www.elsevier.

com/locate/toxcon
Authors Accepted Manuscript
A rapidly diverging superfamily of peptide toxins in
venomous Gemmula species
Francisco M. Heralde III, Julita Imperial, Pradip K.
Bandyopadhyay, Baldomero M. Olivera, Gisela P.
Concepcion, Ameurna D. Santos
PII: S0041-0101(07)00465-5
DOI: doi:10.1016/j.toxicon.2007.12.022
Reference: TOXCON3124
To appear in: Toxcon
Received date: 26 July 2007
Revised date: 14 December 2007
Accepted date: 14 December 2007
Cite this article as: Francisco M. Heralde, Julita Imperial, Pradip K. Bandyopadhyay,
Baldomero M. Olivera, Gisela P. Concepcion and Ameurna D. Santos, A rapidly di-
verging superfamily of peptide toxins in venomous Gemmula species, Toxcon (2007),
doi:10.1016/j.toxicon.2007.12.022
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A Rapidly Diverging Superfamily of Peptide Toxins in Venomous
Gemmula Species

Francisco M. Heralde III
*1,2
, Julita Imperial
*3
, Pradip K. Bandyopadhyay
3
, Baldomero M.
Olivera
**3
, Gisela P. Concepcion
2
and Ameurfina D. Santos
1



1
National Institute of Molecular Biology & Biotechnology and
2
Marine Science Institute,
University of the Philippines, Diliman, Quezon City 1001, Philippines
3
Department of Biology,
University of Utah, 257 South 1400 East, Salt Lake City 84112, UT, USA





* These authors contributed equally to this work.
** Corresponding author. Tel: 1 801 581 8370; Fax: 1 801 585 5010
E-mail address: olivera@biology.utah.edu


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Abstract

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the
family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula
venom ducts was characterized. Gemmula speciosa Reeve, 1843, venom duct cDNA
clones revealed two different conotoxin-like prepropeptide precursors, with identical
signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature
peptide region. The conserved signal sequence was used to successfully amplify
homologous genes from three other Gemmula species; all had the same pattern of Cys
residues in the predicted mature venom peptide. Although the signal sequence and
propeptide regions were highly conserved, the mature toxin regions diverged greatly in
sequence, except that the Cys residues were conserved. We designate this as the Pg-
gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two
members of the family directly from G. speciosa venom was achieved; amino acid
sequence analysis revealed that these peptides are highly posttranslationally modified.
With at least 10-fold as many species of turrids as cone snails, identification of rapidly
diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential
before the facile and systematic discovery and characterization of peptide toxins from
turrid venoms can be achieved.


Keywords: Conoidea, Venom peptides, Exogene superfamilies, Turrids, Drug discovery
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1. Introduction

