Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver

Cirrhosis and Cancer Patients: Implications for a Biomarker of

Hepatocellular Carcinoma: e12419
Comunale, Mary Ann ; Rodemich-Betesh, Lucy; Hafner, Julie; Wang,
Mengjun; Norton, Pamela ; et al. PLoS One 5.8 (Aug 2010).
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Background
We previously reported increased levels of protein-linked fucosylation with the development of liver
cancerand identified many of the proteins containing the altered glycan structures. One such
protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan
analysis on the five major isoformsof A1AT and completed a comprehensive study of the
glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV)
induced liver cirrhosis, and in patients infected with HCV with a diagnosis ofhepatocellular
carcinoma (HCC).
Methodology/Principal Findings
Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing
outer arm (-1,3) fucosylation. Increases in core (-1,6) fucosylation were observed only on A1AT
from patients withcancer. We performed a lectin fluorophore-linked immunosorbent assay using
Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients
with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a
specificity of 86%, which was greater than that observed with the current marker of HCC, alphafetoprotein. Glycosylation analysis of the false positives was performed; results indicated that
these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that
core fucosylation is cancer specific.
Conclusions/Significance
This report details the stepwise change in the glycosylation of A1AT with the progression
from liver cirrhosis tocancer and identifies core fucosylation on A1AT as an HCC specific
modification.

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Abstract
Background

We previously reported increased levels of protein-linked fucosylation with the development of liver
cancerand identified many of the proteins containing the altered glycan structures. One such
protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan
analysis on the five major isoformsof A1AT and completed a comprehensive study of the
glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV)
induced liver cirrhosis, and in patients infected with HCV with a diagnosis ofhepatocellular
carcinoma (HCC).
Methodology/Principal Findings
Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing
outer arm (-1,3) fucosylation. Increases in core (-1,6) fucosylation were observed only on A1AT
from patients withcancer. We performed a lectin fluorophore-linked immunosorbent assay using
Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients
with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a
specificity of 86%, which was greater than that observed with the current marker of HCC, alphafetoprotein. Glycosylation analysis of the false positives was performed; results indicated that
these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that
core fucosylation is cancer specific.
Conclusions/Significance
This report details the stepwise change in the glycosylation of A1AT with the progression
from liver cirrhosis tocancer and identifies core fucosylation on A1AT as an HCC specific
modification.
Citation: Comunale MA, Rodemich-Betesh L, Hafner J, Wang M, Norton P, et al. (2010) Linkage
Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for
a Biomarker ofHepatocellular Carcinoma. PLoS ONE 5(8): e12419.
doi:10.1371/journal.pone.0012419
Editor: Wang-Shick Ryu, Yonsei University, Republic of Korea
Received: June 15, 2010; Accepted: July 22, 2010; Published: August 25, 2010
Copyright: 2010 Comunale et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant R01 CA120206-01 from the National Cancer Institute
(NCI), grant UO1 CA084951-06 from the NCI Early Research Detection Network (EDRN), the

Hepatitis B Foundation, and an appropriation from The Commonwealth of Pennsylvania. The

funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is the major
etiology of hepatocellular cancer(HCC) [1]-[4]. Both HBV and HCV cause acute and
chronic liver infections, and most chronically infected individuals remain asymptomatic for many
years [5]. About 10% to 40% of all chronic HBV carriers eventually develop liver cancer, and it is
estimated that over one million people worldwide die because of HBV- and HCV-associated liver
cancer [2], [6], [7]. Indeed, HBV and HCV infections are associated with over 80% of all
casesof HCC worldwide and can be as high as 96% in regions where HBV is endemic [3].
The progression of liver disease to liver cancer is primarily monitored by serum levels of the
oncofetal glycoprotein, alpha-fetoprotein (AFP), or the core fucosylated glycoform of AFP, AFP-L3.
AFP can, however, be produced in many circumstances, including in relation to other liver diseases
[8]-[10] and is not present in all those with HCC [11]. Therefore the use of AFP as a primary
screen for HCC has been questioned [12], and more sensitive serum biomarkers for HCC are
needed.
The glycosylation of proteins is cell specific. The N-linked glycosylation of a protein reflects
modifications that occurred in the cell from which it came [13]. The glycosylation of the same
protein secreted from diseased tissue, malignant cells or normal cells may, and often do, differ
[14]. We, and others, have observed changes in N-linked glycosylation with the
development of cirrhosis and HCC [15]-[19]. Specifically, the amount of core fucosylated N-linked
glycan derived from total protein preparations isolated from the serum of individuals chronically
infected with HCV and from those with a diagnosis of HCC was consistently greater than that in
healthy patients or in those with HCV and "inactive" disease [19].
Using fucose-specific lectins to identify the proteins that become fucosylated in patients
with liver disease, we identified more than 100 glycoproteins from patients with HCC and/or
cirrhosis that contained increased fucosylation [19]. One of these proteins was alpha-1-antitrypsin
(A1AT). We analyzed the N-linked glycosylation of the five major isoforms of A1AT and discovered,
in addition to increased levels of core fucosylation, significant increases in outer arm fucosylation
with the development of liver cancer. Using a lectin-based assay, we measured this change in over
400 patients with liver disease and found AAL reactive A1AT could detect HCC with a
sensitivity of 70% and a specificity of 86% using a cut-off of 5 relative units. Glycan analysis of the

