal฀portion฀(►)฀
of฀ameloblastin฀persists฀into฀rod฀enamel,฀whereas฀its฀C-terminal฀portion฀is฀reabsorbed฀back฀into฀the฀
ameloblast.฀Amelogenin฀is฀not฀detected฀in฀the฀Tomes’฀process฀either฀because฀its฀synthesis฀into฀the฀
enamel฀ organ฀ (EO)฀ is฀ largely฀ complete฀ before฀ a฀ Tomes’฀ process฀ appears,฀ or฀ it฀ turns฀ over฀ more฀
slowly฀than฀ameloblastin.฀IR฀inter-rod฀enamel,฀R฀–฀rod฀enamel,฀RGS฀rod฀growth฀site฀(Reproduced,฀
with฀permission,฀from฀Nanci฀A,฀Zalzal฀S,฀Lavoie฀P,฀et.฀al.฀(1998)฀“Comparative฀immunochemical฀
analyses฀of฀the฀developmental฀expression฀and฀distribution฀of฀ameloblastin฀and฀amelogenin฀in฀rat฀
incisors.”฀Journal฀of฀Histochemistry฀and฀Cytochemistry฀46(8):930)

9.5.2.
Proteins Involved in Enamel Synthesis

Amelogenin฀ is฀ the฀ major฀ protein฀ product฀ of฀ ameloblasts.฀ It฀ is฀ encoded฀ by฀ two฀ highly฀
homologous฀ genes,฀ Amelx฀ and฀ Amely฀ in฀ the฀ X฀ and฀ Y฀ chromosomes,฀ respectively.฀ The฀
differences฀ in฀ the฀ encoded฀ proteins฀ give฀ rise฀ to฀ minor฀ differences฀ in฀ enamel฀ structure฀
9.5.3. Proposed Mechanism of Enamel Synthesis 147

between฀males฀and฀females.฀Figure฀9.11฀gives฀the฀amino฀acid฀sequence฀of฀the฀amelogenin฀
gene฀encoded฀by฀female฀mice.
The฀N-terminal฀amino฀acid฀of฀secreted฀amelogenin฀is฀number฀17฀of฀the฀encoded฀pro-
tein,฀as฀discussed฀in฀the฀legend฀to฀Fig.฀9.11.฀The฀N-terminal฀domain฀is฀hydrophobic,฀but฀
modiied฀ by฀ a฀ phosphorylated฀ serine฀ residue฀ and฀ a฀ hydrophilic฀ tyrosine-rich฀ domain฀ of฀฀
12฀amino฀acids.฀Except฀for฀11฀amino฀acids฀at฀the฀C-terminus฀(C-terminal฀domain),฀the฀rest฀
of฀ the฀ protein฀ is฀ hydrophobic฀ (Fig.฀ 9.11b).฀ The฀ gene฀ for฀ amelogenin฀ consists฀ of฀ seven฀
exons,฀of฀which฀exon฀6฀encodes฀most฀of฀the฀protein฀as฀a฀single,฀long,฀hydrophobic฀domain.฀
Ameloblasts฀also฀secrete฀small฀amounts฀of฀differentially฀spliced฀products.฀In฀particular,฀
pre-odontoblasts฀and฀pre-ameloblasts฀express฀an฀amelogenin฀variant฀in฀which฀the฀hydro-
phobic฀exon฀is฀missing.฀This฀short,฀hydrophilic฀amelogenin฀may฀interact฀with฀other฀pro-
teins฀or฀DNA฀during฀enamel฀organ฀differentiation.
Enamel฀matrix฀also฀contains฀small฀amounts฀of฀two฀other฀proteins,฀enamelin฀and฀amelo-
blastin,฀both฀hydrophilic.฀Enamelin฀is฀a฀large฀protein฀(1,142฀amino฀acids)฀that฀colocalizes฀
with฀amelogenin฀on฀growing฀enamel฀crystallites฀close฀to฀the฀Tomes’฀process.฀It฀is฀rich฀in฀
glycine,฀ aspartic฀ acid,฀ and฀ serine,฀ and฀ contains฀ two฀ phosphorylated฀ threonine฀ residues.฀
Most฀importantly,฀it฀contains฀many฀N-glycosylated฀sites฀from฀which฀N-acetylglucosamine฀
residues฀protrude.฀These฀residues฀noncovalently฀cross-link฀the฀tyrosine-rich฀region฀of฀dif-
ferent฀ amelogenin฀ molecules฀ within฀ the฀ nanospheres฀ (Figs.฀ 9.10฀ and฀ 9.12).฀ The฀ cross-
linking฀with฀amelogenin฀is฀essential฀for฀enamel฀ribbon฀development.฀Ameloblastin฀(also฀
called฀sheathlin฀or฀amelin;฀mature฀form฀in฀humans฀has฀421฀amino฀acids฀amino฀acids)฀is฀a฀
cell฀adhesion฀molecule฀located฀within฀the฀Tomes’฀process฀(Fig.฀9.10,฀left฀side)฀where฀it฀
likely฀stabilizes฀the฀ameloblasts฀during฀matrix฀secretion.฀During฀maturation,฀its฀extracel-
lular฀N-terminal฀sequence฀of฀almost฀100฀amino฀acids฀becomes฀cleaved฀off฀and฀localizes฀to฀
the฀inter-rod฀space฀where฀its฀functioning฀is฀not฀yet฀understood.
The฀ enamel฀ matrix฀ also฀ possesses฀ two฀ important฀ proteases:฀ enamelysin฀ (MMP20,฀
Sect.฀8.3.3)฀and฀enamel฀matrix฀serine฀proteinase฀(EMSP1).฀EMSP1฀is฀closely฀related฀to฀
kallekreins,฀a฀group฀of฀well-characterized฀serine฀proteases฀found฀in฀blood฀plasma฀and฀other฀
tissues฀ and฀ known฀ by฀ many฀ different฀ names฀ (ARM1;฀ EMSP;฀ PSTS;฀ KLK4;฀ KLK-L1;฀
PRSS17;฀MGC116827;฀MGC116828).฀The฀fragments฀of฀amelogenin฀produced฀by฀enam-
elysin฀and฀EMSP1฀are฀shown฀in฀Fig.฀9.11.

9.5.3.
Proposed Mechanism of Enamel Synthesis

Newly฀secreted฀amelogenin฀aggregates฀into฀20-nm฀nanospheres฀that฀promote฀end-to-end฀
association฀of฀carbonated฀hydroxyapatite฀crystals฀into฀enamel฀ribbons฀(Fig.฀9.12).฀Most฀
amelogenin฀fragments฀in฀immature฀enamel฀have฀already฀been฀cleaved฀by฀enamelysin;฀all฀
or฀part฀of฀the฀C-terminus฀with฀its฀adjacent฀Leucine-Rich฀Amelogenin฀Peptide฀(LRAP)฀are฀
removed฀ (Fig.฀ 9.11).฀ Only฀ the฀ N-terminal฀ Tyrosine-Rich฀ Amelogenin฀ Peptide฀ (TRAP)฀
segment฀with฀its฀N-terminal฀LRAP฀containing฀a฀phosphorylated฀serine฀residue฀(residue฀16฀
in฀Fig.฀9.11)฀remains฀and฀holds฀the฀amelogenin฀nanospheres฀to฀the฀carbonated฀hydroxy-
apatite฀ (enamel)฀ ribbons.฀ The฀ hydrophobic฀ nanospheres฀ accrete฀ more฀ amelogenin฀ and฀
148 9 Biological Mineralization

Fig. 9.11฀ Domains฀and฀exon฀structure฀of฀secreted฀mouse฀amelogenin.฀(a)฀Amino฀acid฀sequence.฀The฀

sequence฀is฀that฀of฀secreted฀mouse฀amelogenin฀encoded฀by฀isoform฀1฀of฀the฀X-chromosome฀(amel-
ogenin฀isoform฀1,฀Accession฀No.฀P63277).฀The฀16฀amino฀acid฀leader฀sequence,฀which฀is฀excised฀in฀
the฀endoplasmic฀reticulum,฀is฀not฀shown.฀(b)฀Functional฀domains:฀The฀red฀line฀and฀dot฀beneath฀the฀
amino฀acid฀sequence฀indicates฀a฀single฀phosphorylation฀site฀(serine-16).฀The฀major฀cleavage฀sites฀
are฀indicated฀above฀the฀uppermost฀horizontal฀bar฀diagram฀and฀the฀functional฀domains฀are฀indicated฀
beneath.฀These฀domains฀were฀identiied฀by฀protein-binding฀studies.฀The฀C-domain฀contains฀evolu-
tionarily฀ conserved฀ tandem฀ repeats฀ whose฀ precise฀ function฀ is฀ uncertain.฀ The฀ N-terminal฀ “A”฀
domain฀includes฀a฀tyrosyl-binding฀motif฀between฀amino฀acids฀34฀and฀45.฀This฀domain฀is฀cleaved฀
off฀ the฀ remainder฀ of฀ the฀ protein฀ by฀ EMSP-1฀ (enamel฀ matrix฀ serine฀ proteinase฀ 1)฀ to฀ give฀ the฀
tyrosine-rich฀amelogenin฀peptide฀(TRAP฀–฀illustrated฀in฀d).฀The฀C-terminal฀B฀domain฀includes฀two฀
of฀ three฀ enamelysin฀ (MMP20฀ metalloproteinase)฀ cleavage฀ sites฀ and฀ the฀ hydrophilic฀ C-terminal฀
domain฀of฀13฀amino฀acids฀that฀facilitates฀hydroxyapatite฀binding฀(Fig.฀9.10).฀(c)฀Exon฀structure.฀
The฀exon฀structure฀bar฀graph฀lies฀beneath฀the฀domain฀graph฀and฀it฀illustrates฀the฀expressed฀margins฀
of฀exons฀2฀and฀7฀as฀well฀as฀exons฀3,฀5,฀and฀6.฀Exon฀1฀is฀not฀expressed฀because฀it฀lies฀5¢฀of฀the฀start-
codon.฀ Exon฀ 6฀ accounts฀ for฀ all฀ but฀ the฀ irst฀ N-terminal฀ 33฀ residues฀ (49฀ residues฀ if฀ the฀ leader฀
sequence฀is฀included)฀and฀the฀amino฀acid฀that฀is฀the฀C-terminus.฀It฀therefore฀accounts฀for฀the฀bulk฀
of฀the฀secreted฀protein.฀(d)฀Cleavage฀products.฀The฀common฀polypeptide฀fragments,฀LRAP฀(leu-
cine-rich฀amelogenin฀peptide)฀and฀TRAP฀(see฀text)฀are฀indicated฀beneath฀the฀exon฀structure฀graph.฀
C-terminal฀LRAP฀peptide฀(bottom฀right)฀is฀cleaved฀by฀enamelysin,฀whereas฀N-terminal฀LRAP฀is฀
cleaved฀from฀TRAP฀(black฀rectangle฀at฀the฀foot฀of฀the฀igure)฀by฀EMSP1฀(see฀text).฀Traces฀of฀other฀
proteases฀then฀release฀the฀N-ter฀leucine-rich฀amelogenin฀peptide,฀LRAP,฀and฀also฀digest฀the฀rest฀of฀
the฀ amelogenin฀ nanospheres฀ to฀ small฀ peptides,฀ which฀ are฀ endocytosed฀ into฀ the฀ ameloblasts฀
(Modiied฀from฀a฀igure฀titled,฀“Architecture฀of฀the฀Mouse฀Amelogenin฀Gene,”฀that฀indicates฀stud-
ies฀being฀conducted฀during฀2005/2006฀by฀The฀Brodie฀Laboratory฀at฀University฀of฀Illinois฀Dental฀
School,฀Chicago,฀IL.฀http://dentistry.uic.edu/CraniofacialGenetics/ResearchTED.htm)
9.5.3. Proposed Mechanism of Enamel Synthesis 149

Fig. 9.12฀ Amelogenin฀ processing฀ in฀ enamel฀ biomineralization.฀ 1฀ –฀ Amelogenins฀ are฀ secreted฀ as฀
monomers฀extracellularly.฀2฀–฀The฀monomers฀assemble฀to฀generate฀nanospheres฀of฀20฀nm฀diameter฀
in฀which฀the฀hydrophilic฀(anionic)฀carboxy-terminals฀are฀externalized.฀3฀–฀The฀nanospheres฀interact฀
electrostatically฀parallel฀to฀the฀c฀axis฀of฀small฀crystals฀(crystallites)฀of฀hydroxyapatite,฀preventing฀
crystal–crystal฀fusions฀and฀acting฀as฀20-nm฀spacers฀that฀keep฀the฀crystallites฀apart.฀4฀–฀Enamelysin฀
processes฀the฀exposed฀amelogenin฀carboxy-terminal฀domains,฀progressively฀reducing฀their฀anionic฀
character.฀5฀–฀Hydrophobic฀nanospheres฀further฀assemble฀by฀the฀TRAP฀region฀binding฀enamelin฀
(see฀text)฀and฀this฀stabilizes฀the฀initial฀enamel฀crystallites.฀6฀–฀EMSP-1฀action฀removes฀the฀N-terminal฀
TRAP,฀which฀detaches฀the฀hydrophobic฀amelogenin฀nanospheres.฀Traces฀of฀other฀proteases฀generate฀
small฀peptides฀that฀are฀eventually฀resorbed฀by฀ameloblasts.฀7฀–฀As฀the฀amelogenin฀nanospheres฀are฀
removed,฀the฀crystallites฀thicken฀and฀fuse฀to฀generate฀the฀mature฀enamel฀(Reprinted฀from฀Fincham฀AG,฀
Moradian-Oldak฀ J,฀ Simmer฀ JP฀ (1999)฀ “The฀ structural฀ biology฀ of฀ the฀ developing฀ dental฀ enamel฀
matrix.”฀Journal฀of฀Structural฀Biology฀126:270–299฀with฀permission฀from฀Elsevier).฀This฀igure฀was฀
modiied฀by฀Dr.฀Wirsig฀Weichmann

grow฀up฀to฀tenfold฀in฀size฀restricting฀crystal฀extension฀to฀the฀long฀axis฀(Fig.฀9.10).฀The฀
TRAP฀region฀of฀amelogenin฀lies฀on฀the฀surface฀of฀the฀nanospheres฀and฀binds฀tightly฀but฀
noncovalently฀ to฀ enamelin฀ by฀ the฀ latter’s฀ N-acetylglucosamine฀ residues.฀ EMSP-1฀ then฀
cuts฀out฀the฀TRAP฀with฀its฀attached฀enamelin,฀causing฀the฀large฀nanospheres฀to฀contain฀
only฀the฀hydrophobic฀central฀region฀of฀amelogenin฀and฀they฀detach฀from฀the฀enamel฀rib-
bon฀ hydroxyapatite฀ crystals.฀ Traces฀ of฀ various฀ other฀ secreted฀ proteases฀ then฀ digest฀ the฀
amelogenin฀(Fig.฀9.12)฀producing฀fragments฀that฀include฀the฀amino-terminal฀leucine-rich฀
150 9 Biological Mineralization

amelogenin฀ peptide฀ (LRAP)฀ from฀ the฀ N-terminal฀ side฀ of฀ the฀ TRAP฀ (Fig.฀ 9.11).฀ The฀
hydroxyapatite฀crystals฀in฀and฀around฀the฀ribbons฀develop฀into฀rod฀and฀inter-rod฀regions฀by฀
lateral฀ expansion฀ and฀ fusion.฀ The฀ small฀ peptides฀ circulate฀ and฀ are฀ endocytosed฀ by฀ the฀
ameloblasts.
Mutations฀that฀inhibit฀any฀of฀the฀interactions฀between฀the฀structural฀proteins฀(amelo-
genin,฀ enamelin,฀ or฀ ameloblastin),฀ or฀ that฀ cause฀ a฀ loss฀ of฀ activity฀ of฀ either฀ MMP20฀ or฀
EMSP1฀protease,฀cause฀primary฀amelogenesis฀imperfecta,฀absence฀or฀incomplete฀forma-
tion฀of฀enamel฀without฀systemic฀effects.฀Secondary฀amelogenesis฀imperfecta฀is฀associated฀
with฀odontogenesis฀imperfecta฀due฀to฀collagen฀mutations฀(Sect.฀7.2.1).฀Abnormal฀enamel฀
ribbons฀form฀due฀to฀the฀altered฀orientation฀of฀the฀mineralized฀collagen฀in฀dentin.฀Mutations฀
of฀laminin-5฀and฀ibrillin-1฀and฀-2฀alter฀stromal฀organization฀and฀may฀also฀result฀in฀abnor-
mal฀or฀absent฀enamel฀(Table฀7.1).

The฀inner฀epithelium฀of฀the฀enamel฀organ฀differentiates฀into฀ameloblasts฀after฀odonto-
blasts฀have฀started฀to฀produce฀dentin.฀The฀ameloblasts฀then:฀(a)฀form฀a฀Tomes’฀process฀
through฀ which฀ the฀ protein฀ matrix฀ and฀ proteases฀ are฀ secreted฀ (secretory฀ stage);฀฀
(b)฀replace฀the฀Tomes’฀process฀with฀a฀rufled฀membrane฀through฀which฀minerals฀are฀
secreted฀and฀peptide฀fragments฀of฀the฀matrix฀are฀reabsorbed฀(maturation฀stage);฀and฀(c)฀
form฀an฀unusual฀basal฀lamina฀that฀becomes฀the฀enamel฀cuticle฀(postmaturation฀stage).฀
Amelogenin฀has฀a฀large,฀central฀hydrophobic฀domain฀that฀aggregates฀into฀small฀nano-
spheres฀attached฀to฀the฀developing฀crystals฀by฀a฀hydrophilic฀C-terminus.฀Enamelysin฀
removes฀the฀C-terminus฀and฀the฀amelogenin฀forms฀large฀nanospheres฀attached฀to฀long฀
ribbons฀of฀carbonated฀hydroxyapatite฀made฀from฀secreted฀bicarbonate,฀phosphate,฀and฀
calcium฀ions.฀The฀TRAP฀region฀of฀amelogenin฀is฀on฀the฀surface฀of฀the฀nanospheres฀
where฀ it฀ binds฀ to฀ N-acetylglucosamine฀ residues฀ of฀ enamelin.฀ This฀ holds฀ the฀ nano-
spheres฀together฀and฀to฀the฀hydroxyapatite.฀A฀second฀protease,฀enamel฀matrix฀serine฀
proteinase-1฀(EMSP1)฀removes฀the฀TRAP฀with฀its฀attached฀enamelysin,฀disintegrating฀
the฀hydrophobic฀nanospheres.฀Amelogenin฀and฀enamelysin฀are฀digested.฀The฀crystals฀
thicken฀into฀rods฀that฀expand฀and฀fuse฀into฀mature฀enamel.฀Genetic฀mutations฀of฀amelo-
genin฀and฀proteases฀cause฀primary฀amelogenesis฀imperfecta.฀Mutations฀of฀mesenchy-
mal฀proteins฀cause฀secondary฀amelogenesis฀imperfects฀by฀causing฀the฀enamel฀ribbons฀
to฀align฀incorrectly.฀Enamel฀formation฀requires฀vitamin฀A฀and฀is฀independent฀of฀vita-
mins฀C,฀D฀and฀K.฀A฀low฀blood฀calcium฀level฀has฀no฀effect฀on฀enamel฀calciication.

9.6.1.
Summary of Ways in Which Enamel and Bone Differ

The฀structure฀of฀enamel฀and฀its฀mechanisms฀of฀formation฀differ฀from฀those฀of฀bone,฀dentin,฀
and฀cementum฀in฀ive฀important฀ways:

1.฀ Mature฀enamel฀is฀more฀than฀95%฀mineral฀(by฀weight),฀with฀only฀1–2%฀matrix฀protein,฀
and฀ 3–4%฀ water.฀ In฀ contrast,฀ bone,฀ dentin,฀ and฀ cementum฀ are฀ about฀ 70%฀ mineral,฀฀
20%฀collagen,฀and฀10%฀water.฀Enamel฀is฀brittle฀compared฀with฀bone.
9.6.2. Summary of the Vitamins for Bone and Enamel Formation 151

2.฀ Phosphorylation฀ initiates฀ crystallization฀ like฀ serine-phosphorylated฀ collagen,฀ but฀ the฀

mineralization฀alters฀enamel฀matrix฀structure฀(nanospheres฀formation฀and฀degradation),฀
unlike฀the฀mineralization฀of฀collagen฀matrix.
3.฀ Enamel฀matrix฀is฀partially฀mineralized฀before฀its฀matrix฀is฀completed,฀whereas฀bone,฀
dentin,฀and฀cementum฀secrete฀a฀preformed฀organic฀(osteoid)฀matrix฀to฀which฀calcium฀
phosphate฀is฀added฀separately฀from฀matrix฀vesicles.
4.฀ Enamel฀matrix฀is฀hydrolyzed฀to฀small฀fragments฀and฀disappears฀after฀mineralization,฀
whereas฀the฀bone฀matrix฀remains.
5.฀ Enamel฀cannot฀be฀remodeled,฀whereas฀bone,฀dentin,฀and฀cementum฀may฀be฀removed฀by฀
osteoclasts฀and฀replaced฀with฀new฀bone฀(remodeled;฀Sect.฀10.1.1).

9.6.2.
Summary of the Vitamins for Bone and Enamel Formation

Osteoblasts,฀odontoblasts,฀and฀cementoblasts฀need฀vitamin฀C฀to฀make฀collagen,฀vitamin฀D฀
for฀uptake฀of฀calcium฀into฀the฀body฀(Chap.฀10),฀and฀vitamins฀A฀and฀K฀to฀synthesize฀and฀
secrete฀the฀active฀form฀of฀osteocalcin.฀Vitamins฀C฀and฀D฀are฀the฀most฀important฀during฀
childhood฀and฀adolescence:฀to฀make฀adequate฀amounts฀of฀type฀I฀collagen฀and฀supply฀a฀net฀
increase฀in฀calcium฀to฀the฀body.฀Ameloblasts,฀like฀other฀cells฀of฀ectodermal฀origin,฀require฀
vitamin฀ A฀ to฀ differentiate฀ and฀ secrete฀ their฀ proteins,฀ but฀ none฀ of฀ the฀ other฀ vitamins.฀
Calciication฀of฀enamel฀appears฀independent฀of฀the฀increase฀in฀blood฀calcium฀level฀medi-
ated฀by฀vitamin฀D.
 Bone Remodeling and Calcium
Metabolism 10

Bones฀ are฀ constantly฀ dissolved฀ by฀ osteoclasts฀ and฀ remineralized฀ by฀ osteoblasts฀ in฀
response฀to฀mechanical฀forces.฀Osteoclasts฀possess฀an฀acidic฀compartment฀and฀pass฀
demineralized฀ bone฀ products฀ to฀ the฀ periosteum฀ (Sect.฀ 1).฀ They฀ develop฀ in฀ stress-
induced฀ bony฀ microcracks฀ and฀ are฀ activated฀ by฀ differentiation฀ factors฀ secreted฀ by฀
osteoblasts,฀ especially฀ after฀ menopause.฀ Menopausal฀ osteoporosis฀ is฀ controlled฀ by฀
drugs฀that฀are฀a฀stable฀form฀of฀pyrophosphate฀(bisphosphonate)฀or฀cathepsin฀K฀inhibi-
tors฀(Sect.฀2).฀The฀calcium฀ion฀concentration฀of฀blood฀is฀raised฀by฀parathyroid฀hor-
mone฀ and฀ a฀ vitamin฀ D฀ derivative฀ called฀ calcitriol.฀ Parathyroid฀ hormone฀ causes฀
kidneys฀ to฀ excrete฀ phosphate,฀ retain฀ calcium,฀ and฀ activate฀ calcitriol฀ production฀
(Sect.฀3).฀Calcitriol฀induces฀calcium฀transporter฀proteins฀in฀osteoclasts฀and฀intestinal฀
epithelium,฀ where฀ they฀ move฀ calcium฀ from฀ bone฀ or฀ diet฀ into฀ blood฀ (Sect.฀ 4).฀ The฀
chapter฀concludes฀with฀a฀discussion฀of฀calcitonin฀which฀lowers฀blood฀calcium฀con-
centrations฀ by฀ reversing฀ parathyroid฀ hormone฀ effects฀ on฀ the฀ kidney฀ and฀ inhibiting฀
osteoclast฀activity฀(Sect.฀5).

10.1.1.
Bone Turnover, Osteoclasts, and Lysosomes

Bones฀ are฀ constantly฀ subjected฀ to฀ forces฀ that฀ cause฀ microscopic฀ cracks.฀ These฀ microc-
racks:฀(1)฀attach฀blood฀monocytes฀circulating฀within฀the฀periosteum฀and฀bone฀marrow฀and฀
(2)฀induce฀adjacent฀osteoblasts฀to฀produce฀cytokines฀(Sect.฀3.3.2)฀that฀cause฀these฀mono-
cytes฀ to฀ proliferate,฀ fuse,฀ and฀ differentiate฀ into฀ large฀ multinucleated฀ cells฀ called฀ osteo-
clasts.฀ Osteoclasts฀ cause฀ bone฀ resorption฀ by฀ acid฀ demineralization฀ and฀ digestion฀ of฀ its฀
proteins฀by฀enzymes฀that฀are฀optimally฀active฀in฀an฀acidic฀environment.฀These฀proteases฀
and฀other฀hydrolytic฀enzymes฀are฀stored฀in฀a฀specialized,฀membrane-sealed฀compartment฀
(lysosomes)฀into฀which฀they฀are฀guided฀by฀possessing฀terminal฀mannose฀6-phosphate฀resi-
dues฀on฀N-linked฀glycans.
In฀osteoclasts,฀a฀tunnel฀or฀lacuna฀in฀the฀bone฀develops฀at฀a฀rate฀of฀~50฀mm/day฀(Fig.฀10.1).฀
The฀bone฀dissolution฀occurs฀in฀an฀acidic฀(demineralizing)฀compartment฀that฀is฀separated฀
from฀the฀rest฀of฀the฀cell฀by฀a฀rufled฀membrane฀whose฀outer฀periphery฀is฀sealed฀to฀the฀bone฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 153
DOI:฀10.1007/978-3-540-88116-2_10,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
154 10 Bone Remodeling and Calcium Metabolism

Fig. 10.1฀ Osteoclasts฀tunneling.฀Osteoclasts฀differentiate฀at฀cracks฀in฀the฀bone฀and฀form฀a฀tunnel฀

(lacuna)฀containing฀a฀central฀capillary฀surrounded฀by฀connective฀tissue.฀Fibroblast-like฀precursors฀
of฀ osteoblasts฀ are฀ activated฀ to฀ become฀ osteoblasts฀ and฀ new฀ bone฀ is฀ formed฀ (Modiied฀ from฀
Fig.฀22.54฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀Garland฀Science,฀
Taylor฀&฀Francis฀Group,฀New฀York)

by฀ integrin฀ aVb3฀ (Fig.฀ 10.2).฀ This฀ integrin฀ attaches฀ to฀ an฀ RGD฀ sequence฀ (arg-gly-asp;฀
Sects.฀ 3.2.1฀ and฀ 8.2.1)฀ on฀ the฀ outer฀ surface฀ of฀ osteopontin,฀ a฀ SIBLING฀ protein฀ that฀ is฀
tightly฀attached฀to฀the฀outer฀surface฀of฀bone฀(Sect.฀9.4.1.).฀The฀rufled฀membrane฀compart-
ment฀is฀therefore฀tightly฀sealed฀from฀the฀cell฀and฀the฀stroma.฀Only฀the฀small฀region฀of฀bone฀
within฀ the฀ compartment฀ is฀ exposed฀ to฀ HCl,฀ lysosomal฀ proteases,฀ and฀ acid฀ phosphatase฀
type฀5฀(Acp5).฀Acp5฀is฀found฀in฀the฀lysosomes฀of฀human฀kidney,฀liver,฀spleen,฀osteoblast,฀
and฀osteoclast฀cells.฀An฀iron฀ion฀(Fe2+)฀at฀the฀catalytic฀center฀is฀responsible฀for฀the฀enzyme’s฀
purple฀color฀and฀also฀a฀protein฀fold฀known฀as฀the฀metallo-phosphoesterase฀domain.฀This฀
domain฀is฀present฀in฀purple฀acid฀phosphatases฀of฀plants฀and฀in฀various฀serine/threonine฀
protein฀ phosphatases.฀ In฀ the฀ bone฀ resorbing฀ compartment฀ of฀ osteoclasts,฀ cathepsin฀ K฀
cleaves฀out฀a฀short฀central฀loop฀from฀lysosomal฀Acp5.฀The฀modiied฀Acp5฀(Acp5b)฀is฀opti-
mally฀active฀at฀pH฀5.0,฀whereas฀the฀unmodiied฀enzyme฀in฀lysosomes฀(Acp5a)฀is฀optimally฀
active฀at฀pH฀5.5.฀
The฀demineralizing฀compartment฀contains฀acidic,฀tissue-degrading฀enzymes฀(Sect.฀3.3.2)฀
that฀are฀secreted฀from฀lysosomes฀through฀the฀rufled฀membrane.฀Demineralization฀and฀pro-
teolysis฀in฀this฀compartment฀produce฀calcium฀ions฀and฀peptides฀that฀are฀endocytosed฀into฀
acidic฀vesicles.฀These฀vesicles฀traverse฀the฀osteoclast฀rufled฀membrane฀฀and฀cytosol฀and฀
10.1.1. Bone Turnover, Osteoclasts, and Lysosomes 155

Fig. 10.2฀ Osteoclast฀membrane฀functions.฀Rufled฀and฀secretory฀membranes฀differentiate฀from฀the฀

outer฀osteoclast฀membrane฀at฀opposite฀ends฀(see฀text).฀Proteases,฀mostly฀cathepsin฀K฀and฀MMP-9฀
are฀ synthesized฀ in฀ the฀ endoplasmic฀ reticulum฀ (ER)฀ (purple฀ dots)฀ and฀ are฀ diverted฀ to฀ lysosomes฀
(blue฀dots฀–฀1)฀because฀the฀proteases฀possess฀N-linked฀glycans฀that฀have฀attached฀mannose฀phos-
phate฀residues.฀Acid฀phosphatase฀(Acp5)฀is฀also฀synthesized฀in฀the฀ER฀and฀it฀passes฀into฀lysosomal฀
trans-Golgi฀vesicles฀(yellow฀dots)฀along฀with฀the฀proteases฀–฀2.฀The฀vesicles฀fuse฀to฀the฀cytosolic฀
side฀of฀the฀rufled฀membrane฀(near฀the฀integrin/osteopontin฀seal)฀and฀release฀their฀contents฀into฀the฀
resorption฀compartment฀–฀3.฀Protons฀and฀chloride฀ions฀are฀separately฀transported฀from฀the฀cytosol฀
to฀the฀bone฀resorption฀compartment฀through฀this฀same฀region฀(Fig.฀10.3).฀Calcium฀but฀not฀phos-
phate฀ions฀from฀demineralized฀bone,฀along฀with฀acid฀phosphatase฀and฀cathepsin฀K฀(red฀dots)฀enter฀
acidic฀vesicles฀in฀the฀center฀of฀the฀rufled฀membrane฀–฀4.฀They฀pass฀through฀the฀cytosol฀(large฀red฀
arrow)฀and฀are฀extruded฀into฀the฀extracellular฀luid฀at฀the฀secretory฀membrane฀–฀5฀(Adapted฀from฀
Fig.฀ 1฀ in฀ HK฀ Väänänen,฀ H฀ Zhao,฀ M฀ Mulari฀ and฀ JM฀ Halleen,฀ “The฀ Cell฀ Biology฀ of฀ Osteoclast฀
Function.”฀Journal฀of฀Cell฀Science.฀113:377–381,฀2000;฀Printed฀in฀Great฀Britain฀©฀The฀Company฀
of฀Biologists฀Limited฀2000฀JCS1073:฀Reproduced฀with฀permission฀of฀the฀Company฀of฀Biologists฀;฀
Figure฀was฀modiied฀by฀Dr.฀Wirsig-Weichmann)

secrete฀their฀contents฀into฀the฀extracellular฀luid฀where฀it฀can฀be฀picked฀up฀by฀blood฀capil-
laries฀(Fig.฀10.2).฀Simultaneously฀(Fig.฀10.3),฀transporter฀proteins฀move฀chloride฀ions฀from฀
the฀extracellular฀luid฀to฀the฀cytosol฀and฀from฀the฀cytosol฀into฀the฀demineralizing฀compart-
ment.฀Other฀transporters฀move฀phosphate฀ions฀in฀the฀opposite฀direction.฀Protons฀from฀car-
bonic฀acid฀in฀the฀cytosol฀are฀exchanged฀for฀released฀sodium฀and฀dihydrogen฀phosphate.฀
Increased฀cytosolic฀acidity฀likely฀restricts฀the฀osteoclast฀half-life฀to฀~1.3฀days;฀new฀osteo-
clasts฀must฀be฀activated฀for฀demineralization฀to฀continue.฀Thus,฀once฀all฀the฀bone฀around฀a฀
microcrack฀ is฀ removed,฀ osteoclasts฀ disappear฀ and฀ osteoblasts฀ lay฀ down฀ new฀ bone฀ in฀
response฀to฀the฀stresses,฀a฀process฀called฀remodeling฀(Sect.฀9.2.1).฀Cementum฀and฀dentin฀
behave฀similarly,฀but฀demineralized฀enamel฀cannot฀be฀remodeled฀because฀new฀ameloblasts฀
cannot฀be฀generated.
156 10 Bone Remodeling and Calcium Metabolism

Fig. 10.3฀ Metabolite฀and฀ion฀exchange฀in฀osteoclasts.฀Glucose฀in฀extracellular฀luid฀(top฀right)฀is฀

transported฀ into฀ osteoclasts฀ by฀ a฀ transporter฀ protein฀ (GLUT2).฀ It฀ is฀ metabolized฀ to฀ pyruvate฀
which฀enters฀mitochondria฀(M฀–฀center฀right)฀to฀provide฀ATP,฀CO2,฀and฀H2O.฀The฀ATP฀mediates฀
a฀transfer฀of฀protons฀(H+)฀from฀carbonic฀acid฀(H2CO3)฀through฀the฀rufled฀membrane฀in฀exchange฀
for฀sodium฀(Na+)฀ions฀from฀demineralized฀bone฀(red).฀Sodium฀ions฀in฀the฀cytosol฀are฀transferred฀
to฀ the฀ periosteum฀ by฀ the฀ ATP-requiring฀ Na+/K+฀ exchanger฀ common฀ to฀ all฀ cells฀ (not฀ shown).฀
Carbonic฀anhydrase฀(CA)฀makes฀bicarbonate฀anions฀(HCO31−)฀from฀CO2฀and฀H2O฀in฀the฀cytosol.฀
The฀bicarbonate฀is฀exchanged฀for฀chloride฀ions฀from฀the฀extracellular฀luid฀(Cl1−;฀top฀left).฀The฀
chloride฀ions฀enter฀the฀sealed฀compartment฀through฀channel฀proteins฀in฀the฀rufled฀membrane฀
(bottom฀left฀and฀right).฀HCl฀accumulates฀in฀the฀demineralizing฀compartment฀and฀dissolves฀bone,฀
releasing฀ calcium฀ (Ca2+)฀ and฀ dihydrogen฀ phosphate฀ ions฀ and฀ collagen฀ polypeptides฀ that฀ are฀
hydrolyzed฀ by฀ cathepsin฀ K.฀ The฀ phosphate฀ ions฀ buffer฀ the฀ demineralizing฀ compartment฀ so฀
that฀the฀pH฀remains฀at฀~4.5.฀A฀proton-dependent฀phosphate฀transporter฀moves฀sodium฀dihy-
drogen฀phosphate฀into฀the฀cytosol฀(purple)฀and฀it฀passes฀through฀the฀cell฀to฀the฀extracellular฀
luid฀ by฀ an฀ unknown฀ mechanism.฀ A฀ sodium฀ ion-dependent฀ type฀ III฀ phosphate฀ transporter฀
(฀ Pit-2)฀ in฀ the฀ basolateral฀ membrane฀ (Fig.฀ 10.2)฀ takes฀ up฀ sodium฀ dihydrogen฀ phosphate฀ (Pi)฀
from฀the฀extracellular฀luid฀around฀and฀transfers฀it฀into฀the฀cytosol฀to฀increase฀pH.฀As฀also฀illus-
trated฀ in฀ Fig.฀ 10.2,฀ calcium฀ ions฀ are฀ transported฀ in฀ acidic฀ vesicles฀ to฀ the฀ extracellular฀ luid฀
(Modiied฀from฀Figure฀2฀in฀Yamashita฀DS฀and฀Dodd฀RB฀(2000)฀“Cathepsin฀K฀and฀the฀Design฀of฀
Inhibitors฀ of฀ Cathepsin฀ K.”฀ Curr.฀ Pharmaceut.฀ Des.฀ 6:1–24).฀ This฀ igure฀ was฀ modiied฀ by฀฀
Dr.฀Wirsig-Weichmann
10.1.3. Demineralization and remineralization 157

10.1.2.
Proteolysis in the Bone Resorbing Compartment

Hydrochloric฀ acid฀ in฀ the฀ demineralizing฀ compartment฀ dissolves฀ the฀ bone฀ mineral฀ and฀
denatures฀exposed฀collagen฀ibers฀for฀hydrolysis฀by฀cathepsin฀K.฀This฀lysosomal฀endopro-
tease฀ (Fig.฀ 10.4a฀ and฀ b)฀ has฀ a฀ cysteine฀ thiol฀ group฀ at฀ its฀ catalytic฀ center฀ (Table฀ 7.1;฀
Fig.฀10.4c).฀Cathepsin฀K฀is฀secreted฀in฀larger฀amounts฀than฀other฀lysosomal฀proteases฀and฀
its฀speciicity฀is฀due฀to฀unique฀amino฀acid฀residues฀around฀the฀peptide฀binding฀site.฀The฀
enzyme฀hydrolyzes฀a1(I)฀and฀a2(I)฀polypeptides฀from฀incompletely฀denatured฀ibers฀in฀the฀
demineralizing฀compartment,฀leaving฀the฀cross-linked,฀telopeptide฀ends฀(Sect.฀4.2.2).
Cathepsin฀ K฀ remains฀ inactive฀ in฀ lysosomes฀ because฀ a฀ propeptide฀ occupies฀ its฀ active฀
site,฀as฀in฀matrilysins฀(Sect.฀8.1.3).฀Once฀secreted฀into฀the฀demineralizing฀compartment฀
along฀with฀smaller฀amounts฀of฀other฀lysosomal฀proteases,฀the฀low฀pH฀loosens฀electrostatic฀
and฀ hydrogen฀ bonds,฀ promoting฀ propeptide฀ cleavage฀ by฀ autocatalysis฀ or฀ other฀ protease฀
action.฀Mutations฀that฀reduce฀or฀abolish฀cathepsin฀K฀activity฀are฀associated฀with฀pycnodys-
ostosis,฀ a฀ rare฀ genetic฀ disease฀ characterized฀ in฀ humans฀ by฀ a฀ reduced฀ stature฀ and฀ brittle฀
(osteosclerotic)฀bones.฀MMP-9฀(neutrophil฀gelatinase;฀Sect.฀8.3.4)฀is฀another฀proteolytic฀
enzyme฀in฀the฀demineralizing฀compartment฀of฀osteoclasts฀where฀it฀degrades฀type฀a1(II)฀
chains฀left฀over฀from฀cartilage฀in฀endochondral-synthesized฀bones฀(Sect.฀9.2.2).

10.1.3.
Demineralization and remineralization

In฀ addition฀ to฀ collagen฀ peptides฀ and฀ calcium฀ ions,฀ demineralized฀ bones฀ release฀ small฀
amounts฀of฀transforming฀growth฀factor-beta,฀TGF-b฀(Sect.฀3.2.2).฀TGF-b฀is฀not฀denatured฀฀
by฀acid฀and฀not฀hydrolyzed฀by฀lysosomal฀proteases฀in฀the฀osteoclast฀demineralizing฀com-
partment.฀Its฀function฀is฀to฀be฀transferred฀transfer฀to฀the฀periosteum฀during฀demineraliza-
tion฀ where฀ it฀ stimulates฀ new฀ bone฀ formation฀ to฀ complete฀ the฀ remodeling฀ process.฀ The฀
TGF-b฀is฀endocytosed฀into฀acidic฀vacuoles฀along฀with฀peptides,฀calcium฀ions,฀and฀Acp5b.฀
The฀acidic฀vacuoles฀pass฀through฀the฀center฀of฀the฀rufled฀membrane฀and฀are฀exocytosed฀
into฀the฀periosteum฀or฀bone฀marrow฀through฀the฀osteoclast฀secretory฀membrane฀(Fig.฀10.2).฀
A฀greater฀blood฀plasma฀content฀of฀Acp5b฀indicates฀increased฀osteoclast฀activity,฀but฀the฀
best฀indicator฀of฀net฀bone฀loss฀is฀an฀increased฀level฀of฀type฀I฀collagen฀telopeptides฀contain-
ing฀pyridinoline฀(Sect.฀4.2.2)฀A฀greater฀amount฀of฀telopeptide฀in฀blood฀plasma฀indicates฀a฀
greater฀rate฀of฀bone฀demineralization,฀whereas฀a฀greater฀amount฀of฀osteocalcin฀indicates฀a฀
greater฀ rate฀ of฀ bone฀ remineralization.฀ A฀ high฀ telopeptide฀ to฀ osteocalcin฀ ratio฀ in฀ blood฀
plasma฀therefore฀indicates฀net฀bone฀demineralization฀in฀the฀body,฀whereas฀a฀low฀such฀ratio฀
indicates฀net฀bone฀mineralization.
158 10 Bone Remodeling and Calcium Metabolism

a Papain Cathepsin K

Prodomain

Active
site
cysteine

b
Cathepsin K

Pre Pro Mature

1−16 115 329

c
Cysteine proteases Metalloproteases
R

C O
O H
O
H H
X
N X C H
O
N O
H S R
O
O

Fig. 10.4฀ Cathepsin฀K฀domain฀structure฀and฀mode฀of฀action.฀(a)฀Ribbon฀diagrams฀of฀papain฀and฀

cathepsin฀K฀structures.฀Left฀–฀papain;฀right฀cathepsin฀K.฀Similarities฀of฀the฀respective฀structures฀
and฀active฀site฀regions฀are฀obvious฀(Diagram฀of฀papain฀is฀part฀of฀Fig.฀10.18฀from฀Berg.,฀et฀al.,฀
Biochemistry,฀5th฀Ed.฀2002,฀W.H.฀Freeman฀&฀Co.,฀New฀York;฀diagram฀of฀cathepsin฀K฀is฀Fig.฀1฀
from฀LaLonde฀(1999)฀“The฀Crystal฀Structure฀of฀Human฀Procathepsin฀K.”฀Biochemistry฀38(3):฀
862–869).฀(b)฀Domain฀structure.฀Signal฀peptide฀for฀protein฀synthesis฀into฀the฀endoplasmic฀reticu-
lum฀(ER)฀–฀light฀blue;฀propeptide฀inhibitor฀region฀–฀dark฀blue;฀mature฀protease฀–฀green.฀Gray฀
bands฀indicate฀exon฀breaks.฀Numbers฀below฀indicate฀the฀number฀of฀amino฀acids฀in฀each฀domain฀
(Modiied฀from฀Fig.฀1฀in฀Donnarumma,฀M.,฀et฀al.฀(2007)฀“Molecular฀analysis฀and฀characterization฀
of฀nine฀novel฀CTSK฀mutations฀in฀twelve฀patients฀affected฀by฀pycnodysostosis.฀Mutation฀in฀brief฀
#961.”฀Human฀Mutat.฀28(5):524).฀(c)฀Catalytic฀mechanism฀of฀cysteine฀(papain฀family)฀proteases฀
and฀metalloproteinases.฀The฀major฀classes฀of฀proteases฀are฀listed฀in฀Table฀8.1.฀They฀use฀two฀fun-
damentally฀different฀catalytic฀mechanisms฀to฀stabilize฀a฀tetrahedral฀intermediate฀during฀polypep
10.1.4. Osteoclast Ion and Proton Transport 159

10.1.4.
Osteoclast Ion and Proton Transport

The฀rufled฀membrane฀is฀mostly฀composed฀of฀two฀chloride฀channel฀proteins:฀a฀chloride฀
intracellular฀channel฀type฀5฀(ClIC-5)฀protein฀found฀in฀the฀endoplasmic฀reticulum฀(ER)฀and฀
outer฀plasma฀membrane;฀and฀ a฀chloride฀channel฀type฀7฀(ClC-7)฀protein฀homologous฀to฀
voltage-gated฀chloride฀channels฀in฀the฀plasma฀membrane฀of฀nerves฀and฀muscles฀(Fig.฀10.3).฀
It฀also฀contains฀two฀other฀transporters:฀an฀ATPase฀that฀moves฀protons฀(H+)฀from฀the฀cytosol฀
in฀exchange฀for฀Na฀ions฀(Na+),฀and฀a฀proton-dependent฀phosphate฀transporter฀that฀moves฀
dihydrogen฀phosphate฀ions฀from฀the฀bone฀resorption฀compartment฀to฀the฀cytosol฀(Fig.฀10.3).฀
The฀calcium฀ions฀are฀moved฀out฀of฀the฀bone฀resorption฀compartment฀in฀acidic฀vesicles฀with฀
digested฀ collagen฀ peptides฀ and฀ Acp5b.฀ They฀ are฀ transported฀ through฀ the฀ cytosol฀ and฀
secreted฀into฀the฀periosteum฀(or฀bone฀marrow).
The฀ATP฀produced฀by฀respiration฀is฀required฀to฀move฀the฀protons฀from฀the฀cytosol฀to฀the฀
bone฀ resorption฀ compartment฀ in฀ exchange฀ for฀ sodium฀ ions,฀ fair฀ amounts฀ of฀ which฀ are฀
released฀from฀demineralizing฀bone฀along฀with฀calcium฀and฀phosphate.฀The฀protons฀are฀pro-
duced฀with฀bicarbonate฀by฀carbonic฀anhydrase฀(CA),฀which฀utilizes฀the฀carbon฀dioxide฀and฀
water฀from฀respiration.฀The฀CA฀catalytic฀site฀utilizes฀a฀zinc฀ion,฀like฀zincin฀proteases฀and฀
alkaline฀phosphatase.฀The฀isozyme฀in฀osteoclast฀is฀identical฀to฀the฀carbonic฀anhydrase฀in฀red฀
blood฀cells฀(CA-II).฀The฀sodium฀and฀bicarbonate฀ions฀in฀the฀cytosol฀are฀exchanged฀for฀extra-
cellular฀ chloride฀ and฀ potassium฀ ions฀ at฀ the฀ periosteal฀ surface.฀ Chloride฀ ions฀ low฀ from฀
periosteum฀or฀bone฀marrow,฀through฀the฀cytosolic฀and฀rufled฀membrane฀chloride฀transport-
ers฀to฀the฀demineralizing฀compartment.฀Sodium฀ions฀low฀in฀the฀opposite฀direction฀to฀the฀
periosteum฀or฀bone฀marrow฀(Fig.฀10.3).฀Protons฀and฀chloride฀ions฀accumulate฀within฀the฀
bone฀resorption฀compartment,฀causing฀the฀pH฀to฀fall฀and฀the฀bone฀to฀demineralize.฀Phosphate฀
and฀sodium฀ions฀from฀the฀bone฀are฀transported฀out฀from฀the฀rufled฀membrane฀each฀at฀the฀
expense฀of฀protons฀moving฀in฀and฀maintaining฀the฀demineralizing฀compartment’s฀acidity.
Despite฀ the฀ substantial฀ amounts฀ of฀ Na+฀ ions฀ released฀ from฀ demineralized฀ bone฀ and฀
entering฀the฀cytosol฀in฀exchange฀for฀protons,฀excessive฀amounts฀of฀dihydrogen฀phosphate฀
(H2PO4)฀also฀enter฀the฀cytosol.฀To฀compensate,฀there฀is฀a฀separate฀inward฀diffusion฀of฀diso-
dium฀ monohydrogen฀ phosphate฀ (Na2HPO4)฀ from฀ the฀ periosteum/bone฀ marrow฀ through฀
Pit-2฀ transporters฀ (Sect.฀ 9.3.5)฀ in฀ the฀ osteoclast฀ basolateral฀ membrane฀ (Fig.฀ 10.2).฀
Nevertheless,฀the฀pH฀eventually฀falls฀below฀levels฀compatible฀with฀mitochondrial฀func-
tion,฀perhaps฀explaining฀the฀osteoclast’s฀short฀half-life.

Fig. 10.4 (continued)฀ tide฀bond฀cleavage.฀In฀the฀cysteine฀(and฀also฀serine฀and฀threonine)฀proteases,฀

the฀nucleophile฀is฀the฀protease฀type฀amino฀acid฀(in฀this฀case฀cysteine)฀which฀forms฀a฀covalent฀bond฀
with฀the฀carbon฀atom฀of฀the฀bond฀to฀be฀cleaved฀(covalent฀catalysis)฀in฀contrast฀to฀the฀metalloprotei-
nases฀and฀aspartic฀proteases฀which฀use฀an฀activated฀water฀molecule฀to฀attack฀the฀carbon฀atom฀to฀be฀
cleaved฀(noncovalent฀catalysis).฀In฀covalent฀catalysis,฀a฀nearby฀histidine฀residue฀normally฀functions฀
as฀a฀base฀to฀activate฀the฀mechanism,฀whereas฀in฀noncovalent฀catalysis,฀the฀protease฀type฀serves฀as฀an฀
acid฀and฀base,฀with฀an฀ancillary฀histidine฀(aspartate฀proteases)฀or฀aspartate฀or฀glutamate฀residue฀acting฀
as฀the฀nucleophile฀(Fig.฀8.2b)฀(Modiied฀from฀Fig.฀9.18฀in฀Berg.,฀et฀al.,฀Biochemistry,฀5th฀Ed.฀2002,฀
W.H.฀Freeman฀&฀Co.,฀New฀York)
160 10 Bone Remodeling and Calcium Metabolism

Stress-induced฀bony฀microcracks฀attract฀circulating฀white฀blood฀cells฀(monocytes)฀that฀
differentiate฀into฀osteoclasts฀possessing฀an฀acidic฀compartment฀in฀contact฀with฀bone.฀
This฀extracellular฀compartment฀is฀separated฀from฀the฀cytosol฀by฀a฀rufled฀membrane฀
attached฀ by฀ an฀ integrin฀ to฀ osteopontin฀ on฀ the฀ bone฀ surface.฀ Protons฀ are฀ produced฀ by฀
carbonic฀anhydrase฀acting฀on฀CO2฀and฀H2O฀from฀respiration฀and฀exchanged฀through฀an฀
ATP-dependent฀proton฀transporter฀for฀Na+฀ions฀from฀the฀compartment.฀Bicarbonate฀ions฀
in฀the฀cytosol฀are฀exchanged฀for฀periosteal฀chloride฀ions฀that฀pass฀on฀to฀the฀acidic฀com-
partment฀through฀chloride฀channel฀proteins฀in฀the฀rufled฀membrane.฀HCl-demineralized฀
collagen฀ibers฀are฀denatured฀and฀digested฀by฀cathepsin฀K,฀an฀acid-activated,฀papain-
related฀gelatinase.฀Calcium฀ions,฀collagen฀peptides,฀and฀TGF-b฀from฀bone฀are฀taken฀up฀
with฀cathepsin฀K-activated฀acid฀phosphatase฀into฀acid฀vesicles฀and฀exocytosed฀into฀the฀
periosteum฀ or฀ bone฀ marrow.฀ Dihydrogen฀ phosphate฀ ions฀ enter฀ from฀ bone฀ through฀ a฀
proton-dependent฀phosphate฀transporter฀and฀pass฀through฀the฀cytosol฀separately฀to฀the฀
periosteum฀or฀bone฀marrow.฀Once฀a฀microcrack฀is฀removed,฀TGF-b฀enhances฀osteoblast฀
differentiation฀and฀a฀stronger฀layer฀of฀bone฀forms฀in฀response฀to฀the฀stress.

10.2.1.
Osteoclast Differentiation

Osteoblasts฀ around฀ bony฀ microcracks฀ are฀ induced฀ to฀ express฀ two฀ cytokines:฀ monocyte฀
Colony฀ Stimulating฀ Factor฀ (mCSF)฀ which฀ is฀ secreted฀ and฀ Osteoclast฀ Differentiating฀
Factor฀ (ODF)฀ which฀ is฀ mainly฀ on฀ the฀ cell฀ surface.฀ The฀ mCSF฀ stimulates฀ microcrack-
adherent฀monocytes฀to฀proliferate฀and฀fuse฀into฀large฀multinucleated฀cells฀(preosteoclasts)฀
that฀express฀Osteoclast฀Differentiation฀and฀Activation฀Receptor฀(ODAR).
When฀ODAR฀attaches฀to฀the฀osteoblast฀surface-bound฀ODF,฀the฀receptor/ligand฀complex฀
activates฀a฀membrane-associated฀tyrosine-protein฀kinase฀to฀induce฀synthesis฀of฀the฀rufled฀
membrane.฀A฀tyrosine฀residue฀on฀ClIC-5฀(Sect.฀10.1.4)฀is฀phosphorylated฀by฀an฀activated฀
protein฀kinase,฀called฀c-src,฀the฀normal฀cytosolic฀homologue฀of฀a฀viral฀tyrosine฀kinase฀which฀
causes฀ a฀ sarcoma฀ (transforms฀ ibroblasts฀ into฀ cancer฀ cells).฀ The฀ phosphorylated฀ ClIC-5฀
interacts฀with฀phospholipids,฀a฀chloride฀channel฀protein฀(ClC-7)฀and฀two฀transporter฀pro-
teins,฀ the฀ ATPase฀ proton฀ transporter,฀ and฀ the฀ proton-dependent฀ phosphate฀ transporter.฀
Mutations฀that฀suppress฀c-src฀or฀prevent฀expression฀or฀functioning฀of฀ClC-7฀or฀ClIC-5฀in฀
mice฀ or฀ humans฀ prevent฀ osteoclast฀ development฀ and฀ cause฀ overly฀ dense,฀ brittle฀ bones฀
(osteopetrosis).
Bone฀resorption฀is฀a฀physiological฀process฀prevented฀by฀the฀osteoblast฀secreting฀osteo-
clast฀inhibition฀factor฀(OCIF),฀more฀commonly฀called฀osteoprotegerin฀(OPG).฀OCIF฀is฀a฀
nonmembrane-bound฀decoy฀receptor฀that฀resembles฀ODAR฀and฀prevents฀ODF฀from฀bind-
ing฀to฀ODAR฀and฀therefore฀causing฀preosteoclasts฀to฀remain฀undifferentiated฀(Fig.฀10.5).฀
Bone฀resorption฀is฀related฀to฀the฀body’s฀response฀to฀injury฀or฀infection,฀a฀complex฀series฀of฀
events฀called฀inlammation.
10.2.2. Osteoclasts and Inflammation 161

Osteoblast OCIF (secreted)

ODF (cell-surface
Inactive ODF/ bound)
OCIF complex sODF sODF(secreted)
OCIF
ODAR (bound)
mCSF (secreted)
Monocyte CSF
(mCSF) mCSF Receptor
Inactive OCIF/ (cell-surface bound)
ODAR
sODF complex
Short life span (1.3 days)
Increased by inflammatory
Differentiation stimuli and glucocorticoids
and Activation (stress induced hormones)
Osteoclast
precursor Mature osteoclast
with ruffled membrane

Fig. 10.5฀ Osteoclast฀differentiation฀factors.฀Osteoblasts฀make฀monocyte฀colony฀stimulating฀factor฀

(mCSF)฀ that฀ induces฀ bone฀ adherent฀ monocytes฀ carrying฀ the฀ corresponding฀ receptor฀ (mCSF฀
Receptor)฀ to฀ fuse฀ into฀ osteoclast฀ precursors฀ (preosteoclasts).฀ Preosteoclasts฀ develop฀ within฀ the฀
periosteum฀and฀are฀detectable฀by฀their฀expression฀of฀the฀osteoclast฀differentiation฀and฀activation฀
receptor฀(ODAR).฀Osteoblasts฀also฀make฀cell-membrane฀bound฀and฀soluble฀osteoclast฀differentia-
tion฀factors฀(ODF฀and฀sODF)฀that฀react฀with฀ODAR฀to฀cause฀preosteoclast฀differentiation.฀Finally,฀
osteoblasts฀make฀osteoclast฀inhibition฀factor฀(OCIF),฀also฀called฀osteoprotegerin,฀which฀acts฀as฀an฀
ODF฀decoy฀receptor฀and฀prevents฀ODF฀or฀sODF฀reacting฀with฀ODAR.฀ODAR฀is฀commonly฀referred฀
to฀as฀RANK฀and฀ODF฀as฀the฀RANK฀ligand฀(RANKL)฀in฀the฀literature฀(see฀text)

10.2.2.
Osteoclasts and Inflammation

Inlammation฀is฀induced฀by฀the฀release฀of฀proinlammatory฀mediators฀from฀the฀cytosol฀of฀
damaged฀or฀infected฀cells฀(Sect.฀13.2.2).฀One฀of฀the฀irst฀identiied฀cytokines฀was฀an฀osteo-
clast฀activation฀factor฀later฀found฀to฀induce฀a฀multitude฀of฀other฀proinlammatory฀events,฀
InterLeukin-1฀(IL-1).฀A฀second฀was฀Tumor฀Necrosis฀Factor฀(TNF),฀more฀formally฀known฀
as฀ tumor฀ necrosis฀ factor-alpha,฀ TNF-a฀ (Sect.฀ 13.2.2).฀ The฀ binding฀ of฀ these฀ proteins฀ to฀
receptors฀on฀adjacent฀cells฀activates฀Nuclear฀Factor฀kappa฀B฀(NFkB),฀a฀protein฀in฀the฀leu-
kocyte฀cytosol.฀The฀ligand-bound฀receptor฀indirectly฀phosphorylates฀an฀NFkB฀partner฀pro-
tein฀ (Inhibitor฀ of฀ NFkB,฀ IkB)฀ in฀ the฀ cytosol.฀ The฀ phosphorylated฀ IkB฀ is฀ targeted฀ for฀
destruction฀and฀its฀loss฀exposes฀a฀nuclear฀localization฀sequence฀on฀the฀NFkB฀protein฀which฀
can฀now฀enter฀the฀nucleus฀where฀it฀induces฀the฀expression฀of฀important฀proteins,฀depending฀
on฀the฀type฀of฀cell฀that฀is฀activated.฀ODAR฀is฀a฀Receptor฀Activator฀of฀Nuclear฀Factor฀kappa฀
B฀(RANK),฀one฀of฀a฀large฀family฀of฀proinlammatory฀ligand฀receptors฀on฀leukocytes.
162 10 Bone Remodeling and Calcium Metabolism

When฀ the฀ ODF/ODAR฀ complex฀ activates฀ c-src฀ to฀ form฀ a฀ rufled฀ membrane,฀ it฀ also฀
activates฀NFkB฀to฀activate฀the฀transcription฀factors฀that฀induce฀cathepsin฀K฀and฀acid฀phos-
phatase฀expression.฀Because฀ODF฀binds฀to฀ODAR/RANK,฀it฀is฀also฀known฀as฀the฀RANK฀
ligand฀ (RANKL).฀ OCIF฀ (OPG),฀ the฀ ODF฀ decoy฀ receptor,฀ is฀ RANK฀ without฀ its฀ trans-
membrane฀ and฀ cytosolic฀ domains.฀ Thus฀ OCIF฀ =฀ OPG฀ and฀ OPG฀ ligand฀ (OPGL)฀ =฀
RANKL฀ =฀ ODF.฀ ODF,฀ ODAR,฀ and฀ OCIF฀ are฀ related฀ to฀ TNFa฀ or฀ its฀ receptor,฀ TNFa฀
family฀molecules฀(Table฀10.1).
Osteoprotegerin฀ is฀ not฀ restricted฀ to฀ osteoclasts;฀ it฀ is฀ produced฀ by฀ many฀ white฀ blood฀
cells.฀During฀lymphocyte฀development฀in฀the฀central฀cavity฀of฀long฀bones฀(Sect.฀11.1.1),฀
OPG฀ prevents฀ OPGL฀ binding฀ to฀ RANK฀ on฀ bone-marrow฀ lymphocytes฀ responsible฀ for฀
antibody฀immunity.฀Because฀NFkB฀is฀not฀activated,฀the฀lymphocytes฀proliferate฀and฀enter฀
the฀systemic฀circulation฀where฀they฀are฀stimulated฀by฀foreign฀antigens฀to฀make฀antibodies.฀
In฀the฀absence฀of฀OPG,฀OPGL฀binds฀to฀RANK฀and฀the฀bone-marrow฀lymphocytes฀undergo฀
apoptosis฀(Sect.฀13.4.2).฀Thus,฀when฀bone-marrow฀lymphocytes฀recognize฀a฀self-antigen,฀
OPG฀secretion฀is฀inhibited฀and฀they฀are฀eliminated.
Injury,฀infection,฀stress-induced฀hormones฀(glucocorticoids),฀parathyroid฀hormone฀(Sect.฀
10.3.1),฀and฀increased฀pressure฀on฀a฀bone฀all฀decrease฀osteoprotegerin฀(OCIF)฀production฀by฀
osteoblasts,฀causing฀greater฀differentiation฀of฀osteoclasts,฀more฀bone฀remodeling,฀and฀more฀

Table 10.1฀ Tumor฀ necrosis฀ factor฀ (TNF)a฀ family฀ molecules฀ in฀ osteoclast฀ development฀ and฀
activation
Type Acronym Synonym
Ligand฀on฀osteoblast฀cell฀surface฀or฀secreted
ODFa Osteoclast฀differentiation฀factor
RANKLb RANK฀ligand
OPGL Osteoprotegerin฀ligand
TRANCE TNF-related฀activation-induced฀
cytokine
SOFA Stromal฀osteoclast-forming฀activity
TNFSF-11 TNF฀superfamily฀11
Osteoclast฀receptor
ODARa Osteoclast฀differentiation฀and฀
activation฀receptor
RANKb Receptor฀activator฀of฀NFkB
TNFRSF-11A TNF฀superfamily฀receptor฀11A
Decoy฀receptor฀secreted฀by฀osteoblast
OCIFa Osteoclastogenesis฀inhibitory฀factor
OPGb Osteoprotegerin
TR-1 TNF฀receptor-like฀molecule฀1
FDCR-1 Follicular฀dendritic฀receptor฀1
TNFRSF-11B TNF฀superfamily฀receptor฀11B
a
Functional฀abbreviation฀and฀name฀used฀in฀this฀book
b
General฀protein฀family฀name฀and฀standard฀name฀since฀2000
(Slightly฀modiied฀from฀“Proposed฀standard฀nomenclature฀for฀new฀tumor฀necrosis฀factor฀family฀
members฀involved฀in฀the฀regulation฀of฀bone฀resorption.”฀Journal฀of฀Bone฀and฀Mineral฀Research฀
15(12):2293–2296,฀2000)
10.2.3. Osteoporosis: Major Causes and Therapies 163

Table 10 2฀ Control฀of฀bone฀metabolism
Causing฀resorption Causing฀calciication
Glucocorticoids฀(stress฀hormones) Growth฀hormones
Inlammation Estrogens฀and฀androgens
Pressure฀on฀bone Tension฀on฀bone
Acidosis฀(cardiac฀or฀lung฀insuficiency)

demineralization฀(Table฀10.2).฀Conversely,฀growth,฀anabolic฀and฀sex฀hormones,฀and฀tension฀
on฀the฀bone,฀all฀increase฀OCIF฀production฀by฀osteoblasts.฀Few฀osteoclasts฀develop฀and฀bone฀
resorption฀is฀decreased.฀Cytokines฀associated฀with฀the฀repair฀of฀tissues฀after฀injury฀or฀infec-
tion,฀speciically฀IL-4฀and฀IL13,฀inhibit฀mechanisms฀that฀stimulate฀osteoclast฀differentiation฀
and฀bone฀resorption.฀They฀decrease฀ODAR฀synthesis฀in฀osteoclasts,฀and฀decrease฀ODF฀syn-
thesis฀ and฀ increase฀ OCIF฀ synthesis฀ in฀ osteoblasts.฀ Table฀10.2฀ lists฀ the฀ environment,฀ hor-
mones,฀ and฀ inlammatory฀ mediators฀ that฀ inluence฀ osteoblast฀ ODF฀ and฀ OCIF฀ (OPG)฀
production฀and฀therefore฀how฀readily฀bone฀resorption฀can฀occur.

10.2.3.
Osteoporosis: Major Causes and Therapies

Just฀as฀inlammation฀enhances฀bone฀resorption฀by฀increasing฀ODF฀production฀locally,฀the฀
cessation฀ of฀ estrogen฀ production฀ at฀ menopause฀ causes฀ osteoporosis฀ systemically฀ (ODF฀
production฀ increases).฀ Menopausal฀ women฀ therefore฀ suffer฀ a฀ net฀ loss฀ of฀ bone฀ because฀
ODAR฀(RANK)฀is฀activated฀by฀ODF฀(RANKL)฀in฀the฀absence฀of฀a฀compensating฀increase฀
in฀OCIF฀(OPG)฀production.฀The฀osteoclasts฀demineralize฀bone฀faster฀than฀the฀osteoblasts฀
deposit฀it.
Mice฀ overexpressing฀ acid฀ phosphatase฀ (Acp5)฀ develop฀ osteoporosis฀ whereas฀ mice฀
lacking฀this฀enzyme฀have฀decreased฀bone฀resorption฀and฀develop฀mild฀osteopetrosis฀(overly฀
dense,฀brittle฀bones).฀Fluoride฀at฀0.12฀mM฀(2.3฀ppm)฀inhibits฀Acp5b฀and฀stimulates฀osteo-
blast฀bone฀deposition฀in฀vitro,฀but฀luoride฀therapy฀does฀not฀inhibit฀human฀osteoporosis฀
(Sect.฀16.2.2).
In฀ 1962,฀ pyrophosphate฀ was฀ found฀ to฀ inhibit฀ the฀ spontaneous฀ calciication฀ of฀ urine.฀
Subsequently,฀the฀degradation฀of฀pyrophosphate฀to฀orthophosphate฀by฀alkaline฀phosphatase฀
(TNAP)฀was฀found฀to฀play฀a฀key฀role฀in฀mineralizing฀collagen฀ibers฀during฀the฀synthesis฀
of฀bone฀(Sect.฀9.3.5).฀Substituting฀a฀carbon฀atom฀for฀the฀central฀oxygen฀atom฀in฀pyrophos-
phate฀(a฀bisphosphonate)฀was฀found฀to฀prevent฀cleavage฀by฀alkaline฀phosphatase.
Because฀bisphosphonates฀remain฀tightly฀bound฀to฀the฀bone฀surface฀and฀cannot฀be฀hydro-
lyzed,฀they฀also฀retard฀demineralization.฀Modiications฀of฀the฀side฀chains฀attached฀to฀the฀
central฀carbon฀atom฀(Fig.฀10.6)฀has฀yielded฀compounds฀such฀as฀alendronate฀(Fosamax),฀
164 10 Bone Remodeling and Calcium Metabolism

Fig. 10.6฀ Structure฀of฀a฀ OH OH OH R1 OH

bisphosphonate฀showing฀its฀
relationship฀to฀pyrophos-
phate.฀Alendronate฀
O=P O P=O O=P C P=O
(Fosamax)฀is฀a฀commonly฀
used฀bisphosphonate฀฀
(R1฀is฀OH฀and฀R2฀is฀
CH2–CH2–CH2–NH2) OH OH OH R2 OH

Pyrophosphate Alendronate

which฀inhibits฀resorption฀at฀doses฀that฀minimally฀block฀mineralization.฀Side฀effects฀include฀
irritation,฀ inlammation,฀ or฀ ulceration฀ of฀ the฀ esophagus฀ and฀ prevent฀ some฀ patients฀ from฀
using฀the฀drug.฀More฀recently,฀the฀longer฀and฀more฀widespread฀use฀of฀bisphosphonates฀has฀
been฀ linked฀ to฀ osteonecrosis฀ of฀ the฀ jaw,฀ especially฀ if฀ used฀ along฀ with฀ some฀ anticancer฀
drugs.฀Symptoms฀include฀jaw฀pain,฀numbness,฀exposed฀oral฀bones,฀loss฀of฀teeth,฀and฀oral฀
infection.฀Bisphosphonates฀are฀the฀current฀drugs฀of฀choice฀to฀control฀osteoporosis,฀but฀have฀
no฀effect฀on฀pathological฀bone฀loss฀which฀is฀mediated฀by฀leukocytes฀(Sect.฀13.3.1).
Cathepsin฀K฀inhibitors฀would฀prevent฀bone฀loss฀without฀inhibiting฀mineralization.฀The฀
compound฀most฀tested,฀Balicatib,฀forms฀a฀covalent฀bond฀with฀the฀cysteine฀thiol฀group฀at฀
the฀ catalytic฀ center฀ of฀ cathepsin฀ K.฀ Unfortunately,฀ Balicatib฀ tends฀ to฀ concentrate฀ in฀ all฀
lysosomes,฀so฀that,฀over฀time,฀amounts฀in฀the฀body฀will฀increase฀and฀inhibit฀the฀catalytic฀
thiol฀group฀of฀other฀cathepsins,฀notably฀cathepsin฀S฀which฀hydrolyzes฀proteins฀to฀peptides฀
for฀antibody฀synthesis฀(antigen฀presentation).฀Nevertheless,฀no฀adverse฀side฀effects฀were฀
reported฀in฀140฀postmenopausal฀women฀receiving฀once-a-day฀treatment฀with฀Balicatib฀for฀
12฀months,฀but฀the฀possibility฀of฀increased฀infections฀after฀some฀years฀of฀taking฀Balicatib฀
is฀a฀potential฀therapeutic฀problem.

Microcracks฀induce฀osteoblasts฀to฀secrete฀monocyte฀colony฀stimulating฀factor฀(mCSF)฀
and฀express฀cell฀surface฀osteoclast฀differentiating฀factor฀(ODF).฀The฀mCSF฀stimulates฀
microcrack฀adherent฀monocytes฀to฀proliferate฀and฀fuse฀into฀preosteoclasts฀expressing฀
osteoclast฀differentiation฀and฀activation฀receptor฀(ODAR).฀The฀ODAR/ODF฀receptor/
ligand฀complex:฀(a)฀activates฀a฀membrane-associated฀tyrosine-protein฀kinase฀to฀phos-
phorylate฀a฀chloride฀transporter฀that฀assembles฀the฀rufled฀membrane;฀and฀b)฀activates฀
nuclear฀ transcription฀ factor฀ (NF-kB)฀ to฀ induce฀ cathepsin฀ K฀ and฀ acid฀ phosphatase.฀
Osteoblasts฀ also฀ secrete฀ osteoclast฀ inhibition฀ factor฀ (OCIF),฀ a฀ nonmembrane-bound฀
decoy฀receptor฀that฀resembles฀ODAR,฀but฀prevents฀ODF฀from฀binding฀to฀it.฀Environment,฀
hormones,฀ and฀ inlammatory฀ mediators฀ inluence฀ ODF฀ and฀ OCIF฀ production,฀ and฀
therefore฀osteoclast-mediated฀bone฀resorption.฀Estrogen฀inhibits฀ODF฀production,฀so฀
that฀osteoclasts฀are฀activated฀during฀menopause,฀when฀estrogen฀production฀falls.฀Drugs฀
to฀treat฀menopausal฀osteoporosis฀include฀derivatives฀of฀pyrophosphate฀and฀cathepsin฀
K.฀Mutations฀of฀acid฀phosphatase฀cause฀thicker฀bones฀(osteopetrosis).฀Fluoride฀inhibits฀
acid฀phosphatase฀but฀not฀osteoporosis.
10.3.1. Calcium Metabolism, Parathyroid Hormone, and Calcitriol 165

10.3.1.
Calcium Metabolism, Parathyroid Hormone, and Calcitriol

Calcium฀is฀the฀most฀abundant฀mineral฀in฀the฀human฀body.฀About฀99%฀is฀in฀the฀bones฀and฀
teeth฀where฀it฀plays฀a฀structural฀role฀and฀the฀remaining฀1%฀is฀in฀the฀body฀tissues฀and฀luids฀
where฀ it฀ is฀ essential฀ for฀ muscle฀ contraction,฀ nerve฀ impulse฀ transmission,฀ and฀ cell฀
metabolism.
Intracellularly,฀calcium฀ions฀are฀retained฀in฀the฀endoplasmic฀reticulum,฀or฀its฀specialized฀
form฀ unique฀ to฀ muscle฀ cells,฀ the฀ sarcoplasmic฀ reticulum.฀ In฀ the฀ endoplasmic฀ reticulum,฀
calcium฀ions฀are฀free,฀chelated฀to฀protein,฀or฀chelated฀to฀phosphatidylserine.฀Free฀calcium฀
ions฀are฀released฀through฀Transient฀Receptor฀Potential฀(TRP)฀calcium฀ion฀channels฀to฀the฀
cytosol฀where฀they฀bind฀and฀stimulate฀second฀messenger฀synthesis.฀Related฀channels฀are฀
involved฀in฀transporting฀calcium฀ions฀from฀the฀small฀intestine฀into฀the฀blood฀plasma,฀into฀
or฀out฀of฀bone฀through฀osteoblasts฀and฀osteoclasts,฀and฀out฀of฀forming฀urine฀through฀the฀
kidney฀collecting฀tubule฀endothelial฀cells฀(see฀Sect.฀10.4.1).฀Calcium฀ions฀in฀the฀cytosol฀
therefore฀mediate฀numerous฀physiological฀processes฀including฀nerve฀and฀muscle฀activity.
Extracellularly,฀ calcium฀ ions฀ circulate฀ in฀ the฀ blood฀ plasma฀ and฀ interstitial฀ luid฀
(Sect.฀3.3.1).฀In฀blood฀plasma,฀calcium฀ions฀are฀chelated฀to฀albumin฀and฀citrate.฀Albumin฀
(mol.฀wt.฀66,700฀kDa)฀is฀present฀at฀50–60฀mg/mL฀in฀plasma,฀corresponding฀to฀0.9฀m฀mol/L.฀
Although฀plasma฀albumin฀has฀many฀different฀sites฀that฀can฀chelate฀calcium฀ions฀in฀vitro,฀
only฀one฀site฀binds฀to฀calcium฀ions฀at฀physiological฀albumin฀concentrations฀and฀pH.฀Thus,฀
albumin฀binds฀0.9฀mmol/L฀of฀free฀plasma฀Ca2+.฀In฀addition,฀citrate฀(Fig.฀10.7),฀a฀tricarboxy-
lic฀acid฀that฀the฀liver฀secretes฀into฀plasma,฀chelates฀a฀free฀calcium฀ion฀to฀two฀of฀its฀three฀
carboxyl฀groups,฀replacing฀two฀Na+฀ions.฀Citrate฀has฀a฀molar฀concentration฀of฀0.08฀mM฀in฀
venous฀blood฀and฀therefore฀binds฀to฀an฀equivalent฀concentration฀of฀free฀calcium.฀Because฀
the฀total฀calcium฀ion฀concentration฀of฀venous฀blood฀is฀1.14฀mmol/L฀(range฀±0.2),฀and฀the฀
free฀calcium฀ion฀concentration฀is฀0.1฀mM,฀it฀appears฀that฀0.15฀mM฀of฀the฀plasma฀calcium฀
ion฀concentration฀is฀bound฀to฀other฀plasma฀components.
The฀free฀calcium฀ions฀in฀blood฀and฀extracellular฀luid฀are฀critical฀for฀building฀and฀main-
taining฀an฀adequate฀bone฀mass,฀and฀also฀for฀preventing฀excessive฀calciication.฀The฀sensor฀
that฀regulates฀the฀free฀calcium฀ion฀concentration฀of฀plasma฀is฀within฀the฀parathyroid฀glands,฀
where฀it฀controls฀the฀secretion฀of฀parathormone฀(PTH).฀This฀84฀amino฀acid฀peptide฀is฀split฀
from฀a฀large,฀precursor฀protein฀and฀retained฀in฀secretory฀vesicles.฀If฀the฀concentration฀of฀
free฀calcium฀ions฀drops฀below฀a฀critical฀level฀in฀blood฀plasma,฀the฀gland฀is฀activated฀to฀
secrete฀PTH฀into฀the฀bloodstream.
Osteoblasts฀and฀kidney฀cells฀respond฀to฀PTH฀by฀possessing฀a฀single฀receptor฀known฀as฀
the฀Parathyroid฀hormone/Parathyroid฀hormone-Related฀protein฀receptor฀(PPR).฀The฀PTH-
mediated฀ activation฀ of฀ the฀ osteoblast฀ PPR฀ receptor฀ reduces฀ OCIF฀ (osteoprotegerin)฀

Fig. 10.7฀ Structure฀of฀sodium฀฀

and฀calcium฀citrate.฀(a)฀Sodium฀฀
citrate.฀(b)฀Sodium฀calcium฀citrate
166 10 Bone Remodeling and Calcium Metabolism

secretion฀and฀increases฀ODF฀secretion฀(RANKL),฀thus฀activating฀osteoclasts฀(Sect.฀10.2.2)฀
which฀do฀not฀possess฀PPR฀receptors.฀The฀greater฀osteoclast฀activity฀increases฀plasma฀Ca2+฀
concentration฀by฀dissolving฀bone฀(Fig.฀10.8).

Fig. 10.8฀ Illustration฀of฀the฀kidney฀showing฀the฀glomerulus,฀the฀proximal฀and฀distal฀collecting฀ducts฀

affected฀by฀parathyroid฀hormone฀and฀calcitriol.฀The฀nephron฀in฀the฀kidney฀glomerulus฀(left)฀con-
sists฀of฀glomeruli฀(detailed฀structure฀shown฀in฀the฀inset฀on฀left)฀and฀collecting฀tubes.฀Parathyroid฀
hormone฀inhibits฀phosphate฀reabsorption฀to฀blood฀plasma฀through฀the฀proximal฀convoluted฀tubules฀
but฀enhances฀that฀of฀calcium฀through฀the฀distal฀tubules฀(Modiied฀from฀Fig.฀20.4฀in฀Hole’s฀Human฀
Anatomy฀ &฀ Physiology,฀ 7th฀ Ed.,฀ Edited฀ by฀ David฀ Shier฀ et฀ al.,฀ 1996:฀ W.฀ C.฀ Brown฀ Publishers,฀
Times-Mirror฀Higher฀Education฀Group฀Inc.,฀2460฀Kerper฀Blvd.,฀Dubuque,฀IA฀52001:฀Copyrighted฀
2008฀by฀McGraw-Hill,฀2฀Penn฀Plaza,฀New฀York,฀NY฀10121–2298.฀The฀inset฀on฀upper฀left฀corner฀
of฀igure฀is฀part฀of฀Fig.฀19-55฀in฀The฀Molecular฀Biology฀of฀the฀Cell.฀B.฀Alberts฀et฀al.,฀4th฀Ed.฀2002.฀
Garland฀Science,฀Taylor฀&฀Francis฀Group,฀New฀York)
10.3.1. Calcium Metabolism, Parathyroid Hormone, and Calcitriol 167

Parathyroid฀hormone-mediated฀PPR฀receptor฀activation฀of฀the฀kidney฀has฀three฀effects฀
(Fig.฀ 10.8).฀ In฀ proximal฀ tubular฀ cells,฀ PTH฀ prevents฀ phosphate฀ reabsorption฀ from฀ the฀
glomerular฀iltrate฀(increasing฀phosphate฀excretion฀in฀urine),฀and฀stimulates฀calcitriol฀pro-
duction฀to฀activate฀calcium฀transport฀in฀intestinal฀epithelial฀cells,฀osteoblasts฀and฀osteo-
clasts฀ (Fig.฀ 10.9).฀ The฀ synthesis฀ and฀ mode฀ of฀ action฀ of฀ calcitriol฀ are฀ discussed฀ in฀ Sect.฀
10.4.1.฀In฀distal฀tubular฀cells,฀parathyroid฀hormone฀activates฀Ca2+฀ion฀reabsorption฀from฀
the฀glomerular฀iltrate฀(decreasing฀calcium฀excretion฀in฀the฀urine).฀PTH฀therefore฀acts฀on฀
the฀kidney฀to฀decrease฀the฀blood฀plasma฀phosphate฀concentration,฀and฀increase฀both฀plasma฀
calcium฀and฀calcitriol฀concentrations.฀The฀PPR฀receptor฀is฀a฀Class฀B฀G฀protein-coupled฀
receptor฀(GPCR).฀An฀activating฀ligand฀such฀as฀PTH฀(an฀agonist)฀binds฀to฀the฀GCPR฀out-
side฀the฀cell฀and฀induces฀changes฀in฀the฀receptor฀that฀activate฀a฀heterotrimeric฀G฀protein฀
(Gs)฀composed฀of฀α-,฀β-฀and฀γ-polypeptide฀subunits฀inside฀the฀cell.฀The฀α-subunit฀(Gs-α,฀
often฀ written฀ Gαs)฀ possesses฀ a฀ non-covalently฀ bound฀ GDP฀ molecule฀ and฀ is฀ tethered฀ by฀
covalently฀attached฀fatty฀acids฀to฀the฀cytoplasmic฀side฀of฀the฀plasma฀membrane฀along฀with฀
the฀β-฀and฀γ-chains.฀The฀changes฀transmitted฀across฀the฀membrane฀by฀the฀agonist-occupied฀
GPCR฀cause฀the฀Gs-α฀chain฀to฀exchange฀its฀bound฀GDP฀for฀GTP฀in฀the฀cytosol.฀The฀Gs-α฀
chain฀containing฀GTP฀is฀activated฀and฀it฀spontaneously฀dissociates฀from฀the฀Gs฀trimer฀and฀
stimulates฀ adenylcyclase฀ to฀ catalyze฀ the฀ formation฀ of฀ the฀ second฀ messenger฀ molecule,฀
cyclic฀AMP฀(cAMP).฀cAMP฀in฀turn฀activates฀the฀cAMP-dependent฀enzyme,฀protein฀kinase฀
A฀(PKA).฀In฀the฀kidney,฀PKA฀is฀the฀effector฀enzyme฀that฀phosphorylates฀the฀Pi฀transporter฀
and฀stops฀Pi฀reabsorption฀into฀blood฀plasma฀across฀proximal฀tubular฀cells.฀It฀also฀phospho-
rylates฀the฀Ca2+฀transporter฀which฀allows฀Ca2+฀reabsorption฀into฀blood฀plasma฀from฀distal฀
tubular฀ cells฀ (see฀ also฀ Sect.฀ 10.5.1).฀ In฀ osteoblasts,฀ PKA฀ reduces฀ OCIF฀ secretion฀ and฀

Low
plasma
calcium

Secretion of 3 Stimulates Kidney

PTH 1,25 dihydroxy-
cholecalciferol
4
Bone Calcitriol
1 (activates osteoclasts Bone
by reducing OCIF and
Kidney secretion) intestine
increases (induces Ca
PO4 excretion carrier protein)
(prevents
reabsorption)
and more bone
dissolves
Ca from bone Ca from diet
2 & decreases
Ca2+ excretion
Increased plasma
calcium

Fig. 10.9฀ How฀parathormone฀raises฀the฀free฀calcium฀ion฀concentration฀of฀blood฀plasma.฀PTH฀para-

thormone.฀Effects฀of฀PTH฀are฀shown฀by฀arrows฀from฀left฀to฀right฀and฀down.฀Effects฀of฀calcitriol฀are฀
shown฀by฀arrows฀running฀from฀right฀to฀left฀and฀down
168 10 Bone Remodeling and Calcium Metabolism

increases฀ODF฀secretion฀to฀activate฀osteoclasts.฀The฀Gs-α฀chain฀slowly฀auto-hydrolyses฀its฀
bound฀GTP,฀shutting฀off฀adenylyl฀cyclase฀activation฀and฀promoting฀reformation฀of฀the฀Gs฀
heterotrimer฀for฀another฀round฀of฀signaling.

