Unraveling the Peptidome of the South African Cone Snails Conus Pictus and Conus Natalis

Peptides 41 (2013) 8–16

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Peptides

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Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis

a a b c a

Steve Peigneur , Annelies Van Der Haegen , Carolina Möller , Etienne Waelkens , Elia Diego-García ,

b d a,∗

Frank Marí , Ryno Naudé , Jan Tytgat

Laboratory of Toxicology, University of Leuven (K.U. Leuven), O&N 2, PO Box 922, Herestraat 49, 3000 Leuven, Belgium

Department of Chemistry and Biochemistry, Florida Atlantic University (FAU), 777 Glades Road, Boca Raton, FL 33431, USA

Laboratory of Protein Phosphorylation and Proteomics, University of Leuven (K.U. Leuven), O&N 1, PO Box 901, Herestraat 49, 3000 Leuven, Belgium

Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active com-

Received 31 May 2012

pounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails

Received in revised form 2 July 2012

are considered to be specialized predators that have evolved the most sophisticated peptide chemistry

Accepted 2 July 2012

and neuropharmacology system for their own biological purposes by producing venoms which contains

Available online 7 July 2012

a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small

cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion chan-

Keywords:

nels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus

Conus pictus

species occurring along the coast of South Africa. Due to their scarceness, these species remain, there-

Conus natalis

fore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus,

Cone snail

Conotoxin a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total,

A-, M-, O-superfamily 26 novel peptides were identiﬁed. Comparing the venoms of both snails, interesting differences were

observed regarding venom composition and molecular characteristics of these components.

1. Introduction precursor that is proteolytically cleaved at speciﬁc sites to

yield the mature toxin. Although hypermutation of peptide

The marine gastropods known as cone snails (Conus) are one sequences is the main cause for conopeptide diversity, post-

of the largest single genus of living marine invertebrates. All cone translational modiﬁcations provide an overlying level of diversity

snails are venomous predators and possess a very complex venom [28].

apparatus. They use their venom for capturing prey, but also to Conopeptides can be classiﬁed into two major groups: the

defend themselves. In addition, it is probably used in other biotic disulﬁde-poor and the disulﬁde-rich conopeptides, the latter

interactions (e.g. interactions with competitors). Based on their also called conotoxins. These are classiﬁed into superfamilies,

prey, cone snails can be divided into three groups: vermivorous based on a conserved signal sequence found in the precur-

(worm-hunting), piscivorous (ﬁsh-hunting), and molluscivorous sor peptide and in their cysteine framework, and subsequently

(mollusc-hunting) cone snails [26]. into pharmacological families based on the targets they interact

More than 700 different cone snail species are known today with. At present, 22 cysteine frameworks have been identiﬁed

and each species produces an unprecedented molecular diversity [16].

of up to 1000 distinct pharmacologically active components. In this work, two cone snails endemic to South Africa were stud-

The venom of each different Conus species has a different set of ied. Conus pictus is a vermivorous cone snail, while Conus natalis

peptides, resulting in an array of more than 150,000 peptides is a molluscivorous cone snail. Their habitats are restricted to the

in total. Each conopeptide is encoded by a single messenger coastal regions of South Africa, in rather shallow water. A mass-

ribonucleic acid (mRNA) and translated as a prepropeptide ﬁngerprint study of the venom of C. pictus was already published

[18] and recently, we reported the ﬁrst characterized peptide from

C. pictus, pc16a [32]. In this work, we identiﬁed 11 novel conopep-

∗ tides in C. pictus venom and 15 novel peptides in the venom of C.

Corresponding author. Tel.: +32 16 32 34 03; fax: +32 16 32 34 05.

natalis, totally or partially characterized by amino acid sequence

E-mail addresses: [email protected],

[email protected] (J. Tytgat). determination. The characterization of novel peptides from both

S. Peigneur et al. / Peptides 41 (2013) 8–16 9

Table 1

venoms expands the knowledge about the South-African cone

Mass spectrometric analysis of puriﬁed Conus pictus and Conus natalis venom.

snails, whose venoms are unexplored to date.

Masses are shown as average masses.