false positives identified outer-arm fucosylation as being the cause of false positivity. In contrast,
increases in core fucosylation were found only in patients with cancer. The reasons for this change
and the clinical usefulness of this change are discussed.
Materials and Methods
Ethics Statement
Both the Drexel University College of Medicine and the Saint Louis University Institutional Review
Boards approved the study protocol, which was consistent with the standards established by the
Helsinki Declarationof 1975. Written informed consent was obtained from each participant.
Patients
Serum samples were obtained from the Saint Louis University School of Medicine (Saint Louis,
MO). Demographic and clinical information along with a blood sample was collected from each
participant in a serum separator tube. The sample was spun within 2 hours, and the serum was
stored at -80C until testing. Patients were enrolled in the Saint Louis University Liver
Cancer Clinic and HCC were diagnosed using the same criteria established for the HALT-C trial
[20]. Participants had HCC identified by biopsy, by a new hepatic defect showing vascular
enhancement on one imaging modality (ultrasound [US], magnetic resonance imaging [MRI], or
computed tomography [CT]) with AFP levels >1000 ng/ml, or by presumed HCC. Participants were
presumed to have HCC if they had a discrete hepatic defect on US with AFP levels <1000 ng/ml
and either two other scans (MRI, CT, angiography) indicating malignancy with at least one of the
following characteristics: hypervascularity, arterial to portal vein shunts, portal vein thrombosis
near the defect, tumor in the portal vein, or one other scan (MRI or CT) showing features
characteristic of HCC and either an increase in size over time after initial discovery (at least
doubling if less than 1 cm) or an increase in the level of AFP to >200 ng/ml. Tumor staging was
determined using the United Network of Organ Sharing-modified (UNOS) TNM staging system for
HCC. For the cirrhosis group, patients with hepatitis C and biopsy-proven cirrhosis were enrolled.
All cirrhotic controls were screened for HCC using US, CT, or MRI prior to enrollment. Patients who
were HBsAg+ (with or without HBV DNA were classified into the HBV group. Similarly, patients who
were HCV RNA positive, with no evidence of cirrhosis, were classified into the HCV group. Patients
with other non viral liver disease but without cirrhosis were classified into the other liver disease
group (OLD). Control patients were healthy patients recruited by the study to act as controls.
Two-Dimensional (2-D) Gel Electrophoresis
A total of 7 l of serum was diluted in 330 l of solubilization buffer containing 7 M urea, 2 M
thiourea, 4% CHAPS, 65 mM DTT, 5 mM tributylphosphine, and a 0.4% mixture of carrier

ampholytes (Servalyt pH 2-4, pH 3-10, pH 9-1, 1:2:1). Samples were vortexed periodically for 1 h
and applied to an 18-cm pH 3-7 NL immobilized pH gradient (IPG) strip (Amersham, Piscataway,
NJ, USA). Gel rehydration was carried out for 14 h at 50 V and focused using the IPGPhor
(Amersham) isoelectric focusing apparatus. After focusing, gel strips were first reduced then
alkylated in 6M urea, 2% SDS, 30% glycerol, 50 mM Tris, pH 6.8, and either 30 mM DTT or 75 mM
iodoacetamide for 10 min each. The second dimension was resolved with an 8% to 18%
acrylamide-0.8% PDA gradient gel on a Protean II xi Cell (Biorad Laboratories Headquarters,
Hercules, CA, USA) with the running conditions set to 20 mA/gel for 20 min and 40 mA/gel for 4 h.
Gels were fixed and stained with colloidal Coomassie. Gels were imaged using the Odyssey
Infrared Imaging System (Li-Cor Biosystems, Lincoln, Nebraska) and analyzed using NonLinear
Dynamics Progenesis Workstation gel imaging software (NonLinear Durham, NC, USA).
Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry
Protein spots were excised from colloidal Coomassie blue stained gels, destained, and digested
with trypsin. Recovered peptides were concentrated and desalted using ZipTip C18 (Millipore,
Bedford, MA, USA) according to the manufacturer's directions and prepared for MALDI-TOF mass
spectrometry by mixing 0.5 mL of peptide mixture with 0.5 mL of 10 mg/mL -cyano-4hydroxycinnamic acid and 1% formic acid in 50% acetonitrile and allowing the droplet to dry on
the MALDI plate. Peptide mass maps were obtained using a Voyager-DE Pro Mass Spectrometer
(Applied Biosystems, Life Biotechnologies, Carlsbad, CA) operated in positive ion reflectron mode.
Proteins were identified from the peptide mass maps using the MASCOT online database
(www.matrixscience.com) to search the nonredundant protein database.
Glycan Analysis
The 2DE gel spots were excised and destained using 40% methanol 7% acetic acid. The gel plugs
were dehydrated with acetonitrile, the acetonitrile was removed and 25mM DTT was allowed to
absorb into the plug. The plug was heated to 100C for 5 min, allowed to cool, and alkylated in the
dark for 30 min with 75mM iodoacetamide. The gel plugs were washed using two
repeats of acetonitrile dehydration and 20 mM ammonium bicarbonate rehydration. After a final
dehydration, the gel plugs were dried in a speed vac. Peptide:N-glycosidase F (PNGase F) was
diluted with 20 mM ammonium bicarbonate pH 7 and allowed to adsorb into the gel plug. The gel
plug was then covered with the same solution and allowed to incubate overnight at 37C. The
glycans were eluted from the gel plug by sonication in Milli-Q water three times; the elutant was
pooled, dried down, and labeled with a 2AB dye (Ludger, Oxford, UK) according to the
manufacturer's instructions. The glycans were then cleaned up using paper chromatography and
filtered using a 0.22-m syringe filter. Fluorescently labeled glycans were subsequently analyzed
using the Waters Alliance high-performance liquid chromatography system with a normal phase

