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Research Article
Received: 8 July 2014
Revised: 22 October 2014
Accepted: 22 October 2014
Published online in Wiley Online Library: 25 November 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2715
Novel M-Superfamily and T-Superfamily
conotoxins and contryphans from the
vermivorous snail Conus figulinus
Rajaian Pushpabai Rajesh*
The venom of Conus figulinus, a vermivorous cone snail, found in the south east coast of India, has been studied in an effort to
identify novel peptide toxins. The amino acid sequences of seven peptides have been established using de novo mass
spectrometric based sequencing methods. Among these, three peptides belong to the M-Superfamily conotoxins, namely,
Fi3a, Fi3b, and Fi3c, and one that belongs to the T-Superfamily, namely, Fi5a. The other three peptides are contryphans,
namely, contryphans fib, fic, and fid. Of these Fi3b, Fi3c, Fi5a, and contryphan fib are novel and are reported for the first
time from venom of C. figulinus. The details of the sequencing methods and the relationship of these peptides with other
‘M’-Superfamily conotoxins from the fish hunting and mollusk hunting clades are discussed. These novel peptides could
serve as a lead compounds for the development of neuropharmacologically important drugs. Copyright © 2014
European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: cone snails; de novosequencing; disulfide rich peptides; venom; M-Superfamily conotoxins; T-Superfamily Conotoxins;
contryphans
Introduction
J. Pept. Sci. 2015; 21: 29–39
* Correspondence to: R. P. Rajesh 207, Molecular Biophysics Unit, Indian Institute of
Science, Bangalore 560012, India. E-mail: rprajesh@mbu.iisc.ernet.in
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
Abbreviations: NMDA, N-methyl-D-aspartate; TCEP, Tris(2-carboxyethyl)phosphine; NEM, N-methylmaleimide; HPLC, high-performance liquid chromatography; ESI, electro spray ionization; LC-ESI, liquid chromatography electro spray
ionization; LC-ESI-MS, liquid chromatography electro spray ionization mass spectrometry; LC-MS, liquid chromatography mass spectrometry; TFA, trifluoroacetic
acid; MALDI-TOF, matrix assisted laser desorption ionization – time of flight;
MALDI-TOF-TOF, matrix assisted laser desorption ionization – time of flight – time
of flight; CCA, alpha-cyano-4-hydroxycinnamic acid; CID, collision induced dissociation; ETD, electron transfer dissociation.
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
29
The venom components of the predatory cone snails (genus
Conus) have been under systematic investigation over the last
two decades to identify neuropharmacologically active compounds [1]. Of specific interest are the peptide components of
the venom. Each conus species is estimated to contain approximately 100–150 small (10–30 amino acids), highly structured, disulfide rich peptides, which are biosynthesized and secreted in
the venom duct [1,2]. These peptides, which are collectively
known as ‘conotoxins’, are classified either on the basis of their
receptor specificities or on the basis of their cysteine framework,
that is, on the basis of the arrangement of cysteines in the amino
acid sequence [3]. The disulfide-pairing pattern is generally
unique for a particular arrangement of cysteines in the primary
structure. However, it has been shown in the case of the ‘M’superfamily of conotoxins, that a single cysteine framework
may accommodate more than one disulfide-pairing pattern
and that this heterogeneity is a consequence of the amino acid
sequence in the ‘inter-cysteine loops’ [4]. M-Superfamily
conotoxins possess distinct disulfide connectivity for each subfamily (M1–M5 superfamily) [5], where M1–M3 are the ‘mini’conotoxins, and M4 and M5 are the ‘maxi’-conotoxins. The
specific pharmacological targets of the ‘maxi’-conotoxins have
been identified. In contrast, the receptor specificities of the
‘mini’-conotoxins are as yet unknown. However, the response
elicited upon direct intracranial injection in animals has been recorded [1]. T-superfamily conotoxins are widely distributed in all
three hunting clades of cone snails. The peptides belonging to
this class of conotoxins consist of residues typically ranging from
9 to 17 amino acids [6]. They are further subcategorized into T1
and T2 superfamily conotoxins. The T1 subfamily has the
cysteine pattern ---CC-----CC----, with disulfide pairing between
cysteines 1–3 and 2–4. The T2 subfamily has the cysteine pattern
---CC----C----C---, with disulfide pairing between cysteines 1–4
and 2–3 [7]. Some members of the T2 subfamily of conotoxins
are known to inhibit the norepinephrine receptors
(e.g., Chi/lambda group [8]), whereas others have been found to
blocks tetrodotoxin sensitive (TTX-S) Na channel (e.g., conotoxin
LtVd isolated from Conus litteratus [9]). The contryphans are a group
of single disulfide containing peptides that are found in the venom
of all cone snails. The contryphans are exceptional in that they are
found to possess numerous post translational modifications such
as epimerization of valine, leucine, and tryptophan, tryptophan bromination, amidation of the C-terminus, and proline hydroxylation
[10]. Contryphans also possess certain biological activities.