ESI-MS [M] (Da) of C. ESI-MS [M] (Da) of C. natalis venom

pictus venom

2. Materials and methods

1062.3 3232.5 1161.5 2546.5 3808.8

1135.6 3312.1 1258.3 2603.1 3980.3

2.1. Materials

1151.8 3325.6 1389.8 2618.4 3985.0

1249.5 3328.8 1428.0 2695.7 4017.6

Specimens were collected from the coastal region of Port

1258.3 3333.8 1631.9 2705.8 4033.6

Elizabeth, South-Africa. Venom ducts and bulbs were dissected, 1259.2 3343.4 1642.5 2723.6 4099.1

◦

lyophilized and stored at −20 C. 1288.2 3376.0 1664.4 2836.1 4127.5

1303.5 3391.5 1680.8 2879.8 4212.9

1324.8 3873.5 1698.6 2890.7 4429.7

1341.0 3901.3 1705.2 3003.2 4627.5

2.2. Extraction of crude venom

1403.5 3953.3 1717.5 3018.8 4627.6

1460.5 3960.8 1721.0 3071.5 4638.6

Venom bulbs and ducts of 6 (C. pictus) and 5 specimens 1509.3 4042.6 1731.8 3087.9 4671.1

(C. natalis) were macerated and extracted with 30% acetonitrile 1566.7 8565.1 1741.8 3168.8 4683.4

1621.3 1742.7 3276.5 4737.7

(ACN) containing 0.1% triﬂuoroacetic acid (TFA) and centrifuged at

◦ 1640.8 1758.4 3305.2 4749.0

16,100 × g at 4 C for 30 min. The supernatant was lyophilized and

1661.8 1798.8 3329.5 4782.7

− ◦

stored at 20 C. 1678.0 1818.8 3431.1 4935.3

1855.0 1825.1 3451.0 4963.5

1932.4 1834.3 3470.7 5039.4

1965.7 1904.4 3546.4 5052.2

2.3. Puriﬁcation

1986.0 1954.8 3553.9 5084.9

2134.3 1998.0 3572.9 5097.2

Lyophilized crude venom extract was dissolved in 30% ACN,

2149.8 2014.6 3585.6 7911.2

containing 0.1% TFA. Size exclusion chromatography was per- 2186.6 2137.9 3590.1 7955.6

formed on a Superdex Peptide 10/300 GL column (GE Healthcare 2259.6 2139.5 3675.7 8008.8

2295.2 2408.7 3701.7 10,460.9

Bio-Sciences AB, Sweden) at room temperature at a ﬂow rate of

2599.6 2453.2 3720.4 11,071.4

0.5 ml/min using an isocratic elution with 30% ACN, containing

2618.5 2467.3 3742.3 11,086.4

0.1% TFA. A second puriﬁcation was performed on a reversed-phase 2922.7 2493.6 3765.1 11,819.2

Vydac C18 column (Grace, USA, 218MS54, 4.6 mm 250 mm, 5 ␮m Total masses = 44 Total masses = 90

particle size) at room temperature at a ﬂow rate of 1 ml/min using

a linear gradient from 0 to 70% solution B in 280 min. Solutions

were as follows: (A) 0.1% (v/v) TFA in water; (B) 0.085% (v/v) TFA 3. Results

in ACN. Absorbance was monitored at 214 nm for all puriﬁcations

and peaks were collected using a PrepFC collector (Gilson, USA). 3.1. Chromatographic separation and mass ﬁngerprinting of C.

pictus and C. natalis venom

2.4. Mass spectrometry

Crude venoms of C. pictus and C. natalis were fractionated

into ﬁve fractions by SE-HPLC using a Superdex Peptide column

Molecular mass analysis was performed on a LCQ Deca XP elec-

(Fig. 1A and Fig. 2A, respectively). Each fraction was lyophilized

trospray ionization – quadrupole ion trap – mass spectrometer

and further puriﬁed by RP-HPLC using a Vydac C18 column

(Thermo Finnigann, USA), using a positive-ionization mode. The

(Fig. 1B–D and Fig. 2B and C). Only the chromatograms of the frac-

sample was dissolved in 50% aqueous ACN containing 0.1% acetic

tions in which several peptides were characterized are shown.