There are >10,000 species of predatory marine snails that use venom as the
primary weapon for prey capture; these are traditionally assigned to a single superfamily,
Conoidea (=suborder Toxoglossa) (Kohn, 1998). Based on shell morphology, the
superfamily is traditionally divided into three groups, cone snails, augers and turrids.
Cone snails and augers are morphologically well defined; species in these groups are
easily recognized through their distinctive shells (Fig. 1A). In most molluscan taxonomic
treatments, cone snails are generally assigned to a single large genus, Conus, while
augers are assigned by most taxonomists to several different genera (Terebra, Hastula,
Duplicaria), in the family Terebridae (Bouchet and Rocroi, 2005, Kohn, 1998).
In contrast, the term turrids has been applied to a group that is much more
diverse (Taylor et al., 1993); it has long been recognized that their biodiversity is greater
than the two other groups (Powell, 1966, Powell, 1967). However, recent field work of
Bouchet and co-workers in New Caledonia and the Philippines has established that the
number of turrid species has been greatly underestimated and that the turrids
comprise the vast majority of conoidean biodiversity (Bouchet et al., 2002). The deep-
water habitat and/or small size of the majority of turrids has made them a significant
component of molluscan biodiversity which has been largely neglected.
We have initiated a broad investigation of the constituents of the venoms of these
marine mollusks, and are systematically analyzing representative venoms from each of
the major Conoidean genera. Among the turrids, we have concentrated on the subfamily
Turrinae (in the family Turridae).
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In this work, we report a combined molecular biological/biochemical
characterization of venom peptides from several species in the genus Gemmula
Weinkauff, 1875, the gem turrids. Gemmula are characterized by the gemmate sculpture
on the peripheral cord of their shells (Fig. 1B), the primary criterion used for taxonomic
assignment to the genus (Powell, 1967). In the classical taxonomy of turrids, Gemmula is
one of the larger groups; Powell remarked on the extensive fossil record of the group and
characterized it as the most vigorous member of the (subfamily) Turrinae and
undoubtedly represents the main stem of the subfamily. Species in the genus are
relatively large compared to most turrids, and like most turrid groups, Gemmula thrive
mostly in the deeper waters of the tropics, primarily in the Indo-Pacific. In this study, we
address whether Gemmula venoms are as complex as those of the cone snails, and
whether the unprecedented rate of interspecific divergence repeatedly noted for cone
snail peptides (Olivera, 2006) also occurs in Gemmula.
An important insight into how the impressive diversity of venom peptides was
generated during the evolutionary history of the genus Conus is that most peptidic
constituents of cone snail venoms are encoded by a relatively small number of rapidly
diversifying gene superfamilies (Olivera, 2006). A major question we have addressed is
whether similar gene superfamilies, characterized by their accelerated evolution, will also
be found in Gemmula venoms.
A final notable characteristic of peptide toxins from Conus venoms is the high
frequency of posttranslational modification found in the mature peptidic gene products.
It is unknown how widespread these posttranslational modifications are within the
superfamily Conoidea; preliminary evidence for the absence of most posttranslational
modifications in one major clade of Conoidea has been obtained (Imperial et al., 2007).
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We demonstrate by purifying and characterizing venom peptides that, as in Conus, the
peptide toxins of Gemmula venoms can be highly posttranslationally modified.

2. Materials and Methods

2.1 Specimen collection and RNA isolation

The snail samples were obtained by trawling between 50-100 m off Manila Bay
and through tangle nets between 300-500 m off the seas of Cebu and Bohol, Philippines.
Live snails were kept in seawater until processed. Samples were dissected and their
venom ducts immediately placed in RNAlater (Ambion, TX, USA) and pooled. The
venom ducts (50-100 mg) were homogenized in 1 mL TRIzol reagent (Invitrogen Life
Technologies, CA, USA) with a disposable microtissue grinder and the total RNA
isolated by phase separation and precipitation following the manufacturers standard
protocol.

2.2 cDNA preparation and sequencing

One microgram of total RNA was utilized as template for the first strand cDNA
preparation, followed by double stranded cDNA synthesis and amplification using long-
distance polymerase chain reaction and subcloning into pDNR-LIB using the Creator
SMART cDNA Library Construction Kit (Clontech Laboratories, CA, USA).
Recombinant plasmids were used to transform TOP10 (Invitrogen Life Technologies,
CA, USA) electrocompetent cells and plasmid libraries were screened for 0.5-1.4 kb
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inserts by performing PCR directly on colonies using M13 forward and reverse primers
provided by the vendor. Plasmids from selected colonies were purified (Wizprep,
Promega, WI, USA) and sequenced using an ABI377 automated sequencer.
Toxin cDNA from other snail samples was obtained by PCR using the gene
specific primers, Ptx1gg01_5 (5-A(G/T)CGAAG(A/C)GCT(C/G)CATTCG-3) and
Ptx1gg01_3 (5-ATC(G/C)A(T/G)(C/T)GAT(C/A)TGTT(T/G)TG-3) adapted with
vector specific sequences for cloning into pNEB206A linearized plasmid vector
following the manufacturers standard protocol (USER Friendly Cloning Kit, New
England Biolabs, MA, USA). The putative toxin gene was amplified from 2 L dsDNA
product as template in 20 L PCR reaction mix containing 2 L PCR buffer (10X),
dNTPs (200 M), primers (0.5 M each), Taq DNA polymerase (1U) and
diethylpyrocarbonate (DEPC)-treated water. The PCR profile used consisted of an initial
denaturation of 1 min at 95
o
C, followed by 40 cycles of 20 sec at 95
o
C, 20 sec at 54
o
C
and 30 sec at 72
o
C, and a final extension of 5 min at 72
o
C. The amplified product was
subcloned into pNEB206A.