Contryphan-M, a highly modified conotoxin isolated from Conus
marmoreus, blocks calcium channel [10].
The diversity in sequence, structure, and pharmacological profile
of these conopeptides has resulted in these molecules to serve as
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leads in the search for neuropharmacological agents with therapeutic or diagnostic potential. The interest in the neurotoxicity of
conotoxins arises from their ability to target ion-channels such as
the Na+, K+, and Ca2+ ion channels and other ligand associated receptors such as the norepinephrine, glutamate, and NMDA, receptors with high specificity [3,11]. The conotoxins that are often able
to distinguish specific subtypes of these receptors have thus been
invaluable for the identification of these cellular targets and for understanding their physiological roles. As a consequence of this
specificity, the conotoxins thereby are thought to have immense
potential as therapeutics for the alleviation of neuromuscular diseases [12].
As part of a program for the systematic characterization of the
peptide components of the cone snail venom [13–18], the characterization of the venom components of Conus figulinus has been
undertaken. C. figulinus snails are commonly found off the northern
coastline of Tamil Nadu and also in the Gulf of Mannar Biosphere
Park, located off the south east coast of Tamil Nadu in India. As of
date, four peptides belonging to the I-Superfamily [19,20] and
one contryphan [ 21] have been reported from the venom of C.
figulinus.
Here, we report the peptide sequences of seven conopeptides
from C. figulinus. These include the‘mini’–M conotoxins, Fi3a, Fi3b,
Fi3c, a novel T-superfamily conotoxin, Fi5a and three contryphans
namely, contryphan fib, contryphan fic, and contryphan fid. Sequence identification was achieved using mass spectrometry based
fragmentation methods [22]. These peptide sequences have been
compared with the sequences of related peptides identified in
the venom from other Conus species.
Mass Spectrometry
ESI mass spectra were recorded on a Bruker Daltonics Esquire 3000
Plus Ion-Trap Mass Spectrometer attached to an Agilent 1100 series
HPLC system. The samples were infused into the mass spectrometer either by direct injection or through an HPLC column (Agilent
Zorbax analytical C18 column, 150 × 4.6 mm, 5 μm, 90 Å pore size)
and eluted using a binary gradient of water (0.1% TFA): acetonitrile
(0.1% TFA) at a flow rate of 0.2 mL min 1. Data were acquired over a
m/z range of 100–2000 in positive ion mode. MALDI-TOF experiments were performed where ever necessary in Ultraflex MALDITOF-TOF (Bruker Daltonics) using CCA as matrix.
LC-ESI-MS of Natural Extract
The lyophillized natural extract of C. figulinus was dissolved in 2 mL methanol and filtered through a 0.2 μM filter. This redissolved sample of the
natural venom formed the stock solution that was then used for mass
spectrometric analysis. The clarified sample was analyzed by LC-ESI-MS
to identify the number of peptide components in the crude mixture.
Global Reduction and Alkylation of Natural Venom and Analysis by LC-ESI-MS
The reducing agent TCEP (final concentration 20 mM) was added to
the crude natural venom extract, and the mixture was incubated at
37 °C for 1.5 h. To this reaction mixture NEM was added to a final
concentration of 40 mM, and the mixture was incubated at room
temperature for 45 min. The reaction mixture was analyzed by LCESI-MS to identify the number of disulfide rich conopeptides and
to establish the number of disulfides in each m/z species.
Materials and Methods
Acetylation of Reduced and Alkylated Peptides
TCEP (tris(2-carboxyethyl)phosphine) was purchased from Pierce
Scientific. NEM was purchased from Sigma-Aldrich chemical company, USA. Acetonitrile and methanol for HPLC were purchased
from Merck India Ltd and purified by distillation prior to use. Analytical grade TFA was purchased from Merck India Ltd.