acid. A ﬂow rate of 7.5 ␮l/min, a spray voltage of 4 kV, a capillary

◦

temperature of 142 C, a capillary voltage of 38.5 V and a tube lens

3.2. Mass spectrometric analysis of puriﬁed C. pictus and C.

offset of 152 V were employed. A full scan from 400 to 2000 m/z was

TM natalis venom

performed and the data were deconvoluted using the ProMass

Deconvolution software giving the monoisotopic mass of the pep-

tide. Due to limited native material, we were not able to determine

the molecular masses of all compounds present in the venom of

C. pictus and C. natalis. In total, we detected 44 components in

the puriﬁed fractions of the venom of C. pictus, with monoisotopic

2.5. N-terminal sequence determination

molecular masses ranging from 1062.3 to 8565.1 Da (Table 1). As

noted previously [18], the venom of C. pictus has an asymmetri-

The N-terminal amino acid sequences of native peptides present

cal distribution i.e. more molecules are being detected in the low

in C. pictus and C. natalis venom were determined by automated

molecular mass range (Fig. 3). The venom of C. natalis contained

Edman degradation using a Shimadzu PPSQ-30 protein sequencer

more components, as compared to C. pictus venom, with many

(Tokyo, Japan) according to the manufacturer’s instructions.

components having a mass of more than 4000 Da (Fig. 3). In total,

90 components were detected in the venom of C. natalis, ranging

2.6. Nomenclature from 1161.5 to 11,819.2 Da (Table 1). The venoms of vermivo-

rous South African cone snails studied before [18], contained more

We followed the nomenclature proposed by Olivera and Cruz components, but MALDI-TOF was used for the mass-spectrometric

[27]. As we do not have information on biological activity, we used analysis, which is more sensitive in terms of detection level.

a two letter code ‘“pc” and “nt” to represent C. pictus and C. natalis, Between 47 and 65% of the components of the South African ven-

respectively, followed by an Arabic number to indicate the cysteine oms studied were in a mass range from 1000 to 3000 Da [18]. For C.

framework, and a small letter for a particular peptide variant. pictus, 75% of the components detected have a mass of less than

10 S. Peigneur et al. / Peptides 41 (2013) 8–16

Fig. 1. Puriﬁcation of several peptides from C. pictus crude venom. (A) Crude venom extract of C. pictus was fractionated by size-exclusion chromatography using a Superdex

Peptide column, with the different fractions indicated. (B–D) Chromatograms of fractions 2, 3 and 4, respectively, further puriﬁed by RP-HPLC using a Vydac C18 column. The

different peptides that were found are indicated in every panel.

3000 Da, whereas for C. natalis, components with a mass up to 3.3.3. Six cysteines

3000 Da constitute only 41% of all components detected. 3.3.3.1. Framework III. One peptide representing framework III,

pc3a, could be identiﬁed in the C. pictus venom. Pc3a is a 18-residue

peptide with framework III (CC-C-C-CC) displaying a monoistopic

3.3. Amino acid sequences of puriﬁed peptides from C. pictus

venom molecular mass of 1985.4 Da (Table 2). The calculated monoisotopic

mass of 1985.7 Da is consistent with the experimental ESI-MS mass.

Pc3a was found in fraction 3 (Fig. 1C) and is very hydrophilic as it

3.3.1. Two cysteines

eluted at 12% ACN. The sequence contains two positively charged

A peptide with only one disulﬁde bridge, named conopeptide-

residues (Lys) and three negatively charged residues (Asp).

pc, with a monoisotopic mass of 1108.6 Da, consisting of 9 amino

acid residues was detected (Table 2). This peptide was present in

size-exclusion fraction 4 and is quite hydrophobic (Fig. 1D). The 3.3.3.2. Framework VI/VII. pc6b and pc6d were found in fractions

calculated monoisotopic mass of 1108.5 Da is consistent with the 2 and 3 (Fig. 1C and D) and have the typical Cys pattern of the