Calcium฀ions฀are฀mostly฀present฀in฀bones฀or฀chelated฀to฀biological฀molecules.฀In฀blood฀
plasma,฀only฀1%฀of฀the฀calcium฀ions฀present฀are฀unbound;฀78%฀is฀bound฀to฀albumin,฀
8%฀to฀citrate,฀and฀13%฀to฀other฀plasma฀proteins.฀The฀free฀calcium฀ions฀are฀prevented฀
from฀precipitating฀by฀plasma฀pyrophosphate.฀Calcium฀ions฀are฀also฀stored฀in฀the฀endo-
plasmic฀reticulum฀(ER),฀mostly฀chelated฀to฀ER-resident฀proteins฀and฀phosphatidylser-
ine.฀Free฀calcium฀ions฀may฀be฀released฀through฀transient฀receptor฀potential฀channels฀to฀
the฀cytosol฀where฀it฀activates฀numerous฀physiological฀processes.฀If฀the฀free฀calcium฀ion฀
concentration฀ of฀ blood฀ plasma฀ falls,฀ parathyroid฀ hormone฀ (PTH)฀ is฀ secreted฀ by฀ the฀
parathyroid฀gland฀cells.฀PTH฀speeds฀up฀the฀transport฀of฀demineralized฀bone฀products฀by฀
osteoclasts.฀ In฀ the฀ kidney,฀ it฀ increases฀ the฀ excretion฀ of฀ phosphate฀ and฀ decreases฀ the฀
excretion฀of฀calcium.฀PTH฀also฀acts฀on฀kidney฀cells฀to฀make฀calcitriol฀from฀vitamin฀D,฀
which฀induces฀calcium฀transporters฀in฀the฀intestine฀and฀osteoclasts.฀PTH฀mediates฀these฀
effects฀by฀activating฀G-protein-coupled฀receptors฀in฀the฀kidney฀and฀osteoclasts.

10.4.1.
Vitamin D, Calcitriol, and Calbindins

Vitamin฀D฀is฀a฀fat฀soluble฀vitamin฀derived฀from฀cholesterol.฀In฀the฀human฀epidermis฀(skin),฀
sunlight฀ spontaneously฀ oxidizes฀ cholesterol฀ to฀ 7-dehydrocholesterol฀ (Fig.฀ 10.10a).฀ The฀
7-dehydrocholesterol฀leaks฀into฀the฀blood฀where฀it฀isomerizes฀to฀cholecalciferol฀(vitamin฀D3,฀
Fig.฀ 10.10b฀ and฀ c).฀ Cholecalciferol฀ is฀ enzymatically฀ hydroxylated฀ at฀ C25฀ in฀ the฀ liver฀
(25-cholecalciferol)฀ and฀ then฀ passes฀ to฀ the฀ kidney฀ where฀ another฀ enzyme฀ is฀ activated฀ by฀
parathyroid฀hormone฀to฀hydroxylate฀it฀at฀C1,฀forming฀calcitriol฀(Fig.฀10.10d).฀The฀kidney฀
hydroxylase฀is฀sensitive฀to฀feedback฀inhibition.฀As฀the฀amount฀of฀calcitriol฀increases,฀it฀binds฀
to฀the฀hydroxylase฀and฀alters฀the฀speciicity฀of฀the฀kidney฀enzyme.฀Additional฀25-cholecal-
ciferol฀is฀hydroxylated฀to฀24,25-dihydroxycholecalciferol฀(inactive฀calcitriol)฀instead฀of฀1,25฀
dihydroxycholecalciferol฀(calcitriol).฀Other฀vitamin฀D฀derivatives฀that฀can฀be฀converted฀to฀
calcitriol฀are฀obtained฀enzymatically฀from฀cholesterol฀in฀other฀vertebrates.฀The฀most฀com-
mon฀of฀these฀are฀vitamin฀D1฀(lamisterol)฀and฀D2฀(ergosterol)฀from฀cold-water฀ish฀such฀as฀
cod,฀where฀their฀presence฀keeps฀membranes฀luid฀at฀low฀body฀temperatures฀10–20°C.
Calcitriol฀behaves฀like฀a฀steroid฀hormone.฀It฀is฀transported฀to฀the฀nucleus฀of฀renal฀distal฀
tubule฀cells,฀intestinal฀epithelial฀cells,฀osteoclasts,฀and฀osteoblasts฀where฀it฀induces฀calbin-
dins,฀vitamin฀D-dependent฀calcium฀binding฀proteins.฀Calbindins฀mediate฀the฀intracellular฀
movement฀of฀calcium,฀from฀diet฀to฀blood,฀from฀blood฀to฀osteoid฀matrix,฀or฀from฀bone฀to฀
blood.฀There฀are฀two฀calbindin฀proteins,฀each฀encoded฀by฀separate฀genes,฀one฀of฀molecular฀
weight฀about฀9฀kDa฀(calbindin-D9K)฀and฀one฀of฀molecular฀weight฀28฀kDa฀(calbindin-D28K).฀
Each฀ binds฀ micromolar฀ amounts฀ of฀ calcium฀ and฀ each฀ disappears฀ from฀ animals฀ that฀ are฀
10.4.1. Vitamin D, Calcitriol, and Calbindins 169

24
a C10 24 26 b 25
23 25 HO
27
1
2 Previtamin D3
Spontaneous 1
3 5 6 isomerization
HO 7 CH3
4
C10
7-Dehydrocholesterol

Alternative hydroxylation at C24

Spontaneous in the kidney, inactive calcitriol.
isomerization (Occurs when PTH is low
or calcitriol is adequate)

c 24 d
24
25
OH

2 Hydroxylations Added in liver

C10 (C25 in liver first)

CH2
CH2 Added in
HO 1 kidney (stimulated
HO OH by PTH)
Vitamin D3
cholecalciferol Calcitriol
(1:25 dihydroxycholecalciferol)

Fig. 10.10฀ Calcitriol฀synthesis฀in฀the฀human฀body.฀(a)฀Numbering฀of฀the฀cholesterol฀carbon฀atoms฀at฀

each฀ end฀ of฀ the฀ molecule฀ and฀ position฀ of฀ oxidation฀ site.฀ (b,฀ c)฀ Isomerization฀ to฀ cholecalciferol.฀฀
(d)฀Calcitriol฀showing฀the฀hydroxylation฀site฀in฀the฀liver฀(top฀right)฀and฀kidney฀(bottom฀left)฀(Modiied฀
from฀Fig.฀27-32฀of฀Biochemistry.฀L.฀Stryer,฀4th฀Ed.฀1995.฀W.H.฀Freeman฀&฀Co.,฀New฀York)

deprived฀ of฀ vitamin฀ D.฀ Knockout฀ mice฀ can฀ mediate฀ the฀ necessary฀ calcium฀ transport฀
฀functions฀associated฀with฀dietary฀uptake,฀bone฀dissolution,฀and฀bone฀formation฀with฀cal-
bindin-D9K฀ alone;฀ calbindin-D28K฀ appears฀ important฀ for฀ maintaining฀ osteoblast฀ viability฀฀
(see฀legend฀to฀Fig.฀10.11).
Vitamin฀D-mediated฀transcellular฀calcium฀transport฀also฀involves฀entry฀and฀extrusion.฀
Calcium฀inlux฀from฀the฀intestine฀or฀bone฀is฀propelled฀by฀a฀steep฀electrochemical฀gradient฀
mediated฀ by฀ the฀ superfamily฀ of฀ Transient฀ Receptor฀ Potential฀ (TRP)฀ ion฀ channels฀ noted฀
above.฀TRP฀channel฀ion฀selectivity฀and฀mode฀of฀activation฀are฀extremely฀variable.฀Some฀
are฀activated฀or฀regulated฀by฀ligands฀฀such฀as฀amino฀acid฀amines฀or฀small฀peptides,฀others฀
by฀physical฀stimuli฀(e.g.,฀heat),฀and฀still฀others฀by฀as฀yet฀unknown฀mechanisms.฀All฀TRP฀
channels฀are฀Ca2+฀and฀Na+฀selective,฀but฀the฀selectivity฀for฀Ca2+฀versus฀Na+฀(Ca/Na฀ratio)฀
varies฀enormously฀from฀>100:1,฀to฀<0.05:1.฀This฀variability฀has฀contributed฀to฀a฀confusing฀
TRP฀channel฀nomenclature.
170 10 Bone Remodeling and Calcium Metabolism

Fig. 10.11฀ Diagram฀ of฀ vitamin-D฀ dependent฀ calcium฀ transport฀ in฀ intestinal฀ epithelial฀ cells.฀ Dietary฀
calcium฀ions฀(illed฀black฀circles)฀in฀the฀intestinal฀lumen฀enter฀the฀epithelial฀cells฀(enterocytes)฀through฀
a฀Transient฀Receptor฀Potential฀calcium฀channel฀family฀V฀subfamily฀6฀transporter฀protein฀(TRPV6,฀
pale฀blue฀ovals).฀Tight฀junctions฀between฀the฀enterocytes฀(black฀rectangles฀on฀each฀side฀of฀the฀entero-
cyte)฀prevent฀cell-free฀diffusion.฀A฀related฀calcium฀ion฀transporter฀(TRPV5)฀is฀used฀in฀osteoclasts฀and฀
kidney฀ (see฀ text).,฀ Osteoclasts฀ also฀ take฀ up฀ calcium฀ ions฀ using฀ Na+/Ca2+-exchanger-1฀ (NCX1).฀
Osteoblasts฀use฀NCX1฀and฀NCX3฀instead฀of฀TRPV฀to฀take฀up฀calcium฀ions฀from฀the฀blood฀vessel฀
periosteal฀stroma.฀Once฀inside฀a฀cell,฀Ca2+฀ions฀attach฀to฀calbindin,฀mostly฀calbindin-D9K,฀which฀dif-
fuses฀through฀the฀cytosol฀to฀the฀basal฀membrane฀side฀that฀contacts฀the฀underlying฀capillary฀stroma฀of฀
enterocytes฀and฀osteoclasts,฀or฀the฀osteoid฀matrix฀of฀osteoblasts.฀Calbindin-D9K-attached฀calcium฀ions฀
are฀extruded฀into฀the฀stromal฀matrix฀of฀enterocytes,฀osteoclasts,฀and฀osteoblasts฀by฀the฀ATP-dependent฀
Plasma฀Membrane฀Ca2+-ATPase฀1b฀(PMCA1b,฀pink฀rectangles).฀Calbindin-D9K฀(pale฀green฀circles)฀
predominates฀in฀enterocytes฀and฀osteoclasts,฀whereas฀calbindin-D28K฀predominates฀in฀osteoblasts฀and฀
may฀protect฀them฀and฀other฀noncalcium฀transporting฀cells฀expressing฀calbindin-D28K฀from฀apoptosis฀
(Sect.฀13.4.2).฀A฀deiciency฀of฀vitamin฀D฀prevents฀cells฀from฀expressing฀either฀calbindin-D9K฀or฀cal-
bindin-D28K฀ and฀ causes฀ rickets฀ in฀ children฀ or฀ osteomalacia฀ in฀ adults฀ (see฀ text)฀ (Modiied฀ from฀ an฀
enterocyte฀ diagram฀ from฀ Wikipedia:฀ http://en.wikipedia.org/wiki/Image:Alpha_Intercalated_Cell_
Cartoon.jpg)

In฀humans,฀TRP฀subfamily฀V฀member฀5฀(TRPV5,฀also฀called฀ECAC1฀or฀CaT2)฀is฀
expressed฀ in฀ osteoclasts฀ and฀ the฀ kidney฀ distal฀ convoluted฀ tubule.฀ TRPV5฀ is฀ closely฀
related฀to฀TRPV6฀(ECAC2฀or฀CaT1)฀expressed฀in฀the฀small฀intestine฀(Fig.฀10.11)฀and฀
other฀tissues.฀Osteoclasts฀endocytose฀Ca2+฀ions฀into฀acidic฀vesicles฀from฀the฀acidic฀com-
partment,฀pass฀the฀vesicles฀through฀the฀rufled฀membrane,฀and฀exocytose฀them฀into฀the฀
periosteum.฀However,฀some฀Ca2+ions฀escape฀into฀the฀cytosol฀where฀calbindin-D9K฀fer-
ries฀ them฀ to฀ where฀ a฀ Na+/Ca2+฀ exchanger฀ and฀ a฀ Ca2+-dependent฀ ATPase฀ on฀ the฀ mem-
brane฀extrude฀it฀into฀the฀stromal฀extracellular฀luid฀and฀blood฀(Fig.฀10.11).฀If฀the฀systemic฀
10.4.2. Rickets and Osteomalacia 171

calcium฀ion฀concentration฀falls,฀calcitriol฀is฀synthesized฀to฀activate฀greater฀expression฀
of฀ cytosolic฀ calbindin-D9K฀ to฀ speed฀ up฀ Ca2+฀ transport฀ from฀ the฀ acidic฀ compartment.฀
TRPV5฀functions฀in฀the฀excretion฀of฀this฀intracellular฀(nonvesicular฀transported)฀cal-
cium฀to฀the฀stromal฀blood฀capillaries฀(of฀the฀periosteum).฀By฀contrast,฀osteoblasts฀use฀
calbindin-D28K฀to฀transfer฀calcium฀from฀the฀periosteal฀stroma฀to฀the฀osteoid฀matrix,฀but฀
as฀ noted฀ above,฀ this฀ function฀ can฀ be฀ subsumed฀ by฀ calbindin-D9K฀ in฀ calbindin-D28K-
knockout฀mice.

10.4.2.
Rickets and Osteomalacia

A฀ lack฀ of฀ vitamin฀ D฀ inhibits฀ the฀ transcellular฀ uptake฀ of฀ calcium฀ from฀ the฀ intestine฀
(Fig.฀10.11).฀In฀growing฀children,฀this฀results฀in฀a฀failure฀of฀the฀osteoid฀tissue฀to฀calcify,฀
causing฀a฀skeletal฀deformity฀called฀rickets.฀In฀rickets,฀the฀most฀obvious฀symptoms฀are฀an฀
outward฀curvature฀of฀the฀long฀bones฀(bowing)฀of฀leg฀and฀insuficiently฀mineralized฀verte-
brae฀that฀produce฀a฀curved฀spine.฀In฀older฀adults,฀intestinal฀TRPV6฀expression฀falls฀and฀
calcitriol฀ loses฀ its฀ ability฀ to฀ induce฀ calbindins.฀ The฀ low฀ blood฀ calcium฀ levels฀ prevent฀
osteoid฀matrix฀from฀calcifying฀during฀bone฀remodeling;฀bones฀incompletely฀mineralized฀
and฀ fragile.฀ This฀ usually฀ elderly-adult฀ form฀ of฀ rickets฀ is฀ called฀ osteomalacia฀ (softened฀
bone).฀Osteoclast฀activity฀is฀normal฀and฀balanced฀with฀osteoblast฀activity,฀but฀the฀osteoid฀
does฀ not฀ mineralize฀ properly.฀ In฀ osteoporosis฀ that฀ occurs฀ at฀ menopause฀ (Sect.฀ 10.2.3),฀
osteoclast฀activity฀is฀over-active฀and฀osteoblast฀activity฀is฀reduced.฀A฀rare฀form฀of฀osteomal-
acia฀ is฀ caused฀ by฀ hypophosphatemia฀ due฀ to฀ PHEX฀ gluzincin฀ mutations฀ Sect.฀ 9.4.1).฀
Osteomalacia฀ is฀ treated฀ by฀ vitamin฀ D฀ or฀ phosphate฀ supplements,฀ depending฀ on฀ the฀
deiciency.

Vitamin฀D฀is฀a฀fat฀soluble฀vitamin฀related฀to฀cholesterol.฀In฀the฀skin,฀sunlight฀spontane-
ously฀ oxidizes฀ cholesterol฀ to฀ 7-dehydrocholesterol.฀ 7-Dehydrocholesterol฀ spontane-
ously฀ isomerizes฀ to฀ cholecalciferol฀ (vitamin฀ D3),฀ which฀ is฀ oxidized฀ in฀ the฀ liver฀ to฀
25-hydroxy฀cholecalciferol฀and,฀under฀the฀inluence฀of฀PTH฀in฀the฀kidney,฀to฀1,25-dihy-
droxy฀cholecalciferol฀(calcitriol),฀the฀active฀form฀of฀vitamin฀D.฀Vitamin฀D฀induces฀the฀
expression฀of฀calcium฀ion฀transport฀proteins฀(calbindins)฀in฀intestinal฀epithelium,฀osteo-
clasts,฀and฀osteoblasts.฀Calbindins฀and฀transient฀receptor฀potential฀channels฀(TRPV)฀are฀
responsible฀for฀the฀uptake฀of฀calcium฀from฀the฀diet.฀In฀children,฀the฀absence฀of฀sunlight฀
provokes฀a฀deiciency฀of฀vitamin฀D,฀causing฀an฀absence฀of฀calbindins฀and฀inadequate฀
blood฀calcium฀levels.฀Osteoid฀tissue฀cannot฀calcify,฀causing฀skeletal฀deformities฀(rick-
ets).฀In฀the฀elderly,฀there฀is฀a฀loss฀of฀intestinal฀TRPV฀receptors฀and฀decreased฀calbindin฀
expression฀by฀vitamin฀D.฀In฀both฀cases,฀the฀resultant฀low฀blood฀calcium฀levels฀cause฀
poor฀mineralization฀during฀bone฀remodeling฀(osteomalacia).฀Rickets฀is฀the฀childhood฀
expression฀of฀osteomalacia.฀Osteoclast฀activity฀is฀normal฀but฀the฀bone฀does฀not฀prop-
erly฀mineralize.฀In฀osteoporosis,฀the฀bone฀is฀properly฀mineralized฀but฀osteoclasts฀are฀
overly฀active.
172 10 Bone Remodeling and Calcium Metabolism

10.5.1.
Actions of Calcitonin

Calcitonin฀ inhibits฀ osteoclasts฀ and฀ decreases฀ blood฀ plasma฀ calcium฀ concentrations฀

(Fig.฀10.12).฀It฀therefore฀opposes฀the฀action฀of฀parathyroid฀hormone.฀It฀is฀a฀32฀amino฀acid฀
hormone฀secreted฀by฀the฀thyroid฀“C”฀cells.฀It฀is฀related฀to฀four฀bioactive฀peptides.฀One฀is฀
an฀alternatively฀spliced฀product฀of฀the฀calcitonin฀gene฀expressed฀in฀tissues฀other฀than฀the฀
thyroid฀ (calcitonin฀ gene-related฀ peptide-1,฀ CGRP-1).฀ The฀ other฀ three:฀ calcitonin฀ gene-
related฀ peptide-2฀ (CGRP-2),฀ amylin,฀ and฀ adrenomedullin฀ are฀ each฀ encoded฀ by฀ separate฀
genes.
The฀effects฀of฀calcitonin฀and฀its฀related฀peptides฀are฀mediated฀by฀the฀calcitonin฀receptor฀
(CTR)฀ or฀ the฀ calcitonin฀ receptor-like฀ receptor฀ (CLR).฀ These฀ family฀ B฀ GPCRs฀ are฀ not฀
exclusive฀to฀osteoclasts.฀The฀speciicity฀for฀ligand฀agonists฀is฀determined฀by฀one฀of฀three฀
receptor฀ activity฀ modifying฀ proteins฀ (RAMPs)฀ which฀ are฀ crucial฀ for฀ chaperoning฀ CLR฀
binding฀to฀a฀targeted฀cell,฀but฀not฀for฀CTR฀binding.฀In฀osteoclasts,฀CTR฀activation฀is฀cou-
pled฀to฀Gs-α฀and฀Gq-α.฀Gq฀is฀homologous฀to฀Gs,฀but฀it฀mediates฀a฀different฀action.฀Activated฀
Gs-α฀ increases฀ cAMP฀ levels฀ and฀ stimulates฀ protein฀ kinase฀ A฀ (Sect.฀ 10.3.1).฀ Activated฀
Gq-α฀increases฀the฀cytosolic฀concentration฀of฀diacylglycerol฀and฀intracellular฀Ca2+฀which฀
stimulate฀protein฀kinase฀C฀to฀retract฀or฀remove฀the฀osteoclast’s฀rufled฀membrane฀(section฀
10.1.1).฀฀
Loss฀of฀the฀thyroid฀gland฀with฀its฀calcitonin-producing฀“C”฀cells฀does฀not฀cause฀osteo-
porosis,฀and฀high฀levels฀of฀calcitonin฀associated฀with฀thyroid฀carcinoma฀do฀not฀cause฀oste-
osclerosis,฀thick,฀well฀mineralized฀bones฀distinct฀from฀the฀dense,฀continuously฀growing฀
bone฀that฀characterizes฀osteopetrosis฀(see฀Sects.฀10.2.1฀and฀10.2.2).฀These฀indings฀indi-
cate฀that฀calcitonin฀does฀not฀increase฀bone฀deposition,฀but฀rather฀inhibits฀osteoclast฀activity฀

Ca2+ in plasma

Bone calcification Parathyroid hormone

Mobilization
Calcitonin from bone

Ca2+ in plasma

Intestine

Vitamin D (1,25-dihydroxy
Cholecalciferol)

Fig. 10.12฀ Combined฀actions฀of฀parathyroid฀hormone,฀calcitriol,฀and฀calcitonin฀on฀the฀calcium฀ion฀

concentration฀of฀blood฀plasma฀(From฀Figure฀13-11฀in฀“Biochemistry:฀a฀case-oriented฀approach,”฀
4th฀ Ed.฀ 1983.฀ Authors:฀ Montgomery฀ R,฀ Dryer฀ RL,฀ Conway,฀ TW฀ and฀ Spector฀ AA.฀ Chap.฀ 13,฀
Hormonal฀regulation฀of฀metabolism,฀page฀675.฀The฀C.V.฀Mosby฀Co.,฀St฀Louis,฀MO.฀Copyright฀
Elsevier฀(2009))
10.5.2. Calcitonin and PTH Therapy for Osteoporosis 173

when฀blood฀calcium฀levels฀are฀high.฀As฀illustrated฀in฀Fig.฀10.12,฀calcitonin฀injections฀lower฀
the฀blood฀calcium฀levels฀back฀to฀baseline฀whereas฀daily฀parathyroid฀hormone฀injections฀
cause฀blood฀Ca2+฀concentrations฀to฀increase.

10.5.2.
Calcitonin and PTH Therapy for Osteoporosis

The฀direct฀inhibitory฀effect฀of฀calcitonin฀on฀osteoclasts฀has฀permitted฀calcitonin฀injections฀
to฀be฀used฀to฀control฀severe฀osteoporosis.฀Surprisingly,฀PTH฀can฀also฀be฀used฀for฀this฀pur-
pose.฀Normally,฀continuous฀exposure฀of฀bone฀cells฀to฀0.001–10฀nmol/L฀PTH฀results฀in฀a฀
dose-dependent฀inhibition฀of฀collagen฀(and฀bone)฀฀synthesis฀by฀osteoclasts฀in฀vitro.฀The฀
PTH-stimulated฀osteoblasts฀reduce฀OCIF฀(osteoprotegerin)฀and฀increase฀ODF฀(RANKL)฀
as฀already฀described฀(Sect.฀10.3.1.).฀Surprisingly,฀a฀single฀daily฀dose฀of฀trace฀amounts฀of฀
PTH฀(0.1-10฀nanomol/liter)฀stimulates฀OCIF฀secretion฀and฀inhibits฀ODF฀action.฀Because฀
menopause฀increases฀ODF฀secretion฀(by฀removing฀estrogen),฀intermittent฀PTH฀may฀make฀฀
OCIF฀secretion฀rebound฀faster.
Daily฀calcitonin฀or฀PTH฀administration฀is฀an฀effective฀alternative฀to฀bisphosphonates฀
for฀treating฀osteoporosis,฀but฀much฀less฀convenient.฀Calcitonin฀and฀PTH฀are฀destroyed฀by฀
proteases฀in฀the฀intestine฀and฀also฀by฀proteases฀in฀the฀bloodstream฀and฀must฀be฀given฀by฀
daily฀injection.฀By฀contrast,฀bisphosphonates฀are฀not฀only฀stable฀after฀ingestion,฀but฀they฀
also฀bind฀to฀the฀surface฀of฀calciied฀tissues฀and฀are฀not฀easily฀removed.฀Bisphosphonates฀
can฀be฀administered฀orally,฀weekly฀or฀even฀monthly.฀(See฀Sect.฀10.2.3).

A฀high฀blood฀calcium฀level฀activates฀secretion฀of฀another฀polypeptide฀hormone,฀calci-
tonin,฀from฀“C”฀cells฀of฀the฀thyroid฀gland.฀Calcitonin฀acts฀on฀G-protein฀receptors฀฀like฀
PRR฀receptors,฀but฀the฀activation฀reverses฀PTH฀action฀in฀the฀kidney฀by฀activating฀phos-
phate฀retention฀and฀inhibiting฀Ca2+฀ion฀reabsorption฀into฀blood฀plasma.฀Although฀calci-
tonin฀inhibits฀osteoclast฀activity,฀its฀loss฀(for฀example,฀from฀thyroid฀carcinoma)฀does฀
not฀cause฀osteoporosis.฀Conversely,฀thyroid฀carcinoma฀is฀associated฀with฀high฀levels฀of฀
calcitonin฀without฀osteopetrosis.฀The฀calcitonin฀receptor฀seems฀more฀central฀to฀bone฀
development฀ and฀ remodeling.฀ Bone฀ mass฀ appears฀ regulated฀ through฀ hormones฀ that฀
induce฀the฀hypothalamus฀to฀produce฀neural฀mediators฀that฀inluence฀RANKL฀(ODF)฀
production฀ by฀ osteoclasts,฀ in฀ part,฀ by฀ activating฀ nerve฀ ibers฀ present฀ throughout฀ the฀
bone.฀Intermittent฀injection฀of฀PTH฀increases฀osteoblast฀OCIF฀secretion฀and฀bone฀depo-
sition฀suggesting฀a฀potential฀new฀mechanism฀for฀preventing฀menopausal฀osteoporosis.
 Blood Coagulation
11

This฀chapter฀describes฀the฀composition฀of฀the฀vascular฀system฀and฀an฀overview฀of฀how฀
common฀bleeding฀problems฀arise฀(Sect.฀1).฀A฀description฀follows฀of฀how฀blood฀vessel฀
injury฀ activates฀ platelets฀ and฀ ibrinogen฀ to฀ plug฀ the฀ clot฀ and฀ attract฀ and฀ activate฀ the฀
blood฀ clotting฀ proteins฀ (Sect.฀ 2),฀ how฀ these฀ proteins฀ activate฀ thrombin฀ by฀ extrinsic,฀
intrinsic,฀ and฀ common฀ pathways฀ and฀ how฀ defects฀ in฀ the฀ thrombin฀ activators฀ lead฀ to฀
different฀forms฀of฀hemophilia฀(Sect.฀3).฀The฀mechanisms฀of฀how฀a฀ibrin฀clot฀is฀made฀
and฀removed฀for฀tissue฀repair฀(Sect.฀4)฀and฀prevented฀from฀occurring฀in฀undamaged฀
(healthy)฀ blood฀ vessels฀ are฀ described฀ (Sect.฀ 5).฀ The฀ chapter฀ concludes฀ with฀ a฀ list฀ of฀
drugs฀that฀affect฀coagulation฀and฀of฀laboratory฀tests฀to฀determine฀the฀condition฀of฀the฀
clotting฀system฀(Sect.฀6).

11.1.1
The Vascular System

The฀vascular฀system฀of฀arteries฀and฀veins฀is฀directed฀toward฀a฀capillary-mediated฀low฀of฀
interstitial฀ luid฀ that฀ provides฀ oxygen฀ and฀ nutrients฀ for฀ stromal฀ cells฀ and฀ removes฀ their฀
carbon฀dioxide฀and฀other฀waste฀products฀(Sect.฀3.3.1.).฀This฀exchange฀is฀effective฀because฀
capillaries฀are฀only฀two฀cell฀layers฀thick,฀a฀layer฀of฀endothelial฀cells฀surrounded฀by฀a฀layer฀
of฀ pericytes.฀ The฀ latter฀ cells฀ have฀ some฀ features฀ of฀ muscle฀ cells฀ and฀ they฀ synthesize฀ a฀
reticular฀iber฀matrix฀which฀includes฀actin฀(a฀muscle฀protein),฀in฀addition฀to฀ibrillin฀(oxyta-
lan฀ibers;฀Sect.฀3.1.3),฀microibrillar฀collagen฀(type฀VI;฀Sect.฀4.3.4)฀and฀delicate฀collagen฀
ibers฀(mixture฀of฀types฀I฀and฀III;฀Sect.฀4.3.2).฀The฀matrix฀is฀anchored฀to฀type฀IV฀collagen฀
of฀the฀endothelial฀cell฀basal฀lamina฀by฀type฀VII฀collagen฀(Sect.฀5.1.1)฀and฀held฀together฀by฀
proteo-glycosaminoglycans฀and฀other฀adhesive฀proteins฀continuous฀with฀a฀loose฀connec-
tive฀tissue฀matrix฀made฀by฀nearby฀stromal฀ibroblasts฀(Fig.฀11.1).฀(Sect.฀5.1.1).฀In฀larger฀
arteries,฀the฀region฀around฀endothelial฀cells,฀basal฀lamina,฀and฀pericytes฀is฀called฀the฀intima฀
(or฀tunica฀intima,฀Latin฀for฀“innermost฀covering”).฀Larger฀blood฀vessels฀are฀surrounded฀by฀
layers฀of฀more฀differentiated฀smooth-muscle฀cells฀that฀secrete฀actin฀and฀myosin฀within฀an฀
elastic฀iber฀matrix฀(Sect.฀6.2.1).฀Histologically,฀the฀outer฀stroma฀is฀called฀“tunica฀media”฀
and฀“tunica฀extrema”฀(middle฀and฀outer฀coverings).

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 175
DOI:฀10.1007/978-3-540-88116-2_11,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
176 11 Blood Coagulation

Capillary
Lumen Endothelial cell

Pericyte
Reticular
fiber matrix
Loose
connective
Basement tissue
membrane

Fibroblast
Collagen
fibers

Fig. 11.1฀ Cross฀ section฀ of฀ blood฀ vessels.฀ Small฀ blood฀ vessels฀ such฀ as฀ capillaries,฀ postcapillary฀
venules฀and฀arterioles฀are฀lined฀by฀a฀single฀layer฀of฀endothelial฀cells฀(ECs),฀surrounded฀by฀a฀continu-
ous฀basement฀membrane฀(BM).฀Pericytes฀(perivascular฀cells)฀surround฀the฀basement฀membrane,฀
and฀are฀themselves฀encircled฀by฀nearby฀stromal฀ibroblasts.฀The฀BM฀underlies฀the฀endothelial฀basal฀
lamina฀histologically฀but฀is฀biochemically฀indistinguishable฀from฀the฀reticular฀iber฀matrix฀synthe-
sized฀ by฀ the฀ pericytes.฀ (Modiied฀ from฀ Laleur฀ MA,฀ Handsley฀ MM฀ and฀ Edwards฀ DR฀ (2003)฀
“Metalloproteinases฀ and฀ their฀ inhibitors฀ in฀ angiogenesis,”฀ Exp.฀ Rev.฀ Mol.฀ Med.฀ 5(23):1–39,฀
Cambridge฀University฀Press:฀Copyright฀permission฀granted฀by฀Cambridge฀University฀Press,฀2008)

The฀cellular฀elements฀of฀blood,฀the฀erythrocytes,฀leukocytes,฀and฀platelets฀are฀immersed฀
in฀blood฀plasma,฀a฀rich฀solution฀of฀proteins,฀small฀molecules,฀and฀minerals.฀Most฀plasma฀
proteins฀are฀made฀and฀secreted฀by฀the฀liver,฀but฀a฀few฀are฀made฀and฀secreted฀by฀the฀blood฀
vessel฀endothelial฀cells.฀The฀cellular฀elements฀of฀blood฀are฀made฀from฀precursors฀in฀the฀red฀
marrow฀of฀long฀bones฀(Sect.฀9.2.1)฀by฀hematopoiesis.฀Aged฀cells฀are฀removed฀from฀the฀
circulation฀by฀macrophages฀in฀the฀spleen,฀liver,฀or฀bone฀marrow.฀Platelets฀are฀derived฀from฀
megakaryocytes,฀large฀cells฀that฀develop฀in฀the฀bone฀marrow฀along฀with฀erythrocytes฀and฀
leukocytes.฀Platelets฀are฀outer฀membrane-covered฀secretion฀granules฀of฀megakaryocytes฀
and฀are฀removed฀by฀macrophages฀passing฀through฀the฀spleen฀after฀only฀8–12฀days฀in฀cir-
culation.฀Launching฀the฀blood฀coagulation฀system฀requires฀an฀interaction฀between฀plasma฀
proteins฀and฀platelets฀at฀a฀site฀of฀injury.

11.1.2
Bleeding and Blood Clotting Problems

Diseases฀or฀drugs฀that฀affect฀the฀production฀of฀platelets฀or฀plasma฀proteins฀responsible฀
for฀clotting฀cause฀excessive฀bleeding฀problems.฀The฀most฀common฀are฀diseases฀of฀the฀
bone฀ marrow฀ and฀ chemotherapy,฀ both฀ of฀ which฀ deplete฀ platelets฀ by฀ inhibiting฀฀
11.1.2 Bleeding and Blood Clotting Problems 177

the฀ production฀ of฀ megakaryocytes.฀ In฀ addition,฀ liver฀ disease,฀ commonly฀ caused฀ by฀
chronic฀alcoholism฀or฀viral฀infections,฀inhibits฀the฀production฀of฀the฀proteins฀respon-
sible฀for฀blood฀coagulation.฀Less฀commonly,฀genetic฀mutations฀of฀these฀proteins฀cause฀
life-long฀ excessive฀ bleeding฀ (hemophilia).฀ Finally,฀ although฀ chronic฀ inlammation฀
such฀ as฀ that฀ associated฀ with฀ chronic฀ periodontitis฀ enhances฀ clotting฀ systemically,฀฀
it฀ often฀ inhibits฀ blood฀ clotting฀ locally฀ (Sects.฀ 13.2.3฀ and฀ 13.3.1).฀ Any฀ of฀ the฀ above฀
conditions฀may฀cause฀an฀unexpectedly฀large฀amount฀of฀bleeding฀from฀a฀tooth฀extrac-
tion฀and฀it฀is฀advisable฀to฀take฀a฀medical฀history฀and฀ensure฀that฀local฀inlammation฀is฀
reduced฀as฀much฀as฀possible฀beforehand฀(Sect.13.5.1).
Capillaries฀are฀fragile.฀Continuous฀movement฀of฀the฀heart฀or฀intermittent฀movements฀
and฀pressure฀changes฀around฀the฀cartilage฀between฀the฀bones฀stresses฀the฀capillaries,฀pre-
disposing฀ them฀ to฀ wear฀ and฀ tear฀ that฀ can฀ activate฀ the฀ clotting฀ system.฀ Micro-size฀ clots฀
continually฀form฀and฀dissolve.฀A฀more฀severe฀injury฀such฀as฀bumping฀into฀a฀hard฀object฀
causes฀bruising฀from฀bleeding฀beneath฀the฀skin.฀Although฀bleeding฀stops฀after฀a฀few฀min-
utes฀because฀a฀clot฀forms,฀the฀bruise฀lasts฀for฀a฀day฀or฀longer฀while฀leukocytes฀attracted฀by฀
proinlammatory฀ cytokines฀ released฀ by฀ the฀ damaged฀ tissues฀ (Sect.฀ 13.3.2)฀ digest฀ the฀
extravascular฀erythrocytes฀(Sect.฀13.2.4).฀As฀the฀bruise฀disappears฀and฀the฀damaged฀tissue฀
begins฀to฀be฀repaired,฀the฀clot฀is฀gradually฀lysed.
In฀older฀adults,฀there฀is฀a฀predisposition฀to฀clotting฀because฀of฀blood฀vessel฀degeneration฀
or฀a฀decreased฀blood฀low.฀The฀clots฀may฀obstruct฀a฀small฀artery฀or฀detach฀as฀an฀embolus฀
that฀obstructs฀a฀arteries฀or฀veins฀elsewhere.฀Possible฀consequences฀are฀heart฀attack,฀stroke,฀
pulmonary฀ obstruction,฀ or฀ peripheral฀ necrosis฀ (Sect.฀ 13.4.3).฀ A฀ tooth฀ extraction฀ in฀ an฀
elderly฀ individual฀ can฀ cause฀ an฀ embolism฀ elsewhere฀ in฀ the฀ body.฀ Elderly฀ patients฀ and฀
patients฀on฀medication฀should฀be฀readied฀for฀oral฀surgery฀only฀after฀taking฀a฀history฀to฀
elicit฀potential฀bleeding฀problems฀and฀consulting฀their฀฀attending฀physician.฀Rarely,฀acute฀
disseminated฀intravascular฀blood฀clotting฀may฀be฀induced฀in฀any฀individual฀by฀a฀severe฀
crushing฀injury,฀a฀severe฀allergic฀reaction฀to฀an฀insect฀bite฀or฀drug,฀or฀uncontrolled฀sys-
temic฀bacterial฀or฀viral฀infections.

Capillaries฀are฀composed฀of฀endothelial฀cells฀and฀an฀outer฀pericyte฀layer฀that฀expresses฀
contractile฀smooth฀muscle฀actin฀within฀a฀reticular฀iber฀matrix฀consisting฀of฀thin฀colla-
gen฀ibers฀surrounded฀by฀collagen฀and฀ibrillin฀microibrils.฀The฀matrix฀is฀held฀together฀
by฀proteo-glycosaminoglycans฀and฀anchored฀to฀the฀capillary฀basal฀lamina.฀Launching฀
the฀ blood฀ coagulation฀ system฀ requires฀ that฀ platelets฀ be฀ present฀ and฀ activated฀ at฀ an฀
injured฀site.฀Most฀blood฀clotting฀proteins฀are฀made฀in฀the฀liver;฀blood฀cells฀and฀platelets฀
are฀made฀in฀the฀red฀bone฀marrow.฀Diseases฀of฀the฀liver฀or฀bone฀marrow฀thus฀enhance฀
bleeding฀ by฀ removing฀ clotting฀ components.฀ Blood฀ vessel฀ degeneration฀ predisposes฀
especially฀ to฀ heart฀ attacks,฀ strokes,฀ pulmonary฀ obstruction,฀ and฀ peripheral฀ necrosis.฀
Disseminated฀ intravascular฀ blood฀ clotting฀ is฀ an฀ often฀ fatal฀ condition฀ induced฀ by฀ a฀
severe฀crushing฀injury,฀allergy฀or฀infection.
178 11 Blood Coagulation

11.2.1
Blood Vessel Injury, von Willebrand Factor, and Platelets

Platelets฀initiate฀coagulation฀by฀interacting฀with฀von฀Willebrand฀Factor฀(VWF),฀a฀multido-
main฀protein฀(Fig.฀11.2)฀synthesized฀and฀secreted฀by฀healthy฀endothelial฀cells.฀Following฀
loss฀ of฀ signal฀ peptide฀ in฀ the฀ endothelial฀ cell฀ endoplasmic฀ reticulum฀ (ER),฀ the฀ proVWF฀
molecule฀dimerizes฀by฀disulide฀bonding฀at฀the฀C-terminal฀cysteine฀knot฀domain,฀which฀is฀
homologous฀to฀the฀domain฀in฀TGF-β฀(Sect.฀3.2.2).฀The฀dimers฀are฀transported฀to฀the฀Golgi,฀
where฀propeptide฀domains฀D1฀and฀D2฀are฀cleaved,฀permitting฀large฀multimers฀to฀assemble฀
by฀disulide฀bond฀exchange฀between฀the฀dimer฀D3฀domains.฀Sites฀of฀stress฀or฀injury฀expose฀
subendothelial฀stromal฀collagen฀that฀binds฀to฀A3฀domains฀in฀the฀multimer.฀The฀collagen฀
interaction฀exposes฀A1฀domains฀that฀tether฀platelets฀from฀the฀blood฀low฀by฀binding฀to฀a฀
platelet฀surface฀glycoprotein฀(Gp)฀called฀GpIbα.฀Tethering฀activates฀the฀platelets฀to฀aggre-
gate฀into฀a฀platelet฀plug฀or฀soft฀clot฀which฀temporarily฀stops฀the฀bleeding฀and฀provides฀a฀
surface฀for฀the฀more฀permanent฀blood฀clot.฀Platelet฀plug฀formation฀is฀enhanced฀mildly฀by฀
epinephrine฀and฀ADP฀(Sect.฀11.6.6),฀and฀strongly฀if฀thrombin฀is฀already฀activated฀(Sect.฀
11.3.4).฀Epinephrine฀is฀released฀into฀the฀blood฀as฀a฀response฀to฀injury,฀and฀ADP฀is฀released฀
passively฀from฀injured฀endothelial฀cells฀and฀erythrocytes฀prior฀to฀activation.฀The฀activated฀
platelets฀form฀a฀plug฀in฀three฀ways:฀First,฀an฀integrin฀on฀the฀platelet฀surface฀(αIIbβ3)฀is฀acti-
vated฀and฀binds฀to฀an฀RGD฀sequence฀(Sect.฀3.2.1)฀on฀the฀VWF฀C1฀domain฀(Fig.฀11.2),฀but฀
is฀displaced฀by฀ibrinogen฀(1.5฀–฀3.0฀mg/ml฀in฀blood฀plasma,฀100฀–฀300฀times฀greater฀than฀
multimeric฀VWF);฀Second,฀phosphatidylserine฀is฀moved฀from฀inner฀to฀outer฀lealet฀of฀the฀
platelet฀membrane;฀Third,฀granules฀whose฀contents฀stimulate฀thrombosis฀(blood฀clotting)฀
and฀wound฀healing฀are฀secreted.฀One฀secreted฀component฀is฀thromboxane฀A2฀(TXA2),฀an฀
eicosanoid฀(Fig.฀13.13.)฀that฀stimulates฀thrombosis.฀TXA2฀recruits฀and฀activates฀additional฀

Fig. 11.2฀ Structure฀ of฀ von฀ Willebrand฀ factor฀ (VWF).฀ The฀ VWF฀ precursor฀ (Expasy฀ accession฀฀
No.฀P04275)฀consists฀of฀a฀signal฀peptide฀(residues฀1–22)฀and฀a฀propeptide฀(residues฀23–763).฀The฀
remaining฀residues฀764–2,813฀comprise฀the฀mature฀protein,฀which฀is฀divided฀into฀A,฀B,฀C,฀D฀and฀
cysteine฀knot฀(CK)฀domains.฀The฀domains฀are฀named฀for฀sequence฀homology฀A1,฀A2฀and฀A3฀are฀
homologous฀(similar)฀but฀unrelated฀to฀B,฀C฀and฀D฀etc.฀The฀CK฀domain฀is฀illustrated฀in฀detail฀in฀
Fig.฀12.4b.฀The฀domains฀were฀identiied฀by฀related฀repeating฀sequences฀in฀this฀and฀many฀other฀
proteins.฀Proteins฀with฀VWF฀domains฀relevant฀to฀topics฀in฀this฀book฀are฀type฀VI฀collagen฀(legend฀
to฀Fig.฀4.10)฀and฀salivary฀mucin฀MG1฀(Sect.฀12.3.1).฀The฀VWF฀binding฀sites฀for฀collagen฀(A3),฀
platelet฀ glycoprotein฀ Ib,฀ GPIb฀ (A1),฀ integrin฀ aIIbb3฀ (C1)฀ and฀ blood฀ coagulation฀ factor฀ VIII฀ are฀
indicated.฀The฀down-pointing฀arrow฀indicates฀the฀site฀of฀degradation฀by฀ADAMTS-13฀between฀
tyr1605฀and฀met1606฀(From฀Sadler฀JE฀(2005)฀“New฀concepts฀in฀von฀Willebrand฀disease.”฀Annu.฀Rev.฀
Med.฀56:173–191.฀Reprinted,฀with฀permission,฀from฀the฀Annual฀Review฀of฀Medicine,฀Volume฀56฀
©2005฀by฀Annual฀Reviews฀(www.annualreviews.org))
11.2.1 Blood Vessel Injury, von Willebrand Factor, and Platelets 179

platelets฀from฀blood฀without฀VWF฀binding.฀Fibrinogen฀binds฀to฀the฀activated฀αIIbβ3฀integ-
rin,฀enlarging฀the฀soft฀clot฀(plug)฀at฀the฀injured฀site฀(Fig.฀11.3).฀The฀activated฀platelets฀fur-
ther฀seal฀capillaries฀by฀contracting฀actin฀in฀the฀pericyte฀cytosol,฀and฀the฀many฀platelets฀in฀
the฀soft฀clot฀provide฀a฀large฀phosphatidylserine฀surface฀for฀coagulation.฀Multimeric฀VWF฀is฀
therefore฀the฀sensor฀of฀blood฀vessel฀injury;฀it฀aggregates฀and฀activates฀platelets฀to฀plug฀the฀
wound฀and฀launch฀the฀blood฀coagulation฀system.
Multimeric฀VWF฀is฀degraded฀by฀an฀adamalysin,฀ADAMTS-13,฀which฀is฀secreted฀into฀the฀
blood฀from฀endothelial฀cells฀along฀with฀VWF.฀ADAMTS-13฀resembles฀procollagen฀N-peptidase฀
(ADAMTS-2;฀Fig.฀8.7b)฀except฀that฀the฀C-terminal฀domain฀of฀ADAMTS-2฀is฀replaced฀by฀four฀

Fig. 11.3฀ Interactions฀ of฀ platelets฀ with฀ a฀ wounded฀ capillary฀ surface.฀ Top฀ half:฀ Resting฀ platelets฀
(white)฀possess฀glycoprotein-Iba.฀This฀glycoprotein฀ligand฀recognizes฀a฀receptor฀on฀multimeric฀von฀
Willebrand฀Factor฀(VWF)฀that฀becomes฀attached฀to฀exposed฀collagen฀(black)฀after฀injury฀has฀broken฀
the฀endothelial฀cell฀layer฀and฀basal฀lamina฀(red).฀Bottom฀half:฀Upon฀contact,฀platelets฀are฀activated฀
(maroon)฀and฀their฀surface฀undergoes฀two฀major฀changes฀during฀activation:฀(i)฀a฀ibrinogen฀receptor฀
(black฀semicircle)฀appears฀and฀(ii)฀the฀phospholipids฀lip-lop฀(not฀shown).฀The฀ibrinogen฀receptor฀
is฀activated฀integrin฀(aIIbb3)฀which฀binds฀to฀a฀domain฀on฀each฀end฀of฀the฀linear฀ibrinogen฀molecule฀
(details฀of฀the฀ibrinogen฀structure฀are฀given฀in฀Fig.฀11.9).฀The฀lipids฀serve฀as฀a฀surface฀for฀localizing฀
and฀activating฀clotting฀ factors฀ (Original฀ igure฀ submitted฀ by฀Dr.฀Paul฀DeAngelis,฀Department฀of฀
Biochemistry,฀University฀of฀Oklahoma฀HSC,฀Oklahoma฀City,฀OK,฀USA)
180 11 Blood Coagulation

additional฀thrombospondin-1฀repeats฀(TS-1฀repeats)฀and฀terminates฀in฀two฀CUB฀repeats฀(like฀
those฀found฀on฀procollagen฀C-terminal฀peptidase,฀PCP,฀Fig.฀8.7a).฀Mutations฀of฀ADAMTS-13฀
prevent฀the฀degradation฀of฀VWF.฀The฀concentration฀of฀multimeric฀VWF฀is฀increased,฀oversen-
sitizing฀the฀blood฀to฀minor฀injuries฀at฀sites฀such฀as฀the฀heart฀where฀capillaries฀are฀stressed.฀The฀
result฀is฀thrombotic฀thrombocytopenic฀purpura,฀a฀life-threatening฀disease฀in฀which฀small฀clots฀
form,฀break฀loose,฀and฀block฀arterioles฀in฀the฀body.฀In฀contrast,฀mutations฀of฀VWF฀prevent฀it฀
from฀binding฀collagen฀and฀do฀not฀stimulate฀platelets.฀The฀blood฀clotting฀system฀is฀underacti-
vated฀and฀injuries฀cause฀excessive฀bleeding฀(hemophilia).

11.2.2
The Gamma-Carboxyglutamate Domain: A Calcium Ion Chelator

Activated฀ platelets฀ possess฀ a฀ phosphatidylserine฀ surface฀ that฀ binds฀ calcium฀ ions฀฀

(as฀in฀matrix฀vesicles;฀Sect.฀9.3.3).฀This฀surface฀attaches฀various฀proteins฀that฀circulate฀in฀
the฀ blood฀ and฀ possess฀ a฀ negatively฀ charged฀ Ca+2฀ binding฀ domain฀ of฀ up฀ to฀ nineγ-
carboxyglutamate฀(gla)฀residues฀(Fig.฀11.4).฀Osteocalcin฀(Sect.฀9.4.2.)฀also฀possesses฀this฀
gla฀domain.
A฀ gla฀ protein฀ is฀ secreted฀ into฀ the฀ endoplasmic฀ reticulum฀ (ER)฀ of฀ the฀ liver฀ where฀ its฀
N-terminal฀domain฀binds฀to฀and฀activates฀an฀enzyme฀that฀converts฀glutamic฀acid฀residues฀
in฀the฀gla฀domain฀to฀γ-carboxyglutamic฀acid.฀The฀activated฀carboxylase฀enzyme฀then฀binds฀
to฀a฀reduced฀form฀of฀vitamin฀K,฀a฀diphenol฀(KH2)฀to฀which฀it฀adds฀molecular฀oxygen.฀The฀

O
Liver H H Bloodstream
N C C
CH2
CH
COC COO
2+ 2+
Ca Ca
2+ Ca
Vit K
2+
Ca

Clotting
Carboxy-
factor
glutamate Activated Platelet

Fig. 11.4฀ Mechanism฀of฀clotting฀factor฀localization฀to฀an฀activated฀platelet฀surface.฀Left:฀After฀syn-

thesis฀in฀the฀liver,฀certain฀blood฀clotting฀proteins฀are฀posttranslationally฀modiied฀in฀the฀endoplas-
mic฀reticulum฀by฀a฀vitamin฀K-dependent฀Vit฀K฀carboxylase.฀This฀enzyme฀forms฀carboxyglutamate฀
residues฀(top฀center)฀that฀chelate฀calcium฀ions.฀Right:฀In฀the฀bloodstream,฀clotting฀factor-bound฀
calcium฀ions฀attach฀to฀negatively฀charged฀phosphatidylserine฀that฀appears฀on฀the฀surface฀of฀acti-
vated฀platelets.฀Certain฀therapeutic฀drugs฀or฀acquired฀deiciencies฀inhibit฀this฀process฀–฀see฀text฀
(Original฀ igure฀ submitted฀ by฀ Dr฀ Paul฀ DeAngelis,฀ Department฀ of฀ Biochemistry,฀ University฀ of฀
Oklahoma฀HSC,฀Oklahoma฀City,฀OK,฀USA)
11.2.2 The Gamma- Carboxyglutamate Domain: A Calcium Ion Chelator 181

product฀ is฀ an฀ epoxide฀ which฀ reacts฀ with฀ a฀ catalytic฀ amine฀ (N:)฀ on฀ the฀ carboxylase฀฀
(Fig.฀11.5a),฀converting฀it฀to฀a฀strong฀epoxide฀base฀(K−)฀that฀abstracts฀a฀proton฀from฀the฀
γ-carbon฀of฀a฀glutamyl฀residue฀in฀the฀gla฀domain.฀The฀resulting฀γ-carbanion฀spontaneously฀
binds฀ to฀ carbon฀ dioxide,฀ converting฀ it฀ to฀ a฀ carboxyglutamate฀ residue฀ (Fig.฀ 11.5b).฀
Simultaneously,฀ the฀ proton฀ (Fig.฀ 5.11a)฀ converts฀ K−฀ into฀ a฀ stable฀ quinone฀ epoxide,฀ KO฀
(Fig.฀11.5b).฀Vitamin฀K฀oxidoreductase,฀a฀separate฀enzyme฀from฀the฀carboxylase฀in฀the฀ER฀
membrane,฀ completes฀ the฀ cycle฀ by฀ regenerating฀ KH2฀ from฀ KO.฀ The฀ carboxylase฀

Fig. 11.5฀ Vitamin฀K-dependent฀carboxylase฀action.฀(a)฀Overall฀view฀of฀the฀carboxylase.฀The฀vitamin฀

K-dependent฀protein฀substrates฀have฀high-฀and฀low-afinity฀domains฀for฀the฀carboxylase฀enzyme.฀
The฀high฀afinity฀site฀is฀the฀propeptide,฀which฀is฀cleaved฀after฀carboxylation,฀and฀the฀low฀afinity฀site฀
is฀ the฀ most฀ N-terminal฀ glutamate฀ residue฀ to฀ be฀ carboxylated.฀ Carboxylation฀ is฀ initiated฀ after฀ the฀
second฀site,฀the฀vitamin฀K฀binding฀site,฀is฀also฀occupied฀by฀reduced฀vitamin฀K฀(KH2).฀A฀catalytic฀
amine฀(N:)฀deprotonates฀KH2,฀hydroquinone.฀A฀strong฀base฀epoxide฀(K−)฀forms฀and฀attacks฀a฀glu฀resi-
due฀in฀the฀substrate฀binding฀domain฀(upward฀pointing฀solid฀arrow).฀Once฀this฀residue฀is฀carboxylated,฀
the฀reactivity฀of฀N:฀is฀increased฀(downward฀pointing฀dotted฀arrow)฀and฀a฀tightly฀coordinated฀complex฀
enables฀multiple฀glutamate฀residues฀in฀the฀substrate฀to฀be฀carboxylated฀by฀a฀processive฀mechanism฀฀
(b):฀Vitamin฀K฀oxidation.฀When฀the฀strong฀base฀epoxide฀(K−)฀generates฀a฀glutamayl฀carbanion฀that฀
reacts฀with฀CO2฀to฀form฀gla,฀the฀base฀K−฀is฀oxidized฀to฀an฀intermediate฀that฀immediately฀loses฀water฀
and฀forms฀a฀quinone฀epoxide฀(KO).฀KO฀regenerates฀KH2฀by฀accepting฀electrons฀from฀glutathione฀via฀
vitamin฀K฀oxidoreductase฀(Sect.฀7.4.1).฀(From฀Figs.฀1฀and฀2฀in฀Berkner฀KL,฀(2005)฀“The฀Vitamin฀
K-Dependent฀ Carboxylase.”฀ Annu.฀ Rev.฀ Nutr.฀ 25:127–149.฀ Reprinted,฀ with฀ permission,฀ from฀ the฀
Annual฀Review฀of฀Nutrition,฀Volume฀25฀©2005฀by฀Annual฀Reviews฀(www.annualreviews.org))
182 11 Blood Coagulation

Table 11.1฀ Major฀coagulation฀factors฀by฀pathway

Extrinsic Intrinsic Common Inhibitory
Tissue฀factor฀(TF)฀(FIII) FXIIb VWF Protein฀Ca
FVII a
FXI b
Ca ฀ions฀(FVa)
2+
Protein฀Sb
FIX a
FX a

FVIII FV฀(FVa฀=฀FVI)
Prothrombin฀(FII)a
Fibrinogen฀(FI)
฀ ฀ Transglutaminase฀
(FXIII)
Factors฀generating฀a฀protease฀and฀with฀a฀gla฀domain
a

Factor฀generating฀a฀protease฀and฀without฀a฀gla฀domain
b

and฀ oxidoreductase฀ iteratively฀ convert฀ all฀ the฀ glutamate฀ residues฀ in฀ a฀ gla-domain.฀ The฀
carboxylase฀then฀catalyzes฀a฀proteolysis฀reaction฀that฀cleaves฀the฀N-terminal฀propeptide,฀
releasing฀both฀carboxylated฀protein฀and฀propeptide฀separately.฀Carboxylation฀of฀glu฀to฀gla฀
resembles฀oxidation฀of฀proline฀to฀hydroxyproline฀by฀vitamin฀C฀during฀collagen฀synthesis฀
(Sect.฀ 7.3.1.).฀ The฀ electrons฀ may฀ be฀ obtained฀ from฀ glutathione฀ and฀ passed฀ through฀ an฀
unknown฀reductase฀to฀a฀cystine฀disulide฀bond฀in฀a฀small฀hydrophilic฀loop฀of฀vitamin฀K฀
oxidoreductase฀protruding฀into฀the฀ER฀lumen.฀The฀electrons฀are฀relayed฀to฀a฀second฀cystine฀
disulide฀bond฀in฀the฀membrane฀of฀the฀oxidoreductase฀before฀being฀passed฀to฀KO.
Wafarin฀is฀one฀of฀a฀family฀of฀vitamin฀K฀mimics฀(coumarins)฀that฀successfully฀compete฀for฀
the฀KH2฀binding฀site฀on฀vitamin฀K฀oxidoreductase.฀The฀diphenol฀cannot฀be฀regenerated฀from฀
KO,฀and฀glutamate฀is฀not฀carboxylated.฀The฀coumarins฀are฀therefore฀anticoagulants฀because฀the฀
gla฀proteins฀are฀secreted฀into฀blood฀as฀precursors฀(Coumarins฀and฀problems฀associated฀with฀
their฀use฀are฀discussed฀in฀Sect.฀11.6.3)฀The฀clotting฀factor฀pro-proteases฀that฀possess฀a฀gla฀
domain฀ are฀ factors฀ II,฀ VII,฀ IX,฀ and฀ X฀ (Table฀ 11.1).฀ These฀ and฀ other฀ factors฀ are฀ carried฀
around฀in฀the฀blood฀until฀they฀touch฀an฀activated฀platelet.฀The฀negative฀charge฀on฀the฀gla฀
domain฀of฀the฀pro-protease฀completes฀a฀calcium฀sandwich฀with฀phosphatidylserine฀on฀the฀
platelet฀ aggregate.฀ The฀ negative฀ lipid฀ is฀ on฀ one฀ side,฀ the฀ g-carboxyglutamate฀ is฀ on฀ the฀
other,฀and฀the฀Ca2+฀ions฀are฀in฀the฀middle฀(Fig.฀11.4).฀Binding฀alters฀the฀conformation฀of฀
the฀pro-protease฀factors฀and฀activates฀proteolysis.

Collagen฀ibers฀exposed฀to฀blood฀after฀an฀injury฀are฀detected฀by฀Von฀Willebrand฀Factor฀
(VWF),฀a฀plasma฀protein฀that฀circulates฀as฀multimeric฀aggregates.฀On฀binding฀collagen,฀
VWF฀tethers฀platelets฀and฀activates฀an฀integrin฀that฀binds฀to฀an฀RGD฀site฀on฀the฀VWF฀
aggregate,฀but฀more฀strongly฀to฀ibrinogen.฀VWF฀is฀degraded฀by฀an฀adamalysin,฀muta-
tions฀of฀which฀enhance฀clotting.฀Mutations฀of฀VWF฀enhance฀bleeding.฀VWF฀adherent฀
platelets฀ release฀ thromboxane,฀ activating฀ by-passing฀ platelets฀ to฀ attach฀ ibrinogen฀ by฀
their฀integrin฀receptors฀and฀a฀platelet฀plug฀or฀soft฀clot฀develops.฀The฀platelets฀in฀the฀soft฀
11.3.2 The Extrinsic Pathway 183

clot฀expose฀a฀phosphatidylserine฀surface฀onto฀which฀calcium฀ions฀attach฀blood฀clotting฀
proteins฀possessing฀γ-carboxyglutamate฀(gla)฀residues.฀Processing฀to฀gla฀occurs฀in฀the฀
liver฀where฀a฀carboxylase฀binds฀to฀the฀N-terminal฀domain฀of฀these฀proteins฀and฀converts฀
an฀internal฀domain฀rich฀in฀glutamate฀residues฀to฀gla฀residues.฀ The฀ carboxylase฀ uses฀ a฀
derivative฀of฀vitamin฀K฀as฀a฀cofactor฀and฀oxidizes฀it฀to฀a฀quinone฀epoxide.฀The฀epoxide฀
is฀reduced฀by฀a฀separate฀enzyme,฀vitamin฀K฀oxidoreductase,฀in฀tandem฀with฀gla฀domain฀
carboxylation.฀ After฀ all฀ of฀ the฀ domain’s฀ glutamate฀ residues฀ are฀ carboxylated,฀ the฀ car-
boxylase฀cleaves฀the฀substrate’s฀N-terminal฀propeptide,฀releasing฀the฀carboxylated฀pro-
tein฀and฀the฀propeptide฀separately.฀The฀oxidoreductase฀is฀inhibited฀by฀vitamin฀K฀analogues฀
(coumarins)฀which฀cause฀the฀precursor฀proteins฀to฀be฀released฀without฀carboxylation฀and฀
unable฀to฀bind฀to฀calcium฀ions฀on฀a฀phosphatidylserine฀surface.฀Large฀concentrations฀of฀
vitamin฀K฀reverse฀the฀coumarin฀inhibition฀by฀providing฀large฀amounts฀of฀the฀initial฀deriv-
ative฀and฀allowing฀the฀oxidized฀quinone฀epoxide฀to฀be฀excreted฀instead฀of฀reduced.

11.3.1
The Extrinsic, Intrinsic, and Common Coagulation Pathways

Following฀the฀recruitment฀and฀activation฀of฀platelets฀by฀injured฀capillaries,฀their฀soft฀clot฀
aggregates฀provide฀a฀surface฀for฀plasma฀proteins฀to฀interact฀and฀activate฀thrombin,฀which฀
transforms฀soluble฀ibrinogen฀to฀a฀ibrin฀clot.฀Thrombin฀is฀activated฀by฀two฀blood฀coagula-
tion฀pathways,฀extrinsic฀and฀intrinsic.฀A฀defective฀extrinsic฀path฀is฀incompatible฀with฀life,฀
whereas฀defects฀of฀the฀intrinsic฀path฀cause฀hemophilia.

11.3.2
The Extrinsic Pathway

The฀extrinsic฀pathway฀is฀mediated฀by฀tissue฀factor฀(TF),฀also฀known฀as฀thromboplastin฀or฀
factor฀III฀(Fig.฀11.6a).฀Tissue฀factor฀is฀not฀present฀in฀blood฀plasma,฀i.e.,฀it฀is฀extrinsic฀or฀
outside฀ of฀ the฀ vascular฀ system.฀ TF฀ is฀ a฀ membrane฀ protein฀ that฀ becomes฀ exposed฀ to฀ the฀
blood฀ by฀ trauma฀ to฀ the฀ endothelial฀ surface฀ of฀ blood฀ vessels.฀ It฀ is฀ especially฀ rich฀ in฀ the฀
membranes฀of฀pericytes,฀platelets,฀and฀leukocytes,฀but฀present฀in฀lesser฀amounts฀in฀most฀
cells.฀It฀is฀absent฀from฀striated฀muscle฀cells฀and฀chondroblasts.
When฀TF฀is฀exposed฀to฀blood,฀it฀spontaneously฀binds฀factor฀VII,฀a฀serine฀protease฀of฀the฀
coagulation฀system฀that฀possesses฀a฀gla฀domain฀(Table฀11.1).฀The฀TF–VII฀complex฀there-
fore฀adheres฀to฀the฀negatively฀charged฀surface฀of฀activated฀platelets.฀About฀0.4%฀of฀factor฀
VII฀is฀activated฀in฀blood฀by฀fatty฀acid-฀or฀triglyceride-mediated฀activation฀of฀factor฀IX฀and฀
binds฀ similarly฀ to฀ TF.฀ The฀ TF–VIIa฀ complex฀ (Fig.฀ 11.6a)฀ has฀ proteolytic฀ activity฀ and฀
cleaves฀(converts)฀another฀gla฀protein฀that฀also฀binds฀to฀the฀activated฀platelets,฀factor฀X.฀
Factor฀Xa฀is฀a฀serine฀protease฀that฀converts฀the฀remainder฀of฀the฀TF–VII฀complex,฀generat-
ing฀additional฀Xa฀proteolytic฀activity฀that฀ultimately฀converts฀prothrombin฀to฀thrombin฀for฀
184 11 Blood Coagulation

a b
Extrinsic X IX Intrinsic
pathway pathway

Tissue factor
VIIIa
Disrupted XII
tissues VIIa IXa
Phospholipids Released
Phospholipids cellular
RNA
c
Prothrombin
Common Activated
pathway Xa Va Platelets

Phospholipids

Thrombin

Fibrinogen Fibrin

Fig. 11.6฀ Extrinsic฀and฀intrinsic฀pathways.฀(a)฀Extrinsic฀pathway฀(top฀left):฀When฀an฀injury฀activates฀

platelets฀to฀form฀a฀phospholipid฀surface฀(white฀rectangle,฀lower฀right),฀disrupted฀tissues฀(magenta,฀
top฀left)฀release฀tissue฀factor฀(TF)฀(green).฀Factor฀VII฀and฀its฀activated฀derivative฀(factor฀VIIa,฀blue)฀
in฀ blood฀ plasma฀ bind฀ to฀ the฀ phospholipid฀ surface฀ by฀ their฀ gla฀ domain฀ and฀ attach฀ tissue฀ factor.฀
Unconverted฀factor฀VII฀is฀activated฀by฀small฀amounts฀of฀VIIa฀present฀and฀bound฀to฀TF.฀Factor฀X฀
(dark฀blue)฀binds฀similarly฀to฀phospholipid฀and฀is฀converted฀by฀the฀TF–VIIa฀complex฀to฀Xa฀(red).฀
(b)฀Intrinsic฀pathway฀(top฀right):฀Injury฀releases฀RNA,฀a฀negatively฀charged฀polymer฀that฀converts฀
factor฀ XII฀ to฀ XIIa฀ (white฀ squares).฀ Factor฀ XIIa฀ activates฀ factor฀ XI฀ (not฀ shown),฀ which฀ remains฀
soluble฀and฀activates฀factor฀IX฀bound฀to฀phospholipids฀(black฀square).฀Factor฀IXa฀activates฀bound฀
factor฀X฀so฀that฀it฀weakly฀converts฀prothrombin฀to฀thrombin.฀The฀thrombin฀then฀activates฀factor฀
VIII฀to฀VIIIa฀(black฀rectangle),฀which฀binds฀to฀factor฀IXa฀and฀accelerates฀its฀conversion฀of฀phos-
pholipid-bound฀factor฀X.฀(c)฀Common฀Pathway฀(bottom฀center):฀Increasing฀amounts฀of฀thrombin฀
activate฀factor฀V,฀converting฀it฀to฀Va.฀Va฀(small฀red฀square)฀binds฀to฀factor฀Xa฀(large฀red฀square)฀on฀
the฀phospholipid฀surface฀and฀greatly฀accelerates฀the฀conversion฀of฀prothrombin฀to฀thrombin฀(red฀
rectangles),฀and฀of฀ibrinogen฀(red฀rectangle)฀to฀ibrin฀(brown฀star).฀Factor฀numbering฀describes฀
discovery,฀not฀interaction.฀More฀abundant฀factors฀were฀discovered฀and฀numbered฀irst,฀ibrinogen฀
(factor฀I),฀thrombin฀and฀prothrombin฀(factors฀IIa฀and฀II).฀Today,฀these฀factors฀are฀usually฀referred฀
to฀by฀their฀names฀for฀clarity.฀The฀later-discovered฀factors฀are฀still฀referred฀to฀by฀their฀numbers.฀
Factor฀XII฀was฀the฀last฀factor฀to฀be฀discovered฀because฀it฀is฀irst฀in฀the฀cascade฀and฀present฀in฀least฀
amounts฀in฀blood.฀A฀few฀molecules฀of฀factor฀XII฀activate฀more฀molecules฀of฀factor฀XI฀and฀factor฀IX,฀
causing฀the฀intrinsic฀path฀to฀activate฀factor฀Xa฀rapidly฀(a,฀b,฀and฀c฀are฀original฀igures฀submitted฀by฀
Dr.฀Paul฀DeAngelis,฀Department฀of฀Biochemistry,฀University฀of฀Oklahoma฀HSC,฀Oklahoma฀City,฀
OK,฀USA)
11.3.4 The Common Pathway 185

ibrin฀generation฀at฀the฀injured฀site.฀TF฀primes฀the฀coagulation฀system฀just฀as฀VWF฀primes฀
platelet฀activation.
Excessive฀ production฀ of฀ factor฀ VIIa฀ is฀ associated฀ with฀ an฀ unusual฀ susceptibility฀ to฀
thrombosis฀in฀heart฀blood฀vessel.฀Unlike฀striated฀muscles,฀smooth฀muscles฀such฀as฀the฀heart฀
possess฀ TF.฀ The฀ amount฀ of฀ factor฀ VIIa฀ in฀ blood฀ may฀ be฀ increased฀ by฀ genetic฀ promoter฀
mutations,฀but฀more฀commonly฀by฀increased฀blood฀triglyceride฀levels.฀The฀conversion฀of฀
circulating฀factor฀IX฀to฀factor฀IXa฀in฀the฀blood฀of฀obese฀or฀diabetic฀individuals฀results฀in฀an฀
increased฀activation฀of฀factor฀VII.฀Because฀shear฀forces฀in฀the฀heart฀continually฀damage฀
arterioles฀and฀capillaries,฀individuals฀with฀more฀factor฀VIIa฀have฀an฀overly฀primed฀extrinsic฀
coagulation฀path,฀making฀them฀susceptible฀to฀thrombosis฀(heart฀attack).฀The฀capillaries฀are฀
similarly฀injured฀around฀the฀joints,฀but฀these฀regions฀are฀protected฀from฀clotting฀because,฀as฀
noted฀above,฀joints฀have฀no฀tissue฀factor.

11.3.3
The Intrinsic Pathway

The฀intrinsic฀pathway฀is฀activated฀by฀blood฀coming฀into฀contact฀with฀a฀negatively฀charged฀
surface.฀In฀vitro,฀glass฀is฀negatively฀charged,฀and฀blood฀in฀contact฀with฀a฀glass฀tube฀or฀glass฀
slide฀readily฀coagulates.฀The฀intrinsic฀pathway฀is฀illustrated฀in฀Fig.฀11.6b.฀The฀key฀initiator฀
is฀factor฀XII฀(Hageman฀factor),฀a฀weak฀serine฀protease฀that฀binds฀to฀all฀negatively฀charged฀
surfaces.
In฀ vivo,฀ Hageman฀ factor฀ appears฀ activated฀ by฀ small฀ amounts฀ of฀ RNA฀ from฀ crushed฀
cells.฀Once฀bound฀to฀a฀negative฀polymer,฀Hageman฀factor฀changes฀conformation฀so฀as฀to฀
greatly฀increase฀its฀protease฀activity฀(×105).฀Nevertheless,฀there฀is฀so฀little฀of฀factor฀XII฀that฀
factor฀XIIa฀has฀little฀absolute฀activity฀–฀only฀enough฀to฀cleave฀factor฀XI฀(listed฀in฀Table฀11.1,฀
but฀not฀shown฀in฀Fig.฀11.6b).฀Factor฀XI฀is฀also฀present฀in฀small฀amounts฀in฀blood,฀and฀its฀
activated฀factor฀remains฀in฀solution.฀Enough฀of฀factor฀XIa฀is฀produced฀to฀cleave฀factor฀IX฀
that฀has฀already฀bound฀by฀its฀gla฀domain฀to฀phosphatidylserine฀on฀activated฀platelets.฀Factor฀
X฀similarly฀binds฀to฀the฀platelet฀aggregate.฀Factor฀IXa฀weakly฀activates฀factor฀X,฀which,฀in฀
turn,฀produces฀small฀amounts฀of฀thrombin฀from฀prothrombin.฀The฀traces฀of฀thrombin฀acti-
vate฀factor฀VIII,฀a฀nonproteolytic฀cofactor฀accelerator฀that฀binds฀to฀factor฀IXa,฀increasing฀
its฀ability฀to฀convert฀factor฀X฀to฀Xa.

11.3.4
The Common Pathway

Irrespective฀of฀whether฀factor฀X฀is฀activated฀by฀extrinsic฀or฀intrinsic฀pathways,฀it฀cata-
lyzes฀the฀activation฀of฀thrombin฀from฀prothrombin฀(Fig.฀11.6c).฀Initially,฀activated฀fac-
tor฀ X฀ (factor฀ Xa)฀ produces฀ small฀ amounts฀ of฀ thrombin฀ that฀ cleave฀ factor฀ V,฀ which฀ is฀
more฀sensitive฀to฀thrombin฀than฀even฀ibrinogen,฀thrombin’s฀major฀substrate.฀Factor฀Va฀
is฀a฀non-proteolytic฀cofactor฀accelerator.฀It฀binds฀to฀factor฀Xa฀and฀greatly฀increases฀the฀
186 11 Blood Coagulation

rate฀of฀Xa฀catalyzed฀conversion฀of฀prothrombin฀to฀thrombin.฀Factor฀Va฀therefore฀resem-
bles฀factor฀VIIIa,฀which฀accelerates฀the฀production฀of฀factor฀IXa฀in฀the฀intrinsic฀path.฀
The฀production฀of฀factor฀Va฀is฀therefore฀the฀key฀activation฀step฀of฀the฀common฀pathway,฀
and฀it฀generates฀large฀amounts฀of฀thrombin฀from฀prothrombin฀(Fig.฀11.6c).
Thrombin฀ mediates฀ blood฀ clotting฀ by฀ catalyzing฀ ibrinogen฀ to฀ form฀ the฀ ibrin฀ blood฀
clot.฀Activation฀by฀factor฀Xa฀involves฀two฀cuts฀of฀prothrombin.฀The฀irst฀cut฀removes฀the฀
g-carboxyglutamate฀domain฀and฀allows฀binding฀to฀glycoprotein-Iba฀of฀platelets฀trapped฀in฀
the฀soft฀clot.฀This฀binding฀positions฀the฀carboxyglutamate-free฀prothrombin฀beside฀factor฀
Xa,฀which฀completes฀the฀conversion฀to฀thrombin฀by฀cutting฀the฀polypeptide฀into฀two.฀The฀
two฀clipped฀segments฀remain฀attached฀by฀disulide฀bonds฀(Fig.฀11.7),฀permitting฀a฀reorien-
tation฀of฀the฀polypeptide฀fragments฀that฀activates฀thrombin’s฀serine฀protease฀activity.฀This฀
protease฀also฀cleaves฀factor฀XI฀to฀factor฀XIa฀and฀enhances฀intrinsic฀path฀activation.฀Once฀
thrombin฀has฀catalyzed฀the฀cleavage฀of฀ibrinogen,฀it฀remains฀ibrin-attached฀where฀it฀also฀
catalyzes฀the฀activation฀of฀factor฀XIII,฀a฀transglutaminase฀cross-linking฀enzyme฀in฀plasma฀
that฀strengthens฀the฀ibrin฀clot.
Thrombin฀ has฀ two฀ binding฀ sites฀ for฀ exogenous฀ partners฀ or฀ ligands฀ called฀ exosites฀
(Fig.฀11.8).฀Exosite฀I฀binds฀to฀ibrinogen,฀converts฀it฀to฀ibrin฀and฀remains฀bound฀to฀the฀
ibrin.฀Conversion฀of฀ibrinogen฀to฀ibrin฀is฀enhanced฀by฀thrombin฀also฀binding฀to฀platelet฀
GpIba฀ glycan฀ at฀ exosite฀ II.฀ The฀ thrombin-platelet฀ complex฀ also฀ activates฀ a฀ plug฀ from฀

Fig. 11.7฀ Cleavage฀of฀prothrombin฀by฀factor฀

Xa.฀The฀g-carboxyglutamate฀(gla)฀domain฀
attaches฀prothrombin฀to฀the฀activated฀
platelets.฀The฀irst฀cleavage฀of฀the฀prothrom-
bin฀polypeptide฀by฀factor฀Xa฀removes฀the฀
g-carboxyglutamate฀domain฀and฀the฀second฀
activates฀the฀protease฀as฀the฀thrombin฀is฀
released฀from฀the฀membrane฀(Original฀igure฀
submitted฀by฀Dr.฀Paul฀DeAngelis,฀Department฀
of฀Biochemistry,฀University฀of฀Oklahoma฀
HSC,฀Oklahoma฀City,฀OK,฀USA)

Fig. 11.8฀ Thrombin–cofactor฀interac-

tions.฀In฀the฀activated฀thrombin฀
molecule,฀exosite฀I฀lies฀at฀right฀angles฀
to฀exosite฀II.฀Exosite฀1฀binds฀either฀
ibrin฀or฀thrombomodulin฀(TM)฀
depending฀on฀what฀glycan฀binds฀to฀
exosite฀II,฀glycoprotein-1ba฀(Gp1ba)฀
or฀heparin.฀See฀Sects.฀11.3.4฀
and฀11.5.1
11.3.5 The Hemophiliac (Excessive Bleeding Diseases) 187

fresh฀ platelets฀ in฀ the฀ blood฀ (Sect.฀ 11.2.1).฀ Otherwise฀ it฀ diffuses฀ into฀ the฀ tissues฀ where฀
exosite฀ II฀ instead฀ binds฀ to฀ glycosaminoglycans฀ that฀ alter฀ exosite฀ I฀ conformation฀ and฀
functions.฀The฀functions฀of฀ligands฀other฀than฀ibrinogen/ibrin฀and฀GpIba฀are฀discussed฀
in฀Sect.฀11.5.2.฀Fibrin฀formation฀is฀discussed฀in฀Sect.฀11.4.1.

