An O-conotoxin from the vermivorous Conus spurius active on mice and mollusks

Here, we report the purification, amino acid sequence and a preliminary biological characterization of a peptide, sr7a, from the venom of Conus spurius, a vermivorous species collected in the Yucatan Channel, Mexico. The peptide consists of 32 amino acid residues (CLQFGSTCFLGDDDICCSGECFYSGGTFGICS&; &, amidated C-terminus) and contains six cysteines arranged in the pattern (C-C-CC-C-C) that characterizes the O-superfamily of conotoxins. This superfamily includes several pharmacological families (omega-, kappa-, muO-, delta- and gamma-conotoxins) that target Ca(2+), K(+), Na(+) and pacemaker voltage-gated ion channels. Compared with other O-conotoxins that were purified from venoms, this peptide displays sequence similarity with omega-SVIA (from Conus striatus), delta-TxVIA/B (from Conus textile), omega-CVID (from Conus catus) and kappa-PVIIA (from Conus purpurascens). At a dose of 250 pmol, peptide sr7a elicited hyperactivity when injected intracranially into mice and produced paralysis when injected into the pedal muscle of freshwater snails, Pomacea paludosa, but it had no apparent effect after intramuscular injection into the limpet Patella opea or the freshwater fish Lebistes reticulatus.     

Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.     

Putative gamma-conotoxins in vermivorous cone snails: the case of Conus delessertii

Peptide de7a was purified from the venom of Conus delessertii, a vermivorous cone snail collected in the Yucatan Channel, Mexico. Its amino acid sequence was determined by automatic Edman degradation after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and several post-translationally modified residues. The determination of its molecular mass by means of laser desorption ionization time-of-flight mass spectrometry (average mass, 3170.0 Da) confirmed the chemical data and suggested amidation of the C-terminus. The primary structure (ACKOKNNLCAITgammaMAgammaCCSGFCLIYRCS*; O, hydroxyproline; gamma, gamma-carboxyglutamate; *, amidated C-terminus; calculated average mass, 3169.66 Da) of de7a contains a motif (gammaCCS) that has previously only been found in two other toxins, both from molluscivorous cone snails: TxVIIA from Conus textile and gamma-PnVIIA from Conus pennaceus. These toxins cause depolarization and increased firing of action potentials in molluscan neuronal systems, and toxin gamma-PnVIIA has been shown to act as an agonist of neuronal pacemaker cation currents. The similarities to toxins TxVIIA and gamma-PnVIIA suggest that peptide de7a might also affect voltage-gated nonspecific cation pacemaker channels.     

A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius

A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68 Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42 Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I-III, II-IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0 nmol) into mice, peptide sr5a caused depressed behavioral activity.     

Characterization of the venom of the vermivorous cone snail Conus fulgetrum

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.     

Intraspecific variation in the venom of the vermivorous cone snail Conus vexillum

A combination of proteomic and biochemical assays was used to examine variations in the venom of Conus vexillum taken from two locations (Hurgada and Sharm El-Shaikh) in the Red Sea, Egypt. Using MALDI/TOF-MS, a remarkable degree of intra-species variation between venom samples from both locations was identified. To evaluate variability in the cytotoxic effects of Conus venom, mice were injected with the same dose from each location. The oxidative stress biomarkers [malondialdehyde (MDA), protein carbonyl content (PCC)], antioxidants [glutathione (GSH), superoxide dismutase (SOD), catalase (CAT)], total antioxidant capacity (TAC) and nitric oxide (NO), were measured 3, 6, 9 and 12 h post venom injection. The venoms induced a significant increase in the levels of PCC, MDA, NO, GSH and CAT. The venoms significantly inhibited the activity of SOD and reduced the TAC. Toxicological data showed that the venom obtained from Hurgada was more potent than that obtained from Sharm El-Shaikh. It can be concluded that: (1) the venom of the same Conus species from different regions is highly diversified (2) the venoms from different locations reflect clear differences in venom potency and (3) the cytotoxic effects of C. vexillum venom can be attributed to its ability to induce oxidative stress.     

Three new species of gnathostomulida from the West Atlantic

Two new species of Gnathostomulida from Bermuda and one from North Carolina are described. All belong to the higher Bursovaginoidae with two occurring in the genus Austrognathia and one in the genus Gnathostomula. Tables of major diagnostic characters showing morphological variability within species and between previously described species in the same genus are included. Clarification in recognition of Gnathostomula jenneri and Gnathostomula brunidens is given under “Remarks on Known Species”.     

Contryphan-Vn: a novel peptide from the venom of the Mediterranean snail Conus ventricosus.

The isolation, purification, and biochemical characterization of the novel peptide Contryphan-Vn, extracted from the venom of the Mediterranean marine snail Conus ventricosus, is reported. Contryphan-Vn is the first Conus peptide described from a vermivorous species and the first purified from the venom of the single Mediterranean Conus species. The amino acid sequence of Contryphan-Vn is As with other contryphans, Contryphan-Vn contains a d-tryptophan residue, is amidated at the C-terminus, and maintains the five-residue intercystine loop size. However, Contryphan-Vn differs from the known contryphans by the insertion of the Asp residue at position 2, by the lack of hydroxylation of Pro(4), and, remarkably, by the presence of the basic residue Lys(6) within the intercystine loop. Although the biological function(s) of contryphans is still unknown, these characteristics suggest distinct molecular target(s) and/or function(s) for Contryphan-Vn.     

Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.     