ESI-MS mass. O-superfamily (C-C-CC-C-C). They both consist of 31 residues and

have a monoisotopic molecular weight of 3311.1 and 3375.0 Da for

3.3.2. Four cysteines pc6b and pc6d, respectively (Table 2). The calculated monoisotopic

3.3.2.1. Framework I. Two peptides with framework I (CC-C- masses of 3310.4 and 3374.4 Da for pc6b and pc6d, respectively,

C), pc1a and pc1b, were identiﬁed in fractions 2 and 3, with are consistent with the ESI-MS masses. Two other peptides with

monoisotopic molecular masses of 1965.2 and 1931.5 Da, respec- framework VI, pc6a and pc6c, were found in the venom (Fig. 1B and

tively (Table 1 and Supplemental Fig. 1). Both peptides have 18 C, respectively). pc6a and pc6c are very similar to pc6b and pc6d,

residues without post-translational modiﬁcations. Their calculated respectively. However, we were not able to completely resolve the

monoisotopic masses of 1964.9 and 1630.8 Da, respectively, are sequence, probably because of post-translation modiﬁcations. A

consistent with the masses obtained by ESI-MS. cDNA library of C. pictus was recently constructed, and degener-

ate primers will be made, in order to ﬁnd the precursor and solve

the puzzle.

3.3.2.2. Framework XVI. Three conotoxins, named pc16a, pc16b

and pc16c (based on their Cys framework C-C-CC) were present

in fractions 3 and 4 (Fig. 1C and D), with a monoisotopic molecular 3.4. Amino acid sequences of puriﬁed peptides from C. natalis

masses of 1257.6 (pc16a), 1258.7 (pc16b) (Supplemental Fig. 2) and venom

1340.8 Da (pc16c), respectively (Table 1). The calculated monoiso-

topic masses of 1257.5 Da, 1258.5 Da and 1342.4 Da, respectively, Due to the high molecular masses and low amount of compo-

are consistent with their ESI-MS masses. nents in C. natalis venom, only one peptide could be fully sequenced.

S. Peigneur et al. / Peptides 41 (2013) 8–16 11

Fig. 2. Puriﬁcation of peptides from C. natalis crude venom. (A) Fractionation by SE-HPLC of C. natalis crude venom, using a Superdex Peptide column, with the different

fractions indicated. (B and C) Chromatograms of fractions 3 and 4, respectively, further puriﬁed by RP-HPLC on a Vydac C18-column.

12 S. Peigneur et al. / Peptides 41 (2013) 8–16

Fig. 3. Histogram of peptide mass abundance in the venom of C. pictus and C. natalis, obtained by ESI-MS. The number of peptides identiﬁed from C. pictus (44) and C. natalis

(90) agree with the number of masses listed in Table 1.

Table 2

Overview of the different peptides characterized from C. pictus and C. natalis. venom. *amidated C-terminus. Masses are shown as monoisotopic (M+H) . For the theoretical

masses, a correction was made for the disulﬁde bridges.

Peptide Exp. mass (Da) Theor. mass (Da) Sequence

Disulﬁde-poor

One S-S conopeptide-pc 1108.6 1108.5 RCLFWSVCP

Disulﬁde-rich

Framework I pc1a 1965.2 1964.9 DECCAIPFCAKIFPGRCP

pc1b 1931.5 1930.8 DECCAIPLCAKIFPGRCP

Framework III pc3a 1985.4 1985.7 HECCKKGFCDPGCDCCDQ

nt3a 1742.7 1742.6 GCCRFPCPDSCRSLCC

Framework VI/VII pc6b 3311.1 3310.4 TCLEIGEFCGKPMMVGSLCCSPGWCFFICVG

pc6d 3375.0 3374.4 KCFEVGEFCGSPMLLGSLCCYPGWCFFVCVG

Framework XVI pc16a 1257.6 1257.5 SCSCKRNFLCC*

pc16b 1258.7 1258.5 SCSCKRNFLCC

pc16c 1340.8 1342.4 SCSCQKHFSCCD*

However, the partial sequences that were obtained show a sig- 3.4.1. Six cysteines

niﬁcant number of very similar, but not identical peptides in C. 3.4.1.1. Framework III. A 16-residue peptide with 6 Cys residues

natalis venom (Table 3). A cDNA library of C. natalis is already con- arranged as framework III was present in the venom of C. natalis,

structed and degenerate primers will be made, in order to obtain with a monoistopic molecular weight of 1742.7 Da (Tables 2 and 4).

full sequences of the peptides. The calculated monoisotopic mass of 1742.6 Da is consistent

with the experimental ESI-MS mass. nt3a contains two positively

charged (Arg), one negatively charged (Asp) and two hydropho-

bic (Phe, Leu) amino acids. Consequently, this peptide is not much

Table 3

retained on the RP column (Fig. 2C).