2.3 Purification of peptides from G. speciosa venom ducts

Venom ducts were dissected from live specimens of G. speciosa collected from
the western Luzon, Philippines. Dissection was done on an ice block; the ducts were
transported with dry ice, and stored at -70 C. Frozen venom ducts from 104 specimens
were thawed by adding 1 mL of 40% acetonitrile with 0.1% trifluoroacetic acid (TFA),
and the mixture homogenized in an Eppendorf tube with a Teflon pestle in 30 strokes.
The homogenate was centrifuged in a Beckmann F650 rotor at 20,000 rpm for 15 min at
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4 C. The supernatant was applied on an analytical C
18
high-pressure liquid
chromatography (HPLC) column (4.6 mm x 250 mm), and the venom components
purified as outlined in Fig. 2. The venom components were separated through sequential
elution with gradients of 4.5% to 54% acetonitrile - 0.1% TFA for 55 min, followed by
54% to 90% acetonitrile - 0.1% TFA for 8 min. The number and masses of major
components of the main peaks were estimated by matrix-assisted laser desorption
ionization spectrometry (MALDI) (Peptide Biology Lab, Salk Institute, La Jolla
California, USA). Peptidic components within 1-6 kDa were reduced and alkylated.
An aliquot of Peak 1 (Fig. 2, Panel A) was reduced and the peptide components
were pyridylethylated following the previously described protocol (Imperial et al., 2003).
The pyridylethylated mixture was fractionated by HPLC on an analytical C
18
column
employing a gradient of 0.45% acetonitrile - 0.1% TFA per minute (Chromatogram is
shown in Fig. 2 Panel B). The mass of the native peptide equivalent to the more
hydrophobic component in Panel B was measured by MALDI (4071.5 Da) and the amino
acid sequence was determined by Edman degradation. The peptide is named gsp9a and
its sequence is shown in Table 2.
Peak 2 (Fig. 2, Panel A) was subfractionated through an analytical C
18
HPLC
column on a gradient of 0.18% acetonitrile - 0.1% TFA per minute (Chromatogram is
shown in Fig 2, Panel C). The components of the more hydrophobic peak in Panel C
were reduced and pyridylethylated. The pyridylethylated mixture was eluted through a
gradient of 0.45% acetonitrile - 0.1% TFA on an analytical C
18
HPLC column (Panel D).
The mass of the main peak in Panel D was determined by MALDI (4522.2 Da) and the
amino acid sequence of the peptide (referred to as gsp9b), was obtained by Edman
degradation.
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Posttranslationally modified hydroxyproline residues were identified directly
from the characteristic peaks in sequencing chromatograms while the posttranslational
modification of glutamic residues to -carboxyglutamic acid was deduced when low
glutamic acid signals were obtained in the respective cycles and confirmed by the
equivalence of calculated and measured molecular masses.