11.3.5
The Hemophiliac (Excessive Bleeding Diseases)

The฀most฀common฀form฀of฀hemophilia฀is฀due฀to฀Von฀Willebrand฀disease฀in฀which฀VWF฀is฀
mutated฀ at฀ the฀ site฀ where฀ it฀ binds฀ to฀ collagen฀ (A3฀ domain)฀ or฀ to฀ platelets฀ (A1฀ or฀ C1฀
domain).฀ The฀ second฀ most฀ common฀ form฀ is฀ classical฀ hemophilia฀ (hemophilia฀ A),฀ long฀
present฀in฀European฀royal฀families.฀Much฀of฀our฀original฀understanding฀of฀blood฀coagula-
tion฀relied฀on฀identifying฀and฀studying฀these฀two฀forms฀of฀hemophilia.
In฀ hemophilia฀ A,฀ the฀ protein฀ sequence฀ of฀ factor฀ VIII฀ is฀ mutated฀ so฀ that฀ it฀ cannot฀ be฀
cleaved,฀resulting฀in฀a฀blocked฀intrinsic฀pathway.฀Therefore,฀tissue฀factor฀must฀be฀activated฀
to฀ stop฀ bleeding฀ (extrinsic฀ pathway).฀ Because฀ the฀ capillaries฀ of฀ joints฀ and฀ muscles฀ are฀
continually฀ damaged฀ by฀ crushing฀ when฀ the฀ surrounding฀ bones฀ and฀ muscles฀ move,฀ they฀
produce฀small฀amounts฀of฀RNA฀that฀activates฀the฀intrinsic฀path.฀Because฀there฀is฀no฀tissue฀
factor฀at฀that฀site฀and฀the฀mutation฀of฀factor฀VIII฀in฀hemophilia฀A฀has฀blocked฀the฀intrinsic฀
path,฀the฀affected฀subjects฀bleed฀into฀the฀joints.฀The฀pressure฀in฀the฀joint฀eventually฀stops฀
the฀bleeding,฀and฀reticuloendothelial฀cells฀are฀recruited฀to฀remove฀the฀blood฀cells.฀The฀pain฀
is฀relieved,฀but฀the฀joint฀structure฀is฀slowly฀destroyed฀and฀over฀time฀surviving฀individuals฀
develop฀arthritis.
Hemophilia฀B฀is฀rarer฀than฀hemophilia฀A฀and฀is฀sometimes฀called฀Christmas฀disease฀
after฀Stephen฀Christmas,฀the฀irst฀patient฀described฀with฀this฀disease.฀It฀is฀due฀to฀a฀mutation฀
that฀prevents฀factor฀IX฀from฀activating฀factor฀VIII.฀However,฀unlike฀hemophilia฀A,฀factor฀
VIII฀can฀be฀activated฀by฀thrombin,฀and฀so฀hemophilia฀B฀is฀less฀severe.
Hemophilia฀C฀occurs฀at฀10%฀of฀the฀frequency฀of฀hemophilia฀A฀and฀is฀the฀least฀common฀
form฀of฀hemophilia฀in฀the฀United฀States,฀where฀it฀mainly฀occurs฀in฀Ashkenazi฀Jews.฀It฀is฀
caused฀by฀a฀deiciency฀of฀factor฀XI.฀Unlike฀hemophilia฀A฀and฀B,฀there฀is฀no฀bleeding฀into฀
joints;฀Factor฀XIIa฀can฀apparently฀activate฀factor฀IX฀directly฀without฀irst฀activating฀factor฀
XI.฀ Nevertheless,฀ aflicted฀ individuals฀ suffer฀ nosebleeds฀ and฀ heavy฀ menstrual฀ bleeding฀
and,฀like฀all฀hemophiliacs,฀require฀a฀clotting฀agent฀to฀prevent฀excessive฀bleeding฀following฀
a฀tooth฀extraction฀(Sect.฀11.6.5).

Two฀ pathways฀ initiate฀ a฀ ibrin฀ clot.฀ Extrinsic฀ path฀ is฀ mediated฀ by฀ tissue฀ factor,฀ also฀
called฀thromboplastin.฀This฀membrane฀protein฀is฀exposed฀when฀pericytes฀are฀damaged.฀
It฀binds฀to฀factor฀VIIa฀in฀blood.฀Factor฀VIIa฀is฀a฀protease฀and฀the฀phospholipid–VIIa–TF฀
complex฀activates฀(converts)฀factor฀X฀by฀cleaving฀it฀to฀Xa.฀Intrinsic฀path฀is฀initiated฀by฀
factor฀XII฀(Hageman฀factor),฀whose฀conformation฀is฀changed฀to฀a฀protease฀(XIIa)฀by฀
contact฀with฀a฀negatively฀charged฀surface฀such฀as฀RNA฀from฀damaged฀or฀necrotic฀cells.฀
188 11 Blood Coagulation

Factor฀XIIa฀activates฀factor฀XI,฀which,฀in฀turn,฀activates฀factors฀IX฀and฀X฀attached฀to฀
platelets฀ aggregated฀ by฀ tissue฀ damage.฀ Xa฀ cleaves฀ prothrombin฀ and฀ makes฀ enough฀
thrombin฀to฀activate฀factor฀VIII฀(IXa฀accelerator).฀The฀phospholipid–IXa–VIIIa฀com-
plex฀activates฀factor฀X.฀Common฀path฀is฀mediated฀by฀Xa฀activating฀factor฀V฀(Xa฀accel-
erator).฀The฀platelet฀membrane฀bound฀Xa–V฀complex฀is฀a฀strong฀activator฀of฀prothrombin฀
and฀ activates฀ enough฀ thrombin฀ to฀ make฀ ibrin฀ from฀ ibrinogen฀ (clot).฀ There฀ are฀ four฀
classes฀of฀hemophilia฀(inadequate฀thrombin฀production):฀(1)฀Von฀Willebrand฀disease฀–฀
mutations฀that฀inhibit฀VWF฀binding฀to฀collagen฀or฀platelets;฀(2)฀Hemophilia฀A฀–฀muta-
tions฀ of฀ factor฀ VIII฀ that฀ inhibit฀ IXa฀ accelerator฀ activation;฀ bleeding฀ occurs฀ mainly฀
because฀of฀intrinsic฀path฀deiciency฀in฀joints;฀(3)฀Hemophilia฀B฀–฀mutations฀of฀factor฀IX฀
conversion฀of฀factor฀VIII,฀but฀factor฀Xa฀conversion฀is฀unaffected;฀side฀effects฀less฀than฀
those฀of฀Hemophilia฀A;฀(4)฀Hemophilia฀C฀–฀deiciency฀of฀factor฀XI;฀side฀effects฀even฀
milder฀than฀Hemophilia฀B฀because฀XIIa฀activates฀some฀factor฀X฀directly.

11.4.1
The Fibrin Blood Clot: Production and Prevention

Blood฀clots฀are฀composed฀of฀ibrinogen,฀an฀abundant฀negatively฀charged฀protein฀in฀blood฀
plasma฀Sect.฀11.2.1.฀Fibrinogen฀is฀also฀secreted฀from฀the฀granules฀of฀activated฀platelets.฀It฀
is฀composed฀of฀three฀interdigitated,฀parallel฀polypeptides,฀Aa,฀Bb,฀and฀g,฀that฀are฀disulide-
bonded.฀ Each฀ trimer฀ is฀ dimerized฀ near฀ its฀ N-terminus฀ by฀ an฀ additional฀ disulide฀ bond฀
between฀two฀Aa฀chains฀(Fig.฀11.9a).฀The฀hexamer฀forms฀a฀dense฀central฀node฀(E฀node,฀
orange)฀and฀two฀on฀either฀side฀(D฀nodes,฀blue;฀Fig.฀11.9b).฀The฀E฀node฀consists฀mainly฀of฀
globular฀ segments฀ of฀ the฀ Aa฀ and฀ Bb฀ polypeptides,฀ whereas฀ the฀ two฀ D฀ nodes฀ are฀ each฀
composed฀of฀the฀Bb,฀and฀g฀chains.฀The฀aIIbb3฀integrin฀of฀activated฀platelets฀attaches฀to฀a฀
single฀site฀on฀a฀D฀node฀to฀aggregate฀them฀(Fig.฀11.3).
Polymerization฀results฀from฀the฀proteolytic฀activity฀of฀thrombin฀speciically฀remov-
ing฀ibrinopeptides฀A฀and฀B฀from฀the฀amino-terminal฀ends฀of฀the฀Aa฀and฀Bb฀peptides฀
(Fig.฀ 11.9b–c).฀ This฀ cleavage฀ is฀ N-terminal฀ to฀ the฀ E฀ node฀ and฀ exposes฀ four฀ knobs฀
(Fig.฀11.9c),฀each฀of฀which฀its฀into฀a฀pit฀present฀in฀the฀D฀node฀of฀ibrinogen฀or฀ibrin.฀
The฀ reaction฀ initially฀ creates฀ a฀ ibrin฀ dimer฀ that฀ polymerizes฀ laterally฀ (Fig.฀ 11.9d–e).฀
The฀ polymer฀ becomes฀ crosslinked฀ by฀ simultaneous,฀ thrombin-induced฀ conversion฀ of฀
factor฀ XIII฀ to฀ transglutaminase฀ (factor฀ XIIIa)฀ that฀ covalently฀ attaches฀ the฀ glutamine฀
amide฀ residues฀ to฀ spatially฀ adjacent฀ lysine฀ residues฀ (Fig.฀ 11.9f)฀ as฀ occurs฀ in฀ ibrillin฀
(Fig.฀6.3).฀At฀irst,฀up฀to฀six฀such฀crosslinks฀form฀between฀g฀chains฀on฀the฀surface฀of฀the฀
D-node.฀Additional฀crosslinks฀form฀later฀between฀a฀chains฀(Fig.฀11.9f)฀that฀lie฀along-
side฀the฀D฀node฀(Fig.฀11.9f).฀Fibronectin,฀which฀is฀secreted฀by฀activated฀platelets฀along฀
with฀ ibrinogen,฀ becomes฀ cross-linked฀ into฀ ibrin,฀ making฀ the฀ clot฀ stronger฀ and฀ more฀
resistant฀ to฀ degradation฀ than฀ fresh฀ ibrin.฀ The฀ ibrin฀ shrinks฀ and฀ a฀ clear฀ luid฀ called฀
serum฀ (blood฀ plasma฀ from฀ which฀ ibrinogen฀ was฀ removed฀ by฀ conversion฀ to฀ ibrin)฀ is฀
released.฀This฀luid฀is฀the฀major฀component฀of฀inlammatory฀exudates฀where฀ibrin฀clot฀
formation฀ is฀ prevented฀ by฀ excessive฀ plasmin฀ activation฀ (see฀ next฀ section฀ and฀
Sect.฀13.1.2).
11.4.1 The Fibrin Blood Clot: Production and Prevention 189

Disulfide bonded
Aa-chain dimer
a Aa 66 kDa
Bb 52 kDa
g 46 kDa
N-ter C-ter C-ter N-ter
Thrombin
cleavage sites

b
Pit E
Fibrin
a quaternary
structure
g
c Thrombin-cleaved
fibrin monomer

D D Self-assembly,
Polymerization
d
Fibrin dimer

e Proto-
fibrin

f
Site of glutaminase
mediated cross-links

Fibrin
strands

Cross-linked fibrin strands

Fig. 11.9฀ Fibrinogen฀and฀ibrin฀structures.฀(a)฀Fibrinogen฀polypeptides:฀Three฀polypeptide฀chains฀are฀

covalently฀linked฀by฀disulide฀bonds.฀Each฀trimer฀is฀further฀head-to-head฀dimerized฀to฀form฀a฀hex-
amer฀whose฀molecular฀weight฀is฀~330,000.฀(b)฀Fibrinogen฀quaternary฀structure:฀The฀N-terminal฀
domains฀of฀the฀two฀Aa฀and฀two฀Bb฀chains฀form฀an฀E฀node฀(letter฀E฀in฀red).฀The฀N-terminal฀30฀or฀
so฀amino฀acids฀of฀each฀of฀these฀polypeptide฀folds฀out฀and฀around฀the฀outside฀of฀the฀E฀domain฀(pink฀
and฀green).฀(c)฀Fibrin฀activation:฀Thrombin฀cleaves฀ibrinopeptides฀A฀(pink;฀16฀amino฀acid฀resi-
dues)฀ and฀ B฀ (green;฀ 14฀ amino฀ acid฀ residues)฀ from฀ ibrinogen.฀ The฀ loss฀ of฀ these฀ ibrinopeptides฀
exposes฀four฀knobs฀(black฀lines)฀that฀protrude฀above฀and฀below฀the฀E฀node.฀(d)฀Fibrin฀dimerization:฀
The฀knobs฀protruding฀from฀the฀four฀cleaved฀polypeptides฀on฀the฀E฀node฀spontaneously฀adhere฀to฀
pits฀in฀the฀D฀node฀of฀another฀molecule฀(blue).฀The฀D฀node฀is฀made฀from฀the฀C-terminal฀portion฀of฀
the฀Bb฀and฀most฀of฀the฀g฀chain.฀The฀Aa฀chain฀forms฀a฀C-terminal฀lexible฀domain฀that฀lies฀away฀
from฀the฀E฀domain฀and฀alongside฀the฀D฀to฀E฀linkage฀(Aa฀chain฀polar฀฀appendage).฀(e)฀Polymerization:฀
The฀ dimer฀ initially฀ lengthens฀ by฀ lateral฀ addition฀ of฀ ibrin฀ monomers฀ to฀ form฀ thin฀ ibrin฀ strands฀
(protoibrin฀ polymers).฀ (f)฀ Covalent฀ cross-linking:฀ The฀ transglutaminase฀ reaction฀ (see฀ text)฀ irst฀
cross-links฀close฀to฀the฀C-terminal฀ends฀of฀the฀g฀chains฀on฀adjacent฀ibrin฀monomers฀(site฀indicated฀
by฀g฀in฀b฀above).฀A฀secondary฀site฀of฀cross-linking฀is฀between฀Aa฀chain฀polar฀appendages฀also฀
indicated฀in฀b.฀This฀slower฀cross-linking฀reaction฀permits฀thick,฀antiparallel฀ibrin฀strands฀to฀develop.฀
The฀chain฀also฀cross-links฀ibrin฀to฀additional฀proteins฀such฀as฀ibronectin฀exposed฀to฀the฀clot฀by฀the฀
tissue฀damage฀(a฀–฀From฀Doug฀Tollefsen฀Lab฀Website,฀Fig.฀10฀slightly฀modiied:฀(http://tollefsen.
wustl.edu/projects/coagulation/coagulation.html#III.D,฀ reproduced฀ here฀ with฀ Dr.฀ Tollefsen’s฀ per-
mission).฀b–f฀–฀Reprinted฀from฀Fig.฀4,฀slightly฀modiied,฀from฀Fuss฀C,฀Palmaz฀JC,฀Sprague฀EA฀
(2001)฀ “Fibrinogen:฀ structure,฀ function,฀ and฀ surface฀ interactions.”฀ J.฀ Vasc.฀ Intervent.฀ Radiol.฀
12(8):677–682.฀Copyright฀2001,฀with฀permission฀from฀Elsevier)
190 11 Blood Coagulation

The฀genes฀for฀all฀three฀chains฀of฀ibrinogen฀are฀found฀within฀a฀50-kb฀length฀of฀DNA฀
on฀chromosome฀4.฀Their฀DNA฀sequences฀show฀a฀high฀degree฀of฀homology,฀suggesting฀
a฀common฀ancestral฀gene.฀The฀homology฀extends฀to฀sites฀upstream฀of฀the฀gene,฀sug-
gesting฀a฀common฀set฀of฀regulatory฀elements฀to฀coordinate฀synthesis.฀The฀thrombin-
cleaved฀ibrinopeptides฀pass฀around฀the฀bloodstream฀to฀the฀liver,฀where฀they฀induce฀the฀
synthesis฀of฀more฀ibrinogen฀by฀inventory฀control฀feedback.฀Thus,฀ibrinogen฀that฀was฀
consumed฀ by฀ clotting฀ signals฀ back฀ to฀ the฀ liver฀ that฀ it฀ needs฀ replenishment฀ in฀ the฀
blood.

11.4.2
Removal of a Blood Clot

A฀ ibrin฀ clot฀ that฀ has฀ sealed฀ an฀ injured฀ blood฀ vessel฀ must฀ be฀ removed฀ (lysed)฀ as฀ the฀
wound฀heals฀and฀new฀capillaries฀develop.฀Lysis฀is฀accomplished฀by฀plasmin,฀a฀serine฀
protease฀ derived฀ from฀ a฀ soluble฀ plasma฀ protein฀ precursor,฀ plasminogen,฀ yet฀ another฀
protein฀ that฀ is฀ made฀ in฀ the฀ liver฀ and฀ secreted฀ into฀ the฀ blood.฀ Plasminogen฀ binds฀ to฀
lysine฀residues฀that฀protrude฀from฀the฀D฀nodes฀of฀ibrin฀but฀not฀ibrinogen.฀The฀binding฀
causes฀plasminogen฀to฀attach฀an฀activator฀from฀the฀surrounding฀stromal฀tissues,฀tissue฀
plasminogen฀activator฀(tPA)฀(Fig.฀11.10a).฀tPA฀is฀a฀serine฀protease฀on฀the฀surface฀of฀
endothelial฀cells฀and฀also฀secreted฀by฀them฀around฀sites฀of฀vascular฀injury.฀The฀attached฀
tPA฀converts฀the฀bound฀plasminogen฀to฀plasmin฀by฀cutting฀its฀single฀polypeptide฀chain฀
into฀ two฀ disulide฀ linked฀ chains,฀ similar฀ to฀ the฀ cutting฀ of฀ prothrombin฀ by฀ factor฀ Xa฀
(Fig.฀11.7).฀The฀newly฀formed฀plasmin฀dissociates฀and฀recognizes฀peptide฀bonds฀down-
stream฀of฀other฀lysine฀residues฀in฀the฀D–E฀node฀connecting฀region฀(Fig.฀11.10a).฀The฀
C-terminal฀ lysine฀ residues฀ of฀ partially฀ hydrolyzed฀ ibrin฀ bind฀ with฀ greater฀ afinity฀ to฀
plasminogen฀ than฀ the฀ D฀ node฀ lysine฀ residues,฀ producing฀ more฀ plasmin฀ and฀ further฀
accelerating฀ibrinolysis.
Plasmin฀is฀soluble฀but฀it฀remains฀active฀in฀the฀location฀of฀a฀clot.฀As฀it฀diffuses฀into฀the฀
blood฀with฀clot฀fragments,฀the฀plasmin฀binds฀to฀a2-antiplasmin,฀a฀serine฀protease฀inhibitor฀
(see฀next฀section).฀In฀addition฀to฀inhibiting฀plasmin฀in฀the฀blood฀(Fig.฀11.10b),฀a2-antiplas-
min฀ inhibits฀ various฀ other฀ serine฀ proteases,฀ especially฀ activated฀ protein฀ C฀ (APC)฀ (next฀
section)฀and฀elastase฀(Sect.฀6.2.1).฀Plasmin฀action฀is฀inhibited฀where฀ibrin฀is฀cross-linked฀
to฀ibronectin,฀but฀the฀large฀ibrin฀fragments฀tend฀to฀promote฀healing.฀The฀fragments฀of฀
ibrin฀are฀named฀as฀shown฀in฀Fig.฀11.10c฀and฀d.฀Factors฀that฀activate฀or฀inhibit฀ibrinolysis฀
are฀summarized฀in฀Table฀11.2.฀
11.4.2 Removal of a Blood Clot 191

Fig. 11.10฀ Plasmin฀dissolution฀of฀ibrin.฀(a)฀Plasminogen฀and฀tissue฀plasminogen฀activator฀(tPA)฀฀

binding:฀The฀D฀node฀in฀proto-ibrin฀and฀ibrin฀strands฀possesses฀lysine฀residues฀that฀protrude฀and฀
bind฀plasma฀plasminogen฀(red฀hexagon).฀The฀attached฀plasminogen฀(but฀not฀free฀plasminogen)฀
binds฀to฀tissue฀plasminogen฀activator฀released฀at฀the฀site฀of฀an฀injury฀(tPA฀–฀green)฀and฀is฀changed฀
to฀ plasmin฀ (pink)฀ by฀ proteolysis.฀ The฀ activated฀ plasmin฀ is฀ released฀ but฀ immediately฀ binds฀ to฀ a฀
nearby฀ motif฀ in฀ the฀ D฀ to฀ E฀ linker฀ region,฀ which฀ it฀ hydrolyzes฀ downstream฀ of฀ lysine฀ residues฀
(slanted฀broken฀lines).฀(b)฀Plasmin฀inactivated:฀The฀plasmin฀is฀now฀free฀to฀diffuse฀away฀and฀is฀
subsequently฀inactivated฀by฀binding฀to฀antiplasmin฀(blue฀crescent)฀in฀the฀blood฀plasma.฀(c)฀Fibrin฀
fragments:฀The฀fragments฀of฀ibrin฀can฀be฀the฀individual฀D฀or฀E฀nodes,฀a฀DE฀combination฀(frag-
ment฀Y),฀or฀an฀E/DD฀combination฀(fragment฀X).฀Usually,฀the฀larger฀fragments฀shown฀are฀obtained฀
because฀of฀incomplete฀fragmentation฀(see฀text)฀(Figure฀is฀derived฀by฀combining฀and฀slightly฀modi-
fying฀Figs.฀3฀and฀5,฀from฀Fuss฀C,฀Palmaz฀JC,฀Sprague฀EA฀(2001)฀“Fibrinogen:฀structure,฀function,฀
and฀surface฀interactions.”฀J.฀Vasc.฀Intervent.฀Radiol.฀12(8):677–682.฀Copyright฀2001,฀with฀permis-
sion฀from฀Elsevier)

Table 11 2฀ Fibrinolysis฀and฀its฀inhibitors
Major฀ibrinolysis฀initiators Major฀ibrinolysis฀inhibitors
Plasminogen/plasmin a2-Antiplasmin
Tissue฀plasminogen฀activator฀(tPA)/urokinase Thrombin-activatable฀ibrinolysis฀inhibitor
192 11 Blood Coagulation

Fibrinogen฀is฀a฀dimer฀of฀each฀of฀three฀interdigitated,฀parallel฀polypeptides฀folded฀into฀a฀
dense฀central฀(N-terminal)฀and฀two฀outer฀(C-terminal)฀nodes.฀The฀central฀(E฀node)฀devel-
ops฀knobs฀after฀thrombin฀hydrolyzes฀off฀N-terminal฀peptides฀from฀two฀of฀the฀three฀poly-
peptide฀chains.฀The฀knobs฀spontaneously฀bind฀to฀pits฀in฀the฀D฀node฀of฀another฀ibrinogen฀
or฀ibrin฀molecule฀and฀simultaneously฀activate฀a฀transglutaminase฀from฀blood฀plasma฀to฀
covalently฀ crosslink฀ adjacent฀ D฀ nodes.฀ The฀ clot฀ includes฀ cross-linked฀ stromal฀ proteins฀
from฀the฀injured฀region,฀especially฀ibronectin,฀and฀it฀shrinks,฀leaving฀a฀clear฀luid฀called฀
serum.฀The฀ibrinopeptides฀activate฀ibrinogen฀expression฀in฀the฀liver฀to฀replace฀the฀ibrin-
ogen฀consumed฀by฀its฀conversion฀to฀ibrin.฀Fibrin฀is฀degraded฀by฀plasmin,฀a฀serine฀pro-
tease.฀Plasminogen฀is฀the฀plasmin฀precursor฀in฀blood฀plasma.฀It฀binds฀to฀lysine฀residues฀
protruding฀ from฀ ibrin฀ D฀ nodes฀ where฀ it฀ attaches฀ tissue฀ plasminogen฀ activator฀ (tPA),฀฀
a฀serine฀proteinase฀secreted฀by฀vascular฀endothelium฀at฀sites฀of฀injury.฀The฀tPA฀cuts฀the฀
bound฀plasminogen฀into฀two฀peptides฀that฀realign฀to฀form฀plasmin,฀which฀loats฀free฀and฀
cuts฀peptide฀bonds฀downstream฀of฀other฀lysine฀residues฀between฀the฀D฀and฀E฀nodes.฀The฀
C-terminal฀lysine฀residues฀activate฀more฀plasminogen,฀accelerating฀plasmin฀production฀
and฀ibrinolysis.฀Plasmin฀that฀diffuses฀away฀from฀a฀clot฀is฀inhibited฀by฀plasma฀a2-antiplas-
min,฀a฀nonspeciic฀protease฀inhibitor.

11.5.1
Prevention of Unwanted Blood Clotting

Capillary฀damage฀causes฀platelets฀to฀secrete฀TXA2,฀an฀eicosanoid฀which฀activates฀them฀to฀
promote฀coagulation฀(Sect.฀11.2.1).฀If฀activated฀platelets฀escape฀the฀damaged฀region,฀they฀
encounter฀ another฀ eicosanoid,฀ prostaglandin฀ I2฀ (PGI2)฀ secreted฀ by฀ undamaged฀ (healthy)฀
endothelial฀ cells.฀ PGI2฀ contains฀ a฀ unique฀ double฀ ring฀ structure฀ (Fig.฀ 13.13)฀ and฀ is฀ also฀
called฀prostacyclin.฀PGI2฀binding฀to฀receptors฀on฀activated฀platelets฀stops฀TXA2฀secretion,฀
collapses฀their฀ibrinogen-binding฀integrin,฀and฀removes฀their฀surface฀phosphatidylserine.฀
Neither฀ calcium฀ ion-binding฀ nor฀ clotting฀ cascade฀ can฀ develop฀ (Fig.฀ 11.11).฀ PGI2฀ also฀
relaxes฀the฀smooth฀muscles฀of฀arterioles,฀keeping฀capillaries฀dilated฀and฀further฀reducing฀
the฀risk฀of฀a฀clot฀forming฀in฀nearby฀undamaged฀regions฀(Sect.฀13.5.5.).
If฀thrombin฀and฀factor฀Xa,฀the฀major฀activated฀blood฀coagulation฀factors฀(Fig.฀11.6),฀
escape฀into฀healthy฀blood฀vessels,฀blood฀clots฀will฀develop฀and฀occlude฀capillaries฀through-
out฀the฀body.฀Direct฀inhibition฀of฀these฀activated฀enzymes฀in฀the฀blood฀low฀utilizes฀serine฀
protease฀inhibitors,฀of฀which฀there฀are฀two฀common฀types:฀a฀Kunitz฀inhibitor฀and฀a฀serpin.฀
The฀former฀possess฀a฀Kunitz฀domain,฀a฀convex฀antiparallel฀β-sheet฀that฀exactly฀its฀into฀the฀
concave฀active฀site฀of฀a฀serine฀protease,฀directly฀blocking฀it฀(lock฀and฀key฀mechanism).฀By฀
contrast,฀serpins฀undergo฀complex฀interactions฀with฀other฀proteins฀to฀cause฀conformational฀
changes฀that฀bait฀and฀block฀the฀catalytic฀action฀(Fig.฀11.12฀shows฀the฀bait).฀Table฀11.3฀lists฀
the฀major฀coagulation฀inhibitors฀and฀cofactors,฀their฀targets฀and฀mechanisms฀of฀action.
Thrombin฀and฀factor฀Xa฀that฀have฀escaped฀into฀the฀blood฀low฀are฀both฀inhibited฀by฀
serpins,฀anti-thrombin฀III฀(ATIII)฀and฀heparin฀cofactor฀II฀(HCII).฀These฀proteins฀bind฀to฀
heparin฀sulfate฀or฀dermatan฀sulfate฀(Sect.฀6.3.1),฀glycosaminoglycans฀which฀are฀secreted฀
onto฀the฀luminal฀surface฀of฀healthy฀endothelial฀cells฀and฀also฀released฀into฀the฀blood฀from฀
mast฀cells฀activated฀by฀an฀injury.฀Among฀the฀heparin฀molecules฀is฀a฀pentaglycan฀sequence฀
11.5.1 Prevention of Unwanted Blood Clotting 193

Va

Blood flow
VIIIa
Protein S
Protein C
Activated
protein C
**Thrombin**
Thrombomodulin Phospholipids

Vaf
Activated
platelets VIIIaf

TxA2
Thrombin Inactivates
PG12

WOUND Activates

Fig. 11.11฀ Regulation฀ of฀ coagulation฀ downstream฀ from฀ a฀ wound฀ Bottom฀ center:฀ Activated฀ platelets฀
secrete฀the฀prostaglandin฀small฀molecule฀thromboxane฀A2฀(TXA2)฀to฀recruit฀and฀activate฀more฀plate-
lets,฀except฀that฀healthy฀intact฀endothelium฀responds฀to฀TXA2฀by฀making฀prostaglandin฀I2฀(PGI2)฀which฀
inhibits฀recruitment฀away฀from฀the฀injury.฀Top:฀Thrombomodulin฀(TM)฀on฀the฀luminal฀surface฀of฀intact฀
blood฀ vessel฀ endothelium฀ binds฀ thrombin฀ and฀ changes฀ its฀ speciicity.฀ Instead฀ of฀ activating฀ ibrin,฀
thrombin฀activates฀protein฀C,฀which฀adheres฀by฀a฀gla฀domain฀to฀activated฀platelets.฀Activated฀protein฀
C฀activates฀plasma฀thrombin฀activatable฀ibrinolysis฀inhibitor฀(see฀text)฀and฀binds฀to฀protein฀S,฀another฀
protease.฀The฀bound฀complex฀with฀protein฀S฀(top฀right฀of฀igure)฀inactivates฀factors฀Va฀and฀VIIIa฀by฀
cutting฀ each฀ of฀ them฀ into฀ two฀ fragments฀ (Vaf฀ and฀ VIIIaf)฀ (Original฀ igure฀ submitted฀ by฀ Dr฀ Paul฀
DeAngelis,฀Department฀of฀Biochemistry,฀University฀of฀Oklahoma฀HSC,฀Oklahoma฀City,฀OK,฀USA)

noted฀in฀the฀legend฀to฀Fig.฀11.12.฀This฀sequence฀attaches฀to฀ATIII฀in฀which฀it฀alters฀the฀
conformation฀of฀a฀surface฀loop฀and฀causes฀an฀arginine฀residue฀to฀protrude฀(Fig.฀11.12).฀
Other฀residues฀on฀the฀same฀heparin฀polymer฀bind฀to฀exosite฀II฀on฀thrombin฀(Fig.฀11.8)฀or฀a฀
similar฀site฀on฀factor฀Xa฀(or฀IXa).฀The฀protruding฀arginine฀residue฀penetrates฀to฀the฀serine฀
residue฀at฀the฀catalytic฀center฀of฀the฀attached฀thrombin฀(or฀factor฀Xa฀or฀factor฀IXa)฀where฀
it฀inactivates฀the฀rate฀of฀proteolysis฀300฀to฀1,000฀fold฀(Fig.฀11.12b).฀Once฀the฀ternary฀com-
plex฀has฀formed,฀conformational฀changes฀keep฀ATIII฀bound฀to฀thrombin฀while฀releasing฀
the฀heparin฀sulfate.฀Heparin฀cofactor฀II฀(HCII)฀binds฀to฀a฀different฀heparin฀motif฀or฀to฀der-
matan฀sulfate.฀It฀undergoes฀similar฀changes,฀but฀a฀different฀amino฀acid฀protrudes฀from฀the฀
homologous฀loop฀to฀inhibit฀thrombin฀or฀factor฀Xa.
The฀ activity฀ of฀ surface-bound฀ factor฀ Xa฀ is฀ controlled฀ by฀ a฀ third฀ serpin,฀ Z-dependent฀
protease฀inhibitor฀(ZPI)฀which฀circulates฀in฀blood฀along฀with฀protein฀Z.฀The฀latter฀has฀a฀gla฀
domain฀and฀it฀attaches฀to฀the฀activated฀platelet฀surface฀alongside฀the฀activated฀clotting฀fac-
tors.฀Protein฀Z฀attaches฀ZPI฀in฀which฀it฀alters฀the฀conformation฀of฀a฀surface฀loop฀and฀causes฀
a฀residue,฀not฀arginine,฀to฀protrude฀and฀inactivate฀the฀bound฀factor฀Xa.฀Protein฀Z฀dissociates฀
from฀the฀ZPI-factor฀Xa฀complex,฀but฀ZPI฀itself฀gets฀cut฀by฀factor฀Xa฀into฀two฀fragments฀
that฀soon฀dissociate฀from฀the฀complex.฀Protein฀Z฀replaces฀a฀glycosaminoglycan฀in฀the฀pre-
vious฀mechanism,฀but฀ZPI฀inhibition฀of฀factor฀Xa฀is฀transient.฀ZPI฀may฀work฀best฀where฀an฀
194 11 Blood Coagulation

Fig. 11.12฀ Heparin฀catalysis฀of฀thrombin฀inhibition฀by฀antithrombin.฀(a)฀Antithrombin฀and฀heparin฀

pentasaccharide฀separately.฀A฀hinge฀region฀(circled)฀expels฀a฀reactive฀center฀loop฀(RCL,฀yellow)฀
from฀the฀bulk฀of฀the฀molecule฀(red,฀green฀and฀gray).฀The฀RCL฀has฀an฀arginine฀residue฀(green)฀that฀
points฀inward,฀towards฀the฀bulk฀of฀the฀molecule.฀Heparin฀is฀to฀the฀right,฀a฀slightly฀curved฀glycan฀chain฀
(blue฀and฀pink).฀(b)฀Antithrombin฀and฀heparin฀pentasaccharide฀bound฀together.฀ATIII฀binds฀strongly฀
to฀heparin฀(large฀forward฀arrow).฀Binding฀causes฀the฀hinge฀region฀of฀ATIII฀to฀expel฀more฀RCL฀loop฀
(yellow)฀from฀the฀central-sheet฀(red฀and฀gray)฀and฀the฀arginine฀residue฀(green)฀now฀points฀outward,฀
away฀from฀central฀sheet฀region.฀(c)฀The฀antithrombin-heparin-thrombin฀ternary฀complex.฀Thrombin฀
(magenta)฀binds฀by฀its฀glycan฀binding฀site฀(Fig.฀11.8)฀to฀the฀heparin฀(blue฀and฀pink)฀attached฀to฀ATIII฀
and฀the฀extended฀RCL฀(yellow)฀with฀its฀outward-pointing฀arginine฀residue฀(green)฀enters฀thrombin’s฀
catalytic฀serine฀residue฀(arrow)฀where฀it฀blocks฀thrombin’s฀proteolytic฀action.฀Once฀the฀ternary฀com-
plex฀has฀formed,฀conformational฀changes฀to฀both฀ATIII฀and฀thrombin฀release฀the฀heparin.฀(Adapted฀
by฀permission฀from฀Li฀W,฀Johnson฀DJ,฀Esmon฀CT฀and฀Huntington฀JA฀(2004฀Sept)฀“Structure฀of฀the฀
antithrombin–thrombin–heparin฀ternary฀complex฀reveals฀the฀antithrombotic฀mechanism฀of฀heparin.”฀
Nat.฀Struct.฀Mol.฀Biol.฀11(9):857–862.฀Copyright฀2004,฀Macmillan฀Publishers฀Ltd.)
11.5.2 Thrombomodulin and Protein C and Protein S 195

Table 11.3฀ Major฀coagulation฀inhibitors

Inhibitor Target
Antithrombin฀IIIa,b Thrombina,฀Factor฀Xaa
Heparin฀cofactor฀IIa,b Thrombina,฀Factor฀Xaa
Tissue฀factor฀pathway฀inhibitor฀(TFPI)c TF-VIIa,฀Xa
Z-dependent฀protease฀inhibitor฀(ZPI) b
Xa฀on฀surface
Protein฀Cd Thrombin
Protein฀Se Protein฀C
Protein฀Ze ZPI
a
Possesses฀a฀glycosaminoglycan฀binding฀site
b
Serpin฀domain
c
Kunitz฀domain
d
Possesses฀a฀gla฀domain฀and฀generates฀a฀protease
e
Possesses฀a฀gla฀domain฀without฀generating฀a฀protease

increased฀low฀of฀blood฀provides฀more฀Z฀and฀ZPI฀proteins฀to฀bind฀and฀inhibit฀factor฀Xa฀
attached฀to฀activated฀platelets฀or฀damaged฀cell฀membrane฀surfaces.
Tissue฀factor฀pathway฀inhibitor฀(TFPI)฀is฀a฀Kunitz฀type฀inhibitor฀composed฀of฀three฀Kunitz฀
domains.฀The฀N-terminal฀Kunitz฀domain฀binds฀to฀and฀inhibits฀the฀VIIa-TF฀complex฀that฀acti-
vates฀factor฀X฀(Fig.฀11.6a),฀whereas฀the฀downstream฀Kunitz฀domain฀binds฀directly฀to฀factor฀
Xa฀and฀inhibits฀it฀strongly฀and฀permanently.฀No฀function฀has฀yet฀been฀demonstrated฀for฀the฀
third฀Kunitz฀domain.฀The฀C-terminal฀region฀of฀TFPI฀is฀basic฀and฀remains฀attached฀to฀endothe-
lial฀cell฀surfaces฀where฀it฀inhibits฀inadvertent฀factor฀Xa฀activation.฀Studies฀in฀mice฀indicate฀
that฀knocking฀out฀the฀gene฀for฀TFPI฀stops฀fetal฀development.฀Indeed,฀clotting฀diseases฀such฀as฀
a฀stroke฀and฀heart฀attacks฀are฀associated฀with฀mutations฀of฀ATIII,฀HCII฀and฀ZPI,฀but฀not฀of฀
TFPI.฀Like฀TF฀(Sect.฀11.3.1),฀diminished฀TFPI฀activity฀may฀be฀incompatible฀with฀life.

11.5.2
Thrombomodulin and Protein C and Protein S

If฀thrombin฀escapes฀the฀damaged฀region฀without฀being฀inactivated฀by฀the฀ATIII฀or฀HCII฀
serpins,฀its฀glycan-binding฀site฀(exosite฀II)฀directly฀interacts฀with฀the฀glycosaminoglycans฀
on฀ the฀ inner฀ surface฀ of฀ an฀ intact฀ blood฀ vessel.฀ This฀ interaction฀ causes฀ conformational฀
changes฀to฀exosite฀I,฀which฀no฀longer฀binds฀ibrinogen,฀but฀instead฀binds฀to฀thrombomodu-
lin,฀Tm฀(Fig฀11.11).฀Tm฀is฀secreted฀by฀endothelial฀cells฀onto฀the฀capillary฀lumen฀surface฀
alongside฀glycosaminoglycans฀and฀tPA฀(tPA฀is฀described฀in฀Sect.฀11.4.2).
Once฀thrombin฀binds฀to฀Tm,฀the฀complex฀cuts฀two฀unrelated฀plasma฀proteins,฀thrombin฀
activatable฀ibrinolysis฀inhibitor฀(TA฀ibrinolysis฀inhibitor)฀and฀protein฀C,฀instead฀of฀ibrino-
gen,฀ transglutaminase฀ and฀ factors฀ VIII฀ and฀ V฀ (Sect.฀ 11.3.4).฀ The฀ thrombin–Tm฀ activated฀
(cleaved)฀TA฀ibrinolysis฀inhibitor฀is฀a฀carboxypeptidase฀that฀removes฀the฀C-terminal฀lysine฀
residues.฀These฀residues฀are฀present฀in฀ibrin฀D-E฀region฀fragments฀(Fig.฀11.10)฀following฀ini-
tial฀plasmin฀action,฀and฀their฀loss฀prevents฀a฀rapid฀acceleration฀of฀ibrinolysis฀(Sect.฀11.4.2).
196 11 Blood Coagulation

Protein฀C฀(listed฀in฀Tables฀11.1฀and฀11.3)฀is฀a฀vitamin฀K-dependent฀(VKD)฀protein฀with฀
a฀ multi-domain฀ structure.฀ It฀ has฀ a฀ serine฀ protease฀ domain,฀ two฀ epidermal฀ growth฀ factor฀
(EGF)฀domains,฀and฀a฀gla฀domain฀at฀its฀N฀terminus.฀The฀gla฀domain฀facilitates฀the฀Ca2+฀
ion-mediated฀ coordination฀ to฀ phospholipids฀ on฀ activated฀ endothelial฀ cells฀ and฀ platelets,฀
albeit฀with฀lower฀afinity฀than฀other฀vitamin฀K-dependent฀proteins.฀The฀thrombin–Tm฀com-
plex฀activates฀protein฀C฀to฀become฀a฀serine฀protease฀(activated฀protein฀C,฀APC),฀which฀
binds฀to฀another฀gla฀domain฀plasma฀protein,฀protein฀S.฀APC฀bound฀to฀protein฀S฀cleaves฀
factors฀VIIIa฀and฀Va฀(making฀Vaf฀and฀VIIIaf),฀reducing฀the฀clotting฀signal฀(Fig.฀11.11).฀The฀
extent฀of฀this฀reduction฀is฀the฀reverse฀of฀its฀boosting฀by฀factors฀VIIIa฀and฀Va,฀~105.฀Thus,฀
the฀thrombin-Tm฀complex฀inhibits฀clot฀formation฀at฀the฀edges฀of฀the฀injured฀area฀by฀chang-
ing฀the฀speciicity฀of฀thrombin฀so฀that฀it฀cleaves฀and฀activates฀plasma฀protein฀C.
Protein฀ S฀ was฀ discovered฀ because฀ bleeding฀ defects฀ occur฀ in฀ some฀ individuals฀ whose฀
blood฀clotting฀factors฀appear฀normal.฀Studies฀of฀their฀blood฀plasma฀proteins฀indicated฀that฀
they฀ were฀ missing฀ what฀ became฀ known฀ as฀ protein฀ S.฀ The฀ absence฀ of฀ protein฀ S฀ prevents฀
activated฀ protein฀ C฀ from฀ degrading฀ factors฀ VIIIa฀ and฀ Va.฀ Because฀ these฀ helper฀ factors฀
remain฀up-regulated,฀individuals฀with฀absent฀or฀mutated฀protein฀S฀are฀unusually฀prone฀to฀
strokes฀and฀heart฀attacks.฀In฀other฀individuals,฀excessive฀clotting฀is฀due฀to฀factor฀V฀and฀fac-
tor฀VIII฀mutations฀that฀prevent฀cleavage฀of฀the฀activated฀forms฀(Va฀and฀VIIIa)฀by฀the฀APC-
protein฀S฀complex.฀The฀activation฀and฀inactivation฀of฀factors฀V฀and฀VIII฀differ฀because฀the฀
respective฀activation฀sites฀use฀different฀proteases฀and฀different฀motifs.฀A฀mutation฀of฀the฀site฀
at฀which฀APC-protein฀S฀cleaves฀factorVa฀(or฀VIIIa)฀results฀in฀excessive฀clotting,฀strokes฀and฀
increased฀susceptibility฀to฀heart฀attacks,฀the฀same฀phenotype฀as฀a฀mutation฀in฀protein฀S.
Figure฀11.13฀summarizes฀the฀entire฀clotting฀process:฀initiation,฀major฀pathways,฀ibrin฀
formation฀and฀ibrinolysis,฀clotting฀control฀pathways,฀and฀major฀associated฀diseases.

There฀are฀three฀processes฀that฀inactivate฀the฀clotting฀process:฀platelet฀inactivation฀by฀
the฀ eicosanoid฀ system;฀ thrombin฀ inactivation฀ by฀ glycosaminoglycan/serpin฀ (ATIII);฀
and฀factors฀Va฀and฀VIIIa฀inactivation฀by฀the฀thrombomodulin/protein฀C/protein฀S฀sys-
tem฀Independently฀of฀their฀binding฀to฀protein฀C,฀thrombin-thrombomodulin฀complexes฀
also฀inhibit฀ibrinolysis฀by฀binding฀to฀and฀activating฀a฀protein฀that฀removes฀the฀ibrin฀
binding฀site฀for฀plasmin.฀The฀absence฀of฀protein฀S,฀or฀mutations฀that฀inhibit฀protein฀S฀
binding฀to฀activated฀protein฀C,฀keep฀factors฀VIIIa฀and฀Va฀upregulated฀and฀cause฀exces-
sive฀clotting฀and฀increased฀susceptibility฀to฀heart฀attacks.฀Mutations฀of฀factors฀VIIIa฀
and฀Va฀that฀prevent฀their฀cleavage฀by฀the฀APC/protein฀S฀complex฀also฀cause฀excessive฀
clotting.฀The฀clotting฀process฀and฀its฀controls฀are฀summarized฀in฀Fig.฀11.13.

11.6.1
Drugs to Remove a Pathogenic Thrombus or Embolus: “Clot Busters”

Age-฀and฀diet-related฀degeneration฀of฀the฀artery฀intima฀(Sect.฀11.1.1)฀results฀in฀fatty฀depos-
its฀that฀narrow฀an฀artery,฀predisposing฀to฀endothelial฀surface฀injury฀and฀clotting,฀patho-
genic฀thrombus฀formation.฀A฀thrombus฀most฀often฀results฀in฀a฀myocardial฀infarction฀(heart฀
attack)฀or฀stroke,฀but฀less฀dramatic฀effects฀occur฀from฀pathogenic฀thrombi฀in฀other฀organs.฀
Alternatively,฀a฀clot฀may฀develop฀because฀of฀an฀irregular฀blood฀low฀caused฀by฀a฀cardiac฀
11.6.1 Drugs to Remove a Pathogenic Thrombus or Embolus: “Clot Busters” 197

Fig. 11.13฀ Coagulation฀low฀

chart.฀Blood฀coagulation฀
events฀are฀divided฀into฀
initiation,฀extrinsic฀and฀
intrinsic฀path฀activation,฀
and฀the฀common฀path฀to฀
ibrin.฀Controls฀are฀
ibrinolysis฀as฀healing฀
begins฀and฀inhibition฀of฀
clotting฀factors฀in฀a฀healthy฀
(uninjured)฀blood฀vessel.฀
Diseases฀are฀excessive฀
bleeding฀(hemophilia)฀and฀
excessive฀clotting฀(Original฀
igure)

arrhythmia,฀or฀an฀excessive฀lack฀of฀movement,฀for฀example,฀of฀the฀legs฀during฀a฀long฀light฀
or฀at฀a฀computer.฀The฀irregular฀blood฀low฀allows฀VWF฀aggregates฀to฀interact฀with฀and฀
activate฀platelets฀away฀from฀the฀endothelium.
A฀clot฀that฀breaks฀free,฀an฀embolus,฀may฀become฀entrapped฀in฀a฀small฀artery฀or฀lodge฀in฀
ine฀ capillary฀ beds,฀ most฀ often฀ within฀ the฀ brain฀ or฀ lung,฀ causing฀ ischemia฀ to฀ the฀ down-
stream฀region.฀Inhibition฀of฀the฀blood฀supply฀to฀the฀brain฀causes฀a฀stroke฀whose฀symptoms฀
depend฀on฀the฀region฀affected.฀Inhibition฀of฀the฀oxygenation฀of฀hemoglobin฀in฀the฀lung฀
causes฀a฀dry฀cough,฀shortness฀of฀breath฀or฀(rarely)฀sudden฀death.฀Moving฀around฀avoids฀a฀
stagnant฀blood฀low฀and฀frequent฀drinks฀of฀water฀keep฀the฀blood฀from฀being฀overconcen-
trated.฀Both฀these฀measures฀prevent฀an฀embolus฀from฀developing.฀Clots฀develop฀rapidly฀
and฀must฀be฀broken฀up฀within฀an฀hour฀or฀2฀for฀serious฀tissue฀damage฀to฀be฀prevented.฀For฀
example,฀the฀clot฀is฀usually฀in฀an฀artery฀and฀blocks฀oxygen฀and฀nutrients฀from฀the฀down-
stream฀region.฀If฀the฀artery฀is฀totally฀obstructed,฀all฀the฀downstream฀tissues฀will฀die฀from฀a฀
lack฀of฀oxygen฀and฀nutrients.฀More฀commonly,฀there฀is฀a฀partial฀closure,฀and฀there฀is฀time฀
to฀dissolve฀the฀clot฀before฀oxygen฀and฀nutrient฀deprivation฀kills฀the฀downstream฀cells.
Treatment฀requires฀a฀“clot฀buster”฀to฀remove฀the฀ibrin฀clot.฀Because฀plasminogen฀cir-
culates฀in฀the฀blood฀in฀relatively฀large฀amounts,฀a฀common฀procedure฀is฀to฀give฀intravenous฀
tissue฀plasminogen฀activator฀(tPA)฀to฀speed฀its฀conversion฀to฀plasmin฀(Fig.฀11.10).฀Thus,฀
intravenous฀tPA฀is฀the฀best฀therapy฀for฀acute฀symptoms฀of฀clot฀development.฀Because฀the฀
half-life฀of฀infused฀tPA฀in฀blood฀plasma฀is฀only฀4–5฀min,฀it฀should฀be฀continuously฀infused฀
for฀about฀an฀hour฀to฀dissolve฀enough฀clot฀to฀limit฀a฀potential฀ischemia.฀Recombinant฀human฀
tPA฀is฀preferred฀to฀animal฀tPA฀because฀it฀is฀the฀natural฀protein฀and฀can฀be฀safely฀injected฀
198 11 Blood Coagulation

into฀the฀blood฀stream฀(nonantigenic).฀Oral฀tPA฀cannot฀be฀absorbed฀because,฀like฀any฀other฀
food,฀it฀is฀irst฀digested฀to฀amino฀acids฀and฀small฀peptides.
Two฀other฀proteins,฀urokinase฀and฀streptokinase,฀are฀cheaper฀alternatives฀for฀tPA,฀but฀
they฀activate฀plasminogen฀throughout฀the฀body,฀not฀just฀the฀ibrin-bound฀plasminogen฀like฀
tPA฀(Fig.฀11.10).฀Urokinase฀is฀a฀plasminogen฀activator฀that฀initiates฀plasmin฀degradation฀
of฀the฀stroma฀following฀injury,฀infection฀or฀environmental฀stress฀(Sect.฀8.1.3).฀Streptokinase฀
is฀made฀by฀various฀pathogenic฀streptococcal฀bacteria฀and฀it฀enhances฀bacterial฀spread฀in฀
the฀tissues฀by฀its฀streptokinase฀acting฀like฀urokinase฀to฀activate฀plasmin฀and฀degrade฀the฀
stroma.฀Because฀urokinase฀and฀streptokinase฀do฀not฀restrict฀the฀plasminogen฀activation฀to฀
ibrin,฀there฀is฀a฀greater฀risk฀of฀uncontrolled฀bleeding฀developing฀because฀of฀clotting฀fail-
ure.฀Subjects฀may฀also฀become฀resistant฀to฀streptokinase฀because฀it฀is฀a฀foreign฀protein.฀
Sequential฀treatments฀will฀quickly฀induce฀antibodies฀that฀bind฀to฀and฀remove฀streptokinase฀
from฀the฀blood฀before฀it฀can฀react฀with฀plasminogen.

11.6.2
Drugs That Inhibit Excessive Clot Formation

Widespread฀uncontrolled฀clotting฀(disseminated฀intravascular฀coagulation,฀DIC)฀is฀associated฀
with฀infection,฀drug฀complications,฀allergies฀or฀various฀other฀diseases฀or฀conditions.฀In฀this฀
instance,฀therapy฀is฀better฀if฀it฀prevents฀the฀widespread฀thrombin฀activation฀instead฀of฀dissolv-
ing฀ the฀ ibrin.฀ As฀ noted฀ above,฀ heparin฀ is฀ a฀ potent฀ anticoagulant฀ that฀ promotes฀ thrombin฀
interaction฀with฀antithrombin฀III฀(see฀Fig.฀11.12฀and฀Sect.฀11.3.4).฀Low-molecular฀weight฀
heparin฀is฀the฀safest฀to฀use฀because฀it฀binds฀to฀only฀one฀molecule฀of฀thrombin฀(factor฀IIa).฀The฀
heparin฀is฀injected฀because,฀like฀tPA,฀it฀is฀not฀absorbed฀in฀intact฀form฀from฀the฀gut.฀Heparin฀is฀
also฀ used฀ during฀ surgery฀ to฀ prevent฀ incisions฀ from฀ clotting฀ until฀ after฀ stent฀ placement฀ or฀
stitching฀is฀completed.฀An฀antidote฀to฀heparin฀is฀protamine฀sulfate,฀a฀positively฀charged฀small฀
protein฀that฀complexes฀with฀heparin฀and฀prevents฀ATIII฀or฀thrombin฀binding.

11.6.3
Drugs That Retard Clot Formation

Vitamin฀K฀antagonists,฀most฀commonly฀the฀coumarins฀(coumadin฀and฀warfarin),฀are฀a฀set฀of฀
anticoagulant฀drugs฀often฀referred฀to฀as฀“blood฀thinners.”฀These฀drugs฀structurally฀mimic฀vita-
min฀K฀and฀prevent฀g-carboxyglutamate฀(gla)฀formation฀(Sect.฀11.2.2).฀The฀gla-containing฀coag-
ulation฀factors฀localize฀by฀binding฀to฀Ca2+฀ions฀on฀the฀surface฀of฀activated฀platelets฀(Fig.฀11.4).
Coumarins฀act฀slowly฀because฀proteins฀that฀have฀already฀been฀synthesized฀with฀a฀gla฀
domain฀(Table฀11.1)฀must฀irst฀be฀eliminated.฀The฀clotting฀factor฀half-life฀is฀1–5฀days,฀and฀
so฀it฀takes฀about฀a฀week฀for฀the฀gla฀residue-containing฀proteins฀to฀decrease฀signiicantly.฀In฀
the฀presence฀of฀a฀high฀concentration฀of฀vitamin฀K,฀high฀levels฀of฀dietary฀quinone฀(KH2)฀are฀
made฀ and฀ oxidized฀ without฀ an฀ epoxide฀ intermediate.฀ Vitamin฀ K฀ therefore฀ promotes฀ gla฀
formation฀in฀the฀presence฀of฀coumarins.฀Nevertheless,฀new฀protein฀has฀to฀be฀synthesized,฀
post-translationally฀modiied฀at฀the฀appropriate฀glutamate฀residues฀and฀secreted.฀Increasing฀
the฀blood฀concentration฀of฀gla฀clotting฀factors฀is฀therefore฀slow฀also.
11.6.5 Drugs That Promote Clotting 199

Coumarins฀are฀potentially฀highly฀toxic.฀Blood฀levels฀must฀be฀monitored฀carefully,฀and฀vita-
min฀K฀must฀be฀given฀as฀necessary฀if฀overdosing฀is฀detected.฀An฀important฀concern฀is฀dehydra-
tion,฀ which฀ increases฀ the฀ drug฀ concentration.฀ Dehydration฀ is฀ especially฀ common฀ in฀ elderly฀
patients฀who฀are฀also฀often฀on฀a฀diuretic.฀In฀addition,฀a฀change฀in฀multivitamins฀may฀shift฀the฀
correct฀dose฀of฀a฀coumarin.฀It฀is฀therefore฀especially฀important฀to฀communicate฀with฀both฀patient฀
and฀supervising฀doctor฀before฀undertaking฀any฀dental฀surgery฀procedures฀on฀these฀patients.

11.6.4
Drugs That Inhibit Platelet Activation

Platelets฀use฀cyclooxygenases฀to฀make฀thromboxane฀A2฀for฀recruiting฀and฀activating฀other฀plate-
lets฀(Sect.฀11.2.1).฀Intact฀endothelial฀cells฀cyclooxygenases฀to฀make฀prostaglandin฀PGI2฀(Sect.฀
11.5.1).฀Because฀platelets฀are฀cell฀remnants฀and฀have฀no฀DNA฀or฀RNA,฀they฀cannot฀transcribe฀
or฀translate฀new฀proteins.฀Thus,฀once฀the฀platelet฀cyclooxygenases฀are฀inhibited,฀the฀platelets฀
cannot฀ make฀ more฀ thromboxane฀ to฀ promote฀ their฀ activation.฀ By฀ contrast,฀ PGI2-producing฀
endothelial฀cells฀simply฀synthesize฀additional฀enzyme฀to฀make฀more฀PGI2฀(Fig.฀11.11).
All฀ cyclooxygenases฀ (Classes฀ 1฀ and฀ 2)฀ are฀ irreversibly฀ inhibited฀ by฀ nonsteroidal฀ anti-
inlammatory฀drugs฀(NSAIDS)฀such฀as฀aspirin฀and฀ibuprofen.฀Thus,฀a฀daily฀low฀dose฀of฀aspi-
rin฀inhibits฀clotting฀by฀reducing฀thromboxane฀levels฀from฀platelets,฀while฀interfering฀less฀with฀
PGI2฀production.฀Because฀platelets฀are฀almost฀completely฀renewed฀weekly,฀aspirin฀dosage฀
must฀be฀maintained.฀Ibuprofen฀does฀not฀inhibit฀platelet฀thromboxane฀production฀as฀effec-
tively฀as฀aspirin฀(Sect.฀13.5.5).฀Low฀doses฀of฀aspirin฀are฀preferable฀because฀high฀doses฀inter-
fere฀with฀vitamin฀K฀uptake฀and฀promote฀excessive฀intestinal฀bleeding.฀Spontaneous฀bleeding฀
can฀occur฀due฀to฀a฀lack฀of฀vitamin฀K,฀just฀as฀when฀an฀individual฀is฀given฀coumarin.

11.6.5
Drugs That Promote Clotting

After฀surgery,฀an฀excessive฀activation฀of฀plasmin฀can฀cause฀blood฀loss฀and฀inhibitors฀of฀plas-
min฀are฀used฀to฀retard฀ibrinolysis.฀The฀drugs฀of฀choice฀are฀epsilon-aminocaproic฀acid฀and฀
tranexamic฀acid,฀but฀the฀latter฀is฀preferred฀because฀smaller฀doses฀inhibit฀plasmin.฀Plasmin฀
interacts฀with฀an฀amino฀acid฀motif฀between฀the฀D฀and฀E฀domains฀of฀ibrin฀(see฀Fig.฀11.10).฀
The฀motif฀contains฀a฀lysine฀residue฀that฀is฀N-terminal฀to฀the฀plasmin฀cleavage฀point฀and฀a฀
substrate฀for฀plasmin฀binding.฀Thus,฀plasmin฀binds฀to฀ibrin฀by฀a฀lysine฀motif฀and฀cuts฀down-
stream฀(C-terminal)฀to฀its฀binding฀site.฀Tranexamic฀acid฀binds฀to฀plasmin฀and฀prevents฀it฀from฀
binding฀to฀lysine฀residues฀on฀the฀motif;฀the฀clot฀is฀stabilized฀and฀no฀longer฀hydrolyzed.
Although฀slowing฀clot฀removal฀might฀seem฀dangerous,฀tranexamic฀acid฀does฀not฀affect฀
the฀clotting฀process฀and฀is฀therefore฀relatively฀safe.฀If฀given฀intravenously฀for฀uncontrolled฀
bleeding฀after฀surgery,฀a฀correct฀amount฀of฀antidotal฀heparin฀should฀be฀available฀and฀ready฀
to฀inject฀intravenously฀in฀a฀syringe.฀Minutes฀may฀be฀important฀after฀an฀intravenous฀injec-
tion.฀Tranexamic฀acid฀is฀given฀orally฀when฀postsurgical฀bleeding฀is฀a฀problem฀or฀antici-
pated฀after฀surgery฀as฀in฀hemophiliacs.฀Tranexamic฀acid฀is฀rapidly฀absorbed฀and฀excreted,฀
preventing฀the฀plasma฀concentration฀rising฀to฀dangerous฀levels.
200 11 Blood Coagulation

Drugs฀to฀remove฀a฀pathogenic฀thrombus฀or฀embolus:฀Recombinant฀tissue฀plasmino-
gen฀activator฀(tPA)฀breaks฀up฀ibrin฀clots฀by฀activating฀plasmin฀when฀intravenously฀
infused฀for฀an฀hour.฀Urokinase฀and฀streptokinase฀are฀cheaper฀alternatives,฀but฀their฀
global฀activation฀of฀plasminogen฀may฀cause฀uncontrolled฀bleeding.฀Subjects฀previ-
ously฀exposed฀to฀streptokinase฀may฀have฀antibodies฀that฀prevent฀its฀activation฀of฀plas-
minogen.฀ Drugs฀ that฀ inhibit฀ excessive฀ clotting:฀ Intravenous฀ heparin฀ possessing฀ the฀
pentaglycan฀ activates฀ ATIII฀ to฀ inactivate฀ thrombin,฀ factor฀ Xa฀ and฀ IXa.฀ Drugs฀ that฀
retard฀clotting:฀clot฀Coumarins฀and฀vitamin฀K฀antagonists฀slow฀thrombin฀production฀
by฀inhibiting฀gla฀formation.฀Because฀inhibition฀and฀antidote฀(vitamin฀K)฀take฀a฀week฀
to฀be฀effective,฀blood฀clotting฀eficacy฀must฀be฀monitored.฀Drugs฀that฀inhibit฀platelet฀
activation:฀Cyclo-oxygenase฀inhibitors฀such฀as฀aspirin฀inhibit฀thromboxane฀A4฀pro-
duction,฀slowing฀platelet฀activation.฀Prostaglandin฀I2฀production฀is฀also฀reduced,฀but฀
the฀enzyme฀in฀endothelial฀cells฀is฀constantly฀being฀synthesized฀de฀novo,฀unlike฀the฀
platelet฀enzyme฀which฀takes฀a฀week฀to฀replace.฀High฀doses฀of฀cyclooxygenase฀inhibi-
tors฀interfere฀with฀vitamin฀K฀uptake฀and฀promote฀intestinal฀bleeding.฀Drugs฀that฀pro-
mote฀ clotting:฀ Major฀ surgery,฀ severe฀ menstruation,฀ and฀ hemophilia฀ overactivate฀
plasmin,฀ destabilizing฀ ibrin฀ clots.฀ Inhibitors฀ of฀ plasmin฀ are฀ epsilon-aminocaproic฀
acid฀ or฀ tranexamic฀ acid฀ which฀ retard฀ ibrin฀ digestion฀ by฀ covering฀ extruding฀ lysine฀
residues฀that฀otherwise฀promote฀plasminogen฀activation.

11.6.6
Laboratory Tests to Determine the State of the Blood Clotting System

Laboratory฀tests฀can฀identify฀a฀faulty฀component฀of฀clotting฀or฀whether฀an฀elderly฀patient฀
is฀required฀to฀alter฀coumarin฀drug฀or฀vitamin฀K฀dosages฀before฀a฀tooth฀is฀extracted.฀Samples฀
of฀the฀patient’s฀blood฀are฀taken฀and฀treated฀in฀various฀ways฀to฀allow฀the฀observation฀of฀the฀
appropriate฀steps฀in฀the฀hemostasis฀pathways.฀For฀example,฀tissue฀factor฀is฀added฀to฀the฀
blood฀ sample฀ for฀ the฀ prothrombin฀ time฀ (PT)฀ test.฀ Alternatively,฀ certain฀ clotting฀ system฀
components฀in฀the฀blood฀sample฀are฀removed฀or฀inactivated.฀These฀tests฀can฀be฀run฀quickly฀
on฀small฀samples฀of฀blood.

1.฀ The฀prothrombin฀time฀(PT)฀test฀is฀measured฀by฀how฀long฀fresh฀blood฀clots฀after฀adding฀
a฀ixed฀amount฀of฀tissue฀factor.฀This฀time฀(normally฀11–14฀s)฀is฀a฀measure฀of฀the฀rate฀
of฀factor฀VII฀conversion฀to฀factor฀VIIa฀(extrinsic฀pathway).฀The฀test฀is฀usually฀done฀in฀
children฀to฀determine฀a฀coagulation฀factor฀deiciency,฀or฀in฀adults฀monitor฀the฀dose฀of฀
a฀ coumarin฀ blood฀ thinner฀ such฀ as฀ wafarin.฀ Time฀ is฀ increased฀ in฀ individuals฀ taking฀
coumarins฀or฀exhibiting฀liver฀disease.฀Specialized฀tests฀are฀required฀to฀exclude฀liver฀
disease.฀Time฀is฀shortened฀in฀disseminated฀intravascular฀coagulation,฀but฀again฀further฀
tests฀are฀required฀(see฀item฀4).
2.฀ The฀activated฀partial฀thromboplastin฀time฀(APTT)฀measures฀the฀eficacy฀of฀the฀intrinsic฀
and฀common฀pathways.฀Blood฀is฀collected฀and฀oxalate฀or฀citrate฀is฀added฀to฀arrest฀coag-
ulation฀by฀binding฀calcium.฀In฀the฀laboratory,฀calcium฀is฀added฀to฀reverse฀the฀anticoagu-
11.6.6 Laboratory Tests to Determine the State of the Blood Clotting System 201

lant฀ effect฀ of฀ the฀ oxalate฀ and฀ phospholipid.฀ Silica,฀ a฀ synthetic฀ contact฀ phase,฀ is฀ then฀
added฀to฀the฀plasma฀sample฀and฀mixed.฀The฀time฀for฀a฀thrombus฀(clot)฀to฀form฀takes฀
25–35฀s.฀A฀shorter฀time฀has฀no฀meaning.฀A฀prolonged฀time฀occurs฀in฀hemophiliacs,฀but฀
a฀bleeding฀time฀(BT)฀test฀(below)฀is฀also฀required฀to฀separate฀von฀Willebrand’s฀disease฀
from฀hemophilia฀A฀through฀C.฀If฀the฀patient฀is฀taking฀blood฀thinners,฀or฀an฀anticoagu-
lant฀such฀as฀heparin,฀clotting฀takes฀up฀to฀two฀and฀a฀half฀times฀longer.
3.฀ The฀ bleeding฀ time฀ (BT)฀ test฀ is฀ made฀ from฀ how฀ long฀ a฀ small,฀ precise฀ skin฀ wound฀
requires฀to฀stop฀bleeding฀and฀measures฀platelet฀functions.฀A฀nick฀is฀made฀in฀the฀ear,฀
and฀touched฀with฀a฀fresh฀piece฀of฀ilter฀paper฀every฀15฀s฀until฀the฀bleeding฀stops.฀The฀
count฀of฀how฀many฀15฀s฀intervals฀have฀passed฀indicates฀the฀bleeding฀time.฀Normal฀is฀
six฀intervals฀of฀15฀s.฀More฀intervals฀indicate฀von฀Willebrand’s฀disease,฀or฀thrombo-
cytopenia฀(deiciency฀of฀platelets),฀or฀a฀patient฀taking฀aspirin฀or฀ibuprofen.฀Patients฀
with฀hemophila฀A฀through฀C฀or฀taking฀coumarin฀have฀a฀normal฀number฀of฀intervals฀
because฀these฀conditions฀do฀not฀affect฀platelet฀plug฀formation.

A฀new฀method฀is฀replacing฀the฀BT฀assay.฀A฀platelet฀function฀analyzer฀simulates฀the฀expo-
sure฀of฀blood฀to฀a฀vascular฀wall฀injury.฀Instead฀of฀a฀nick฀in฀the฀ear,฀blood฀is฀collected฀by฀
venipuncture฀into฀a฀tube฀containing฀3.8%฀citrate฀(anticoagulant)฀and฀aspirated฀from฀a฀res-
ervoir฀through฀a฀capillary฀to฀a฀membrane฀coated฀with฀collagen฀and฀epinephrine฀or฀ADP฀
(Sect.11.2.1).฀The฀membrane฀contains฀a฀central฀aperture฀that฀simulates฀a฀blood฀vessel฀wall฀
injury.฀As฀blood฀lows฀through฀the฀aperture,฀the฀platelets฀adhere฀and฀aggregate.฀The฀time฀
until฀the฀aperture฀is฀completely฀occluded฀by฀the฀platelet/red฀blood฀cell฀thrombus,฀is฀the฀
closure฀time.฀The฀membrane฀coat฀and฀amount฀of฀citrate฀affects฀the฀closure฀time.฀For฀a฀col-
lagen/epinephrine฀aperture,฀the฀bleeding฀time฀is฀normally฀about฀2฀min฀for฀3.2%฀citrated฀
blood฀and฀2.5฀min฀for฀3.8%.

(1)฀Prothrombin฀time฀test฀determines฀intrinsic฀factor฀VIIa฀activation,฀which฀is฀gla-acti-
฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀
vated฀so฀that฀the฀time฀of฀the฀test฀increased฀by฀coumarin;฀(2)฀The฀activated฀partial฀throm-
฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀
boplastin฀time฀measures฀the฀eficacy฀of฀the฀intrinsic฀and฀common฀pathways,฀which฀are฀
฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀
prolonged฀ in฀฀hemophiliacs,฀ ฀ von฀ Willebrand’s฀
฀ ฀ disease฀
฀ ฀ or฀ when฀ ฀ ฀heparin฀ ฀or฀ coumarin฀
฀ ฀ ฀
therapy฀ ฀is฀ ฀given;฀ (3)฀฀The฀฀bleeding฀ time฀฀ is฀ longer฀
฀ ฀ in฀฀ von฀฀Willebrand’s฀
฀ ฀ disease฀
฀ and฀ ฀
thrombocytopenia,฀or฀during฀low฀dose฀NSAIDS฀therapy,฀but฀normal฀in฀the฀presence฀of฀
฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀
coumarin฀ ฀and฀ ฀ in฀ hemophilia฀
฀ ฀ ฀ A฀ through฀ ฀ ฀ C;฀ (4)฀ An฀
฀ ฀increased฀ thrombin฀
฀ ฀ clotting฀
฀ ฀ time฀
indicates฀a฀lack฀of฀ibrinogen฀or฀slower฀rate฀of฀ibrin฀clot฀development,฀usually฀due฀to฀
฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀
liver฀disease฀(detectable฀in฀other฀ways),฀but฀occasionally฀to฀genetic฀mutations.฀A฀faster฀
฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀ ฀
thrombin฀clotting฀time฀may฀indicate฀disseminated฀vascular฀thrombosis฀and฀is฀conirmed฀
฀ ฀ ฀ ฀ ฀ ฀
by฀the฀presence฀of฀abnormally฀high฀ibrinopeptide฀levels.
 Saliva
12
฀

This฀chapter฀briely฀describes฀the฀key฀elements฀of฀salivary฀gland฀structure,฀their฀prod-
ucts,฀and฀their฀various฀functions,฀concluding฀with฀a฀discussion฀of฀how฀they฀control฀the฀
oral฀microbiota฀in฀conjunction฀with฀oral฀hygiene฀(Sect.฀1).฀Following฀a฀brief฀discussion฀
of฀secretion฀(Sect.฀2),฀there฀is฀a฀detailed฀description฀of฀the฀mucous฀proteins฀and฀their฀
glycans฀(Sect.฀3)฀and฀their฀relationship฀to฀glycans฀at฀the฀red฀blood฀cell฀surface฀(Sect.฀4).฀
The฀chapter฀concludes฀with฀a฀detailed฀description฀of฀the฀structure฀and฀functioning฀of฀
the฀ salivary฀ amylase฀ (Sect.฀ 5)฀ and฀ its฀ proline-rich฀ proteins฀ and฀ salivary฀ agglutinin฀
(Sect.฀6).

12.1.1.
Cell Biology of Salivary Glands

Saliva฀is฀secreted฀into฀the฀oral฀cavity฀by฀pairs฀of฀major฀glands,฀the฀parotid,฀submandibular,฀
and฀sublingual฀(Fig.฀12.1a),฀and฀by฀many฀minor฀glands฀scattered฀throughout฀the฀oral฀epi-
thelium.฀Each฀gland฀is฀composed฀of฀clusters฀of฀epithelial-like฀cells,฀acinar฀cells฀secreting฀
a฀ serous฀ (watery)฀ luid฀ or฀ tubular฀ cells฀ secreting฀ a฀ mucous฀ (viscous)฀ luid฀ (Fig.฀ 12.1b).฀
Parotid฀glands฀are฀composed฀only฀of฀acinar฀cells;฀sublingual฀and฀submandibular฀glands฀
have฀both฀types฀(mixed).฀Each฀cluster฀ends฀with฀a฀small฀duct฀that,฀in฀the฀major฀glands,฀
joins฀up฀to฀a฀common฀collection฀duct฀and฀empties฀into฀the฀oral฀cavity฀from฀the฀upper,฀pos-
terior฀region฀of฀each฀cheek฀(parotid),฀or฀on฀either฀side฀of฀the฀frenum฀beneath฀the฀tongue฀
(submandibular฀and฀sublingual).฀Minor฀glands฀are฀composed฀of฀a฀single฀acinus฀or฀tubule฀
cluster,฀mostly฀serous฀at฀the฀lips฀and฀increasingly฀mucous฀towards฀the฀back฀of฀the฀mouth.

12.1.2.
Whole Saliva: Collection and Composition

Whole฀saliva฀is฀collected฀by฀asking฀volunteers฀to฀spit฀into฀an฀ice-cooled฀vial.฀If฀necessary,฀
the฀low฀is฀stimulated฀by฀chewing฀parafin฀wax฀or฀washed฀elastic฀bands.฀Pure฀parotid฀gland฀
secretions฀ are฀ collected฀ using฀ a฀ Lashley฀ Cup,฀ a฀ small฀ plastic฀ cup฀ held฀ against฀ the฀ duct฀
oriice฀inside฀the฀cheek฀by฀a฀vacuum.฀The฀secretion฀drains฀into฀a฀vial฀through฀a฀tube฀that฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 203
DOI:฀10.1007/978-3-540-88116-2_12,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
204 12 Saliva

Accessory
parotid gland

Parotid duct

Opening
of sub-
mandibular
(Wharton’s)
duct

Parotid
Sublingual gland
gland
Body of mandible
Submandibular gland
Submandibular
(Wharton’s) duct

(1) (2) (3)

Fig. 12.1฀ The฀major฀salivary฀glands:฀(a)฀Anatomy:฀The฀parotid฀gland฀lies฀under฀the฀ear฀and฀behind฀

the฀back฀of฀the฀mandible.฀The฀duct฀excretes฀its฀contents฀bilaterally฀into฀the฀buccal฀mucosa฀oppo-
site฀the฀second฀upper฀molars.฀The฀sublingual฀gland฀lies฀beneath฀the฀tongue฀and฀the฀submandibular฀
gland฀curls฀around฀the฀lower฀inside฀region฀of฀the฀mandible,฀mostly฀forward฀of฀the฀angle.฀The฀
ducts฀from฀these฀two฀glands฀release฀their฀secretions฀close฀together฀bilaterally฀beneath฀the฀tongue.฀
(b)฀ Composition:฀ The฀ salivary฀ glands฀ are฀ exocrine฀ glands฀ that฀ are฀ composed฀ of฀ serous฀ cells฀
around฀a฀single฀acinus฀as฀in฀a฀minor฀gland฀(1),฀or฀group฀of฀acini฀sharing฀a฀common฀collection฀duct฀
as฀in฀a฀major฀gland฀(2),฀or฀mucus฀cells฀around฀a฀single฀tubule฀(3),฀or฀group฀of฀tubules฀(similar฀to฀
acini฀ except฀ that฀ cells฀ around฀ the฀ collection฀ duct฀ are฀ in฀ a฀ tubular฀ arrangement;฀ not฀ shown).฀฀
(a:฀ Figure฀ from฀ Dorland’s฀ Illustrated฀ Medical฀ Dictionary.฀ 31st฀ ed.฀ Elsevier฀ Saunders:฀ 2007,฀฀
p฀790.฀Figure,฀“Salivary฀Glands,”฀under฀“Glands”฀Online฀at฀http://medicaldictionary.thefreedic-
tionary.com/_/viewer.aspx?path=dorland&name=gland_salivary.jpg;฀b:฀Figure฀from฀http://www.
siumed.edu/~dking2/erg/glands.htm#acini)

its฀into฀a฀hole฀in฀the฀cup.฀The฀low฀is฀stimulated฀by฀sucking฀a฀sour฀lemon฀drop฀or฀by฀apply-
ing฀a฀drop฀of฀sodium฀citrate฀to฀the฀tongue.฀The฀collection฀of฀pure฀submandibular,฀sublin-
gual,฀or฀minor฀gland฀secretions฀requires฀individually฀designed฀equipment.
Whole฀saliva฀is฀a฀dilute,฀viscous฀solution฀containing฀electrolytes,฀proteins,฀and฀epi-
thelial฀ cells฀ from฀ the฀ oral฀ mucosa.฀ The฀ major฀ electrolytes฀ are฀ sodium฀ chloride฀ and฀
12.1.3. Functions of the Salivary Components 205

sodium฀bicarbonate.฀Mucin฀proteins฀mediate฀the฀viscosity,฀saliva’s฀most฀obvious฀char-
acteristic.฀Mucins฀are฀proteoglycans฀with฀many฀short,฀negatively฀charged฀side-chains,฀
and฀they฀are฀made฀only฀by฀mucous฀cells:฀5-15%฀of฀proteins฀from฀the฀submandibular฀
gland,฀10-30%฀of฀proteins฀from฀the฀sublingual฀gland,฀and฀virtually฀all฀the฀proteins฀from฀
minor฀mucous฀glands.฀Besides฀mucins,฀the฀major฀proteins฀in฀saliva฀are฀a-amylase฀(a1,4฀
glucan฀endohydrolase)฀and฀proline-rich฀proteins.฀The฀salivary฀glands฀secrete฀a-amy-
lase฀into฀blood฀as฀well฀as฀saliva.฀Salivary฀a-amylase฀is฀encoded฀by฀a฀single฀gene฀(Amyl)฀
on฀ chromosome฀ 1,฀ but฀ traces฀ of฀ enzymes฀ in฀ saliva฀ modify฀ it,฀ so฀ that฀ a฀ mixture฀ of฀
฀proteins฀of฀similar฀size฀and฀charge฀(a-amylase฀isozyme฀families)฀appears฀on฀gel฀elec-
trophoresis.฀ The฀ proline-rich฀ proteins฀ are฀ encoded฀ as฀ one฀ of฀ two฀ or฀ three฀ different฀
alleles฀on฀each฀of฀six฀adjacent฀genes฀on฀chromosome฀12฀and฀also฀undergo฀proteolysis฀
in฀saliva.฀They฀therefore฀give฀rise฀to฀an฀extremely฀complex฀mixture฀of฀small฀polypep-
tides฀on฀gel฀electrophoresis.฀Mucins฀are฀encoded฀by฀MUC5B฀on฀chromosome฀11฀and฀
MUC7฀on฀chromosome฀4.
In฀an฀individual฀maintaining฀oral฀hygiene,฀the฀total฀protein฀content฀of฀saliva฀is฀~1.6฀mg/
mL,฀ much฀ less฀ than฀ in฀ blood฀ plasma.฀ About฀ half฀ the฀ proteins฀ are฀ amylase,฀ 40-45%฀ are฀
proline-rich฀proteins,฀and฀5-10%฀are฀mucins.฀Stimulated฀saliva฀has฀20%฀less฀protein,฀but฀
up฀to฀a฀tenfold฀greater฀sodium฀chloride฀content฀and฀up฀to฀double฀the฀sodium฀bicarbonate฀
content.฀Bicarbonate฀is฀produced฀by฀the฀same฀intracellular฀carbonic฀anhydrase฀in฀acinar฀
cells฀as฀in฀osteoclasts฀and฀red฀blood฀cells฀(Chap.฀10).฀The฀greater฀sodium฀bicarbonate฀con-
tent฀of฀stimulated฀saliva฀makes฀its฀pH฀more฀alkaline฀(7.4–7.8)฀than฀unstimulated฀saliva฀
(6.8–7.2).
The฀salivary฀glands฀also฀secrete฀urea,฀which฀some฀oral฀bacteria฀convert฀to฀ammonia฀
and฀carbon฀dioxide฀with฀an฀enzyme,฀urease.฀The฀greater฀content฀of฀ammonia฀results฀in฀
the฀oral฀cavity฀being฀better฀buffered฀to฀acids฀and฀better฀protection฀from฀caries฀(Chap.฀
15,฀Sect.฀3).฀Calcium฀and฀phosphate฀are฀also฀present฀in฀saliva฀at฀supersaturating฀con-
centrations฀but฀do฀not฀precipitate฀due฀to฀protein฀chelation.฀Whole฀saliva฀also฀contains฀
small฀amounts฀of฀various฀other฀proteins:฀proteases,฀protease฀inhibitors฀(cystatins),฀type฀
IV฀carbonic฀anhydrase,฀statherin,฀histatins,฀lysozyme,฀salivary฀agglutinin,฀and฀immu-
noglobulin฀A.
Finally,฀ the฀ salivary฀ glands฀ concentrate฀ sodium฀ nitrate฀ from฀ fruit฀ and฀ vegetables.฀
The฀sodium฀nitrate฀is฀absorbed฀into฀the฀blood฀plasma฀and฀concentrated฀10฀–฀20฀fold฀by฀
salivary฀gland฀cells฀before฀secretion.฀The฀nitrate฀in฀saliva฀is฀reduced฀to฀sodium฀nitrite฀
by฀ Eikenella฀ corrodens,฀ a฀ common฀ commensal฀ bacterium฀ in฀ human฀ saliva฀ (Sect.฀
12.1.5).฀Sodium฀nitrite฀is฀an฀important฀vasodilator,฀but฀it฀cannot฀cross฀healthy฀tissues.