Partial amino acid sequences of peptides obtained in C. natalis venom. “X” indicates

uncertainties within the sequence, probably due to post-translation modiﬁcations.

Masses are shown as monoisotopic (M+H) .

4. Discussion

Fraction Mass exp Sequence

name (Da)

4.1. Peptides from C. pictus venom

N1 4671.1 DCTKSCEXXDNFCQGTCHCSGXANCYC. . .

N2 4627.9 DCTKSCEXXDNFCQGT. . .

4.1.1. Two cysteines

N3 4683.4 DCTISCEFXDNKCQGSCYCSGXANCYCTSDTXNCGCGCA. . .

To date, conopeptides with two Cys residues have been classiﬁed

DCTISCEFXDNKCQGSCYCSGXANCYC. . .

N4 4639.4 2+ 2+ +

into contryphans (which target Ca channels or Ca -activated K

N5 4017.0 ECPVTGCPYPFXDCMXA. . .

N6 3354.0 XWCXPGFAYNVALGTCTI. . . channels) [21], conopressins (which target the vasopressin recep-

LWCXPGFAYNVALGTCTISLQXIKYPGLYE. . .

N7 3347.2 tor) [9] and conoCAPs [24]. Contryphans are characterized by a large

N8 3572.9 LWCXPGFAYNPVLGTCTISLQXIKYPGLYE. . .

number of post-translational modiﬁcations, such as epimerization

N9 1954.8 XPNYEDT. . .

of Trp or Leu, bromination of Trp, ␥-carboxylation of Glu, hydroxy-

N10 1731.8 VCCPFGGCHEL. . .

lation of Pro and C-terminal amidation, whereas conopressins are

N11 1641.8 VCCXFG. . .

SCTDXWQACSYOTQCXTXNXDGY. . .

N12 2964.3 vasopressin-like peptides (Table 5). The C. pictus peptide with two

N13 1258.3 GCSIFDNSCCG. . .

Cys residues was provisionally named conopeptide-pc as it can-

N14 1814.9 XLAKGDYCNLISQD. . .

not be classiﬁed within the contryphans or conopressins. Sequence

S. Peigneur et al. / Peptides 41 (2013) 8–16 13

Table 4

Sequence comparison of nt3a with mini-M3-conotoxins. Sequences were aligned using ClustalW2 [4]. *C-terminal amidation, m = molluscivorous, SA = South Africa, and

IP = Indo-Paciﬁc. Reference for tx3f and mr3.5 [5].

Peptide Sequence Species Loop size distribution Similarity

nt3a -GCCRFPCPDSCRSLCC C. natalis (m, SA) 3/3/3 100%

tx3f -RCCKFPCPDSCRYLCC* C. textile (m, IP) 3/3/3 87.5%

mr3.5 MGCCPFPCKTSCTTLCC C. marmoreus (m, IP) 3/3/3 75.0%

Table 5

Alignment of Conopeptide-pc with some contryphans and conopressins. Sequences were aligned using ClustalW2 [4]. *amidated C-terminus, W = d-tryptophan, L = d-leucine,

v = vermivorous, m = molluscivorous, p = piscivorous, SA = South Africa, IP = Indo-Paciﬁc, EA = Eastern Atlantic and M = Mediterranean.

