α-Conotoxin recombinant protein ImI-AFP3 efficiently inhibits the growth and migration of lung cancer cells.

α-Conotoxin ImI is a selective antagonist of alpha7 nicotinic acetylcholine receptor (α7 nAChR) that is involved in cancer development. Human alpha fetoprotein domain 3 (AFP3) is a prototype of anticancer agents. In an effort to design drugs for anticancer treatments, we fused the ImI peptide to AFP3 as a fusion protein for testing. The fusion protein (ImI-AFP3) was highly expressed in the insect Bac-to-Bac system. The purified fusion protein was found to have improved anticancer activity and synergized with the drug gefitinib to inhibit the growth and migration of A549 and NCI-H1299 lung cancer cells. Our data have demonstrated that the recombinant protein ImI-AFP3 is a promising candidate for drug development to suppress lung cancer cell growth, especially to suppress hepatoid adenocarcinoma of the lung (HAL) cell growth.

Collaborative Expression: Transcriptomics of Conus virgo Suggests Contribution of Multiple Secretory Glands to Venom Production.

Venomous marine gastropods of the family Conidae are among the most diversified predators in marine realm-in large due to their complex venoms. Besides being a valuable source of bioactive neuropeptides conotoxins, cone-snails venoms are an excellent model for molecular evolution studies, addressing origin of key innovations. However, these studies are handicapped by scarce current knowledge on the tissues involved in venom production, as it is generally assumed the sole prerogative of the venom gland (VG). The role of other secretory glands that are present in all Conus species (salivary gland, SG) or only in some species (accessory salivary gland, ASG) remains poorly understood. Here, for the first time, we carry out a detailed analysis of the VG, SG, and ASG transcriptomes in the vermivorous Conus virgo. We detect multiple transcripts clusters in both the SG and ASG, whose annotations imply venom-related functions. Despite the subsets of transcripts highly-expressed in the VG, SG, and ASG being very distinct, SG expresses an L-, and ASG-Cerm08-, and MEFRR- superfamily conotoxins, all previously considered specific for VG. We corroborate our results with the analysis of published SG and VG transcriptomes from unrelated fish-hunting C. geographus, and C. striatus, possibly fish-hunting C. rolani, and worm-hunting Conus quercinus. In spite of low expression levels of conotoxins, some other specific clusters of putative venom-related peptides are present and may be highly expressed in the SG of these species. Further functional studies are necessary to determine the role that these peptides play in envenomation. In the meantime, our results show importance of routine multi-tissue sampling both for accurate interpretation of tissue-specific venom composition in cone-snails, and for better understanding origin and evolution of venom peptides genes.

Nicotinic acetylcholine receptor subtype expression, function, and pharmacology: Therapeutic potential of α-conotoxins.

The pentameric nicotinic acetylcholine receptors (nAChRs) are typically classed as muscle- or neuronal-type, however, the latter has also been reported in non-neuronal cells. Given their broad distribution, nAChRs mediate numerous physiological and pathological processes including synaptic transmission, presynaptic modulation of transmitter release, neuropathic pain, inflammation, and cancer. There are 17 different nAChR subunits and combinations of these subunits produce subtypes with diverse pharmacological properties. The expression and role of some nAChR subtypes have been extensively deciphered with the aid of knock-out models. Many nAChR subtypes expressed in heterologous systems are selectively targeted by the disulfide-rich α-conotoxins. α-Conotoxins are small peptides isolated from the venom of cone snails, and a number of them have potential pharmaceutical value.

Analgesic α-Conotoxin Binding Site on the Human GABAB Receptor.