Collection
The C. figulinus samples were collected from the waste at fish landing sites located in Mandapam(9°16′38.98″N, 79°09′19.49″E ) and
Rameshwaram(9°16′44.61″N, 79°12′19.99″E), Tamil Nadu, India
[23]. As these species (C. figulinus) are not listed under endangered
or protected species, we continued to work with this species without prior permission with the wild life authorities.
Identification of the Cone Snail
The collected cone snail was identified following standard keys.
Extraction of Natural Peptides
30
The venom ducts of approximately 40 C. figulinus specimens were
dissected and stored in 50% HPLC grade acetonitrile at the collection site. The samples were transported to the laboratory, frozen
in liquid nitrogen, and ground well using a mortar and pestle. The
finely ground sample was extracted with HPLC grade 50% Acetonitrile. The natural extract was filtered through Whatman No.1 filter
paper. The filtrate was concentrated in vacuo using a rotary vacuum
evaporator. The concentrated sample was lyophilized and stored at
20 °C till further use.
wileyonlinelibrary.com/journal/jpepsci
To 5 μL of a methanolic solution of reduced and alkylated natural venom,
2 μL acetic anhydride was added and the volume was made up to 20 μL
with distilled water. The reaction mixture was incubated for 1 h at 25 °C,
and the product was analyzed by LC-ESI-MS as described earlier to identify
the conopeptide sequences that had a free amino-terminus and/or have
lysine residues in the sequence. The acetylation reaction also enabled
distinction between lysine and glutamine residues.
Esterification Reduced and Alkylated Peptides
Methanolic HCl was prepared by drop wise addition of 20 μL acetyl
chloride to 100 μL of ice cold, anhydrous methanol. Added to 16 μL of
methanolicHCl was 4 μL of reduced and alkylated peptide. The esterification was allowed to proceed at 25 °C. The reaction mixture was analyzed
by MALDI-MS at different incubation times of 0 min, 30 min, and 6 h.
CID and ETD Fragmentation Of Chemically Modified Natural
Extract
Auto MS(n) experiments (CID and ETD fragmentation) were performed
for the reduced and alkylated peptide molecules in the natural venom.
The chemically modified peptides were chromatographically separated based on their polarity using a reverse phase C18column. The
peptides eluting from the column were fragmented using nitrogen
gas (CID fragmentation) and by collision of these peptides with thermal excited electrons in a methane atmosphere (ETD fragmentation).
The daughter ion spectra were analyzed to derive the sequence of
the peptides. The derived peptide sequences are compared with
known peptide sequences of conotoxins.
J. Pept. Sci. 2015; 21: 29–39
CONE SNAIL VENOM PEPTIDE SEQUENCING
90 A pore size) using methanol (0.1% trifluoroacetic acid) /water
(0.1% trifluoroacetic acid) that was applied as a linear gradient.
The flow-rate was maintained at 1 mL/min, and the fractions were
detected at 226 and 280 nm.
The sequencing of peptides was aided by use of chemical modification reactions and the mass spectrometric methods described
for the peptides in the natural extract.
The peptide Fi3b was custom synthesized (Thermo Scientific,
Germany). The crude peptide was refolded in solution-phase using
a Glutathione-based redox system. The linear crude peptide was incubated with a 2 : 1 mixture of reduced (2 mM) and oxidized (1 mM)
Glutathione at room temperature for 12 h and subsequently purified by HPLC. The purified synthetic peptide was analyzed by mass
spectrometry. The daughter ion spectra obtained from fragmentation of the intact, refolded peptide was compared with that obtained for the peptide purified from natural venom.
Figure 1. The shell of the vermivorous cone snail Conus figulinus.
Purification and Sequencing Fi3a, Fi3b and Fi5a.
The peptides Fi3a, Fi3b, and Fi5a were purified from the natural
venom extract of C. figulinus. Peptides were purified by HPLC using
a Agilent ZORBAX C18 semi preparative column (9.4 mm × 250 mm,
Results
Identification
The collected cone snail was identified using standard keys as described and identified as C. figulinus Linnaeus, 1758 (Figure 1) [ 23].
J. Pept. Sci. 2015; 21: 29–39
31
Figure 2. The liquid chromatography mass spectrometry profile of the (A) natural and (B) reduced and alkylated venom extract from Conus figulinus. ‘Insets
show the isotope cluster distribution of the singly and doubly charged species of peptides’.