12.1.3.
Functions of the Salivary Components

If฀a฀major฀salivary฀gland฀is฀lost฀from฀trauma฀or฀disease,฀or฀if฀nasal฀allergies฀or฀sinus฀infec-
tions฀ cause฀ persistent฀ mouth-breathing,฀ or฀ if฀ tobacco฀ smoking฀ persists,฀ the฀ oral฀ cavity฀
becomes฀dry฀(xerostomia).฀The฀oral฀mucosa฀and฀teeth฀become฀covered฀with฀bacteria฀and฀
dental฀caries฀and฀periodontal฀disease฀become฀dificult฀to฀control.฀The฀functions฀of฀whole฀
206 12 Saliva

saliva฀are฀(1)฀lubricating฀the฀oral฀tissues฀and฀food฀particles;฀(2)฀promoting฀the฀clearance฀of฀
food฀particles;฀(3)฀protecting฀the฀oral฀mucosa฀and฀teeth฀from฀excessive฀bacterial฀coloniza-
tion;฀(4)฀forming฀an฀acquired฀pellicle฀that฀protects฀teeth฀surfaces฀from฀dissolution฀or฀over-
accretion;฀and฀(5)฀stabilizing฀teeth฀surfaces฀from฀bacterial฀acid฀dissolution.฀The฀irst฀three฀
functions฀are฀performed฀mainly฀by฀the฀mucin฀and฀water฀content,฀aided฀by฀amylase฀and฀
immunity฀ proteins฀ described฀ later,฀ the฀ fourth฀ is฀ associated฀ with฀ other฀ secreted฀ salivary฀
proteins,฀statherin,฀and฀the฀proline฀rich฀proteins,฀and฀the฀last฀is฀caused฀by฀buffers฀in฀saliva,฀
mostly฀sodium฀bicarbonate฀(Table฀12.1).
The฀stability฀of฀the฀tooth฀surface฀to฀spontaneous฀dissolution฀and฀accretion฀is฀primarily฀
mediated฀by฀statherin,฀a฀43฀amino฀acid฀polypeptide฀encoded฀by฀a฀single฀gene฀(Stath)฀on฀
chromosome฀ 4.฀ Its฀ two฀ N-terminal฀ serine฀ residues฀ spontaneously฀ become฀ phosphory-
lated฀in฀saliva,฀and฀they฀attach฀statherin฀tightly฀to฀exposed฀tooth฀enamel,฀preventing฀dis-
solution฀or฀calcium฀phosphate฀accretion.฀Nevertheless,฀all฀proteins฀in฀whole฀saliva฀adhere฀
to฀ some฀ extent฀ to฀ enamel,฀ forming฀ an฀ acquired฀ pellicle฀ that฀ replaces฀ abraded฀ enamel฀
cuticle฀(Chap.฀9).฀Differences฀in฀amount฀or฀composition฀of฀the฀proteins฀in฀whole฀saliva฀
cause฀differences฀in฀acquired฀pellicle฀composition.
Carbonic฀anhydrase฀and฀sodium฀bicarbonate฀together฀neutralize฀the฀acids฀produced฀by฀
bacterial฀metabolism฀of฀dietary฀carbohydrate฀(Fig.฀12.2).฀When฀salivary฀carbonic฀anhy-
drase฀ is฀ swallowed,฀ it฀ adheres฀ to฀ the฀ mucosal฀ surface฀ of฀ the฀ stomach฀ where฀ it฀ remains฀
active฀and฀forms฀carbonic฀acid฀from฀sodium฀bicarbonate฀in฀the฀gastric฀mucosa.฀A฀lack฀of฀
salivary฀carbonic฀anhydrase฀causes฀acid฀to฀remain฀longer฀in฀the฀stomach,฀contributing฀to฀
peptic฀disease฀in฀addition฀to฀dental฀caries฀(Sect.฀15.3.3).

Table 12.1฀ Functions฀of฀saliva฀effector฀molecules

Functions Effector฀molecules
Lubricate฀oral฀mucosa฀and฀food฀particles฀฀ Mucins
and฀inhibit฀bacterial฀colonization
Facilitates฀clearance฀and฀inhibits฀฀ Water฀(dilute฀secretion)
bacterial฀colonization
Digests฀starch฀in฀food฀particles,฀฀ Amylase
promoting฀bolus฀cohesion
Stabilize฀tooth฀surface Sodium฀bicarbonate,฀carbonic฀anhydrase,฀
and฀statherin
Form฀acquired฀enamel฀pellicle Proteins
Innate฀immunity฀that฀inhibits฀฀ Peroxidases,฀histatins,฀lysozyme,฀and฀
bacterial฀growth lactoferrin
Innate฀immunity฀that฀enhances฀฀ Salivary฀agglutinin฀and฀mucin
bacterial฀clearance
Acquired฀immunity฀that฀enhances฀฀ Secretory฀immunoglobulin฀(sIgA)
bacterial฀clearance
12.1.4. Innate and Acquired Immune Proteins in Saliva 207

Acquired pellicle
Saccharolytic bacteria
Saliva

Sodium
Lactic acid bicarbonate
CH3 CHOH COO− H+ + Na+ HCO3−

Reversible

Sodium lactate Carbonic acid

CH3 CHOH COO− Na+ + H+ HCO3−
Carbonic
Irreversible
anhydrase

Water and carbon dioxide

H2O + CO2

Fig. 12.2฀ Neutralization฀of฀bacterial฀acids฀by฀salivary฀carbonic฀anhydrase:฀Saliva฀contains฀proteins฀

that฀form฀the฀acquired฀pellicle฀to฀which฀saccharolytic฀bacteria฀attach฀and฀grow฀anaerobically฀using฀
dietary฀ carbohydrate฀ as฀ substrate.฀ Lactic฀ acid฀ is฀ produced฀ and฀ reacts฀ reversibly฀ with฀ salivary฀
sodium฀bicarbonate฀to฀make฀sodium฀lactate฀and฀carbonic฀acid.฀Traces฀of฀salivary฀carbonic฀anhy-
drase฀in฀saliva฀contribute฀to฀the฀acquired฀pellicle฀and฀also฀remain฀in฀solution.฀The฀enzyme฀converts฀
carbonic฀acid฀to฀water฀and฀carbon฀dioxide.฀This฀step฀is฀irreversible฀because฀carbon฀dioxide฀is฀lost฀
to฀the฀environment฀whenever฀the฀mouth฀is฀opened.฀Thus,฀lactic฀acid฀is฀transformed฀into฀its฀salt,฀
sodium฀lactate.฀(Original฀Figure)

12.1.4.
Innate and Acquired Immune Proteins in Saliva

Innate฀immunity฀in฀saliva฀is฀mediated฀by฀enzymes,฀peroxidase,฀lactoferrin฀and฀lysozyme,฀by฀
small฀peptides฀histatins฀and฀proline-rich฀proteins,฀and฀by฀two฀proteins,฀salivary฀agglutinin฀
and฀mucin฀MG2.฀All฀are฀secreted,฀but฀the฀enzymes฀may฀be฀added฀independently฀฀from฀leuko-
cytes฀at฀the฀gingival฀sulcus฀(Sects.฀13.2.3.฀and฀13.2.4).฀The฀peroxidase฀reaction฀is฀discussed฀
Chap.฀16,฀Fig.฀16.8b.฀In฀saliva฀the฀cofactor฀(glutathione)฀is฀absent,฀and฀the฀electron-deicient฀
oxygen฀species฀released฀by฀enzyme฀action฀convert฀chloride฀(Cl-1)฀and฀thiocyanate฀(SCN-1)฀
ions฀to฀hypochlorite฀(OCl-1)฀and฀hypothiocyanate฀(OSCN-1)฀ions฀which฀diffuse฀into฀bacteria฀
and฀inhibit฀their฀growth฀or฀kill฀them฀directly.฀Lactoferrin฀is฀encoded฀by฀chromosome฀3฀and฀
secreted฀as฀an฀aggregate฀of฀four฀identical฀polypeptides฀surrounding฀two฀ferric฀(Fe3+)฀ions.฀It฀
is฀ electron฀ deicient฀ like฀ hypochlorite฀ or฀ hypothiocyanate฀ and฀ retards฀ the฀ colonization฀ of฀
Gram-negative฀bacteria฀and฀fungi฀on฀the฀oral฀mucosa.฀Lysozyme฀is฀a฀glycoside฀hydrolase฀
encoded฀by฀Lyz฀on฀chromosome฀12.฀It฀lyses฀bacteria฀by฀hydrolyzing฀a฀glycan฀bond฀of฀pepti-
doglycan฀on฀the฀surface฀of฀Gram-positive฀bacteria฀(Sect.฀1.4.1)฀and฀is฀distantly฀related฀to฀
amylase฀(GH฀family฀22,฀Sect.฀12.5.1.).฀Gram-negative฀bacteria฀are฀not฀readily฀hydrolyzed฀by฀
this฀enzyme฀because฀a฀second,฀outer฀membrane฀covers฀the฀peptidoglycan฀(Sect.฀1.4.1).
Histatins฀are฀positively฀charged฀small฀polypeptides฀encoded฀by฀two฀genes฀on฀chromo-
some฀2,฀His1฀and฀His2.฀They฀bind฀to฀the฀oral฀mucosa฀and฀teeth฀where฀they฀inhibit฀bacterial฀
208 12 Saliva

growth.฀ The฀ proline-rich฀ proteins฀ (PRPs)฀ may฀ be฀ positively฀ or฀ negatively฀ charged.฀ The฀
latter,฀the฀acidic฀PRPs,฀bind฀to฀teeth฀where฀they฀likely฀alter฀dental฀bioilm฀development฀
(Sect.฀ 12.6.1).฀ Salivary฀ agglutinin฀ binds฀ to฀ various฀ oral฀ bacteria฀ and฀ causes฀ them฀ to฀ be฀
swallowed฀instead฀of฀forming฀bioilms.฀Mucin฀MG2฀(Sect.฀12.3.1)฀clears฀fungi฀similarly.
Acquired฀immunity฀is฀mediated฀by฀lymphocytes฀directly฀or฀by฀their฀secreting฀immun-
globulins฀(Ig).฀An฀acquired฀immune฀response฀in฀the฀oral฀cavity฀is฀induced฀by฀antigens,฀
usually฀a฀foreign฀molecule฀from฀a฀bacterium฀or฀fungus฀that฀colonizes฀the฀oral฀cavity฀from฀
the฀environment.฀High฀molecular฀weight฀surface฀components฀of฀these฀organisms฀activate฀
gut-associated฀lymphoid฀tracts฀(GALT),฀which฀include฀the฀tonsils฀of฀the฀pharynx.฀Lymphoid฀
cells฀within฀these฀tissues฀are฀induced฀to฀secrete฀IgA฀into฀the฀surrounding฀blood฀plasma,฀
which฀passes฀into฀saliva฀and฀other฀secretions.฀In฀saliva,฀secreted฀IgA฀(sIgA)฀binds฀to฀the฀
antigen฀that฀induced฀its฀production.฀The฀IgA–antigen฀complex฀is฀swallowed฀and฀digested฀
in฀the฀stomach฀or฀small฀intestine.

12.1.5.
Poor Oral Hygiene Adds Bacteria and Host Leukocyte Products to Saliva

The฀bacteria฀in฀a฀healthy฀oral฀cavity,฀the฀commensal฀oral฀microbiota,฀protect฀it฀from฀dis-
ease฀by฀repelling฀disease-causing฀(pathogenic)฀bacteria.฀A฀weak฀point฀in฀this฀protection฀is฀
adherence฀of฀the฀commensal฀microbiota฀to฀the฀acquired฀pellicle.฀Microbial฀bioilms฀attach฀
to฀the฀teeth,฀and฀differences฀ in฀ salivary฀ protein฀ composition฀ cause฀ differences฀ in฀ which฀
bacteria฀make฀up฀the฀commensal฀oral฀microbiota.฀This฀microbiota฀extends฀into฀the฀gingi-
val฀sulcus฀and฀induces฀gingival฀crevicular฀luid฀(GCF),฀an฀inlammatory฀exudate฀derived฀
from฀ blood฀ plasma฀ containing฀ activated฀ leukocytes฀ (Sect.฀ 13.1.2).฀ Thus,฀ differences฀ in฀
salivary฀composition฀inluence฀how฀much฀GCF฀is฀produced.฀The฀GCF฀is฀a฀better฀substrate฀
for฀microbial฀growth฀than฀saliva,฀and฀the฀presence฀of฀commensal฀bioilms฀on฀teeth฀surfaces฀
provides฀a฀more฀favorable฀environment฀for฀the฀colonization฀by฀pathogenic฀bacteria฀that฀are฀
otherwise฀present฀in฀only฀trace฀amounts.฀These฀bacteria฀are฀gram฀negative฀and฀asaccharo-
lytic.฀They฀release฀proteases฀and฀cell฀surface฀components฀into฀the฀oral฀cavity฀along฀with฀
asaccharolytic฀metabolic฀end฀products,฀ammonia,฀amines,฀and฀sulides฀(Sect.฀1.3.1),฀which฀
give฀mixed฀whole฀saliva฀(spit)฀an฀offensive฀odor.

Whole฀saliva฀(spit)฀is฀a฀dilute,฀viscous฀solution฀of฀proteins฀and฀shed฀epithelial฀cells.฀The฀
major฀electrolytes฀are฀sodium,฀chloride,฀and฀bicarbonate.฀Calcium฀and฀phosphate฀are฀
present฀ at฀ a฀ supersaturated฀ concentration.฀ Viscosity฀ is฀ due฀ to฀ mucins,฀ proteoglycans฀
with฀numerous฀short฀glycan฀chains฀that฀lubricate฀the฀oral฀cavity,฀hold฀a฀bolus฀of฀chewed฀
food฀together,฀and฀reduce฀bacterial฀adherence฀to฀teeth.฀Besides฀mucins,฀the฀major฀pro-
teins฀secreted฀in฀saliva฀are฀amylase฀and฀proline-rich฀proteins.฀The฀major฀electrolytes,฀
sodium฀chloride฀and฀sodium฀bicarbonate,฀increase฀with฀stimulation฀of฀salivary฀low,฀but฀
the฀ protein฀ content฀ decreases.฀ All฀ proteins฀ in฀ whole฀ saliva฀ adhere฀ to฀ some฀ extent฀ to฀
12.2.1. Physiology and Biochemistry of Saliva Secretion 209

enamel,฀forming฀an฀acquired฀pellicle฀that฀replaces฀abraded฀enamel฀cuticle.฀Differences฀
in฀the฀amount฀or฀composition฀of฀whole฀saliva฀may฀result฀in฀individual,฀saliva-associated฀
differences฀ in฀ dental฀ caries.฀ Small฀ amounts฀ of฀ carbonic฀ anhydrase฀ protect฀ the฀ teeth฀
from฀bacterial฀acid฀dissolution.฀Small฀amounts฀of฀statherin฀are฀secreted฀to฀protect฀the฀
teeth฀from฀accreting฀salivary฀calcium฀phosphate.฀Various฀innate฀and฀acquired฀immunity฀
proteins฀protect฀from฀bacterial฀infections.฀Innate฀immunity฀in฀secreted฀saliva฀is฀medi-
ated฀by฀enzymes,฀peroxidase,฀lactoferrin฀and฀lysozyme,฀by฀small฀peptides,฀histatins฀and฀
proline-rich฀ proteins,฀ and฀ by฀ two฀ proteins,฀ salivary฀ agglutinin฀ and฀ mucin฀ MG2.฀
Acquired฀immunity฀is฀mediated฀by฀IgA.฀A฀bacterial฀bioilm฀(plaque)฀develops฀on฀the฀
acquired฀pellicle฀where฀it฀induces฀the฀exudation฀of฀gingival฀crevicular฀luid฀from฀under-
lying฀capillaries.฀Asaccharolytic฀bacteria฀grow฀on฀this฀luid฀and฀add฀malodorous฀meta-
bolic฀end฀products฀to฀whole฀saliva.

12.2.1.
Physiology and Biochemistry of Saliva Secretion

In฀the฀cytosol,฀polypeptides฀destined฀for฀secretion฀in฀saliva฀are฀steered฀to฀the฀cytosolic฀side฀
of฀the฀rough฀endoplasmic฀reticulum฀(ER)฀by฀the฀amino฀acid฀sequence฀of฀their฀N-terminal฀
domains.฀This฀domain฀interacts฀with฀a฀signal฀recognition฀particle฀(SRP)฀in฀the฀cytosol฀and฀
stops฀ translation฀ until฀ the฀ ribosomal/SRP฀ complex฀ moves฀ to฀ a฀ ribosomal฀ receptor.฀
Placement฀on฀the฀receptor฀releases฀the฀SRP฀particle฀and฀allows฀the฀ribosome฀to฀resume฀
translation.฀Polypeptide฀synthesis฀is฀completed฀in฀the฀ER฀lumen฀instead฀of฀the฀cytosol,฀and฀
moved฀ to฀ the฀ smooth฀ ER,฀ where฀ certain฀ asparagine฀ amide฀ residues฀ are฀ glycosylated฀
(N-linked฀glycosylation).฀The฀glycan฀is฀preformed฀attached฀to฀dolichol฀diphosphate฀in฀the฀
cytosol฀and฀transferred฀to฀the฀asparagine฀amide฀acceptor฀after฀switching฀from฀cytosolic฀to฀
luminal฀face฀of฀the฀ER฀(Fig.฀12.3,฀upper฀third).฀Protein-rich฀vesicles฀form฀and฀are฀trans-
ported฀ to฀ the฀ cis฀ face฀ of฀ the฀ Golgi฀ (Fig.฀ 12.3,฀ lower฀ two-thirds).฀ As฀ the฀ proteins฀ pass฀
through฀the฀Golgi,฀glycosidases฀and฀glycan฀synthetases฀greatly฀alter฀the฀N-linked฀glycans฀
and฀attach฀other฀glycans฀to฀the฀−OH฀group฀of฀certain฀serine฀and฀threonine฀residues฀in฀the฀
protein฀(O-linked฀glycosylation,฀Fig.฀12.3,฀lower฀two-thirds).
Vesicles฀containing฀proteins฀destined฀for฀intracellular฀use฀in฀lysosomes฀or฀outer฀mem-
branes฀bud฀off฀from฀the฀trans฀face฀of฀the฀Golgi.฀Depending฀on฀their฀contents,฀some฀of฀the฀
vesicles฀are฀diverted฀to฀nonsecretory฀vesicles฀by฀the฀presence฀of฀phosphomannose฀residues฀
on฀their฀N-linked฀glycans฀or฀by฀possessing฀domains฀rich฀in฀hydrophobic฀amino฀acids.฀At฀the฀
apical฀surface฀of฀salivary฀acini,฀the฀remaining฀vesicles฀accumulate฀as฀secretory฀vesicles.฀
These฀vesicles฀become฀surrounded฀by฀myoibrils,฀which฀move฀the฀secretory฀vesicles฀to฀the฀
cell฀membrane฀where฀they฀fuse฀and฀expel฀the฀saliva฀secretion฀into฀a฀small฀duct.฀The฀odor฀or฀
taste฀of฀food฀provides฀a฀neuronal฀stimulus฀to฀the฀gland’s฀myoibrils,฀and฀this฀stimulates฀sali-
vary฀secretion.
Water฀is฀secreted฀separately.฀Neurotransmitters฀activate฀a฀28-kDa฀integral฀membrane฀
protein,฀ aquaporin-5฀ (AQP5),฀ one฀ of฀ 13฀ aquaporins฀ in฀ mammals.฀ AQP5฀ is฀ localized฀ in฀
lipid฀rafts฀in฀the฀plasma฀membrane฀of฀salivary฀cells฀and฀translocates฀to฀the฀apical฀plasma฀
210 12 Saliva

Fig. 12.3฀ How฀salivary฀proteins฀are฀secreted฀(a):฀N-linked฀glycans฀are฀synthesized฀and฀attached฀to฀dolichol฀

diphosphate฀(orange฀diamond)฀in฀the฀cytosol.฀The฀completed฀glycan฀is฀translocated฀to฀the฀lumen฀of฀the฀
endoplasmic฀reticulum฀(blue).฀The฀glycan฀attached฀to฀dolichol฀diphosphate฀by฀its฀C1฀(anomeric฀or฀reduc-
ing฀OH฀group)฀is฀N-acetyl฀glucosamine฀(GlcNAc;฀small฀red฀hexagon).฀The฀completed฀glycan฀also฀con-
tains฀mannose฀and฀glucose฀residues฀(small฀pink฀and฀blue฀hexagons).฀(1)฀The฀glycan฀(oligopolysaccharide)฀
is฀transferred฀to฀an฀asparagine฀residue฀of฀the฀growing฀peptide฀also฀in฀the฀lumen฀of฀the฀rough฀endoplasmic฀
reticulum฀(ER).฀(2)฀Synthesis฀of฀the฀glycan-attached฀polypeptide฀amino฀acid฀sequence฀is฀completed.฀(3)฀
The฀free฀dolichol฀diphosphate฀translocates฀back฀into฀the฀cytosol฀where฀a฀phosphatase฀removes฀its฀outer฀
phosphate฀residue.฀(4)฀In฀the฀cytosol,฀a฀molecule฀of฀UDP-GlcNAc฀reattaches฀to฀give฀dolichol฀diphosphate฀
GlcNAc฀(not฀shown)฀onto฀which฀an฀identical฀set฀of฀monosaccharides฀are฀added฀to฀give฀another฀glycan฀to฀
be฀transferred฀as฀shown฀in฀1.฀(b):฀The฀released฀glycan-attached฀polypeptide฀buds฀off฀into฀vesicles฀(smooth฀
endoplasmic฀reticulum,฀ER)฀that฀discharge฀their฀contents฀into฀the฀cis-Golgi฀where฀the฀glycans฀are฀pro-
cessed฀by฀removing฀most฀of฀the฀mannose฀and฀adding฀fucose,฀sialic฀acid,฀galactose,฀and฀other฀glycan฀resi-
dues.฀Mucin฀polypeptides฀have฀a฀sequence฀that฀activates฀an฀addition฀of฀glycans฀beginning฀with฀N-acetyl฀
galactosamine฀(GalNAc)฀to฀serine฀and฀threonine฀–OH฀groups฀in฀the฀Golgi.฀In฀the฀trans-Golgi,฀the฀contents฀
are฀either฀moved฀to฀lysosomal฀vesicles฀or฀secreted.฀(Modiied฀from฀Figs.฀27 34฀and฀27.35฀in฀Lehninger฀
Principles฀of฀Biochemistry.฀D.L.฀Nelson฀&฀M M.฀Cox,฀4th฀Ed.฀2005.฀W.H.฀Freeman฀&฀Co ,฀NY)
12.3.1. Salivary Mucin Composition 211

membrane฀where฀the฀secretory฀vesicles฀have฀accumulated.฀The฀extent฀of฀secretory฀vesicle฀
and฀AQP5฀translocation฀and฀fusion฀with฀the฀apical฀membrane฀depends฀on฀the฀extent฀of฀
stimulation฀ by฀ cholinergic฀ (acetylcholine)฀ receptors,฀ or฀ alpha-1฀ (adrenergic)฀ receptors฀
coupled฀to฀G฀proteins฀and฀phospholipase฀C.฀Cholinergic฀receptors฀activate฀Ca2+฀mobilizing฀
receptors฀ and฀ signal฀ both฀ secretory฀ vesicles฀ (protein)฀ and฀ water฀ secretion฀ from฀ salivary฀
glands.฀ Adrenergic฀ stimulation฀ of฀ cyclic฀ adenosine฀ monophosphate฀ (cAMP-dependent฀
protein฀ kinase฀ system)฀ increases฀ secretory฀ vesicle฀ exocytosis฀ immediately฀ and฀ the฀ Ca2+฀
mobilizing฀receptors฀for฀water฀secretion฀afterward.฀The฀reason฀why฀these฀two฀neuronal฀
activation฀responses฀are฀linked฀is฀not฀known.฀Eating฀and฀smelling฀food,฀or฀even฀just฀think-
ing฀of฀these฀things,฀transports฀water฀faster฀than฀the฀secretory฀vesicle฀contents;฀stimulated฀
saliva฀has฀more฀water฀and฀less฀protein.

Salivary฀proteins฀are฀secreted฀like฀other฀proteins.฀Polypeptides฀destined฀for฀secretion฀
are฀relocated฀by฀their฀N-terminal฀sequence฀to฀a฀ribosomal฀receptor฀on฀the฀cytosolic฀side฀
of฀the฀endoplasmic฀reticular฀(ER)฀membrane.฀The฀polypeptides฀enter฀the฀ER฀lumen฀and฀
move฀from฀rough฀to฀smooth฀ER฀where฀asparagine฀amide฀residues฀are฀glycosylated฀with฀
a฀preformed฀glycan฀(N-linked฀glycosylation).฀Vesicles฀bud฀off฀and฀are฀transported฀to฀
the฀Golgi฀where฀glycosidases฀and฀glycan฀synthetases฀modify฀the฀N-linked฀glycans฀and฀
attach฀glycans฀to฀the฀-OH฀groups฀of฀serine฀and฀threonine฀residues฀(O-linked฀glycosyla-
tion).฀Water฀is฀separately฀secreted฀through฀a฀28-kDa฀integral฀membrane฀protein,฀aqua-
porin฀(AQP5).฀AQP5฀translocates฀from฀lipid฀rafts฀in฀the฀plasma฀membrane฀to฀the฀apical฀
membrane฀where฀the฀secretory฀vesicles฀have฀accumulated฀and฀become฀surrounded฀by฀
myoibrils.฀ Smelling฀ or฀ ingesting฀ food฀ stimulates฀ saliva,฀ making฀ it฀ more฀ dilute.฀ The฀
smell฀or฀ingestion฀activates฀cholinergic฀and฀adrenergic฀receptors฀at฀autonomic฀nerve฀
endings฀around฀the฀salivary฀acini,฀causing฀faster฀AQP5฀translocation฀through฀the฀cell฀
membrane.฀Water฀is฀thus฀transported฀faster฀and฀independently฀of฀secretory฀vesicle฀฀con-
tents,฀which฀฀are฀more฀forcibly฀expelled฀by฀the฀stronger฀myoibril฀contractions.

12.3.1.
Salivary Mucin Composition

Mucin฀proteins฀account฀for฀the฀high฀viscosity฀of฀saliva฀in฀the฀oral฀cavity฀and,฀like฀the฀connec-
tive฀tissue฀proteoglycans,฀have฀many฀serine฀and฀threonine-linked฀glycan฀residues฀that฀absorb฀
a฀large฀volume฀of฀water.฀A฀sulfate฀(-SO4)−1฀group฀is฀also฀attached฀in฀the฀Golgi฀to฀the฀-OH฀
group฀ of฀ certain฀ glycan฀ residues฀ following฀ synthesis฀ of฀ the฀ chain,฀ usually฀ galactose,฀
N-acetylgalactosamine฀ or฀ N-acetylglucosamine.฀ Sulfation฀ also฀ accounts฀ for฀ the฀ negative฀
charge฀ of฀ the฀ connective฀ tissue฀ proteoglycans฀ (dermatan฀ sulfate,฀ chondroitin฀ sulfate,฀ etc.).฀
Sialic฀acid฀residues฀terminate฀some฀of฀the฀short฀mucin฀glycan฀chains฀and฀contribute฀also฀to฀the฀
strong฀net฀negative฀charge฀of฀salivary฀and฀other฀mucins.฀The฀connective฀tissue฀proteoglycan฀
chains฀are฀longer฀and฀fewer฀and฀use฀uronate฀instead฀of฀sialate฀as฀the฀negatively฀charged฀glycan.
Human฀submandibular฀and฀sublingual฀glands฀secrete฀two฀mucin฀glycoproteins,฀MG1฀and฀
MG2.฀MG1฀is฀a฀very฀large฀polypeptide฀composed฀of฀more฀than฀5,500฀amino฀acid฀residues฀
(mol฀wt฀~590฀kDa).฀Its฀N-฀and฀C-terminal฀regions฀each฀contain฀two฀N-glycosylated฀sites฀
(Fig.฀12.4a)฀and฀a฀long฀central฀region฀of฀72฀repeating฀domains฀that฀are฀rich฀in฀threonine฀and฀
212 12 Saliva

N C
a MG1
5678 aa
355 aa
MG2 VWF CK
355 aa
VWF D1
Repeats
VWF D2
VWF D4
VWF C1
VWF D3 VWF C2
VWF C3
Other Cys-rich
Other domains

b
VWF Cysteine Knot (CK) domain
1 6 29 33 50 52 53 67 83 85 89
1 a 2 3 b X 4 c 5 6 d

AsN-linked
c glycan
N
C O-linked
glycans
C
C

N
N

Endoplasmic Golgi
reticulum

Fig. 12.4฀ Human฀ salivary฀ mucin฀ composition.฀ (a)฀ Polypeptide฀ domains฀ of฀ MG1฀ and฀ MG2.฀
Numbering฀begins฀after฀the฀signal฀sequence฀is฀removed฀in฀the฀endoplasmic฀reticulum฀before฀pro-
cessing฀(see฀Expasy฀sequence฀Q9HC84,฀MUC5B_HUMAN).฀Both฀mucins฀have฀a฀central฀domain฀
consisting฀of฀multiple฀short฀tandem฀repeats฀of฀a฀sequence฀that฀is฀rich฀in฀serine,฀threonine,฀and฀pro-
line฀(yellow).฀Many฀of฀the฀serine฀and฀threonine฀residues฀become฀O-glycosylated฀(illustrated฀in฀C)฀
and฀the฀proline฀residues฀keep฀the฀chain฀extended.฀The฀nonrepeat฀domains฀of฀MG1฀at฀the฀N-฀and฀
C-termini฀are฀mostly฀derived฀from฀domains฀of฀Von฀Willebrand฀factor฀(VWF),฀a฀protein฀associated฀
with฀the฀blood฀clotting฀system.฀MG2฀is฀much฀smaller,฀as฀illustrated฀by฀the฀double-headed฀arrow฀
below฀the฀N-terminal฀region฀of฀MG1฀(see฀text)฀and฀has฀no฀VWF฀domains.฀(b)฀Cysteine฀knot฀(CK)฀
domain฀of฀MG1.฀In฀MG1,฀this฀domain฀consists฀of฀89฀amino฀acids฀that฀terminate฀shortly฀before฀the฀
C-terminus.฀It฀includes฀six฀cysteines฀arranged฀in฀a฀knot-like฀topology.฀All฀of฀the฀cysteine฀residues฀
in฀the฀domain฀are฀circled฀and฀numbered.฀Disulide฀bonds฀between฀the฀second฀and฀ifth฀cysteines,฀
and฀between฀the฀third฀and฀sixth฀cysteines,฀form฀a฀double฀ring฀that฀is฀penetrated฀by฀a฀disulide฀bond฀
between฀the฀irst฀and฀fourth฀cysteines฀(the฀knot,฀dotted฀lines).฀Other฀cysteine฀residues฀(labeled฀a,฀b,฀
c,฀and฀d)฀are฀present฀in฀MG1,฀VWF,฀and฀mucins฀from฀the฀pulmonary฀and฀digestive฀tracts,฀but฀absent฀
from฀cystine฀knot-proteins.฀Within฀the฀knot,฀there฀is฀always฀a฀seventh฀unpaired฀cysteine฀residue฀(CX฀
red)฀that฀causes฀dimerization.฀In฀MG1,฀the฀knot฀domain฀(dark฀blue฀in฀A)฀is฀at฀the฀C-terminus.฀Two฀
additional฀cysteine฀residues฀(not฀shown)฀are฀present฀in฀the฀cysteine฀knot฀of฀platelet฀derived฀growth฀
12.3.2. Glycan Composition of Salivary Mucins 213

serine฀residues.฀Within฀this฀central฀region,฀the฀sequence฀TXXP฀(X฀=฀any฀small฀side-chain฀
amino฀acid),฀recurs฀as฀a฀common฀threonine฀attachment฀site.฀The฀polypeptide฀is฀extended,฀
due฀to฀the฀proline฀residues฀in฀the฀sequence,฀but฀lexible฀due฀to฀absence฀of฀collagen฀helix.฀The฀
terminal฀-OH฀groups฀of฀the฀threonine฀and฀serine฀residues฀stick฀out฀and,฀in฀the฀Golgi,฀bind฀to฀
a฀small฀family฀of฀enzymes฀that฀attach฀monosaccharides฀in฀an฀orderly฀manner.
The฀N-฀and฀C-terminal฀regions฀are฀less฀extended.฀They฀each฀possess฀a฀single฀N-linked฀
glycan฀and฀are฀free฀of฀O-linked฀glycans.฀The฀C-terminus฀possesses฀a฀cysteine฀knot฀domain฀
identical฀ to฀ that฀ in฀ von฀ Willebrand฀ factor฀ and฀ TGF-b฀ (Fig.฀ 12.4b)฀ and฀ therefore฀ a฀ free฀
cysteine฀residue฀(Chapter฀3,฀section฀3.2.2.)฀which฀causes฀dimerization฀at฀the฀C-terminus฀of฀
MG1฀molecules฀(Fig.฀12.4c).฀The฀D1,฀D2฀and฀D3฀domains฀near฀the฀N-terminus฀each฀possess฀
a฀free฀cysteine฀residue,฀enabling฀each฀MG1฀dimer฀to฀form฀a฀cystine฀bond฀with฀two฀other฀
dimers฀until฀the฀molecule฀attains฀a฀molecular฀mass฀of฀about฀25฀million฀daltons฀(Fig.฀12.4c)฀
Although฀cysteine฀knot-aggregated฀VWF฀plays฀an฀important฀role฀in฀initiating฀blood฀clotting฀
(Sect.฀11.2.1),฀salivary฀mucins฀do฀not฀seem฀to฀enhance฀blood฀clotting฀in฀humans.
Mucin฀MG2฀is฀less฀than฀a฀tenth฀the฀size฀of฀MG1,฀355฀amino฀acids;฀mol฀wt฀~39฀kDa.฀It฀
consists฀of฀a฀single฀repeat฀domain฀with฀separate฀N-฀and฀C-terminal฀domains฀that฀are฀not฀
VWF-related.฀ The฀ N-terminal฀ domain฀ has฀ ive฀ cysteine฀ residues,฀ but฀ the฀ C-terminal฀
domain฀ has฀ none฀ and฀ the฀ secreted฀ protein฀ is฀ probably฀ a฀ mixture฀ of฀ monomers฀ and฀
head-฀to-head฀disulide฀dimers.
The฀gene฀for฀MG1฀is฀called฀MUCSB฀(MUC฀for฀mucin)฀and฀the฀gene฀for฀MG2฀is฀called฀
MUCT.฀The฀gene฀for฀MG1฀is฀called฀MUC5B฀(MUC฀for฀mucin)฀and฀the฀gene฀for฀MG2฀is฀called฀
MUC7.฀MG1฀is฀made฀by฀mucous฀cells,฀whereas฀MG2฀is฀made฀only฀by฀serous฀cells฀within฀
mixed฀ glands,฀ i.e.฀ only฀ the฀ submandibular฀ and฀ sublingual฀ glands.฀ The฀ parotid฀ and฀ minor฀
glands฀that฀contain฀only฀serous฀cells฀do฀not฀secrete฀either฀MG1฀or฀MG2,฀whereas฀goblet฀cells฀
of฀the฀intestinal฀and฀bronchial฀tracts฀secrete฀both฀MG1฀and฀MG2.฀Other฀MUC฀genes฀encode฀
similar฀mucins฀that฀protect฀the฀stomach฀from฀acid฀and฀the฀lungs฀from฀inhaled฀particles.

12.3.2.
Glycan Composition of Salivary Mucins

MG1฀and฀MG2฀account฀for฀the฀smooth฀surface฀of฀the฀oral฀mucosa฀and฀the฀viscosity฀of฀
whole฀ saliva.฀ There฀ are฀ four฀ monosaccharides฀ associated฀ with฀ these฀ mucins,฀ of฀ which฀
three฀are฀illustrated฀in฀Fig.฀12.5:฀N-acetyl฀galactosamine,฀l-fucose฀(6-deoxy-l-galactose),฀

Fig. 12.4฀ (continued)฀ factor,฀another฀protein฀associated฀with฀blood฀clotting฀like฀VWF฀as฀discussed฀

in฀Chap.฀11.฀(c)฀Assembly฀of฀human฀MG1.฀MG1฀is฀glycosylated฀and฀forms฀C-terminus฀to฀C-terminus฀
cystine฀cross-links฀in฀the฀smooth฀ER฀(left฀two฀diagrams).฀It฀is฀then฀transported฀to฀the฀Golgi฀where฀฀
its฀many฀repeat฀domains฀are฀glycosylated.฀In฀addition,฀the฀dimers฀develop฀crosslinked฀multimers฀
using฀the฀free฀cysteine฀residues฀at฀its฀various฀N-terminal฀VWF฀D฀domains฀(฀฀far฀right฀diagram).฀(a)฀
and฀(c)฀฀Modiied฀Figs.฀1฀and฀2฀in฀Perez-Vilar฀J,฀Hill฀RL฀(1999฀Nov฀5)฀“The฀Structure฀and฀Assembly฀
of฀Secreted฀Mucins.”฀J.฀Biol.฀Chem.฀271(45),฀31751–4.฀(b)฀Slightly฀modiied฀from฀Fig.฀1฀in฀Bell฀
SL,฀Xu,฀G฀and฀Forstner,฀JF.฀(2001)฀“Role฀of฀the฀cystine-knot฀motif฀at฀the฀C-terminus฀of฀rat฀mucin฀
protein.”฀Biochem.฀J.฀357:฀203–209.฀฀Reproduced฀with฀permission฀from฀Portland฀Press).
214 12 Saliva

a CH2OH
O-link to
b H
O O
HO H H ser or thr H CH3 OH
OH H H OH
H OH HO H
H HN C CH3 OH H
O
N-acetyl-a-galactosamine a-L-fucose
(GaINAc) 6-deoxy-L-galactose
c
HO OH
C C OH
H H CH
O H
C N H O COOH
H 3C
H H H
OH
OH H
Sialic acid
N-acetyl a-D-neuraminic acid

Fig. 12.5฀ Chemical฀ structures฀ of฀ the฀ major฀ monosaccharides฀ of฀ mucins.฀ (a)฀ N-Acetyl-α-
galactosamine.฀ (b)฀ α-L-Fucose฀ (6-deoxy-฀ α-L-galactose).฀ The฀ α-anomeric฀ bond฀ points฀ up฀ in฀
L-sugars.฀(c)฀Sialic฀acid฀(N-Acetyl฀neuraminic฀acid).฀(a:฀and฀b:฀Molecular฀structures฀from฀http://
www.webbooks.com/MoBio/Free/Ch1B4.htm.฀ With฀ permission฀ from฀ Web฀ Books฀ Publishing.฀
Email:฀info2008@web-books.com;฀c:฀Molecular฀structures฀modiied฀from฀an฀online฀catalogue฀pic-
ture฀ published฀ by฀ Sigma-Aldrich,฀ St฀ Louis฀ MO฀ and฀ reproduced฀ with฀ permission฀ from฀ Sigma-
Aldrich฀Co.฀http://www.sigmaaldrich.com/catalog/search/ProductDetail/ALDRICH/855650)

and฀sialic฀acid฀(also฀called฀N-acetyl฀neuraminic฀acid).฀D-galactose฀is฀not฀illustrated.฀Fucose฀
has฀a฀methyl฀group฀at฀the฀C6฀position฀instead฀of฀an฀alcohol฀(-CH2OH)฀or฀acid฀(-COOH)฀
group฀and฀all฀hydroxyl฀groups฀attached฀to฀the฀ring฀point฀in฀the฀opposite฀direction฀(relative฀
to฀the฀respective฀hydrogen฀atoms)฀from฀those฀in฀d-galactose.฀Within฀the฀Golgi,฀all฀O-linked฀
glycans฀are฀made฀using฀enzymes฀that฀irst฀recognize฀a฀nucleotide-activated฀monosaccha-
ride฀and฀incorporate฀it฀at฀the฀appropriate฀place฀in฀a฀glycan฀chain.฀Fucose฀and฀sialic฀acid฀
always฀terminate฀a฀glycan฀chain.
Sialic฀acid฀consists฀of฀six฀carbon฀atoms฀from฀N-acetyl฀mannosamine฀and฀three฀from฀
pyruvate฀(Fig.฀12.6)฀and฀is฀formed฀by฀sialic฀acid฀synthetase.฀This฀enzyme฀condenses฀the฀฀
C1฀atom฀of฀mannosamine฀with฀the฀C3฀atom฀of฀phosphoenolpyruvate฀(PEP)฀in฀the฀cytosol.฀
The฀9-carbon฀chain฀product฀is฀numbered฀from฀the฀carboxylic฀acid฀group,฀the฀C1฀atom.฀
The฀C2฀atom,฀the฀enol-phosphate฀of฀phosphoenolpyruvate฀becomes฀a฀keto฀group฀as฀in฀
pyruvate.฀The฀C2฀ketone฀spontaneously฀forms฀a฀ketal฀with฀the฀OH฀group฀of฀C6฀(originally฀
C3฀ of฀ N-acetyl฀ mannosamine)฀ to฀ form฀ a฀ six-membered฀ pyranose฀ ring฀ (right฀ side฀ of฀
Fig.฀12.6).฀The฀C2฀atom฀of฀sialic฀acid฀is฀therefore฀the฀anomeric฀carbon฀atom.฀The฀C3฀atom฀
(originally฀C3฀of฀PEP)฀is฀connected฀to฀the฀C1฀atom฀of฀N-acetyl฀mannosamine฀(C4฀of฀sialic฀
acid).฀The฀C2฀atom฀of฀mannosamine฀(C5฀of฀sialic฀acid)฀carries฀the฀N-acetyl฀group฀(like฀
glucosamine฀ and฀ galactosamine).฀ Sialic฀ acid฀ is฀ therefore฀ a฀ multisubstituted฀ pyranose฀
ring฀ that฀ has฀ only฀ two฀ hydroxyl฀ groups฀ available฀ to฀ form฀ glycoside฀ bonds,฀ C2฀ and฀ C4฀
(Fig.฀12.5c).
12.3.2. Glycan Composition of Salivary Mucins 215

Fig. 12.6฀ Sialic฀acid฀ Phosphoenol pyruvate H PO -2

+H
+

synthetase.฀Right:฀ 2 4
COOH +
Reaction฀of฀the฀substrates,฀฀
phopshoenolpyruvate฀and฀ C O PO3H2 COOH
N-acetyl฀mannosamine.฀ CH2 H-OH C OH
Left:฀Sialic฀acid฀(product).฀
CH
(Original฀Figure) 2
H C O O H C OH
O 1

CH C HN C H CH C HN C H
3 3
O C H
H O C H
H C OH
H C H C OH

H C OH C8 formerly CH OH
2
C5 in N-acetyl mannosamine
CH2OH
N-acetyl mannosamine Sialic acid

The฀ C2-OH฀ group฀ is฀ shown฀ as฀ an฀ a-anomer,฀ because฀ it฀ becomes฀ attached฀ to฀ a฀
n฀ on-anomeric฀OH฀group฀of฀another฀glycan,฀most฀commonly฀the฀C6฀position฀of฀N-acetyl฀
galactosamine฀in฀an฀a2฀→฀6฀bond฀(Fig.฀12.7).฀Substitution฀at฀the฀OH฀group฀of฀C4฀of฀sialic฀
acid฀is฀never฀found;฀apparently฀there฀is฀no฀enzyme฀glycoside฀synthetase฀to฀recognize฀the฀
unique฀coniguration฀around฀the฀sialic฀acid฀C4฀residue.฀

HO OH
C C OH
H H CH
O H
C N H O
COOH
H3C
H H H
Oα
Polypeptide
OH H
G

chain
lyc

CH2
an

Sialic acid O
ch

HO H
(

a2 H NH
ai
n

OH H
6 H Oα CH2 CH Serine
(

residue
GaINAc-a-Ser
H HN C CH3 C O
O

Fig. 12.7฀ An฀O-linked฀glycan฀containing฀sialic฀acid฀Serine฀(or฀threonine)฀residues฀are฀O-linked฀to฀

the฀a-anomeric฀form฀of฀N-acetylgalactosamine฀(GalNAc).฀The฀anomeric฀–OH฀group฀of฀sialic฀acid฀
(which฀is฀attached฀to฀C2)฀becomes฀a-linked฀to฀the฀C6–OH฀group฀of฀GalNAc.฀More฀complex฀gly-
cans฀with฀or฀without฀sialic฀acid฀are฀shown฀in฀Fig.฀12.8.฀(Original฀igure฀using฀structures฀modiied฀
from฀Fig.฀12.5)
216 12 Saliva

Salivary฀mucins฀are฀N-acetylgalactosamine฀linked฀to฀certain฀serine฀and฀threonine฀resi-
dues฀on฀polypeptides฀(O-linked)฀within฀the฀Golgi.฀The฀oligosaccharides฀possess฀a฀net฀
negative฀charge฀from฀terminal฀sialic฀acid฀residues,฀or฀sulfate฀residues฀esteriied฀to฀gly-
can฀-OH฀groups.฀The฀salivary฀mucin฀polypeptides,฀MG1฀and฀MG2,฀are฀heavily฀glyco-
sylated฀within฀an฀extended,฀lexible฀domain฀that฀is฀rich฀in฀serine,฀threonine,฀and฀proline.฀
MGI฀ is฀ large฀ and฀ forms฀ large,฀ disulide฀ bonded฀ multimers,฀ whereas฀ MG2฀ is฀ much฀
smaller฀ and฀ forms฀ only฀ N-terminal฀ head-to-head฀ cystine-linked฀ dimers.฀ Mucins฀ are฀
only฀secreted฀by฀glands฀possessing฀mucous฀cells,฀although฀MG2฀is฀in฀fact฀secreted฀by฀
the฀ serous฀ cells฀ of฀ these฀ glands.฀ d-Galactose,฀ N-acetyl฀ galactosamine฀ (GalNAc),฀
l-fucose฀(6-deoxy-l-galactose),฀and฀sialic฀acid฀(N-acetyl฀neuraminic฀acid)฀are฀the฀most฀
common฀sugars.฀Sialic฀acid฀is฀the฀most฀complex,฀possessing฀nine฀carbon฀atoms฀derived฀
from฀a฀condensation฀of฀the฀C1฀aldehyde฀atom฀of฀acetyl฀mannosamine฀with฀the฀C3-OH฀
group฀ of฀ phosphoenolpyruvate.฀ C2฀ through฀ C6฀ form฀ a฀ multisubstituted฀ six-member฀
pyran฀ring฀in฀which฀the฀anomeric฀carbon฀OH฀group฀is฀always฀in฀the฀alpha฀orientation.฀
Its฀single฀carboxyl฀group฀is฀attached฀to฀the฀anomeric฀carbon฀and฀possesses฀a฀strong฀net฀
negative฀charge.฀Sialic฀acid฀and฀fucose฀are฀not฀found฀as฀intermediate฀sugars฀in฀an฀oli-
gosaccharide฀chain.

12.4.1.
Mucin Glycans, ABO Antigens, and Forensic Dentistry

Mucin-type฀linkages฀(GalNAc-a1-O-Ser/Thr)฀are฀initiated฀by฀a฀family฀of฀glycosyl฀trans-
ferases฀that฀transfer฀GalNAc฀from฀the฀sugar฀donor฀UDP-GalNAc฀to฀serine฀and฀threonine฀
residues.฀Because฀O-linked฀glycosylation฀proceeds฀step-wise,฀addition฀of฀GalNAc฀to฀serine฀
or฀threonine฀represents฀the฀irst฀committed฀step฀in฀mucin฀biosynthesis.฀This฀initiating฀gly-
cosyl฀transferase฀bonds฀the฀GalNAc฀anomeric฀(reducing)฀residue฀(C1)฀as฀a฀a-glycoside฀to฀a฀
recognized฀serine฀or฀threonine฀−OH฀group฀on฀the฀polypeptide.฀The฀simplest฀oligosaccha-
ride฀(short฀glycan)฀is฀formed฀by฀a฀second฀enzyme฀bonding฀sialic฀acid฀to฀the฀GalNAc฀C6฀
atom฀ (a2฀ →฀ 6฀ bond;฀ Fig.฀ 12.7).฀ More฀ often,฀ an฀ appropriate฀ enzyme฀ adds฀ a฀ second฀
N-acetylglucosamine฀(GalNAc),฀or฀instead฀a฀GlcNAc฀residue,฀to฀the฀C4฀OH฀group฀of฀the฀
serine฀or฀threonine-attached฀GalNAc,฀forming฀a฀(b1฀→฀4฀glycoside฀bond.฀Enzymes฀then฀
add฀additional฀GalNAc฀or฀GlcNAc฀residues,฀or฀sialic฀acid฀and฀fucose฀residues฀to฀generate฀a฀
diverse฀group฀of฀oligosaccharide฀structures฀to฀the฀central฀threonine/serine฀rich฀domains฀of฀
MG1฀and฀MG2.
The฀enzymes฀that฀synthesize฀these฀oligosaccharides฀also฀synthesize฀short฀glycans฀that฀
protrude฀from฀the฀red฀blood฀cell฀surface฀and฀specify฀the฀ABO฀blood฀group฀antigens.฀In฀
developing฀red฀blood฀cells,฀these฀oligosaccharides฀are฀synthesized฀attached฀to฀ceramide,฀a฀
derivative฀of฀sphingosine฀that฀contains฀a฀saturated฀or฀monounsaturated฀fatty฀acid฀(Fig.฀12.8).฀
12.4.1. Mucin Glycans, ABO Antigens, and Forensic Dentistry 217

a A antigen GIcNAc
specificity
Gal
Fuc

Outer cell surface GIcNAc Gal

β-1,3
GaINAc
β
Lipid layer O CH2
O H
C N CH
Attached fatty acid (C18)

CH3 (CH2)12 CH CH CH OH
Sphingosine
b
Group A Group B Group AB Group O

Red blood
cell type A B AB O

Antigens
present A antigen B antigen A and B No antigens
antigens

Antibodies
present
Anti-B Anti-A None Anti-A and Anti-B

Fig. 12.8฀ Blood฀group฀antigen.฀(a)฀Red฀blood฀cell฀attachment:฀Ceramide,฀a฀derivative฀of฀sphin-

gosine฀(purple฀highlight),฀is฀a฀common฀component฀of฀the฀outer฀lealet฀of฀the฀cell฀lipid฀layer฀and฀
possesses฀a฀free฀OH฀group.฀As฀each฀ceramide฀molecule฀passes฀through฀the฀Golgi฀on฀its฀way฀to฀
insertion฀in฀the฀outer฀membrane฀of฀a฀developing฀red฀blood฀cell,฀certain฀enzymes฀recognize฀this฀
structure฀ and฀ form฀ the฀ oligosaccharide฀ (short฀ glycan)฀ shown.฀ This฀ oligosaccharide฀ sticks฀ out฀
from฀the฀cell฀membrane฀and฀its฀terminal฀(sixth)฀glycan฀residue฀confers฀red฀cell฀antigen฀type.฀The฀
igure฀shows฀the฀red฀cell฀type฀A฀speciicity,฀in฀which฀the฀sixth฀monosaccharide฀is฀a฀GalNAc฀resi-
due.฀ (b)฀ ABO฀ speciicity฀ of฀ red฀ blood฀ cells:฀ Different฀ enzymes฀ add฀ the฀ 6th฀ monosaccharide.฀
GalNAc฀confers฀A฀antigen฀speciicity;฀Gal฀confers฀B฀antigen฀speciicity;฀both฀enzymes฀present฀
confers฀AB฀speciicity฀and฀the฀absence฀of฀both฀enzymes฀confers฀O฀speciicity฀in฀which฀there฀are฀
only฀ive฀residues฀in฀the฀oligosaccharide฀(a:฀Figure฀is฀a฀pastische฀of฀the฀diagram฀of฀the฀red฀blood฀
cell฀A฀antigen฀in฀Fig.฀11.17฀from฀Berg฀et฀al.,฀Biochemistry,฀5th฀Ed.฀2002,฀W.H.฀Freeman฀&฀Co.,฀
NY.฀Uppermost฀part฀is฀from฀Fig.฀10.12฀in฀Nelson฀&฀Cox,฀Lehninger’s฀Principles฀of฀Biochemistry,฀
4th฀Ed.฀2005,฀W.H.฀Freeman฀&฀Co.,฀New฀York;฀b:฀Wikipedia฀public฀domain฀image;฀http://en.
wikipedia.org/wiki/Image:ABO_blood_type.svg)
218 12 Saliva

Ceramide฀ is฀ synthesized฀ de฀ novo฀ in฀ the฀ endoplasmic฀ reticulum฀ and฀ subsequently฀ trans-
ported฀to฀the฀Golgi฀where฀it฀is฀linked฀to฀glycan฀as฀a฀glycosphingolipid.฀In฀the฀Golgi,฀the฀
ceramide฀-OH฀group฀is฀recognized฀by฀the฀same฀UDP-GalNAc฀transferases฀that฀O-glycosylate฀
MG1฀and฀MG2.฀Following฀attachment,฀the฀non-reducing฀end฀of฀the฀O-linked฀GalNAc฀is฀
attached฀to฀galactose,฀followed฀by฀a฀second฀GalNAc,฀a฀second฀galactose฀and฀then฀fucose฀
(Fig.฀ 12.8a).฀ This฀ sequence,฀ GalNAc-Gal-GalNAc-Gal-Fuc฀ is฀ called฀ H฀ antigen,฀ and฀ it฀
speciies฀the฀blood฀group฀O฀speciicity฀(Fig.฀12.9).
Some฀individuals฀have฀a฀monosaccharide฀transferase฀enzyme฀that฀adds฀a฀GalNAc฀resi-
due฀as฀a฀second฀substituent฀to฀the฀preterminal฀galactose฀that฀is฀attached฀to฀fucose.฀That฀
addition฀ speciies฀ the฀ red฀ cell฀ A-antigen฀ speciicity฀ (yellow฀ highlight฀ in฀ Fig฀ 12.8).฀
Alternatively,฀a฀related฀enzyme฀adds฀a฀galactose฀residue฀(green)฀instead,฀and฀this฀addition฀
speciies฀the฀red฀cell฀B-antigen฀(Fig.฀12.9).฀Individuals฀who฀express฀both฀transferases฀are฀
type฀AB,฀and฀each฀red฀cell฀has฀a฀mixture฀of฀A฀and฀B฀antigens฀(Fig.฀12.8b).฀AB฀individuals฀

a
Gal Fuc

GlcNAc
Gal
GlcNAc Gal
Gal Gal α-1,3 Gal α-1,3
α-1,2 α-1,2 α-1,2
β-1,3 Fuc β-1,3 Fuc β-1,3 Fuc
GlcNAc GlcNAc GlcNAc
β-1,3 β-1,3 β-1,3 b
Gal Gal Gal Gal
β-1,3 α-2,3
β-1,3 β-1,3 β-1,3 Sial
GalNAc GalNAc GalNAc GalNAc
α α α α
Ser Ser Ser Ser
O (H) antigen A antigen B antigen S. mutans-binding
antigen (MG2)

Fig. 12.9฀ Mucin฀glycan฀sequences.฀(a)฀Blood฀group฀antigens:฀The฀precursor฀sequence฀of฀sugars฀is฀

built฀up฀on฀a฀serine฀or฀threonine฀−OH฀residue฀of฀a฀mucin฀protein฀and฀becomes฀O-antigen฀(also฀
called฀H฀antigen)฀when฀the฀ifth฀monosaccharide,฀fucose,฀becomes฀attached฀to฀the฀2-OH฀position฀
of฀the฀previous฀residue,฀galactose฀[Fuc(a1฀→฀2)Gal].฀This฀sequence฀becomes฀A฀antigen฀if฀another฀
enzyme฀(GalNAc฀transferase)฀recognizes฀the฀3-OH฀group฀of฀the฀galactose฀with฀fucose฀attached฀
and฀attaches฀a฀sixth฀monosaccharide,฀GalNAc฀(terminal฀yellow฀residue).฀The฀B฀antigen฀forms฀if฀a฀
different฀ enzyme฀ (Gal฀ transferase)฀ attaches฀ a฀ galactose฀ residue฀ (green฀ residue)฀ instead.฀ If฀ both฀
enzymes฀are฀expressed,฀an฀individual฀has฀50%฀of฀glycan฀sequences฀with฀GalNAc฀and฀50%฀with฀
Gal฀(AB฀antigen),฀and฀if฀neither฀enzyme฀is฀present,฀the฀individual฀has฀the฀O฀antigen.฀(b)฀Bacterial฀
binding฀ antigen:฀ In฀ addition฀ to฀ blood฀ group฀ antigens,฀ there฀ are฀ many฀ other฀ different฀ glycan฀
sequences฀attached฀to฀a฀salivary฀mucin฀serine/threonine฀rich฀domain.฀One฀such฀sequence฀is฀unique฀
to฀the฀MG2฀mucin฀and฀binds฀to฀Streptococcus฀mutans,฀an฀organism฀associated฀with฀dental฀caries฀
development,฀Chap.฀15,฀Sect.฀2฀(Modiied฀from฀Fig.฀11.17฀in฀Berg.฀et฀al.,฀Biochemistry,฀5th฀Ed.฀
2002.฀W.H.฀Freeman฀&฀Co.,฀New฀York)
12.5.1. Amylase: Substrates, Products, and Mode of Action 219

cannot฀make฀antibodies฀to฀A฀or฀B฀antigen฀on฀transfused฀red฀cells฀because฀these฀antigens฀
are฀not฀foreign.฀Only฀individuals฀lacking฀these฀antigens฀can฀make฀these฀antibodies฀after฀
transfusion฀with฀donor฀AB฀cells.฀Donated฀O฀blood฀cannot฀induce฀either฀antibody,฀because฀
the฀necessary฀terminal฀additions฀are฀absent.฀Thus,฀O-blood฀can฀be฀given฀to฀any฀individual฀
in฀an฀emergency,฀whereas฀AB฀blood฀given฀to฀other฀than฀an฀AB฀individual฀is฀potentially฀
dangerous.
The฀ ABO฀ glycan฀ sequences฀ comprise฀ a฀ fraction฀ of฀ the฀ O-linked฀ glycans฀ of฀ salivary฀
mucins฀in฀80%฀of฀individuals.฀It฀is฀not฀clear฀why฀the฀remaining฀20%฀of฀individuals฀do฀not฀
secrete฀these฀antigens.฀The฀presence฀of฀ABO฀antigens฀in฀salivary฀mucins฀from฀bite฀wounds฀
in฀a฀victim฀who฀was฀raped฀or฀murdered฀is฀used฀to฀eliminate฀suspects.฀DNA฀is฀also฀present฀
in฀cells฀that฀are฀shed฀into฀saliva฀and฀polymerase฀chain฀reaction฀can฀enhance฀and฀sequence฀
certain฀fragments฀of฀the฀DNA฀from฀these฀cells฀as฀an฀accurate฀and฀preferred฀identiication฀
method,฀but฀it฀is฀expensive฀to฀perform.฀The฀presence฀of฀ABO฀antigens฀in฀salivary฀mucins฀
often฀provides฀a฀rapid฀and฀relatively฀inexpensive฀initial฀screen฀to฀determine฀if฀a฀suspect฀can฀
be฀eliminated฀without฀needing฀DNA฀testing.

Mucin-type฀ linkages฀ (GalNAc-a1-O-Ser/Thr)฀ are฀ initiated฀ by฀ a฀ family฀ of฀ glycosyl฀

transferases฀ that฀ transfer฀ GalNAc฀ from฀ the฀ sugar฀ donor฀ UDP-GalNAc฀ to฀ serine฀ and฀
threonine฀residues.฀Enzymes฀then฀add฀additional฀GalNAc฀or฀GlcNAc฀residues,฀or฀sialic฀
acid฀and฀fucose฀residues฀to฀generate฀a฀diverse฀group฀of฀oligosaccharide฀structures฀to฀the฀
central฀threonine/serine฀rich฀domains฀of฀MG1฀and฀MG2.฀The฀same฀enzymes฀also฀add฀
glycans฀to฀ceramide,฀a฀derivative฀of฀sphingosine฀on฀the฀outer฀lipid฀lealet฀of฀red฀blood฀
cells.฀These฀sequences฀stick฀out฀from฀the฀red฀blood฀cell฀surface฀and฀specify฀ABO฀red฀
blood฀cell฀type.฀The฀same฀oligosaccharide฀sequences฀are฀also฀present฀attached฀to฀serine฀
or฀threonine฀residues฀of฀salivary฀mucins฀in฀80%฀of฀the฀population.฀The฀ABO฀system฀
depends฀on฀the฀presence฀of฀one,฀other฀or฀both฀glycan฀transferases฀that฀add฀a฀terminal฀
sugar฀ to฀ one฀ of฀ these฀ short฀ oligosaccharides.฀ Individuals฀ with฀ one฀ or฀ other฀ enzyme฀
missing฀or฀inactive฀express฀the฀O฀antigen฀in฀which฀the฀terminal฀monosaccharide฀reside฀
is฀missing.฀The฀mucin฀ABO฀speciicity฀of฀saliva฀provides฀a฀rapid฀initial฀screen฀of฀saliva฀
from฀bites฀on฀victims฀to฀exclude฀suspects฀of฀rape฀or฀murder.

12.5.1.
Amylase: Substrates, Products, and Mode of Action

Salivary฀alpha-amylase฀(a-amylase)฀is฀the฀major฀protein฀secreted฀by฀saliva฀and฀is฀encoded฀
by฀ the฀ Amy1฀ gene฀ on฀ chromosome฀ 1.฀ It฀ is฀ an฀ endoglycohydrolase,฀ which฀ means฀ that฀ it฀
hydrolyzes฀internal฀a1฀→฀4฀glucoside฀linkages.฀Its฀primary฀substrate฀is฀starch,฀which฀is฀
composed฀exclusively฀of฀glucose฀in฀two฀forms,฀amylose฀and฀amylopectin.฀Amylose฀is฀a฀
linear฀ polymer฀ of฀ glucose฀ connected฀ by฀ a1฀ →฀ 4฀ bonds฀ (Fig.฀ 12.10a).฀ The฀ a-amylase฀
220 12 Saliva

hydrolyzes฀from฀the฀nonreducing฀end฀(left฀side฀in฀Fig.฀12.10a)฀to฀give฀maltose฀(a1฀→฀4฀
bonded฀glucose฀disaccharide)฀and฀a฀limit฀dextrin฀(Fig.฀12.10b).฀Glucose฀is฀never฀a฀product฀
of฀a-amylase฀digestion.฀Amylopectin฀resembles฀amylose฀but฀with฀a1฀→฀6฀cross-linked฀
bonds฀(Fig.฀12.10c).฀Amylase฀cuts฀amylopectin฀every฀two฀residues฀from฀the฀nonreducing฀

CH2OH CH2OH CH2OH CH2OH CH2OH

H O H H O H H O H H O H H O H
a H H H H H
OH H OH H OH H OH H OH H α
HO O O O O OH
Amylose
H OH H OH H OH H OH H OH
Non-reducing
α1→4 Reducing
end
Hydrolysis by end
salivary amylase
b
Maltose amylose limit dextrin
CH2OH CH2OH CH2OH CH2OH CH2OH
H O H H O H + H O H H O H H O H
H H H H H
OH H OH H OH H OH H OH H α
O OH O O
HO HO OH
H OH H OH H OH H OH H OH
Non-reducing Reducing Non-reducing Reducing
end end end end
Non-reducing
c Non-reducing end
end
sites
lea vage
lto se c Reducing
r ma
Othe end
Amelopectin

2nd Branch
point 1st Branch
point
s
ite
g e s int
va o
lea h p
s e c ranc
lt o t b
Non-reducing end Ma firs
to

Branch
d CH2OH
O H
H
H
Portion of OH H
O
amylopectin HO
H OH
α1→6
limit dextrin
CH2
CH2OH CH2OH CH2OH CH2OH
H O H H O H H O H H O H H O H
Non-reducing H H H H H
OH H OH H OH H
end O OH H OH H α
HO O O O
H OH H OH OH
H OH H OH H OH
12.5.2. Mechanism of Action of Salivary Amylase 221

end฀(Fig.฀12.10d)฀to฀give฀maltose฀and฀various฀sized฀oligosaccharides,฀limit฀dextrins฀con-
taining฀ a1฀ →฀ 4฀ and฀ a1฀ →฀ 6฀ bonds.฀ Limit฀ dextrins฀ are฀ sticky฀ and฀ help฀ hold฀ the฀ bolus฀
together฀for฀swallowing.฀Glycogen฀is฀a฀third฀substrate฀for฀amylase,฀but฀it฀is฀digested฀less฀
eficiently฀because฀of฀its฀greater฀branching฀(more฀a1฀→฀6฀bonds).
When฀salivary฀a-amylase฀digests฀starch฀following฀a฀meal,฀it฀produces฀maltose฀and฀large฀
limit฀dextrins฀that฀pass฀through฀the฀stomach฀to฀the฀small฀intestine.฀There฀they฀are฀further฀
hydrolyzed฀by฀pancreatic฀a-amylase,฀which฀is฀97%฀identical฀to฀salivary฀amylase฀but฀arises฀
from฀a฀separate฀gene฀on฀chromosome฀1฀(Amy2).฀The฀limit฀dextrins฀arising฀from฀salivary฀
and฀ pancreatic฀ amylase฀ digestion฀ pass฀ with฀ maltose฀ through฀ the฀ mucous฀ outer฀ layer฀ of฀
intestinal฀ epithelial฀ cells฀ to฀ the฀ brush฀ border฀ where฀ a-glucosidases฀ degrade฀ them฀ all฀ to฀
glucose.฀The฀glucose฀is฀then฀transported฀through฀the฀cells฀and฀is฀absorbed฀into฀the฀blood-
stream฀where฀it฀raises฀the฀blood฀sugar฀levels.
The฀name฀a-amylase฀implies฀additional฀amylases.฀b-Amylase฀is฀synthesized฀by฀micro-
organisms฀in฀the฀digestive฀tract฀of฀animals฀and฀also฀by฀plants.฀It฀resembles฀salivary฀and฀
pancreatic฀a-amylase฀in฀activity.฀In฀plants,฀it฀breaks฀the฀starch฀of฀ripening฀fruits฀into฀malt-
ose฀to฀give฀the฀sweet฀lavor.฀g-Amylases฀are฀found฀in฀a฀few฀yeasts฀and฀bacteria฀and฀is฀more฀
powerful฀than฀a-฀or฀b-amylases.฀It฀hydrolyzes฀every฀second฀a1฀→฀4฀bond฀to฀maltose,฀but฀
it฀also฀hydrolyzes฀a1฀→฀6฀glycoside฀bonds฀in฀limit฀dextrins฀to฀a฀mixture฀of฀maltose฀and฀
glucose.฀Cellulose,฀a฀polymer฀of฀b1฀→฀4฀linked฀glucose,฀is฀hydrolyzed฀by฀the฀cellulases,฀฀
a฀set฀enzymes฀with฀poor฀homology฀to฀the฀amylases฀and฀absent฀from฀the฀human฀genome.

12.5.2.
Mechanism of Action of Salivary Amylase

Glycosidases฀ such฀ as฀ a-amylases฀ are฀ very฀ common฀ in฀ nature.฀ The฀ numerous฀ variants฀
expressed฀by฀different฀organisms฀show฀poor฀amino฀acid฀sequence฀homology,฀but฀neverthe-
less฀share฀a฀well-conserved฀three-dimensional฀structure.฀Moreover,฀this฀same฀three-dimen-
sional฀ structure฀ is฀ shared฀ by฀ a-glycosyl฀ transferases฀ such฀ as฀ the฀ glucosyl-฀ and฀

Fig. 12.10฀ Structure฀of฀starch฀in฀relation฀to฀salivary฀amylase฀action฀(a)฀Amylose฀in฀starch,฀Amylose฀is฀

an฀α1→4฀polymer฀of฀glucose.฀A฀stretch฀of฀5฀glucopyranose฀residues฀from฀non-reducing฀end฀(red)฀to฀
reducing฀end฀(green)฀is฀shown.฀The฀C1฀of฀glucose฀is฀the฀reducing฀end฀and฀it฀possesses฀the฀anomeric฀
OH฀group฀which฀is฀shown฀in฀green฀and฀pointing฀down฀in฀the฀more฀common฀α-coniguration฀(See฀
Fig.฀6.7฀and฀especially฀Fig.฀15.6).฀(b)฀Amylase฀products฀of฀amylose,฀Amylase฀adds฀a฀molecule฀of฀
water฀to฀every฀second฀α-1→4฀glycoside฀bond฀from฀the฀non-reducing฀end฀(left,฀red).฀The฀products฀
are฀maltose฀(left)฀and฀a฀limit฀dextrin฀(right),฀never฀free฀glucose.฀The฀water฀molecule฀provides฀the฀OH฀
group฀attached฀to฀the฀reducing฀end฀of฀maltose฀(green).฀The฀proton฀of฀that฀water฀molecule฀becomes฀
attached฀to฀glutamate฀residue฀233฀on฀the฀enzyme฀(See฀Fig฀12.11).฀(c)฀Large฀scale฀amylopectin฀draw-
ing฀showing฀amylase฀action,฀Black฀circles฀indicate฀each฀glucopyranose฀residue.฀Short฀red฀arrows฀
indicate฀sites฀of฀amylase฀hydrolysis฀at฀every฀second฀α1→4฀bond฀(maltose฀cleavage฀sites)฀beginning฀
at฀the฀non-reducing฀end฀of฀each฀branch฀point.฀Glycogen฀is฀also฀a฀substrate,฀but฀it฀is฀more฀branched฀
than฀amylopectin.฀(d)฀Portion฀of฀an฀amylopectin฀limit฀dextrin,฀Amylopectin฀has฀a฀single฀free฀ano-
meric฀OH฀group฀(one฀reducing฀end)฀like฀amylose.฀(Original฀Figures)
222 12 Saliva

fructosyl-transferases฀of฀oral฀streptococci฀that฀metabolize฀sucrose฀and฀cause฀dental฀caries฀
(Chap.฀ 15).฀ Homologies฀ in฀ amino฀ acid฀ sequence฀ have฀ resulted฀ in฀ these฀ enzymes฀ being฀
assigned฀to฀one฀of฀three฀glycan฀hydrolase฀(GH)฀families.฀Family฀GH13฀includes฀the฀mam-
malian฀ a-amylases.฀ Family฀ GH70฀ includes฀ the฀ streptococcal฀ a-glucosy฀ltransferases฀
(Chap.฀15),฀and฀family฀GH77฀includes฀other฀a-glycohydrolases฀and฀transferases.
Hydrolysis฀ of฀ substrates฀ by฀ the฀ GH13฀ family฀ begins฀ two฀ glucose฀ residues฀ from฀ a฀
nonreducing฀end.฀Catalysis฀occurs฀in฀two฀steps:฀the฀formation฀of฀a฀β-diglucosyl฀interme-
diate฀ covalently฀ attached฀ to฀ the฀ enzyme,฀ followed฀by฀ hydrolysis฀ of฀the฀ intermediate฀to฀
maltose฀ with฀ inversion฀ of฀ the฀ β-coniguration฀ (Fig.฀ 12.11a).฀ These฀ steps฀ are฀ reiterated฀

a
Non-reducing Reducing
end Asp197 end Asp197
Asp197
OH O O O
R OH R OH
Nucleophile R
O O O HO-R’ Oβ O O O
O O
HO HO α
α HO H H
OH O R’ O OH O
OH
H H α-Maltose
O
O OH
Acid/base catalyst
O
O O
Glu233
Glu233 Glu233
1 2 3
b
H101
wat
Q63 W59 D197 Wat
*S163
OH
Wat
OH OH OH
O O O O
-3 -2 -1 +1 +2
HO OH O OH O OH N OH O OH OH
HO HO HO HO HO
H

H305 H299 Wat

E240
D300 E233 K200
H201
R195 Wat
*

Fig. 12.11฀ Mechanism฀of฀salivary฀amylase฀catalysis.฀(a)฀Formation฀and฀breakdown฀of฀a฀covalent฀

glycosyl-enzyme฀intermediate.฀Red:฀glucose฀a1฀→฀4฀polymer.฀Black:฀catalytic฀aspartate฀and฀gluta-
mate฀residues.฀Green:฀water฀molecule.฀(1):฀Segments฀of฀at฀least฀5–6฀glucose฀a1฀→฀4฀residues฀in฀
starch฀(amylose฀and฀amylopectin)฀bind฀to฀the฀enzyme฀such฀that฀the฀negatively฀charged฀carboxyl฀
group฀ of฀ aspartate฀ residue฀ 197฀ attacks฀ the฀ glycoside฀ bond฀ in฀ the฀ center฀ of฀ the฀ bound฀ sequence,฀
displacing฀its฀a-anomeric฀glycoside฀bond.฀This฀displacement฀is฀enhanced฀by฀a฀glutamate฀residue฀
233,฀whose฀side฀chain฀is฀nearby฀and฀accepts฀the฀electrons฀from฀the฀glycoside฀OH฀bond.฀(2):฀Asp197฀
and฀glu฀233฀make฀a฀nucleophilic฀attack฀on฀the฀glycoside฀bond฀two฀glucose฀residues฀downstream฀
12.5.2. Mechanism of Action of Salivary Amylase 223

until฀all฀possible฀maltose฀units฀have฀been฀removed฀and฀the฀residue฀containing฀the฀reduc-
ing฀end฀is฀the฀limit฀dextrin.฀The฀mechanism฀is฀facilitated฀by฀how฀the฀substrate฀is฀bound฀by฀
the฀amino฀acid฀residues฀of฀the฀enzyme฀and฀the฀aqueous฀environment฀(Fig.฀12.11b).฀The฀
substrate฀in฀Fig.฀12.11b฀is฀an฀inhibitor฀(acarbose)฀that฀binds฀like฀starch฀but฀contains฀an฀
aminoglycoside฀link฀that฀cannot฀be฀hydrolyzed฀(red).
The฀enzymes฀of฀the฀GH13,฀GH70,฀and฀GH77฀families฀all฀have฀a฀catalytic฀domain฀in฀
the฀form฀of฀a฀barrel฀despite฀sequence฀variability.฀This฀domain฀was฀irst฀observed฀in฀tri-
ose-phosphate฀ isomerase,฀ an฀ enzyme฀ that฀ interconverts฀ glyceraldehyde฀ 3-phosphate฀
with฀dihydroxyacetone฀3-phosphate฀(Chap.฀1,฀Fig.฀2.10).฀The฀barrel฀is฀assembled฀from฀
consecutive฀ beta-strand/alpha-helix฀ units:฀ eight฀ parallel฀ beta-strands฀ each฀ surrounded฀
by฀an฀alpha-helix฀(Fig.฀12.12a).฀A฀simpliied฀diagram฀of฀the฀overall฀structure฀is฀pictured฀
in฀ Fig.฀ 12.12b.฀ In฀ all฀ three฀ glycoside฀ hydrolase฀ family฀ enzymes฀ (GH13,฀ GH70,฀ and฀
GH77),฀the฀catalytic฀site฀is฀formed฀from฀four฀conserved฀sequences฀(I฀through฀IV)฀that฀
come฀together฀at฀the฀top฀of฀the฀barrel฀in฀the฀tertiary฀structure฀(Fig.฀12.12a;฀sequences฀
numbered฀for฀salivary฀amylase).฀Sequences฀II฀and฀III฀include฀the฀catalytic฀aspartate฀or฀
glutamate฀residues฀described฀in฀Fig.฀12.11a.฀Sequence฀IV฀contains฀a฀second฀essential฀
aspartate฀residue฀that฀acts฀with฀its฀adjacent฀histidine฀residue฀and฀the฀conserved฀histidine฀
residue฀in฀sequence฀I฀to฀position฀the฀glycoside฀bond฀for฀hydrolysis฀(Fig.฀12.11b).฀Other฀
residues฀that฀are฀important฀for฀salivary฀amylase฀positioning฀the฀substrate฀are฀shown฀in฀
Fig.฀12.11b.

Fig. 12.11฀ (continued)฀ from฀a฀nonreducing฀end฀(R).฀A฀large฀fragment฀containing฀the฀reducing฀end฀

(R¢)฀is฀hydrolyzed฀and฀the฀remaining฀disaccharide฀moiety฀forms฀a฀covalent฀b-glycoside฀bond฀with฀
asp197.฀(3):฀The฀covalent฀intermediate฀is฀immediately฀attacked฀by฀water,฀restoring฀the฀a-conigu-
ration.฀As฀it฀does฀so,฀its฀proton฀is฀removed฀by฀glu฀233.฀The฀cleaved฀disaccharide฀(maltose)฀cannot฀
be฀retained฀on฀the฀enzyme฀and฀it฀falls฀away฀as฀a฀maltose฀residue.฀This฀mechanism฀applies฀to฀all฀
a-glucosyl฀ transferases฀ including฀ the฀ bacterial฀ glucosyl฀ transferase฀ that฀ synthesizes฀ mutan฀ (see฀
Sect.฀15.2.1).฀(b)฀Diagram฀of฀enzyme฀bound฀to฀the฀catalytic฀inhibitor฀acarbose.฀This฀diagram฀is฀
enlarged฀from฀the฀X-ray฀structure฀with฀a฀bound฀acarbose฀inhibitor฀molecule฀shown฀in฀Fig.฀12.11a.฀
The฀acarbose฀inhibitor฀is฀a฀ive-membered฀sugar-like฀structure฀in฀which฀there฀is฀an฀N-linked฀glyco-
sidic฀bond฀that฀cannot฀be฀cleaved฀and฀binds฀to฀the฀enzyme฀irreversibly.฀It฀is฀formed฀from฀naturally฀
occurring฀acarbose฀by฀salivary฀or฀pancreatic฀amylase฀hydrolyzing฀and฀transferring฀residues฀among฀
related฀structures.฀Red,฀glycoside฀amide฀bond฀of฀acarbose;฀Green,฀catalytic฀residues฀that฀function฀
as฀shown฀in฀Fig.฀12.10a;฀Blue:฀invariant฀pair฀of฀histidine฀residues฀that฀position฀the฀substrate฀cor-
rectly฀(see฀text);฀Large฀green฀asterisk฀(bottom฀center),฀the฀catalytic฀water฀molecule;฀Black฀asterisk฀
(top฀ left)฀ a฀ serine฀ residue฀ in฀ salivary฀ amylase฀ that฀ is฀ threonine฀ in฀ pancreatic฀ amylase.฀ Negative฀
numbers฀indicate฀glucosyl฀residue฀binding฀sites฀on฀the฀nonreducing฀side฀of฀the฀glycoside฀bond฀that฀
would฀normally฀be฀cleaved฀and฀positive฀numbers฀indicate฀these฀sites฀on฀the฀reducing฀side.฀When฀
amylase฀or฀amylopectin฀bind฀instead฀of฀the฀acarbose฀inhibitor,฀a฀+3฀residue฀site฀is฀detectable฀(a:฀
Figure฀modiied฀from฀Fig.฀2฀in฀Brayer฀GD,฀et฀al.฀(2000)฀“Subsite฀mapping฀of฀the฀human฀pancreatic฀
alpha-amylase฀active฀site฀through฀structural,฀kinetic,฀and฀mutagenesis฀techniques.”฀Biochemistry฀
39(16):4778–4791;฀b:฀Modiied฀from฀Fig.฀6฀in฀Maurus,฀R.฀et฀al.฀(2005)฀“Structural฀and฀mechanistic฀
studies฀ of฀ chloride฀ induced฀ activation฀ of฀ human฀ pancreatic฀ alpha-amylase.”฀ Protein฀ Science฀
14(3):743–755)
224 12 Saliva

Fig. 12.12฀ Structure฀of฀salivary฀a-amylase฀(GH13฀family).฀(a)฀Topology฀diagram:฀The฀secreted฀enzyme฀

is฀a฀single฀polypeptide฀of฀496฀residues฀that฀establishes฀three฀domains,฀A,฀B,฀and฀C.฀The฀A฀domain฀
consists฀of฀8฀b-sheet/a-helical฀domain฀that฀forms฀a฀barrel.฀The฀b-sheets฀(green)฀are฀labeled฀b1฀through฀
b8฀with฀each฀succeeding฀a-helix฀(light฀blue)฀not฀labeled.฀The฀B฀domain฀(blue)฀interrupts฀the฀A฀domain฀
between฀b3฀and฀its฀corresponding฀a-helix฀(residues฀100–168).฀The฀(C฀domain,฀red)฀is฀a฀small,฀structur-
ally฀ independent฀ antiparallel฀ b฀ barrel฀ close฀ to฀ the฀ C-terminus฀ (residues฀ 405–496).฀ There฀ are฀ four฀
conserved฀regions฀(I–IV)฀of฀which฀two฀contain฀the฀residues฀essential฀for฀catalysis฀shown฀in฀Fig.฀12.11฀
(Asp฀197฀and฀Glu฀233;฀green).฀An฀additional฀invariant฀residue฀in฀conserved฀region฀I฀(His฀101)฀and฀two฀
in฀conserved฀region฀II฀(His฀299฀and฀Asp฀300)฀hold฀the฀substrate฀so฀that฀the฀catalytic฀residues฀are฀cor-
rectly฀positioned฀(see฀text).฀In฀sequences฀I฀through฀IV,฀residue฀X฀=฀hydrophobic;฀B฀=฀hydrophilic;฀฀
Z฀=฀amino฀acid฀sequence฀that฀determines฀enzyme฀speciicity฀(Adapted฀from฀Fig.฀1฀in฀Kralj฀SE฀et฀al.฀
(2005)฀“Rational฀transformation฀of฀Lactobacillus฀reuteri฀121฀reuteransucrase฀into฀a฀dextransucrase.”฀
Biochemistry฀44(25):9206–9216;฀sequences฀from฀Macgregor฀EA฀et฀al.฀(2001฀Mar฀9)฀“Relationship฀of฀
sequence฀and฀structure฀to฀speciicity฀in฀the฀alpha-amylase฀family฀of฀enzymes.”฀Biochim.฀Biophys.฀
Acta฀ 1546(1):1–20,฀ with฀ residue฀ numbers฀ altered฀ to฀ accord฀ with฀ salivary฀ amylase)฀ (b)฀ Diagram:฀฀
A฀barrel฀is฀drawn฀for฀simplicity.฀It฀is฀formed฀in฀the฀order฀of฀the฀b-sheet/a-helix฀sequences฀in฀the฀poly-
peptide,฀N-terminal฀to฀C-terminal.฀The฀view฀is฀looking฀down฀from฀the฀top฀of฀a฀tilted฀(b/a)8-helix฀
barrel฀(A฀domain)฀with฀the฀B฀domain฀protruding฀to฀the฀left.฀The฀calcium฀ion฀(red)฀is฀required฀for฀the฀
B฀domain฀structure฀to฀interfaces฀correctly฀with฀the฀substrate฀binding฀region฀on฀the฀A฀domain฀at฀the฀top฀
of฀ the฀ barrel฀ (pictured฀ on฀ the฀ far฀ right).฀ The฀ acarbose฀ inhibitor฀ (substrate฀ analogue;฀ see฀ legend฀ to฀
Fig.฀12.10b)฀its฀in฀a฀cleft฀between฀the฀A฀and฀B฀domains฀(red).฀The฀C-domain฀is฀loosely฀associated฀
with฀the฀A฀domain฀and฀perhaps฀enhances฀enzyme฀solubility฀(see฀text)฀
12.5.3. Detection of Salivary and Pancreatic Amylase 225

The฀A฀domains฀of฀all฀mammalian฀and฀some฀other฀GH13฀a-amylases฀have฀a฀chloride-
binding฀site฀that฀is฀essential฀for฀activity฀together฀with฀a฀short,฀inserted฀downstream฀amino฀
acid฀loop฀(residues฀304฀through฀311฀in฀salivary฀amylase).฀This฀loop฀is฀lexible฀and฀interacts฀
with฀substrate฀after฀binding฀so฀that฀the฀catalytic฀residues฀are฀correctly฀positioned฀for฀hydro-
lysis.฀It฀is฀absent฀from฀enzymes฀that฀do฀not฀require฀chloride฀ions฀for฀activity.฀The฀C฀domain฀
has฀no฀known฀function,฀but฀it฀may฀facilitate฀solubility,฀in฀part,฀because฀of฀glycan฀attached฀
to฀asparagine-413฀in฀this฀domain.