column (TSK amide 80 columns) complemented with a Waters fluorescence detector and quantified
using the Millennium Chromatography Manager (Waters Corporation, Milford, MA). The mobile
phase consisted of solvent A (50 mM ammonium formate, pH 4.4) and solvent B (acetonitrile). The
gradient used was as follows: linear gradient from 20% to 58% solvent A at 0.4 mL/minute for
152 min followed by a linear gradient from 58% to 100% solvent A for the next 3 min. The flow
rate was increased to 1.0 mL/minute; the column was washed in 100% solvent A for 5 min.
Following the wash step, the column was equilibrated in 20% solvent A for 22 min in preparation
for the next sample. Glycan structures were identified by calculating the glucose uptake value and
exoglycosidase digestion, as described previously [21].
Lectin-Fluorophore-linked Immunosorbent Assay (FLISA)
The capture antibody (mouse antihuman A1AT, AbD Serotec, Raleigh NC, USA), was incubated with
10 mM sodium periodate for 1 h at 4C. This treatment ensures the lectin is unable to react with
the glycosylation ofthe antibody and does not affect antibody substrate binding. An equal
volume of ethylene glycol was added, and the oxidized antibody was brought to a
concentration of 10 g/mL with sodium carbonate buffer, pH 9.5. Antibody (5 g/well) was added
to the plate and, following incubation, was washed with 0.1% Tween 20mM phosphate buffered
saline pH 7.4. The plate was blocked overnight with 3% bovine serum albumen/phosphate
buffered saline. For analysis, 5 l of serum was diluted in 95 l blocking reagent in Heterophilic
Blocking Tubestm (Scantibodies Laboratory, Inc. Santee, CA, USA) and was incubated at room
temperature for 1 h. Subsequently, samples were added to the plates for 2 h and washed 5 times
in lectin incubation buffer (10 mM Tris pH 8.0, 0.15M NaCl, 0.1% Tween 20). Fucosylated A1AT
was detected with a biotin conjugated Aleuria aurantia lectin (AAL) (Vector Laboratories,
Burlingame, CA). Bound lectin was detected using IRDye 800 conjugated streptavidin; the signal
intensity was measured using the Odyssey infrared imaging system (LI-COR Biotechnology,
Lincoln, Nebraska). In all cases, signal intensities were compared to those of signals detected with
commercially purchased human serum (Sigma Chemicals). The lectin-FLISA detects the
amount offucosylation present on an equal quantity of molecules captured from each patient
sample and is performed in a manner independent of the total amount of protein in any given
patient.
Statistical Analysis
Descriptive statistics for staged patients were compared by scatter plots that included the outliers.
All values were reported as mean values plus or minus the standard error unless otherwise stated.
Because the data did not follow typical Gaussian distribution, a nonparametrical test (two-tailed,
95% confidence, Mann-Whitney test) was used to determine statistical difference between the
groups. To determine the optimal cutoff value for each marker, the receiver operating characteristic