3. Results and Discussion

3.1. Identification of toxin sequences by molecular cloning

Specimens of Gemmula speciosa Reeve, 1843, the splendid turrid (Fig. 1) were
dissected and cDNA from venom ducts prepared as described under Methods. Five out
of 40 random clones sequenced from the cDNA gave conotoxin-like sequences; two of
these showed considerable sequence identity (Table 1). The translated open reading
frame encoded by these two cDNAs exhibit characteristics typical of the venom peptides
from Conus: at the N-terminal end there is a signal sequence; at the C-terminal end, after
a proteolytic processing at a predictable site (boxed), there is a cysteine-rich mature
peptide sequence with an intervening region (the pro region) found between the signal
sequence and the mature toxin regions. These two cDNA sequences, Gsp9.1 and Gsp9.2
have extensive sequence identity in the signal sequence and propeptide regions (they
differ at only one position). However, the predicted mature peptide regions are much
more divergent from each other than the N-terminal prepro sequences; although the Cys
residues are completely conserved, almost half of the non-Cys residues (14/30) have
diverged. This striking juxtaposition of highly conserved and hypervariable regions is
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characteristic of gene superfamilies encoding the toxin peptides found in Conus venoms,
a feature repeatedly noted ever since the first conopeptide gene superfamily was
characterized (Conticello et al., 2001, Olivera, 1999, Woodward et al., 1990).
In the last 15 years, since the first Conus peptide was characterized directly from
its gene sequence (Hillyard et al., 1992), the presence of conserved sequences in Conus
gene superfamilies has led to a strategy for new peptide discovery using PCR. We
explored using the same strategy for venom peptide discovery from Gemmula. We used
conserved sequences from Gsp9.1 and 9.2 to design PCR primers that were used to scan
for related venom peptide cDNAs from other Gemmula species. The results of the PCR
analysis of mRNA isolated from three other Gemmula species, Gemmula sogodensis
Olivera, 2004, Gemmula diomedea Powell, 1964, and Gemmula kieneri Doumet, 1840
are shown in Table 1. An alignment of all five sequences shows a striking conservation
of signal sequences and the same pattern of cysteine residues in all five mature toxin
regions. Thus, as in Conus, once one member of a gene superfamily has been identified,
the conserved signal sequence can be used to identify related toxin sequences that belong
to the same superfamily from other species through the use of PCR primers based on the
conserved sequences. A notable feature of the sequences in Table 1 is that the two
sequences obtained from Gemmula speciosa (Gsp9.1 and Gsp9.2) are more divergent
from each other than Gsp9.1 is from the sequences obtained from two of the other
Gemmula species, Gsg9.1 and Gdm9.1.
The results presented above provide the first compelling evidence for a rapidly
diversifying gene superfamily that encodes peptide toxins in venom ducts of Gemmula,
the gem turrids. Gemmula is one of the well-known genera in a major family of
venomous molluscs, the Turridae. Gem turrids occur in deeper waters of tropical oceans,
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with the bulk of species found in the Indo-Pacific. A variety of criteria, including both
morphological and molecular data, suggest that Gemmula and Conus are on branches of
the Conoidean superfamily tree that diverged early in the evolutionary history of
Conoidea; thus, Conus uses a hypodermic needle-like radular tooth to inject venom while
Gemmula apparently stabs its prey and injects venom at the wound site (Gemmula sp. do
not have hollow, hypodermic-type teeth). However, little is known regarding the detailed
biology of any Gemmula.
This study demonstrates that the biosynthesis of Gemmula venom peptides is very
similar to the pattern established for Conus: each toxin gene is translated as a
prepropeptide precursor and proteolytic cleavage of the precursor releases the mature
peptide toxin.