12.5.3.
Detection of Salivary and Pancreatic Amylase

Amylase฀isoenzymes฀are฀detected฀after฀separation฀over฀an฀isoelectric฀pH฀gradient.฀They฀
then฀ diffuse฀ into฀ an฀ overlying,฀ insoluble฀ starch฀ polymer฀ containing฀ a฀ covalently฀ bound฀
dye.฀Release฀of฀the฀dye฀indicates฀where฀the฀isoenzymes฀are฀on฀the฀gel.฀Salivary฀and฀pan-
creatic฀ amylases฀ are฀ secreted฀ into฀ blood฀ as฀ well฀ as฀ saliva฀ and฀ small฀ intestine฀ and฀ each฀
accounts฀for฀about฀half฀the฀amylase฀content฀of฀blood฀plasma.฀More฀recently,฀monoclonal฀
antibodies฀speciic฀for฀human฀salivary฀amylase฀have฀facilitated฀the฀measurement฀of฀amy-
lases฀in฀serum฀during฀salivary฀gland฀malfunction,฀and฀facilitated฀the฀differentiation฀of฀sali-
vary฀from฀pancreatic฀amylases฀(Fig.฀12.13).

Pancreatic
Normal Pancreatitis
insufficiency
Anode
(+)
S

P
Cathode

Fig. 12.13฀ Amylase฀ families฀ on฀ electrophoresis:฀ The฀ appearance฀ of฀ multiple฀ amylase฀ enzymes฀
(isoenzymes฀or฀isozymes)฀is฀due฀to฀variable฀loss฀of฀glycan฀and฀variable฀deamidation฀of฀up฀to฀three฀
asparagine฀residues.฀Salivary฀amylase฀(S)฀is฀secreted฀into฀blood฀plasma฀as฀well฀as฀into฀the฀oral฀
cavity.฀It฀is฀more฀negatively฀charged฀(has฀a฀lower฀pI)฀and฀therefore฀moves฀more฀to฀the฀anode฀than฀
pancreatic฀amylase฀(P),฀which฀is฀also฀in฀blood฀plasma.฀The฀bands฀that฀move฀most฀to฀the฀anode฀are฀
the฀most฀modiied,฀but฀the฀salivary฀amylases฀are฀always฀more฀negatively฀charged฀(see฀text).฀In฀
pancreatitis,฀pancreatic฀amylase฀isozymes฀are฀increased฀and฀in฀pancreatic฀insuficiency,฀the฀sali-
vary฀isozymes฀are฀increased.฀Dense฀hatching—glycan-containing฀isozymes:฀light฀hatching—gly-
can-free฀isozymes฀(Part฀of฀Figure฀1฀in฀Pieper-Bigelow฀C,฀Strocchi฀A฀and฀Levitt฀MD฀(1990)฀“Where฀
does฀serum฀amylase฀come฀from฀and฀where฀does฀it฀go?”฀Gastroenterology฀Clinics฀of฀North฀America฀
19:793–810.฀Copyright฀Elsevier,฀1990)
226 12 Saliva

In฀parotid฀and฀whole฀saliva,฀the฀glycan฀linked฀to฀asp-413฀is฀often฀removed฀by฀enzymes฀
in฀the฀oral฀cavity.฀In฀addition,฀this฀and฀two฀nearby฀asparagine฀residues฀may฀be฀deamidated฀
(asn฀→฀asp)฀by฀other฀enzymes฀(deamidases).฀The฀resultant฀increase฀in฀net฀negative฀charge฀
causes฀the฀appearance฀of฀amylase฀isozyme฀families฀after฀isoelectric฀focusing฀or฀gel฀elec-
trophoresis฀(Fig.฀12.13b).฀Loss฀of฀the฀N-terminal฀positive฀charge฀and฀deamidation฀of฀all฀
three฀asparagines฀decrease฀the฀calculated฀isoelectric฀pH฀(pI)฀of฀salivary฀amylase฀from฀6.34฀
to฀5.98,฀similar฀to฀observed฀values฀(6.4฀and฀6.0).฀Secreted฀pancreatic฀amylase฀has฀one฀less฀
negative฀charge฀and฀therefore฀migrates฀less฀to฀the฀anode.฀However,฀its฀observed฀pI฀is฀7.0,฀
considerably฀greater฀than฀its฀calculated฀pI฀of฀6.45฀(Fig.฀12.13).฀Figure฀12.13฀shows฀also฀
that฀salivary฀amylase฀is฀more฀diverse฀than฀pancreatic฀amylase,฀possibly฀due฀to฀cosecreted฀
deamidases฀that฀are฀absent฀from฀the฀pancreatic฀secretion.
As฀noted฀previously,฀the฀N-terminal฀methionine฀residue฀was฀lost฀when฀the฀15฀amino฀
acid฀secretory฀signal฀sequence฀was฀removed฀in฀the฀endoplasmic฀reticulum.฀The฀N-terminus฀
of฀secreted฀salivary฀amylase฀is฀glutamine,฀which฀is฀slowly฀deamidated฀to฀glutamate฀by฀the฀
deamidase฀ that฀ also฀ converts฀ the฀ asparagine฀ residues฀ to฀ aspartate฀ as฀ noted฀ above.฀ The฀
N-terminal฀glutamate฀then฀slowly฀and฀spontaneously฀forms฀pyroglutamate฀(Fig.฀12.14),฀
which฀ prevents฀ amino฀ acid฀ sequencing฀ by฀ Edman฀ degradation,฀ interferes฀ with฀ protein฀
identiication฀by฀shotgun฀and฀top–down฀techniques,฀and฀prevents฀a฀determination฀of฀the฀
biological฀functions฀associated฀with฀this฀change.

The฀ major฀ protein฀ of฀ saliva฀ is฀ a-amylase,฀ an฀ endoglycohydrolase฀ encoded฀ by฀ the฀
gene฀ Amy1฀ on฀ chromosome฀ 1.฀ It฀ hydrolyzes฀ internal฀ a1฀ →฀ 4฀ glucoside฀ bonds฀ of฀
starch฀to฀the฀disaccharide฀maltose฀and฀moderate฀length฀oligosaccharides฀called฀limit฀
dextrins.฀These฀products฀adhere฀to฀chewed฀food฀and฀hold฀the฀bolus฀together฀for฀swal-
lowing.฀ In฀ the฀ intestine,฀ limit฀ dextrins฀ are฀ digested฀ to฀ smaller฀ oligosaccharides฀ by฀
pancreatic฀amylase฀(Amy2),฀an฀homologous฀enzyme,฀and฀then฀to฀glucose฀on฀the฀sur-
face฀of฀intestinal฀epithelial฀cells.฀The฀glucose฀is฀then฀transported฀through฀these฀cells฀
to฀ the฀ bloodstream.฀ Catalysis฀ by฀ amylase฀ involves฀ an฀ internal฀ a-glucosyl฀ bond฀
becoming฀esteriied฀to฀an฀asp฀residue฀on฀the฀enzyme฀with฀immediate฀hydrolysis฀by฀
water฀with฀assistance฀from฀a฀nearby฀glu฀residue.฀The฀catalytic฀site฀lies฀at฀the฀top฀of฀a฀
barrel-shaped฀ domain฀ on฀ the฀ enzyme.฀ A฀ tightly฀ linked฀ second฀ domain฀ containing฀
Ca2+฀stabilizes฀the฀catalytic฀portion฀of฀the฀domain฀and฀a฀Cl−1฀ion฀within฀the฀barrel฀
domain฀interacts฀with฀a฀short,฀lexible฀amino฀acid฀loop฀to฀position฀the฀substrate฀and฀
catalytic฀residues฀correctly.฀Amylase฀is฀detected฀by฀isoelectric฀focusing฀followed฀by฀
diffusion฀ of฀ the฀ protein฀ into฀ an฀ overlying,฀ insoluble฀ starch฀ polymer฀ containing฀ a฀
covalently฀bound฀dye.฀Release฀of฀the฀dye฀indicates฀the฀enzyme฀on฀the฀gel.฀Saliva฀and฀
blood฀ contain฀ amylase฀ isozyme฀ families฀ due฀ to฀ other฀ enzymes฀ removing฀ variable฀
amounts฀of฀attached฀glycan฀and฀amide฀groups฀from฀up฀to฀three฀asn฀residues฀near฀the฀
C-terminus.฀ Salivary฀ and฀ pancreatic฀ amylases฀ are฀ also฀ secreted฀ into฀ blood,฀ where฀
they฀each฀account฀for฀about฀half฀of฀the฀amylase฀content฀and฀differ฀by฀the฀salivary฀
amylase฀family฀having฀a฀greater฀negative฀charge.
12.6.1. Proline-Rich Proteins 227

O O
+฀− O
H O
R
R −HOH NH
NH2 O +
H
H+
Glutamate Pyroglutamate

Fig. 12.14฀ Formation฀of฀pyroglutamate฀from฀N-terminal฀glutamate.฀The฀N-terminal฀gamma-amide฀

of฀ glutamine฀ is฀ deamidated฀ to฀ form฀ glutamate฀ (left).฀ The฀ carboxyl฀ group฀ of฀ the฀ newly฀ formed฀
glutamate฀then฀slowly฀attacks฀the฀free฀N-terminal฀amino฀group,฀removing฀a฀water฀molecule฀to฀
form฀a฀secondary฀amide฀(see฀text).฀R฀is฀the฀primary฀amide฀bond฀of฀the฀alpha-carboxyl฀group฀of฀
glutamine฀(or฀glutamate)฀to฀the฀alpha฀amino฀group฀of฀the฀next฀amino฀acid฀of฀the฀primary฀sequence฀
(tyrosine)฀(Slightly฀modiied฀from฀Fig.฀1฀of฀Chelius฀D,฀et฀al.฀(2006)฀“Formation฀of฀Pyroglutamic฀
Acid฀from฀N-terminal฀glutamic฀acid฀in฀immunoglobulin฀gamma฀antibodies.”฀Anal.฀Chem.฀78(7):฀
2370–2376)

12.6.1.
Proline-Rich Proteins

About฀ 40%฀ of฀ the฀ non-mucous฀ proteins฀ in฀ salivary฀ secretions฀ are฀ proline-rich฀ proteins฀
(PRPs),฀which฀are฀about฀equally฀divided฀into,฀acidic,฀basic,฀and฀glycosylated฀proteins.฀The฀
N-terminal฀domain฀of฀the฀acidic฀and฀basic฀PRPs฀is฀dominated฀by฀either฀negatively฀or฀posi-
tively฀charged฀amino฀acids฀and฀their฀C-terminal฀domain฀by฀many฀proline฀residues.฀This฀
simple฀ 2-domain฀ primary฀ structure,฀ in฀ which฀ each฀ half฀ has฀ a฀ predominance฀ of฀ speciic฀
amino฀acids,฀is฀diagrammed฀in฀Fig.฀12.15.฀The฀acidic฀PRPs฀are฀rich฀in฀aspartate฀and฀gluta-
mate฀residues฀at฀the฀N-terminal฀domain฀and฀are฀encoded฀by฀two฀genes,฀PRH1฀and฀PRH2.฀
The฀ basic฀ PRPs฀ are฀ rich฀ in฀ lysine,฀ arginine,฀ and฀ histidine฀ residues฀ at฀ their฀ N-terminal฀
domain฀and฀encoded฀by฀four฀genes,฀PRB1,฀2,฀3,฀and฀4.฀Each฀gene฀has฀several฀alleles฀that฀
vary฀in฀size฀and฀their฀products฀account฀for฀about฀20฀proteins,฀giving฀rise฀to฀complex฀poly-
morphic฀patterns฀between฀individuals.฀During฀and฀after฀secretion,฀various฀proteins฀arise฀by฀
partial฀proteolytic฀cleavage฀at฀sites฀with฀sequence฀arg-pro-pro-arg฀in฀acidic฀PRPs฀and฀arg-
ser-X-arg฀in฀basic฀PRPs฀(where฀X฀stands฀for฀any฀amino฀acid).฀The฀glycosylated฀PRPs฀are฀
all฀basic฀and฀the฀glycan฀is฀asparagine-linked฀as฀in฀amylase.
As฀noted฀earlier฀(Sect.฀12.1.3),฀the฀reduced฀enamel฀epithelium฀is฀replaced฀with฀adherent฀
salivary฀proteins,฀mostly฀amylase฀and฀acidic฀proline-rich฀proteins.฀The฀acidic฀PRPs฀are฀at฀
about฀ a฀ three฀ times฀ greater฀ concentration฀ than฀ in฀ secreted฀ saliva.฀ The฀ acidic฀ domain฀ of฀
these฀proteins฀especially฀has฀a฀high฀afinity฀for฀hydroxyapatite.฀Once฀bound฀to฀pellicle,฀the฀
N-terminus฀of฀acidic-PRPs฀forms฀an฀attachment฀site฀for฀the฀major฀classes฀of฀commensal฀
bacteria฀ (viridans฀ streptococci฀ and฀ Actinomyces฀ spp.)฀ that฀ occupy฀ the฀ mucosal฀ surfaces฀
and฀saliva฀of฀a฀healthy฀oral฀cavity.฀Some฀streptococci฀grow฀by฀themselves฀in฀dental฀bio-
ilms,฀but฀many฀others฀grow฀better฀if฀certain฀Actinomyces฀spp.฀are฀also฀present.
228 12 Saliva

a Acidic proline rich protein PRP-1

(PRH1 locus)
N-terminal C-terminal
domain domain
4 81 106

Acidic residues Proline-rich

(Hydroxyapatite binding) (Bacterial binding)
b
Residue 4 26 50 81 103 106 127 150 171
Primary polypeptide products
PRH1 Db-8 Asn Leu Asp [rpt 61-81] Arg Gln
PIF-8 Asn lie Asp Arg Gln
Pa Asp
1mer Asn Leu Cys Arg Gln
PRH2 PRP-1 Asp lie Asn Arg Gln
PRP-2 Asp lie Asp Arg Gln

Derived molecules
PRH1 Db-f Asn Leu Asp [rpt 61-81] Arg
PIF-f Asn lie Asp Arg
Asn Leu Asp Cys Arg Gln
Pa2mer
Asn Leu Asp Cys Arg Gln
PRH2 PRP-3 Asp lie Asn Arg
PRP-4 Asp lie Asp Arg

Fig. 12.15฀ The฀acidic฀proline-rich฀proteins.฀(a)฀Domain฀organization.฀The฀proline฀rich฀domain฀(black)฀

comprises฀residues฀82฀through฀150฀in฀most฀acidic฀PRPs฀and฀the฀terminal฀sequence฀Pro-Gln฀is฀respon-
sible฀for฀bacterial฀binding.฀The฀irst฀81฀amino฀acids฀are฀rich฀in฀aspartate฀and฀glutamate,฀and,฀if฀the฀amino฀
acid฀in฀position฀4฀is฀asparagine,฀it฀is฀phosphorylated,฀adding฀to฀the฀negative฀charge.฀All฀acidic฀PRPs฀are฀
hydrolyzed฀at฀arginine฀106฀and฀the฀short฀C-terminal฀fragment฀is฀further฀degraded.฀Residues฀1–106,฀the฀
large฀fragment฀(f)฀is฀usually฀seen฀on฀gels฀together฀with฀the฀whole฀secreted฀protein฀(s)฀(b)฀Alleles.฀The฀
secreted฀ N-terminal฀ amino฀ acid฀ (numbered฀ 1)฀ follows฀ a฀ 16฀ amino฀ acid฀ signal฀ sequence฀ that฀ was฀
removed฀in฀the฀endoplasmic฀reticulum.฀It฀is฀therefore฀residue฀17฀of฀the฀encoded฀sequence.฀The฀acidic฀
PRPs฀are฀encoded฀at฀one฀of฀two฀genetic฀loci฀PRH1฀or฀PRH2.฀The฀primary฀and฀secondary฀allelic฀prod-
ucts฀are฀similar฀except฀for฀protein฀Db,฀a฀PRH1฀allelic฀product฀whose฀greater฀length฀is฀due฀to฀a฀repeat฀of฀
amino฀acids฀61฀through฀81.฀It฀is฀therefore฀21฀amino฀acids฀longer฀than฀the฀other฀alleles.฀Allele฀Pa฀has฀an฀
encoded฀cysteine฀residue,฀and฀it฀spontaneously฀forms฀a฀Pa฀dimer.฀Subscripts฀for฀Db฀and฀Pif฀indicate฀the฀
secreted฀sequence฀(s)฀or฀the฀large฀fragment฀(f)฀(a:฀original฀igure;฀b:฀Copy฀of฀Fig.฀1฀in฀Hay฀DI฀(1994฀
Nov)฀“Human฀salivary฀acidic฀proline-rich฀protein฀polymorphisms฀and฀biosynthesis฀studied฀by฀high-
performance฀liquid฀chromatography.”฀J.฀Dent.฀Res.฀73(11):1717–1726)

12.6.2.
Salivary Agglutinin

Salivary฀agglutinin,฀identical฀to฀lung฀glycoprotein-340฀(gGP-340)฀and฀a฀protein฀with฀an฀
unrelated฀function฀in฀nervous฀tissue฀(DMBT1),฀is฀a฀highly฀conserved฀bacterial฀receptor.฀In฀
the฀lungs฀and฀saliva฀where฀it฀is฀secreted,฀it฀attaches฀to฀many฀different฀bacteria,฀including฀
12.6.2. Salivary Agglutinin 229

many฀oral฀streptococci.฀All฀bacterial฀binding฀to฀salivary฀agglutinin฀is฀believed฀to฀occur฀at฀
a฀ single฀ site฀ in฀ SRCR฀ domain฀ 2฀ near฀ the฀ N-terminus฀ (Fig.฀ 12.16).฀ Salivary฀ agglutinin฀
remains฀soluble฀after฀binding฀bacteria,฀and฀it฀is฀part฀of฀the฀innate฀immunity฀system฀that฀
clears฀bacteria฀from฀the฀oral฀cavity฀(Table฀12.1฀and฀Sect.฀12.1.4).฀Use฀of฀a฀monoclonal฀
antibody฀to฀quantify฀the฀amounts฀of฀salivary฀agglutinin฀have฀shown฀that฀it฀is฀present฀in฀
small฀but฀highly฀variable฀amounts.฀Because฀salivary฀agglutinin฀contributes฀also฀to฀acquired฀
pellicle,฀individuals฀who฀secrete฀more฀agglutinin฀may฀bind฀a฀greater฀complexity฀of฀bacte-
ria฀ to฀ teeth฀ surfaces.฀ Indeed,฀ the฀ presence฀ of฀ certain฀ acidic฀ PRPs฀ along฀ with฀ a฀ greater฀
amounts฀of฀salivary฀agglutinin฀increase฀an฀adult’s฀susceptibility฀to฀caries฀(Sect.฀15.3.3).
Salivary฀agglutinin฀is฀a฀member฀of฀the฀scavenger฀receptor฀cysteine-rich฀(SRCR)฀super-
family,฀a฀group฀of฀large฀(~2,400฀amino฀acids;฀mol฀wt฀262฀kDa)฀cell฀surface฀and฀secreted฀
glycoproteins.฀ These฀ glycoproteins฀ are฀ characterized฀ by฀ SRCR฀ and฀ SRCR-interspersed฀
(SID)฀ domains฀ illustrated฀ in฀ Fig.฀ 12.16.฀ Other฀ proteins฀ in฀ this฀ family฀ include:฀ (1)฀ the฀

N-terminus
*
1 2 3 4 5 6 7 8 9

Unique
SID
Signal
peptide
(aa 1 - 19)
Lysine
1812
C-terminus
10 11 12 13 14
CUB CUB ZP

STP- Unique
rich

Fig. 12.16฀ Salivary฀agglutinin.฀Salivary฀agglutinin฀is฀characterized฀by฀14฀SRCR฀domains฀(gray฀and฀

numbered)฀and฀many฀SRCR-interspersed-domains฀(SID).฀SRCR฀and฀SID฀domains฀are฀~100฀and฀~30฀
amino฀acids฀in฀length,฀respectively,฀and฀the฀secreted฀molecule฀consists฀of฀a฀total฀of฀2,394฀amino฀acids.฀
SRCR฀domains฀are฀described฀in฀the฀text.฀The฀N-terminal฀methionine฀and฀following฀19฀amino฀acids฀
(signal฀ sequence,฀ black)฀ is฀ cleaved฀ in฀ the฀ endoplasmic฀ reticulum.฀ The฀ N-terminal฀ domain฀ of฀ the฀
secreted฀protein฀(purple)฀has฀a฀short฀unique฀domain฀before฀the฀irst฀SRCR฀domain.฀The฀asterisk฀above฀
SRCR฀domain฀2฀indicates฀a฀unique฀bacterial฀binding฀site฀(see฀text).฀Like฀the฀N-terminal฀domain฀of฀the฀
secreted฀protein,฀the฀C-terminal฀domain฀(purple)฀is฀also฀short฀and฀unique.฀Immediately฀upstream฀of฀the฀
C-terminus฀is฀a฀zona฀pellucida฀(ZP)฀domain฀(blue)฀and฀2฀CUB฀domains฀(yellow,฀see฀Chap.฀8)฀before฀
and฀after฀the฀14th฀SRCR฀domain.฀Immediately฀upstream฀of฀each฀CUB฀domain฀is฀a฀short฀serine-threo-
nine-proline-rich฀(STP-rich)฀domain฀(green).฀The฀ZP฀domain฀consists฀of฀260฀amino฀acids฀just฀upstream฀
of฀the฀C-terminus฀and฀is฀common฀in฀membrane-anchored฀eukaryotic฀glycoproteins฀possessing฀a฀large฀
multidomain,฀extracellular฀region.฀However,฀salivary฀agglutinin฀is฀secreted,฀not฀membrane฀anchored.฀
The฀STP-rich฀domain฀consists฀of฀25฀amino฀acids฀of฀which฀19฀are฀serine,฀threonine,฀or฀proline฀residues฀
forming฀ive฀possible฀O-linked฀glycosylation฀sites.฀(Figure฀is฀simpliied฀from฀Bikker฀et฀al.฀(2002฀Aug฀
30)฀“Identiication฀of฀the฀bacteria-binding฀peptide฀domain฀on฀salivary฀agglutinin฀(gp-340/DMBT1),฀a฀
member฀of฀the฀scavenger฀receptor฀cysteine-rich฀superfamily.”฀J.฀Biol.฀Chem.฀277(35):32109–32115)
230 12 Saliva

macrophage฀scavenger฀receptor฀involved฀in฀the฀endocytosis฀of฀low-density฀lipoproteins฀by฀
macrophages;฀ (2)฀ human฀ macrophage-associated฀ galectin3-binding฀ protein฀ also฀ called฀
Mac-2฀(a฀lectin฀is฀a฀carbohydrate฀binding฀protein฀and฀a฀galectin฀is฀a฀protein฀that฀binds฀to฀
b-galactosides);฀and฀(3)฀cell฀differentiation฀(CD)฀antigens฀CD5฀and฀CD6฀involved฀in฀the฀
activation฀and฀differentiation฀of฀thymus-derived฀lymphocytes฀in฀response฀to฀foreign฀sub-
stances฀(antigens).

The฀salivary฀proline-rich฀proteins฀have฀a฀two-domain฀structure,฀a฀proline฀poor฀N-terminal฀
domain฀ that฀ is฀ acidic฀ or฀ basic฀ and฀ determines฀ enamel฀ binding,฀ and฀ a฀ proline-rich฀
C-terminal฀ domain฀ that฀ determines฀ bacterial฀ binding.฀ Individual฀ variations฀ in฀ acidic฀
proline-rich฀ proline฀ allelic฀ composition฀ and฀ in฀ the฀ amount฀ of฀ salivary฀ agglutinin,฀ a฀
secreted฀ innate฀ immunity฀ protein฀ that฀ binds฀ bacteria,฀ may฀ account฀ for฀ differences฀ in฀
bioilm฀composition฀and฀dental฀caries฀susceptibility.
 Chronic Periodontitis
13

Periodontal฀ disease฀ is฀ a฀ mixture฀ of฀ diseases฀ in฀ which฀ the฀ periodontal฀ attachment฀ is฀
destroyed,฀ resulting฀ in฀ loose฀ teeth฀ that฀ may฀ exfoliate.฀ This฀ chapter฀ describes฀ how฀
chronic฀ periodontitis,฀ the฀ common฀ form฀ of฀ this฀ disease,฀ begins฀ as฀ gingivitis฀ and฀ is฀
induced฀by฀bacteria฀(Sect.฀1),฀how฀in฀turn฀the฀bacteria฀induce฀the฀neutrophil฀iniltrates฀
responsible฀ for฀ gingivitis฀ (Sect.฀ 2),฀ and฀ how฀ the฀ inlammatory฀ response฀ becomes฀
destructive฀ with฀ macrophage฀ activation฀ (Sect.฀ 3),฀ concluding฀ with฀ a฀ discussion฀ of฀
mechanisms฀of฀cell฀death฀in฀chronic฀periodontitis฀(Sect.฀4).฀The฀last฀section฀discusses฀
the฀roles฀of฀eicosanoids฀in฀promoting฀and฀resolving฀periodontal฀inlammation,฀and฀the฀
effects฀of฀nonsteroidal฀antiinlammatory฀drugs฀on฀these฀processes฀(Sect.฀5).

13.1.1
Detecting Periodontal Disease

Periodontal฀disease฀describes฀a฀mixture฀of฀diseases฀in฀which฀the฀periodontal฀attachment฀is฀
destroyed,฀resulting฀in฀loose฀teeth฀that฀may฀exfoliate.฀Periodontitis฀is฀divided฀into฀chronic฀
and฀aggressive฀forms฀that฀are฀localized฀or฀generalized฀(affect฀few฀or฀many฀teeth).฀Chronic฀
periodontitis฀is฀very฀common฀(Sects.฀13.1.2–13.4.3),฀whereas฀aggressive฀periodontitis฀is฀
rare฀ (Chap.฀ 14).฀ The฀ collagen฀ ibers฀ of฀ the฀ gingiva฀ and฀ periodontium฀ are฀ described฀ in฀
Chap.฀3฀(Sect.฀3.1.5),฀and฀its฀epithelial฀cover฀in฀Chap.฀5฀(Sect.฀5.2.3).
Chronic฀periodontitis฀irst฀appears฀at฀the฀gingival฀sulcus,฀the฀tooth-soft฀tissue฀interface฀
(Fig.฀13.1a).฀The฀marginal฀gingiva฀becomes฀red,฀swells฀with฀loss฀of฀collagen,฀and฀bleeds฀on฀
gentle฀probing฀(gingival฀inlammation฀or฀gingivitis,฀Fig.฀13.1b).฀If฀the฀gingivitis฀and฀collagen฀
loss฀extends฀into฀the฀underlying฀periodontium,฀periodontitis฀is฀present฀(Fig.฀13.1c).฀Gingival฀
inlammation฀ is฀ more฀ prominent฀ in฀ chronic฀ than฀ aggressive฀ periodontitis.฀ The฀ severity฀ of฀
periodontitis฀is฀measured฀by฀periodontal฀attachment฀loss:฀distance฀in฀millimeters฀from฀the฀
enamel–cemental฀junction฀to฀the฀base฀of฀the฀gingival฀sulcus฀or฀pocket฀(deepened฀sulcus).฀
Measurements฀are฀made฀at฀six฀sites฀on฀each฀tooth:฀at฀mesial,฀central,฀and฀distal฀surfaces฀of฀the฀
buccal฀and฀lingual฀sides.฀A฀mean฀attachment฀loss฀of฀up฀to฀2฀mm฀indicates฀mild฀periodontitis;฀
2–4฀mm฀indicates฀moderate฀periodontitis;฀and฀more฀than฀4฀mm฀advanced฀periodontitis.

M. Levine, Topics in Dental Biochemistry, 231

DOI: 10.1007/978-3-540-88116-2_13, © Springer-Verlag Berlin Heidelberg 2011
232 13 Chronic Periodontitis

a b c

Fig. 13.1฀ Healthy฀gingiva,฀gingivitis,฀and฀periodontitis฀in฀the฀oral฀cavity.฀(a)฀Healthy฀gingiva.฀The฀

pale-pink,฀ stippled฀ gingiva฀ is฀ attached฀ tightly฀ to฀ clean฀ teeth.฀ The฀ gingival฀ sulcus฀ is฀ indicated฀
(arrow).฀(b)฀Gingivitis.฀The฀gingiva฀is฀red,฀especially฀at฀the฀free฀gingival฀margins฀(upper฀arrow)฀
and฀plaque฀is฀present฀(lower฀arrow).฀Patients฀with฀moderate฀to฀severe฀gingivitis฀(illustrated฀in฀this฀
slide)฀complain฀that฀their฀gums฀bleed฀on฀toothbrushing.฀(c)฀Periodontitis:฀The฀gingiva฀has฀receded฀
and฀the฀cementum฀has฀been฀abraided,฀exposing฀the฀dentinal฀surface฀of฀the฀tooth฀root฀(recession,฀
arrow).฀The฀gingiva฀is฀inlamed฀(red)฀and฀the฀sulcus฀is฀deepened.฀Periodontal฀pockets฀have฀formed฀
between฀the฀exposed฀dentin฀and฀the฀inlamed฀(red)฀gingival

13.1.2
Gingivitis and Chronic Periodontitis in Humans and Animals

The฀ present-day฀ understanding฀ of฀ how฀ the฀ commensal฀ microbiota฀ causes฀ gingivitis฀ began฀
with฀a฀human฀study฀of฀experimental฀gingivitis฀by฀Harald฀Löe฀and฀his฀colleagues฀in฀Denmark฀
in฀1965.฀Adults฀with฀no฀periodontal฀attachment฀loss฀are฀selected,฀and฀their฀teeth฀are฀thoroughly฀
cleaned฀for฀a฀few฀days.฀They฀then฀abstain฀from฀oral฀hygiene฀procedures฀for฀3฀weeks.฀A฀micro-
bial฀bioilm฀(plaque)฀adheres฀to฀teeth,฀and฀an฀inlammatory฀exudate฀appears฀at฀the฀sulcus,฀the฀
Gingival฀Crevicular฀Fluid฀(GCF;฀Table฀13.1).฀This฀luid฀is฀derived฀from฀serum,฀blood฀plasma฀
in฀which฀ibrin฀clot฀formation฀is฀prevented฀by฀plasmin฀activation฀(Sect.฀13.2.3).฀After฀a฀week,฀
gingivitis฀appears฀and฀extends฀to฀all฀teeth฀within฀about฀3฀weeks.฀The฀severity฀of฀gingivitis฀is฀
measured฀by฀gingival฀index฀(Tables฀13.2)฀averaged฀across฀the฀sites฀described฀above฀for฀attach-
ment฀level.฀Dogs฀develop฀periodontal฀disease฀as฀in฀humans,฀except฀that฀a฀hard฀diet฀cleans฀the฀
teeth฀instead฀of฀oral฀hygiene.฀Changing฀dogs฀from฀hard฀to฀soft฀diets฀causes฀bioilm฀accumula-
tion฀and฀gingival฀inlammation,฀which฀progress฀to฀periodontitis.฀Other฀animal฀models฀are฀less฀
satisfactory.฀Gingivitis฀and฀periodontitis฀are฀induced฀in฀non-human฀primates฀only฀if฀silk฀liga-
tures฀are฀placed฀at฀the฀gingival฀margins.฀In฀rodents,฀periodontitis฀only฀develops฀if฀the฀endog-
enous฀ commensal฀ microbiota฀ is฀ depressed฀ by฀ antibiotics฀ and฀ the฀ oral฀ cavity฀ monoinfected฀
13.1.3 Microbiota of Gingivitis and Chronic Periodontitis in Man 233

Table 13.1฀ Interstitial฀and฀gingival฀crevicular฀luid฀composition

ISF GCF
Transudate Exudate
No฀proteins Rich฀in฀proteins
No฀neutrophils Many฀neutrophils

Table 13 2฀ The฀gingival฀index
Marginal฀gingiva Score
Pink฀and฀stippled a
0
Red฀and฀edematousb 1
Bleeds ฀within฀10฀s
c
2
Bleedsc฀immediately 3
a
Fig.฀13.1a
b
Fig.฀13.1b
c
After฀gentle฀probing

with฀an฀oral฀bacterial฀species.฀Hair฀and฀bedding฀become฀trapped฀in฀gingival฀pockets฀and฀may฀
enhance฀periodontitis฀development.฀Gingival฀inlammation฀is฀minimal.

13.1.3
Microbiota of Gingivitis and Chronic Periodontitis in Man

Because฀animals฀require฀sedation฀before฀sampling฀the฀oral฀cavity,฀most฀studies฀have฀been฀of฀
the฀ human฀ oral฀ microbiota.฀ These฀ studies฀ indicate฀ that,฀ in฀ a฀ healthy฀ oral฀ cavity,฀ bacteria฀
attach฀to฀the฀oral฀mucosa฀and฀are฀continually฀shed฀into฀saliva฀(Sect.฀12.1.5).฀This฀commensal฀
microbiota฀is฀present฀in฀saliva฀(Sect฀12.1.5)฀and฀loosely฀adherent฀to฀the฀oral฀mucosa.฀It฀is฀
mostly฀composed฀of฀viridans฀streptococci฀and฀Actinomyces฀species฀(Fig.฀13.2a,฀b).฀These฀
bacteria฀hydrolyze฀sialic฀acid฀and฀glycans฀from฀mucins฀and฀glycoproteins฀on฀the฀mucosal฀
surface฀or฀in฀saliva฀(Sect.฀12.3.1),฀and฀they฀grow฀by฀metabolizing฀the฀glycans฀to฀carbon฀
dioxide฀and฀water฀(respiration,฀Sect.฀1.3.1).฀They฀also฀attach฀to฀teeth฀surfaces฀where฀mutu-
alistic฀interactions,฀characterized฀by฀luxuriant฀mixed฀culture฀growth฀on฀saliva฀alone฀in฀vitro฀
or฀in฀vivo,฀generate฀a฀bioilm.฀These฀interactions฀are฀called฀quorum฀sensing,฀and฀they฀are฀
mediated฀by฀4,5-dihydroxy-2,3-pentanedione฀(DPD;฀Fig.฀13.3a).
DPD฀is฀also฀called฀autoinducer-2฀(AI-2;฀AI-1฀is฀another,฀less฀common฀inducer).฀AI-2฀is฀
derived฀from฀S-ribosyl฀homocysteine฀which฀is฀hydrolyzed฀to฀homocysteine฀and฀1-deoxy-
3-dehydro-d-ribulose฀in฀bacteria.฀The฀d-ribulose฀derivative฀is฀extruded฀as฀a฀boric฀acid฀ester฀
(boron฀is฀a฀trace฀element,฀Chap.฀1),฀which฀spontaneously฀rearranges฀into฀numerous฀iso-
mers฀that,฀together,฀comprise฀AI-2.฀Each฀isomer฀binds฀with฀a฀structurally฀and฀functionally฀
distinct฀receptor.฀Thus,฀quorum฀sensing฀through฀AI-2฀secretion฀activates฀the฀expression฀of฀
genes฀required฀for฀growth฀by฀mutualistic฀interactions฀within฀bioilms.฀For฀example,฀coag-
gregation฀ with฀ an฀ actinomyces฀ species฀ enables฀ a฀ streptococcus฀ species฀ to฀ switch฀ from฀
quiescence฀to฀active฀growth฀when฀arginine฀is฀scarce฀as฀it฀is฀in฀saliva.
234 13 Chronic Periodontitis

a b

c d

Fig. 13.2฀ Common฀bacteria฀of฀plaque.฀Slides฀show฀typical฀gram฀stain฀for:฀(a)฀viridans฀streptococci;฀

(b)฀Actinomyces฀naeslundii,฀previously฀A.฀viscosus;฀(c)฀Eikenella฀corrodens,฀and฀(d)฀Fusobacterium฀
nucleatum฀(From฀Public฀Health฀Image฀Library฀(PHIL)฀at฀the฀CDC฀–฀Bacteria฀Site:฀http://phil.cdc.
gov/phil/home.asp)

Another฀ bacterium฀ that฀ contributes฀ to฀ commensal฀ bioilms฀ is฀ Eikenella฀ corrodens,฀฀
a฀gram฀negative,฀small฀rod฀(Fig.฀13.2c).฀E.฀corrodens฀grows฀in฀a฀healthy฀oral฀cavity฀by฀
reducing฀nitrate฀in฀saliva฀to฀nitrite฀(Sects.฀1.3.2฀and฀12.1.3).฀It฀is฀an฀important฀contributor฀
to฀bite฀wound฀infections฀and฀is฀also฀the฀major฀known฀producer฀of฀lysine฀decarboxylase,฀
which฀converts฀lysine฀to฀cadaverine฀and฀carbon฀dioxide฀(Fig.฀13.4).฀Lysine฀is฀a฀nutrition-
ally฀essential฀amino฀acid,฀whereas฀cadaverine฀is฀not.฀In฀humans,฀E.฀corrodens฀and฀lysine฀
13.1.3 Microbiota of Gingivitis and Chronic Periodontitis in Man 235

a
HO OH
B
O O

HO CH3

O
HO

b
Commensal Microbial Reciprocal interactions
microbiota succession (Climax microbiota)

Purple
Actinomyces sp. Increasing
Orange Red Gingivitis
Yellow
GCF exudation
Green

Oral hygiene

(Decreasing GCF exudation)

Fig. 13.3฀ Bacterial฀ colonization฀ and฀ succession฀ in฀ gingivitis.฀ (a)฀ Structure฀ of฀ autoinducer-2.฀ All฀
possible฀conigurations฀of฀the฀OH฀groups฀occur฀spontaneously฀in฀solution฀due฀to฀boron-catalyzed฀
hydrolysis฀and฀rearrangement.฀Different฀bacterial฀species฀have฀receptors฀that฀only฀recognize฀one฀
or฀ a฀ limited฀ number฀ of฀ these฀ conigurations฀ (b)฀ Bacterial฀ complexes.฀ The฀ commensal฀ bacteria฀
attach฀to฀teeth฀as฀distinct฀complexes฀within฀a฀bioilm฀(plaque).฀The฀most฀prominent฀complexes฀are฀
Actinomyces฀spp.฀(blue),฀Streptococcus฀spp.฀(yellow)฀and฀a฀mixture฀of฀Capnocytophaga฀spp.฀with฀
E.฀ corrodens฀ (green).฀ The฀ successor฀ microbiota฀ attaches฀ to฀ these฀ commensal฀ bioilms฀ as฀ com-
plexes฀of฀Fusobacteria฀spp.฀with฀many฀other฀gram฀negative฀bacteria฀(orange)฀and฀a฀climax฀com-
plex฀ of฀ just฀ three฀ species:฀ Porphyromonas฀ gingivalis,฀ Treponema฀ denticola,฀ and฀ Tannerella฀
forsythia฀(red)฀(a:฀Public฀domain฀image:฀http://en.wikipedia.org/wiki/Autoinducer-2;฀b:฀Modiied฀
from฀Fig.฀1.฀in฀Socransky฀and฀Haffajee,฀“Periodontal฀microbial฀ecology.”฀Periodontology฀2000,฀
38:135–187,฀Blackwell฀Munksgaard฀2005)

decarboxylase฀activity฀increase฀markedly฀in฀teeth-adherent฀bioilms฀during฀the฀irst฀week฀
of฀experimental฀human฀gingivitis.
Lysine฀decarboxylase฀may฀play฀a฀critical฀role฀in฀initiating฀gingivitis฀because฀basal฀cells฀
of฀the฀epithelial฀attachment฀at฀the฀base฀of฀gingival฀sulci฀must฀proliferate฀continuously฀to฀
maintain฀a฀basal฀lamina฀seal฀(Sect.฀5.2.3).฀By฀starving฀these฀epithelial฀cells฀of฀lysine,฀their฀
proliferation฀and฀ability฀to฀seal฀the฀base฀of฀a฀sulcus฀are฀impaired.฀This฀means฀that฀com-
mensal฀microbial฀products฀could฀better฀access฀the฀gingiva฀where฀they฀induce฀inlammation฀
and฀GCF฀exudation฀(Sect.฀13.2.1).฀E.฀corrodens฀is฀a฀common฀cause฀of฀dog฀bite฀infections,฀
and฀if฀dogs฀are฀fed฀a฀soft฀diet฀to฀accumulate฀bioilm฀faster฀(Sect.฀13.1.2),฀immunization฀
with฀puriied฀lysine฀decarboxylase฀could฀induce฀antibodies฀that฀slow฀gingivitis฀develop-
ment.฀The฀GCF฀is฀a฀richer฀substrate฀for฀bacterial฀growth฀than฀saliva,฀and฀it฀promotes฀the฀
development฀ of฀ a฀ predominantly฀ gram฀ negative฀ successor฀ and฀ climax฀ microbiota฀ in฀
236 13 Chronic Periodontitis

gingival฀ sulci฀ (Fig.฀ 13.3b).฀ Individual฀ bacteria฀ contributing฀ to฀ the฀ successor฀ microbiota฀
constantly฀enter฀the฀oral฀cavity฀from฀the฀environment.฀Some฀coaggregate฀within฀the฀bio-
ilms฀and฀generate฀autoinducer-2฀levels฀that฀promote฀their฀growth฀in฀GCF฀at฀the฀expense฀
of฀the฀commensal฀microbiota.฀It฀is฀generally฀accepted฀that฀the฀successor฀microbiota฀are฀
mostly฀responsible฀for฀gingivitis฀and฀chronic฀periodontitis.
H

NH2 CH2 CH2 CH2 CH2 C COOH NH2 CH2 CH2 CH2 CH2 CH2 NH2
Cadaverine
NH2
+
Lysine CO2

Fig. 13.4฀ Reaction฀catalyzed฀by฀lysine฀decarboxylase

The฀ metabolism฀ of฀ the฀ successor฀ microbiota฀ is฀ asaccharolytic.฀ Its฀ various฀ bacteria฀
release฀ammonia฀from฀all฀amino฀acids฀and฀sulides฀that฀cause฀oral฀malodor฀from฀cysteine฀
and฀methionine฀amino฀acids฀(Sect.฀1.3.2).฀When฀albumin,฀citrate,฀and฀pyrophosphate฀in฀the฀
GCF฀are฀hydrolyzed,฀calcium฀ions฀(Sect.฀9.1.4)฀become฀free฀to฀precipitate฀with฀phosphate฀
ions฀and฀form฀calculus.฀Calculus฀protects฀the฀bioilm฀at฀the฀base฀of฀a฀gingival฀sulcus,฀pro-
moting฀persistent฀inlammation฀that฀impairs฀the฀viability฀of฀the฀junctional฀epithelial฀attach-
ment฀(Sect.฀13.4.1).฀Salivary฀gland฀loss฀or฀malfunction,฀or฀persistent฀mouth-breathing฀or฀
tobacco฀smoking,฀dry฀up฀the฀mouth฀and฀enhance฀bacterial฀colonization฀and฀disease฀(Section฀
12.1.3).฀In฀periodontitis,฀bacterial฀products฀pass฀from฀gingival฀capillaries฀into฀the฀systemic฀
circulation฀and฀promote฀inlammation฀that฀worsens฀some฀preexisting฀diseases,฀notably฀car-
diovascular฀disease,฀and฀adult-onset฀diabetes.฀Periodontitis฀also฀predisposes฀to฀bacterial฀
endocarditis฀(bacteria฀attaching฀to฀heart฀valves)฀and฀low฀birth-weight฀babies.

13.1.4
Drugs to Prevent Gingivitis

In฀ addition฀ to฀ toothbrushes฀ and฀ dental฀ loss฀ for฀ mechanical฀ oral฀ hygiene฀ (Sect.฀ 13.5.1),฀
antiseptic฀ mouthwashes฀ exert฀ chemical฀ control฀ of฀ the฀ microbiota.฀ The฀ most฀ commonly฀
sold฀mouthwashes฀in฀the฀United฀States฀are฀made฀from฀mixtures฀of฀volatile฀aromatic฀com-
pounds฀from฀plants.฀Different฀mixtures฀of฀these฀plant฀compounds฀possess฀a฀unique฀scent฀
or฀essence฀and฀are฀commonly฀called฀“essential฀oil”฀mixtures.฀They฀have฀antiseptic฀proper-
ties฀ that฀ control฀ the฀ successor฀ microbiota,฀ but฀ a฀ need฀ for฀ 15–20%฀ alcohol฀ to฀ maintain฀
“essential฀oil”฀solubility฀has฀been฀linked฀to฀an฀increased฀risk฀of฀oral฀cancer฀in฀long-time฀
daily฀human฀users.฀Less฀popular฀mouthwashes฀contain฀an฀antiseptic฀called฀chlorhexidine฀
(Peridex)฀or฀a฀detergent฀that฀interferes฀with฀bacterial฀bioilm฀aggregation฀called฀delpolmi-
nol฀(Decapinol).฀Long-term฀use฀of฀all฀of฀these฀mouthwashes฀interferes฀with฀taste฀and฀pro-
biosis,฀protection฀by฀the฀commensal฀microbiota฀from฀pathogenic฀organisms.
The฀next฀generation฀of฀drugs฀to฀supplement฀oral฀hygiene฀may฀interfere฀more฀specii-
cally฀with฀one฀of฀four฀actions:฀(a)฀initial฀stages฀of฀bacterial฀attachment฀to฀teeth฀surfaces;฀฀
(b)฀coaggregation฀of฀successor฀bacteria฀into฀a฀bioilm;฀(c)฀quorum฀sensing;฀and฀(d)฀lysine฀
13.2.1 Mammalian Cells Recognize Prokaryotic Molecules 237

decarboxylase฀impairment฀of฀the฀epithelial฀attachment฀barrier.฀Recent฀studies฀by฀the฀author฀
suggest฀ that฀ immunological฀ inhibition฀ of฀ E.฀ corrodens฀ lysine฀ decarboxylase฀ in฀ beagle฀
dogs,฀or฀chemical฀inhibition฀of฀this฀enzyme฀in฀humans,฀retards฀gingivitis฀development฀and฀
therefore฀the฀eventual฀development฀of฀chronic฀periodontitis.

Periodontal฀disease฀describes฀a฀mixture฀of฀diseases฀in฀which฀periodontal฀attachment฀is฀
destroyed.฀It฀may฀be฀chronic฀or฀aggressive,฀and฀localized฀or฀generalized.฀Chronic฀perio-
dontitis฀ irst฀ appears฀ as฀ gingival฀ inlammation฀ (gingivitis)฀ accompanied฀ by฀ a฀ teeth฀
adherent฀ microbial฀ bioilm฀ (plaque)฀ and฀ calculus.฀ Bacteria฀ are฀ always฀ present฀ in฀ the฀
oral฀cavity.฀They฀grow฀on฀mucosal฀surfaces,฀in฀saliva,฀and฀adhere฀to฀teeth฀as฀bioilms฀
within฀which฀quorum฀sensing฀generates฀chemicals฀that฀activate฀mutualistic฀interactions฀
leading฀ to฀ luxuriant฀ mixed฀ culture฀ growth.฀ Eikenella฀ corrodens฀ in฀ the฀ commensal฀
microbiota฀ is฀ a฀ major฀ source฀ of฀ lysine฀ decarboxylase,฀ which฀ depletes฀ the฀ interstitial฀
luid฀of฀lysine฀necessary฀to฀maintain฀an฀intact฀epithelial฀barrier฀at฀the฀base฀of฀gingival฀
sulci.฀Loss฀of฀this฀barrier฀likely฀increases฀exposure฀of฀the฀gingiva฀to฀individual฀mem-
bers฀ of฀ a฀ gram฀ negative,฀ successor฀ microbiota฀ that฀ induce฀ an฀ inlammatory฀ exudate฀
derived฀from฀blood฀plasma,฀the฀gingival฀crevicular฀luid,฀which฀is฀richer฀in฀bacterial฀
substrates฀than฀saliva.฀The฀GCF฀promotes฀quorum฀sensing฀by฀the฀successor฀microbiota฀
at฀ the฀ expense฀ of฀ the฀ commensal฀ microbiota฀ and฀ gingivitis฀ appears.฀ Current฀ mouth-
washes฀contain฀antiseptics฀or฀detergents฀that฀supplement฀oral฀hygiene฀but฀interfere฀with฀
probiosis,฀protection฀of฀the฀oral฀cavity฀by฀the฀commensal฀microbiota.฀New฀drugs฀that฀
maintain฀ probiosis฀ would฀ inhibit฀ bacterial฀ attachment฀ to฀ teeth,฀ quorum฀ sensing,฀ or฀
lysine฀decarboxylase฀impairment฀of฀the฀epithelial฀barrier.

13.2.1
Mammalian Cells Recognize Prokaryotic Molecules

The฀successor฀microbiota฀is฀a฀major฀source฀of฀products฀containing฀conserved฀prokaryotic฀
motifs,฀ pathogen-associated฀ molecular฀ patterns฀ (PAMPs).฀ The฀ best-known฀ PAMPs฀ are฀
lipopolysaccharides฀of฀gram฀negative฀bacteria฀(Sect.฀1.4.2).฀Lipopolysaccharides฀are฀rec-
ognized฀by฀pattern-recognition฀receptors฀(PRRs)฀known฀as฀toll-like฀receptors฀(TLRs)฀on฀
the฀mammalian฀cell฀surface.฀[TLRs฀received฀their฀name฀from฀their฀homology฀to฀a฀protein฀
encoded฀by฀a฀gene฀identiied฀in฀1985฀from฀Drosophila฀lies.฀Mutations฀of฀this฀gene฀made฀the฀
lies฀look฀so฀unusual,฀that฀German฀researchers฀spontaneously฀called฀out฀“toll,”฀their฀word฀
for฀“amazing.”].฀Other฀PAMPs฀are฀membrane฀permeable,฀for฀example,฀bacterial฀peptido-
glycan฀ fragments฀ (Sect.฀ 1.4.1),฀ fatty฀ acids฀ hydrolyzed฀ from฀ lipopolysaccharides฀ (Sect.฀
1.4.2),฀bacterial฀DNA฀fragments,฀and฀various฀other฀products฀of฀lysed฀bacteria.฀These฀agents฀
are฀ recognized฀ by฀ PRR฀ proteins฀ that฀ are฀ soluble฀ in฀ the฀ cytosol฀ and฀ have฀ an฀ amino฀ acid฀
sequence฀homology฀to฀nucleotide-binding฀oligomerization฀domain฀(NOD)฀proteins.฀There฀
are฀23฀such฀NOD-like฀receptor฀proteins฀(NLRs)฀in฀humans,฀some฀of฀which฀have฀alternative฀
238 13 Chronic Periodontitis

names฀derived฀from฀indings฀before฀this฀family฀and฀its฀functions฀were฀recognized.฀PAMP-
bound฀TLRs฀mediate฀inlammation,฀whereas฀PAMP-bound฀NLRs฀mediate฀either฀inlam-
mation฀ or฀ tissue฀ destruction,฀ depending฀ on฀ the฀ state฀ of฀ the฀ cell฀ and฀ type฀ of฀ molecule฀
recognized฀(Sect.฀13.4.1).

13.2.2
PAMPs Induce PRRs to Release Cytokines That Attract Leukocytes

Innate฀immunity฀to฀infections฀is฀mediated฀by฀many฀of฀the฀same฀enzymes฀that฀function฀in฀
bone฀ demineralization,฀ i.e.,฀ acid-activated฀ lysosomal฀ enzymes฀ (Sect.฀ 10.1.1).฀ These฀
enzymes฀ are฀ brought฀ to฀ infected฀ regions฀ by฀ leukocytes฀ (neutrophils฀ and฀ macrophages).฀
PAMP-bound฀ PRRs฀ release฀ two฀ endogenous฀ ligands฀ (proinlammatory฀ cytokines)฀ that฀
attract฀and฀activate฀leukocytes฀from฀the฀cytosol฀of฀junctional฀epithelial฀cells฀and฀gingival฀
ibroblasts.฀One฀important฀cytokine,฀interleukin-1฀(IL-1),฀is฀released฀from฀the฀cell฀cytosol฀
by฀ligand-bound฀PRRs฀causing฀changes฀in฀membrane฀associated฀proteins฀without฀involv-
ing฀the฀Golgi฀or฀secretory฀pathways.
IL-1฀is฀stable,฀and฀it฀diffuses฀to฀and฀activates฀receptors฀(IL-1฀receptors)฀on฀adjacent฀and฀
distant฀cells.฀The฀IL-1฀receptor฀of฀endothelial฀cells฀is฀especially฀important฀(Fig.฀13.5a).฀In฀
all฀ cells,฀ the฀ IL-1฀ ligand฀ bound฀ to฀ its฀ receptor฀ causes฀ surface฀ expression฀ of฀ a฀ second฀
cytokine,฀tumor฀necrosis฀factor-a฀(TNF-a).฀TNF-a฀was฀originally฀described฀as฀a฀factor฀
that฀kills฀(causes฀necrosis฀of)฀mouse฀ibrosarcoma฀(cancer)฀cells฀but฀not฀normal฀mouse฀
ibroblasts.฀TNF-a฀is฀released฀from฀the฀cell฀surface฀by฀an฀adamalysin฀protease,฀TNF฀alpha฀
converting฀enzyme฀(TACE).฀TACE฀is฀also฀known฀as฀ADAM17,฀one฀of฀more฀than฀40฀cell฀
surface฀ bound฀ adamalysins฀ related฀ to฀ the฀ ADAM-TS2฀ subfamily฀ (Sect.฀ 8.2.1).฀ Unlike฀
IL-1,฀TNF-a฀is฀unstable฀and฀can฀only฀activate฀TNF฀receptors฀on฀nearby฀cells.฀It฀usually฀
produces฀responses฀that฀supplement฀those฀from฀IL-1฀activation฀(Fig.฀13.5b).
IL-1฀is฀released฀as฀two฀forms,฀IL-1a฀and฀IL-1b,฀encoded฀by฀separate฀genes฀that฀are฀only฀
28%฀homologous.฀The฀expressed฀polypeptides฀form฀a฀12-stranded฀b-sheet฀around฀a฀central฀
axis฀ such฀ that฀ two฀ sets฀ of฀ six฀ b-sheets฀ come฀ together฀ to฀ form฀ an฀ antiparallel฀ b-barrel฀
(Fig.฀13.6a).฀Similar฀b-barrel฀structures฀are฀present฀in฀a-amylase฀and฀many฀other฀proteins฀
that฀recognize฀carbohydrates฀(Sect.฀12.5.2).฀IL-1฀is฀guided฀to฀its฀receptor฀by฀interacting฀
irst฀with฀glycans฀on฀the฀cell฀surface.฀The฀receptor-binding฀domain,฀the฀C-terminal฀domain฀
(Fig.฀ 13.6b),฀ is฀ correctly฀ folded฀ in฀ IL-1a,฀ but฀ that฀ of฀ IL-1b฀ must฀ be฀ cleaved฀ from฀ its฀
N-terminal฀domain฀by฀interleukin-1฀converting฀enzyme฀(ICE).฀This฀calcium฀ion-activated฀
serine฀protease,฀also฀called฀caspase-1฀(Sect.฀13.4.1),฀is฀secreted฀by฀ibroblasts฀and฀mac-
rophages.฀The฀active฀form฀of฀IL-1b฀is฀therefore฀one฀third฀smaller฀than฀IL-1a.฀Other฀pro-
teases฀ secreted฀ by฀ the฀ successor฀ microbiota,฀ for฀ example,฀ T.฀ denticola฀ in฀ the฀ climax฀
microbial฀ complex฀ (Fig.฀ 13.3),฀ also฀ activate฀ IL-1b.฀ Keratinocytes,฀ including฀ junctional฀
epithelial฀basal฀cells฀exposed฀to฀PAMPs,฀secrete฀IL-1b฀along฀with฀IL-1a,฀whereas฀ibro-
blasts฀and฀macrophages฀secrete฀IL-1b฀and฀ICE.
13.2.2 PAMPs Induce PRRs to Release Cytokines That Attract Leukocytes 239

PRR activation, stress, DNA or oxidative

a IL-1 damage

IL-1

Distant Local
Distant TNF-α
Proliferation

ÝEndothelial Adhesion Molecules,

Synthesis of Chemokines: e.g. IL-8

Neutrophil Endothelial Adhesion, Emigration of Neutrophils,

Activation of Neutrophils, Plasminogen activator

GCF, Inflammation,
Tissue Destruction, Loss of Function

b TNF

Proinflammatory cytokines INCREASED

Macrophages Chemokines INFLAMMATION

Adhesion molecules
INCREASED
(e.g. ICAM-1)
Endothelium CELL INFILTRATION

Vascular endothelial
growth factor (VEGF) INCREASED
ANGIOGENESIS

Keratinocytes Proinflammatory cytokines

HYPERPROLIFERATION

Fig. 13.5฀ How฀IL-1฀and฀TNF-a฀cause฀inlammation.฀(a)฀IL-1฀function.฀IL-1฀induces฀the฀secretion฀of฀

TNF-a฀to฀enhance฀its฀actions.฀The฀major฀effect฀of฀IL-1฀is฀on฀capillary฀endothelial฀cells฀(see฀text).฀
IL-1฀causes฀keratinocytes฀and฀endothelial฀cells฀to฀proliferate฀and฀secrete฀other฀interleukins,฀espe-
cially฀IL-8,฀which฀acts฀like฀IL-1฀on฀capillary฀endothelial฀cells฀(b)฀TNF-a฀function.฀TNF-a฀usually฀
acts฀as฀a฀proinlammatory฀growth฀and฀survival฀factor฀(see฀text).฀Its฀induction฀of฀vascular฀endothe-
lial฀growth฀factor฀(VEGF)฀promotes฀greater฀capillary฀proliferation฀and฀permeability.฀VEGF฀con-
tains฀a฀cysteine฀knot฀domain฀like฀von฀Willebrand฀factor฀(Fig.฀11.2)฀[a:฀Based฀on฀Fig.฀1฀in฀Dinarello฀
CA฀(2000)฀“Proinlammatory฀Cytokines.”฀Chest฀118(2):503–508;฀b:฀Slightly฀modiied฀Fig.฀1฀from฀
Jackson฀JM฀(2007);฀TNF-a฀inhibitors,฀Dermatologic฀Therapy,฀20:251–261]
240 13 Chronic Periodontitis

Fig. 13.6฀ Structures฀of฀IL-1a฀and฀IL-8.฀(a)฀Structures฀of฀Interleukin-1.฀Interleukins฀are฀a฀family฀of฀

intercellular฀signaling฀molecules฀(cytokines)฀that฀are฀ligands฀for฀a฀speciic฀receptor,฀the฀IL-1฀recep-
tor.฀ IL-1฀ forms฀ a฀ 12-stranded฀ b-sheet฀ structure,฀ several฀ regions฀ of฀ which,฀ but฀ especially฀ a฀ loop฀
between฀strands฀4฀and฀5,฀are฀implicated฀in฀binding฀to฀the฀IL-1฀receptor.฀(b)฀Domain฀structure฀of฀
IL-1.฀IL-1a฀is฀composed฀of฀an฀N-terminal฀prodomain฀(residues฀1–109),฀a฀short฀unfolded฀connector฀
region฀ (residues฀ 110–128)฀ and฀ a฀ large฀ C-terminal฀ domain฀ (residues฀ 129–267)฀ containing฀ the฀
b-sheet฀conformation฀shown฀in฀(a)฀above.฀IL-1b฀is฀similarly฀constructed,฀but฀two฀residues฀shorter฀
than฀IL-1a.฀IL-1a฀is฀active฀without฀cleavage,฀whereas฀the฀prodomain฀of฀IL-1b,฀which฀ends฀at฀resi-
due฀103,฀prevents฀receptor-binding฀unless฀it฀is฀cleaved฀in฀the฀connector฀domain฀(residues฀104฀–฀
118)฀ C-terminal฀ to฀ aspartate฀ residue฀ 116฀ (see฀ text).฀ The฀ large฀ fragment฀ contains฀ a฀ homologous฀
b-sheet฀domain฀(residues฀119–265).฀(c)฀Interleukin-8฀(IL-8).฀IL-8฀is฀not฀of฀the฀interleukin฀family.฀
It฀is฀one฀of฀a฀family฀of฀13฀CXC฀chemokines,฀small,฀positively฀charged฀proteins฀that฀attract฀and฀
activate฀leukocytes฀(see฀text).฀The฀many฀chemokines฀in฀the฀body฀are฀divided฀into฀CXC฀and฀CC฀
subfamilies,฀based฀on฀the฀arrangement฀of฀the฀irst฀two฀of฀four฀conserved฀cysteine฀residues;฀the฀two฀
cysteines฀are฀separated฀by฀a฀single฀amino฀acid฀in฀CXC฀chemokines฀but฀lie฀next฀to฀each฀other฀in฀CC฀
chemokines.฀Chemokine฀structures฀all฀have฀a฀double฀a-helix฀that฀interacts฀with฀the฀receptor฀fol-
lowing฀heparin฀binding฀(a:฀Figure฀is฀public฀domain;฀http://en.wikipedia.org/wiki/Image:2ILA.png;฀
b:฀ Figure฀ is฀ modiied฀ from฀ an฀ EMBO฀ SMART฀ diagram฀ of฀ IL-1฀ at฀ http://smart.embl.de/smart/
show_motifs.pl?ID=P01583;฀c:฀Figure฀constructed฀by฀Ramin฀Herati,฀12-10-฀2006฀as฀a฀Wikipedia฀
public฀domain฀image฀http://en.wikipedia.org/wiki/Image:IL8_Solution_Structure.rsh.png)

Ligand-bound฀ IL-1฀ and฀ TNF฀ receptors฀ activate฀ intracellular฀ protein฀ kinases฀ called฀
mitogen-activated฀protein฀ (MAP)฀ kinases.฀ These฀ kinases฀ phosphorylate฀ serine฀ or฀ threo-
nine฀residues฀on฀adjacent฀proteins฀in฀response฀to฀extracellular฀stimuli฀that฀were฀irst฀identi-
ied฀as฀mitogenic฀because฀they฀induced฀ibroblast฀growth฀factors฀(Sect.฀13.2.5).฀IL-1฀or฀
13.2.4 Neutrophils Function in Tissue Destruction 241

TNF฀ receptor฀ activation฀ of฀ these฀ kinases฀ stimulates฀ NFkB,฀ a฀ transcription฀ factor฀ that฀
induces฀ the฀ expression฀ of฀ proteins฀ responsible฀ for฀ inlammation฀ and฀ demineralization฀
(Sect.฀ 10.2.2),฀ not฀ growth.฀ Expressed฀ NFkB฀ gene฀ products฀ cause฀ the฀ IL-1฀ and฀ TNF-a฀
activities฀listed฀in฀Fig.฀13.5.

13.2.3
IL-1: A Host Mediator of Gingival Inflammation

In฀gingivitis,฀IL-1฀production฀by฀PAMP฀activation฀of฀external฀and฀cytosolic฀PRRs฀is฀maxi-
mal฀within฀the฀junctional฀epithelium.฀As฀little฀as฀0.1฀ng/mL฀of฀IL-1฀will฀bind฀to฀and฀acti-
vate฀IL-1฀receptors฀in฀capillary฀endothelium฀where฀they:฀(a)฀Open฀intercellular฀junctions฀
allowing฀plasma฀to฀exude;฀(b)฀Promote฀secretion฀of฀plasminogen฀activator฀to฀prevent฀the฀
plasma฀luid฀exudate฀from฀clotting฀in฀the฀stroma฀(Sect.฀11.4.2);฀(c)฀Induce฀intercellular฀
adhesion฀molecule-1฀(ICAM-1)฀to฀arrest฀and฀transfer฀neutrophils฀from฀blood฀to฀the฀exu-
date;฀and฀D)฀Induce฀TNF-a฀to฀enhance฀ICAM-1฀and฀VEGF฀production.
Neutrophils฀are฀slowed฀within฀the฀blood฀low฀by฀possessing฀a฀receptor฀that฀binds฀to฀an฀
endothelial฀protein฀protruding฀into฀the฀capillary฀lumen฀(E-selectin).฀The฀ligand-receptor฀
complex฀ activates฀ cytosolic฀ proteins฀ to฀ transmit฀ a฀ signal฀ that฀ extends฀ a฀ b2฀ integrin,฀
Lymphocyte฀function-associated฀antigen-1฀(LFA-1)฀by฀inside–out฀signaling฀(Sect.฀4.4.1).฀
The฀extended฀LFA-1฀binds฀to฀the฀ICAM-1฀induced฀by฀IL-1฀and฀the฀double฀receptor-ligand฀
binding฀(Fig.฀13.7)฀arrests฀neutrophils฀in฀the฀blood฀low.฀It฀also฀activates฀a฀respiratory฀burst฀
that฀provides฀energy฀for฀the฀neutrophils฀to฀migrate฀through฀the฀opened฀intercellular฀junc-
tions฀with฀a฀luid฀exudate฀that฀becomes฀the฀GCF.
IL-1฀ also฀ induces฀ epithelial฀ and฀ endothelial฀ cells฀ to฀ secrete฀ IL-8,฀ a฀ chemokine฀ mis-
named฀an฀interleukin฀(Fig.฀13.6c),฀and฀neutrophils฀to฀secrete฀leukotriene฀B4,฀an฀eicosanoid฀
(Sects.฀13.5.2฀and฀13.5.3).฀IL-8฀and฀leukotriene฀B4฀enhance฀ICAM-1฀expression฀from฀the฀
capillary฀endothelium,฀cause฀neutrophils฀to฀emigrate฀and฀accumulate฀on฀the฀outer฀surface฀
of฀ the฀ opened฀ capillaries,฀ and฀ migrate฀ with฀ the฀ GCF฀ through฀ the฀ junctional฀ epithelium฀
where฀ IL-1฀ production฀ is฀ greatest.฀ The฀ expanded,฀ leaky฀ capillary฀ plexus฀ in฀ the฀ stroma฀
beneath฀the฀mucosal฀covering฀of฀the฀gingival฀sulcus฀(section฀3.3.1)฀swells฀the฀gingiva฀and฀
may฀cause฀spontaneous฀sulcular฀bleeding฀on฀probing.

13.2.4
Neutrophils Function in Tissue Destruction

Neutrophils฀contain฀secretory฀vesicles฀and฀granules.฀The฀former฀deliver฀their฀contents฀dur-
ing฀passage฀though฀the฀capillary฀wall,฀whereas฀granule฀secretion฀is฀activated฀in฀the฀stroma฀
by฀a฀b2฀integrin฀that฀binds฀to฀type฀I฀collagen฀and฀other฀stromal฀ligands.฀This฀promiscuous฀
receptor,฀aMb2฀(Fig.฀4.12)฀is฀named฀Mac-1฀because฀it฀was฀originally฀discovered฀in฀mac-
rophages.฀The฀outside-in฀activation฀of฀this฀integrin฀stimulates฀a฀neutrophil฀to฀releases฀pri-
mary฀ and฀ secondary฀ granule฀ contents฀ into฀ the฀ infected฀ stroma.฀ The฀ granules฀ contain฀
242 13 Chronic Periodontitis

Fig. 13.7฀ Neutrophil฀activation.฀(a)฀Neutrophil฀in฀the฀blood.฀Neutrophils฀express฀P-selectin฀glyco-

protein฀ ligand-1฀ (PSGL-1;฀ blue)฀ and฀ integrin฀ aLb2฀ (lymphocyte฀ function-associated฀ antigen-1,฀
LFA-1;฀pink฀and฀blue).฀Endothelial฀cells฀constitutively฀express฀E-฀and฀P-selectins฀(dark฀green)฀and฀
intercellular฀adhesion฀molecule-1,฀ICAM-1฀(light฀green)฀when฀activated฀by฀IL-1,฀IL-8฀and฀other฀
cytokines.(b)฀Neutrophil฀arrest฀and฀activation.฀The฀PSGL-1/P-selectin฀interaction฀slows฀neutro-
phil฀rolling฀and฀activates฀its฀LFA-1฀(aLb2฀integrin)฀to฀extend฀to฀an฀intermediate฀afinity฀conforma-
tion฀(i.e.,฀inside-out฀integrin฀activation฀discussed฀in฀Chap.฀4).฀If฀ICAM-1฀is฀present,฀the฀LFA-1฀
extends฀fully฀and฀its฀ICAM-1฀interaction฀arrests฀neutrophil฀at฀the฀endothelial฀cell฀surface฀(Figure฀
from฀left฀side฀of฀Fig.฀6฀in฀Miner฀JJ,฀Xia,฀L,฀Yago฀T,฀et฀al.฀(2008฀Sept฀1)฀“Separable฀requirements฀
for฀ cytoplasmic฀ domain฀ of฀ PSGL-1฀ in฀ leukocyte฀ rolling฀ and฀ signaling฀ under฀ low.”฀ Blood,฀
112(5):2035–45).฀This฀igure฀was฀modiied฀by฀Dr.฀Wirsig-Weichmann

defensins,฀a฀group฀of฀small,฀cysteine-rich฀cationic฀proteins฀that฀destabilize฀bacterial฀cell฀
membranes,฀peroxidase฀and฀lactoferrin฀(See฀Sect.฀12.1.4),฀and฀proteins฀that฀activate฀mac-
rophages฀and฀tissue-hydrolyzing฀lysosomal฀enzymes฀(Sect.฀13.3.1).
During฀passage฀through฀the฀capillary฀wall,฀the฀neutrophil฀tertiary฀granules฀release฀neu-
tral฀gelatinase฀(MMP-9)฀with฀which฀the฀neutrophils฀cut฀through฀type฀IV฀collagen฀in฀the฀
13.2.5 Gingivitis is Reversible; Antiinflammatory Cytokines Mediate Repair 243

endothelial฀basement฀membrane.฀The฀neutrophils฀also฀secrete฀neutral฀collagenase฀(MMP8)฀
and฀ metalloelastase฀ (MMP12)฀ with฀ which฀ they฀ cut฀ through฀ the฀ collagen฀ and฀ elastin฀ of฀
capillary฀walls฀(Sect.฀11.1.1)฀and฀the฀infected฀stroma.฀These฀matrilysins฀are฀all฀activated฀
by฀plasmin,฀which฀is฀already฀present฀to฀prevent฀the฀clotting฀of฀plasma฀exuding฀from฀capil-
laries฀ with฀ the฀ neutrophils.฀ A฀ tetracycline฀ antibiotic฀ derivative฀ (doxycycline฀ sold฀ as฀
Periostat)฀ inhibits฀ neutrophil฀ collagenase฀ activity฀ at฀ a฀ tenth฀ of฀ the฀ amount฀ required฀ for฀
bacterial฀growth฀inhibition.฀Unfortunately,฀Periostat’s฀effect฀is฀limited฀because฀most฀col-
lagen฀iber฀loss฀is฀caused฀by฀acid-activated฀cathepsin฀K฀from฀macrophages฀(Sect.฀13.3.1).฀
Periostat฀is฀used฀in฀conjunction฀with฀therapy฀for฀periodontitis฀to฀facilitate฀reattachment฀and฀
healing฀by฀limiting฀neutrophil฀collagenase฀action฀in฀the฀absence฀of฀cathepsin฀K.

13.2.5
Gingivitis is Reversible; Antiinflammatory Cytokines Mediate Repair

If฀oral฀hygiene฀is฀restored,฀the฀PAMPs฀mediating฀proinlammatory฀stimuli฀decrease.฀The฀
surrounding,฀healthy฀keratinocytes฀produce฀a฀natural฀inhibitor฀of฀IL-1,฀IL-1฀receptor฀antag-
onist฀(IL-1ra).฀IL-1ra฀competes฀with฀IL-1฀for฀binding฀to฀the฀IL-1฀receptor,฀closing฀capillary฀
endothelial฀spaces.฀More฀importantly,฀the฀iniltrated฀leukocytes฀age฀and฀change฀to฀secreting฀
antiinlammatory฀cytokines฀(lipoxins,฀resolvins,฀and฀protectins;฀Sect.฀13.5.4)฀that฀promote฀
healing฀and฀tissue฀repair.฀Nearby฀healthy฀cells฀respond฀to฀the฀antiinlammatory฀cytokines฀
by฀releasing฀ibroblast฀growth฀factor฀(FGF)฀proteins.฀These฀proteins฀act฀on฀receptors฀that฀
activate฀ capillary฀ growth฀ (angiogenesis)฀ and฀ ibroblast฀ secretion฀ of฀ hyaluronan฀ into฀ the฀
spaces฀created฀by฀lost฀gingival฀collagen฀ibers฀(Sect.฀6.3.1.).฀Collagenase฀action฀is฀inhibited฀
by฀an฀anti-inlammatory฀cytokine-mediated฀increase฀in฀TIMPs฀(Sect.฀8.1.3).฀Fresh฀collagen฀
ibers฀are฀laid฀down,฀and฀a฀healthy฀gingival฀architecture฀is฀restored.
Interestingly,฀the฀secretion฀of฀FGF฀family฀members฀that฀mediate฀tissue฀repair฀resembles฀
IL-1฀secretion,฀that฀is,฀not฀involving฀the฀Golgi.฀The฀FGF฀family฀proteins฀also฀bind฀to฀their฀
receptors฀by฀b-barrel฀structures฀that฀interact฀with฀a฀glycan฀(in฀this฀case฀known฀to฀be฀hepa-
rin)฀during฀activation.฀Hyaluronan฀and฀heparin฀are฀glycosaminoglycans฀(Sect.฀6.3.1).

The฀successor฀microbiota฀is฀a฀major฀source฀of฀pathogen-associated฀molecular฀patterns฀
(PAMPs),฀ conserved฀ prokaryotic฀ motifs฀ that฀ are฀ recognized฀ by฀ pattern฀ recognition฀
receptors฀(PRRs)฀in฀mammalian฀cells.฀These฀ligand-bound฀PRRs฀release฀interleukin-1฀
from฀the฀junctional฀epithelium฀and฀underlying฀tissues฀at฀the฀base฀of฀a฀sulcus.฀All฀cells฀
have฀a฀receptor฀that฀is฀activated฀by฀binding฀to฀IL-1.฀The฀capillary฀endothelium฀receptor฀
is฀ especially฀ sensitive.฀ IL-1฀ receptors฀ activate฀ NFkB฀ to฀ express฀ a฀ group฀ of฀ proteins฀
which,฀in฀endothelial฀cells:฀(a)฀open฀intercellular฀junctions฀allowing฀plasma฀proteins฀to฀
exude฀ into฀ the฀ stroma;฀ (b)฀ secrete฀ plasminogen฀ activator฀ to฀ prevent฀ the฀ plasma฀ from฀
clotting;฀(c)฀induce฀ICAM-1฀to฀arrest฀neutrophils฀and฀activate฀their฀extrusion฀from฀the฀
capillaries;฀and฀(d)฀induce฀secretion฀of฀other฀proinlammatory฀cytokines.฀ICAM-1฀binds฀
to฀LFA-1,฀a฀b2฀integrin฀on฀the฀neutrophil฀surface฀where฀it฀induces฀a฀respiratory฀burst฀that฀
244 13 Chronic Periodontitis

stimulates฀neutrophil฀migration฀into฀the฀stroma.฀A฀second฀integrin,฀Mac-1,฀is฀activated฀
when฀the฀neutrophils฀contact฀collagen฀ibers฀in฀the฀stroma฀Mac-1฀activation฀facilitates฀
the฀release฀of฀defensins฀and฀lysosomal฀enzymes฀that฀destroy฀bacteria.฀If฀oral฀hygiene฀is฀
restored,฀ the฀ PAMPs฀ are฀ removed฀ and฀ the฀ capillaries฀ stop฀ exuding฀ neutrophils.฀ The฀
remaining฀neutrophils฀age฀and฀start฀releasing฀anti-inlammatory฀cytokines฀that฀stimulate฀
the฀surrounding฀healthy฀cells฀into฀releasing฀ibroblast฀growth฀factor฀(FGF).฀Eventually,฀
fresh฀collagen฀ibers฀are฀laid฀down,฀and฀a฀healthy฀gingival฀architecture฀is฀restored.