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Table 1. Conopeptides and their cysteine composition as identified from the natural venom extract of Conus figulinus
Sl. No
+
Massa [M + H]
1
2
3
4
5
6
7
8
9
10
11
2+
Massa [M + 2H]
1685.6
1701.8
1410.6
1428.6
1626.6
1767.4
1417.8
960.5
976.5
992.5
903.4
+
R/A Massb [M + H]
843.9
851.9
—
715.3
814.4
884.7
—
—
—
—
—
2444.0
2460.0
2166.7
2186.8
2384.8
2524.4
1924.2
1212.5
1228.9
1244.5
1155.2
2+
R/A Massb [M + 2H]
1222.5
1230.5
—
1093.9
1192.9
1262.7
—
—
—
—
—
Disulfides
3-S-S (6 C)
3-S-S (6 C)
3-S-S (6 C)
3-S-S (6 C)
3-S-S (6 C)
3-S-S (6 C)
2-S-S (4 C)
1-S-S (2 C)
1-S-S (2 C)
1-S-S (2 C)
1-S-S (2 C)
a
Unmodified natural peptides
N-ethylmaleimide alkylated peptides
b
Analysis of Peptides in the Natural Venom
Mass Spectrometry
LC-MS of Natural Venom and Reduced Alkylated Natural Venom
The LC-MS profile of the natural venom extract from C. figulinus reveals
the presence of several peptide components. The mass spectra shown
in Figure 2A and 2B reveal the presence of several singly charged
species. Interestingly, doubly [M + 2H]2+charged m/z species could also
be observed in the spectrum in the 800 to 900 Da mass range. The reduction of disulfide bonds in conopeptides will convert the persulfides
into free thiols. Subsequent alkylation with NEM will result in peptides
with an added mass of 126 Da for each cysteine in the molecule. From
the change in mass, the number of cysteine residues in the peptide
can be determined in a facile manner. As a control experiment, the
alkylation reactions were also carried out on the unmodified form of
the molecules in the natural venom. Using this protocol, the cysteine
content of eleven peptides was unequivocally established. The
observed unmodified mass, the reduced and alkylated mass, and the
number of experimentally determined disulfide bonds for these
eleven peptides in the natural venom are given in Table 1. In all, six
peptides were shown to possess three disulfide bonds, one to possess
two disulfide bonds, and four to possess one disulfide bond.
HPLC Purification of Peptides
mass of 756 Da (126 Da × 6). This is conclusive proof of the fact that
Fi3b contains six cysteine residues that are paired to form three disulfide bonds. Acetylation of the reduced alkylated peptide resulted
in a 42 Da increase in mass, suggestive of the presence of free
amino terminal group (Supplementary Figures 2A and 2B). Reduced, alkylated, and esterified Fi3b shows presence of two major
peaks at 2472 and 2485(Supplementary Figures 3A and 3B) indicative of the presence of two carboxylic acid functional groups in the
molecule.
Amino acid sequencing of Fi3b was carried out from analysis of
LC-ESI-MS-MS spectra obtained from the reduced and alkylated
peptide. Figures 4 and 5 show the daughter ion spectra obtained
from fragmentation of the doubly charged ([M + 2H]+2,
m/z = 1230.4) and triply charged [M + 3H]+3, m/z = 820.7) species, respectively. Rich fragment ion spectra were obtained with intense
peaks being distributed over the m/z region 250 to 2200, which is
presented in Table 2.
The ‘y2’ ion (m/z 279.2) of high intensity, observed in the
MS2spectrum of the triply charged parent ion (Figure 5), was
assigned tentatively to the dipeptide sequence PY (proline-tyrosine). The difference in mass between the y4 and y5 ions is
113 Da, suggesting the presence of either of the isobaric residues isoleucine, leucine, or hydroxyproline (O) in sequence. Detailed analysis of the daughter ion spectra (Figures 4 and 5) in
conjunction with the data obtained from reduction and
Figure 3 shows the HPLC chromatogram of the natural venom. The
peptides eluting at 51.8 min, 55.1 min, and 56.3 min showed masses
of 1701.8, 1685.7, and 1417.6 Da, respectively. These peptides were
purified to homogeneity and were correspondingly designated as
Fi3b, Fi3a, and Fi5a.
De novo sequencing by Mass Spectrometry
Mass spectrometry based sequencing was undertaken of the peptides in the natural venom as well as of the peptides that were purified by HPLC.
Three Disulfide Peptides
Sequence of Fi3b (1701 Da)
32
Mass spectra of the intact and the reduced and alkylated Fi3b peptide (Supplementary Figures 1A and 1B) showed an increase in
Figure 3. High-performance liquid chromatography spectrum of crude
venom of C. figulinus. Indicated on the chromatogram are the masses of
the peptides identified in each fraction.