curves were constructed using all possible cutoffs for each assay. The area under the receiver
operating characteristic curve was constructed and compared as described previously. A two-tailed
P value of 0.05 was used to determine statistical significance. All analyses were performed using
GraphPad Prism (San Diego, CA, USA).
Results
Levels of A1AT and Degree of Sialyation in Patients with Liver Cirrhosis and HCC
Pooled sera from healthy patients (n = 20), patients with liver cirrhosis (n = 20) and patients with
HCC with a background of cirrhosis (n = 20) were resolved via 2-D gel electrophoresis (2DE)
(Table 1). Figure 1A shows a representative 2-DE of normal human serum and Figures 1B, 1C and
1D show a focus on the 5 isoforms (M1, M2, M4, M6, and M7) of A1AT from the three patient
groups. Three major and two minor A1AT isotypes are commonly seen when human serum is
isoelectrically focused and run on a 2-D gel [22], [23] and these are not altered in the three
patient groups. The A1AT concentrations in each patient group were comparable (Figures 1E-1G)
and fell within normal serum concentrations. This result was confirmed by analyzing A1AT an
Enzyme-linked immunosorbent assay (ELISA), whereby the A1AT levels were 3.2 mg/mL in the
composite from the healthy patients, 3.3 mg/mL in the composite from the cirrhotic patients, and
3.3 mg/mL in the composite from the HCC patients.
[Figure omitted, see PDF]
Figure 1. Two-dimensional gel purification of alpha-1-antitrypsin isoforms.
Sera from pools of healthy controls (A, B, E) and cirrhosis (C, F) and cancer (D, G) patients were
focused using IPGPhor 3-7 NL first dimension strips followed by SDS-PAGE separation on 8% to
18% acrylamide gels. The pIof selected gel spots are M1 = 4.91, M2 = 4.95, M4 = 5.00, M6 =
5.05, and M7 = 5.10. Panels E, F, and G show the relative abundance of each gel spot.
Table 1. Patients Utilized in Study.
We performed glycan analysis on the M1, M2, M4, M6, and M7 isotypes from the pools of sera from
normal participants and cirrhotic and HCC patients (Figures 1B-1D) and examined the sialylation
patterns of each isotype. Figure S1 shows a representative glycan profile for the M4 isotype from
the controls. Consistent with results of previous studies [24], the biantennary glycan is the most
abundant species. Figure S1-B shows the level of the sialyated biantennary and triantennary
glycan associated with each isoforms from each patient group. As S1-B shows, the
level of sialyation on the bi-anntennery glycan (A2G2S1 or A2G2S2) was similar in the different
patient groups. Similarly, the level of sialyation was not altered on the triantennary glycan.
However, there was an increase in the level of the tri-sialyated -1,3 linked fucosylated outer arm

fucosylated glycan (A3F(3)1G3S3) on A1AT from the cancer patients, as compared to the healthy
and cirrhotic patients. These peak are indicated in figure S1A & B with an asterisk.
Increased Levels of Core and Outer Arm Fucosylation Are Observed on A1AT from Patients with
HCC
To test if the increased level of tri-sialyated -1,3 linked fucosylated outer arm fucosylated glycan
(A3F(3)1G3S3) was the result of an increase in sialyation or an increase in the total
amount of parent glycan, sialic acid was removed enzymatically and the sample re-analyzed.
Figure 2 shows the simplified desialylated glycoprofile of the five major isoforms for each of patient
group following treatment with neuraminidase (Arthrobacter ureafaciens). Three peaks of interest,
indicated with a star in Figure 2, are reproducibly altered in the M1, M2, and M4 isoforms as the
diseased liver progresses from cirrhosis to cancer. Sequential exoglycosidase digestion (data not
shown) showed these peaks to be a core fucosylated biantennary glycan (F(6)A2G2), a
triantennary glycan (A3G3), and a triantennary glycan with a single 1,3 linked outer arm fucose
residue (A3F(3)1G3). Figure 3A shows a representative desialylated profile with the corresponding
glycan structure identified for each peak. Table 2 is a quantitation of each glycan structure present
on the five major isoforms for each patient group. Specific changes in glycosylation are observed
in the A1AT isoforms with the progression to cirrhosis and HCC. On M6, the isoform with very little
tri and tetra-antennary structures, the biantennary core fucosylated glycan (F(6)A2G2), represents
4.48% in normal patients, 4.37% in patients with cirrhosis, and 7.04% in patients with cancer, a
trend seen across each isoforms (Table 2, glycan 3). The percent change between healthy and
cirrhosis and between cirrhosis and cancer is given in Figure 3C and shows that this structure
changes with cancer only. In M4, the triantennary glycan with an outer arm fucose residue
(A3F(3)1G3) increases from 4.34% in normal participants to 6.78% in patients with cirrhosis, and
13.18% in patients with cirrhosis plus cancer. Again, this trend is seen in each of the isoforms,
with the exception of the M6 due to the total lack of triantennary structures (Table 2, glycan 8).
The relative increase in each of these fucosylated glycan structures as a function of disease is
shown in Figure 3B and shows that this structures changes in both liver cirrhosis and HCC. The
increase in outer arm fucosylation was also associated with a decrease in the parent N-linked
glycan. That is, the increase in the outer arm fucosylated triantennary glycan, A3F(3)1G3, was
associated with a decrease in triantennary glycan A3G3. (Table 2, glycan 6).
[Figure omitted, see PDF]
Figure 2. The desialylated N-linked glycan profile for each of the five A1AT isoforms from normal
controls (top) and cirrhotic (middle) or HCC patients (bottom).
The major peaks that are altered are indicated with an asterisk and are (from left to right) a core
fucosylated bianntennary glycan (F96)A2G2), a trianntennary N-linked glycan, and a trianntennary