The analgesic α-conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABAB receptors (GABABRs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABABR and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic α-conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABABR subunits predicted through computational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the α-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABABR ligand-binding site. The effects of baclofen and α-conotoxins on the peak Ba2+ current (IBa) amplitude were investigated on wild-type and 15 GABABR mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABABR subunits attenuated baclofen-sensitive IBa inhibition by the analgesic α-conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABABR residues involved in interactions with the α-conotoxins are K168 and R207 on the B2 subunit and S130, S153, R162, E200, F227, and E253 on the B1 subunit. The double mutant, S130A + S153A, abolished inhibition by both baclofen and the α-conotoxins. Depolarization-activated IBa mediated by both wild-type and all GABABR mutants were inhibited by the selective GABABR antagonist CGP 55845. This study identifies specific residues of GABABR involved in the binding of the analgesic α-conotoxins to the VFT domains of the GABABR. SIGNIFICANCE STATEMENT: This study defines the binding site of the analgesic α-conotoxins Vc1.1, RgIA, and PeIA on the human GABAB receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and molecular dynamics simulations of GABABR identified amino acids of the Venus flytrap (VFT) domains with which the α-conotoxins interact. GABABR alanine mutants attenuated baclofen-sensitive Cav2.2 inhibition by the α-conotoxins. We identify an allosteric binding site at the interface of the VFT domains of the GABABR subunits for the analgesic α-conotoxins.

A fluorescent screening method for optimization of conotoxin expression in Pichia pastoris.

Conotoxins are small cysteine-rich peptides secreted by the Conus venom glands, which act on ion channels or membrane receptors with high specificity and potency. Conotoxins are invaluable sources for neuroscience research and drug leads, but their application is hindered by the limited successes in quantitative engineering using either chemical or biotechnological approaches. Here, we explore the Pichia pastoris to express 23 selected conopeptides using a GFP-based fluorescence screen. We found that, in a protease-deficient strain PichiaPink™ Strain 4 (ade2 prb1 pep4), most of the recombinant conopeptides were expressed as two major folding variants including a compact form that was somehow resistant to reduction and high temperature. The GFP-αTxIA was the only one displaying a single band that showed a dose-dependent neurotoxicity on larvae of the insect Plutella xylostella, with a 48-h LD50 lower than 1.12 pmol mg-1 body weight. Furthermore, the recombinant αTxIA after cleavage from the fusion was able to inhibit cell proliferation of the LYCT and HEK293T cell lines with an appearance IC50 of 341 ± 8 and 235 ± 15 nM, respectively. This screening method is straightforward and easy to scale up, providing a versatile tool for further optimization of conotoxin production in the yeast cell.

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Conotoxins: Chemistry and Biology.

The venom of the marine predatory cone snails (genus Conus) has evolved for prey capture and defense, providing the basis for survival and rapid diversification of the now estimated 750+ species. A typical Conus venom contains hundreds to thousands of bioactive peptides known as conotoxins. These mostly disulfide-rich and well-structured peptides act on a wide range of targets such as ion channels, G protein-coupled receptors, transporters, and enzymes. Conotoxins are of interest to neuroscientists as well as drug developers due to their exquisite potency and selectivity, not just against prey but also mammalian targets, thereby providing a rich source of molecular probes and therapeutic leads. The rise of integrated venomics has accelerated conotoxin discovery with now well over 10,000 conotoxin sequences published. However, their structural and pharmacological characterization lags considerably behind. In this review, we highlight the diversity of new conotoxins uncovered since 2014, their three-dimensional structures and folds, novel chemical approaches to their syntheses, and their value as pharmacological tools to unravel complex biology. Additionally, we discuss challenges and future directions for the field.

Diversity of Conopeptides and Conoenzymes from the Venom Duct of the Marine Cone Snail Conus bayani as Determined from Transcriptomic and Proteomic Analyses.