13.3.1
Long-term Effects of Persistent PAMP Stimulation

If฀oral฀hygiene฀remains฀poor,฀PAMPs฀keep฀stimulating฀IL-1฀secretion฀and฀neutrophils฀con-
tinue฀to฀iniltrate฀the฀region.฀Another฀neutrophil฀product,฀protease-3,฀induces฀endothelial฀
cells฀to฀secrete฀monocyte฀chemotactic฀protein-1฀(MCP-1),฀a฀chemokine฀resembling฀IL-8.฀It฀
binds฀to฀a฀receptor฀on฀passer-by฀monocytes฀and฀induces฀an฀adhesion฀molecule฀similar฀to฀
ICAM.฀Monocytes฀are฀arrested฀by฀double฀binding฀similar฀to฀neutrophils.฀MCP-3฀functions฀
like฀ MCP-1,฀ but฀ it฀ is฀ secreted฀ instead฀ of฀ MCP-1฀ by฀ ibroblasts฀ exposed฀ to฀ IL-1.฀ MCP3฀
remains฀intact฀and฀functional฀in฀gingivitis฀because฀its฀cleavage฀requires฀ibroblast฀gelati-
nase,฀not฀leukocyte฀gelatinase฀(see฀Sect.฀8.3.4);฀ibroblast฀gelatinase฀is฀not฀secreted.
The฀ monocytes฀ differentiate฀ into฀ macrophages฀ and฀ osteoclasts.฀ The฀ former฀ enhance฀
leukocyte฀(i.e.,฀macrophage฀and฀neutrophil)฀destruction฀of฀the฀stroma฀by฀adding฀to฀neutral฀
collagenase฀(MMP8)฀activity.฀MMP8฀cleaves฀collagen฀ibers฀into฀one-quarter฀and฀three-
quarter฀ length฀ tropocollagen฀ fragments฀ (Sect.฀ 8.3.4).฀ The฀ leukocytes฀ endocytose฀ many฀
partially฀digested฀collagen฀fragments฀into฀sealed฀compartments฀called฀phagosomes,฀which฀
then฀fuse฀with฀lysosomal฀vesicles฀as฀in฀osteoclasts฀(Sect.฀10.1.2).฀Cathepsin฀K฀is฀likely฀
responsible฀for฀most฀of฀the฀gingival฀collagen฀lost฀in฀periodontitis,฀explaining฀why฀Periostat฀
is฀only฀effective฀with฀other฀therapy฀(Sect.฀13.2.4).฀Within฀phagolysosomes,฀lysosomal฀per-
oxidases฀and฀oxidases฀make฀reactive฀oxygen฀species฀(ROS)฀such฀as฀hydrogen฀peroxide฀that฀
kills฀ viable฀ bacteria฀ but฀ also฀ damages฀ the฀ surrounding฀ gingival฀ and฀ periodontal฀ tissues.฀
Gingival฀and฀periodontal฀membrane฀cells฀restrict฀ROS฀production฀and฀repair฀the฀damaged฀
cells฀ (Sect.฀ 16.3.2),฀ but฀ the฀ ROS฀ production฀ from฀ persistent฀ leukocyte฀ activation฀ is฀ so฀
excessive฀that฀host฀cell฀and฀tissue฀damage฀cannot฀be฀prevented฀(Sect.฀16.3.2).
Activated฀leukocytes฀also฀express฀IL-6,฀which฀increases฀the฀proliferation฀of฀antibody-
producing฀lymphocytes฀(B-lymphocytes)฀locally฀and฀the฀replenishment฀of฀bone฀marrow฀
neutrophils฀globally฀(Fig.฀13.8).฀Another฀function฀of฀IL-6฀is฀to฀act฀with฀IL-1฀to฀increase฀the฀
hepatic฀expression฀and฀plasma฀concentration฀of฀most฀blood฀clotting฀proteins฀(Sect.฀11.1.1).฀
This฀stimulation฀of฀blood฀clotting฀protein฀synthesis฀may฀partially฀explain฀the฀association฀
of฀ periodontitis฀ with฀ an฀ increased฀ likelihood฀ of฀ vascular฀ clotting฀ events,฀ notably฀ heart฀
attacks฀and฀strokes฀(Sect.฀13.1.2).
Activated฀leukocytes฀induce฀the฀synthesis฀and฀secretion฀from฀the฀liver฀of฀an฀unrelated฀
set฀ of฀ proteins฀ called฀ acute-phase฀ proteins.฀ One฀ such฀ protein,฀ C-reactive฀ protein฀ (CRP)฀
was฀ originally฀ identiied฀ as฀ binding฀ to฀ the฀ phosphocholine฀ attachment฀ site฀ of฀ capsular฀
polysaccharide฀(C-polysaccharide)฀from฀Streptococcus฀pneumoniae.฀CRP฀in฀blood฀has฀a฀
half฀life฀of฀less฀than฀a฀day,฀compared฀with฀4฀days฀for฀ibrinogen.฀A฀continuously฀elevated฀
13.3.1 Long-term Effects of Persistent PAMP Stimulation 245

Fig. 13.8฀ Neutrophil฀activation฀products.฀An฀activated฀neutrophil฀secretes฀monocyte฀chemotactic฀

factor฀(MCP)฀and฀an฀interleukin,฀IL-6.฀Top฀right:฀MCP฀attracts฀monocytes฀that฀differentiate฀into฀
macrophages฀and฀osteoclasts.฀Lower฀right:฀MCP฀independently฀attracts฀thymic฀(T-)฀lymphocytes฀
which฀are฀responsible฀for฀acquired,฀cell-mediated฀immunity.฀Lower฀left:฀IL-6฀has฀systemic฀effects฀
on฀blood฀plasma฀proteins฀and฀local฀effects฀on฀keratinocytes฀(Figure฀made฀up฀from฀Wikipedia฀pub-
lic฀domain฀images฀except฀for฀osteoclasts฀and฀reorganized฀into฀an฀original฀igure฀http://en.wikipe-
dia.org/wiki/File:Neutrophil.jpg;฀ http://en.wikipedia.org/wiki/File:Lymphocyte2.jpg;฀ http://en.
wikipedia.org/wiki/File:PBMonozyt.jpg;http://en.wikipedia.org/wiki/File:Macrophage.
png;Osteoclasts฀igure฀is฀from฀Fig.฀4,฀p.฀645฀in฀Teitelbaum฀SL,฀Ross฀FP฀(2003฀Aug)฀“Genetic฀regu-
lation฀ of฀ osteoclast฀ development฀ and฀ function.”฀ Nature฀ Reviews—Genetics฀ 8:638฀ –฀ 649).฀ This฀
igure฀was฀modiied฀by฀Dr.฀Wirsig-Weichmann

CRP฀content฀indicates฀a฀persistent฀proinlammatory฀stimulus฀in฀the฀body.฀CRP฀binds฀to฀
host฀or฀bacterial฀phosphocholine,฀and฀the฀complex฀activates฀a฀group฀of฀plasma฀proteins฀
called฀ complement฀ (Sect.฀ 3.3.2).฀ The฀ complement฀ system฀ resembles฀ the฀ blood฀ clotting฀
system,฀except฀that฀proteolytic฀cleavage฀of฀its฀components฀results฀in฀peptide฀fragments฀that฀
to฀attract฀and฀enhance฀the฀phagocytosis฀(opsonization)฀of฀CRP-฀or฀antibody-bound฀mate-
rial฀by฀macrophages฀(like฀IL-1฀or฀IL-8).฀CRP฀is฀part฀of฀the฀innate฀immune฀response,฀and฀
the฀antibody฀response฀is฀part฀of฀the฀acquired฀immune฀response฀(Sect.฀12.1.4).฀Many฀bacte-
ria฀of฀the฀successor฀microbiota฀induce฀an฀antibody฀response฀that฀is฀increased฀in฀periodon-
titis฀compared฀with฀gingivitis฀or฀healthy฀subjects.

If฀oral฀hygiene฀remains฀poor,฀the฀neutrophils฀make฀a฀protease฀(protease-3)฀that฀releases฀
monocyte฀chemotactic฀protein-1฀(MCP-1)฀from฀endothelial฀cells.฀This฀protein฀behaves฀
like฀IL-1฀or฀IL-8,฀except฀that฀it฀acts฀on฀monocytes฀instead฀of฀neutrophils.฀In฀the฀tissues,฀
the฀monocytes฀develop฀into฀macrophages฀and฀monocytes.฀Together฀with฀neutrophils,฀
the฀macrophages฀engulf฀(phagocytose฀or฀opsonize)฀infected฀tissues฀into฀vesicles฀called฀
phagosomes,฀ which฀ fuse฀ with฀ lysosomes฀ to฀ form฀ phagolysosomes฀ whose฀ lysosomal฀
component฀secretes฀acid฀and฀acid฀proteases฀as฀in฀osteoclasts.฀The฀phagolysosomes฀also฀
246 13 Chronic Periodontitis

secrete฀defensins฀which฀disrupt฀the฀inner฀membrane฀of฀bacteria,฀and฀peroxidases฀and฀
oxidases฀that฀make฀reactive฀oxygen฀species฀(ROS).฀The฀osteoclasts฀cause฀alveolar฀bone฀
loss.฀ Thus,฀ periodontal฀ pocket฀ development฀ and฀ periodontitis฀ are฀ mostly฀ due฀ to฀ the฀
release฀ of฀ phagolysosome฀ contents฀ that฀ remove฀ gingival฀ and฀ periodontal฀ membrane฀
collagen฀ibers,฀demineralize฀alveolar฀bone,฀and฀reduce฀the฀viability฀of฀the฀junctional฀
epithelium฀and฀฀surrounding฀cells.฀In฀addition,฀IL-6฀secreted฀by฀neutrophils฀increases฀
the฀ plasma฀ content฀ of฀ clotting฀ proteins฀ and฀ C-reactive฀ protein฀ (CRP).฀ CRP฀ binds฀ to฀
phosphocholine฀ on฀ the฀ surface฀ of฀ bacterial฀ cells฀ or฀ of฀ phagolysosome-damaged฀ host฀
cells฀and฀activates฀complement,฀a฀group฀of฀plasma฀proteins฀that฀undergo฀partial฀prote-
olysis฀into฀fragments.฀Some฀complement฀fragments฀attract฀and฀activate฀neutrophils฀like฀
IL-8;฀ others฀ enhance฀ the฀ phagocytosis฀ of฀ CRP-฀ or฀ antibody-bound฀ material฀ by฀
macrophages.

13.4.1
Apoptosis in Chronic Periodontitis

Mammalian฀cell฀death฀is฀classiied฀as฀autophagy,฀apoptosis,฀or฀necrosis.฀Autophagy฀occurs฀
when฀cells฀are฀starved฀of฀one฀or฀more฀essential฀metabolites฀and฀have฀to฀obtain฀them฀by฀
internal฀digestion฀of฀their฀organelles฀and฀macromolecules.฀The฀cell฀shrinks฀until฀its฀con-
tents฀are฀so฀depleted฀that฀it฀undergoes฀apoptosis,฀self-induced฀cell฀death฀due฀to฀internally฀
activated฀cathepsin฀proteases฀(Table฀8.1).฀In฀apoptosis,฀these฀cathepsin฀proteases฀are฀acti-
vated฀from฀within฀or฀externally฀and฀the฀cytosol฀is฀completely฀digested,฀followed฀by฀the฀cell฀
membrane.฀Apoptosis฀is฀a฀biologically฀controlled฀procedure฀for฀getting฀rid฀of฀unwanted,฀
damaged฀or฀infected฀cells฀which฀disappear฀without฀trace.฀Apoptosis฀occurs฀during฀organ-
ism฀development฀and฀is฀also฀activated฀by฀infection฀and฀various฀physicochemical฀agents.
By฀contrast,฀in฀necrosis,฀severe฀physical,฀chemical,฀or฀bacterial฀damage฀causes฀a฀cell฀
membrane฀ to฀ burst,฀ releasing฀ apoptotic฀ mediators฀ (Sect.฀ 13.4.3)฀ and฀ proinlammatory฀
cytokines฀into฀the฀stroma฀(Sect.฀13.2.2).฀The฀cytosolic฀enzymes฀continue฀to฀make฀lactic฀
acid฀in฀the฀absence฀of฀mitochondrial฀function,฀making฀the฀necrotic฀environment฀strongly฀
acidic฀ and฀ activating฀ lysosomal฀ enzymes฀ (Sect.฀ 13.2.1)฀ to฀ digest฀ the฀ released฀ cytosolic฀
contents.฀Necrosis฀is฀discussed฀in฀relation฀to฀aggressive฀periodontitis฀(Chap.฀14).
In฀gingivitis,฀lysine฀decarboxylase-induced฀autophagy฀may฀impair฀the฀junctional฀epi-
thelial฀attachment฀barrier฀to฀bacterial฀products฀(PAMPs)฀as฀gingivitis฀develops฀de฀novo฀or฀
following฀therapy฀(Sect.฀13.1.2).฀The฀dentally฀attached฀cells฀become฀autophagic฀and฀stop฀
dividing฀if฀their฀lysine฀content฀becomes฀insuficient฀to฀support฀protein฀synthesis.฀A฀loss฀of฀
basal฀laminar฀attachment฀to฀the฀tooth฀increases฀the฀permeability฀of฀junctional฀epithelium฀
to฀PAMPs,฀and฀this฀activates฀PRRs฀(TLRs฀and฀NLRs)฀to฀release฀IL-1฀(Sect.฀13.2.1).฀Both฀
TLRs฀ and฀ NLRs฀ activate฀ caspase฀ 1฀ (ICE,฀ Sect.฀ 13.2.2)฀ by฀ creating฀ a฀ cytosolic฀ inlam-
masome,฀similar฀to฀the฀creation฀of฀a฀cytosolic฀apoptosome฀which฀activates฀different฀cas-
pases฀to฀mediate฀apoptosis฀(Sect.฀13.4.3).
13.4.2 Intracellular Induction of Apoptosis 247

In฀chronic฀periodontitis,฀apoptosis฀is฀caused฀by฀bioilm฀products.฀Products฀of฀asac-
charolytic฀ metabolism฀ by฀ the฀ successor฀ microbiota,฀ most฀ notably฀ butyrate฀ (Sect.฀
1.3.2),฀ permeate฀ the฀ cell฀ membrane฀ of฀ junctional฀ epithelial฀ cells฀ at฀ the฀ base฀ of฀ the฀
sulcus฀and฀induce฀apoptosis฀by฀speciic฀intracellular฀interactions.฀Other฀PAMPs฀bind฀
to฀ NLRs฀ in฀ the฀ cytosol฀ and฀ may฀ mediate฀ either฀ apoptosis฀ or฀ inlammation฀ (Sect.฀
13.2.1).฀In฀addition,฀leukocytes฀encountering฀teeth฀adherent฀microbial฀bioilm฀at฀the฀
base฀of฀a฀sulcus฀undergo฀phagolysosome฀degranulation.฀The฀phagolysosome฀contents฀
kill฀ the฀ bacteria฀ (Sect.฀ 13.3.1),฀ but฀ also฀ junctional฀ epithelial฀ cells฀ in฀ the฀ immediate฀
vicinity.฀Apoptosis฀or฀mild฀necrosis฀of฀junctional฀epithelium฀at฀the฀base฀of฀the฀gingival฀
sulcus฀is฀therefore฀reported฀in฀chronic฀periodontitis,฀but฀not฀in฀gingivitis.฀Necrosis฀of฀
the฀ gingiva,฀ acute฀ necrotizing฀ gingivitis฀ (ANUG),฀ is฀ discussed฀ under฀ aggressive฀฀
periodontitis฀(Sect.฀14.1.1).

13.4.2
Intracellular Induction of Apoptosis

Apoptosis฀is฀controlled฀by฀the฀Bcl-2฀family฀of฀proteins฀whose฀nomenclature฀was฀derived฀
from฀the฀irst฀discovered฀protein฀of฀the฀family.฀This฀protein฀was฀encoded฀as฀the฀second฀
gene฀within฀a฀segment฀of฀DNA฀that฀translocates฀from฀chromosome฀11฀to฀chromosome฀14฀
in฀certain฀B฀cell฀lymphomas,฀B฀cell฀lymphoma฀protein-2฀(Bcl-2).฀The฀Bcl-2฀proteins฀pos-
sess฀a฀tertiary฀structure฀that฀is฀common฀to฀pore-forming฀bacterial฀toxins,฀a฀hydrophobic฀
helix฀surrounded฀by฀amphipathic฀helices฀(Fig.฀13.9a).฀Each฀Bcl-2฀family฀member฀shares฀at฀
least฀one฀of฀four฀domains฀that฀bind฀with฀other฀family฀members.฀These฀Bcl-2฀Homology฀
domains฀are฀named฀BH1฀through฀BH4฀(Figs.฀13.9a,฀b).฀Bcl-2฀proteins฀that฀effect฀apoptosis฀
such฀as฀BAX฀and฀BAK฀possess฀domains฀BH1฀through฀BH3฀(BH4-missing฀effector฀pro-
teins).฀Proteins฀that฀initiate฀or฀activate฀the฀effector฀proteins฀possess฀a฀BH3฀domain฀only฀
(BH3฀ domain-only฀ proteins),฀ and฀ those฀ that฀ prevent฀ apoptosis,฀ antiapoptotic฀ like฀ the฀
founder฀member,฀possess฀all฀four฀domains.
Apoptosis฀is฀caused฀by฀the฀release฀or฀activation฀of฀one฀or฀more฀BH3฀domain-only฀pro-
teins฀(Fig.฀13.9c,฀step฀1).฀These฀initiating฀BH3-only฀proteins฀bind฀to฀a฀cytosolic฀heterodi-
mer฀containing฀a฀Bcl-2฀protein฀(step฀2)฀and฀displace฀a฀previously฀bound฀BH3-only฀protein,฀
Bid,฀BIM฀or฀PUMA฀(step฀3).฀In฀the฀case฀of฀Bid,฀dissociation฀from฀a฀partner฀such฀as฀Bcl-2฀
may฀be฀mediated฀by฀proteolysis฀of฀the฀Bid฀N-terminal฀60฀amino฀acid฀residues,฀creating฀
truncated฀Bid฀(tBid),฀which฀activates฀an฀effector฀protein.฀The฀BH3-only฀proteins฀are฀there-
fore฀ activators฀ of฀ apoptosis฀ that฀ displace฀ effector฀ proteins฀ (BAK฀ or฀ BAX)฀ from฀ a฀ non-
Bcl-2฀partner฀on฀the฀cytosolic฀surface฀of฀mitochondria฀and฀endoplasmic฀reticulum฀(step฀4).฀
For฀example,฀BAK฀is฀attached฀to฀an฀outer฀mitochondrial฀membrane฀channel฀protein฀in฀a฀
healthy฀cell.฀An฀activating฀BH3-only฀protein฀displaces฀BAK,฀which฀then฀self-aggregates฀
into฀homodimers฀that฀burrow฀a฀hole฀in฀the฀mitochondrial฀or฀endoplasmic฀reticular฀mem-
brane,฀releasing฀the฀contents฀(step฀5).
Induction฀by฀interactions฀between฀Bcl-2฀family฀proteins฀may฀be฀induced฀by฀cytokine-
receptor฀binding฀(extrinsic)฀or฀by฀intracellular฀changes฀(intrinsic).
248 13 Chronic Periodontitis

a BH2

BH1 a7
N C
76

a6 a2

a4
BH3
a3

a1
a5
BH4

26

b
Anti-Apoptotic
N-ter Bcl-2, Bcl-XL
Mcl-1, CED-9
BH4 BH3 BH1 BH2 Tm

Pro-Apoptotic
N-ter Bax, Bak

BH3 only - initiating or activating

N-ter Bim
Bad, Bid,
N-ter Noxa, Puma

Fig. 13.9฀ Domain฀structure฀and฀interactions฀among฀Bcl-2฀family฀proteins.฀(a)฀Structure฀of฀a฀Bcl-2฀

family฀member,฀Bcl-XL.฀There฀are฀six฀a-helices฀(blue,฀numbered฀1฀through฀6)฀whose฀side฀chains฀
mediate฀ partner฀ binding฀ and/or฀ insertion฀ into฀ a฀ membrane.฀ The฀ Bcl-2฀ homology฀ domains฀ (BH฀
domains)฀are฀labeled฀BH1,฀BH2,฀and฀BH3฀regions฀(yellow,฀red,฀and฀green฀colored฀ribbon,฀respec-
tively,฀corresponding฀to฀amino฀acid฀regions฀86–100,฀129–148฀and฀180–195).฀The฀BH4฀region฀is฀
coincident฀with฀the฀a1฀helix฀(amino฀acids฀4–24)฀(b)฀Bcl-2฀family฀member฀domain฀organization.฀
Bcl2฀family฀members฀have฀four,฀three฀or฀one฀of฀the฀Bcl-2฀Homology฀(BH)฀domains.฀A฀common฀
sequence฀motif฀identiies฀each฀domain฀and฀a฀unique฀sequence฀motif฀each฀domain฀number.฀Bcl-2฀
family฀members฀with฀only฀three฀BH฀domains฀are฀BH4฀domain-lacking฀proteins฀(the฀apoptosis฀effec-
tor฀proteins).฀Bcl-2฀family฀member฀with฀only฀one฀domain฀are฀BH3฀domain฀only฀proteins฀and฀divided฀
into฀initiating฀and฀activating฀as฀indicated฀below฀(a:฀Figure฀1c฀from฀Muchmore฀SW,฀Sattlert฀M,฀Liang฀
H,฀et฀al.฀1996,฀May฀23,฀“X-ray฀and฀NMR฀structure฀of฀human฀Bcl-XL,฀an฀inhibitor฀of฀programmed฀
cell฀death.”฀Nature฀381:335–341;฀b:฀Wikipedia฀public฀domain฀image฀(http://en.wikipedia.org/wiki/
Image:Bcl-2_Family.jpg)
13.4.3 Mechanisms of Apoptosis 249

13.4.3
Mechanisms of Apoptosis

The฀ extrinsic฀ pathway฀ mechanism฀ is฀ mediated฀ by฀ cytokine฀ ligands:฀ the฀ TNF-related฀฀
apoptosis-inducing฀ligand฀(TRAIL),฀the฀Fas฀ligand฀(FasL),฀and฀other฀homologous฀cytokines.฀
All฀these฀ligands฀bind฀to฀receptors฀homologous฀to฀the฀TNF-a฀receptor.฀Like฀TNF-a,฀FasL฀and฀
TRAIL฀are฀present฀in฀the฀cytosol฀of฀most฀cells฀and฀are฀released฀by฀the฀ligand-bound฀receptor฀
as฀unstable฀homotrimers฀(Sect.฀3.2.2).฀For฀apoptosis฀to฀occur,฀the฀bound฀ligand฀must฀pool฀the฀
membrane฀domain฀of฀the฀receptor฀into฀clusters฀of฀lipids฀(lipid฀rafts)฀on฀the฀cell฀surface.฀The฀
clustering฀allows฀the฀C-terminal฀domains฀of฀the฀receptors฀to฀associate฀and฀form฀TNF฀receptor-
associated฀death฀domains฀(TRADD)฀or฀FAS฀receptor-associated฀death฀domains฀(FADD).
The฀TRADD฀or฀FADD฀domains฀bind฀to฀adapter฀proteins฀in฀the฀cytosol฀and฀together฀
they฀form฀of฀a฀death฀inducing฀signaling฀complex฀(DISC).฀This฀complex฀recruits฀caspase-8,฀
one฀of฀a฀family฀of฀calcium฀ion-activated฀serine฀proteases฀(caspases)฀which฀cleave฀poly-
peptides฀on฀the฀C-terminal฀side฀of฀their฀aspartate฀residues.฀DISC฀activates฀caspase-8฀to฀
activate฀a฀Bcl-2฀activator,฀BID฀(Fig.฀13.10a).฀The฀resultant฀tBid฀fragment฀activates฀effector฀
Bcl-2฀proteins฀on฀the฀endoplasmic฀reticular฀membrane฀and฀mitochondrial฀outer฀membrane.฀

Fig. 13.10฀ Caspase฀activation฀by฀extrinsic฀and฀intrinsic฀apoptotic฀pathways.฀(a)฀Extrinsic฀path.฀This฀

path฀ is฀ mediated฀ by฀ members฀ of฀ the฀ tumor฀ necrosis฀ factor฀ (TNF)฀ superfamily฀ (see฀ text).฀฀
(b)฀Intrinsic฀pathway.฀This฀path฀may฀be฀mediated฀by฀various฀physiological฀or฀pathological฀changes,฀
but฀only฀the฀modes฀of฀induction฀of฀this฀path฀in฀periodontitis฀are฀discussed฀(see฀text).฀Details฀of฀
cytochrome฀c฀release฀and฀caspase฀activation฀are฀shown฀in฀Fig.฀13.11฀(This฀igure฀is฀modiied฀from฀
Fig.฀ 1฀ in฀ Schimmer฀ AD฀ (2004฀ Oct฀ 15)฀ “Inhibitor฀ of฀ Apoptosis฀ Proteins:฀ Translating฀ Basic฀
Knowledge฀into฀Clinical฀Practice.”฀Cancer฀Research,฀64:7183–7190)
250 13 Chronic Periodontitis

Calcium฀ions฀are฀released฀into฀the฀cytosol฀from฀the฀endoplasmic฀reticulum฀and฀cytochrome฀
c฀and฀other฀proteins฀from฀the฀mitochondrial฀intermembrane฀space.฀The฀calcium฀ions฀acti-
vate฀caspase-9,฀enhancing฀the฀activation฀of฀caspase-3,฀which฀is฀activated฀by฀caspase-8฀in฀
addition฀to฀BID.
In฀chronic฀periodontitis,฀the฀intrinsic฀apoptotic฀pathway฀(Fig.฀13.10b)฀may฀involve฀short-
chain฀ fatty฀ acids฀ and฀ other฀ bacterial฀ products฀ penetrating฀ into฀ the฀ cytosol฀ of฀ junctional฀
epithelial฀cells฀at฀the฀base฀of฀a฀sulcus.฀Some฀NLR฀proteins฀may฀activate฀Bcl-2฀effector฀pro-
teins,฀or฀exposure฀to฀the฀contents฀of฀phagolysosomes฀that฀have฀degranulated฀(Sect.฀13.3.1)฀
may฀cause฀membrane฀leakage.฀In฀either฀case,฀soluble฀proteins฀from฀the฀mitochondrial฀inter-
membrane฀space฀and฀calcium฀ions฀from฀the฀endoplasmic฀reticulum฀and฀extracellular฀luid฀
pass฀into฀the฀cytosol.฀Cytochrome฀c฀is฀released฀into฀the฀cytosol฀where฀it฀reacts฀with฀apop-
totic฀protease฀activating฀factor-1฀(apaf-1)฀and฀calcium฀ions฀to฀activate฀caspase-9฀and฀cas-
pase฀3฀(Fig.฀13.11a).฀Apaf-1฀possesses฀an฀N-terminal฀caspase฀recruitment฀domain฀(CARD)฀
that฀ interacts฀ with฀ a฀ matching฀ CARD฀ on฀ caspase฀ 9฀ (Fig.฀ 13.11a).฀ The฀ exposed฀ CARD฀
domains฀in฀the฀cytochrome฀c/Apaf-1฀complex฀aggregate฀to฀form฀an฀apoptosome฀(Fig.฀13.11b)฀
whose฀ central฀ hub฀ contains฀ attached฀ caspase-9.฀ The฀ bound฀ caspase-9฀ auto-activates฀ and฀
frees฀itself฀from฀the฀apoptosome฀by฀cleaving฀off฀its฀pro-฀and฀CARD฀domains.
In฀ addition฀ to฀ cytochrome฀ c,฀ a฀ second฀ mitochondria-derived฀ activator฀ of฀ caspases฀
(SMAC)฀is฀also฀released฀from฀the฀mitochondrial฀intermembrane฀space.฀SMAC฀binds฀to฀an฀
inhibitor฀of฀apoptosis฀protein฀(IAP)฀to฀prevent฀spontaneous฀caspase฀activation฀in฀a฀healthy฀
cell.฀The฀IAP฀that฀binds฀to฀caspase-9฀is฀called฀X-IAP.฀When฀SMAC฀binds฀to฀X-IAP,฀X-IAP฀
is฀ released฀ from฀ caspase-9.฀ Autocleavage฀ of฀ the฀ caspase-9฀ prodomain฀ then฀ exposes฀ the฀
catalytic฀site.฀Caspase-9฀then฀activates฀caspase-3฀which฀immediately฀cuts฀out฀the฀X-IAP฀
binding฀domain฀from฀procaspase-9,฀preventing฀X-IAP฀inhibition฀and฀accelerating฀activa-
tion฀of฀both฀caspase-3฀and฀caspase-9฀(Fig.฀13.11a),฀which฀digest฀all฀of฀the฀cell’s฀proteins.฀
The฀apoptotic฀cells฀also฀possess฀a฀caspase-activated฀DNase฀(CAD)฀that฀fragments฀chro-
mosomal฀DNA.฀Other฀proteins฀from฀the฀mitochondrial฀intermembrane฀space฀can฀activate฀
caspases฀by฀related฀mechanisms.฀The฀combination฀of฀mechanisms,฀or฀the฀overwhelming฀
activation฀of฀any฀one฀of฀them,฀irreversibly฀propels฀a฀cell฀into฀apoptosis.

Mammalian฀cell฀death฀is฀classiied฀as฀autophagy,฀apoptosis,฀or฀necrosis.฀In฀gingivitis,฀
autophagy฀may฀impair฀the฀barrier฀to฀bacterial฀products฀(PAMPs).฀In฀chronic฀periodon-
titis,฀apoptosis฀occurs฀in฀junctional฀epithelium.฀Bacterially฀produced฀short฀chain฀fatty฀
acids฀permeate฀the฀cell฀membrane฀and฀activate฀NOD-like฀proteins฀that฀mediate฀apopto-
sis,฀or฀phagolysosomes฀degranulate฀on฀contacting฀the฀bioilm.฀Both฀mechanisms฀acti-
vate฀Bcl-2฀family฀proteins฀to฀punch฀holes฀in฀endoplasmic฀reticular฀and฀mitochondrial฀
membranes,฀releasing฀calcium฀ions฀into฀the฀cytosol฀along฀with฀cytochrome฀c฀and฀other฀
proteins฀from฀the฀mitochondrial฀intermembrane฀space.฀Cytochrome฀c฀reacts฀with฀apop-
totic฀protease฀activating฀factor-1฀in฀the฀cytosol฀to฀form฀an฀apoptosome฀complex฀that฀
binds฀to฀and฀activates฀caspases,฀calcium฀ion-activated฀serine฀proteases฀that฀cleave฀on฀
the฀C-terminal฀side฀of฀aspartic฀acid฀residues฀in฀polypeptides.฀In฀healthy฀cells,฀inhibitors฀
of฀ apoptosis฀ proteins฀ (IAPs)฀ block฀ the฀ catalytic฀ site฀ of฀ spontaneously฀ activated฀
caspases.
13.4.3 Mechanisms of Apoptosis 251

a ‘Death’ receptors Stress or PAMPs

in the cytosol

Caspase-8

Bid
tBid
(or BIM or PUMA)
Catalytic Mitochondrial
site Cyt c inter-membrane
CARD BAK/OMP space
Apaf-1 complex
Caspase-3

BA
BA

OMP
BAK
K
Caspase-9
K
K
BA

X-IAP/
K
BA

SMAC
complex X-IAP/
caspase-9 SMAC
complex

b Apaf-1

Cyt c

CARD
domains

Fig 13.11฀ Mitochondrial฀intermembrane฀space฀molecules฀in฀apoptosis฀(a)฀Activation฀creates฀a฀pore฀

in฀the฀mitochondrial฀membrane:฀BAK฀or฀BAX฀create฀a฀pore฀(hydrophilic฀hole)฀in฀the฀membrane฀
allowing฀cytochrome฀c฀(red฀dots)฀or฀SMAC฀(purple฀crescent)฀to฀enter฀the฀cytosol฀from฀the฀mito-
chondrial฀intermembrane฀space.฀BAK฀and฀BAX฀are฀also฀present฀with฀a฀partner฀on฀the฀endoplasmic฀
reticulum฀ and฀ their฀ activation฀ similarly฀ releases฀ Ca2+฀ ions฀ into฀ the฀ cytosol฀ (not฀ illustrated).฀
Cytochrome฀c฀interacts฀with฀Apaf-1฀(orange฀oval)฀whose฀CARD฀domains฀(blue฀rectangles)฀bind฀
to฀caspase-9.฀Caspases฀also฀bind฀to฀cytosolic฀inhibitor฀of฀activation฀proteins฀called฀IAPs฀(see฀text).฀
X-IAP฀is฀the฀speciic฀IAP฀that฀binds฀to฀caspase-9.฀(b)฀The฀apoptosome:฀The฀central฀CARD฀domains฀
of฀aggregated฀Apaf-1฀proteins฀form฀a฀pinwheel-like฀apoptosome฀that฀activates฀caspase-9฀(see฀text)฀
(a:฀Figure฀is฀modiied฀from฀Fig.฀2฀in฀Joza฀N,฀Kroemer฀G฀and฀Penninger฀JM฀(2002)฀“Genetic฀analy-
sis฀of฀the฀mammalian฀cell฀death฀machinery.”฀Trends฀in฀Genetics฀18฀(3):142–149;฀b:฀Figure฀is฀taken฀
from฀Fig.฀1฀in฀Danial฀NN,฀Korsmeyer฀SJ฀(2004฀Jan฀23)฀“Cell฀death:฀critical฀control฀points.”฀Cell฀
116(2):205–219).฀This฀igure฀was฀modiied฀by฀Dr.฀Wirsig-Weichmann
252 13 Chronic Periodontitis

13.5.1
Eicosanoids and Periodontal Repair

Correct฀ tooth฀ brushing,฀ mouth฀ cleansing,฀ and฀ lossing฀ is฀ the฀ primary฀ defense฀ against฀
chronic฀ periodontitis,฀ but฀ nearly฀ always฀ insuficient.฀ Regular฀ visits฀ to฀ a฀ dentist฀ are฀
extremely฀important฀to฀remove฀calculus฀from฀the฀tooth฀surface.฀Once฀periodontitis฀is฀pres-
ent,฀therapy฀begins฀with฀a฀procedure,฀called฀scaling฀and฀root฀planning฀in฀which฀the฀gingival฀
and฀root฀surfaces฀of฀the฀teeth฀are฀scraped฀clean฀using฀ultrasonic฀and฀hand฀instruments.฀The฀
teeth฀are฀then฀polished฀and฀the฀patient฀is฀instructed฀in฀oral฀hygiene฀to฀slow฀the฀redevelop-
ment฀of฀plaque฀and฀calculus.฀If฀the฀disease฀is฀moderate฀or฀severe,฀one฀quadrant฀is฀treated฀
per฀week฀and฀the฀scaling฀and฀root฀planning฀is฀accompanied฀by฀minor฀surgery฀to฀reconsti-
tute฀the฀gingival฀architecture.฀This฀therapy฀facilitates฀the฀disappearance฀(resolution)฀of฀the฀
inlammation฀and฀the฀restoration฀of฀a฀healthy฀sulcus.฀Removing฀the฀successor฀microbiota฀
immediately฀stops฀any฀apoptosis฀or฀necrosis฀of฀DAT฀cells฀and฀reduces฀the฀bacterial฀inlam-
matory฀stimulus฀(Sect.฀13.4.1).
The฀process฀of฀resolution฀(described฀in฀Sect.฀13.2.5)฀is฀directed฀by฀a฀subgroup฀of฀eico-
sanoids,฀a฀family฀of฀signaling฀molecules฀found฀in฀virtually฀all฀tissues฀and฀organs.฀They฀are฀
synthesized฀ from฀ polyunsaturated฀ fatty฀ acids,฀ fatty฀ acids฀ with฀ multiple฀ double฀ bonds฀
(Fig.฀13.12)฀that฀are฀essential฀components฀of฀cell฀membranes.฀These฀fatty฀acids฀must฀be฀
obtained฀ from฀ the฀ diet฀ because฀ they฀ cannot฀ be฀ synthesized฀ de฀ novo฀ in฀ the฀ mammalian฀
body.฀Their฀function฀is฀to฀prevent฀membrane฀hardening,฀especially฀in฀cold฀water฀ish฀for฀
the฀ same฀ reason฀ that฀ less฀ hydroxyproline฀ allows฀ physiological฀ dissociation฀ of฀ collagen฀
ibers฀(Chap.฀4).฀In฀polyunsaturated฀fatty฀acids,฀the฀carboxyl฀group฀(COO–)฀is฀always฀num-
bered฀carbon฀atom฀1฀(C1),฀and฀the฀double฀bond฀nearest฀to฀the฀terminal฀(omega,฀w)฀carbon฀
atom฀is฀3฀or฀6฀carbon฀atoms฀distant฀(omega-3฀or฀omega-6;฀Fig.฀13.12).

13.5.2
Eicosanoid Structure

Eicosanoids฀are฀made฀up฀of฀six฀families:฀prostaglandins,฀thromboxanes,฀leukotrienes,฀and฀
lipoxins฀from฀arachidonic฀acid฀(omega-6);฀resolvins฀from฀eicosapentaenoate฀or฀docosa-
hexaenoate฀(omega-3);฀and฀protectins฀from฀docosahexaenoate฀(omega-6).฀Prostaglandins฀
were฀originally฀identiied฀as฀hormone-like฀compounds฀secreted฀by฀the฀prostate฀gland฀and฀
thromboxanes฀as฀blood฀coagulation฀factors฀secreted฀by฀activated฀platelets.฀Their฀inlam-
mation฀ promoting฀ and฀ resolving฀ activity฀ were฀ subsequently฀ discovered฀ in฀ related฀ com-
pounds.฀ The฀ arachidonate-derived฀ eicosanoids฀ are฀ made฀ by฀ reacting฀ arachidonate฀ with฀
cyclooxygenases,฀ which฀ are฀ present฀ in฀ most฀ cells,฀ or฀ lipoxygenases,฀ which฀ are฀ present฀
mostly฀ in฀ leukocytes฀ (granulocytes฀ and฀ macrophages).฀ There฀ are฀ two฀ cyclooxygenase฀
(COX)฀isoenzymes,฀one฀constitutive฀(COX-1)฀and฀the฀other฀induced฀by฀cytokines฀(COX-
2),฀and฀together฀they฀give฀rise฀to฀the฀prostaglandins฀and฀thromboxanes.฀There฀are฀also฀three฀
lipoxygenase฀isoenzymes,฀each฀with฀slightly฀different฀speciicity฀and฀expressed฀mainly฀by฀
granulocytes฀and฀macrophages.
13.5.2 Eicosanoid Structure 253

Fig. 13.12฀ Polyunsaturated฀fatty฀acids฀required฀for฀eicosanoid฀synthesis.฀Oleic฀acid฀is฀the฀only฀fatty฀acid฀

synthesized฀by฀mammals฀de฀novo.฀Linoleic฀(w-3)฀and฀a-linolenic฀acid฀(w-6)฀cannot฀be฀synthesized,฀
because฀mammals฀have฀a฀desaturase฀enzyme฀that฀only฀makes฀w-9฀or฀greater฀fatty฀acids.฀Ingested฀w-3฀
fatty฀acids฀are฀metabolized฀to฀other฀w-3฀fatty฀acids฀with฀w-9฀double฀bonds.฀The฀same฀applies฀to฀w-6฀fatty฀
acids.฀The฀major฀dietary฀sources฀of฀polyunsaturated฀fatty฀acids฀are฀ish฀and฀plants฀oils

The฀two฀COX฀enzymes฀have฀~60%฀identical฀amino฀acid฀sequences.฀They฀both฀exhibit฀
two฀ enzymatic฀ activities:฀ cyclooxygenase฀ activity,฀ which฀ converts฀ arachidonic฀ acid฀ to฀
prostaglandin฀G2;฀and฀peroxidase฀activity฀which฀reduces฀a฀hydroperoxide฀on฀prostaglandin฀
G2฀to฀the฀corresponding฀alcohol,฀prostaglandin฀H2฀(Fig.฀13.13a).฀The฀COX฀enzymes฀pos-
sess฀dioxygenase฀and฀peroxidase฀centers฀that฀are฀interdependent.฀The฀peroxidase฀center฀
254 13 Chronic Periodontitis

a b
COO- COO−
CH3 CH3

Arachidonate Arachidonate
Cyclo-oxygenase 2 O2
activity
O OH
O2
COO−
O COO− Lipoxygenase-5
O CH3 activity 5-HPETE

O
HO
COO−
Prostaglandin G2 O2

H
O
O
2 H+ + 2e−
Myeloperoxidase Lipoxygenase-12
activity activity 12-HPETE
H2O

O2
O COO− COO−
O CH3
Lipoxygenase-15
OH activity O OH
15-HPETE
Prostaglandin H2

Fig. 13.13฀ Cyclooxygenase฀ and฀ lipoxygenase฀ action฀ on฀ arachidonic฀ acid.฀ (a)฀ Cyclooxygenase฀
mechanism.฀There฀are฀two฀forms฀of฀cyclooxygenase,฀constitutive฀and฀inducible.฀Both฀possess฀a฀
cyclooxygenase฀ reaction฀ center,฀ which฀ adds฀ two฀ oxygen฀ molecules฀ (in฀ red)฀ per฀ molecule฀ of฀
arachidonate.฀One฀oxygen฀molecule฀forms฀a฀cyclic฀ring฀involving฀three฀carbon฀atoms฀and฀the฀
other฀forms฀a฀peroxide฀at฀carbon฀atom฀15฀(C15).฀The฀product฀(prostaglandin฀G2)฀is฀immediately฀
attacked฀by฀a฀separate,฀interactive฀myeloperoxidase฀reaction฀center฀on฀the฀cyclooxygenase฀poly-
peptide.฀The฀myeloperoxidase฀reduces฀the฀C15฀peroxide฀to฀a฀hydroxy฀group.฀(b)฀Lipoxygenase฀
action.฀Leukocytes฀may฀oxidize฀arachidonate฀at฀C5,฀or฀C12฀or฀C15฀with฀one฀of฀three฀lipoxyge-
nase฀ isoenzymes฀ to฀ give฀ arachidonate฀ 5฀ hydroperoxide฀ (hydrodoperoxyeicosatetroenoate,฀
5-HPETE,฀etc.).฀The฀peroxy฀group฀is฀converted฀to฀the฀corresponding฀hydroxyeicosatetroenoate฀
(5-HETE฀etc.)฀by฀a฀peroxidase฀enzyme฀and฀is฀then฀oxidized฀further฀by฀an฀appropriate฀lipoxyge-
nase฀ and/or฀ hydrated฀ by฀ a฀ hydrolase฀ to฀ give฀ the฀ intermediates฀ and฀ end-products฀ indicated฀ in฀
Fig.฀13.10฀(a:฀Figure฀from฀http://www.jungfreudlich.de/wp-content/uploads/2007/08/formation-
of-prostaglandin-h2.jpg;฀b:฀Figure฀from฀http://www.benbest.com/health/Lipoxygenase.jpg)

possesses฀a฀Fe2+/heme฀cofactor฀related฀to฀myoglobin฀that฀attaches฀two฀molecules฀of฀molec-
ular฀ oxygen฀ to฀ activate฀ the฀ dioxygenase-catalyzed฀ oxidation฀ and฀ reduction฀ of฀ bound฀
arachidonic฀acid.฀One฀molecule฀of฀oxygen฀(O2)฀forms฀a฀ring฀between฀carbons฀9฀and฀11฀(C9฀
and฀C11)฀of฀arachidonate฀and฀the฀other฀attaches฀as฀a฀peroxy฀group฀to฀carbon฀15฀(C15).฀
Because฀the฀two฀reactions฀are฀inseparable,฀COX1฀and฀COX฀2฀are฀also฀known฀as฀the฀pros-
taglandin฀H฀synthetases฀(PGHSs).฀Prostaglandin฀H2฀is฀the฀precursor฀of฀all฀other฀prosta-
glandins฀or฀thromboxanes฀(Fig.฀13.13a),฀which฀are฀made฀by฀further฀cyclooxygenase฀action฀
determined฀by฀tissue฀type.
13.5.4 Lipoxygenase-Mediated Resolution of Inflammation 255

Lipoxygenases฀are฀Fe2+-containing฀dioxygenases฀that฀catalyze฀the฀hydroperoxidation฀of฀
arachidonate฀and฀other฀unsaturated฀fatty฀acids฀to฀form฀leukotrienes,฀lipoxins,฀resolvins,฀or฀
protectins.฀They฀are฀attached฀to฀the฀cytosolic฀side฀of฀membranes฀by฀calcium฀ions.฀There฀
are฀ various฀ lipoxygenase฀ isoenzymes,฀ of฀ which฀ three฀ make฀ leukotrienes฀ or฀ lipoxins฀ by฀
adding฀a฀peroxy฀group฀to฀a฀speciic฀carbon฀atom฀of฀a฀double฀bond฀in฀arachidonate,฀C5,฀C12฀
or฀ C15฀ (Fig.฀ 13.13b).฀ The฀ peroxy฀ group฀ is฀ enzymatically฀ reduced฀ to฀ a฀ hydroxyl฀ group฀
(−OH)฀by฀a฀separate฀peroxidase฀enzyme.฀Lipoxygenases฀are฀unusual฀in฀requiring฀two฀iden-
tical฀substrate฀molecules฀for฀activity.฀The฀active฀site฀of฀lipoxygenases฀contains฀a฀single฀
iron฀atom฀bound฀(coordinated)฀to฀three฀histidine฀residues.฀The฀iron฀attaches฀a฀molecule฀of฀
oxygen฀to฀each฀of฀two฀polyunsaturated฀fatty฀acid฀substrates฀and฀then฀transfers฀two฀elec-
trons฀to฀form฀two฀peroxy฀groups,฀one฀on฀each฀substrate.฀Lipoxygenases฀are฀regenerated฀by฀
electrons฀from฀glutathione฀by฀the฀pathway฀described฀in฀Chap.฀6฀(Fig.฀6.8).

13.5.3
Functions of the Proinflammatory Eicosanoids

Cytokines฀or฀hormones฀release฀arachidonate฀or฀other฀fatty฀acid฀substrate฀from฀the฀cytosolic฀
side฀of฀phospholipids฀by฀activating฀phospholipase฀A2.฀For฀example,฀leukotriene฀B4฀(LTB4)฀
is฀synthesized฀in฀neutrophils฀in฀which฀arachidonate฀is฀released฀and฀lipoxygenases฀induced฀
by฀IL-1฀or฀IL-8.฀Like฀IL-8,฀LTB4฀induces฀the฀adhesion฀of฀leukocytes฀to฀capillary฀endothe-
lium,฀activates฀them,฀and฀promotes฀their฀movement฀into฀the฀infected฀stroma฀(Sect.฀13.2.3).฀
LTB4฀is฀also฀a฀potent฀chemoattractant฀for฀neutrophils฀in฀which฀it฀induces฀the฀formation฀of฀
reactive฀ oxygen฀ species฀ and฀ the฀ secretion฀ of฀ lysosomal฀ (acid-activated)฀ enzymes.฀
Macrophages฀secrete฀prostaglandin฀E2,฀the฀osteoclast฀activating฀factor฀responsible฀for฀alve-
olar฀bone฀loss฀in฀periodontitis.฀The฀varied฀actions฀of฀eicosanoids฀on฀target฀cells฀are฀medi-
ated฀by฀receptors฀that฀functionally฀resemble฀cytokine฀receptors.
Prostaglandins฀are฀secreted฀by฀a฀prostaglandin฀transporter฀(PGT)฀protein.฀This฀single-
polypeptide฀contains฀12฀transmembrane฀regions฀within฀the฀plasma฀membrane฀of฀most฀cells฀
and฀tissues.฀PGT฀proteins฀transport฀small฀negatively฀charged฀solutes฀through฀a฀membrane฀in฀
response฀ to฀ chemiosmotic฀ ion฀ gradients.฀ These฀ transporters฀ belong฀ to฀ the฀ organic-anion-
transporting฀polypeptide฀(OATP)฀class฀of฀proteins,฀a฀subgroup฀of฀the฀major฀(transport)฀facili-
tator฀superfamily฀(MFS).฀These฀transporters฀are฀unrelated฀to฀the฀ABC฀or฀drug฀export฀proteins฀
that฀are฀involved฀in฀transporting฀large฀proteins฀such฀as฀Aa฀leukotoxin฀(Sect.฀14.2.2).

13.5.4
Lipoxygenase-Mediated Resolution of Inflammation

Resolution฀is฀accompanied฀by฀a฀switch฀away฀from฀the฀cyclooxygenases฀(prostaglandins,฀
thromboxanes)฀to฀lipoxygenases฀(lipoxins,฀resolvins,฀and฀protectins).฀Each฀has฀a฀different฀
substrate:฀arachidonate฀for฀lipoxins,฀eicosapentaenoate฀for฀resolvins,฀and฀docosahexaeno-
ate฀for฀protectins฀(Fig.฀13.12).฀Each฀class฀therefore฀requires฀a฀different฀lipoxygenase฀isoen-
zyme.฀The฀irst฀peroxy฀group฀is฀always฀added฀to฀the฀15C฀atom฀and฀then฀peroxy฀groups฀are฀
256 13 Chronic Periodontitis

attached฀to฀other฀positions฀(Fig.฀13.14).฀Indeed,฀as฀the฀neutrophils฀age,฀their฀lipoxygenase฀
speciicity฀spontaneously฀changes฀from฀5C฀to฀15C฀to฀promote฀the฀synthesis฀of฀lipoxins,฀
resolvins,฀and฀protectins฀over฀leukotrienes฀(Fig.฀13.14).

Arachidonic acid in
cell membrane phospholipids

Phosphoipase A2 or
Diacylglycerol lipase

Free arachidonic acid

a COX-1 & COX-2 b lipoxygenase

isoenzymes isoenzymes

PGG2 5-HPETE 12-HETE 15-HPETE

PGH2 15-HETE
LTA4 12-HETE
5-HETE

LTC4
PGD2 PGE2 PGI2 TXA2 PGF2α LTB4 Lipoxin A4
LTD4

PGJ2 LTE4 ?? Resolution

Proinflammatory

Fig. 13.14฀ Arachidonate฀conversion฀to฀eicosanoids฀Boxed฀names฀identify฀eicosanoids:฀prostaglan-

din฀(PG),฀thromboxane฀(TX),฀leukotrienes฀(LT),฀and฀lipoxin฀whose฀functions฀are฀described฀in฀the฀
text.฀Each฀eicosanoid฀is฀indicated฀by฀letters฀and฀numbers฀(PGG2,฀PGH2฀LTA4,฀LTB4,฀lipoxin฀A4,฀
etc.).฀Eicosanoid฀structures฀and฀enzyme฀actions฀are฀given฀in฀Fig.฀13.13.฀(a)฀Cyclooxygenase฀con-
version.฀Cyclooxygenase฀(COX)฀produces฀PGH2฀and฀then฀makes฀the฀various฀PGs฀and฀TXs฀from฀
PGH2.฀The฀various฀end฀products฀are฀cell-type฀speciic฀and฀transported฀by฀carriers฀to฀the฀extracellular฀
luid฀(see฀text).฀Boxed฀names฀indicate฀end-products฀that฀are฀commonly฀found฀in฀chronic฀periodonti-
tis.฀(b)฀Lipoxygenase฀conversion.฀The฀5-lipoxygenase฀(5-LX)฀makes฀5-HPETE฀from฀arachidonate฀
(shown฀in฀Fig.฀13.13b)฀and฀converts฀it฀to฀leukotriene฀A4฀(LTA4).฀The฀latter฀is฀then฀acted฀on฀by฀other฀
lipoxygenases฀and฀peroxidases฀to฀produce฀the฀various฀illustrated฀leukotrienes.฀PGs,฀TXs฀and฀LTs฀
made฀by฀the฀induced฀COX฀enzyme฀are฀proinlammatory.฀Lipoxins,฀resolvins฀and฀protectins฀made฀
by฀various฀lipoxygenases฀from฀arachidonate฀and฀larger฀w-3฀and฀w-6฀fatty฀acids฀are฀antiinlamma-
tory฀[Modiied฀from฀Fig.฀3฀of฀Sala-Vila฀A,฀Miles฀EA฀and฀Calder฀PC฀(2008฀Sep)฀“Fatty฀acid฀com-
position฀ abnormalities฀ in฀ atopic฀ disease:฀ evidence฀ explored฀ and฀ role฀ in฀ the฀ disease฀ process฀
examined.”฀Clinical฀and฀Experimental฀Allergy,฀38(9):1432–1450]
13.5.5 Antiinflammatory Drugs 257

In฀chronic฀periodontitis,฀a฀proinlammatory฀response฀is฀maintained฀by฀the฀continued฀
binding฀of฀PAMPs฀in฀the฀junctional฀epithelium฀and฀gingival฀stroma.฀If฀PAMPs฀are฀not฀
entirely฀ removed,฀ a฀ fresh฀ neutrophil฀ iniltrate฀ replaces฀ any฀ aged฀ neutrophils฀ that฀ are฀
synthesizing฀lipoxins,฀resolvins,฀and฀protectins.฀Thus,฀a฀balance฀between฀resolution฀and฀
enhancement฀ of฀ inlammation฀ develops,฀ depending฀ on฀ how฀ much฀ of฀ the฀ microbiota฀
remains฀at฀any฀time.฀Resolvins฀and฀protectins฀have฀three฀major฀roles.฀(a)฀Inhibit฀nuclear฀
factor-kB฀activation฀(Sect.10.2.2);฀(b)฀Recruit฀monocytes฀that฀differentiate฀into฀modi-
ied฀macrophages฀that฀exit฀into฀the฀lymphatic฀system฀after฀phagocytosis฀of฀microorgan-
isms,฀ apoptotic฀ neutrophils฀ and฀ other฀ cell฀ debris฀ without฀ producing฀ proinlammatory฀
mediators;฀and฀(c)฀Stimulate฀the฀expression฀of฀acquired฀and฀innate฀immunity฀molecules฀
(Sect.฀12.1.4).

13.5.5
Antiinflammatory Drugs

The฀inhibitors฀of฀COX฀enzymes฀are฀called฀nonsteroidal฀antiinlammatory฀drugs฀(NSAIDs)฀
that฀are฀prescribed฀to฀relieve฀pain฀and฀fever.฀They฀stop฀prostaglandin฀and฀thromboxane฀
production.฀Acetylsalicylic฀acid฀(aspirin)฀was฀used฀for฀this฀for฀many฀years฀and฀it฀was฀even-
tually฀ discovered฀ to฀ acetylate฀ a฀ serine฀ residue฀ involved฀ in฀ the฀ dioxygenase฀ action฀ of฀
cyclooxygenases.฀ A฀ second฀ class฀ of฀ NSAIDs,฀ typiied฀ by฀ ibuprofen฀ (commonly฀ called฀
Advil฀or฀Motrin),฀inhibits฀catalysis฀by฀attaching฀irreversibly฀to฀cyclooxygenases.฀Aspirin฀
and฀ibuprofen฀inhibit฀all฀prostaglandin฀and฀thromboxane฀synthesis.
Both฀classes฀of฀these฀drugs฀inhibit฀important฀functions฀of฀constitutive฀prostaglan-
dins฀and฀thromboxanes:฀an฀adequate฀secretion฀of฀stomach฀mucus฀and฀blood฀clotting.฀
Mucous฀protects฀the฀stomach฀epithelium฀from฀acid฀ulceration฀and฀bleeding.฀Ibuprofen฀
causes฀stomach฀bleeding฀by฀stopping฀mucus฀secretion฀and฀aspirin฀inhibits฀thromboxane฀
A2฀synthesis,฀which฀down-regulates฀the฀platelet฀activation฀required฀for฀blood฀clotting฀
(Sect.฀11.2.1).฀Unfortunately,฀a฀combination฀of฀aspirin฀and฀ibuprofen฀reduces฀the฀use-
fulness฀of฀aspirin฀in฀preventing฀clotting฀without฀decreasing฀the฀incidence฀of฀stomach฀
bleeding.
NSAIDs฀ that฀ preferentially฀ inhibit฀ COX-2฀ more฀ than฀ COX-1฀ (Vioxx฀ and฀ Celebrex)฀
target฀ the฀ pain฀ associated฀ with฀ inlammation฀ such฀ as฀ rheumatoid฀ or฀ osteoarthritis฀ with฀
fewer฀stomach฀problems฀because฀prostaglandin฀I2฀is฀synthesized฀by฀COX-1฀in฀the฀stomach.฀
In฀ the฀ heart,฀ continuous฀ muscular฀ movements฀ cause฀ capillary฀ wear.฀ Cytokines฀ induce฀
COX-2฀to฀make฀prostaglandin฀I2฀which฀dilates฀the฀capillaries฀and฀prevents฀excessive฀blood฀
clotting฀by฀thromboxane฀A4,฀a฀COX-1฀enzyme฀product.฀A฀COX-2฀inhibitor฀stops฀prosta-
glandin฀I2฀synthesis.฀The฀heart฀capillaries฀do฀not฀dilate฀and฀there฀is฀a฀greater฀risk฀of฀coro-
nary฀artery฀obstruction฀(heart฀attacks).
Some฀ studies฀ suggest฀ that฀ COX฀ inhibitors฀ might฀ control฀ chronic฀ periodontitis฀ after฀
therapy.฀The฀inhibition฀of฀COX฀would฀reduce฀neutrophil฀activation฀and฀emigration฀to฀the฀
affected฀site.
258 13 Chronic Periodontitis

By฀removing฀the฀successor฀microbiota,฀therapy฀and฀oral฀hygiene฀facilitate฀the฀disap-
pearance฀(resolution)฀of฀inlammation,฀the฀regeneration฀of฀the฀apical฀portion฀of฀a฀dam-
aged฀ periodontium฀ and฀ the฀ restoration฀ of฀ a฀ healthy฀ junctional฀ epithelial฀ attachment.฀
Resolution฀is฀directed฀by฀eicosanoids,฀derived฀from฀omega-3฀or฀omega-6฀polyunsatu-
rated฀ fatty฀ acids฀ which฀ are฀ dietary฀ essential฀ components฀ of฀ cell฀ membranes.฀
Proinlammatory฀cytokines฀release฀free฀polyunsaturated฀fatty฀acids฀that฀activate฀induc-
ible฀ and฀ noninducible฀ classes฀ of฀ cyclooxygenase฀ (COX-1฀ and฀ COX฀ 2)฀ to฀ synthesize฀
proinlammatory฀prostaglandins฀and฀thromboxanes.฀In฀addition,฀lipoxygenases฀or฀per-
oxidases฀ add฀ peroxy฀ groups฀ to฀ arachidonate,฀ giving฀ rise฀ to฀ proinlammatory฀ leukot-
rienes.฀ As฀ leukocytes฀ age,฀ lipoxygenases฀ change฀ their฀ speciicity฀ to฀ making฀ lipoxins฀
instead฀of฀leukotrienes฀from฀arachidonate.฀Omega-3฀fatty฀acids฀larger฀than฀arachidonate฀
are฀also฀released฀as฀additional฀lipoxygenase฀substrates฀for฀the฀synthesis฀of฀additional฀
antiinlammatory฀eicosanoids฀(resolvins฀and฀protectins).฀Lipoxins฀resolvins฀and฀protec-
tins฀stop฀neutrophil฀iniltration฀by฀inhibiting฀nuclear฀factor-kB฀activation฀and฀activate฀
the฀ recruitment฀ and฀ differentiation฀ of฀ monocytes฀ into฀ phagocytes฀ (modiied฀ mac-
rophages)฀that:฀(1)฀remove฀cell฀debris฀without฀stimulating฀proinlammatory฀mediators;฀
(2)฀increase฀exit฀of฀these฀phagocytes฀from฀the฀inlamed฀site฀to฀the฀lymphatics;฀and฀(3)฀
stimulate฀the฀expression฀of฀innate฀and฀acquired฀immunity.฀Nonsteroidal฀antiinlamma-
tory฀drugs฀(NSAIDs)฀reduce฀prostaglandin฀and฀thromboxane฀production฀by฀irreversibly฀
binding฀to฀or฀acetylating฀a฀COX฀enzyme.฀Inhibition฀of฀COX-1฀stops฀stomach฀mucus฀
secretion฀and฀promotes฀erosions฀that฀bleed฀because฀of฀the฀simultaneous฀inhibition฀of฀
thromboxane฀A4.฀Other฀NSAIDs฀inhibit฀COX-2฀enzyme฀activity฀but฀also฀prostaglandin฀
I2,฀which฀dilates฀physiologically฀damaged฀capillaries฀in฀the฀heart.฀Capillary฀obstruction฀
occurs฀ because฀ thromboxane฀ A4,฀ from฀ COX-1฀ remains฀ active.฀ The฀ antiinlammatory฀
effects฀of฀NSAIDs฀might฀control฀chronic฀periodontitis.
 Aggressive Periodontitis
14

This฀chapter฀discusses฀generalized฀and฀localized฀aggressive฀periodontitis.฀The฀general-
ized฀form฀is฀mediated฀by฀genetic฀defects฀or฀perhaps฀by฀altered฀cytokine฀or฀hormonal฀
responses฀to฀stress฀making฀the฀gingiva฀exceptionally฀sensitive฀to฀products฀from฀the฀suc-
cessor฀microbiota฀(Sect.฀1).฀The฀localized฀form฀is฀primarily฀caused฀by฀infection฀of฀the฀
gingival฀sulci฀by฀Aggregatibacter฀actinomycetemcomitans฀(Sect.฀2).

14.1.1.
Generalized Aggressive Periodontitis

Aggressive฀periodontitis฀occurs฀in฀less฀than฀0.1%฀of฀the฀US฀population.฀Some฀forms฀of฀
aggressive฀periodontitis฀are฀clinically฀different฀from฀chronic฀periodontitis,฀whereas฀others฀
differ฀mainly฀in฀the฀rapid฀rate฀at฀which฀the฀attachment฀loss฀progresses.฀Like฀chronic฀perio-
dontitis,฀aggressive฀periodontitis฀may฀be฀localized฀or฀generalized.฀The฀microbial฀etiology฀
of฀the฀localized฀disease฀is฀well฀understood,฀whereas฀that฀of฀generalized฀aggressive฀perio-
dontitis฀is฀not.฀The฀latter฀may฀accompany฀certain฀collagen-฀or฀iber-maturation฀enzyme-
related฀mutations฀(Table฀7.1),฀or฀acquired฀or฀genetic฀defects฀of฀neutrophil฀function฀such฀as฀
genetic฀mutations฀of฀the฀integrin฀aL฀or฀aM฀subunits฀(Sects.฀13.2.3฀and฀13.2.4),฀or฀alterations฀
in฀leukocyte฀responsiveness฀associated฀with฀systemic฀cytokine฀stimulation฀in฀stress,฀diabe-
tes,฀or฀heart฀disease.
One฀ form฀ of฀ generalized฀ aggressive฀ periodontitis฀ is฀ Acute฀ Necrotizing฀ Ulcerative฀
Gingivitis฀ (ANUG),฀ clearly฀ a฀ misnomer.฀ The฀ junctional฀ epithelial฀ attachment฀ with฀ its฀
underlying฀cells฀and฀collagen฀ibers฀is฀rapidly฀destroyed฀along฀with฀coronal฀alveolar฀bone฀
(see฀deinition฀of฀coronal฀in฀Sect.฀3.1.5).฀The฀amount฀of฀attachment฀lost฀in฀10฀days฀may฀
take฀many฀years฀to฀occur฀in฀chronic฀periodontitis.
A฀key฀feature฀of฀ANUG฀is฀necrosis฀of฀sulcular฀and฀junctional฀epithelial฀cells฀and฀of฀
ibroblasts฀and฀osteoblasts฀in฀the฀underlying฀stroma.฀Alveolar฀bone฀may฀be฀exposed฀in฀the฀
oral฀ cavity฀ and฀ the฀ resulting฀ pain฀ usually฀ leads฀ a฀ patient฀ to฀ seek฀ help.฀ In฀ necrosis,฀ the฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 259
DOI:฀10.1007/978-3-540-88116-2_14,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
260 14 Aggressive Periodontitis

plasma฀membrane฀ruptures฀(Sect.฀3.4.1),฀causing฀a฀release฀of฀apoptotic฀and฀inlammatory฀
agents฀into฀the฀extracellular฀medium.฀TRAIL฀and฀FAS฀mediate฀extrinsic฀apoptosis฀of฀adja-
cent฀cells฀(Sect.฀13.4.2)฀and฀TNFa฀and฀IL-1฀mediate฀a฀powerful฀release฀of฀pro-inlammatory฀
cytokines฀from฀more฀distant฀cells.฀The฀exact฀cause฀of฀ANUG฀is฀unknown,฀but฀it฀usually฀
appears฀ in฀ individuals฀ who฀ have฀ been฀ severely฀ stressed.฀ Hormonal฀ responses฀ to฀ severe฀
stress฀alter฀the฀junctional฀epithelial฀cells฀response฀to฀TLR฀or฀NOD฀receptor฀activation฀by฀
PAMPs฀from฀the฀successor฀microbiota฀(Sect.฀13.2.1).
During฀ANUG,฀acid฀and฀lysosomal฀enzymes฀are฀released฀into฀the฀extracellular฀environ-
ment฀and฀acid฀activated.฀Cathepsin฀L฀hydrolyzes฀uncalciied฀collagen฀ibers,฀and฀cathepsin฀
K฀hydrolyzes฀calciied฀collagen฀ibers฀due฀to฀osteoclast฀activation.฀Cathepsin฀L฀is฀homolo-
gous฀to฀cathepsin฀K฀but฀has฀a฀different฀speciicity.฀In฀necrotic฀tissues,฀cathepsin฀L฀cleaves฀
the฀nonhelical฀telopeptides฀regions฀of฀types฀I฀and฀II฀collagen.฀These฀collagen฀peptides฀are฀
absorbed฀and฀passed฀to฀the฀bloodstream฀where฀they฀provide฀a฀unique฀blood฀plasma฀marker฀
for฀necrotic฀bone฀destruction฀(Sect.฀4.2.2).฀Cathepsin฀L฀is฀also฀involved฀in฀the฀macrophage฀
digestion฀(processing)฀of฀foreign฀material฀(antigens)฀during฀the฀acquisition฀of฀immunity.

Aggressive฀periodontitis฀occurs฀in฀less฀than฀0.1%฀of฀the฀US฀population.฀ANUG฀is฀a฀form฀
of฀aggressive฀periodontitis฀in฀which฀stress฀hormones฀may฀overactivate฀TLRs฀to฀PAMPs฀
around฀ and฀ within฀ cells฀ at฀ the฀ base฀ of฀ gingival฀ sulci.฀ The฀ junctional฀ epithelium฀ frag-
ments฀and฀its฀cells฀release฀their฀contents฀(necrosis).฀The฀necrotic฀cell฀contents฀induce฀a฀
massive฀release฀of฀pro-inlammatory฀cytokines฀that฀cause฀apoptosis฀of฀adjacent฀cells฀and฀
a฀ heavy฀ iniltrate฀ of฀ leukocytes฀ from฀ more฀ distant฀ cells.฀ Neutrophils฀ and฀ monocytes฀
invade฀and฀destroy฀the฀surrounding฀periodontal฀attachment฀and฀alveolar฀bone.

14.2.1.
Localized Aggressive Periodontitis

Localized฀aggressive฀periodontitis฀(LAP)฀occurs฀as฀deep฀pockets฀around฀the฀irst฀erupted฀
teeth฀of฀children฀and฀young฀adults,฀i.e.฀their฀permanent฀central฀incisors฀and฀irst฀molars.฀The฀
LAP฀pockets฀extend฀well฀into฀the฀periodontium฀and฀contain฀large฀amounts฀of฀a฀small฀gram-
negative฀rod,฀Aggregatibacter฀(formerly฀Actinobacillus)฀actinomycetemcomitans฀(Aa).฀This฀
bacterium฀expresses฀two฀soluble฀protein฀toxins:฀a฀Leukotoxin฀(Ltx)฀and฀a฀Cytolethal฀dis-
tending฀toxin฀(Cdt).฀Most฀Aa฀isolates฀express฀relatively฀low฀levels฀of฀Ltx฀and฀have฀been฀
designated฀minimally฀leukotoxic,฀but฀several฀express฀elevated฀levels.฀Aa฀is฀present฀in฀plaque฀
(bioilm)฀from฀about฀75%฀of฀East฀Asian฀populations฀(China฀and฀Thailand),฀about฀50%฀in฀
Africans฀and฀Hispanics,฀and฀about฀25%฀in฀Caucasians.฀The฀greater฀prevalence฀of฀LAP฀in฀
these฀ populations฀ is฀ believed฀ due฀ to฀ a฀ cultural฀ environment฀ that฀ promotes฀ increased฀ Aa฀
exposure,฀ although฀ genetic฀ (racial)฀ differences฀ cannot฀ be฀ discounted.฀ About฀ half฀ of฀ US฀
individuals฀with฀LAP฀have฀an฀otherwise฀symptomless฀defect฀of฀neutrophil฀function.
Aa฀leukotoxin฀lyses฀leukocytes฀(neutrophils,฀monocytes,฀and฀macrophages)฀on฀contact,฀
whereas฀Cdt฀kills฀lymphocytes฀whose฀surface฀antigen฀receptor฀has฀bound฀to฀an฀antigen,฀or฀
macrophages฀whose฀Mac1฀integrin฀has฀bound฀to฀one฀of฀its฀many฀ligands฀(Sect.฀13.2.4).฀฀In฀
LAP,฀antibodies฀that฀bind฀to฀both฀these฀toxins฀appear฀in฀the฀blood฀and฀they฀may฀facilitate฀
14.2.2. Aa Leukotoxin Composition and Properties 261

the฀rapid฀removal฀of฀these฀toxins฀after฀infection,฀preventing฀attachment฀loss฀from฀spread-
ing฀beyond฀the฀irst฀infected฀sites฀(the฀irst฀six฀permanent฀teeth฀to฀appear฀in฀the฀oral฀cavity).฀
These฀antibodies฀inhibit฀leukotoxin฀but฀not฀cytolethal฀distending฀toxin฀activity฀in฀vitro.

14.2.2.
Aa Leukotoxin Composition and Properties

Aa฀leukotoxin฀belongs฀to฀the฀RTX฀(Repeats฀in฀toxin)฀family฀of฀proteins,฀important฀viru-
lence฀ (damaging฀ or฀ poisonous)฀ agents฀ secreted฀ by฀ Gram-negative฀ bacteria.฀ The฀ repeat฀
sequence฀in฀these฀proteins฀is฀a฀domain฀of฀6–40฀repeats฀of฀a฀9฀amino฀acid฀sequence.฀In฀Aa฀
leukotoxin,฀14฀such฀repeats฀form฀7฀calcium฀ion฀binding฀domains฀(Fig.฀14.1a).฀Removing฀
calcium฀ions฀from฀the฀extracellular฀medium,฀or฀expressing฀a฀mutant฀in฀which฀the฀repeat฀
regions฀are฀absent฀results฀in฀an฀inactive฀leukotoxin.฀Two฀classes฀of฀RTX฀toxins฀are฀cytolytic฀
(i.e.฀they฀lyse฀the฀cells฀of฀an฀infected฀host):฀hemolysins฀and฀leukotoxins.฀Hemolysins฀and฀
leukotoxins฀cause฀erythrocytes฀and฀leukocytes฀to฀lyse฀(necrosis),฀hemolysins฀primarily฀on฀
erythrocytes฀ and฀ leukotoxins฀ primarily฀ on฀ leukocytes.฀ In฀ both฀ cases฀ the฀ mechanism฀ is฀
similar.฀The฀toxin฀causes฀the฀membrane฀of฀the฀affected฀cell฀to฀burst฀and฀cytosolic฀contents฀
are฀released฀into฀the฀extracellular฀luid,฀stimulating฀necrosis฀of฀the฀target฀cells฀and฀inlam-
mation฀of฀surrounding฀cells.฀Aa฀leukotoxin฀mainly฀targets฀human฀neutrophils.฀
The฀repeats฀which฀characterize฀Aa฀leukotoxin฀and฀other฀RTX฀family฀members฀are฀
present฀in฀lipases฀and฀metallopeptidases฀related฀to฀serralysin฀in฀various฀Gram-negative฀
bacteria฀(Sect.฀8.1.1),฀and฀in฀anthrax฀toxin฀lethal฀factor฀(Fig.฀8.1)฀and฀diphtheria฀toxin฀
in฀Gram-positive฀bacteria.฀The฀Gram฀negative฀RTX฀toxins฀are฀all฀secreted฀using฀a฀type฀
I฀secretion฀path฀(Chapter฀1,฀Sect.฀1.4.2).฀This฀path฀involves฀two฀other฀proteins฀(protein฀
B฀and฀protein฀D)฀and฀an฀activating฀enzyme฀(protein฀C),฀all฀encoded฀with฀the฀leukotoxin฀
(protein฀A)฀in฀an฀operon,฀the฀ltx฀operon.฀The฀activating฀enzyme฀is฀an฀acyl฀transferase฀
enzyme,฀which฀transfers฀a฀fatty฀acid฀to฀an฀internal฀lysine฀residue.฀Transfer฀of฀this฀fatty฀
acid฀is฀from฀an฀acylated฀Acyl-Carrier฀Protein฀(acylACP)฀in฀the฀bacterial฀cytosol฀to฀the฀
leukotoxin฀(Fig.฀14b).฀Mutations฀or฀inhibitors฀of฀the฀transferase฀cause฀the฀protein฀to฀be฀
secreted฀without฀leukotoxic฀activity.
Leukotoxin฀secretion฀is฀mediated฀by฀proteins฀B฀and฀D฀assisted฀by฀protein฀TdeA,฀which฀is฀
encoded฀elsewhere฀in฀the฀genome฀(Fig.฀14.1b).฀TdeA฀and฀TolC,฀its฀homologue฀in฀E.฀coli฀strains฀
possessing฀an฀LTX฀hemolysin,฀are฀members฀of฀a฀family฀of฀proteins฀that฀enhance฀drug฀resis-
tance฀by฀transporting฀antibiotics฀out฀of฀bacterial฀cells.฀The฀name฀of฀the฀Aa฀protein฀hints฀at฀the฀
drug-export฀function,฀Toxin฀and฀drug฀export฀protein฀A฀(TdeA).฀Protein฀B฀is฀an฀ATP-Binding฀
Cassette฀transporter฀(ABC-transporter),฀one฀of฀a฀large฀and฀ancient฀family฀of฀transmembrane฀
proteins฀that฀bind฀to฀ATP฀on฀the฀cytosolic฀face฀of฀a฀membrane฀and฀use฀the฀energy฀of฀ATP฀hydro-
lysis฀ to฀ transport฀ a฀ substrate฀ to฀ the฀ extracellular฀ face.฀ Protein฀ D฀ is฀ a฀ multimeric฀ membrane฀
fusion฀protein฀whose฀channel฀spans฀the฀entire฀inner฀membrane฀and฀periplasm฀(intermembrane฀
space).฀The฀protein฀D฀multimers฀therefore฀attach฀protein฀B฀on฀the฀cytosolic฀side฀to฀a฀TdeA-
formed฀channel฀in฀the฀outer฀membrane฀(Fig.฀14.1a).
As฀noted฀above,฀proteins฀B฀and฀D฀are฀encoded฀downstream฀of฀the฀acylase฀(protein฀C)฀
and฀the฀leukotoxin฀(protein฀A)฀genes฀and฀their฀expression฀is฀attenuated฀in฀comparison฀with฀
upstream-encoded฀proteins.฀The฀amounts฀of฀proteins฀B฀and฀D฀are฀reduced฀compared฀with฀
262 14 Aggressive Periodontitis

a
N-ter C-ter

Hydrophobic pore-forming
domain
Common Ca binding C-terminal
domain domain domain

b
RfaH

ops
C A B D TdeA
p synthesis secretion secretion

C C BACTERIAL CELL
CYTOSOL
proA
acylACP

Pi
+
C C ATP ADP

B OUT
B D
IM D
OM TdeA
LPS

TARGET
CELL

Fig. 14.1฀ Structure฀ and฀ synthesis฀ of฀ Aa฀ leukotoxin.฀ (a)฀ Polypeptide฀ domain฀ structure.฀ The฀
p฀ olypeptide฀ is฀ 1,050฀ amino฀ acid฀ residues฀ in฀ length.฀ The฀ pore-forming฀ region฀ is฀ the฀ large฀
N-terminal฀domain฀(dark฀green;฀residues฀5–654)฀plus฀the฀short฀C-terminal฀domain฀(light฀green;฀
residues฀757–774).฀The฀large฀N-terminal฀domain฀is฀followed฀by฀a฀region฀that฀is฀common฀to฀all฀
RTX฀ proteins.฀ It฀ consists฀ of฀ two฀ domains,฀ one฀ multicolored฀ (yellow,฀ red,฀ and฀ blue;฀ residues฀
660–695)฀and฀one฀colored฀blue฀only฀(residues฀696–720)฀and฀a฀tandem฀repeat฀sequence฀(red;฀resi-
dues฀721–846)฀from฀which฀the฀protein฀family฀takes฀its฀name฀(see฀text).฀Each฀repeat฀contains฀the฀
Ca2+฀ion฀binding฀consensus฀sequence:฀GGXGXDXФX฀where฀Ф฀is฀a฀hydrophobic฀residue฀and฀X฀
is฀any฀amino฀acid฀(see฀text).฀(b)฀Synthesis฀and฀secretion.฀The฀lktCABD฀operon฀is฀responsible฀for฀
the฀ synthesis,฀ maturation,฀ and฀ secretion฀ of฀ Aa฀ leukotoxin฀ (protein฀ A;฀ green).฀ The฀ promoter฀
sequence฀(p)฀is฀shown฀along฀with฀an฀operon฀polarity฀suppressor฀sequence฀(ops)฀and฀a฀termina-
tion฀suppressor฀protein฀(RfaH)฀that฀binds฀to฀the฀ops฀nucleotide฀sequence฀and฀facilitates฀expres-
sion฀of฀the฀downstream฀proteins฀B฀(white)฀and฀D฀(yellow).฀Proteins฀B฀and฀D฀are฀expressed฀at฀
lower฀levels฀due฀to฀transcription฀termination฀within฀the฀operon฀and฀are฀required฀to฀secrete฀the฀
14.2.3. Mutations Enhance Aa Ltx and LAP Severity 263

the฀amounts฀of฀proteins฀A฀and฀C.฀Less฀proteins฀B฀and฀D฀are฀required฀because฀they฀induce฀
secretion฀of฀the฀acylated฀leukotoxin฀by฀forming฀a฀complex฀spanning฀the฀inner฀and฀outer฀
cell฀membranes.฀This฀complex฀recruits฀TdeA฀by฀conformational฀changes฀and฀then฀trans-
ports฀the฀acylated฀leukotoxin,฀unfolded฀and฀rapidly,฀to฀the฀outer฀the฀cell฀surface฀where฀it฀
folds฀to฀a฀native฀conformation,฀is฀released฀and฀binds฀calcium฀ions฀(Fig.฀14.1b).
After฀secretion,฀the฀acylated,฀calcium-adherent฀leukotoxin฀attaches฀to฀the฀b2฀integrin฀of฀
LFA-1฀that฀extends฀out฀from฀the฀surface฀of฀activated฀neutrophils.฀The฀leukotoxin฀binding฀
occurs฀ near฀ the฀ N-terminus฀ at฀ the฀ propeller฀ region฀ (Chapter฀ 4,฀ Sect.฀ 4.4.1),฀ possibly฀ to฀
N-linked฀oligosaccharides.฀The฀bound฀leukotoxin฀undergoes฀a฀conformational฀change฀that฀
exposes฀its฀hydrophobic฀surface฀and฀causes฀its฀insertion฀into฀the฀neutrophil฀membrane.฀At฀
low฀doses,฀the฀leukotoxin฀increases฀calcium฀levels฀in฀the฀cytosol,฀activating฀caspases฀and฀
apoptosis฀by฀the฀intrinsic฀pathway฀(Fig.฀13.10).฀At฀high฀concentrations,฀pores฀form฀in฀the฀
cell฀membrane฀and฀the฀cell฀contents฀empty฀into฀the฀stroma฀(necrosis).

14.2.3.
Mutations Enhance Aa Ltx and LAP Severity

In฀the฀operon฀encoding฀the฀leukotoxin฀(ltx฀operon),฀proteins฀B฀and฀D฀are฀encoded฀downstream฀
of฀the฀acylase฀(protein฀C)฀and฀the฀leukotoxin฀(protein฀A)฀genes.฀In฀long฀bacterial฀RNA฀tran-
scripts,฀such฀as฀the฀hemolysin฀(hly)฀operon฀in฀E.฀coli,฀the฀expression฀of฀proteins฀B฀and฀D฀is฀
normally฀attenuated฀when฀compared฀with฀that฀of฀the฀irst฀encoded฀proteins฀C฀and฀A.฀A฀similar฀
attenuation฀occurs฀in฀the฀Aa฀ltx฀operon,฀except฀in฀LAP฀strains,฀which฀possess.฀In฀Aa฀strains฀
isolated฀from฀subjects฀with฀localized฀juvenile฀periodontitis,฀there฀is฀often฀a฀530฀base฀pair฀dele-
tion฀between฀the฀ltx฀promoter฀sequence฀and฀the฀irst฀nucleotide฀of฀the฀RNA฀transcript.฀This฀
deletion฀causes฀the฀promoter฀sequence฀(TATAAT)฀to฀lie฀much฀closer฀to฀the฀translation฀start฀site฀
(ATG฀encoding฀the฀irst฀amino฀acid฀of฀protein฀C).฀The฀530฀base฀pair฀deletion฀includes฀the฀
C-terminal฀part฀of฀an฀open฀reading฀frame,฀orfA,฀a฀ifth฀protein฀expressed฀from฀the฀transcript฀
along฀with฀the฀4฀proteins฀of฀the฀ltx฀operon฀(Fig.฀14.1b,฀up-arrow฀on฀the฀far฀left฀side).฀The฀

Fig. 14.1฀ (continued)฀ leukotoxin.฀Prior฀to฀secretion,฀a฀lysine฀residue฀near฀the฀C-terminal฀end฀of฀

the฀pore-forming฀domain฀(possibly฀lys฀555,฀which฀corresponds฀to฀lys฀565฀in฀the฀homologous฀E.฀
coli฀hemolysin)฀is฀acylated฀with฀an฀unknown฀fatty฀acid.฀A฀second฀acylation฀site฀(lys฀691฀in฀the฀
E.฀coli฀hemolysin)฀is฀absent฀from฀Aa฀leukotoxin.฀Acylation฀precedes฀secretion฀into฀the฀extracel-
lular฀ medium,฀ after฀ which฀ Ca2+฀ ion฀ binding฀ to฀ the฀ leukotoxin฀ repeat฀ sequences฀ activates฀ the฀
toxin.฀TdeA฀is฀an฀outer฀membrane฀protein฀homologous฀to฀TolC฀and฀encoded฀by฀a฀gene฀outside฀the฀
lktCABD฀ operon฀ (see฀ text)฀ (a:฀ From฀ http://pfam.sanger.ac.uk/protein?acc=P16462;฀ b:฀ Adapted฀
from฀ Fig.฀ 1฀ in฀ Stanley฀ P,฀ Koronakis฀ V,฀ and฀ Hughes฀ C฀ (1998)฀ “Acylation฀ of฀ Escherichia฀ coli฀
hemolysin:฀A฀unique฀protein฀lipidation฀mechanism฀underlying฀toxin฀function.”฀Microbiology฀and฀
Molecular฀ Biology฀ Reviews฀ 62(2):309–333.฀ Modiied฀ for฀ Aa฀ as฀ described฀ by฀ Crosby฀ JA฀ and฀
Kachlany฀SC฀(2007)฀“TdeA,฀a฀TolC-like฀protein฀required฀for฀toxin฀and฀drug฀export฀in฀Aggregatibacter฀
(Actinobacillus)฀actinomycetemcomitans.”฀Gene฀388:83–92.฀With฀permission)
264 14 Aggressive Periodontitis

deleted฀portion฀of฀the฀OrfA฀protein฀encodes฀a฀basic฀amino฀acid฀sequence฀that฀likely฀binds฀to฀
DNA.฀In฀the฀many฀Aa฀strains฀without฀this฀deletion,฀the฀OrfA฀protein฀may฀control฀ltx฀operon฀
protein฀expression฀by฀an฀unknown฀mechanism.฀Deletion฀of฀this฀protein฀moves฀the฀ltx฀operon฀
promoter฀region฀closer฀to฀the฀translation฀start฀site฀and฀enhances฀leukotoxin฀expression.
Unlike฀chronic฀periodontitis,฀Aa฀does฀not฀form฀bioilms฀(plaques)฀around฀the฀affected฀
teeth.฀Aa฀strains฀have฀a฀“rough”฀or฀“smooth”฀appearance฀when฀grown฀on฀blood฀agar฀plates.฀
When฀grown฀on฀liquid฀medium,฀the฀“rough”฀strains฀adhere฀tightly฀to฀the฀glass฀surface฀of฀
the฀cultivation฀tube฀whereas฀the฀“smooth”฀strains฀adhere฀only฀slightly.฀The฀lack฀of฀adher-
ence฀is฀due฀to฀mutations฀in฀tad฀genes฀that฀encode฀proteins฀homologous฀to฀the฀type฀II฀and฀
type฀IV฀secretion฀proteins฀required฀for฀pilus฀formation฀(Sect.฀1.4.2).฀These฀nonadherent฀
tad฀mutants฀secrete฀copious฀amounts฀of฀leukotoxin฀in฀vitro,฀whereas฀the฀wild-type฀strains฀
(with฀ “rough”฀ morphology฀ and฀ tight฀ adherence)฀ secrete฀ very฀ little฀ leukotoxin฀ (which฀
remains฀attached฀to฀the฀outer฀cell฀membrane).฀Tad฀gene฀mutations฀and฀the฀530฀base฀pair฀
deletion฀of฀OrfA฀protein฀in฀the฀ltx฀operon฀may฀have฀a฀common฀cause.฀Both฀types฀of฀muta-
tions฀enhance฀leukotoxin฀expression฀and/or฀secretion฀and฀associate฀with฀severe฀LAP.