J. Pept. Sci. 2015; 21: 29–39
Figure 4. De novo sequencing of HPLC purified Fi3b. Mass spectrum resulting from fragmentation of reduced and alkylated doubly charged peptide (m/z [M
2+
+ 2H] = 1230.3).
alkylation, esterification and acetylation experiments yielded
CCSQDCRVC(I/L/O)PCCPY as the sequence of the Fi3b. Further
support for this assignment comes from the sequence analysis
of the peptide Fi3a (vide infra).
Sequence of Fi3a
Figures 6A and 6B show the isotopic cluster of the singly protonated m/z species of Fi3b and Fi3a peptides, respectively. A difference in mass of 16 Da corresponds to the mass of an oxygen
atom. Hydroxylation of an amino acid in Fi3a, as a posttranslational
modification, will result in a peptide of mass 1701.8. Proline hydroxylation is a commonly occurring posttranslational modification in
conus peptides [24–26].
Figures 7 and 8 show the daughter ion spectra obtained from
fragmentation of doubly ([M + 2H]+2; m/z = 1222.4) and triply ([M
+ 3H]+3; m/z = 815.3) charged species. Well-defined fragment ion
spectra were obtained with intense peaks being distributed over
the m/z region 250 to 2200, which is populated in Table 2.
The presence of a ‘y2’ ion at m/z 279, of identical mass to that
observed in the MS2 spectrum of Fi3b, indicates that the sequences at the C-terminii of these peptides are identical, that
is, proline-tyrosine (PY). The difference in mass between the y5
and y4 ions in the spectrum of Fi3a (ΔM = 97) and that of the
same ions in the spectrum of Fi3b (ΔM = 113) indicates that
Pro11 in Fi3a undergoes hydroxylation. Complete analysis of
the daughter ion spectra (Figures 7 and 8) in conjunction with
the data obtained from the reduction and alkylation, esterification and acetylation experiments yielded CCSQDCRVCIPCCPY
as the sequence of the Fi3a (Table 3).
Thus, the sequence assigned to the peptide Fi3b may be corrected
to CCSQDCRVC(I/L)OCCPY. It is interesting to note that the sequence
CCSQDCRVCIPCCPY has earlier been identified from cDNA analysis of
the venom libraries in C. tessulatus and C. ventricosus. Thus, there is a
high probability that residue 10 in Fi3b and Fi3a is an isoleucine
(supplementary table 1). The clustal W analysis (Table 4) of Fi3b
and Fi3a with available sequence in database confirms the presence
of isoleucine in both Fi3b and Fi3a and proline and hydroxyl proline
in 11th position of Fi3b and Fi3a, respectively.
Comparison of Natural and Synthetic Fi3b
To verify the results of the mass spectrometry based analysis described
earlier, a peptide corresponding to the sequence, CCSQDCRVCIOCCPY,
of Fi3b was chemically synthesized. Supplementary Figures 4A and 4B
shows the mass spectra of the reduced peptide and the fully oxidized,
refolded peptide. A change in mass of 6 Da upon oxidation suggests
that three disulfide bonds have formed. Figure 9 shows the HPLC
co-elution experiment of the natural and refolded synthetic peptides.
The single peak at 21.7 min strongly suggests that the two molecules
have identical conformational properties. This further asserts the
assignment of residue 10 being an isoleucine.
Sequence of Fi3c
J. Pept. Sci. 2015; 21: 29–39
33
Figure 5. De novo sequencing of Fi3b. Mass spectrum resulting from
collision induced dissociation fragmentation of reduced and alkylated
3+
triply charged peptide (m/z [M + 3H] = 820.7).
The sequence of Fi3c was obtained by fragmentation of the
reduced and alkylated peptide in the natural venom. Esterification and acetylation showed that this peptide had free
amino-terminus and carboxyl-terminus. Figure 10 shows the
daughter ion spectra obtained from fragmentation of the
doubly charged ([M + 2H]+2; m/z = 1083.4) species. The b and
y ions are presented in Table 2.