Novel M‐Superfamily and T‐Superfamily conotoxins and contryphans from the vermivorous snail Conus figulinus

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.     

Purification and sequence of a presynaptic peptide toxin from Conus geographus venom

A novel toxin, omega-conotoxin (omega-CgTX), from the venom of the fish-eating marine mollusc Conus geographus has been purified and biochemically characterized. Recently, this omega-conotoxin has been shown to inhibit the voltage-activated entry of Ca2+, thus providing a potentially powerful probe for exploring the vertebrate presynaptic terminal [Kerr, L. M., & Yoshikami, D. (1984) Nature (London) 308, 282-284]. The toxin is a basic 27 amino acid peptide amide with three disulfide bridges. An unusual feature is a remarkable preponderance of hydroxylated amino acids. The sequence of omega-CgTx GVIA is Cys-Lys-Ser- Hyp-Gly5-Ser-Ser-Cys-Ser-Hyp10-Thr-Ser-Tyr-Asn-Cys15-C ys-Arg-Ser- Cys-Asn20-Hyp-Tyr-Thr-Lys-Arg25-Cys-Tyr-NH2.     

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture.     

Omega Conus geographus toxin: a peptide that blocks calcium channels

We previously reported that omega Conus geographus toxin (omega CgTX), blocks evoked-release of transmitter at synapses in frog and attenuates the Ca2+ component of the action potential of chick dorsal root ganglion neurons. We report here voltage-clamp experiments on cultured chick dorsal root ganglion neurons which demonstrate that omega CgTX produces a persistent block of voltage-gated Ca2+ currents. Thus, we conclude that omega CgTX inhibits synaptic transmission by blocking Ca2+ channels in the presynaptic nerve terminal. The toxin had no effect on K+ currents; however, in some but not all neurons, omega CgTX reduced Na+ currents by 10-25%. These findings suggest that omega CgTX should be useful as a probe to examine synaptic Ca2+ channels.     

Alpha-conotoxin GIC from Conus geographus,a novel peptide antagonist of nicotinic acetylcholine receptors

Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, alpha-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. alpha-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 microm, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC(50) approximately 1.1 nm) for the human alpha3beta2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although alpha-conotoxin GIC shares some sequence similarity with alpha-conotoxin MII, which is also a potent alpha3beta2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the alpha3beta2 nAChR subtype (possibly inhibiting the fight-or-flight response).     

Amino acid sequence and biological activity of a gamma-conotoxin-like peptide from the worm-hunting snail Conus austini

A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.     

Pc16a, the first characterized peptide from Conus pictus venom, shows a novel disulfide connectivity

A novel conotoxin, pc16a, was isolated from the venom of Conus pictus. This is the first peptide characterized from this South-African cone snail and it has only 11 amino acid residues, SCSCKRNFLCC*, with the rare cysteine framework XVI and a monoisotopic mass of 1257.6Da. Two peptides were synthesized with two possible conformations: globular (pc16a_1) and ribbon (pc16a_2). pc16a_1 co-eluted with the native peptide, which indicates a disulfide connectivity I-III, II-IV. The structure of pc16a_1 was determined by NMR. Both synthetic peptides were used to elucidate the biological activity. Bioassays were performed on crickets, ghost shrimps, larvae of the mealworm beetle and mice, but no effect was seen. Using two-electrode voltage clamp, a range of voltage-gated ion channels (Na(v) and K(v)) and nicotinic acetylcholine receptors were screened, but again no activity was found. Hence, the specific target of pc16a still remains to be discovered.     

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 2. Three-dimensional solution structure.

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D 1H NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and muO-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18+/-0.05 A for the backbone atoms and 1.39+/-0.33 A for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.     

A novel D-leucine-containing Conus peptide: diverse conformational dynamics in the contryphan family.

A Conus peptide family (the contryphans) is noteworthy because of the presence of a post-translationally modified D-amino acid in all members of the family. A new contryphan peptide, Leu-contryphan-P, was isolated from the venom of Conus purpurascens; the sequence of this peptide is: Gly-Cys-Val-D-Leu-Leu-Pro-Trp-Cys-OH. This is the first known occurrence of D-leucine in a Conus peptide. The discovery of Leu-contryphan-P suggests that there may be branches of the contryphan peptide family that diverge much more in sequence than previously anticipated. Several natural contryphans provide dramatic examples of interconversion between multiple conformational states in small constrained peptides. The contryphans that have 4-trans-hydroxyproline and D-tryptophan in positions 3 and 4, respectively, exhibit two peaks under reverse-phase HPLC conditions, indicating interconversion between two discrete conformations. However, [L-Trp4]contryphan-Sm (with L-Trp substituted for D-Trp) exhibits a single, broad peak that elutes later than the natural peptide, suggesting that D-Trp stabilizes a conformation in which hydrophobic residues are buried. Leucontryphan-P which has valine and D-leucine instead of 4-trans-hydroxyproline and D-tryptophan shows only a single peak that elutes much later than the other contryphans.     

Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha 3- and chimeric alpha 6-containing nAChRs; BuIA blocks alpha 6/alpha 3 beta 2 nAChRs with a 40,000-fold lower IC(50) than alpha 4 beta 2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta 4 subunit have very slow off-times, compared with the corresponding beta 2 subunit-containing nAChR. In each instance, rat alpha x beta 4 may be distinguished from rat alpha x beta 2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha 3 beta 2 to mouse alpha 3 beta 4, and human alpha 3 beta2 to human alpha 3 beta 4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta 2- and beta 4-containing nAChRs.