14.2.4.
Cytolethal Distending Toxin (Cdt)

Cdt฀ is฀ related฀ to฀ a฀ eukaryotic฀ cytosolic฀ enzyme,฀ phosphatidyl฀ inositol-3,4,5,฀ triphosphate฀
5-phosphatase฀which฀removes฀the฀5-phosphate฀group฀from฀phosphatidyl฀inositol-3,4,5,฀triphos-
phate.฀This฀activity฀is฀part฀of฀an฀intracellular฀signaling฀cascade฀induced฀by฀a฀ligand฀binding฀to฀
a฀ nearby฀ receptor.฀ Phosphatidyl฀ inositol-3,4,5-triphosphate฀ 5-phosphatase฀ possesses฀ an฀ Src฀
Homology฀2฀(SH2)฀domain฀in฀addition฀to฀its฀Inositol฀Phosphatase฀activity฀(SHIP).
The฀SH2฀domain฀is฀a฀conserved฀sequence฀in฀a฀viral฀tyrosine฀kinase฀responsible฀for฀trans-
forming฀fibroblasts฀into฀neoplastic฀sarcoma฀cells฀(the฀viral฀src฀gene,฀v-src;฀see฀sect.฀10.2.1.฀
for฀the฀normal฀cell฀counterpart,฀c-src).฀SH1฀is฀a฀tyrosine฀kinase฀domain;฀SH2฀is฀the฀domain฀
that฀binds฀to฀phosphotyrosine฀residues;฀and฀SH3฀is฀a฀v-src฀domain฀that฀binds฀to฀proline-rich฀
sequences฀in฀proteins.฀SH2฀and฀SH3฀domains฀are฀found฀in฀many฀proteins฀involved฀in฀signal฀
transduction.

SHIP฀is฀primarily฀expressed฀in฀bone฀marrow฀hemopoietic฀and฀lymphoid฀precursor฀cells.฀It฀is฀
especially฀important฀in฀lymphocytes฀whose฀cell฀surface฀receptors฀have฀bound฀to฀an฀antigen,฀
and฀ in฀ macrophages฀ that฀ have฀ bound฀ to฀ lipopolysaccharide฀ by฀ TLR-4฀ (Sect.฀ 13.2.1).฀ The฀
phosphatidylinositol-3฀ kinase฀ (PI3฀ kinase)฀ pathway฀ generates฀ PI-3,4,5-triphosphate.฀ As฀ it฀
accumulates,฀ PI-3,4,5-triphosphate฀ activates฀ SHIP฀ in฀ a฀ feedback฀ loop฀ to฀ reduce฀ PI-3,4,5-
triphosphate฀concentration฀on฀the฀inner฀membrane.฀In฀nonleukocytes,฀antigen฀and฀TLR฀recep-
tors฀are฀absent,฀and฀PTEN฀(Phosphatase฀of฀the฀tensin฀family฀of฀phospholipid฀phosphatases)฀is฀
activated฀instead฀of฀SHIP.฀The฀name฀PTEN฀is฀derived฀by฀its฀irst฀being฀found฀deleted฀from฀
chromosome฀10฀in฀certain฀cancer฀cells.฀PTEN฀reduces฀membrane฀PI-3,4,5-triphosphate฀by฀
removing฀the฀3-phosphate฀instead฀of฀the฀5-phosphate฀(Fig.฀14.2).฀PI-3,4,5-triphosphate฀is฀a฀
second฀ messenger฀ molecule฀ that฀ stimulates฀ cell฀ survival,฀ adhesion,฀ migration,฀ metabolic฀
activity,฀proliferation,฀differentiation,฀and฀end฀cell฀activation฀by฀various฀pathways฀(not฀shown฀
in฀Fig.฀14.2)฀and฀its฀amount฀is฀continuously฀adjusted฀in฀response฀to฀the฀environment.
14.2.4. Cytolethal Distending Toxin (Cdt) 265

Fatty acid
ester Pl-4,5-P2 Pl-3,4,5-P3 Pl-3,4-P2

Inner bilayer
of membrane

6
P
5
Pl-3 Kinase 6
P
5 6
OH
5 Cytosol
P P P
C-3 atom 1
OH
OH OH 4 1
OH
OH P 4 1
OH
OH P
4

of glycerol 2 3 P
Pl-3,4,5-P3 2 3 P 2 3 P

3-Phosphatase Pl-3,4,5-P3
(PTEN) 5-Phosphatase
(SHIP)

Fig. 14.2฀ Mode฀of฀action฀of฀cytolethal฀distending฀toxin.฀Phosphatidyl-inositol฀is฀attached฀to฀the฀

inner฀layer฀of฀a฀cell฀membrane฀by฀its฀fatty฀acids.฀Inositol฀is฀a฀six-membered฀carbon฀ring฀with฀an฀
OH฀group฀on฀each฀carbon฀atom.฀The฀atoms฀of฀the฀inositol฀ring฀are฀numbered฀counter-clockwise฀
from฀the฀C1฀atom,฀which฀is฀attached฀to฀a฀membrane฀diacylglycerol฀phosphate฀by฀the฀C3฀atom฀of฀
the฀latter’s฀glycerol฀moiety.฀The฀C2฀and฀C1฀atoms฀are฀attached฀to฀fatty฀acids.฀Inositol฀phosphate฀is฀
phosphorylated฀by฀membrane฀kinases฀at฀the฀-4฀and฀-5฀positions฀and฀then฀the฀-3฀position฀to฀make฀
phosphatidyl฀inositol-3,4,5-trisphosphate฀(PI-3,4,5-P3),฀which฀activates฀growth฀and฀various฀bio-
logical฀processes.฀In฀most฀cells,฀production฀of฀PI-3,4,5-P3฀is฀kept฀low฀by฀the฀enzyme฀PTEN฀(see฀
text),฀but฀in฀macrophages฀SHIP฀is฀activated฀to฀remove฀the฀5-phosphate฀(see฀text).฀Cytolethal฀dis-
tending฀toxin฀(Cdt)฀is฀a฀homologue฀of฀SHIP฀and฀therefore฀competes฀for฀PI-3,4,5-P3฀only฀in฀acti-
vated฀leukocytes;฀other฀cells฀are฀less฀sensitive฀(see฀text)฀(Modiied฀from฀Fig.฀1฀(p.฀1171)฀in฀Sly฀LM,฀
Rauh฀MJ,฀Kalesnikoff฀J,฀et฀al.฀(2003)฀“SHIP,฀SHIP2,฀and฀PTEN฀activities฀are฀regulated฀in฀vivo฀by฀
modulation฀of฀their฀protein฀levels:฀SHIP฀is฀up-regulated฀in฀macrophages฀and฀mast฀cells฀by฀lipopoly-
saccharide.”฀Experimental฀Hematology฀31:1170–1181.฀With฀permission)฀Edited฀by฀Foxit฀Ready฀
Copyright฀(c)฀by฀foxit฀software฀company,฀2005–2008.฀for฀Evaluation฀only.

Cdt฀is฀composed฀of฀three฀proteins,฀all฀encoded฀by฀the฀cdt฀operon.฀Two฀of฀these฀proteins฀
bind฀to฀human฀cell฀membranes฀where฀they฀facilitate฀the฀translocation฀of฀the฀third฀subunit,฀a฀
SHIP-like฀ phosphatase฀ PI-3,4,5-triphosphate฀ 5-phosphatase.฀ This฀ bacterial฀ phosphatase฀
passes฀ through฀ the฀ membrane฀ and฀ removes฀ the฀ 5-phosphate฀ from฀ PI-3,4,5-triphosphate,฀
causing฀stimulated฀lymphocytes฀and฀macrophages฀to฀undergo฀apoptosis฀instead฀of฀growth.฀
In฀ nonleukocytic฀ cells฀ such฀ as฀ epidermal฀ cells฀ or฀ ibroblasts,฀ PI-3,4,5-phosphatase฀ is฀
exposed฀to฀PTEN,฀which฀is฀10฀times฀more฀active฀on฀PI-3,4,5-triphosphate฀than฀Cdt.฀The฀
SHIP-like฀bacterial฀enzyme฀therefore฀cannot฀compete฀with฀PTEN.฀The฀sensitivity฀of฀non-
leukocytic฀cells฀to฀Cdt฀is฀much฀less฀than฀that฀of฀stimulated฀lymphocytes฀and฀macrophages.฀

Localized฀ aggressive฀ periodontitis฀ is฀ self-limiting฀ but฀ it฀ predisposes฀ to฀ generalized฀
chronic฀periodontitis฀if฀untreated.฀It฀appears฀around฀the฀gingival฀sulci฀of฀the฀irst฀erupt-
ing฀permanent฀teeth฀due฀to฀infection฀with฀Aggregatibacter฀(formerly฀Actinobacillus)฀
actinomycetemcomitans฀(Aa).฀This฀bacterium฀expresses฀two฀soluble฀protein฀toxins,฀a฀
leukotoxin฀(Ltx)฀and฀a฀cytolethal฀distending฀toxin฀(Cdt).฀Both฀affect฀leukocytes,฀but฀
induce฀systemic฀antibodies฀that฀may฀prevent฀the฀periodontitis฀from฀spreading฀to฀later฀
266 14 Aggressive Periodontitis

erupting฀teeth.฀Aa฀leukotoxin฀belongs฀to฀the฀RTX฀family฀of฀proteins,฀found฀in฀other฀
Gram-negative฀bacteria.฀Ltx฀is฀encoded฀along฀with฀other฀proteins฀by฀the฀ltx฀operon:฀
these฀are฀OrfA,฀a฀putative฀protein฀that฀controls฀operon฀expression;฀an฀acyl฀transferase฀
that฀ adds฀ a฀ fatty฀ acyl฀ group฀ essential฀ for฀ leukotoxin฀ activity;฀ and฀ two฀ proteins฀ that฀
facilitate฀secretion.฀Aa฀leukotoxin฀is฀activated฀by฀its฀repeat฀regions฀binding฀Ca2+฀ions฀in฀
the฀extracellular฀medium.฀It฀binds฀to฀the฀LFA-1฀integrin฀of฀neutrophils฀and฀forms฀pores฀
in฀the฀neutrophil฀cell฀membrane.฀High฀expression฀is฀associated฀with฀deletion฀mutations฀
of฀OrfA,฀or฀point฀mutations฀of฀tad฀genes฀responsible฀for฀adherence฀and฀bioilm฀forma-
tion.฀LAP฀is฀associated฀with฀an฀absence฀of฀tooth฀adherent฀bioilms฀and฀high฀Ltx฀expres-
sion.฀Cytolethal฀toxin฀(Cdt)฀is฀encoded฀by฀Aa฀along฀with฀two฀other฀proteins฀in฀the฀Cdt฀
operon.฀In฀humans,฀two฀of฀the฀encoded฀proteins฀bind฀to฀cell฀membranes฀and฀translocate฀
the฀third,฀a฀phosphatase฀that฀inactivates฀a฀growth฀promoter,฀phosphatidyl฀inositol-3,4,5฀
triphosphate฀(PI-3,4,5-P3)฀by฀removing฀its฀5-phosphate฀group.฀Lymphocytes฀and฀mac-
rophages฀are฀unable฀to฀grow฀and฀ultimately฀apoptose.฀Nonleukocytic฀cells฀possess฀a฀
3-phosphatase,฀which฀competes฀for฀this฀substrate฀better฀than฀Cdt,฀making฀nonleuko-
cytes฀resistant.฀
 Dental Caries
15

This฀ chapter฀ describes฀ dental฀ caries฀ (tooth฀ decay)฀ and฀ its฀ causes.฀ Sucrose฀ and฀ other฀
mono-฀and฀disaccharides฀are฀metabolized฀to฀acid฀(lactate)฀by฀bacteria฀that฀remain฀in฀
“stagnation”฀areas฀of฀the฀teeth.฀Rats฀and฀hamsters฀fed฀a฀50%฀sucrose฀diet฀developed฀a฀
caries-sensitive,฀predominantly฀gram-positive฀microbiota฀that฀became฀caries฀resistant฀
when฀the฀rodents฀were฀fed฀penicillin฀(Sect.฀1).฀Further฀studies฀identiied฀Streptococcus฀
mutans฀ (S.฀ mutans)฀ as฀ the฀ etiological฀ agent.฀ This฀ organism฀ synthesizes฀ an฀ insoluble฀
polysaccharide฀capsule฀that฀is฀stable฀and฀retains฀lactate฀at฀the฀enamel฀surface฀(Sect.฀2).฀
The฀key฀enzyme,฀glucosyl฀transferase,฀is฀related฀to฀salivary฀amylase฀which฀adheres฀to฀
oral฀ bacteria฀ and฀ enhances฀ bacterial฀ acid฀ production.฀ The฀ chapter฀ concludes฀ with฀ a฀
discussion฀of฀salivary฀and฀other฀factors฀responsible฀for฀the฀marked฀variation฀observed฀
in฀individual฀caries฀experience฀(Sect.฀3).

15.1.1.
Dental Caries: Definition and Measurement

Dental฀caries,฀cavities฀or฀tooth฀decay,฀provides฀dentists฀with฀an฀ongoing฀demand฀for฀preventive฀
and฀reconstructive฀services.฀Caries฀is฀of฀Greek฀origin;฀it฀means฀destruction฀or฀decay.฀Dental฀
caries฀refers฀to฀the฀dissolution฀of฀tooth฀enamel฀and฀dentin.฀It฀starts฀in฀the฀pits,฀issures,฀and฀
interdental฀regions฀of฀the฀teeth,฀“stagnation฀areas”฀from฀which฀bacteria฀are฀dificult฀to฀remove฀
(Fig.฀15.1).฀The฀extent฀of฀caries฀is฀measured฀as฀the฀number฀of฀teeth฀diagnosed฀as฀decayed,฀
missing,฀or฀illed฀due฀to฀caries,฀the฀Decayed,฀Missing฀and฀Filled฀Teeth฀(DMFT)฀index.

15.1.2.
Sugar, Dental Caries, and the Dental Profession

Because฀teeth฀are฀preserved฀long฀after฀death,฀it฀was฀evident฀that฀European฀populations฀suf-
fered฀little฀from฀caries฀before฀the฀late฀eighteenth฀century.฀The฀rise฀of฀caries฀coincided฀with฀
increased฀ sucrose฀ consumption;฀ perhaps,฀ the฀ revenge฀ of฀ black฀ slaves฀ on฀ their฀ masters.฀
Before฀the฀civil฀war฀ended฀in฀1863,฀slaves฀from฀Africa฀were฀forced฀to฀cut฀cane฀sugar฀in฀the฀

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 267
DOI:฀10.1007/978-3-540-88116-2_15,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
268 15 Dental Caries

Fig. 15.1฀ Severe,฀untreated฀

dental฀caries.฀Dental฀caries฀
initially฀affects฀the฀occlusal฀
pits฀and฀issures฀of฀posterior฀
teeth.฀In฀severe฀caries,฀the฀
smooth฀surfaces,฀especially฀
the฀interdental฀surfaces฀of฀
all฀teeth฀may฀be฀involved

West฀ Indies฀ and฀ far฀ Southern฀ US.฀ From฀ the฀ late฀ eighteenth฀ century฀ and฀ throughout฀ the฀
nineteenth฀century,฀increasing฀amounts฀of฀the฀raw฀cane฀were฀exported฀to฀Atlantic-facing฀
ports฀of฀England฀and฀Scotland.฀There,฀in฀sugar฀reineries,฀it฀was฀crushed,฀cleaned,฀and฀the฀
luid฀ crystallized฀ to฀ give฀ pure฀ sucrose,฀ reined฀ sugar.฀ During฀ this฀ period,฀ new฀ methods฀
increased฀the฀yields฀of฀corn,฀wheat,฀and฀other฀grains฀and฀their฀starch฀was฀puriied฀(reined)฀
to฀add฀to฀various฀foods.฀Pure฀starch฀and฀sucrose฀are฀called฀reined฀carbohydrate.฀Populations฀
in฀Europe,฀Canada,฀and฀the฀Northern฀US฀became฀addicted฀to฀reined฀carbohydrate฀because฀
of฀the฀cold฀climate.฀The฀dramatic฀increase฀in฀caries฀throughout฀the฀nineteenth฀and฀early฀
twentieth฀centuries฀led฀to฀the฀development฀of฀local฀and฀general฀anesthetics฀and฀of฀the฀dental฀
profession฀to฀treat฀the฀associated฀infections,฀pain,฀and฀discomfort.
In฀1945,฀the฀Vipeholm฀study฀was฀set฀up฀to฀determine฀whether฀increasing฀sucrose฀intake฀
actually฀ increased฀ human฀ caries฀ experience.฀ The฀ marked฀ variation฀ in฀ caries฀ severity฀
between฀individuals฀(Sect.฀15.2.1)฀had฀led฀to฀doubts฀that฀increased฀sucrose฀consumption฀
was฀really฀responsible.฀The฀study฀was฀conducted฀at฀the฀Vipeholm฀Hospital฀for฀individuals฀
with฀mental฀handicaps฀outside฀the฀University฀City฀of฀Lund,฀Sweden.฀The฀study฀examined฀
the฀ effects฀ of฀ different฀ diets฀ on฀ dental฀ caries฀ in฀ the฀ inmates฀ and฀ it฀ ended฀ in฀ 1951.฀ The฀
results฀indicated฀that฀sticky฀sugar฀candies฀(toffees)฀between฀meals฀and฀popular฀with฀the฀
inmates฀increased฀their฀DMFT฀by฀an฀average฀of฀one฀cavity฀per฀year.฀The฀use฀of฀mentally฀
handicapped฀subjects฀was฀criticized฀in฀the฀Swedish฀press฀and฀all฀studies฀on฀mentally฀hand-
icapped฀individuals฀were฀stopped฀in฀1954.
Another฀series฀of฀studies฀was฀conducted฀on฀the฀inmates฀of฀Hopewood฀House,฀Bowral,฀
New฀South฀Wales,฀Australia.฀This฀home฀for฀children฀of฀unmarried฀mothers฀opened฀in฀1942฀
and฀closed฀in฀1965.฀The฀children฀were฀made฀to฀eat฀a฀diet฀of฀locally฀grown฀raw฀vegetables฀
with฀a฀minimal฀amount฀of฀protein฀in฀the฀form฀of฀milk฀and฀raw฀soybeans.฀The฀almost฀com-
plete฀absence฀of฀dental฀caries฀compared฀with฀other฀Australian฀children฀was฀noted฀in฀studies฀
conducted฀between฀1955฀and฀1960.฀Unfortunately,฀as฀soon฀as฀they฀entered฀the฀general฀popu-
lation฀as฀adults,฀all฀former฀inmates฀developed฀caries฀rapidly.฀Moreover,฀a฀harrowing฀descrip-
tion฀of฀what฀this฀diet฀was฀like,฀and฀of฀many฀other฀violations฀by฀the฀owners฀and฀employees฀of฀
this฀ orphanage,฀ was฀ given฀ to฀ the฀ Australian฀ Senate฀ by฀ Sandra฀ Pendergast฀ in฀ 2003.฀ In฀
November฀2009,฀the฀Australian฀government฀issued฀a฀formal฀apology฀to฀those฀who฀had฀grown฀
up฀suffering฀abuses฀that฀were฀common฀to฀this฀and฀many฀other฀orphanages฀in฀Australia.
15.1.3. Sucrose and the Appearance of Acid in Dental Biofilms 269

12

DMFT of 12-year children

10

0
0 25 50 75 100 125 150
Sugar (g/person/day)

Fig. 15.2฀ Relationship฀ between฀ dietary฀ sucrose฀ intake฀ and฀ dental฀ caries฀ severity.฀ The฀ number฀ of฀
teeth฀ that฀ were฀ decayed,฀ missing,฀ and฀ illed฀ due฀ to฀ caries฀ (decayed,฀ missing,฀ and฀ illed฀ teeth฀
[DMFT])฀is฀graphed฀against฀mean฀sugar฀consumption฀in฀12-year฀old฀children.฀Each฀point฀on฀the฀
graph฀represents฀a฀different฀country.฀The฀indings฀were฀available฀from฀World฀Health฀Organization฀
activities฀ in฀ oral฀ epidemiology฀ (The฀ graph฀ was฀ assembled฀ by฀ the฀ author฀ from฀ Table฀ 3฀ in฀฀
L.฀ M.฀ Sreebny฀ (1982)฀ “Sugar฀ availability,฀ sugar฀ consumption฀ and฀ dental฀ caries.”฀ Community฀
Dentistry฀and฀Oral฀Epidemiology฀10:1–7)

Today,฀cane฀sugar฀is฀mostly฀grown฀and฀exported฀throughout฀the฀world฀from฀the฀eastern฀
half฀of฀Africa฀south฀of฀the฀Equator,฀and฀from฀India฀and฀Indonesia.฀The฀most฀compelling฀
evidence฀ for฀ sucrose฀ causing฀ caries฀ was฀ obtained฀ by฀ comparing฀ the฀ sucrose฀ intake฀ in฀
12-year฀old฀children฀from฀47฀countries฀(obtained฀from฀the฀World฀Health฀Organization’s฀
Global฀ Oral฀ Epidemiology฀ Bank฀ during฀ the฀ 1970s)฀ with฀ the฀ DMFT฀ during฀ this฀ period฀
(Fig.฀15.2).฀The฀number฀of฀affected฀teeth฀increased฀by฀about฀one฀DMFT฀for฀every฀25฀g฀of฀
sugar฀consumed฀daily.฀About฀50%฀of฀the฀variance฀in฀DMFT฀between฀populations฀is฀due฀to฀
the฀daily฀sucrose฀intake.

15.1.3.
Sucrose and the Appearance of Acid in Dental Biofilms

Bones฀and฀teeth฀dissolve฀in฀acid.฀The฀insoluble฀calcium฀monophosphate฀salt,฀from฀which฀
hydroxyapatite฀is฀made,฀is฀converted฀to฀the฀more฀soluble฀calcium฀dihydrogen฀phosphate฀
salt฀in฀an฀environment฀whose฀pH฀is฀less฀than฀6.2฀(Sect.฀9.1.1).฀The฀severity฀of฀caries฀was฀
related฀to฀the฀pH฀produced฀in฀dental฀bioilms฀(plaques)฀after฀ingesting฀sucrose฀and฀other฀
sugars฀ by฀ Richard฀ M฀ Stephan.฀ The฀ pH฀ response฀ he฀ identiied฀ is฀ referred฀ to฀ as฀ Stephan฀
Curve.฀He฀found฀that฀the฀starting฀pH,฀the฀extent฀of฀its฀drop,฀and฀the฀time฀for฀recovery฀to฀the฀
starting฀pH฀were฀all฀related฀to฀caries฀severity.฀The฀pH฀drop฀was฀later฀associated฀with฀lactic฀
acid฀ production฀ due฀ to฀ bacterial฀ carbohydrate฀ fermentation฀ (saccharolytic฀ fermentation,฀
Sect.฀1.3.2).฀The฀subsequent฀rise฀in฀pH฀was฀due฀to฀the฀production฀of฀ammonia฀by฀bacterial฀
270 15 Dental Caries

7.0

6.5

Plaque pH
6.0

5.5

5.0
0 10 20 30 40
Time (min)

Fig. 15.3฀ Bioilm฀(plaque)฀pH฀in฀caries-resistant฀and฀caries-susceptible฀subjects.฀The฀pH฀decreased฀

and฀returned฀to฀initial฀values฀more฀rapidly฀following฀a฀10%฀sucrose฀rinse฀in฀caries-resistant฀subjects฀
(○)฀than฀in฀caries-susceptible฀subjects฀(●).฀Each฀group฀had฀ive฀subjects฀(Figure฀2฀in฀Mandel฀and฀
Zengo฀(1973)฀“Genetic฀and฀chemical฀aspects฀in฀caries฀resistance.”฀Comparative฀immunology฀of฀the฀
oral฀cavity,฀ed.฀H.฀Scherp฀and฀S.฀Mergenhagen,฀pp.฀118฀–฀137,฀US฀Government฀Printing฀Ofice)

asaccharolytic฀fermentation฀after฀the฀sucrose฀was฀depleted฀(see฀Sect.฀15.3.1).฀The฀curve฀
was฀originally฀shown฀to฀occur฀after฀a฀10%฀glucose฀mouth฀rinse,฀but฀a฀sucrose฀mouth฀rinse฀
produces฀a฀similar฀result฀(Fig.฀15.3).

15.1.4.
Cavities in Animals and Streptococcus mutans

In฀rodents,฀caries฀induction฀was฀unpredictable฀until฀a฀high-carbohydrate฀low-fat฀diet฀(~60%฀
sucrose฀plus฀soy฀and฀milk฀proteins฀plus฀vitamins฀and฀minerals)฀was฀developed.฀Germ-free฀
rodents฀fed฀this฀diet฀did฀not฀develop฀caries฀and฀normal฀rodents฀(rats,฀hamsters,฀and฀mice)฀
also฀did฀not฀develop฀caries฀when฀their฀diet฀was฀supplemented฀with฀penicillin฀(0.01–0.05%฀
in฀the฀drinking฀water).฀Penicillin฀inhibits฀the฀growth฀of฀gram-positive฀bacteria฀by฀interfer-
ing฀with฀cell฀wall฀formation฀(Sect.฀1.4.1).฀Young฀mice฀fed฀the฀diet฀without฀penicillin฀devel-
oped฀ rampant฀ caries฀ within฀ 35฀ days,฀ but฀ no฀ caries฀ in฀ the฀ presence฀ of฀ penicillin.฀ If฀ the฀
penicillin฀was฀removed฀from฀the฀drinking฀water฀after฀one฀generation,฀the฀mice฀produced฀
several฀generations฀of฀progeny฀that฀developed฀little฀caries฀despite฀the฀diet,฀suggesting฀that฀
the฀absence฀of฀gram-positive฀bacteria฀resulted฀in฀a฀stable฀microbiota฀associated฀with฀caries฀
resistance.฀These฀experiments฀are฀illustrated฀in฀Fig.฀15.4.
In฀1960,฀Robert฀Fitzgerald฀and฀Paul฀Keyes฀demonstrated฀the฀types฀of฀bacteria฀respon-
sible฀for฀caries.฀They฀isolated฀streptococci฀from฀a฀carious฀lesion฀and฀made฀several฀of฀the฀
isolates฀resistant฀to฀a฀non-penicillin฀antibiotic฀(streptomycin)฀which฀provided฀them฀with฀a฀
means฀of฀isolating฀and฀identifying฀these฀bacteria฀subsequently.฀The฀streptomycin-resistant฀
strains฀were฀used฀to฀infect฀young฀hamsters฀that฀were฀resistant฀to฀caries฀despite฀the฀diet.฀The฀
infected฀ hamsters฀ developed฀ caries฀ and฀ the฀ streptomycin-resistant฀ bacteria฀ appeared฀ in฀
15.1.4. Cavities in Animals and Streptococcus mutans 271

Infected females

Antibiotic Transmission of
a (Penicillin) cariogenic flora
depresses flora to offspring

Same cage

b
Noninfected Both carious Carious

Cariogenic
Caries
Streptococci
c
Non “Labelled” strains “Labelled’
carious Streptococci caries

Plaque with
d Non "Labelled" “Labelled”
carious Streptococci caries Carious

Same cage

e Non “Labelled” “Labelled”

carious caries caries Carious

Fig. 15.4฀ Identiication฀ of฀ a฀ cariogenic฀ microbiota฀ –฀ the฀ Fitzgerald–Keyes฀ experiments.฀฀

(a)฀ Penicillin฀ inhibits฀ bacterial฀ colonization.฀ When฀ penicillin฀ was฀ added฀ to฀ the฀ drinking฀ water,฀
gram-positive฀bacteria฀were฀depleted฀(depressed)฀and฀caries฀did฀not฀develop.฀(b)฀Caries฀is฀trans-
missible.฀After฀a฀generation,฀penicillin฀was฀no฀longer฀provided,฀but฀the฀offspring฀remained฀caries฀
resistant฀to฀the฀sucrose฀diet฀for฀many฀generations฀(left฀side฀of฀igure).฀The฀caries-resistant฀rodents฀
developed฀caries฀only฀if฀they฀were฀placed฀in฀the฀same฀cage฀as฀the฀animals฀that฀did฀develop฀cavities฀
(not฀receiving฀penicillin).฀(c)฀Adding฀streptococci฀to฀the฀caries-resistant฀microbiota฀causes฀caries.฀
Strains฀of฀what฀were฀later฀called฀mutans฀streptococci฀were฀isolated.฀Some฀were฀labeled฀by฀being฀
made฀streptomycin฀resistant.฀Oral฀inoculation฀of฀the฀unlabeled฀or฀ streptomycin-resistant฀ strains฀
into฀ caries-resistant฀ animals฀ caused฀ them฀ to฀ develop฀ caries.฀ (d)฀ Adding฀ streptomycin-resistant฀
“labeled”฀strains฀to฀bioilm฀(plaque)฀from฀caries-resistant฀males฀or฀females฀cause฀caries.฀(e)฀These฀
streptococci฀cause฀caries฀even฀if฀obtained฀from฀feces.฀The฀streptomycin-resistant฀streptococci฀in฀
feces฀smeared฀on฀the฀body฀of฀a฀caries-resistant฀rodent฀caused฀the฀animal฀to฀develop฀caries฀(Figure฀3฀
in฀Fitzgerald฀RJ฀and฀Keyes฀PH฀(1965฀Oct)฀“Dental฀caries฀as฀a฀major฀disease฀problem.”฀Medical฀
Annals฀of฀the฀District฀of฀Columbia฀34(10):463–467)

their฀oral฀cavity฀and฀feces.฀These฀bacteria฀remained฀absent฀from฀separately฀caged฀caries-
resistant฀animals฀fed฀the฀same฀diet฀in฀the฀same฀room.฀Thus,฀the฀caries-susceptible฀micro-
biota฀ is฀ composed฀ of฀ streptococci฀ and฀ is฀ transmitted฀ by฀ direct฀ contact,฀ not฀ aerosols฀
(Fig.฀ 15.4).฀ The฀ streptococci฀ were฀ later฀ identiied฀ as฀ Streptococcus฀ mutans.฀ Although฀ a฀
caries-resistant฀ microbiota฀ is฀ stable฀ in฀ the฀ presence฀ of฀ a฀ sucrose฀ diet,฀ the฀ procedures฀
required฀to฀develop฀and฀maintain฀it฀are฀impracticable฀for฀human฀populations.฀Removing฀
sucrose฀from฀the฀diet฀or฀adding฀luoride฀to฀the฀diet฀markedly฀decreased฀caries฀development฀
in฀rodents฀possessing฀the฀cariogenic฀microbiota฀(lora).
272 15 Dental Caries

Dental฀caries฀(cavities฀or฀tooth฀decay)฀led฀to฀the฀development฀of฀the฀dental฀profession฀
by฀the฀late฀nineteenth฀century.฀It฀is฀caused฀by฀reined฀carbohydrates,฀pure฀sucrose,฀and฀
starch฀in฀the฀diet.฀Sucrose฀between฀meals,฀especially฀sticky฀candies,฀increased฀cavities฀
in฀most฀adults,฀and฀raising฀children฀on฀a฀diet฀without฀reined฀carbohydrate฀prevented฀
caries฀development.฀A฀World฀Health฀Organization฀study฀showed฀that฀the฀average฀num-
ber฀of฀carious฀teeth฀in฀populations฀of฀12-year฀old฀children฀from฀47฀countries฀increased฀
by฀one฀for฀every฀25฀g฀of฀sugar฀consumed฀daily.฀Experiments฀in฀rodents฀conirmed฀that฀
a฀high฀sucrose฀diet฀caused฀caries฀and฀that฀the฀responsible฀microbiota฀was฀sensitive฀to฀
penicillin.฀Administering฀penicillin฀to฀the฀drinking฀water฀protected฀the฀rodents฀from฀
developing฀ caries฀ despite฀ the฀ diet.฀ Caries฀ resistance฀ was฀ transmitted฀ after฀ removing฀
penicillin฀by฀keeping฀the฀offspring฀isolated฀from฀caries-susceptible฀rodents฀despite฀the฀
diet.฀The฀caries-resistant฀rodents฀developed฀caries฀if฀they฀came฀into฀direct฀contact฀with฀
penicillin-sensitive฀streptococci฀subsequently฀identiied฀as฀Streptococcus฀mutans฀from฀
caries-susceptible฀rodents.

15.2.1.
How Sucrose Connects S. mutans to the Oral Microbiota and Dental Caries

By฀the฀late฀1960s,฀the฀rodent฀studies฀had฀led฀to฀dental฀caries฀being฀generally฀accepted฀as฀
caused฀ by฀ reined฀ carbohydrate฀ promoting฀ a฀ saccharolytic฀ gram-positive฀ microbiota฀ that฀
adhered฀ to฀ teeth฀ surfaces.฀ Mutans฀ streptococci฀ were฀ considered฀ key฀ components฀ of฀ this฀
microbiota.฀The฀name฀“mutans”฀is฀derived฀from฀the฀Latin฀word฀for฀change,฀“mutatio.”฀On฀
agar,฀Streptococcus฀mutans฀grows฀as฀small฀round฀colonies฀in฀the฀presence฀of฀glucose,฀but฀as฀
large,฀ sticky,฀ gelatinous฀ colonies฀ in฀ the฀ presence฀ of฀ sucrose.฀ The฀ gelatinous฀ colonies฀ are฀
caused฀by฀a฀capsule฀around฀the฀bacteria:฀mutan,฀an฀a1→3฀glucose฀polymer฀(Fig.฀15.5),฀and฀
dextran,฀an฀a1→6฀glucose฀polymer.฀Mutan฀and฀dextran฀are฀glucans฀(made฀from฀glucose)฀like฀
starch,฀glycogen,฀and฀cellulose.฀Functionally,฀the฀most฀important฀portion฀of฀the฀S.฀mutans฀
capsule฀is฀its฀mutan.฀The฀aqueous฀solubility฀of฀a฀glucan฀depends฀on฀its฀major฀bonds;฀cellulose฀
(b1→4)฀and฀mutan฀(a1→3)฀are฀mostly฀insoluble,฀whereas฀dextran฀(a1→6),฀glycogen,฀and฀
starch฀(a1→4)฀are฀mostly฀soluble.
15.2.1. How Sucrose Connects S. mutans to the Oral Microbiota and Dental Caries 273

6
HO-CH2
O OH
1
a1 6 branch 6 Redusing
6 HO-CH2 end
Mutan HO-CH2 3 2
O O
O 1
1
6 6
HO-CH2 O CH2 3
3 2
one O O O O
kb 1 1
ac
3b 6 6
HO-CH2
HO-CH2 3 3
a1 O O
O O 1
1
6 6
HO-CH2 HO-CH2 3 2
3
O O O
O 1
1
6
HO-CH2 3
3 e
HO O O b on
1 ack
6 3b
HO-CH2 3 a1
O O
1

3
HO

Fig. 15.5฀ Structure฀of฀mutan.฀Diagram฀shows฀the฀glucose฀pyranose฀ring฀with฀extensions฀indicating฀

the฀ OH฀ group฀ positions.฀ The฀ carbon฀ atoms฀ are฀ numbered.฀ The฀ residues฀ are฀ linked฀ together฀ by฀
a1→3฀bonds฀with฀a1→6฀branching฀(shown)฀and฀occasional฀a1→2฀branching฀(not฀shown).฀The฀
reducing฀end฀is฀at฀the฀top฀right.฀Mutan฀is฀the฀product฀of฀GTF-I

In฀ sucrose,฀ the฀ glucose฀ and฀ fructose฀ moieties฀ are฀ connected฀ by฀ their฀ respective฀ ano-
meric฀carbon฀atoms฀(Fig.฀15.6)฀The฀alpha฀anomer฀predominates฀in฀glucose,฀whereas฀the฀
beta฀anomer฀predominates฀in฀fructose฀(indicated฀by฀the฀larger฀size฀of฀the฀predominant฀form฀
in฀Fig.฀15.6฀c฀compared฀with฀b).฀The฀hydrolysis฀of฀sucrose฀releases฀two฀anomeric฀carbon฀
atoms฀that฀provide฀the฀energy฀for฀bacterial฀capsule฀formation฀at฀the฀stagnation฀areas฀where฀
caries฀ develops.฀ Glucan฀ capsules฀ (i.e.฀ mutan฀ and฀ dextran)฀ are฀ made฀ from฀ the฀ glucosyl฀
moiety฀ of฀ sucrose.฀ Some฀ oral฀ bacteria฀ produce฀ instead฀ a฀ capsule฀ called฀ levan฀ from฀ the฀
fructose฀moiety฀of฀sucrose฀(Fig.฀15.7).฀The฀enzymes฀that฀synthesize฀these฀polysaccharides,฀
glucosyl-฀and฀fructosyl-transferases,฀are฀not฀exclusive฀to฀oral฀bacteria.
274 15 Dental Caries

Fig. 15.6฀ Structure฀of฀sucrose.฀Sucrose฀is฀a฀disaccharide฀composed฀of฀glucose฀and฀fructose.฀(a)฀Ring฀

structures.฀In฀glucose,฀the฀ring฀is฀formed฀from฀an฀aldehyde฀at฀carbon฀1฀of฀the฀molecule฀(top).฀The฀C1฀
carbon฀is฀the฀reducing฀end฀and฀it฀possesses฀the฀anomeric฀–OH฀group,฀a฀–฀pointing฀down฀or฀b฀–฀
pointing฀up฀in฀the฀D฀series฀sugars฀(see฀legend฀to฀Fig.฀6.7).฀In฀fructose,฀the฀ring฀is฀formed฀from฀a฀
ketone฀at฀C2฀of฀the฀molecule฀(bottom).฀The฀reducing฀carbon฀is฀C2฀and฀the฀anomeric฀–OH฀group฀is฀
attached฀to฀C2.฀(b)฀Anomers.฀The฀usual฀glucose฀anomer฀is฀the฀a-form฀and฀the฀usual฀fructose฀ano-
mer฀is฀the฀b-form.฀These฀dominant฀anomers฀are฀drawn฀larger฀than฀the฀respective฀uncommon฀ano-
mers.฀(c)฀Anomeric฀forms฀incorporated฀into฀sucrose.฀The฀usual฀anomeric฀forms฀of฀glucose฀and฀
fructose฀create฀the฀sucrose฀glycoside฀(anomeric฀–OH฀groups฀in฀red).฀(d)฀Structure฀of฀sucrose.฀The฀
two฀anomeric฀–OH฀groups฀form฀the฀glycoside฀bond฀of฀sucrose฀(O-glycoside฀atom฀colored฀red).฀
The฀structure฀is฀drawn฀to฀make฀the฀fructose฀glycoside฀comparable฀with฀maltose฀(see฀Fig.฀12.10).฀
The฀ sucrose฀ diagram฀ in฀ Fig.฀ 2.9฀ has฀ the฀ fructose฀ rotated,฀ causing฀ its฀ glycoside฀ bond฀ to฀ appear฀
a-anomeric,฀whereas฀it฀is฀in฀fact฀a฀b-anomer฀as฀in฀this฀diagram

6 β2 6
6
HOH2C O CH2 O CH2 O CH2 O
O O O O
2
5
4 CH OH CH2OH CH2 CH2OH
3 1 2 1
1
O
HOH2C O CH2 O CH2
O O O β2
2

CH2OH CH2OH CH2OH

Fig. 15.7฀ Levan.฀Levan฀is฀a฀fructan฀(from฀fructose).฀It฀consists฀of฀a฀b2→6฀backbone฀and฀a฀b2→1฀

crosslinks.฀Carbon฀atoms฀are฀numbered฀(top฀left)฀and฀vertical฀lines฀up฀or฀down฀from฀the฀ring฀indi-
cate฀the฀OH฀group฀position.฀The฀enzyme,฀fructosyl฀transferase,฀is฀present฀in฀various฀oral฀bacteria฀
but฀not฀S.฀mutans฀(Extensively฀modiied฀from฀Fig.฀XI฀in฀US฀patent฀7,528,100฀B2฀http://www.bing.
com/images/search?q=Levan+polysaccharide&go=&form=QBIR&qs=n&sk=#focal=92a464cd0
0c520daee834d27a0b2c34c&furl=http%3A%2F%2Fwww.uspto.gov%2Fweb%2Fpatents%2Fpat
og%2Fweek18%2FOG%2Fhtml%2F1342–1%2FUS07528100–20090505–C00021.gif)
15.2.1. How Sucrose Connects S. mutans to the Oral Microbiota and Dental Caries 275

Fig. 15.8฀ Glucosyl฀ transferase฀ structure.฀ Virtually,฀ all฀ carbohydrate฀ synthesizing฀ and฀ degrading฀
enzymes฀ are฀ structurally฀ related฀ (in฀ families)฀ because฀ polysaccharides฀ (glycans)฀ are฀ much฀ less฀
diverse฀than฀polypeptides.฀Glucosyl฀transferases฀(family฀70)฀are฀related฀to฀the฀amylases฀(family฀13)฀
and฀the฀catalytic฀structure฀is฀a฀putative฀barrel฀composed฀of฀eight฀a-helices฀and฀eight฀b-strands฀like฀
a-amylase฀(depicted฀in฀Fig฀12.12a)฀and฀containing฀a฀similar฀catalytic฀site.฀This฀site฀allows฀glucosyl฀
residues฀to฀become฀transiently฀attached฀to฀an฀aspartate฀residue฀corresponding฀to฀the฀aspartate฀resi-
due฀ in฀ Fig.฀ 12.11.฀ The฀ barrel฀ forms฀ from฀ b-sheet/a-helix฀ sequences฀ in฀ the฀ same฀ order฀ as฀ they฀
appear฀in฀the฀above฀polypeptide฀N-terminal฀to฀C-terminal.฀This฀order฀of฀strands฀and฀of฀conserved฀
regions฀differs฀from฀those฀in฀salivary฀amylase฀(Fig.฀12.12a).฀The฀barrel฀starts฀at฀a-helix3฀and฀ends฀
at฀b-strand3,฀whereas฀in฀salivary฀amylase,฀it฀starts฀at฀b-strand1,฀and฀ends฀at฀a-helix1.฀Domain฀B,฀
between฀b-strand3฀and฀a-helix4฀of฀salivary฀a-amylase,฀is฀replaced฀with฀a฀large฀stretch฀of฀sequence฀
of฀ unknown฀ function฀ between฀ a-helix8฀ and฀ b-strand1.฀ The฀ barrel฀ region฀ is฀ preceded฀ by฀ a฀ long฀
N-terminal฀region฀of฀variable฀length฀(Variable฀Region,฀VR)฀that฀is฀absent฀from฀salivary฀amylase.฀
At฀the฀C-terminal฀end,฀domain฀C฀(which฀enhances฀the฀solubility฀of฀salivary฀amylase)฀is฀replaced฀
with฀a฀Glucan฀Binding฀Domain฀(GBD)฀(Adapted฀from฀Fig.฀2B฀in฀van฀Hijum฀SAFT,฀Kralj฀S,฀Ozimek฀
LK,฀et฀al.฀(2006).฀Structure-function฀relationships฀of฀glucansucrase฀and฀fructansucrase฀enzymes฀
from฀lactic฀acid฀bacteria.฀Microbiology฀and฀Molecular฀Biology฀Reviews,฀70(1):157–176)

The฀ glucosyl฀ transferases฀ have฀ a฀ domain฀ structure฀ (Fig.฀ 15.8)฀ similar฀ to฀ a-amylase฀
(Sect.฀ 12.5.1).฀ The฀ S.฀ mutans฀ genome฀ possesses฀ three฀ glucosyltransferase฀ genes฀ (gtfB,฀
gtfC฀and฀gtfD)฀whose฀products฀are฀expressed฀constitutively฀and฀form฀the฀capsule฀at฀the฀
cell฀surface.฀The฀GTF-I฀(gtfB฀gene)฀mostly฀makes฀the฀water-insoluble฀mutan.฀The฀GTF-S฀
(gtfD฀gene)฀mostly฀makes฀the฀water-soluble฀dextran,฀and฀the฀GTF-SI฀(gtfC฀gene)฀makes฀
a฀฀mixture฀of฀water-soluble฀short฀chain฀mutan฀and฀dextran.฀All฀three฀GTF฀proteins฀have฀
two฀functional฀domains฀(Fig.฀15.8):฀a฀large,฀central฀barrel-like฀domain฀that฀is฀catalytic฀
and฀ a฀ C-terminal฀ domain฀ that฀ binds฀ to฀ the฀ glucan฀ polymer฀ (Glucan฀ Binding฀ Domain,฀
GBD).
The฀reaction฀catalyzed฀by฀glucosyl฀transferase฀(Fig.฀15.9a)฀is฀likewise฀similar฀to฀that฀
catalyzed฀ by฀ a-amylase฀ (Sect.฀ 12.5.1).฀ The฀ glucosyl฀ moiety฀ of฀ a฀ sucrose฀ molecule฀
(Fig.฀15.9b-i฀and฀c-i)฀is฀transferred฀to฀an฀aspartyl฀ester฀on฀GTF-I฀or฀GTF-S฀(analogous฀to฀
Asp฀197฀in฀saliva฀amylase;฀Fig.฀12.11)฀and฀a฀fructose฀residue฀is฀released฀(Fig.฀15.9b-ii฀and฀
c-ii).฀The฀release฀of฀fructose฀frees฀the฀enzyme฀to฀bind฀to฀a฀second฀sucrose฀molecule฀whose฀
glucosyl฀residue฀displaces฀the฀irst฀glucosyl฀residue฀on฀the฀activating฀aspartate฀residue.฀The฀
displaced,฀irst฀glucosyl฀moiety฀immediately฀forms฀a฀glycoside฀bond฀with฀the฀3-OH฀group฀
of฀the฀new฀aspartate-bound฀glucosyl฀residue฀(in฀GTF-I)฀or฀the฀6-OH฀group฀of฀that฀residue฀
(in฀ GTF-S).฀ The฀ glucan฀ chain฀ extends฀ at฀ the฀ C3฀ or฀ C6฀ end฀ by฀ one฀ glucose฀ residue฀
(Fig.฀15.9c-iii).
276 15 Dental Caries

Fig. 15.9฀ Synthesis฀ of฀ mutan฀ from฀ sucrose฀ by฀ S.฀ mutans.฀ (a)฀ Reaction฀ of฀ sucrose฀ with฀ glycosyl฀
transferase฀(see฀text).฀(b)฀Fates฀of฀the฀products.฀(i)฀The฀sucrose฀is฀hydrolyzed฀at฀its฀glycoside฀bond฀
(red฀O฀atom฀also฀shown฀in฀c-i).฀The฀glucose฀moiety฀b-i฀is฀briely฀enzyme฀bound฀as฀indicated฀in฀
[c(ii)]฀and฀polymerized฀to฀mutan฀by฀the฀enzyme฀interacting฀at฀the฀C1฀and฀C3฀positions.฀The฀fruc-
tose฀is฀transferred฀intracellularly฀and฀metabolized฀to฀lactose฀which฀is฀secreted.฀The฀fructose฀metab-
olism฀provides฀energy.฀(c)฀Glucosyl฀transferase฀mode฀of฀action฀(see฀text)฀

This฀process฀resembles฀the฀synthesis฀of฀starch฀and฀glycogen.฀Instead฀of฀nucleoside฀diphos-
phate฀glucose฀(UDP-glucose)฀being฀hydrolyzed฀to฀release฀UDP฀as฀the฀glucosyl฀residues฀form฀
starch฀or฀glycogen,฀sucrose฀releases฀fructose฀and฀the฀enzyme-bound฀activated฀glucosyl฀resi-
dues฀form฀a฀mutan฀or฀dextran.฀The฀process฀is฀repeated฀until฀stopped฀by฀lack฀of฀sucrose.฀The฀
polymer฀may฀then฀be฀transferred฀to฀water฀or฀to฀make฀a฀branch:฀i.e.,฀to฀a฀C6–OH฀group฀on฀an฀
adjacent฀mutan฀chain;฀or฀to฀a฀C3–฀or฀C4–OH฀group฀on฀an฀adjacent฀dextran฀chain.
15.2.3. Dentinal (Advanced) Dental Caries 277

15.2.2.
Sources of Bacterial Lactic Acid in Caries

The฀fructose฀released฀during฀this฀process฀(Fig.฀15.9b-ii)฀binds฀to฀a฀glycan฀phosphotrans-
ferase฀system฀(PTS)฀of฀enzymes฀on฀the฀cell฀surface.฀This฀binding฀activates฀an฀enzyme฀on฀
the฀cytosolic฀side฀of฀the฀PTS฀to฀attach฀a฀phosphate฀residue฀from฀phosphoenolpyruvate฀in฀
the฀cytosol฀to฀a฀histidine฀residue฀of฀a฀PTS฀protein฀on฀the฀cytosoic฀side฀of฀the฀inner฀mem-
brane.฀This฀activates฀the฀PTS฀enzyme฀system฀to฀transfer฀fructose฀6-phosphate฀to฀the฀cyto-
sol฀where฀it฀is฀phosphorylated฀and฀metabolized฀to฀lactate฀by฀glycolysis฀(Fig.฀1.7).฀The฀
PTS฀returns฀to฀its฀original฀conformation฀and฀another฀fructose฀molecule฀attaches฀and฀is฀
transferred.฀ The฀ lactic฀ acid฀ is฀ excreted,฀ but฀ trapped฀ at฀ the฀ tooth฀ surface฀ by฀ the฀ glucan฀
capsule.
Salivary฀a-amylase฀is฀a฀protein฀that฀contributes฀to฀the฀enamel฀pellicle฀(Sect.฀12.1.3).฀More฀
importantly,฀it฀attaches฀bacteria,฀especially฀streptococci,฀to฀teeth฀surfaces.฀Thus,฀following฀a฀
meal฀rich฀in฀carbohydrates,฀amylopectin,฀amylase,฀and฀glycogen฀are฀digested฀to฀maltose฀at฀the฀
surface฀of฀many฀oral฀bacteria.฀The฀maltose฀is฀taken฀into฀the฀cytosol฀by฀a฀phosphoenolpyruvate฀
transporter฀homologous฀to฀the฀fructose฀transporter฀of฀S.฀mutans.฀Within฀these฀bacteria,฀the฀
maltose฀is฀digested฀to฀two฀molecules฀of฀glucose฀6-phosphate฀and฀metabolized฀to฀lactic฀acid.฀
Thus,฀twice฀as฀much฀acid฀is฀produced฀per฀mole฀maltose฀than฀per฀mole฀sucrose฀and฀it฀contrib-
utes฀to฀tooth฀demineralization฀even฀if฀less฀sucrose฀is฀consumed.
Actinomyces฀spp.฀and฀the฀various฀viridans฀species฀of฀streptococci฀are฀more฀common฀in฀
the฀oral฀cavity฀than฀the฀mutans฀species฀of฀streptococci.฀They฀bind฀to฀teeth฀surface-adherent฀
salivary฀proteins฀in฀the฀absence฀of฀sucrose฀(Sect.฀12.6.1)฀and฀make฀glucans฀and฀fructans฀
that฀ enhance฀ mutans฀ streptococcal฀ adherence฀ and฀ acid฀ production฀ in฀ the฀ presence฀ of฀
sucrose.฀S฀mutans฀and฀other฀oral฀streptococci฀express฀glucan฀binding฀proteins฀that฀stabilize฀
the฀S.฀mutans฀capsule฀in฀vitro฀and฀enhance฀its฀adherence฀to฀these฀other฀bacterial฀glucans฀on฀
teeth฀ surfaces฀ in฀ vivo.฀ Fructan฀ and฀ dextran฀ polymers฀ that฀ are฀ not฀ bound฀ to฀ mutan฀ are฀
digested฀by฀bacterial฀enzymes,฀dextranase฀and฀fructanase.฀As฀mutan฀cannot฀be฀digested฀
(Sect.฀15.2.1),฀its฀stability฀promotes฀lactic฀acid฀retention฀and฀caries.

15.2.3.
Dentinal (Advanced) Dental Caries

Because฀only฀S.฀mutans฀survives฀in฀acid฀(below฀pH฀5.5),฀repeated฀sucrose฀intake฀tends฀to฀
increase฀the฀S.฀mutans฀colonization฀of฀sheltered฀“stagnation”฀regions฀where฀cavities฀mostly฀
develop.฀As฀the฀region฀becomes฀more฀acidic,฀the฀developing฀cavity฀promotes฀the฀growth฀
of฀yet฀more฀acid-tolerant฀bacteria,฀the฀Lactobacillus฀spp.,฀especially฀Lactobacillus฀casei฀
(L.฀casei)฀which฀can฀survive฀for฀a฀few฀hours฀in฀dilute฀hydrochloric฀acid฀(pH฀1).฀Lactobacilli฀
are฀short฀fat฀rods฀similar฀in฀shape฀to฀E.฀corrodens฀(Fig.฀13.3d),฀but฀larger฀and฀gram฀positive฀
like฀S.฀mutans฀(blue฀staining)฀instead฀of฀gram฀negative฀(red฀staining).฀Lactobacilli฀coloni-
zation฀is฀associated฀with฀the฀expansion฀of฀a฀cavity฀from฀enamel฀into฀dentin.
278 15 Dental Caries

Outside฀a฀carious฀cavity,฀S.฀mutans฀accounts฀for฀less฀than฀1%฀of฀the฀microbiota฀on฀teeth฀
surfaces,฀and฀lactobacilli฀for฀less฀than฀0.1%.฀Within฀a฀developing฀cavity,฀S.฀mutans฀often฀
accounts฀for฀5%฀or฀more฀of฀the฀microbiota.฀Once฀a฀cavity฀is฀established,฀the฀local฀pH฀often฀
falls฀ below฀ 4.0,฀ which฀ stops฀ the฀ growth฀ of฀ S.฀ mutans฀ and฀ all฀ other฀ oral฀ bacteria฀ except฀฀
L.฀ casei.฀ The฀ L.฀ casei฀ grows฀ on฀ the฀ monosaccharide฀ residues฀ of฀ proteoglycans฀ at฀ the฀
enamel–dentinal฀ junction฀ and฀ in฀ the฀ non-mineralized฀ portions฀ of฀ dentinal฀ sheaths฀ and฀
tubules.฀S.฀mutans฀therefore฀initiates฀enamel฀dissolution฀and฀lactobacilli฀dissolve฀dentin.฀
The฀lactobacilli฀excrete฀lactic฀acid฀which฀dissolves฀dentinal฀hydroxyapatite,฀releasing฀col-
lagen.฀ Asaccharolytic฀ bacteria฀ follow฀ the฀ lactobacilli,฀ infect฀ the฀ pulp฀ and฀ spread฀ to฀ the฀
bone฀at฀the฀apex฀of฀the฀infected฀tooth.

S.฀mutans฀possesses฀glucosyltransferases฀that฀make฀a฀metabolically฀stable฀mutan฀capsule฀
from฀the฀glucosyl฀moiety฀of฀sucrose.฀The฀fructose฀moiety฀of฀the฀sucrose฀is฀metabolized฀to฀
lactic฀acid.฀Other฀bacteria฀possess฀a฀fructosyltransferase฀that฀transfers฀the฀fructosyl฀moi-
ety฀ to฀ a฀ fructan฀ and฀ metabolizes฀ the฀ glucosyl฀ moiety฀ to฀ lactate.฀ Mutan฀ interacts฀ with฀
dextrans฀and฀fructans฀through฀glucan฀binding฀proteins.฀The฀result฀is฀a฀sticky฀matrix฀that฀
retains฀lactic฀acid฀at฀the฀tooth฀surface.฀Many฀oral฀bacteria฀bind฀salivary฀amylase,฀which฀
converts฀ starch฀ to฀ maltose.฀ Maltose฀ is฀ transported฀ into฀ the฀ cytosol฀ and฀ metabolized฀ to฀
lactate.฀The฀transport฀of฀maltose,฀fructose,฀or฀glucose฀into฀the฀cytosol฀involves฀a฀group฀of฀
enzymes฀ that฀ transfer฀ the฀ phosphate฀ residue฀ of฀ cytosolic฀ phosphoenolpyruvate฀ to฀ the฀
sugar,฀ the฀ phosphoenolpyruvate฀ transport฀ (PTS)฀ system.฀ As฀ the฀ region฀ becomes฀ more฀
acidic,฀the฀developing฀cavity฀promotes฀greater฀colonization฀by฀S.฀mutans฀and฀even฀more฀
acid-tolerant฀ bacteria,฀ Lactobacillus฀ spp.฀ The฀ latter฀ expand฀ the฀ cavity฀ into฀ dentin.฀
Asaccharolytic฀bacteria฀of฀the฀oral฀microbiota฀become฀protected฀from฀the฀acid฀by฀metab-
olizing฀the฀exposed฀dentinal฀proteins฀in฀the฀demineralized฀dentin.฀These฀bacteria฀induce฀
painful฀ inlammation฀ and฀ abscesses฀ within฀ the฀ pulp฀ and฀ periapical฀ regions฀ of฀ a฀ tooth฀
affected฀with฀untreated฀late-stage฀dental฀caries.

15.3.1.
Variation in Individual Human Caries Experience

As฀in฀rodents฀and฀other฀animals,฀there฀is฀much฀individual฀variation฀in฀human฀caries฀experi-
ence.฀In฀the฀Vipeholm฀study,฀25%฀of฀subjects฀taking฀the฀sticky฀candies฀did฀not฀develop฀any฀
cavities฀over฀6฀years,฀whereas฀a฀few฀cavities฀appeared฀in฀control฀subjects฀who฀received฀a฀
diet฀ that฀ contained฀ little฀ carbohydrate฀ and฀ no฀ reined฀ carbohydrate.฀ A฀ few฀ cavities฀ also฀
appeared฀in฀children฀of฀the฀Hopewood฀House฀study฀who฀received฀a฀similar฀diet.฀Within฀74฀
junior฀and฀senior฀dental฀students฀attending฀the฀College฀of฀Dentistry฀at฀the฀University฀of฀
Oklahoma฀in฀1985฀(mean฀age฀26฀years),฀the฀mean฀DMFT฀was฀8.4฀with฀a฀variability฀of฀40%฀
about฀the฀mean฀(Fig.฀15.10).฀Two฀had฀only฀one฀tooth฀affected฀and฀two฀others฀had,฀respec-
tively฀15฀and฀16฀teeth฀affected.฀The฀variation฀is฀due฀to฀differences฀in฀microbiota,฀dietary฀
carbohydrate฀intake,฀saliva฀low,฀luoride฀exposure,฀and฀acquired฀immunity฀(Table฀15.1).฀
15.3.2. Bacterial Causes of the Variation in Caries Susceptibility 279

14

12

Number of students
10

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
DMFT

Fig. 15.10฀ Distribution฀of฀caries฀in฀a฀dental฀student฀population.฀The฀number฀of฀decayed,฀missing,฀and฀

illed฀teeth฀due฀to฀caries฀(excluding฀third฀molars)฀was฀obtained฀for฀74฀junior฀and฀senior฀dental฀students฀
attending฀the฀University฀of฀Oklahoma฀College฀of฀Dentistry฀in฀1985฀(M.฀Levine:฀Unpublished฀data)

Table 15.1฀ Factors฀determining฀dental฀caries฀severity

1. Differences฀in฀the฀gram-positive฀composition฀of฀supragingival฀฀
microbial฀bioilms
2. Dietary฀carbohydrate฀ingestion,฀especially฀its฀sucrose฀content,฀฀
and฀also฀its฀physical฀consistency฀and฀frequency฀of฀ingestion
3. Saliva฀access฀to฀teeth฀surfaces฀and฀its฀low฀rate
4. Fluoride฀in฀the฀drinking฀water฀and฀toothpastes
5. Antibodies฀to฀Streptococcus฀mutans฀in฀blood฀฀
and฀the฀oral฀cavity

Saliva฀low฀and฀its฀acquired฀immunity฀proteins฀are฀discussed฀in฀Chap.฀12,฀Sects.฀12.1.3.฀
and฀12.1.4.฀Fluoride฀is฀discussed฀in฀Chap.฀16.฀It฀should฀be฀noted฀that฀despite฀considerable฀
efforts,฀an฀association฀between฀sucrose฀intake฀and฀caries฀experience฀(Fig.฀15.2)฀was฀only฀
established฀for฀large฀populations,฀not฀individuals.

15.3.2.
Bacterial Causes of the Variation in Caries Susceptibility

The฀microbiota฀of฀the฀oral฀cavity฀forms฀within฀a฀few฀months฀of฀birth฀and฀is฀stable฀thereaf-
ter.฀The฀types฀of฀bacteria฀present฀in฀the฀bioilm฀adhering฀to฀teeth,฀and฀therefore฀the฀extent฀
of฀caries฀development฀depends฀at฀least฀in฀part฀on฀that฀microbiota.฀Individual฀differences฀in฀
microbial฀composition฀cause฀individual฀variation฀in฀caries฀development.
In฀addition฀to฀variations฀in฀a฀bioilm’s฀polymer฀content฀related฀to฀sucrose฀consumption,฀
bacteria฀ that฀ metabolize฀ salivary฀ urea฀ or฀ dietary฀ arginine฀ also฀ affect฀ bioilm฀ pH.฀ The฀
enzyme฀ urease฀ converts฀ salivary฀ urea฀ to฀ ammonia฀ and฀ carbon฀ dioxide฀ (Sect.฀ 12.1.2),฀฀
280 15 Dental Caries

Fig. 15.11฀ Factors฀affecting฀pH฀after฀a฀10%฀glucose฀or฀sucrose฀mouth฀rinse.฀Dashed฀line฀indicates฀

pH฀7.฀Vectors฀1฀and฀4฀increase฀the฀pH;฀vectors฀2฀and฀3฀decrease฀the฀pH.฀The฀short-chain฀carboxylic฀
acids฀(SCCA)฀may฀be฀produced฀from฀amino฀acids฀along฀with฀ammonia,฀or฀from฀the฀catabolism฀of฀
lactic฀acid฀(see฀text฀and฀also฀Fig.฀1.8).฀(From฀Fig.฀9฀in฀Kleinberg฀I,฀2002.฀“A฀mixed-bacterial฀eco-
logical฀approach฀to฀understanding฀the฀role฀of฀the฀oral฀bacteria฀in฀dental฀caries฀causation:฀an฀alterna-
tive฀ to฀ Streptococcus฀ mutans฀ and฀ the฀ speciic-plaque฀ hypothesis.”฀ Crit฀ Rev฀ Oral฀ Biol฀ Med฀
13:108–125)

promoting฀a฀faster฀pH฀rise฀after฀consuming฀glucose฀or฀sucrose฀(Fig.฀15.11).฀Arginase฀con-
verts฀dietary฀arginine฀to฀ornithine฀and฀urea฀which฀is฀acted฀on฀by฀urease.฀The฀ornithine฀is฀
ultimately฀converted฀to฀ammonia฀and฀acetate.
The฀ short-chain฀ carboxylic฀ acids฀ (SCCA)฀ produced฀ by฀ asaccharolytic฀ metabolism฀
(Sect.฀1.3.2)฀are฀less฀acidic฀than฀lactate,฀and฀more฀common฀at฀the฀base฀of฀a฀gingival฀sul-
cus฀ or฀ pocket฀ than฀ coronally.฀ Correspondingly,฀ the฀ pH฀ is฀ alkaline฀ in฀ this฀ region฀
(Sect.฀1.3.2)฀because฀of฀the฀greater฀ammonia฀production,฀and฀a฀carious฀cavity฀does฀not฀
normally฀ extend฀ into฀ the฀ gingival฀ sulcus.฀ This฀ observation฀ gave฀ rise฀ to฀ a฀ clinical฀ rule฀
called,฀“extension฀prevention,”฀which฀is฀no฀longer฀applied.฀Extension฀beneath฀the฀sulcus฀
predisposes฀to฀periodontitis฀and฀a฀root฀surface฀exposed฀in฀the฀oral฀cavity฀where฀caries฀can฀
redevelop.
Counts฀of฀the฀number฀of฀S.฀mutans฀or฀L.฀casei฀are฀a฀poor฀measure฀of฀caries฀susceptibil-
ity฀because฀as฀noted฀above,฀they฀cannot฀account฀for฀ammonia฀production฀or฀differences฀in฀
glycan฀production฀by฀other฀bacteria฀in฀the฀bioilms.฀Acid฀production฀after฀a฀10%฀sucrose฀
or฀glucose฀rinse฀after฀a฀6฀h฀or฀longer฀fast฀is฀a฀better฀measure฀of฀caries฀susceptibility,฀because฀
it฀represents฀the฀balanced฀outcome฀of฀the฀factors฀that฀promote฀or฀prevent฀caries.฀Because฀
this฀ method฀ requires฀ time฀ and฀ skill,฀ it฀ is฀ dificult฀ to฀ apply฀ to฀ many฀ subjects.฀ A฀ simpler฀
method฀of฀measuring฀caries฀susceptibility฀is฀past฀caries฀experience฀and฀age.฀The฀more฀teeth฀
affected฀by฀caries฀in฀a฀child฀or฀adolescent,฀the฀greater฀is฀the฀likelihood฀that฀a฀new฀cavity฀
will฀develop฀and,฀therefore,฀the฀greater฀is฀the฀need฀for฀preventive฀measures.฀Unfortunately,฀
this฀ measure฀ is฀ completely฀ uninformative฀ about฀ what฀ factors฀ are฀ present฀ to฀ promote฀ or฀
prevent฀caries.
15.3.3. Saliva Causes of Caries Susceptibility 281

15.3.3.
Saliva Causes of Caries Susceptibility

A฀major฀factor฀mediating฀resistance฀to฀caries฀is฀the฀low฀of฀saliva฀over฀the฀teeth฀and฀its฀bicar-
bonate฀content.฀Thus,฀a฀low฀salivary฀bicarbonate฀content,฀or฀restriction฀of฀low฀due฀to฀excessive฀
mouth฀breathing฀or฀salivary฀gland฀disease฀causes฀a฀steeper฀Stephan฀curve.฀The฀pH฀takes฀longer฀
to฀return฀to฀resting฀levels฀and฀associates฀clinically฀with฀severe฀caries.฀In฀addition,฀the฀shape฀and฀
spacing฀of฀the฀teeth฀affects฀saliva฀access,฀and฀individuals฀who฀have฀large,฀overcrowded฀teeth฀
that฀are฀in฀tight฀contact฀with฀each฀other฀develop฀more฀cavities฀than฀individuals฀with฀small,฀well-
spaced฀ teeth.฀ Orthodontic฀ treatment฀ prevents฀ caries฀ by฀ alleviating฀ the฀ crowding,฀ which฀
enhances฀the฀access฀of฀saliva฀to฀teeth฀surfaces.฀Orthodontic฀therapy฀is฀an฀important฀preventive฀
measure฀to฀ensure฀that฀as฀much฀saliva฀circulates฀over฀the฀tooth฀surface฀as฀possible.
A฀controversial฀area฀of฀salivary฀inluence฀on฀caries฀involves฀the฀acidic฀proline-rich฀pro-
teins฀(Sect.฀12.6.1).฀There฀are฀ive฀such฀proteins,฀Pa,฀Pif,฀Db,฀Pr1,฀and฀Pr2.฀Pr1฀and฀Pr2฀are฀
encoded฀at฀the฀PRH2฀locus,฀whereas฀the฀irst฀three฀are฀encoded฀at฀the฀PRH1฀locus.฀These฀
two฀loci฀encode฀all฀possible฀alleles฀of฀the฀salivary฀acidic฀PRPs฀in฀Caucasians฀(Fig.฀15.12).฀
Knowledge฀of฀the฀human฀genome฀sequence฀has฀permitted฀the฀genes฀encoding฀each฀of฀the฀
respective฀ PRP฀ proteins฀ to฀ be฀ ampliied฀ by฀ polymerase฀ chain฀ reaction฀ (PCR).฀ Because฀
protein฀Db฀is฀larger฀than฀any฀other฀(allelic)฀protein฀encoded฀by฀the฀PRH1฀or฀PRH2฀locus,฀
the฀PCR฀product฀indicates฀the฀presence฀of฀Db฀in฀genomic฀DNA,฀and฀therefore฀in฀saliva.
In฀3฀–฀5฀year-old฀children฀of฀low฀socio-economic฀status,฀Caucasian฀children฀not฀express-
ing฀Db฀exhibit฀twice฀the฀severity฀of฀caries฀than฀do฀African-American฀children฀who฀have฀a฀

Fig. 15.12฀ The฀PRP฀protein฀alleles.฀The฀three฀proteins฀encoded฀by฀the฀PRH1฀locus฀are฀on฀the฀left฀

two฀columns฀and฀the฀two฀proteins฀by฀the฀PRH2฀locus฀on฀the฀right฀two฀columns.฀Because฀this฀locus฀
is฀expressed฀from฀both฀parental฀genes฀(A฀and฀B),฀there฀are฀six฀possible฀protein฀(allelic)฀combina-
tions฀of฀Pa,฀Pif,฀and฀Db฀and฀three฀possible฀combinations฀of฀Pr1฀and฀Pr2.฀This฀gives฀a฀total฀of฀18฀
combinations฀for฀each฀of฀the฀six฀combinations฀encoded฀by฀the฀PRH1฀locus฀is฀paired฀with฀one฀of฀
the฀three฀combinations฀encoded฀by฀the฀PRH2฀locus
282 15 Dental Caries

2.5-times฀greater฀frequency฀of฀Db฀in฀saliva.฀Yet฀at฀age฀12,฀the฀Caucasian฀children฀without฀
Db฀have฀less฀caries฀experience฀than฀Caucasian฀children฀with฀Db.฀This฀latter฀inding฀was฀
associated฀with฀greater฀S.฀mutans฀adhesion฀to฀Db,฀which฀is฀part฀of฀the฀acquired฀pellicle฀
(Sect.฀12.6.1).฀The฀different฀mixtures฀of฀acidic฀proline-rich฀proteins฀in฀African-Americans฀
may฀inhibit฀this฀Db-associated฀process,฀protecting฀them฀from฀caries.฀Because฀60%฀of฀cavi-
ties฀ occur฀ in฀ only฀ 20%฀ of฀ individuals,฀ identifying฀ caries-susceptible฀ individuals฀ early฀
improves฀the฀targeting฀of฀procedures฀to฀prevent฀caries฀before฀it฀is฀severe.฀Comparing฀Db฀
within฀the฀full฀complements฀of฀acidic฀proline-rich฀proteins฀in฀young฀children฀of฀African-
American฀and฀Caucasian฀descent฀may฀better฀identify฀sub-populations฀who฀are฀biologically฀
more฀susceptible฀to฀caries.

15.3.4.
Caries Immunity and Susceptibility

Immunizing฀caries-susceptible฀rats฀and฀mice฀with฀S.฀mutans฀prevented฀caries฀from฀devel-
oping฀when฀these฀animals฀were฀fed฀a฀caries-promoting฀diet฀(see฀Sect.฀15.1.4).฀Subsequently,฀
the฀rodents฀were฀also฀protected฀from฀caries฀if฀they฀were฀immunized฀with฀the฀glucosyltrans-
ferases฀puriied฀from฀S.฀mutans.
Similar฀systemic฀immunization฀experiments฀were฀carried฀out฀in฀monkeys.฀Monkeys฀fed฀
candies฀and฀cookies฀and฀given฀water฀containing฀2%฀sucrose฀developed฀many฀cavities฀after฀
3–4฀years฀unless฀immunized฀with฀whole฀cells฀of฀S.฀mutans.฀Various฀puriied฀protein฀anti-
gens฀given฀by฀subcutaneous฀vaccination฀induced฀a฀blood฀plasma฀immunoglobulin฀response฀
that฀protected฀monkeys฀from฀caries,฀whereas฀the฀glucosyl฀transferases฀protected฀only฀if฀
vaccination฀methods฀that฀promoted฀salivary฀IgA฀immunity฀Sect.฀12.1.4฀were฀used.
The฀ major฀ protective฀ antigen฀ of฀ S.฀ mutans฀ was฀ a฀ cell-surface฀ adhesin฀ named฀ AgI/II฀
(Fig.฀15.13).฀Antigen฀I/II฀attaches฀S.฀mutans฀to฀teeth฀surfaces฀by฀binding฀to฀salivary฀agglu-
tinin฀(Sect.฀12.6.2).฀Variants฀of฀antigen฀I/II฀are฀present฀in฀many฀streptococci,฀not฀just฀S.฀
mutans.฀ Attachment฀ to฀ salivary฀ agglutinin฀ may฀ explain฀ the฀ predominance฀ of฀ viridans฀
streptococci฀in฀the฀initial฀bioilms฀that฀form฀on฀the฀acquired฀pellicle฀in฀experimental฀human฀
gingivitis฀(Sect.฀12.1.5).฀Antigen฀I฀was฀puriied฀by฀1984฀to฀vaccinate฀children฀against฀car-
ies,฀unfortunately฀coinciding฀with฀reports฀that฀this฀antigen฀cross-reacted฀with฀human฀heart฀
proteins.฀It฀was฀subsequently฀found฀that฀an฀impurity฀not฀present฀in฀the฀puriied฀antigen฀had฀
induced฀the฀heart฀cross-reactivity.฀Nevertheless,฀a฀fear฀of฀problems฀led฀to฀the฀vaccination฀
proposal฀being฀dropped.฀Indeed,฀most฀individuals฀do฀not฀require฀immunization฀because฀of฀
almost฀ universal฀ access฀ to฀ luoridated฀ drinking฀ water฀ and฀ toothpastes฀ (Sect.฀ 16.2.1).฀ In฀
regions฀such฀as฀the฀US,฀Western฀Europe,฀and฀Australia/New฀Zealand,฀no฀more฀than฀20%฀
of฀the฀population฀suffers฀from฀severe฀caries.
Severe฀caries฀tends฀to฀occur฀in฀rural฀or฀economically฀disadvantaged฀children฀and฀can฀lead฀
to฀oral฀and฀or/health฀problems฀later฀in฀life.฀These฀children฀usually฀have฀no฀access฀to฀luori-
dated฀water฀or฀toothpastes,฀let฀alone฀regular฀dental฀care.฀Vaccinating฀such฀at-risk฀populations฀
with฀antigen฀I/II฀or฀glucosyltransferase฀could฀provide฀effective฀caries฀protection฀at฀little฀risk.฀
Unfortunately,฀there฀is฀currently฀no฀biologically฀based฀susceptibility฀test฀to฀diagnose฀a฀child฀at฀
risk฀for฀severe฀caries.฀Identifying฀which฀acidic฀PRP฀alleles฀are฀present฀in฀genomic฀DNA฀along฀
with฀measurements฀of฀salivary฀agglutinin฀levels฀in฀saliva฀might฀provide฀a฀suitable฀test.
15.3.4. Caries Immunity and Susceptibility 283

LPXTG

38 203 448 848 964

S A A A V PPP C

996 Ag II

Fig. 15.13฀ Major฀adhesion฀antigen฀of฀S.฀mutans.฀The฀amino฀acid฀sequence฀of฀antigen฀I/II฀(1,561฀

residues;฀mol.฀wt.฀185,000฀Da)฀begins฀with฀a฀putative฀signal฀peptide฀(S).฀The฀N-terminal฀region฀
(residues฀157–448)฀is฀an฀alanine-rich฀domain฀whose฀three฀subdomains฀are฀made฀up฀of฀a฀7-amino฀
acid฀ tandem฀ repeating฀ sequence฀ that฀ possesses฀ at฀ least฀ one฀ alanine฀ residue฀ (A).฀ The฀ tandem฀
repeats฀begin฀at฀residue฀203฀and฀end฀at฀residue฀448,฀each฀set฀of฀repeats฀being฀82฀residues.฀This฀
domain฀likely฀forms฀a฀set฀of฀juxtaposed฀a-helices.฀Following฀a฀short฀linker฀sequence,฀the฀third฀
domain฀(461–834)฀is฀a฀lectin-like฀variable฀region฀(V)฀that฀may฀bind฀glycan.฀A฀fourth฀domain฀has฀
three฀homologous฀proline-rich฀domains฀(residues฀848–964)฀that฀favor฀an฀extended฀conformation.฀
The฀ remaining฀ sequence฀ is฀ a฀ C-terminal฀ domain฀ (C)฀ within฀ which฀ residue฀ 1535฀ near฀ the฀
C-terminus฀ (within฀ the฀ black฀ region)฀ is฀ a฀ conserved฀ threonine฀ residue฀ (not฀ shown)฀ whose฀
hydroxyl฀group฀is฀covalently฀bonded฀by฀an฀amide฀link฀to฀cell฀wall฀peptidoglycan฀(Sect.฀1.4.1).฀At฀
the฀N-terminal฀end฀of฀this฀region฀is฀a฀strongly฀conserved฀sequence฀(LPXTG)฀that฀is฀a฀cleavage฀
site฀for฀amidases฀which฀release฀the฀antigen฀from฀the฀cell฀wall.฀X฀stands฀for฀any฀amino฀acid.฀It฀
varies฀between฀genus฀and฀species฀and฀is฀always฀N-terminal฀on฀the฀fragment฀that฀remains฀cova-
lently฀attached฀to฀murein.฀Antigen฀II฀is฀antigen฀I฀truncated฀at฀residue฀996฀and฀has฀therefore฀lost฀all฀
the฀upstream฀N-terminal฀regions฀(Derived฀from฀Fig.฀1฀of฀Younson฀J฀and฀Kelly฀C฀(2004)฀“The฀
rational฀ design฀ of฀ an฀ anti-caries฀ peptide฀ against฀ Streptococcus฀ mutans.”฀ Molecular฀ Diversity฀
8:121–126.฀Domain฀and฀repeating฀sequences฀taken฀from฀UniProtKB/Swiss-Prot฀P11657฀(PAC_
STRMU)฀modiied฀September฀22,฀2009฀(Version฀74))

Caries฀experience฀depends฀on฀the฀microbial฀composition฀of฀bioilms,฀amount฀of฀sucrose฀
and฀reined฀carbohydrate฀in฀the฀diet,฀saliva฀low,฀luoride฀in฀drinking฀water฀and฀in฀tooth-
pastes,฀ and฀ immunity฀ to฀ S.฀ mutans.฀ Bioilms฀ are฀ a฀ mixture฀ of฀ symbiotic฀ bacteria,฀
entrapped฀ in฀ an฀ adhesive฀ capsule.฀ Individual฀ differences฀ in฀ microbial฀ composition฀
cause฀variations฀in฀polymer฀content.฀The฀extent฀to฀which฀a฀microbiota฀utilizes฀salivary฀
urea฀or฀dietary฀arginine฀to฀make฀ammonia฀affects฀bioilm฀pH฀and฀caries฀experience.฀
Diseased฀salivary฀glands,฀excessive฀breathing฀through฀the฀mouth฀instead฀of฀the฀nose,฀
and฀teeth฀that฀are฀tightly฀squeezed฀together฀impede฀access฀of฀saliva฀and฀promote฀caries.฀
Greater฀ bioilm฀ colonization฀ and฀ caries฀ may฀ be฀ promoted฀ by฀ high฀ levels฀ of฀ salivary฀
agglutinin฀in฀subjects฀whose฀saliva฀also฀secretes฀a฀speciic฀acidic฀proline-rich฀protein฀
allele,฀Db.฀Nasal฀or฀oral฀spray฀immunization฀with฀glucosyl฀transferase฀induces฀salivary฀
IgA฀that฀protects฀rodents฀from฀caries฀despite฀a฀cariogenic฀diet.฀Subcutaneous฀immuni-
zation฀with฀mutans฀adhesion฀antigen฀I฀protects฀primates฀by฀reducing฀S.฀mutans฀coloni-
zation฀ of฀ the฀ oral฀ cavity.฀ Severe฀ caries฀ tends฀ to฀ occur฀ in฀ rural฀ or฀ economically฀
disadvantaged฀ children฀ and฀ it฀ can฀ lead฀ to฀ oral฀ and฀ or/health฀ problems฀ later฀ in฀ life.฀
Vaccinating฀such฀children฀seems฀an฀attractive฀therapy฀that฀could฀be฀explored฀further.
 Fluoride
16

This฀chapter฀describes฀how฀individuals฀with฀severe฀enamel฀luorosis฀(mottled฀tooth฀
enamel)฀became฀associated฀with฀luoride฀in฀the฀public฀water฀supply฀and฀protection฀
from฀dental฀caries.฀A฀comparison฀of฀caries฀experience฀with฀the฀luoride฀content฀of฀
public฀water฀supplies฀and฀enamel฀luorosis฀in฀adolescents฀indicated฀that฀1฀μg฀luoride/mL฀
(1฀part/million)฀in฀the฀water฀provides฀caries฀protection฀with฀minimal฀enamel฀luorosis฀
(sect.฀ 1).฀ One฀ mechanism฀ is฀ the฀ spontaneous฀ isomorphic฀ replacement฀ of฀ apatite’s฀
hydroxide฀anions฀with฀luoride,฀which฀reduces฀enamel฀solubility.฀A฀second฀is฀luoride-
฀mediated฀inhibition฀of฀enolase,฀which฀retards฀bacterial฀acid฀production฀at฀teeth฀sur-
faces.฀These฀indings฀led฀to฀the฀use฀of฀luoride฀in฀toothpastes,฀which฀provides฀better฀
protection฀from฀caries฀at฀tooth฀surfaces฀than฀water฀luoridation฀alone฀(sect.฀2).฀The฀
chapter฀concludes฀with฀a฀discussion฀of฀potentially฀harmful฀effects฀of฀luoride฀inges-
tion฀(sect.฀3).