Marine cone snails are predatory gastropods characterized by a well-developed venom apparatus and highly evolved hunting strategies that utilize toxins to paralyze prey and defend against predators. The venom of each species of cone snail has a large number of pharmacologically active peptides known as conopeptides or conotoxins that are usually unique in each species. Nevertheless, venoms of only very few species have been characterized so far by transcriptomic approaches. In this study, we used transcriptome sequencing technologies and mass spectrometric methods to describe the diversity of venom components expressed by a worm-hunting species, Conus bayani. A total of 82 conotoxin sequences were retrieved from transcriptomic data that contain 54 validated conotoxin sequences clustered into 21 gene superfamilies including divergent gene family, 17 sequences clustered to 6 different conotoxin classes, and 11 conotoxins classified as unassigned gene family. Seven new conotoxin sequences showed unusual cysteine patterns. We were also able to identify 19 peptide sequences using mass spectrometry that completely overlapped with the conotoxin sequences obtained from transcriptome analysis. Importantly, herein we document the presence of 16 proteins that include five post-translational modifying enzymes obtained from transcriptomic data. Our results revealed diverse and novel conopeptides of an unexplored species that could be used extensively in biomedical research due to their therapeutic potentials.

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.

Conformational dynamics of [Formula: see text]-conotoxin PnIB in complex solvent systems.

Cone snails are slow-moving animals that secure survival by injecting to their prey a concoction of highly potent and stable neurotoxic peptides called conotoxins. These small toxins (~ 10-30 AA) interact with ion channels and their diverse structures account for various variables such as the environment and the prey of preference. This study probed the conformational space of α-conotoxin PnIB from Conus pennaceus by performing all-atom molecular dynamics simulations on the conotoxin in complex solvent systems of water and octanol. Secondary structure analyses showed a uniform conformation for the pure (C100Oc, C100W) and minute (C95Oc, C5Oc) systems. In C50Oc, however, structural changes were observed. The original helices were converted to turns and were shown to happen simultaneously with the elongation of the helix and shortening of end-to-end distance. The transitions complement the orientation of the peptide at the interface. The shift to the broken helix conformation is marked by the rearrangement of solvent molecules to a framework that favors the accumulation of water molecules at residues 6-11 of the H2 region. This promotes specific protein-solvent interactions that facilitate secondary structure transitions. As PnIB has shown favorable binding toward neuronal nicotinic acetylcholine receptors, this study may provide insights on this conotoxin's therapeutic potential. Description: Structural changes in PnIB are accompanied by a simultaneous change in solvent density.

Diversity of Conopeptides and Their Precursor Genes of Conus Litteratus.

The venom of various Conus species is composed of a rich variety of unique bioactive peptides, commonly referred to as conotoxins (conopeptides). Most conopeptides have specific receptors or ion channels as physiologically relevant targets. In this paper, high-throughput transcriptome sequencing was performed to analyze putative conotoxin transcripts from the venom duct of a vermivorous cone snail species, Conus litteratus native to the South China Sea. A total of 128 putative conotoxins were identified, most of them belonging to 22 known superfamilies, with 43 conotoxins being regarded as belonging to new superfamilies. Notably, the M superfamily was the most abundant in conotoxins among the known superfamilies. A total of 15 known cysteine frameworks were also described. The largest proportion of cysteine frameworks were VI/VII (C-C-CC-C-C), IX (C-C-C-C-C-C) and XIV (C-C-C-C). In addition, five novel cysteine patterns were also discovered. Simple sequence repeat detection results showed that di-nucleotide was the major type of repetition, and the codon usage bias results indicated that the codon usage bias of the conotoxin genes was weak, but the M, O1, O2 superfamilies differed in codon preference. Gene cloning indicated that there was no intron in conotoxins of the B1- or J superfamily, one intron with 1273-1339 bp existed in a mature region of the F superfamily, which is different from the previously reported gene structure of conotoxins from other superfamilies. This study will enhance our understanding of conotoxin diversity, and the new conotoxins discovered in this paper will provide more potential candidates for the development of pharmacological probes and marine peptide drugs.

Binding of κ-Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are Differentially Affected by the Ionic Strength.

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey's excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4-5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.

α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli.

α7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer's and schizophrenia disease. The mutant ArIB (V11L, V16A) of α-conotoxin ArIB with 17-amino acid residues specifically targets α7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.

Blockers of Nicotinic Acetylcholine Receptors Delay Tumor Growth and Increase Antitumor Activity of Mouse Splenocytes.