N-linked glycan with a single outer arm fucose residue (A3F[3]3). The percent of each of these
peaks in the different isoforms and in the different patient groups is shown in Table 2.
[Figure omitted, see PDF]
Figure 3. Specific changes in glycosylation on A1AT can be observed with the progression
from liver cirrhosis toliver cancer.
(A) A representative N-linked glycan profile from a normal control patient. The 11 major glycan
structures identified are indicated and given a number. This number is used in Table 2 with
structure names provided. The relative change in the level of the trianntennary N-linked glycan
with a single outer arm fucose residue (A3F[3]G3) (B) and the core fucosylated bianntennary
glycan (F[6]A2G2) (C) in normal to cirrhotic and cirrhotic to HCC is shown as percent change. As
this figure shows, increases in outer arm fucosylation are associated with both cirrhosis and HCC,
whereas increased core fucosylation is only observed with HCC.
Table 2. The N-linked Glycans Found on A1AT Isoforms from Controls, Patients with Cirrhosis, or
Patients with HCC.
Analysis of Fucosylated A1AT by Lectin-FLISA in a Cohort of 458 Patients
To further examine if the changes in both core and outer arm fucosylation could be seen in
individual patients and potentially used as a diagnostic marker of cancer, we analyzed a patient
cohort consisting of 458 patients for the level of fucosylated A1AT using a lectin-FLISA based
assay. In this assay, A1AT was captured using a monoclonal antibody and the level of fucosylation
was determined using the fucose-binding AAL. AAL recognizes both outer arm and core fucosylated
glycan. Twenty patients with no evidence of liver disease were used as controls; 33 patients
infected with HBV had an unknown level of liver fibrosis; 215 patients infected with HCV had an
unknown level of liver fibrosis; 65 patients had liver cirrhosis; 62 patients had otherliver diseases;
and 63 patients had liver cancer (Table 1). Figure 3A shows the relative level of fucose lectinreactive A1AT in the six patient groups. Values are given as -fold increase in relation to the level in
commercially purchased "normal" sera. The mean and 95% confidence interval of the mean are
shown for each group. We found a clear statistical difference between the HCC group and all other
groups (P<0.0001) and between the cirrhotic group and the control group (P = 0.0173) but not
between any other groups (Figure 3A). The mean level of lectin-reactive A1AT was 1.4-fold
(0.80) above sigma in the control group, 1.7-fold (0.1.8) in the group infected with HBV, 1.9fold (1.8) in the group infected with HCV, 2.6-fold (2.3) in the group with cirrhosis, 2.6-fold
(2.0) in the group with other liver diseases, and 7.70-fold (4.45) in the group with HCC.
Surprisingly, there was no difference in the mean level of AAL-reactive A1AT in HCC patients in
regards to the stage of HCC (data not shown). That is patients with stage 1 HCC, as defined as a

single lesion of less than 2cm [11], had a 9.1 fold (4.70) increase in the level of AAL reactive
A1AT while patients with stage 4 HCC (those with multiple lesions >6cm in diameter) had a
mean of 6.7 fold (5.54), which was not different than that observed in stage 1 patients (p =
0.114).
In this cohort, fucosylated A1AT could distinguish between HCC and non-HCC cases with an area
under the receiver operator curve (AUROC) of 0.871. When comparing only HCC versus cirrhosis,
the discriminatory ability was 0.867. Using a cut-off of 5 relative units AAL reactive A1AT could
differentiate HCC from cirrhosis with a sensitivity of 70% and a specificity of 86%. In contrast,
AFP, when analyzed in this cohort, had an AUROC of0.764 and could differentiate HCC from
cirrhosis with a sensitivity of 59% and a specificity of 93% using a cut-off of 20 ng/mL.
False Positives Have Increased Levels of Outer Arm Fucosylation Whereas Core Fucosylation Is
Specific for HCC
Some patients do not have cancer but have elevated levels of lectin-reactive A1AT (Figure 4A).
Because we observed changes in outer arm fucosylation on A1AT from patients with cirrhosis, it
was of interest to determine if those with false positive results had core or outer arm fucosylation.
Figure 4A shows the results ofglycan analysis from purified A1AT from three cirrhotic patients
(out of nine) and three patients with stage 1 or 2 HCC (out of nine) and analyzed as before. The
results from these patients are shown in Figure 4A, and a representative glycan
profile of one of these patients is shown in Figure 4B, along with the level of 4 major glycan
structures indicated. As Figure 4B shows, consistent with the results presented in Table 2 and in
Figures 3A-3C, the three cirrhotic patients have levels of core fucosylation (F(6)A2G2) similar to
those observed in healthy controls. However, these patients have significant increases in outer arm
fucosylation (A3F(3)1G3), similar to the highest levels seen in patients with HCC. That is, the
level of outer arm fucosylation in these patients ranged from 10% to 17%, which is similar to the
level observed in patients with HCC (13%). In contrast, three patients with HCC who had very
strong positive results from the lectin FLISA test had increased core and outer arm fucosylation.
For example, the core fucosylated bianntennary glycan represented 9.55% on A1AT purified from
patient H27. Similarly, this glycan represented more than 9% on A1AT purified from patients H18
and H23. Increases in outer arm fucosylation ranging from 14.03% to 15.97% of the total glycan
on A1AT were also observed in these patients. Figure 4D shows the results of all 18 patients with a
focus on the levelof outer arm (-1,3) and core (-1,6) fucosylation. When examining all 9
cirrhotic false positives the mean levelof core fucosylation was 3.77%0.25 and the mean
level of outer arm fucosyalation was 11.88%2.79. In the case of the cancers the mean
level of core fucosylation was 8.62%1.2 and the mean level of outer arm fucosylation was
14.26%1.9. There was statistical difference between the level of core -1,6 fucosylation between
these nine cirrhotic and nine HCC patients (p<0.0001) but not with -1,3 linked fucosylation (p =