16.1.1.
Properties of Fluorine and Fluoride

The฀element฀luorine฀has฀nine฀protons,฀ten฀neutrons,฀and฀nine฀electrons,฀giving฀a฀mass฀of฀
19฀Da.฀Fluorine฀is฀the฀smallest฀halide฀element฀(Sect.฀1.1.5)฀and฀it฀forms฀a฀gas฀that฀is฀so฀
electron฀deicient฀that฀it฀has฀all฀reacted฀with฀metals฀and฀only฀its฀ions฀(F-1)฀are฀found฀on฀
earth.฀Fluoride฀and฀hydroxide฀ions฀have฀almost฀the฀same฀mass,฀19฀Da฀and฀17฀Da฀respec-
tively.฀฀Because฀luoride฀is฀more฀electronegative,฀it฀replaces฀the฀hydroxide฀ion฀on฀crystals฀
without฀altering฀their฀overall฀structure.฀This฀type฀of฀alteration฀is฀referred฀to฀as฀an฀isomor-
phous฀ion฀replacement.
The฀luoride฀ion฀content฀of฀solutions฀is฀measured฀using฀a฀luoride฀electrode.฀The฀sensor฀
is฀ a฀ crystal฀ of฀ lanthanum฀ triluoride฀ containing฀ small฀ amounts฀ of฀ europium฀ diluoride฀฀
(Sect.฀1.1.5).฀Because฀lanthanum฀forms฀a฀tri-luoride฀lattice,฀but฀europium฀forms฀di-฀or฀tri-
luoride฀lattices,฀vacancies฀of฀luoride฀ions฀develop฀in฀a฀mixed฀lanthanum-europium฀crys-
tal.฀If฀a฀solid฀electrochemical฀cell฀is฀constructed฀from฀this฀mixed฀crystal,฀differences฀in฀the฀
transference฀of฀charge฀are฀exclusively฀due฀to฀differences฀in฀luoride฀availability,฀provided฀
the฀samples฀are฀acidic.฀Hydroxide฀ions฀interfere฀with฀luoride฀transfer.฀The฀cell฀is฀standard-
ized฀by฀inserting฀it฀into฀known฀concentrations฀of฀luoride฀solutions฀and฀the฀voltage฀mea-
sured.฀A฀graph฀of฀voltage฀against฀luoride฀concentration฀then฀allows฀the฀luoride฀content฀of฀
an฀unknown฀solution฀to฀be฀calculated.

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 285
DOI:฀10.1007/978-3-540-88116-2_16,฀©฀Springer฀Verlag-Berlin฀Heidelberg฀2011
286 16 Fluoride

16.1.2.
How Mottled Enamel Relates to Fluoride in the Water Supply

The฀studies฀that฀identiied฀luoride฀in฀the฀water฀supply฀eventually฀led฀to฀its฀use฀in฀caries฀
prevention.฀ These฀ studies฀ were฀ mostly฀ due฀ to฀ the฀ extraordinary฀ abilities฀ of฀ one฀ man,฀
Frederick฀ Sumner฀ McKay฀ (Fig.฀ 16.1).฀ McKay฀ graduated฀ in฀ dentistry฀ in฀ Boston฀ but฀ in฀
1901,฀he฀moved฀to฀Colorado฀Springs฀because฀of฀health฀problems.฀There,฀mottled฀enamel฀
was฀ common฀ and฀ he฀ soon฀ became฀ acquainted฀ with฀ this฀ problem฀ because฀ many฀ of฀ his฀
patients฀were฀seeking฀treatment฀for฀unsightly฀teeth฀due฀to฀mottling,฀not฀dental฀caries฀as฀in฀
Boston.฀ In฀ 1908,฀ McKay฀ sent฀ samples฀ of฀ mottled฀ teeth฀ to฀ G.฀ V.฀ Black,฀ Dean฀ of฀ the฀
Northwestern฀University฀Dental฀School฀of฀Chicago,฀for฀advice.฀Beginning฀in฀1916,฀he฀and฀
Black฀published฀a฀number฀of฀reports฀in฀The฀Dental฀Cosmos,฀which฀later฀merged฀with฀the฀
Journal฀of฀the฀American฀Dental฀Association.฀McKay฀outlined฀the฀geographic฀boundaries฀
of฀the฀areas฀where฀mottled฀enamel฀was฀found,฀determined฀the฀percentage฀of฀aflicted฀indi-
viduals,฀and฀compiled฀evidence฀that฀pointed฀to฀the฀water฀supply฀as฀a฀cause.฀Black฀detailed฀
histological฀ studies฀ that฀ compared฀ the฀ enamel฀ structure฀ of฀ mottled฀ and฀ normal฀ teeth.฀
Between฀1925฀and฀1931,฀similar฀changes฀were฀described฀in฀the฀teeth฀of฀rats฀fed฀sodium฀
luoride฀in฀their฀diet,฀ but฀ these฀ changes฀ were฀ not฀ compared฀ with฀ Black’s฀ description฀ of฀
mottled฀human฀enamel.
At฀the฀same฀time,฀McKay฀was฀informed฀of฀an฀outbreak฀of฀mottled฀enamel฀in฀children฀
who฀lived฀in฀Bauxite,฀a฀town฀near฀Colorado฀Springs.฀Bauxite฀was฀named฀for฀large฀depos-
its฀ of฀ bauxite,฀ which฀ the฀ Aluminum฀ Company฀ of฀ America฀ (ALCOA)฀ mined฀ there.฀
Children฀who฀drank฀spring-฀or฀shallow-well฀water฀had฀normal฀teeth,฀but฀children฀drinking฀
deep-well฀ water฀ had฀ severe฀ enamel฀ mottling.฀ In฀ 1931,฀ ALCOA’s฀ chief฀ chemist,฀

Fig. 16.1฀ Dr฀ Frederick฀ Sumner฀ McKay฀ (From฀

“Obituary:฀ Frederick฀ Sumner฀ McKay.”฀ (1960)฀
American฀Journal฀of฀Orthodontics฀46(9):฀695–699)
16.1.3. Mottled Enamel Is Moderate to Severe Enamel Fluorosis 287

Fig. 16.2฀ Mottled฀enamel.฀(a)฀Severe฀mottling.฀Note฀the฀wear฀at฀the฀coronal฀edge฀of฀the฀central฀inci-

sors,฀the฀brown฀color,฀and฀holes฀on฀the฀coronal฀half฀of฀the฀crowns฀and฀the฀opaque฀white฀color฀inter-
spersed฀with฀brown฀spots฀on฀the฀apical฀half฀(Figure฀downloaded฀from฀The฀Free฀Dictionary฀http://img.
tfd.com/mosby/thumbs/500087-fx12.jpg).฀ (b)฀ Mild฀ mottling.฀ Note฀ the฀ speckles฀ of฀ opaque฀ white฀
color฀on฀the฀labial฀surface฀of฀the฀central฀incisors.฀Other฀teeth฀may฀be฀similarly฀affected฀(Figure฀is฀a฀
gift฀from฀Dr.฀Kenneth฀W.฀Stephens,฀University฀of฀Glasgow฀College฀of฀Dentistry)

Dr.฀Churchill,฀read฀McKay’s฀reports฀and฀ordered฀a฀test฀that฀identiied฀trace฀elements฀in฀
water฀ using฀ a฀ new฀ quartz฀ spectroscope.฀ The฀ instrument฀ identiied฀ luoride฀ in฀ the฀ deep฀
wells฀only.฀McKay฀then฀obtained฀samples฀of฀drinking฀water฀from฀the฀many฀communities฀
where฀he฀knew฀mottled฀enamel฀was฀found฀and฀Churchill฀conirmed฀the฀presence฀of฀luo-
ride.฀ The฀ association฀ of฀ luoride฀ with฀ mottled฀ enamel฀ was฀ published฀ in฀ the฀ Scientiic฀
American฀in฀1931.
Dr.฀ H.฀ Trendley฀ Dean,฀ a฀ dental฀ oficer฀ of฀ the฀ U.S.฀ Public฀ Health฀ Service,฀ advanced฀
McKay’s฀indings,฀identifying฀various฀amounts฀of฀naturally฀occurring฀luoride฀in฀the฀pub-
lic฀water฀supplies฀from฀certain฀parts฀of฀the฀US฀(Fig.฀16.3).฀By฀1936,฀Dean฀had฀found฀that฀
luoride฀levels฀of฀up฀to฀1.0฀part฀per฀million฀(1฀ppm฀=฀1฀mg/L)฀in฀the฀drinking฀water฀did฀not฀
cause฀mottling,฀or฀obvious฀dental฀luorosis.฀More฀importantly,฀he฀also฀reported฀a฀correla-
tion฀between฀the฀luoride฀content฀of฀the฀water฀and฀a฀reduced฀incidence฀of฀dental฀decay.฀
Community-wide฀studies฀were฀carried฀out฀to฀evaluate฀the฀effect฀of฀luoride฀in฀the฀water฀
supply฀ on฀ caries฀ by฀ adding฀ sodium฀ luoride฀ to฀ luoride-deicient฀ water฀ supplies.฀ These฀
studies฀began฀in฀Grand฀Rapids,฀Michigan,฀in฀1945.

16.1.3.
Mottled Enamel Is Moderate to Severe Enamel Fluorosis

Fluoride฀is฀both฀incorporated฀into฀enamel฀crystals฀and฀also฀affects฀the฀enzymes฀involved฀in฀
enamel฀formation฀(Sect.฀16.2.2),฀causing฀mottled฀enamel,฀a฀severe฀example฀of฀enamel฀luo-
rosis.฀Enamel฀luorosis฀is฀evident฀as฀specks฀or฀white฀lecks฀on฀the฀enamel฀surface฀(Fig.฀16.2b).฀
In฀1941,฀the฀public฀water฀supply฀of฀Aurora฀(IL)฀contained฀1.2฀ppm฀of฀luoride฀(F),฀but฀only฀
5%฀of฀teeth฀exhibited฀luorosis,฀mostly฀premolars฀and฀second฀molars.฀A฀sensitive฀index฀of฀
288 16 Fluoride

Fig. 16 3฀ Geographic฀location฀of฀mottled฀enamel฀in฀the฀US฀(1941)฀(Figure฀is฀a฀reproduction฀of฀Fig.฀2฀
from฀Dean฀HT,฀Kitchin฀P,฀Moulton฀FR฀(eds)฀Fluorine฀and฀dental฀health.฀American฀Association฀of฀the฀
Advancement฀of฀Science฀Publication฀No฀19:฀Washington฀DC฀1942฀©฀AAAS฀1942,฀pp.฀6–11)

luorosis฀ requires฀ children฀ aged฀ 12–13฀ in฀ whom฀ the฀ premolars฀ and฀ second฀ molars฀ have฀
erupted.฀The฀mottled฀enamel฀irst฀drawn฀to฀McKay’s฀attention฀in฀Colorado฀is฀moderate฀and฀
severe฀luorosis฀(Fig.฀16.4).
The฀level฀of฀luorosis฀in฀an฀individual฀is฀determined฀by฀observing฀the฀buccal฀surface฀
of฀each฀tooth฀and฀classifying฀it฀as฀one฀of฀the฀following:฀Normal฀(score฀0)฀Enamel฀pres-
ents฀the฀usual฀translucent,฀appearance.฀The฀surface฀is฀smooth,฀glossy,฀and฀usually฀of฀a฀
pale฀creamy฀white฀color;฀Questionable฀(score฀1)฀The฀enamel฀has฀slight฀aberrations฀from฀
normal฀ translucency,฀ ranging฀ from฀ a฀ few฀ white฀ lecks฀ to฀ occasional฀ white฀ spots;฀ Very฀
mild฀(score฀2)฀Small,฀opaque,฀paper-white฀areas฀scattered฀over฀less฀than฀25%฀of฀the฀tooth฀
surface฀or฀a฀premolar฀or฀molar฀tooth฀showing฀no฀more฀than฀2฀mm฀of฀white฀opacity฀at฀the฀
tip฀of฀the฀cusps;฀Mild฀(score฀3)฀White฀opaque฀areas฀are฀discontinuous฀or฀a฀continuous฀
white฀opacity฀affects฀less฀than฀50%฀of฀the฀tooth฀surface฀(Central฀incisors฀in฀Fig.฀16.1b);฀
Moderate฀(score฀4)฀More฀than฀50%฀but฀not฀all฀of฀the฀enamel฀surface฀is฀affected฀with฀a฀
continuous฀white฀opacity฀and฀little฀or฀no฀brown฀is฀present฀(Fig.฀16.4);฀Severe฀(score฀5);฀
All฀of฀the฀enamel฀surface฀displays฀a฀continuous฀white฀opacity฀or฀more฀than฀half฀of฀the฀
tooth฀surface฀is฀stained฀brown฀and฀surfaces฀are฀subject฀to฀mild฀attrition฀(Figs.฀16.1a฀and฀
16.4).฀An฀individual฀is฀assigned฀a฀number฀(0฀through฀5)฀which฀represents฀two฀or฀more฀
teeth฀with฀the฀greatest฀severity฀score.฀The฀index฀of฀luorosis฀is฀the฀mean฀of฀these฀indi-
vidual฀scores฀within฀a฀community.
16.1.4. Identification of 1 ppm Fluoride in the Water as Optimal for Cavity Protection 289

Fig. 16.4฀ Index฀of฀luorosis.฀Figure฀depicts฀moderate฀to฀severe฀luorosis฀of฀the฀lateral฀incisor,฀canine,฀

and฀second฀molar฀teeth:฀index฀values฀of฀4฀and฀5฀(Copied฀from฀the฀University฀of฀Oklahoma฀Library฀
Collection฀circa฀1990,฀Source฀unknown)

16.1.4.
Identification of 1 ppm Fluoride in the Water as Optimal for Cavity Protection

The฀greater฀the฀luoride฀content฀of฀the฀public฀water฀supply,฀the฀greater฀is฀the฀index฀of฀luo-
rosis.฀At฀less฀than฀1฀ppm฀luoride฀in฀the฀drinking฀water,฀the฀index฀of฀luorosis฀in฀the฀com-
munity฀is฀essentially฀zero฀(no฀luorosis)฀and฀increases฀to฀severe฀at฀8–10฀ppm฀luoride฀in฀the฀
drinking฀water.฀Fig.฀16.5฀illustrates฀how฀the฀severity฀of฀luorosis฀changes฀with฀amount฀of฀
luoride฀in฀the฀community฀water฀supply฀and฀also฀the฀average฀number฀of฀decayed,฀missing,฀
and฀illed฀teeth฀(DMFT,฀Sect.฀15.1.1).฀The฀subjects฀were฀children฀aged฀12–14฀years฀in฀vari-
ous฀US฀cities฀and฀communities฀and฀their฀DMFT฀is฀compared฀in฀Fig.฀16.5฀with฀the฀ppm฀
luoride฀in฀their฀water฀supply.
If฀the฀water฀supply฀had฀no฀luoride,฀the฀population฀displayed฀6–8฀DMFT.฀As฀the฀luoride฀
concentration฀increased฀to฀1฀ppm,฀the฀mean฀DMFT฀of฀the฀population฀decreased฀by฀about฀
50%,฀but฀the฀index฀of฀luorosis฀remained฀well฀below฀1.฀Increasing฀the฀luoride฀above฀1฀ppm฀
290 16 Fluoride

Fig. 16.5฀ Relationship฀of฀DMFT฀and฀index฀of฀luorosis฀to฀water฀luoride฀content.฀The฀left฀y-axis฀

indicates฀the฀number฀of฀decayed฀missing฀and฀illed฀teeth฀from฀caries฀(DMFT)฀and฀the฀right฀y-axis฀
indicates฀the฀index฀of฀luorosis,฀a฀measure฀of฀the฀deleterious฀effect฀of฀luoride฀on฀the฀enamel฀sur-
face฀(see฀text).฀The฀x-axis฀indicates฀the฀ppm฀luoride฀found฀naturally฀in฀the฀drinking฀water฀supply.฀
Triangles฀ indicate฀ DMFT฀ and฀ circles฀ indicate฀ the฀ luorosis฀ index฀ in฀ the฀ same฀ populations.฀ The฀
curves฀showing฀the฀decrease฀in฀caries฀and฀decrease฀in฀luorosis฀intersect฀at฀~1฀ppm฀luoride฀in฀the฀
water฀supply฀on฀the฀x-axis฀(Copy฀of฀Fig.฀3฀from฀Hodge฀HC,฀Smith฀FA.฀(1954).฀Some฀public฀health฀
aspects฀of฀water฀luoridation.฀American฀Association฀of฀the฀Advancement฀of฀Science฀Publication฀
No฀19:฀Washington฀DC฀1954฀©฀AAAS฀1954,฀pp.฀79–109)

slightly฀decreased฀the฀DMFT฀further,฀but฀markedly฀increased฀the฀prevalence฀of฀luorosis.฀
The฀line฀of฀decreasing฀luorosis฀intersects฀with฀the฀line฀of฀decreasing฀caries฀at฀just฀over฀
1฀ppm฀luoride฀as฀seen฀in฀the฀center฀of฀Fig.฀16.5.

The฀luoride฀ion฀can฀replace฀the฀hydroxide฀ion฀in฀a฀crystal฀without฀signiicantly฀altering฀
its฀ structure,฀ an฀ isomorphous฀ ion฀ replacement.฀ Fluoride฀ also฀ affects฀ the฀ enzymes฀
involved฀in฀enamel฀formation,฀causing฀mottled฀enamel,฀a฀severe฀example฀of฀enamel฀
luorosis.฀White฀opaque฀patches฀on฀the฀normally฀translucent฀enamel฀indicate฀mild฀luo-
rosis.฀Fluorosis฀is฀measured฀on฀a฀grade฀of฀0–5฀where฀1฀through฀3฀indicate฀an฀increased฀
cover฀of฀opaque฀white฀patches฀on฀the฀tooth฀surface,฀and฀4฀and฀5฀indicate฀an฀increased฀
mottling.฀The฀two฀worst฀affected฀teeth฀make฀up฀an฀individual’s฀score.฀The฀community’s฀
index฀of฀luoridation฀is฀the฀mean฀score฀for฀all฀individuals.฀As฀the฀natural฀or฀artiicial฀
luoride฀concentration฀of฀the฀water฀supply฀increases฀to฀1฀ppm,฀the฀mean฀number฀of฀cavi-
ties฀in฀10–12฀year-old฀children฀decreases฀from฀7฀to฀3.฀Above฀1฀ppm฀luoride,฀caries฀does฀
not฀decrease฀much฀more,฀but฀the฀index฀of฀luorosis฀increases฀markedly.฀This฀is฀the฀rea-
son฀why฀public฀water฀supplies฀are฀luoridated฀to฀only฀1฀ppm฀and฀not฀more฀or฀less.
16.2.1. Mechanisms of Fluoride Protection from Caries 291

16.2.1.
Mechanisms of Fluoride Protection from Caries

Dental฀caries฀occurs฀when฀bacterial฀acids฀dissolve฀the฀hydroxyapatite฀crystals฀of฀enamel฀
and฀produce฀amorphous฀sodium฀monohydrogen฀phosphate฀from฀hydroxyapatite฀by฀revers-
ing฀ the฀ solid-state฀ reactions฀ shown฀ in฀ Fig฀ 16.6.฀ This฀ process฀ occurs฀ when฀ bacteria฀ in฀ a฀
bioilm฀produce฀lactic฀acid฀by฀saccharolytic฀fermentation฀(Sect.฀15.1.3).฀Protons฀(H+)฀dif-
fuse฀into฀the฀crystal฀where฀they฀react฀with฀hydroxyl฀groups฀in฀hydroxyapatite฀and฀change฀
the฀crystal฀to฀an฀amorphous฀calcium฀monohydrogen฀phosphate฀solid฀that฀slowly฀dissolves฀
(Fig฀16.6b).฀An฀increased฀lactic฀acid฀concentration฀(pH฀<฀6.2)฀makes฀the฀dissolution฀pro-
ceed฀faster฀by฀converting฀calcium฀monohydrogen฀phosphate฀to฀calcium฀dihydrogen฀phos-
phate฀which฀is฀10฀times฀more฀soluble.฀The฀critical฀pH฀for฀apatite฀dissolution฀is฀pH฀6.2.฀
Apatite฀dissolution฀is฀slow฀above฀pH฀6.2฀and฀fast฀below฀pH฀6.2.฀Thus,฀after฀a฀10%฀sucrose฀
rinse,฀subjects฀with฀severe฀caries฀exhibit฀a฀pH฀fall฀that฀is฀further฀and฀longer฀below฀6.2฀than฀
subjects฀with฀mild฀caries฀(Fig.฀15.3).
The฀presence฀of฀even฀one฀luoride฀ion฀in฀the฀crystal฀slows฀the฀transformation฀to฀amor-
phous฀ calcium฀ monohydrogen฀ phosphate.฀ Thus,฀ in฀ the฀ presence฀ of฀ luoride฀ (e.g.,฀ after฀
using฀luoridated฀toothpastes),฀luoroapatite฀forms฀at฀the฀tooth฀surface฀and฀reduces฀the฀rate฀
of฀caries฀development.฀The฀increased฀luoride฀concentration฀at฀the฀tooth฀surface฀also฀inhib-
its฀lactate฀production.฀These฀observations฀explain฀why฀cleaning฀the฀teeth฀with฀luoridated฀
toothpaste฀prevent฀caries.฀Cleaning฀the฀teeth฀exposes฀the฀apatite฀at฀the฀enamel฀surface.฀In฀
the฀ absence฀ of฀ luoride,฀ there฀ is฀ no฀ protection฀ because฀ the฀ bioilm฀ forms฀ within฀ a฀ few฀

Fig. 16.6฀ Hydroxyapatite฀and฀luoroapatite:฀formation฀and฀dissolution.฀(a)฀Hydroxyapatite฀is฀trans-

formed฀to฀luoroapatite฀by฀isomorphous฀replacement.฀Fluoride฀ions฀diffuse฀into฀a฀hydroxyapatite฀
crystal฀ where฀ they฀ replace฀ the฀ hydroxide฀ ions.฀ (b)฀ Fluoroapatite฀ cannot฀ dissolve฀ as฀ easily฀ as฀
hydroxyapatite.฀ Right฀ to฀ left฀ shows฀ the฀ solid-state฀ rearrangement฀ of฀ hydroxyapatite฀ to฀ calcium฀
monohydrogen฀phosphate,฀free฀calcium฀ions,฀and฀monohydrogen฀phosphate.฀The฀latter฀becomes฀
mostly฀dihydrogen฀phosphate฀above฀pH฀6.2.฀Arrows฀between฀(b)฀and฀(a)฀indicate฀enhanced฀apatite฀
formation฀or฀slower฀changes฀to฀the฀amorphous฀solid฀if฀luoride฀is฀present.฀Left฀to฀right฀shows฀the฀
precipitation฀of฀calcium฀monohydrogen฀phosphate฀and฀its฀change฀to฀hydroxyapatite฀if฀an฀acid฀solu-
tion฀is฀made฀alkaline.
292 16 Fluoride

minutes฀and฀dissolves฀the฀apatite฀as฀before฀after฀sugars฀are฀consumed.฀In฀the฀presence฀of฀
toothpaste฀containing฀1,000฀ppm฀luoride,฀luoroapatite฀forms฀on฀the฀exposed฀apatite฀and฀
cannot฀dissolve฀as฀rapidly฀in฀the฀acid฀after฀sugars฀are฀consumed.฀Beyond฀this,฀luoridated฀฀
water฀provides฀protection฀independently฀of฀toothbrushing฀by฀increasing฀the฀luoride฀con-
centration฀within฀enamel฀as฀it฀develops.
Before฀ luoride฀ was฀ added฀ to฀ toothpastes฀ (1950–1970),฀ luoridated฀ water฀ provided฀
considerable฀ protection฀ from฀ caries,฀ but฀ now฀ that฀ virtually฀ all฀ toothpastes฀ are฀ luori-
dated,฀ some฀ have฀ questioned฀ the฀ need฀ for฀ water฀ luoridation.฀ Yet฀ carefully฀ controlled฀
studies฀in฀Scotland,฀where฀public฀pressure฀to฀remove฀luoride฀from฀the฀water฀supply฀has฀
been฀extremely฀strong,฀indicate฀a฀marked฀increase฀in฀caries฀despite฀the฀continued฀use฀of฀
luoridated฀toothpaste฀(see฀Sect.฀16.3.2).฀Fluoride฀toothpaste-mediated฀protection฀from฀
caries฀is฀dependent฀on฀oral฀hygiene฀eficacy,฀whereas฀protection฀from฀caries฀by฀luoride฀
in฀the฀water฀supply฀appears฀independent฀of฀oral฀hygiene.฀Protection฀from฀caries฀by฀arti-
icial฀ luoridation฀ of฀ water฀ supplies฀ and฀ luoridated฀ toothpaste฀ is฀ independent฀ and฀
additive.
In฀ the฀ USA,฀ water฀ luoridation฀ became฀ widely฀ available฀ after฀ 1955฀ and฀ luoridated฀
toothpastes฀ after฀ 1975.฀ In฀ 1954,฀ few฀ locations฀ had฀ naturally฀ or฀ artiicially฀ luoridated฀
drinking฀ water฀ and฀ luoridated฀ toothpastes฀ did฀ not฀ exist฀ and฀ the฀ mean฀ DMFT฀ in฀ US฀
12–13-year-old฀ children฀ was฀ about฀ 7.฀ By฀ 2004,฀ according฀ to฀ the฀ National฀ Health฀ &฀
Nutrition฀ Examination฀ Survey,฀ most฀ locations฀ had฀ luoridated฀ water฀ and฀ luoridated฀
toothpaste฀ and฀ the฀ mean฀ DMFT฀ in฀ 11–15-year-old฀ children฀ had฀ fallen฀ from฀ 8.0฀ in฀ the฀
mid-1950s฀to฀less฀than฀2.0.

16.2.2.
How Fluoride Protects from Caries

The฀ irst฀ and฀ primary฀ protective฀ effect฀ of฀ luoride฀ is฀ due฀ to฀ its฀ strong,฀ spontaneous฀
reaction฀with฀metal฀ions.฀Biologically,฀the฀most฀important฀of฀these฀ions฀is฀the฀calcium฀
ion,฀large฀amounts฀of฀which฀interact฀with฀phosphate฀to฀form฀bones฀and฀teeth.฀Studies฀
show฀that฀luoride฀reduces฀apatite฀solubility฀in฀acids฀by฀an฀isomorphic฀replacement฀of฀
hydroxide฀ions฀with฀luoride฀ions฀to฀form฀luoro-hydroxyapatite฀and฀diluoro-apatite฀
(Fig.฀16.6a).
Apatites฀must฀undergo฀a฀solid-state฀transition฀to฀amorphous฀calcium฀phosphate฀before฀
they฀can฀dissolve฀and฀the฀spontaneous฀replacement฀of฀hydroxide฀with฀luoride฀ions฀slows฀
the฀rate฀at฀which฀this฀transition฀occurs฀(Fig.฀16.6b).฀Conversely,฀as฀an฀acid฀environment฀
becomes฀ more฀ alkaline,฀ luoride฀ ions฀ promote฀ the฀ precipitation฀ and฀ crystallization฀ of฀
amorphous฀calcium฀monohydrogen฀phosphate/calcium฀luoride฀into฀luoro-฀and฀diluoro-
apatites฀faster฀than฀amorphous฀calcium฀phosphate฀would฀crystallize฀into฀hydroxyapatite.฀
Thus,฀luoride฀ions฀have฀two฀effects฀on฀enamel฀that฀protect฀from฀caries:฀they฀slow฀enamel฀
dissolution฀in฀lactic฀acid฀and฀promote฀its฀re-precipitation฀and฀crystallization฀when฀the฀lac-
tic฀acid฀is฀neutralized.
16.2.2. How Fluoride Protects from Caries 293

Dihydroxyacetone
phosphate
H O-
H C O P O
H O− C OO
H C O P O HO C H
C OO H
HO C H
O
H C OH Pi −O P O
H C OHO− +
H + O
H C O P O C O NAD+ NADH + H
C O
H O H C OH O H C OHO
H C O P O H C O P O
Fructose 1,6− H O H O
biphosphate
3-Phospho- 1,3-Bisphospho-
glyceraldehyde glycerate

ADP

Enolase ATP
H2O
ATP ADP O O O O O O
O O
C C O C O C
H C O P O H C O P O H C OH O
H C O
C O H C OH O H C O P O
H CH
H H H H O
H
Pyruvate Phosphoenol- 2-Phospho- 3-Phospho-
pyruvate (PEP) glycerate glycerate

Fig. 16.7฀ Glycolysis฀showing฀enolase.฀Saccharides฀are฀processed฀intracellularly฀into฀UDP-sugars฀

and฀metabolized฀to฀fructose฀6-phosphate฀and฀then฀1,6-bisphosphate฀if฀required฀for฀energy.฀The฀
various฀steps฀preceding฀fructose฀1,6-bisphosphate฀formation฀are฀omitted฀in฀this฀igure฀(Wikipedia฀
public฀ domain฀ image฀ slightly฀ modiied.฀ Accessed฀ at:฀ http://commons.wikimedia.org/wiki/
File:GlycolysisPathway.svg).฀This฀igure฀was฀modiied฀by฀Dr฀Wirsig-Weichmann

A฀ second฀ mechanism฀ of฀ protection฀ from฀ caries฀ is฀ the฀ incorporation฀ of฀ luoride฀ into฀
b฀ acterial฀bioilms฀where฀it฀inhibits฀enolase.฀Enolase฀catalyzes฀the฀production฀of฀phospho-
enolpyruvate,฀the฀precursor฀of฀lactate฀in฀glycolysis,฀from฀2-phosphoglycerate฀during฀gly-
colysis฀ (Fig.฀ 16.7฀ –฀ see฀ also฀ Fig.฀ 1.7).฀ In฀ addition,฀ oral฀ bacterial฀ uptake฀ of฀ mono-฀ and฀
disaccharides฀mostly฀utilizes฀the฀phosphoenolpyruvate฀transport฀system฀to฀transfer฀them฀
into฀the฀cytosol฀(Sect.฀15.2.2).฀Fluoride฀therefore฀inhibits฀not฀only฀lactic฀acid฀production,฀
but฀ also฀ the฀ phosphoenolpyruvate฀ transport฀ system-mediated฀ uptake฀ of฀ saccharide฀ sub-
strates.฀In฀short,฀luoride฀inhibits฀saccharolytic฀fermentation฀by฀many฀oral฀bacteria.
Although฀high฀levels฀of฀luoride฀(<3,000฀ppm)฀kill฀most฀bacteria,฀there฀is฀little฀evidence฀
that฀common฀levels฀of฀luoride฀(1–10฀ppm)฀alter฀the฀types฀of฀bacterial฀species฀or฀their฀rela-
tive฀concentrations฀in฀bioilms.฀Mutans฀and฀other฀streptococci฀in฀the฀bioilm฀may฀switch฀to฀
asaccharolytic฀fermentation฀(Sect.฀1.3.2).฀A฀฀luoride-mediated฀reduction฀in฀bacterial฀acid฀
production,฀ ensues฀ without฀ a฀ detectable฀ change฀ in฀ bacterial฀ bioilm฀ composition.฀
Correspondingly,฀luoride฀has฀no฀effect฀on฀the฀development฀of฀gingivitis฀or฀its฀progression฀
to฀periodontitis.
294 16 Fluoride

NOTE: Studies in the 1980s determined that large doses of fluoride do not protect from osteo-
porosis (Sect. 10.2.3), or decrease the incidence of bone fractures. It appears that increased
fluoride in the diet inhibits osteoblast activity more than osteoclast activity; women on fluo-
ride supplements suffer from more bone fractures, not less. Fluoride therapy for osteoporosis
was popular in the 1980s, but the reports published after 1990 reduced enthusiasm for this
treatment and it is not now recommended for post-menopausal bone loss.

In฀ the฀ USA,฀ water฀ luoridation฀ became฀ widely฀ available฀ after฀ 1955฀ and฀ luoridated฀
toothpastes฀ after฀ 1975฀ and฀ caries฀ in฀ adolescent฀ children฀ has฀ decreased฀ by฀ 66%.฀ The฀
effects฀of฀luoride฀on฀caries฀are฀topical฀from฀the฀surface฀to฀the฀interior.฀Water฀luorida-
tion฀ensures฀small฀amounts฀of฀luoride฀throughout฀a฀tooth฀and฀luoridated฀toothpaste฀
enhances฀the฀luoride฀concentration฀at฀the฀tooth฀surface.฀Protection฀from฀caries฀by฀arti-
icial฀ luoridation฀ of฀ water฀ supplies฀ and฀ luoridated฀ toothpaste฀ is฀ cumulative.฀
Investigations฀as฀to฀how฀luoridation฀protects฀from฀caries฀has฀identiied฀three฀mecha-
nisms฀ of฀ caries฀ protection:฀ (1)฀ inhibition฀ of฀ demineralization,฀ (2)฀ enhancement฀ of฀
remineralization,฀and฀(3)฀inhibition฀of฀bacterial฀enolase฀activity฀reducing฀lactate฀pro-
duction฀from฀ingested฀carbohydrates.฀Fluoride฀has฀little฀effect฀on฀bacterial฀growth,฀and฀
gives฀no฀direct฀protection฀from฀gingivitis,฀periodontitis,฀or฀osteoporosis

16.3.1.
Systemic Effects of Fluoride

At฀levels฀of฀1฀ppm,฀luoride฀shows฀no฀skeletal฀or฀other฀poisonous฀effects.฀Children฀who฀
drink฀5–10฀ppm฀luoridated฀water฀develop฀enamel฀luorosis฀and฀mottled฀enamel฀because฀
that฀is฀when฀the฀permanent฀teeth฀are฀developing.฀Adolescents฀or฀adults฀who฀start฀drinking฀
5–10฀ppm฀luoridated฀water฀show฀no฀signs฀of฀enamel฀luorosis฀and฀no฀other฀deleterious฀
health฀effects.฀Fluoride฀ions฀are฀excreted฀rapidly฀from฀the฀body฀and฀individual฀cells฀excrete฀
about฀30%฀of฀the฀blood฀plasma฀concentration.฀In฀fact,฀acute฀deleterious฀effects฀require฀very฀
high฀doses฀of฀luoride฀>฀1,000฀ppm฀(>1฀mg/mL)฀in฀the฀water฀or฀air฀(as฀occurred฀near฀Fort฀
William.฀Scotland).฀Chronic฀(persistent)฀deleterious฀effects฀likely฀require฀a฀daily฀exposure฀
to฀<10฀ppm฀for฀some฀months฀and฀primarily฀show฀up฀in฀children฀and฀young฀adults฀as฀mot-
tled฀enamel฀and฀skeletal฀defects.฀Reports฀of฀luorosis฀being฀associated฀with฀an฀increased฀
cancer฀risk฀in฀humans฀are฀probably฀due฀to฀high฀amounts฀of฀heavy฀metals฀that฀associate฀
with฀an฀increased฀luoride฀content฀of฀the฀water฀supply.฀Indeed,฀the฀need฀to฀purify฀luoride฀
from฀poisonous฀metals฀likely฀requires฀more฀careful฀oversight฀than฀is฀currently฀practiced฀
before฀adding฀it฀to฀public฀water฀supplies฀in฀the฀USA฀and฀elsewhere.
In฀children,฀enamel฀mottling฀appears฀at฀>1฀ppm฀luoride฀in฀the฀water฀supply฀and฀bone฀
mineralization฀ is฀ affected฀ at฀ >10฀ ppm.฀ In฀ rats฀ exposed฀ to฀ >10฀ ppm฀ luoride฀ (>0.5฀ mM),฀
radiolabeled฀amelogenin฀matrix฀fails฀to฀calcify,฀despite฀luoride฀not฀affecting฀enamelysin฀
or฀EMSP1฀proteolysis฀of฀amelogenin฀in฀vitro.฀Amelogenin฀binds฀better฀to฀luoridated,฀car-
bonated฀hydroxyapatite฀than฀to฀the฀non-luoridated฀form,฀perhaps฀inhibiting฀protease฀bind-
ing฀ or฀ cleavage฀ in฀ vivo.฀ If฀ so,฀ the฀ serine-phosphorylated฀ tyrosine฀ rich฀ peptide฀ (TRAP฀
peptide)฀ remains฀ on฀ the฀ large฀ amelogenin฀ nanospheres฀ (Fig.฀ 9.12;฀ Chap.฀ 9).฀ Enamel฀
16.3.2. Fluoride Toxicity 295

ribbons฀cannot฀calcify;฀enamel฀cannot฀mature฀and฀mottling฀results.฀In฀mottled฀enamel,฀the฀
lack฀of฀calciied฀enamel฀rods฀leave฀the฀enamel฀with฀holes฀and฀the฀retention฀of฀amelogenin฀
nanospheres฀give฀it฀a฀brown฀color฀(Fig.฀16.1a).฀In฀mild฀enamel฀luorosis,฀these฀defects฀are฀
less฀severe,฀resulting฀only฀in฀opaque฀white฀regions฀(Fig.฀16.1b).
The฀luoride-mediated฀inhibition฀of฀enolase฀in฀the฀glycolysis฀pathway฀was฀discussed฀in฀
relation฀to฀bacterial฀acid฀production฀(Sect.฀16.2.2).

16.3.2.
Fluoride Toxicity

The฀extraction฀of฀aluminum฀from฀bauxite,฀a฀naturally฀occurring฀mineral,฀causes฀luoride-
rich฀particles฀and฀luoride฀gas฀to฀be฀released฀into฀the฀environment.฀Because฀it฀utilizes฀lots฀
of฀electricity,฀the฀process฀is฀carried฀out฀in฀regions฀where฀waterfalls฀abound.฀The฀postindus-
trial฀ revolution฀ greatly฀ increased฀ the฀ need฀ for฀ aluminum฀ and฀ a฀ major฀ processing฀ plant฀
developed฀in฀the฀Fort฀William฀area฀and฀continues฀there฀today.฀Fort฀William฀is฀a฀port฀on฀the฀
mainland฀opposite฀the฀Isle฀of฀Skye฀to฀the฀west.฀To฀the฀northeast฀of฀Fort฀William฀is฀a฀long,฀
narrow฀valley.฀In฀the฀1940s,฀the฀plant฀expanded฀and฀uncontrolled฀luoride฀emissions฀blew฀
up฀this฀valley฀due฀to฀the฀prevailing฀southwesterly฀winds.฀The฀dust฀settled฀on฀the฀pasture฀
where฀its฀high฀luoride฀content฀poisoned฀cattle฀in฀the฀area.฀The฀factory฀was฀made฀to฀control฀
its฀luoride฀emissions,฀but฀luoride฀became฀entrenched฀in฀the฀Scottish฀political฀psyche฀as฀a฀
dangerous฀poison฀and฀adding฀it฀to฀water฀was฀vigorously฀opposed฀as฀potentially฀dangerous.฀
As฀a฀result,฀many฀Scottish฀cities฀do฀not฀have฀luoridated฀water฀despite฀the฀dental฀caries฀rate฀
being฀among฀the฀highest฀in฀the฀world.฀Indeed,฀in฀the฀early฀1990s,฀a฀moderate-sized฀city฀in฀
Scotland,฀Kilmarnock฀actually฀ceased฀adding฀luoride฀to฀their฀water฀supply.
The฀case฀for฀discontinuing฀the฀addition฀of฀luoride฀to฀public฀water฀supplies฀has฀been฀
growing฀in฀the฀USA,฀partly฀because฀of฀a฀lack฀of฀government฀control฀of฀the฀trace฀metal฀
problem.฀The฀CDC฀has฀also฀documented฀a฀large฀increase฀in฀mild฀luorosis฀now฀that฀water฀
luoride฀is฀combined฀with฀toothpastes.฀Nowadays,฀the฀ability฀to฀control฀luoride฀dosage฀by฀
tablets,฀toothpastes฀and฀professional฀administration฀has฀improved฀enormously฀in฀the฀USA.฀
Studies฀on฀the฀Kilmarnock฀population฀since฀1990฀nevertheless฀indicate฀an฀increasing฀inci-
dence฀of฀dental฀caries,฀indicating฀that฀self-dosage฀is฀inadequate฀and฀there฀is฀a฀balance฀to฀
safety฀ with฀ disease฀ prevention.฀ One฀ solution฀ would฀ be฀ to฀ reduce฀ water฀ luoridation฀ to฀฀
0.7–0.9฀ppm฀in฀the฀USA฀and฀better฀enforce฀luoride฀manufacture.
Adults฀exposed฀for฀long฀periods฀to฀greater฀luoride฀concentrations฀in฀the฀aluminum฀and฀
glass฀industries฀exhibit฀skeletal฀luorosis.฀In฀the฀early฀1980s,฀there฀were฀reports฀of฀crip-
pling฀ bone฀ diseases฀ caused฀ by฀ 2฀ or฀ more฀ ppm฀ luoride฀ in฀ the฀ water฀ in฀ India฀ and฀ other฀
countries.฀Naturally฀occurring฀luoridated฀water฀often฀contains฀increased฀amounts฀of฀heavy฀
metals฀and฀these฀could฀have฀caused฀the฀skeletal฀effects.฀In฀the฀USA฀in฀the฀early฀twentieth฀
century,฀children฀drinking฀2–10฀ppm฀water฀exhibited฀enamel฀luorosis฀and฀enamel฀mottling฀
but฀no฀bone฀disease.
If฀the฀luoride฀intake฀is฀maintained฀above฀10฀ppm,฀the฀inhibition฀of฀enolase฀(Sects.฀16.2.2฀
and฀ 16.3.1)฀ inhibits฀ both฀ anaerobic฀ and฀ aerobic฀ glycolysis.฀ In฀ addition,฀ skeletal฀ defects฀
may฀be฀caused฀by฀improper฀collagen฀synthesis฀inhibiting฀proper฀bone฀formation฀in฀฀children.฀
Skeletal฀luorosis฀in฀children฀and฀luoride฀poisoning฀in฀adults฀is฀therefore฀accompanied฀by฀
296 16 Fluoride

an฀extreme฀lack฀of฀energy฀partly฀due฀to฀the฀inhibition฀of฀enolase,฀but฀also฀to฀other฀effects฀
indicated฀below.
The฀irst฀is฀the฀inhibition฀of฀gluconeogenesis.฀Mg+2฀ions฀activate฀fructose฀1,6-bisphos-
phate฀phosphatase฀and฀convert฀it฀to฀phosphate฀and฀fructose฀6-phosphate.฀The฀latter฀is฀a฀
precursor฀of฀glucose฀and฀glycogen฀from฀glycogenic฀amino฀acids฀and฀of฀monosaccharides฀
other฀than฀glucose.฀Fluoride฀chelates฀Mg+2฀and฀inhibits฀fructose฀6-phosphate฀production฀
and฀gluconeogenesis.฀The฀resultant฀inability฀to฀make฀glucose฀and฀glycogen฀from฀noncar-
bohydrate฀precursors฀is฀probably฀a฀second฀factor฀contributing฀to฀the฀lack฀of฀energy฀associ-
ated฀with฀luoride฀poisoning.
The฀second฀is฀an฀inhibition฀of฀protein฀synthesis.฀Fluoride฀interacts฀with฀magnesium฀
ions฀ a฀ cofactor฀ for฀ binding฀ of฀ the฀ large฀ (60฀ S)฀ ribosomal฀ subunit฀ to฀ the฀ small฀ subunit฀
tRNA/mRNA฀complex฀during฀translation฀initiation฀in฀eukaryotes.฀Fluoride฀chelates฀Mg+2฀
ions฀and฀prevents฀the฀protein฀synthesis฀initiation฀complex฀from฀forming.฀The฀inhibition฀
probably฀occurs฀in฀association฀with฀the฀Mg+2฀catalyzed฀GTPase-mediated฀hydrolysis฀of฀
eukaryotic฀initiation฀factor-2฀(eIF2)฀by฀the฀large฀ribosomal฀subunit.
A฀third,฀more฀controversial฀effect฀is฀the฀luoride-mediated฀inhibition฀of฀enzymes฀that฀
remove฀ reactive฀ oxygen฀ species฀ from฀ the฀ body.฀ The฀ enzymes฀ catalase,฀ peroxidase,฀ and฀
superoxide฀ dismutase฀ are฀ primarily฀ responsible฀ for฀ eliminating฀ harmful฀ oxygen฀ species฀
during฀respiration.฀The฀reaction฀of฀oxygen฀with฀cytochrome฀oxidase฀is฀only฀50–70%฀efi-
cient฀and฀the฀remaining฀30–50%฀of฀oxygen฀is฀released฀in฀an฀incompletely฀reduced฀form฀
known฀as฀the฀Reactive฀Oxygen฀Species฀(ROS).฀All฀these฀products฀are฀highly฀reactive฀in฀
the฀cell฀despite฀some฀being฀relatively฀stable.฀These฀three฀enzymes฀prevent฀the฀more฀stable฀
ROS฀ compounds฀ from฀ being฀ available฀ to฀ react฀ with฀ proteins,฀ lipids,฀ carbohydrates,฀ or฀
nucleic฀acids฀within฀a฀cell.฀Ascorbate฀is฀secondary฀to฀this฀protection฀(Sect.฀7.4.1).฀At฀high฀
concentrations฀of฀luoride฀in฀the฀water฀supply฀(>50฀ppm)฀these฀protective฀enzymes,฀are฀
inhibited,฀but฀there฀is฀no฀evidence฀for฀this฀at฀1฀ppm.
Hydrogen฀peroxide฀is฀the฀most฀common,฀stable฀by-product฀of฀respiration.฀Each฀oxygen฀
atom฀has฀received฀only฀one฀electron฀instead฀of฀two฀and฀it฀shares฀the฀second฀electron฀with฀
a฀hydrogen฀atom฀to฀give฀H2O2฀(hydrogen฀peroxide)฀instead฀of฀water฀(Fig.฀16.8a).฀Catalase฀
produces฀a฀molecule฀of฀water฀and฀a฀molecule฀of฀oxygen฀from฀hydrogen฀peroxide.฀A฀luo-
ride฀ion฀(F−)฀competes฀with฀a฀peroxide฀ion฀(−OOH)฀for฀the฀iron฀atom฀within฀a฀heme฀mole-
cule฀at฀the฀catalytic฀center฀of฀catalase.฀The฀catalase฀can฀no฀longer฀bind฀peroxide฀anions฀and฀
peroxide฀ions฀from฀hydrogen฀peroxide฀accumulate฀in฀the฀tissues.
Peroxide฀and฀other฀partially฀reduced฀oxygen฀species฀often฀attach฀to฀an฀organic฀group,฀per-
haps฀the฀amino฀acid฀side฀chain฀of฀a฀nearby฀protein฀or฀fatty฀acid,฀and฀form฀organic฀peroxides.฀
Peroxidase฀binds฀to฀such฀peroxides฀and฀converts฀them฀to฀an฀alcohol฀by฀releasing฀the฀second฀
oxygen฀atom฀that฀is฀reduced฀to฀water฀by฀2฀molecules฀of฀glutathione฀(Fig.฀16.8b).฀The฀oxidized฀
glutathione฀(GSSG)฀is฀reduced฀(Chap.฀7,฀Fig.฀7.8฀bottom฀half).฀Peroxidase฀functions฀like฀cata-
lase,฀but฀utilizes฀selenium฀(Se+2)฀instead฀of฀iron฀at฀its฀active฀site.฀Fluoride฀competes฀for฀Se+2,฀
preventing฀the฀peroxide฀substrate฀from฀binding.฀Organic฀peroxides฀accumulate฀in฀the฀tissues฀
and฀mediate฀spontaneous฀reactions฀that฀interfere฀with฀many฀biological฀processes.
Superoxide฀dismutase฀converts฀many฀other฀types฀of฀reactive฀oxygen฀species฀to฀oxygen฀gas,฀
hydrogen฀peroxide,฀or฀water.฀This฀enzyme฀binds฀to฀these฀various฀reactive฀oxygen฀species฀by฀a฀
molybdenum฀ion฀(Mo+2)฀at฀its฀active฀center.฀Again,฀luoride฀will฀bind฀to฀the฀molybdenum฀ion฀
and฀ inhibit฀ this฀ enzyme’s฀ activity.฀ Reactive฀ oxygen฀ species฀ accumulate฀ in฀ the฀ tissues฀ and฀
mediate฀rapid,฀spontaneous฀reactions฀that฀interfere฀with฀many฀biological฀processes.
16.3.2. Fluoride Toxicity 297

a
H2O2 (Hydrogen peroxide)
HOOH H+฀+฀−OOH
peroxide
anion
Catalase reaction
2H2O2 H2O +฀O2
b
Peroxidase reaction
R−OOH +฀2GSH
peroxide
species
R−OH +฀GSSG
฀฀฀฀฀฀฀฀฀฀฀฀+
H2O

Fig. 16.8฀ Fluoride฀promotes฀retention฀of฀reactive฀oxygen฀species.฀The฀peroxide฀ion฀(−OOH)฀is฀a฀

reactive฀oxygen฀species฀derived฀from฀hydrogen฀peroxide฀or฀attached฀to฀an฀organic฀molecule฀(R−).฀
(a)฀ Hydrogen฀ peroxide฀ is฀ eliminated฀ by฀ catalase฀ that฀ converts฀ it฀ to฀ oxygen฀ gas฀ and฀ water.฀฀
(b)฀Organic฀peroxide฀is฀eliminated฀by฀peroxidase฀converting฀it฀to฀the฀corresponding฀organic฀alco-
hol฀and฀water฀with฀assistance฀from฀glutathione฀(GSH),฀which฀loses฀its฀electrons฀and฀is฀oxidized฀
(GSSG).฀Fluoride฀inhibits฀both฀enzymes,฀causing฀organic฀peroxides฀to฀accumulate,฀see฀text

Fluoride฀causes฀mottled฀enamel฀at฀1฀ppm,฀toxic฀effects฀on฀bone฀at฀>10฀ppm฀and฀more฀
general฀effects฀at฀>50฀ppm.฀Fluoride฀poisoning฀is฀especially฀associated฀with฀a฀lack฀of฀
energy฀due฀to฀its฀inhibiting฀enolase฀and฀therefore฀of฀glycolysis.฀If฀taken฀at฀high฀levels฀
to฀ control฀ osteoporosis,฀ it฀ inhibits฀ osteoblast฀ activity฀ more฀ than฀ osteoclast฀ activity,฀
resulting฀in฀an฀increased฀frequency฀of฀bone฀fractures.฀In฀the฀1940s,฀a฀bauxite฀plant฀to฀
extract฀aluminum฀in฀the฀Fort฀William฀area฀of฀rural฀west฀Scotland฀poisoned฀local฀cattle฀
by฀emitting฀luoride฀at฀50–100฀ppm.฀These฀high฀levels฀of฀luoride฀inhibit฀gluconeogen-
esis฀by฀binding฀to฀Mg+2฀ions฀that฀activate฀fructose฀bisphosphate฀phosphatase฀and฀pro-
tein฀ synthesis฀ by฀ inhibiting฀ the฀ Mg+2฀ ion฀ association฀ of฀ ribosomal฀ subunits฀ during฀
eukaryotic฀protein฀synthesis฀initiation.฀These฀high฀levels฀of฀luoride฀also฀inhibit฀perox-
ide฀ and฀ other฀ reactive฀ oxygen฀ species฀ elimination฀ by฀ binding฀ to฀ iron,฀ selenium฀ and฀
molybdenum฀ions฀at฀the฀respective฀catalytic฀centers฀of฀catalase,฀peroxidase,฀and฀super-
oxide฀dismutase.฀At฀levels฀of฀1฀ppm,฀luoride฀shows฀none฀of฀these฀toxic฀effects฀and฀is฀
safe฀and฀effective฀in฀the฀water฀supply฀and฀toothpastes฀to฀prevent฀caries.
 Index

A Amylase
Aa฀leukotoxin฀(Aa฀Ltx) a-amylase,฀219–220
binding,฀263 b-amylase฀and฀g-amylase,฀221
Repeats฀in฀toxin฀(RTX)฀family,฀261 detection
secretion,฀261 electrophoresis,฀225
structure฀and฀synthesis,฀261–262 pyroglutamate฀formation,฀226–227
Activated฀partial฀thromboplastin฀time฀(APTT),฀ mechanism฀of฀action
200–201 catalysis,฀222
Acute฀necrotizing฀ulcerative฀gingivitis฀ domains,฀224–225
(ANUG),฀259–260 enzymes,฀223
Adamalysin,฀179–180 Annexins
Agglutinin,฀salivary crystal฀structure฀and฀membrane฀insertion฀
binding,฀228–229 model,฀138–139
SRCR฀domains,฀229–230 isolation,฀140
Aggrecan Apoptosis
fetal฀bovine฀cartilage,฀96 chronic฀periodontitis,฀246–247
N-terminal฀and฀C-terminal฀domains,฀98 intracellular฀induction,฀247–248
peptide฀domains,฀95 mechanisms
sources,฀95 extrinsic฀and฀intrinsic฀pathways,฀
Aggregatibacter฀actinomycetemcomitans฀฀ 249–250
(Aa),฀260 mitochondrial฀intermembrane฀space฀
Aggressive฀periodontitis molecules,฀251
Aa฀leukotoxin Ascorbate
binding,฀263 antioxidants฀and,฀109–112
LAP฀severity฀enhancement,฀mutations,฀ depletion,฀scurvy,฀110
263–265 norepinephrine฀synthesis,฀111
Repeats฀in฀toxin฀(RTX)฀family,฀261 oxidation,฀107,฀112
secretion,฀261 structure,฀109
structure฀and฀synthesis,฀261,฀262 synthesis,฀110,฀111
cytolethal฀distending฀toxin฀(Cdt),฀฀ uses,฀111
260,฀264–266 Atomic฀structure
generalized,฀259–260 chemical฀bonds
localized,฀260–261 covalent,฀7
Amelogenin฀biomineralization electrostatic,฀6–7
domains฀and฀exon฀structure,฀147,฀148 hydrophobic,฀8
mutations,฀150 polarized฀covalent,฀7–8
processing,฀147,฀149 periodic฀table,฀1–2

M.฀Levine,฀Topics฀in฀Dental฀Biochemistry, 299
DOI:฀10.1007/978-3-540-88116-2,฀©฀Springer-Verlag฀Berlin฀Heidelberg฀2011
300 Index

elements,฀5 Bleeding฀time฀(BT),฀201
energy฀production฀mechanism Blood฀coagulation
fermentation,฀10 bleeding฀and฀clotting฀problems,฀176–177
NAD+฀and฀NADP+,฀9 blood฀vessel฀injury,฀178–180
respiration,฀9–10 determination,฀laboratory฀tests,฀200–201
Autoinducer-2฀(AI-2),฀233 drugs
clot฀formation,฀retardation,฀198–199
clotting฀enhancement,฀199–200
B inordinate฀clot฀formation,฀inhibition,฀198
Bacterial฀cell pathogenic฀thrombus/embolus฀฀
outer฀surface removal,฀196–198
imbriae/pili,฀14 platelet฀activation,฀inhibition,฀199
lipopolysaccharide฀(LPS),฀15–16 extrinsic฀pathway,฀183–185
polysaccharide฀(glycan)฀capsule,฀14 factors,฀182
structure,฀13–14 ibrin฀blood฀clot
Basal฀laminas production฀and฀prevention,฀188–190
dental฀epithelial฀attachment removal,฀190–192
epidermolysis฀bullosa฀(EB),฀72–73 gamma-carboxyglutamate฀domain,฀180–183
gingival฀composition,฀72 hemophiliacs,฀187–188
desmosomes inhibitors,฀192,฀195
differentiation,฀74 intrinsic฀pathway,฀184,฀185
molecular฀composition,฀76 mechanism,฀184–186
plaques,฀75 platelets
epithelium capillary฀surface฀interactions,฀179–180
skin,฀74–75 plug฀formation,฀178–179
stratiied,฀73 protein฀C,฀196
transitional,฀73 protein฀S,฀196
type฀IV฀collagen,฀66 prothrombin฀cleavage,฀186
type฀VII฀collagen,฀65–66 thrombin–cofactor฀interactions,฀186–187
hemidesmosomes฀(HDs) thrombomodulin,฀195
ilaments,฀67–68 unwanted฀clotting฀prevention,฀192–195
laminin-5฀ilament,฀71 vascular฀system,฀175–176
model,฀70 von฀Willebrand฀factor฀(VWF)
protein฀composition,฀68–69 multimeric,฀179–180
type฀XVII฀collagen,฀70–71 structure,฀178
keratin Blood฀thinners,฀198
junctional฀and฀gingival฀epithelia,฀฀ Bone฀morphogenic฀proteins฀(BMPs),฀82–83
78 Bone฀remodeling
molecular฀composition,฀75–76 calcitonin
structure,฀77 effects,฀172–173
laminins osteoporosis,฀173
composition,฀65 calcium฀metabolism฀and
structure,฀67 parathyroid฀hormone฀and฀calcitriol,฀
oral฀and฀junctional฀epithelium 165–168
biochemistry฀and฀metabolism,฀79 vitamin฀D,฀calcitriol,฀and฀calbindins,฀
gingivectomy,฀78 168–171
integrin,฀80 demineralization฀and฀remineralization,฀
keratin฀composition,฀78 157–158
periodontium,฀78–79 osteoclasts
structure,฀66 differentiation,฀160–161
Beaded฀collagen฀ilaments inlammation฀and,฀161–163
ibrillin,฀56 ion฀and฀proton฀transport,฀159–160
type฀VI,฀56,฀57 membrane฀functions,฀154–155
Biological฀mineralization.฀See฀Mineralization metabolite฀and฀ion฀exchange,฀155–156
Index 301

microcracks,฀153 cell฀surface฀binding,฀41
tunneling,฀153–154 and฀elastin฀content,฀tissues,฀34
osteoporosis iber฀arrangements,฀34
calcitonin฀and฀PTH฀therapy,฀173 ibrillar,฀32
causes฀and฀therapies,฀163–164 non-ibrillar,฀34
proteolysis,฀bone฀resorbing฀compartment,฀157 striated฀appearance,฀32,฀33
rickets฀and฀osteomalacia,฀171 types,฀30,฀31
components,฀29–30
elastic฀iber฀system
C elastin,฀36
Calcitonin oxytalan฀ibers,฀35
effects,฀172–173 types,฀34,฀35
osteoporosis,฀173 ibronectin,฀40,฀41
Cartilage฀collagens gingiva
N-and฀C-terminal฀noncollagenous฀domains,฀ ibres,฀38–40
96,฀98 free฀and฀attached,฀37,฀38
structure,฀97 glycosaminoglycans,฀36
Catalase,฀296 integrins,฀40
Cathepsin฀L,฀260 periodontium,฀36–37
Cdt.฀See฀Cytolethal฀distending฀toxin฀(Cdt) thrombospondins
Cellulose,฀27 gene฀family฀domain฀organization,฀
Cementum,฀36–37 41–42
Chondroitin฀sulfate groups,฀41–43
structure,฀93 TSP-1,฀41
synthesis,฀linker฀glycan,฀94 TSP-2,฀42–43
Collagen transforming฀growth฀factor-b฀(TGF-b),฀
cartilage 41–42
N-and฀C-terminal฀noncollagenous฀ Coumarins,฀198–199
domains,฀96,฀98 Cyclooxygenase฀(COX),฀253–254
structure,฀97 Cytolethal฀distending฀toxin฀(Cdt),฀260,฀
cell฀surface฀binding,฀41 264–266
Ehlers-Danlos฀syndrome฀(EDS)
mixed฀function฀oxidases,฀107–108
types,฀105,฀106 D
elastin฀content,฀tissues,฀34 Dark฀reaction
iber฀arrangements,฀34 Calvin฀cycle,฀23
ibrillar,฀32 carbon฀dioxide฀assimilation,฀22
glycosaminoglycan฀interactions,฀98–99 Decayed,฀missing฀and฀illed฀teeth฀(DMFT)฀
intracellular฀synthesis,฀101–103 index,฀289,฀290
non-ibrillar,฀34 Dental฀bioilms,฀269–270
polypeptide฀mutations฀effects,฀104–105 Dental฀caries
propeptides,฀50 advanced฀(see฀Dentinal฀dental฀caries)
striated฀appearance,฀32,฀33 bacterial฀lactic฀acid,฀sources฀of,฀277
stromal฀proteins฀and bioilms,฀269–270
extracellular฀matrix฀protein฀฀ cavities฀in฀animals,฀270–272
components,฀124 deinition,฀267,฀268
matrilysin฀connective฀tissue฀degrading฀ diet฀effects฀on,฀268
enzyme,฀126 distribution฀in฀dental฀students,฀278,฀279
matrix฀metalloproteases,฀124–125 experience฀variation฀in฀humans,฀278–279
types,฀30,฀31 immunity฀and฀susceptibility,฀282–283
Collagenases฀and฀gelatinases,฀127–128 period฀of฀incease,฀268
Connective฀tissue฀extracellular฀matrix saliva฀causes,฀281–282
basal฀lamina฀organizations,฀30,฀31 severity฀factors,฀278,฀279
collagen Streptococcus฀mutans
302 Index

glucosyl฀transferase,฀275 Enamel฀biomineralization
identiication,฀cariogenic฀microbiota,฀ amelogenin
270,฀271 domains฀and฀exon฀structure,฀147,฀148
mutan,฀structure฀and฀synthesis,฀273,฀276 mutations,฀150
sucrose฀and฀levan,฀274 processing,฀147,฀149
sucrose฀consumption,฀267–269 matrix฀development,฀144–146
susceptibility฀variation,฀bacterial฀causes,฀ proteins฀involved,฀146–147
279–280 vitamins,฀151
Dental฀epithelial฀attachment vs.฀bone,฀150–151
epidermolysis฀bullosa฀(EB),฀72–73 Enamel฀luorosis
gingival,฀composition,฀72 DMFT฀index,฀289,฀290
Dentin,฀36–37 luoride
Dentinal฀dental฀caries,฀277–278 cavity฀protection,฀289–290
Desmosomes incorporation,฀287–289
differentiation,฀74 properties,฀285
molecular฀composition,฀76 protection฀mechanism,฀291–294
plaques,฀75 systemic฀effects,฀294–295
Dihydroxyacetone฀phosphate฀(DHAP),฀฀ toxicity,฀295–297
24–25 in฀water฀supply,฀286–287
Disintegrins,฀40–41 geographic฀location,฀US,฀287,฀288
levels,฀288,฀289
Enamelysin,฀126–127
E Enolase,฀293
EDS.฀See฀Ehlers-Danlos฀syndrome฀(EDS) Epidermolysis฀bullosa฀(EB),฀72–73
Ehlers-Danlos฀syndrome฀(EDS) Epithelium
mixed฀function฀oxidases,฀107–108 dental฀epithelial฀attachment
types,฀105,฀106 epidermolysis฀bullosa฀(EB),฀72–73
Eicosanoids gingival,฀composition,฀72
functions,฀proinlammatory,฀255 oral฀and฀junctional
periodontal฀repair฀and,฀252 biochemistry฀and฀metabolism,฀79
structure gingivectomy,฀78
cyclooxygenase฀(COX),฀253–254 integrin,฀80
families,฀252 keratin฀composition,฀78
lipoxygenase,฀254–255 periodontium,฀78–79
Elastic฀ibers฀and฀proteoglycans skin,฀74–75
ibrillin stratiied,฀73
bone฀morphogenic฀proteins฀(BMPs),฀ transitional,฀73
82–83
calcium-binding฀domains,฀85
cbEGF฀domains,฀81–82 F
characteristics,฀81 Fibrillar฀collagens
hinged฀model,฀83 array,฀gap฀functions,฀51
individual฀domains,฀82 collagen฀helix,฀46,฀48
mutations,฀84–85 iber฀cross-linking฀(see฀Tropocollagen)
transglutaminase฀reaction,฀84 formation
structure฀and฀synthesis,฀88 helical฀wrapping,฀51
tropoelastin procollagen,฀precursor,฀50
cross-linking,฀86–87 hydroxyproline฀and฀hydroxylysine฀
human฀domains,฀86 structures,฀46,฀47
synthesis,฀85 polypeptide฀composition,฀45,฀47
Elastic฀iber฀system propeptides,฀50
elastin,฀36 source,฀52
oxytalan฀ibers,฀35 superfamily,฀55
types,฀34,฀35 triple฀helix฀formation,฀46,฀48
Index 303

tropocollagen Eikenella฀corrodens,฀234
aminoacid฀composition,฀45,฀46 lysine฀decarboxylase฀reaction,฀235–236
b-polypeptides฀formation,฀52,฀53 Glutathione฀(GSH),฀111
pyridinoline฀formation,฀51,฀54 Glycosaminoglycans฀(GAGs)
standard฀model,฀49 chondroitin฀sulfate
super-arrays,฀51 structures,฀93
triple฀helix,฀46,฀49 synthesis,฀linker฀glycan,฀94
Fibrillar฀procollagen฀processing collagen฀interactions,฀98–99
astacins,฀121 connective฀tissue฀extracellular฀matrix,฀36
integrin฀and฀disintegrin,฀122–123 hyaluronan
N-and฀C-proteinases฀activation,฀122 hyaluronidase,฀92
PCP฀and฀PNP,฀domain฀organization,฀ structure,฀89
122–123 synthesis,฀90–91
polypeptide฀motifs,฀120–121 UDP-monosaccharide,฀94
Fibrillin volume฀comparison,฀92
bone฀morphogenic฀proteins฀(BMPs),฀82–83 proteo-GAGs
calcium-binding฀domains,฀85 aggrecan,฀95–96
cbEGF฀domains,฀81–82 cartilage฀collagen฀synthesis,฀97–98
characteristics,฀81 versican,฀95
hinged฀model,฀83 synthesis,฀93–94
individual฀domains,฀82 synthetases,฀94
mutations,฀84–85
transglutaminase฀reaction,฀84
Fibronectin,฀40,฀41,฀188 H
Fluoride.฀See฀also฀Enamel฀luorosis Hemidesmosomes฀(HDs)
luorides,฀6 ilaments,฀67–68
identiication฀in฀water,฀289–290 laminin-5฀ilament,฀71
mottled฀enamel,฀relationship฀with,฀฀ model,฀70
286–287 protein฀composition,฀68–69
properties,฀285 type฀XVII฀collagen,฀70–71
protection฀mechanism,฀291–294 Hemostasis.฀See฀Blood฀coagulation
systemic฀effects,฀294–295 Heparin฀cofactor฀II฀(HCII),฀192,฀193
toxicity Hyaluronan
inhibition,฀296 connective฀tissue฀extracellular฀matrix,฀36
skeletal฀luorosis,฀295 hyaluronidase,฀92
Fluoroapatite,฀291 structure,฀89
synthesis,฀90–91
UDP-monosaccharide,฀94
G volume฀comparison,฀92
Gelatinases,฀127–128 Hydrogen฀peroxide,฀296
Gingiva Hydroxyapatite,฀291
ibres,฀38–40
free฀and฀attached,฀37,฀38
Gingival฀crevicular฀luid฀(GCF),฀208 I
Gingivitis Integrins,฀40
antiinlammatory฀cytokines,฀243–244 collagen฀binding
detection,฀231–232 functions,฀61,฀63
drugs,฀236–237 sites,฀61
humans฀and฀animals,฀232–233 domain฀structure฀and฀conformations,฀59–61
IL-1,฀host฀mediator,฀241 functions,฀58
microbiota ligand-integrin฀receptor฀interactions,฀59,฀62
autoinducer-2฀(AI-2),฀233 subunit฀combinations,฀58,฀59
bacterial฀colonization฀and฀฀ Interleukin-1฀(IL-1)
succession,฀235 forms,฀238
304 Index

host฀mediator,฀241 synthesis,฀134–135
inlammation vitamins,฀151
causes,฀239 vs.฀enamel,฀150–151
gingival,฀241 calcium฀and฀phosphate฀ions
structures,฀240 osteoblast฀transport,฀136–138
Interstitial฀luid,฀stromal฀matrix,฀43,฀44 rupture฀and฀precipitation,฀138
Intracellular฀collagen฀synthesis calcium฀phosphate฀precipitation,฀129–130
processing฀in฀endoplasmic฀reticulum,฀101,฀102 calcium฀transporter฀proteins,฀138–140
procollagen cementum,฀133
cis-Golgi฀cisternae,฀functions฀in,฀102,฀103 deinition,฀129
formation,฀101,฀102 dentin,฀133
enamel฀synthesis
matrix฀development,฀144–146
K mechanism,฀147–150
Keratin proteins฀involved,฀146–147
junctional฀and฀gingival฀epithelia,฀78 vitamins,฀151
molecular฀composition,฀75–76 vs.฀bone,฀150–151
structure,฀77 endochondral฀ossiication,฀134
Kunitz฀inhibitor,฀192 intramembranous฀ossiication,฀134
non-collagenous฀bone฀proteins,฀฀
142–143
L nucleation,฀131–132
Laminins osteoblast฀transport,฀136–138
composition,฀65 osteocalcin,฀143–144
structure,฀67 osteoid฀matrix฀secretion
LAP.฀See฀Localized฀aggressive฀periodontitis bone฀synthesis,฀135,฀136
Leukotoxin฀(Ltx),฀260,฀261,฀263–264 vesicle฀composition,฀136,฀137
Light฀reaction phosphate฀transporter฀proteins฀and฀
ATPase฀orientation,฀19,฀21 pyrophosphate
phosphorylation฀and฀electron฀transport,฀19,฀20 PiT-2฀transporter,฀140–141
process,฀18–19 pyrophosphate฀(PPi)฀removal,฀142
Lipoxygenase Mottled฀enamel.฀See฀Enamel฀luorosis
characteristics,฀254–255 Mucin,฀salivary
inlammation฀mediated฀resolution,฀255–257 ABO฀glycan฀sequences,฀218–219
Localized฀aggressive฀periodontitis฀(LAP),฀ composition
260–261 MG1฀and฀MG2,฀212–213
Lysine฀hydroxylase฀(LH),฀107–108 sulfation,฀211
Lysosomes,฀44 glycan฀composition
chemical฀structures,฀214
sialic฀acid฀synthetase,฀214–215
M Mutan
Matrilysin฀(MMP) structure,฀272,฀273
connective฀tissue฀degrading฀enzyme,฀126 synthesis,฀275,฀276
domain฀arrangement,฀124–125
Metal฀ion฀dependent฀adhesion฀site฀(MIDAS),฀
59 N
Metzincin฀family Neutrophils
activation,฀117–119 activation,฀241–242
catalysis,฀115–117 Periostat,฀243
Mineralization Non-ibrillar฀collagens
apatite฀crystal฀substitutions,฀131 iber-modifying,฀55–56
apatite฀precipitate,฀130–131 structure,฀56
bone฀tissue Nonsteroidal฀antiinlammatory฀drugs฀
structure,฀132–133 (NSAIDs),฀257–258
Index 305

O asaccharolytic฀fermentation,฀12–13
Oral฀and฀junctional฀epithelium saccharolytic฀fermentation,฀11
biochemistry฀and฀metabolism,฀79 detection,฀231–232
gingivectomy,฀78 eicosanoids
integrin,฀80 functions,฀proinlammatory,฀255
keratin฀composition,฀78 periodontal฀repair฀and,฀252
periodontium,฀78–79 structure,฀252–255
Osteocalcin,฀143–144 gingivitis
Osteoclasts antiinlammatory฀cytokines,฀243–244
differentiation,฀160–161 drugs,฀236–237
inlammation฀and humans฀and฀animals,฀232–233
bone฀metabolism฀control,฀162,฀163 IL-1,฀host฀mediator,฀241
cytokines,฀161–162 microbiota,฀233–236
TNF-a,฀162 lipoxygenase-mediated฀resolution,฀
ion฀and฀proton฀transport,฀159–160 inlammation,฀255–257
membrane฀functions,฀154–155 mammalian฀cells฀recognition,฀237–238
metabolite฀and฀ion฀exchange,฀155–156 neutrophils
microcracks,฀153 activation,฀241–242
tunneling,฀153–154 Periostat,฀243
Osteoporosis PAMP
calcitonin฀and฀PTH฀therapy,฀173 induce฀PRRs,฀238–241
causes฀and฀therapies,฀163–164 stimulation,฀244–246
Oxytalan฀ibers,฀35 Periodontal฀ligaments,฀37
Periodontitis
aggressive฀(see฀Aggressive฀periodontitis)
P chronic
Pancreatic฀amylase,฀225–227 apoptosis,฀246–247
Pathogen-associated฀molecular฀patterns฀ detection,฀231–232
(PAMPs) humans฀and฀animals,฀232–233
induce฀PRRs intrinsic฀apoptotic฀pathway,฀249–250
forms,฀IL-1,฀238 microbiota,฀233–236
gingival฀inlammation,฀241 Phosphate฀(Pi)
structure,฀IL-1,฀240 calcium฀phosphate฀precipitation,฀฀
TNF-a,฀238–239 129–130
stimulation,฀244–246 Dihydroxyacetone฀phosphate฀(DHAP),฀฀
Pattern-recognition฀receptors฀(PRRs) 24–25
mammalian฀cells฀recognition,฀237–238 Phosphatase฀of฀the฀tensin฀family฀of฀
PAMPs phospholipid฀phosphatases฀(PTEN),฀
forms,฀IL-1,฀238 264
gingival฀inlammation,฀241 PiT-2฀transporter,฀140–141
structure,฀IL-1,฀240 pyrophosphate฀(PPi)฀removal,฀142
TNF-a,฀238–239 Src฀homology฀2฀(SH2)฀domain฀in฀addition฀to฀
Periodic฀table,฀1–2 its฀inositol฀phosphatase฀activity฀
Periodontal฀disease (SHIP),฀264–265
antiinlammatory฀drugs,฀257–258 Phosphotransferase฀system฀(PTS),฀277
apoptosis Photosynthesis
chronic฀periodontitis,฀246–247 dark฀reaction
intracellular฀induction,฀247–248 Calvin฀cycle,฀23
mechanisms,฀249–251 carbon฀dioxide฀assimilation,฀22
chronic฀periodontitis light฀reaction
apoptosis,฀246–247 ATPase฀orientation,฀19,฀21
humans฀and฀animals,฀232–233 phosphorylation฀and฀electron฀transport,฀
microbiota,฀233–236 19,฀20
and฀dental฀caries process,฀18–19
306 Index

phosphoglycerate฀utilization,฀24–26 amylase
and฀respiration,฀17,฀18 a-amylase,฀219–220
role,฀living฀organisms,฀17,฀18 b-amylase฀and฀g-amylase,฀221
sucrose detection฀of,฀225–227
role,฀plant฀growth฀and฀development,฀ mechanism฀of฀action,฀221–225
26–27 bacteria฀and฀host฀leukocyte฀products,฀
synthesis,฀24,฀25 208–209
Plasmin blood฀group฀antigens,฀216–219
ibrin฀dissolution,฀190,฀191 cell฀biology,฀salivary฀glands,฀203,฀204
functions,฀190 collection,฀203–204
Platelet฀function฀analyzer,฀201 composition,฀204–205
Platelet฀plug,฀178–179 functions,฀205–207
Procollagen,฀101,฀102 innate฀immunity,฀207–208
Proline฀hydroxylase฀(PH),฀107 mucin฀glycan฀sequences,฀216–219
Prostaglandin฀I2฀(PGI2),฀192 pancreatic฀amylase,฀225–227
Protein฀disulide฀isomerase฀(PDI),฀107,฀฀ physiology฀and฀biochemistry,฀209–211
108,฀111 proline-rich฀proteins,฀227–228
Proteo-glycosaminoglycan฀(Proteo-GAG) salivary฀mucin
aggrecan composition,฀211–213
fetal฀bovine฀cartilage,฀96 glycan฀composition,฀213–216
N-terminal฀and฀C-terminal฀domains,฀98 Scurvy,฀110–111.฀See฀also฀Ascorbate
peptide฀domains,฀95 Sharpey’s฀ibers,฀39
sources,฀95 Short-chain฀carboxylic฀acids฀(SCCA),฀280
cartilage฀collagens Signal฀recognition฀particle฀(SRP),฀209
N-and฀C-terminal฀noncollagenous฀ Src฀homology฀2฀(SH2)฀domain฀in฀addition฀
domains,฀96,฀98 to฀its฀inositol฀phosphatase฀activity฀
structure,฀97 (SHIP),฀264–265
versican,฀95 Streptococcus฀mutans
Proteolysis,฀bone฀resorbing฀compartment,฀157 adhesion฀antigen,฀282,฀283
Prothrombin฀time฀(PT)฀test,฀200 glucosyl฀transferase,฀275
identiication,฀cariogenic฀microbiota,฀฀
270,฀271
Q mutan,฀structure฀and฀synthesis,฀273,฀276
Quorum฀sensing,฀233 sucrose฀and฀levan,฀274
Streptokinase,฀198
Stroma
R matrix฀(see฀Connective฀tissue฀extracellular฀
Radioactive฀isotopes matrix)
ancient฀life฀forms฀and฀climate฀changes,฀5 nutrition,฀43–44
importance,฀4 turnover,฀inlammation,฀and฀bone฀loss,฀44
isotopes฀date฀paleontology฀sample,฀4 Stromelysins,฀126
Reactive฀oxygen฀species฀(ROS) Sucrose
production,฀296 production฀(see฀Photosynthesis)
retention,฀296,฀297 role,฀plant฀growth฀and฀development,฀26–27
Repeats฀in฀toxin฀(RTX),฀261 structure,฀273,฀274
RTX.฀See฀Repeats฀in฀toxin฀(RTX) synthesis,฀24,฀25
Superoxide฀dismutase,฀296

S
Saliva T
acquired฀immunity,฀208 TdeA.฀See฀Toxin฀and฀drug฀export฀protein฀฀
agglutinin A฀(TdeA)
binding,฀228–229 Thrombomodulin฀(Tm),฀195
SRCR฀domains,฀229–230 Thromboplastin,฀183–185
Index 307

Thrombospondins von฀Willebrand฀factor฀(VWF)
gene฀family฀domain฀organization,฀41–42 multimeric,฀179–180
groups,฀41–43 structure,฀178
TSP-1,฀41
TSP-2,฀42–43
Thromboxane฀A2฀(TXA2),฀178–179 W
Tissue฀factor฀(TF).฀See฀Thromboplastin Wafarin,฀182
Tissue฀factor฀pathway฀inhibitor฀(TFPI),฀195
Tissue฀plasminogen฀activator฀(tPA),฀190,฀191
Toxin฀and฀drug฀export฀protein฀A฀(TdeA),฀261 Z
Tranexamic฀acid,฀199 Z-dependent฀protease฀inhibitor฀(ZPI),฀฀
Transforming฀growth฀factor-b฀(TGF-b),฀41–42 193,฀195
Tropocollagen Zincins
aminoacid฀composition,฀45,฀46 collagen฀and฀stromal฀proteins
b-polypeptides฀formation,฀52,฀53 extracellular฀matrix฀protein฀compo-
model,฀49 nents,฀124
N-and฀C-terminals,฀54–55 matrilysin฀connective฀tissue฀degrading฀
pyridinoline฀formation,฀51,฀54 enzyme,฀126
super-arrays,฀51 matrix฀metalloproteases,฀124–125
telopeptide฀domains,฀50–51 collagenases฀and฀gelatinases,฀127–128
triple฀helix,฀46,฀49 enamelysin,฀126–127
Tropoelastin enzyme฀family,฀113–115
cross-linking,฀86–87 ibrillar฀procollagen฀processing
human฀domains,฀86 astacins,฀121
synthesis,฀85 integrin฀and฀disintegrin,฀122–123
Tumor฀necrosis฀factor-a฀(TNF-a),฀238–239 N-and฀C-proteinases฀activation,฀122
Type฀IV฀collagen,฀66 PCP฀and฀PNP,฀domain฀organization,฀
Type฀VII฀collagen,฀65–66 122–123
Type฀XVII฀collagen,฀70–71 polypeptide฀motifs,฀120–121
metzincin฀family
activation,฀117–119
V catalysis,฀115–117
Vitamin฀K฀oxidoreductase,฀181–182 stromelysins,฀126