Peptide Sequence Species Similarity Reference

pc1a –DECCAIPFCAKIFPGRCP C. pictus (v, SA) 100% This work

pc1b –DECCAIPLCAKIFPGRCP C. pictus (v, SA) 100% This work

␣ —GCCSLPPCAANNPDY

-PnIA C* C. pennaceus (m, IP) 50.0% [11]

␣

-SrIA –RTCCSROTCRM␥YP␥LCG* C. spurius (v, WA and C) 44.4% [20]

␣-MII —GCCSNPVCHLEHSNLC* C. magus (p, IP) 38.9% [3]

␳-TIA FNWRCCLIPACRRNHKKFC* C. tulipa (p, IP) 44.6% [30]

comparison with contryphans and conopressins shows that, even the physiological targets of these conotoxins have not been found

though the Cys pattern is conserved, their primary structures vary yet. cal16a [2] and qc16a [33] are active on Cav and Kv channels,

considerably (Table 5). Conopeptide-P has 5 intercysteine residues, respectively, but only in very high concentrations, therefore, it can

whereas conopressins only have 4. Even though contryphans also be questioned whether these channels are the physiological tar-

have an intercysteine loop consisting of 5 residues, there is no gets. We tested pc16a on a wide panel of voltage-gated channels

similarity in sequence between contryphans and conopeptide-pc. and nAChRs but were unable to identify the target [32]. pc16a and

Consequently, this peptide might belong to a new family of one- pc16b have the same sequence consisting of 11 amino acid residues,

disulﬁde bridge peptides. but their monoisotopic molecular mass differs by 1 Da, indicating

that pc16a is amidated at the C-terminus, while pc16b is not. pc16c

4.1.2. Four cysteines has 12 amino acid residues and has the same intercysteine loop

4.1.2.1. Framework I. Conotoxins with framework I can be clas- spacing as pc16a and pc16b. The difference between the theoreti-

␣ ␳

siﬁed into either - or -conotoxins, which both belong to the cal mass of pc16c without amidation (1342.4 Da) and experimental

A-superfamily. They share their disulﬁde connectivity (I–III, II–IV), mass (1340.8 Da) indicates that the C-terminus of pc16c is ami-

␣

but have different targets. -Conotoxins are antagonists of nAChRs dated. Three non-Cys amino acid residues are conserved among

␣

[25] and are classiﬁed into subtypes ( -m/n) based on the num- pc16a, pc16b and pc16c: Ser1, Ser3 and Phe8 (Table 7). Besides

␳

ber of residues between the Cys loops (CCXmCXnC). -Conotoxins the Cys residues they have in common, pc16a and pc16b have

␣ ␳

are antagonists of the 1-adrenoreceptor and to date, only one - two hydrophobic residues (Phe and Leu), while pc16c has only one

␳

conotoxin, -TIA from the venom of C. tulipa, has been identiﬁed (Phe). Consequently, pc16c is less retained on the RP column (Fig. 1

[30]. pc1a and pc1b have a 4/7 loop size distribution (CCX4CX7C) D).

␣

␳

and might therefore be 4/7-conotoxins, however, -TIA also has Sequence comparison between these framework XVI-

a 4/7 loop size distribution (Table 6). Sequence comparison with conotoxins from C. pictus and other framework XVI-conotoxins,

␣ ␳

other 4/7-conotoxins and -TIA identiﬁed in cone snail venom, shows that only the Cys pattern is conserved (Table 7).

shows that, besides the conserved Cys residues and one con-

served Pro (hydroxylated or not hydroxylated) at position 7, the 4.1.3. Six cysteines

primary structures are quite different (Table 6). Thus, based on 4.1.3.1. Framework III. Framework III-conotoxins belong

their sequences, it is not clear whether pc1a and pc1b are ␣- or to the M-superfamily, which is divided into 5 branches

␳-conotoxins. (M1–M5) according to the number of residues between the

fourth and the ﬁfth Cys residue. The M4-and M5-branches

4.1.2.2. Framework XVI. The framework XVI (C-C-CC) is rare among are called maxi-Ms and contain more than 22 residues.

␮ ␬ ␺

conotoxins, since only 4 other conotoxins with this Cys pattern have They are further classiﬁed into -, M- and -conotoxins

␮

been identiﬁed (Table 7). For qc16a, the structure was determined based on their targets. -Conotoxins block Nav channels,

␬ ␺

and the disulﬁde connectivity was determined to be I–IV, II–III [33]. M-conotoxins block Kv channels and -conotoxins block nAChRs.