Blockade of α6, α3ß2, α9α10, and α7 subtypes of nicotinic acetylcholine receptors slows tumor growth in vivo, increases cytotoxic activity of splenocytes from tumor-bearing mice, and, to some extent, reduces the viability of Ehrlich carcinoma cells in vitro. These data indicate that nicotinic acetylcholine receptors are involved in oncogenesis, affecting the survival of tumor cells, inter alia, via modulation of the antitumor immunity.

Molecular determinants of α-conotoxin potency for inhibition of human and rat α6ß4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and ß4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat α6/α3ß4 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to α-conotoxins. Sequence homology comparisons of human and rat α6ß4 nAChR subunits indicated that α6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of ß4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of α-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro13 in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu119 of human ß4 contributes to higher sensitivity of human α6/α3ß4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat α6/α3ß4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 and residue 119 of the ß4 subunit. These results highlight the potential significance of species differences in α6ß4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.

Targeted Sequencing of Venom Genes from Cone Snail Genomes Improves Understanding of Conotoxin Molecular Evolution.

To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and nontoxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100× coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of one to six exons and are typically short in length (mean = ∼85 bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: 1) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, 2) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24-63%), and 3) extensive gene turnover, where Conidae species varied from 120 to 859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification.

Rapid Photolysis-Mediated Folding of Disulfide-Rich Peptides.

Structure-activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated "one-pot" strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin.

Structural studies of Staphylococcus aureus Sortase inhibiton via Conus venom peptides.

Sortases (Srts) are transpeptidase enzymes that anchor plethora of surface proteins with LPXTG and NPQTN motifs to the cell wall of Gram-positive bacteria, and they are potential targets for the development of anti-infective agents. Despite the existence of numerous inhibitors for Staphylococcus aureus SrtA (SaSrtA), only two inhibitors are known for Staphylococcus aureus SrtB (SaSrtB). Moreover, no study has yet documented the anti-virulence potential of cone snail venom conopeptides against these virulence factors. Here we report M2-conotoxin and contryphan-R as effective inhibitory agents that may competitively block the binding of natural substrates with SaSrtA and SrtB, respectively, via molecular docking and dynamic simulation assays. M2-conotoxin also exhibited strong binding inside the catalytic grooves of distantly related SrtA homologs from Streptococcus mutans (SmSrtA) and Streptococcus agalactiae (SgSrtA). On the other hand, contryphan-R failed to occupy the substrate binding site of closely related Bacillus anthracis SrtB (BaSrtB), but successfully blocked the catalytic site of very divergent C. Perfringens SrtB (CpSrtB). Hence, these naturally existing venom peptides and their mimetics may serve as promising candidates for further development of therapeutically useful anti-infectives for the treatment of infections caused by multi-drug resistant bacterial pathogens having SrtA and SrtB in their enzymatic set.

High-Throughput Identification and Analysis of Novel Conotoxins from Three Vermivorous Cone Snails by Transcriptome Sequencing.

The venom of each Conus species consists of a diverse array of neurophysiologically active peptides, which are mostly unique to the examined species. In this study, we performed high-throughput transcriptome sequencing to extract and analyze putative conotoxin transcripts from the venom ducts of 3 vermivorous cone snails (C. caracteristicus, C. generalis, and C. quercinus), which are resident in offshore waters of the South China Sea. In total, 118, 61, and 48 putative conotoxins (across 22 superfamilies) were identified from the 3 Conus species, respectively; most of them are novel, and some possess new cysteine patterns. Interestingly, a series of 45 unassigned conotoxins presented with a new framework of C-C-C-C-C-C, and their mature regions were sufficiently distinct from any other known conotoxins, most likely representing a new superfamily. O- and M-superfamily conotoxins were the most abundant in transcript number and transcription level, suggesting their critical roles in the venom functions of these vermivorous cone snails. In addition, we identified numerous functional proteins with potential involvement in the biosynthesis, modification, and delivery process of conotoxins, which may shed light on the fundamental mechanisms for the generation of these important conotoxins within the venom duct of cone snails.

Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa.

The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, µ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.