0.07). Importantly, the maximum level of core fucosylation observed in the false positive cirrhotics
was 4.01%, similar to what was observed in healthy controls (3.62%).
[Figure omitted, see PDF]
Figure 4. Increase in lectin-reactive A1AT with the development of HCC and identification of core
fucose as a specific marker of liver cancer.
(A) The level of lectin-reactive A1AT in patients with HCC, HBV infection, HCV infection, or
other liver diseases (OLD) and in controls. The solid line represents the mean value. The x-axis
represents the patient group. The y-axis shows the -fold increase in lectin-reactive A1AT compared
with that in commercially purchased serum. (B) Glycan analysis of A1AT isoform M4 from patient
21. (C) Quantification of glycan analysis from three cirrhotic patients (01,21 and 27) and three
patients with stage 1 or 2 HCC (HCC-23, HCC 27 and HCC 18). The levels of4 major glycan
structures are shown. As this figure shows, in cirrhotic false positives, there is an increase in outer
arm but not core fucosylation. Consistent with data shown in Figures 2 and 3, increases in core
fucosylation on AAT were observed only from patients with HCC. (D) Scatter plot of the level of 1,3 or 1,6 linked fucose from 9 cirrhotic false positives and 9 patients with either stage 1 or 2
HCC.
Discussion
Recent reports have indicated that increased core and outer-arm fucosylation could be observed
following glycomic analysis of either total human serum or serum depleted of immunoglobulin
[18], [19], [25]. We examined the glycosylation of a single protein, A1AT, as a function of
liver cirrhosis and liver cancer. To this end, we found some changes that were consistent with our
previous findings such as an increase in core fucosylation as well as changes in outer arm
fucosylation.
The observation that changes in both core and outer arm fucosylation occur may provide clues to
the molecular basis of this change. That is, although the exact mechanisms for increased core
fucosylation in HCC are unknown, they are thought to involve increases in both the levels of the
enzyme and the substrates involved in core fucosylation [17].
It is also possible that these markers reflect some alteration in the Golgi apparatus. Recent reports
have suggested that, in regards to the liver, the fucosylation of proteins is involved in protein
sorting to the bile [26]. Thus, it is conceivable that the appearance of fucosylated proteins in the
serum may reflect a common defect in protein sorting. It is interesting to note that the
glycosylation of A1AT in the serum of patients withliver cancer (Figures 2, 3 and 4) is similar to
that observed in the bile of healthy individuals [26].

It is also interesting to note that lectin reactivity was greater than the total change observed in
fucosylation. For example, patient 27 had either core or outer arm fucose on 22.82% of his or her
N-linked glycans. In contrast, A1AT from a healthy individual had fucose (core or outer arm) on
9% of the N-linked glycans. Hence, using the lectin FLISA, we should have observed closer to a
2.5-fold increase and not the 11-fold increase that was obtained. This difference may be an
artifact of the lectin-FLISA, the result of other proteins attached to A1AT, increased or modified Oglycosylation, or increased accessibility of the lectin to the fucose residues. Allof these possible
scenarios are under investigation.
It is also important to note that increased outer arm fucosylation was observed both in patients
with cirrhosis and in those with HCC. This finding implies that outer arm fucosylation was not
specific to cancer but rather was probably associated with inflammation, first from
the liver cirrhosis and then from the presence of the HCC lesion. Indeed, the level of outer arm
fucosylation may be a more specific marker of inflammation and may be predictive of
cancer development [27], [28]. In contrast, increases in core fucosylation are observed only in
patients with HCC, suggesting that this change is cancer specific.
As Figure 4A shows, many patients in the non-HCC groups have elevated levels of lectin-reactive
AAL. Analysisof three patients with elevated AAL levels from the cirrhosis group indicated that they
had changes primarily in outer arm fucosylation, not in core fucosylation. To that end, core
fucosylation may be a more specific target for the detection of cancer [29]. Efforts to use lectins
such as lens culinaris and Pisum sativum agglutinin, which will not bind outer arm fucosylated
glycan, in the lectin FLISA proved problematic because these lectins have much weaker binding
affinities and do not actually bind fucose directly. Efforts to make recombinant AAL with specificity
only to core-linked -1,6 fucose are currently underway.
In summary, we have analyzed the glycosylation of A1AT as a function of HCC and used a lectin
FLISA to measure this change in a cohort of more than 400 patients. These data need to be
confirmed in larger cohortsof patients to determine if these markers are truly reliable serum
markers of early HCC, to compare their accuracy with AFP in patients of diverse gender, ethnicity,
etiologies of liver disease, and to determine their role in HCC surveillance. Future studies should
also test the benefit of combinatorial analysis with other potential markers of HCC, such as desgamma-carboxy prothrombin as well as to examine how the level ofcore and outer arm
fucosylation varies as a function of anti-cancer treatment.
Supporting Information
Figure S1.