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Table 2. ‘b’ and ‘y’ ions of peptides Fi3b, Fi3a, Fi3c, Fi5a, Contryphanfid, Contryphanfic and Contryphanfib are presented
b ions
B ions
b1
b2
b3
b4
b5
b6
b7
b8
b9
b10
b11
b12
b13
m/z Fi3b
m/z Fi3a
m/z Fi3c
m/z Fi5a
457.3
457
544.1
672.2
787.2
1015.3
1171.3
1270.5
1498.5
1611.6
1708.7
1936.8
2164.7
457
344
572.1
758.2
855.3
912.3
1068.5
1165.5
1280.6
1508.6
1736.6
1807.6
672.2
787.2
1270.5
1498.5
1611.6
1724.9
1952.6
2180.7
646.0
759.8
987.4
1058.5
1157.5
1385.6
1498.8
1595.7
1823.7
2051.8
m/z Contryphan fid
627.6
724.7
910.5
1138.5
m/z Contryphan fic
m/z Contryphan fib
383.3
399.0
684.9
781.8
967.5
714.9
811.7
997.9
y ions
Y ions
y1
y2
y3
y4
y5
y6
y7
y8
y9
y10
y11
y12
y13
m/z Fi3b
m/z Fi3a
279.2
507.1
735.3
848.3
961.3
1189.4
279
507.1
735.3
832.2
945.4
1173.4
1272.4
1428.7
1656.7
1771.6
1898.9
1986.9
1672.6
2002.5
m/z Fi3c
669.9
1010.5
1109.6
1180.7
1408.7
1522.7
1623.6
m/z Fi5a
415.2
643.3
758.3
855.3
1011.1
1068.2
1165.4
1351.5
1580.6
1807.7
The peak at m/z = 457 may be confidently assigned to the b2 ion
arising from cleavage of a tandem CC ([228 × 2] + 1) residues at the
N-terminus. Similarly, the intense peak at m/z = 670 (y4) ion, arises
from the preferential cleavage at a X-Pro bond. Interestingly, the corresponding ‘b’ ion, namely, b10, also appears as an intense peak at
m/z = 1498.9, further supporting the assignment of the sequence
PCCP as the sequence at the C-terminus. The peak at m/z = 2051.8
(b13) ion arises from the loss of a C-terminal proline residue.
Detailed analysis of other prominent ‘b’ and ‘y’ ions enabled establishment of the sequence of Fi3c to be CCSTNCAVC(I/L)PCCP.
34
Figure 6. Comparison of mass spectra of Fi3a (A) and Fi3b (B). The
difference in mass of 16 Da (one oxygen atom) indicates that Fi3b is most
probably the hydroxylated form of Fi3a.
m/z Contryphan fid
m/z Contryphan fic
644.9
830.7
927.5
m/z Contryphan fib
644.9
659.0
927.5
959.5
Attempts to distinguish I from L were unsuccessful. Comparison of
the sequence of Fi3c with other M-2 branch peptides (table S-1)
shows that the sequence CIPCCP predominates at the C-terminus.
Two-Disulfide Peptide
Sequence of Fi5a
The peptide Fi5a was purified by HPLC (Figure 3). Reduction and alkylation using NEM caused a shift in mass shift of 504 Da (Supplementary Figures 5A and 5B), indicating the presence of four
cysteines. Esterification and acetylation yielded molecules with an
increase in mass by 28 Da (Supplementary Figures 6A and 6B) and
42 Da (Supplementary Figures 7A and 7B), respectively. This
strongly suggests the presence of two acidic groups and a free
amino group. Figure 11 shows the daughter ion spectrum obtained
upon fragmentation of the doubly charged reduced alkylated peptide ([M + 2H]+2; m/z = 962.0). In the spectrum, it is clear that the
‘b12’ ion arises from a loss of 115 Da from the molecular ion. This
mass difference can be reconciled if the C-terminal residue is a proline residue, the loss of which will give rise to the ‘b12’ ion of m/z
1807.7. Analysis of the MS/MS spectrum (Supplementary Figure 8)
of the reduced, alkylated and esterified Fi5a peptide, shows a shift
in mass of 14 Da the ‘b9’, ‘b10’, and ‘b11’ ions (the ‘b12’ ions are not
observed in here), when compared with the reduced and alkylated
Fi5a (Figure 11). The ‘b8’ ion on the other hand shows a mass
J. Pept. Sci. 2015; 21: 29–39
Figure 7. De novo sequencing of HPLC purified Fi3a. Mass spectrum resulting from collision induced dissociation fragmentation of reduced and alkylated
2+
doubly charged peptide (m/z [M + 2H] = 1222.4).
Figure 8. De novo sequencing of Fi3a. Mass spectrum resulting from collision induced dissociation fragmentation of reduced and alkylated triply charged
3+
peptide (m/z [M + 3H] = 815.3).