3.2 Biochemical purification of venom peptides

The venom ducts of Gemmula speciosa are extremely fine, and it was a challenge
to obtain enough specimens to perform biochemical purification (a detailed account of
the field collecting that had to be carried out is presented elsewhere Heralde, et al.
manuscript in preparation). We purified major components of the venom, and two
peptides that are encoded by the gene superfamily characterized by molecular cloning
described in the preceding section were identified. The purification of these two peptides
is detailed in Fig. 2.
The purified peptides were analyzed utilizing Edman sequencing and mass
spectrometry. The identity of the posttranslationally modified amino acids was
established as outlined in Methods; the final sequence assignments are shown in Table 2.
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These sequences were compared to the mature sequences predicted from the putative
precursors obtained by molecular cloning (Table 1).
The two peptides characterized from Gemmula speciosa venom, gsp9a and gsp9b
appear to be polymorphic variants of the venom peptides encoded by the two clones from
Gemmula speciosa, Gsp9.1 and Gsp9.2. A comparison of the peptide sequences of the
components purified from venom with the predicted amino acid sequences from the
clones reveals that the purified venom peptides are highly posttranslationally modified.
Apparently, in peptides of this gene superfamily, proline is posttranslationally modified
to 4-transhydroxyproline and glutamate residues are posttranslationally carboxylated to -
carboxyglutamate. Both the final posttranslationally modified mature toxin sequences
predicted from the clones, as well as the directly sequenced venom peptides are
summarized in Table 2.
The sequences fall into two classes, those that display greater homology to gsp9a
comprise a major class, and two peptides, gsp9b and its polymorphic variant encoded by
Gsp9.2. The sequence from Gemmula kieneri, Gkn9.1, is quite divergent from the others.
Although the physiological function of these peptides is unknown, a reasonable working
hypothesis is that the different groups are differentially targeted (possibly to different
molecular isoforms of homologous targets encoded by the same gene family), and that
the predicted venom peptides from Gemmula sogodensis and Gemmula diomedea will
have a physiological function homologous to that of gsp9a, but divergent from that of
gsp9b. The results suggest that Gemmula venom peptides have good potential to be
subtype-specific ligands, and be useful as are conotoxins to functionally characterize
different ion channels and receptors in nervous systems.
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Members of the gene superfamily directly purified from venom have proven to be
highly posttranslationally modified. Two characteristic Conus posttranslational
modifications, the hydroxylation of proline residues and the -carboxylation of glutamate
(Buczek et al., 2005) were found in the Gemmula speciosa peptides directly isolated from
venom all of the proline and glutamate residues in gsp9a and gsp9b were
posttranslationally modified. Thus, these unusual posttranslational modifications are not
restricted to Conus and may be widely distributed across the superfamily Conoidea.
The arrangement of Cys residues in the primary sequence of the venom peptides
gsp9a and gsp9b, is similar to that of the peptides from Conus in the P-superfamily.
However, as is shown in Table 1, there is no obvious homology between the sequence of
P-superfamily conopeptides and the presently characterized superfamily members from
Gemmula. We propose to call this peptide superfamily the Pg-superfamily, in accordance
with the nomenclature described in the Appendix. This superfamily of venom peptides
was detected in all four Gemmula species investigated; how widely distributed the gene
superfamily is within the family Turridae and across the superfamily Conoidea remains to
be established.
As is evident from Fig. 2, the venom of Gemmula speciosa is comparable in
complexity to Conus venoms. A much more extensive characterization of this venom is
in progress (J. Imperial and B. Olivera unpublished results). Gemmula venoms have
many of the characteristics now well established for Conus: venom constituents that are
disulfide rich peptides, many with extensive posttranslational modifications. Initial mass
measurements of native and reduced major peak components demonstrated the presence
of peptides within the 1-6 kDa, the size range of most conotoxins. The results of this
study suggest that the different constituents of these complex Gemmula venoms are likely
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to be generated by the rapid diversification of gene superfamilies such as the Pg-
superfamily, the first gene superfamily shown to be expressed in Gemmula venom ducts.
We note that the only previous study of any Gemmula venom yielded a peptide toxin that
was strikingly different in its biochemical properties (Lopez-Vera et al., 2004).
The larger significance of establishing for the first time in the presence of rapidly
diversifying gene superfamilies in the turrid genus Gemmula is that the general strategy
used to discover, identify and characterize peptides from the venoms of Conus has been
demonstrated to be applicable to turrids. An important component of the discovery
strategy developed for Conus is the systematic examination of venom peptide sequences
obtained by PCR amplification of gene superfamily homologs from different Conus
species. We have provided proof-of-principle that this strategy is effective for the
Gemmula Pg-superfamily; not only were we able to obtain five sequences by PCR
amplification, but the availability of the gsp9a and gsp9b peptide sequences allows us to
predict with some confidence which amino acid residues are posttranslationally modified.
Conus peptides have proven not only to provide ligands that are important tools in
neuroscience, but compounds that are used for diagnostic and therapeutic purposes as
well. One of the peptides found in Conus venoms is now Prialt, a commercial drug for
intractable pain. There are 70,000 venom peptides in the venoms of the 500-700 living
Conus species; the total biodiversity of turrids is still undetermined, but present estimates
indicate that there should be an order of magnitude more turrid species, thereby
increasing the total pharmacological resource that can theoretically be accessed from the
superfamily Conoidea by at least tenfold. The problem is that unlike Conus, turrids tend
to be smaller, live in deeper water habitats (such as the Gemmula species described in this
report) and have proportionally smaller venom ducts. However, the exogene-based
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discovery strategy developed for Conus (Olivera, 2006) makes such differences less of a
barrier to discovery.
It would be virtually impossible to access enough venom from most turrid species
to do traditional biochemical characterization of venom components. However, by
defining gene superfamilies as has been accomplished here, even tiny and rare Gemmula
species can now theoretically be examined for the members of the Pg-gene superfamily
expressed in their venoms. Thus, the definition of venom constituents can bypass the
tedious purification procedures involved in traditional discovery. Clearly, the Pg-
superfamily will be only the first of the gene superfamilies that generate the chemical
diversity found in turrid venoms.