The sialylated N-linked glycan profile for each of the five A1AT isoforms from normal, cirrhotic, or
HCC patients. (A) A representative sialyated profile of the M4 A1AT isoform from healthy
individuals. (B) The relative percentof sialyated bi-antennary or tri-antennary glycan in each A1AT
isoform.
(3.00 MB TIF)
Author Contributions
Conceived and designed the experiments: MAC AM. Performed the experiments: MAC LRB JH MW
AM. Analyzed the data: MAC MW PN AMDB TB AM. Contributed reagents/materials/analysis tools:
AMDB AM. Wrote the paper: MAC AM.
References
1. Di Bisceglie AM (1989) Hepatocellular carcinoma: molecular biology of its growth and
relationship to hepatitis B virus infection. Med Clin North Am 73: 985-997. Find this article online
2. Block TM, Mehta AS, Fimmel CJ, Jordan R (2003) Molecular viral oncology of hepatocellular
carcinoma. Oncogene 22: 5093-5107. Find this article online
3. Marrero JA (2006) Hepatocellular carcinoma. Curr Opin Gastroenterol 22: 248-253. Find this
article online
4. Sallie R, Di Bisceglie AM (1994) Viral hepatitis and hepatocellular carcinoma. Gastroenterol Clin
North Am 23: 567-579. Find this article online
5. Lok A, McMahon B (2001) Chronic hepatitis B. Hepatology 34: 1225-1241. Find this article
online
6. El Serag HB, Mason AC, Key C (2001) Trends in survival of patients with hepatocellular
carcinoma between 1977 and 1996 in the United States. Hepatology 33: 62-65. Find this article
online
7. Sarbah SA, Gramlich T, Younoszai A, Osmack P, Goormastic M, et al. (2004) Risk factors for
hepatocellular carcinoma in patients with cirrhosis. Dig Dis Sci 49: 850-853. Find this article online
8. Alpert ME, Uriel J, de Nechaud B (1968) alpha fetogloblin in the diagnosis of human hepatoma.
N Engl J Med 278: 984-986. Find this article online
9. Ruoslahti E, Salaspuro M, Pihko H, Andersson L, Seppala M (1974) Serum alpha-fetoprotein:
diagnostic significance in liver disease. Br Med J 2: 527-529. Find this article online

10. Di Bisceglie AM, Hoofnagle JH (1989) Elevations in serum alpha-fetoprotein levels in patients
with chronic hepatitis B. Cancer 64: 2117-2120. Find this article online
11. Marrero JA, Romano PR, Nikolaeva O, Steel L, Mehta A, et al. (2005) GP73, a resident Golgi
glycoprotein, is a novel serum marker for hepatocellular carcinoma. J Hepatol 43: 1007-1012. Find
this article online
12. Sherman M (2005) Hepatocellular carcinoma: epidemiology, risk factors, and screening.
Semin Liver Dis 25: 143-154. Find this article online
13. Rudd PM, Dwek RA (1997) Glycosylation: heterogeneity and the 3D structure of proteins. Crit
Rev Biochem Mol Biol 32: 1-100. Find this article online
14. Aoyagi Y, Suzuki Y, Igarashi K, Yokota T, Mori S, et al. (1993) Highly enhanced
fucosylation of alpha-fetoprotein in patients with germ cell tumor. Cancer 72: 615-618. Find this
article online
15. Naitoh A, Aoyagi Y, Asakura H (1999) Highly enhanced fucosylation of serum glycoproteins in
patients with hepatocellular carcinoma. J Gastroenterol Hepatol 14: 436-445. Find this article
online
16. Aoyagi Y, Isokawa O, Suda T, Watanabe M, Suzuki Y, et al. (1998) The fucosylation
index of alpha-fetoprotein as a possible prognostic indicator for patients with hepatocellular
carcinoma. Cancer 83: 2076-2082. Find this article online
17. Noda K, Miyoshi E, Gu J, Gao CX, Nakahara S, et al. (2003) Relationship between elevated FX
expression and increased production of GDP-L-fucose, a common donor substrate for fucosylation
in human hepatocellular carcinoma and hepatoma cell lines. Cancer Res 63: 6282-6289. Find this
article online
18. Block TM, Comunale MA, Lowman M, Steel LF, Romano PR, et al. (2005) Use of targeted
glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and
humans. Proc Natl Acad Sci U S A 102: 779-784. Find this article online
19. Comunale MA, Lowman M, Long RE, Krakover J, Philip R, Seeholzer S, Evans AA, Hann HWL,
Block TM, Mehta AS (2006) Proteomic analysis of serum associated fucosylated glycoproteins in
the development of primary hepatocellular carcinoma. Journal of Proteome Research 6: 308-315.
Find this article online