Recently, we published the sequence and three-dimensional struc- The M1-, M2- and M3-branches are called mini-Ms and contain

ture of pc16a [32]. Interestingly, its disulﬁde connectivity differs less than 22 residues. Unfortunately, little is known about their

from qc16a as pc16a has a I–III and II–IV conﬁguration. To date, targets [14].

Table 6

Sequence comparison of pc1a and pc1b with the amino acid sequences of ␣4/7-conotoxins. Sequences were aligned with ClustalW2 [4]. *amidated C-terminus,

␥

O = hydroxyproline, = gamma carboxylic glutamic acid, Y = sulfotyrosine, v = vermivorous, p = piscivorous, m = molluscivorous, SA = South Africa, IP = Indo-Paciﬁc,

WA = Western Atlantic and C = Caribbean.

Peptide Sequence Species Similarity Reference

Conopeptide-pc -RCLFWSVCP C. pictus (v, SA) 100% This work

Contryphan-In -GCVLYPWC* C. inscriptus (v, IP) 55.6% [12]

Contryphan-Tx -GCOWQPYC* C. textile (m, IP) 33.3% [15]

GDCPW

Contryphan-Vn KPWC* C. ventricosus (p, EA and M) 33.3% [21]

–C-FIRNCPKG*

Lys-Conopressin-G C. geographus (p, IP) 55.6% [7]

Conopressin-T –C-YIQNCLRV* C. tulipa (p, IP) 33.3% [9]

14 S. Peigneur et al. / Peptides 41 (2013) 8–16

Table 7

Alignment of pc16a, pc16b and pc16c with other framework XVI-conotoxins. Sequences were aligned using ClustalW2 [4]. *C-terminal amidation, Z = pyroglutamic acid,

v = vermivorous, p = piscivorous, SA = South Africa, IP = Indo-Paciﬁc, EP = Eastern Paciﬁc, and n.d. = not determined. References for these peptides are: qc16a [33], cal16a [10],

cl16av [2] and lt16a [29].

Peptide Sequence Species Structure Similarity

pc16a ——–SCS–CKRN-FLCC* C. pictus (v, SA) 100%

pc16b ——–SCS–CKRN-FLCC C. pictus (v, SA) Identical to pc16a, but without amidation 100%

pc16c ——–SCS–CQKH-FSCCD* C. pictus (v, SA) n.d. 90.9%

qc16a ——–DCQP-CGHN–VCC C. quercinus (v, IP) 63.6%

cal16a ——-ZGCV–CNANAKFCCGE* C. californicus (p, EP) n.d. 63.6%

cal16a ——–NCPAGCRSQ–GCCM C. californicus (p, EP) n.d. 54.5%

lt16a RTGEDFLEECMGGCAFD–FCCK C. litteratus (v, IP) n.d. 54.5%

Based on the M-superfamily classiﬁcation, pc3a is a mini-M- ␬-conotoxins block Kv channels, ␻-conotoxins block Cav channels

conotoxin, belonging to the M1-branch, as there is a single residue and ␥-conotoxins antagonize pacemaker channels [31]. The O-

between the fourth and the ﬁfth Cys residue. Sequence comparison superfamily toxins have about 25–35 residues and a C-C-CC-C-C

with other M1-conotoxins, shows that, besides the Cys framework, Cys pattern. These Cys residues form three disulﬁde bridges (I–IV,

loop size distribution is conserved, except for tx3a (Table 8). Struc- II–V and III–VI) resulting in an ICK motif. Two disulﬁde bridges and

ture determination of three M1-conotoxins, mr3a and mr3e from the backbone form a ring, which is threaded by the third disulﬁde

C. marmoreus and tx3a from C. textile showed that they have the bond forming a knotted structure, which makes the structure very

same disulﬁde connectivity (I–V, II–IV and III–VI) but their sec- stable [6].

ondary structures vary considerably, mr3e having a double-turn pc6b and pc6d have several hydrophobic amino acid residues

motif [8] while mr3a and tx3a have a triple-turn motif [22,23] and they elute at high concentrations of ACN (40–50%) (Fig. 1C