Table 3. Conopeptides of Conus figulinus
Sl.No Gene superfamily
M superfamily
M superfamily
M superfamily
T-Superfamily
Contryphan
Contryphan
Contryphan
Contryphan
I2 Superfamily
I1 Superfamily
I1 Superfamily
I1 Superfamily
J. Pept. Sci. 2015; 21: 29–39
Fi3a
Fi3b
Fi3c
Fi5a
Contryphan fib
Contryphan fic
Contryphan fid
Contryphan fia
Fi11.11
Fi11.1a
Fi11.6
Fi11.8
Sequence evidence
Protein level
Protein level
Protein level
Protein level
Protein level
Protein level
Protein level
Protein level
Nucleic acid level
Nucleic acid level
Nucleic acid level
Nucleic acid level
Sequence
Mass
Reference
CCSQDCRVCIPCCPY
CCSQDCRVCIOCCPY
CCSTNCAVCIPCCP
DCCWPGRPDCCAP
GCOWMPWC-NH2
GCPWDPWC
CPWDPWC
GCODWQPWC
CHHEGLPCTSGDGCCGMECCGGVCSSHCGN(NH2)
GHVSCGKDGRACDYHADCCNCCLGGICKPSTSWIGCSTNVFlTR
GCKKDRKPCSYHADCCNCCLSGICAPSTNWILPGCSTSSFfKI
GPSSCKADEEPCEYHADCCNCCLSGICAPSTNWILGCSTSSFfKI
1685.8
1701.6
1409.8
1417.6
991.5
959.5
902.4
1187
2932.91
4628.91
4634.98
4864.94
This work
This work
This work
This work
This work
This work
This work
[21]
[20]
[20]
[19]
[19]
35
1
2
3
4
5
6
7
8
9
10
11
12
Name
Rajesh
Table 4. Clustal W analysis of Fi3b and Fi3a with similar sequence in
database
C.tessulatus CCSQDCRVCIPCCPY cDNA level Conticello et al., 2001
C.ventricosus CCSQDCRVCIPCCPY cDNA level Conticello et al., 2001
C.figulinus(Fi3a)CCSQDCRVCIPCCPY Protein level This work
C.figulinus(Fi3b)CCSQDCRVCIOCCPY Protein level This work
observed MS/MS spectrum. The intense ‘b7’ peak at m/z 1068.5
arises from the cleavage of the Arg7–Pro8 peptide bond, which is
consistent with earlier observations.
Single Disulfide Peptides
The ESI-MS-MS spectrum of contryphan fid, contryphan fic and
contryphan fib is shown in Figures 12A, 12B, and 12C, respectively.
Figure 9. High-performance liquid chromatography chromatograms of co eluted natural and synthetic Fi3b which shows single peak at 21.798 min.
Figure 10. De novo sequencing of Fi3c. Mass spectrum resulting from collision induced dissociation fragmentation of reduced and alkylated doubly charged
2+
peptide (m/z [M + 2H] = 1083.5).
36
increase of 14 Da, indicating that residue 9 is an aspartate. Similarly,
increase in mass of the ‘b2’ ion, indicates that the second carboxylic
acid bearing amino acid is present at the N-terminus. In principle,
the C-terminal proline ought also to have been converted to an
ester. However this is not observed.
Near complete ‘b’ and ‘y’ series ions can be observed in the spectra shown in Figure 11. The b and y ions are presented in Table 2.
Detailed analysis of ‘b’ and ‘y’ led to the conclusion that the
sequence DCCWPGRPDCCAP may be derived directly from the
From an analysis of ‘b’ ions and ‘y’ ions in the daughter ion spectra
(Table 2), the sequence of all three contryphans was assigned as
CPWDPWC for contryphan fid, GCPWDPWC for contryphan fic,
and GCOWMPWC-NH2 for contryphan fib.
Table 3 lists the peptides identified either in the venom of C.
figulinus or from analysis of cDNA libraries derived from the venom
glands of this organism. This study has identified seven peptides,
which makes the total number peptides identified in the venom
of C. figulinus to eight. Of these seven, three peptides Fi3b, Fi3a,
J. Pept. Sci. 2015; 21: 29–39
Figure 11. De novo sequencing of Fi5a. Mass spectrum resulting from collision induced dissociation fragmentation of reduced and alkylated doubly charged
2+
peptide (m/z [M + 2H] = 962.0).