20. Di Bisceglie AM, Shiffman ML, Everson GT, Lindsay KL, Everhart JE, et al. (2008) Prolonged
therapy ofadvanced chronic hepatitis C with low-dose peginterferon. N Engl J Med 359: 24292441. Find this article online
21. Guile GR, Rudd PM, Wing DR, Prime SB, Dwek RA (1996) A rapid high-resolution highperformance liquid chromatographic method for separating glycan mixtures and analyzing
oligosaccharide profiles. Anal Biochem 240: 210-226. Find this article online
22. Jeppsson JO, Einarsson R (1992) Genetic variants of alpha 1-antitrypsin and hemoglobin typed
by isoelectric focusing in preselected narrow pH gradients with PhastSystem. Clin Chem 38: 577580. Find this article online
23. Brooks KP, Iammarino RM (1985) Determination of alpha-1-antitrypsin phenotypes and M
subtypes by isoelectric focusing in immobilized pH gradients. Clin Biochem 18: 280-284. Find this
article online
24. Kolarich D, Weber A, Turecek PL, Schwarz HP, Altmann F (2006) Comprehensive glycoproteomic analysis ofhuman alpha1-antitrypsin and its charge isoforms. Proteomics 6: 3369-3380.
Find this article online
25. Liu XE, Desmyter L, Gao CF, Laroy W, Dewaele S, et al. (2007) N-glycomic changes in
hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus. Hepatology 46:
1426-1435. Find this article online
26. Nakagawa T, Uozumi N, Nakano M, Mizuno-Horikawa Y, Okuyama N, et al. (2006)
Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts. J Biol
Chem 281: 29797-29806. Find this article online
27. Abd Hamid UM, Royle L, Saldova R, Radcliffe CM, Harvey DJ, et al. (2008) A strategy to reveal
potential glycan markers from serum glycoproteins associated with breast cancer progression.
Glycobiology 18: 1105-1118. Find this article online
28. Arnold JN, Saldova R, Hamid UM, Rudd PM (2008) Evaluation of the serum N-linked glycome
for the diagnosis of cancer and chronic inflammation. Proteomics 8: 3284-3293. Find this article
online
29. Peracaula R, Sarrats A, Saldova R, Pla E, Fort E, et al. (2010) Glycosylation of liver acutephase proteins in pancreatic cancer and chronic pancreatitis. PROTEOMICS - Clinical Applications.
In press. Find this article online
Word count: 6061

 2010 Comunale et al. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited: Comunale MA, Rodemich-Betesh L,
Hafner J, Wang M, Norton P, et al. (2010) Linkage Specific Fucosylation of Alpha-1-Antitrypsin in
Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma.
PLoS ONE 5(8): e12419. doi:10.1371/journal.pone.0012419

Indexing (details)
Cite
Subject
Liver cirrhosis;
Liver cancer;
Hepatitis;
Disease;
Liver diseases;
Proteins
Title
Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis andCancer Patients:
Implications for a Biomarker of Hepatocellular Carcinoma: e12419
Author
Comunale, Mary Ann; Rodemich-Betesh, Lucy; Hafner, Julie; Wang, Mengjun;Norton,
Pamela; Bisceglie, Adrian MDi; Block, Timothy; Mehta, Anand
Publication title
PLoS One
Volume
5
Issue
8
Publication year
2010
Publication date
Aug 2010
Year
2010
Section
Research Article
Publisher

Public Library of Science

Place of publication
San Francisco
Country of publication
United States
Publication subject
MEDICAL SCIENCES, SCIENCES: COMPREHENSIVE WORKS
Source type
Scholarly Journals
Language of publication
English
Document type
Journal Article
DOI
http://dx.doi.org/10.1371/journal.pone.0012419
ProQuest document ID
1318937316
Document URL
http://search.proquest.com/docview/1318937316?accountid=62690
Copyright
2010 Comunale et al. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited: Comunale MA, Rodemich-Betesh L,
Hafner J, Wang M, Norton P, et al. (2010) Linkage Specific Fucosylation ofAlpha-1-Antitrypsin
in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma.
PLoS ONE 5(8): e12419. doi:10.1371/journal.pone.0012419
Last updated
2013-03-23
Database
ProQuest Agriculture Journals