(Table 8). It is likely that pc3a also has a double-turn motif, as it has and D, respectively). Besides the Cys residues, pc6b has 12

the same loop size distribution as mr3e (Table 8). However, because hydrophobic residues, while pc6d is slightly more hydropho-

of the structural plasticity displayed by these mini-M-conotoxins, bic with 13 hydrophobic residues and, hence, has a higher

structure determination of pc3a is required to verify its secondary retention on the column. Both have the same loop size

structure. distribution: CX6CX9CX0CX4CX3C (Table 2). According to the

KNOTTIN database, the consensus sequence for an ICK motif is

4.1.3.2. Framework VI/VII. Framework VI/VII-conotoxins belong to CX2–7CX2–10CX0–7CX1–17CX2–19C. Consequently, pc6b and pc6d are

the O-superfamily, which is further divided into families based likely to form ICK structures.

␮ ␦ ␻ ␬

on the targets they interact with. ␮O-conotoxins block Nav chan- To date, O-, -, - and -conotoxins have not been phar-

nels whereas ␦-conotoxins inhibit the inactivation of Nav channels, macologically described in the venom of vermivorous cone

Table 8

Alignment of pc3a and nt3a with mini M1-conotoxins and mini M3-conotoxins, respectively. Sequences were aligned using ClustalW2 [4]. *C-terminal amidation,

v = vermivorous, m = molluscivorous, SA = South Africa, and IP = Indo-Paciﬁc. References for these peptides are: mr3e [13] and tx3a [5].

Peptide Sequence Species Loop size and structure Similarity

pc3a HECCKKGFCDPG-CDCCD C. pictus (v, SA) 4/3/1 100%

mr3e -VCCPFGGCHEL-CYCCD* C. marmoreus (m, IP) 4/3/1 47.1%

tx3a –CCSWDVCDHPSCTCCG C. textile (m, IP) 4/4/1 47.1%

S. Peigneur et al. / Peptides 41 (2013) 8–16 15

Table 9

Alignment of pc6a, pc6b, pc6c and pc6d with other framework VI/VII-conotoxins. Sequences were aligned using ClustalW2 [4]. v = vermivorous, p = piscivorous,

m = molluscivorous, SA = South Africa, EA = Eastern Atlantic, M = Mediterranean, and IP = Indo-Paciﬁc. References for these peptides are: Vn6.8 [16], conotoxin-5 [17], ␻-PnVIA

␻

[19] and -PnVIB [19].

Peptide Sequence Species Similarity

pc6b –TCLEIGEFCGKPMMVGSLCCSPGWCFFICVG C. pictus (v, SA) 100%

–KCFEVGEFCGSPMLLGSLCCYPGWCFFVCVG

pc6d C. pictus (v, SA) 90.3%

Vn6.8 –DCVAGGHFCGFPKI-GGPCCS-GWCFFVCA C. ventricosus (p, EA and M) 61.3%

Conotoxin-5 –GCREGGEFCGTL–YEERCCS-GWCFFVCV C. imperialis (v, IP) 61.3%

␻

-PnVIA –GCLEVDYFCGIPFANNGLCCS-GNCVFVCTPQ C. pennaceus (m, IP) 58.1%

␻

DDDCEPPGNFCGM-IKIGPPCCS-GWCFFACA

-PnVIB C. pennaceus (m, IP) 58.1%

␥

snails [16]. Only two -conotoxins, ␥-as7a [34] and ␥-de7a [1], Appendix A. Supplementary data

have been identiﬁed from the venom of respectively C. aus-

tini and C. delessertii, respectively, two vermivorous cone snails. Supplementary data associated with this article can be found,

Sequence comparison with framework VI/VII conotoxins reveals in the online version, at http://dx.doi.org/10.1016/j.peptides.

that pc6b and pc6d have similar sequences to two putative tox- 2012.07.002.

ins Vn6.8 [16] and conotoxin-5 [17] from C. ventricosus and from

C. imperialis, respectively (Table 9). Pc6b and pc6d also have

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