Figure 12. De novo sequencing of contryphans. Mass spectrum resulting from collision induced dissociation fragmentation of (A) reduced and alkylated
+
+
Contryphan fid (m/z [M + H] = 1155.0); (B) reduced and alkylated Contryphan fic (m/z [M + H] = 1212.0); and (C) reduced and alkylated Contryphan fib
+
(m/z [M + H] = 1243.0).
and Fi3c belong to three disulfide bonded Mini M-Superfamily
conotoxin, one peptide Fi5a belong to 2 disulfide bonded TSuperfamily conotoxin, and the other three peptides contryphan
fib, contryphan fic, and contryphan fid belongs to single disulfide
bonded contryphan. Among the aforementioned seven peptides
Fi3b, Fi3c, Fi5a, and contryphan fib are novel peptides.
Discussion
J. Pept. Sci. 2015; 21: 29–39
37
C. figulinus is abundantly distributed along the coast of the Southeastern state of Tamil Nadu in India. Despite its abundance, the
venom components of this species have not been studied. To date,
only one peptide sequence has been determined from the venom
of this animal. Analysis of nucleotide sequences from cDNA libraries
has yielded the translated peptide sequences of four peptides.
These peptides are thought to belong to the I-superfamily of
conotoxins [19,20].
Here, we report the peptide sequences of two novel ‘M’superfamily conotoxins [4], which are named as Fi3b and Fi3c (following the nomenclature suggested by Kalyana et al. 2014) and one ‘T’superfamily conotoxin named as Fi5a [27]. In addition, we also report
the sequence of three contryphan peptides. The novel ‘M’-superfamily
Rajesh
peptides belong to the M-2 branch of the ‘mini-M’ – conotoxins. Fi3b
contains a proline hydroxylation as the sole post-translational modification. It is important to note that the unmodified peptide (Fi3a) had
also been identified in the venom. The peptide Fi3c, however, does
not carry any posttranslational modifications [4]. Supplementary
Table 1 compares the sequences of the known M-2 branch mini-M
conotoxins. Interestingly, Fi3c is the only peptide that does not have
charged residues in its sequence. Fi3b and Fi3c are similar to the
‘mini-M’ conotoxins from C. quercinus [28] in that these peptides have
the sequence CIOCCP as part of loop-2 and the C-terminus. The M-2
branch conotoxins from vermivorous snails can be distinguished from
their molluscivorous counterparts in having a non-polar residue adjacent to the consensus O/P in loop 2.
The peptide Fi3a has been identified from cDNA libraries of two
other vermivorous cone snails namely, C. ventricosus and C.
tessulatus [7]. However, identification of this peptide in the venom of
C. figulinus has established that the peptide does undergo proline
hydroxylation. This data may prove to be useful in studies aimed at
establishing genetic and evolutionary relationships among cone snails.
Two contryphans, contryphan fic and contryphan fid, identified
in this study have been earlier identified in other cone snails venom
[16]. One contryphan, contryphan fib is a novel toxin to the
conopeptide library. Few contryphans like Am975 and Lo959 act
on calcium channels. Am975 inhibits calcium current whereas
Lo959 increases calcium current [16].
Although the current focus in Cone snail ‘venomomics’ has been
targeted toward gene sequencing, it is still important to retain focus
on the expressed and post-translationally modified peptides. Here,
we have identified seven peptides from the venom of the predatory,
wormivorous cone snail, C. figulinus. The sequences have been determined by high-resolution Mass spectrometric based sequencing
methods of the peptides directly in the crude as well as purified, natural venom. Nucleotide sequences can help in resolving overlap in
the case of isobaric residues such as isoleucine and leucine. The
presence of the codons for proline will resolve the ambiguity
between isoleucine, leucine, and the post-translationally modified
proline to hydroxyproline. However, the position of such a posttranslationally modified prolines can only be ascertained by
sequencing protocols, of which mass spectrometry occupies a preeminent position, because of its applicability even to crude mixtures.
Acknowledgements
Rajesh R. P. acknowledges the University Grants Commission, Government of India for the Dr D. S. Kothari Post-Doctoral Fellowship
(F.4-2/2006(BSR)/13-263/2008(BSR). The author thanks the DBT supported Proteomics Facility at the Indian Institute of Science. The author would like to thank Prof Siddhartha P. Sarma, MBU, IISc., for
many helpful discussions.
References
38
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Supporting Information
Additional supporting information may be found in the online version of this article at the publisher’s web site.
39
J. Pept. Sci. 2015; 21: 29–39

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