Acknowledgements
This work was supported by grant GM48677 from the U.S. National Institute of General
Medical Sciences. FMH would like to acknowledge PCASTRD-DOST and Cavite State
University for his PhD Scholarship. We thank Carlo Lapid for his assistance in the
preparation of G. speciosa cDNA library and Maren Watkins for annotating the cDNA
library sequences.

Appendix

A proposal for the nomenclature of Conoidean venom peptides
In this manuscript, we adopt a nomenclature that we propose be applied to all
Conoidean venoms. It builds on the widely used nomenclature for Conus peptides. We
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propose that the term conopeptide be generalized for peptidic constituents in all
Conoidean venoms, not just for Conus.
In Conus, when the mechanism of action for a peptide is unknown, it is referred to
by one or two lower case letters followed by an Arabic number and a small letter. Thus,
the peptides might be initially called rg1a or r11c: the letters refer to the Conus species,
while the Arabic number provides information about the pattern of cysteine residues in
the primary sequence. Thus, rg refers to Conus regius, r to Conus radiatus; and the
number 1 indicates the characteristic Cys pattern of the A-superfamily (CC---C---C)
while 11 suggests the Cys pattern of the I-superfamily (C---C---CC---CC---C---C). The
last lower case letter following the number simply refers to the order in which the
peptides have been either characterized from the venom or synthesized. Thus, r11a, r11b
and r11c all come from Conus radiatus with r11c the third peptide to be characterized in
that series, (different in sequence from r11a and r11b, but sharing the same cysteine
pattern).
We propose that basically the same nomenclature be used for all conoidean
venoms, excepts that peptides that come from taxa other than Conus will have either
three or four letters. In the present work, we use three letters, with the first letter always
being G or g (for Gemmula), and the two following derived from the species name.
Thus, we refer to the two peptides isolated from Gemmula speciosa venom as gsp9a and
gsp9b.
Similarly, references to specific clones should follow the Conus nomenclature; in
Conus, the first letter is capitalized and instead of using an Arabic number followed by a
letter, the clones use the same number with the designation 1.1, 1.2, 1.3, etc. for those
that encode peptides with Cys pattern 1 and 11.1, 11.2, 11.3, etc. for clones encoding
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peptides with Cys pattern 11. Thus, in the present work, we use this nomenclature, with
the first letter capitalized, when clones are being referred to. The clones from Gemmula
speciosa are designated Gsp9.1, Gsp9.2, while the clone from Gemmula sogodensis is
Gsg9.1 and from Gemmula diomedea, Gdm9.1.
We further propose that superfamilies be referred to in the same manner as
in Conus except that the name of the superfamily will now have a capital letter and a
small letter with the small letter referring to the genus from which the superfamily was
first characterized. Thus, in the present work, since the superfamily has a Cys pattern
similar to that of the P-superfamily of Conus peptides, but it has been characterized from
Gemmula, we refer to the gene superfamily as the Pg-superfamily.
We propose that the physiological mechanisms of actions, once clarified, use the
same Greek letters as in the Conus nomenclature. Thus, if peptide gsp9a in the Pg-
superfamily proves to be nicotinic receptor antagonist, then this will be referred to as -
conopeptide GspIXA (abbreviated as -GspIXA). This reveals a nicotinic antagonist
() from Gemmula speciosa (Gsp) that has the Cys pattern C--C--C--C--C--C
(1X) characteristic of the P-like superfamilies. A second nicotinic antagonist in the
same superfamily would be -GspIXB; note that GspIXA and GspIXB may target
different subtypes of nicotinic receptors.
Thus, all the conopeptides will have the same basic nomenclature, and the 10
6

diverse peptides from multiple genetic families with diverse structure, functional
mechanisms and biological origins will be named to provide information about the
species the conopeptide was derived from, structural information based on the Cys
pattern, and a general mechanism of action (based on the Greek letter designation).

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References

BOUCHET, P., LOZOUET, P., MAESTRATI, P. & HEROS, V. (2002) Assessing the
magnitude of species richness in tropical marine environments: high numbers of
molluscs at a New Caledonia site. Biol. J. Linnean Soc., 75, 421-436.
BOUCHET, P. & ROCROI, J. P. (2005) Malacologia: International Journal of
Malacology, Classification and nomenclature of gastropod families, ConchBooks.
BUCZEK, O., BULAJ, G. & OLIVERA, B. M. (2005) Conotoxins and the
posttranslational modification of secreted gene products. Cell Mol Life Sci, 62,
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CONTICELLO, S. G., GILAD, Y., AVIDAN, N., BEN-ASHER, E., LEVY, Z. &
FAINZILBER, M. (2001) Mechanisms for evolving hypervariability: the case of
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HILLYARD, D. R., MONJE, V. D., MINTZ, I. M., BEAN, B. P., NADASDI, L.,
RAMACHANDRAN, J., MILJANICH, G., AZIMI-ZOONOOZ, A.,
MCINTOSH, J. M., CRUZ, L. J., IMPERIAL, J. S. & OLIVERA, B. M. (1992) A
new Conus peptide ligand for mammalian presynaptic Ca
2+
channels. Neuron, 9,
69-77.
IMPERIAL, J., KANTOR, Y., WATKINS, M., HERALDE, F., STEVENSON, B. J.,
CHEN, P., OWNBY, J.-P., BOUCHET, P. & OLIVERA, B. M. (2007) The
Venomous Auger Snail Hastula (impages) hectica (Linnaeus, 1758): Molecular
Phylogeny, Foregut Anatomy and Comparative Toxinology. Journal of
Experimental Zoology, Part B, In press.
IMPERIAL, J. S., WATKINS, M., CHEN, P., HILLYARD, D. R., CRUZ, L. J. &
OLIVERA, B. M. (2003) The augertoxins: biochemical characterization of
venom components from the toxoglossate gastropod Terebra subulata. Toxicon,
41, 391-398.
KOHN, A. J. (1998) Superfamily Conoidea, Pp 846-854. IN BEESLEY, P. L., ROSS, G.
J. B. & WELLS, A. (Eds.) Mollusca: Southern Synthesis. Fauna of Australia, Vol.
5. Melbourne, CSIRO Publishing, Part A xvi 563 pp.
LOPEZ-VERA, E., HEIMER DE LA COTERA, E. P., MAILLO, M., RIESGO-
ESCOVAR, J. R., OLIVERA, B. M. & AGUILAR, M. B. (2004) A novel
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1020.








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Figure Captions

Fig. 1. A. Shells of Conus, Terebra, Turrids. Top, Terebra subulata (Terebridae);
Middle row: left, Hastula hectica (Terebridae); right, Conus marmoreus; Bottom row:
left, Gemmula speciosa; right, Lophiotoma olangoensis (both Turridae).
B. Left to right: Gemmula speciosa, G. diomedea, G. sogodensis, and G. kieneri.

Fig. 2. Purification of gsp9a and gsp9b. The components of the G. speciosa venom duct
extract prepared as described under Methods were separated by sequential elution with
gradients of 4.5% to 54% acetonitrile in 55 min and 54% to 90% acetonitrile in 8 min in
the presence of 0.1% TFA. The absorbance profile at 220 nm is shown in Fig. 2, Panel
A. An aliquot of Peak 1 in Panel A was reduced and pyridylethylated. The
pyridylethylated mixture was fractionated on an analytical C
18
column employing a
gradient of 0.45% acetonitrile/min in 0/1% TFA, (Panel B). The more hydrophobic peak
was determined as described in Methods to be gsp9a and the sequence is shown in Table
2. Peak 2 in Panel A was subfractionated on an analytical C
18
HPLC column with a
gradient of 0.18% acetonitrile/min in 0.1% TFA (Panel C). The components of the more
hydrophobic peak were reduced, pyridylethylated, and then fractionated with a gradient
of 0.45% acetonitrile/min in 0.1% TFA on an analytical C
18
HPLC column (Panel D).
The main peak in Panel D was determined by MALDI and amino acid sequencing to be
gsp9b (Table 2).

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Fig. 1

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Fig. 2