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Mollusk-hunting (molluscivorous) cone snails belong to a monophyletic group in Conus, a genus of venomous marine snails. The molluscivorous lineage evolved from ancestral worm-hunting (vermivorous) snails ∼18 Ma. To enable the shift to a molluscivorous lifestyle, molluscivorous cone snails must solve biological problems encountered when hunting other gastropods, namely: (i) preventing prey escape and (ii) overcoming the formidable defense of the prey in the form of the molluscan shell, a problem unique to molluscivorous Conus. Here, we show that χ-conotoxins, peptides exclusively expressed in the venoms of molluscivorous Conus, provide solutions to the above problems. Injecting χ-conotoxins into the gastropod mollusk Aplysia californica results in impaired locomotion and uncoordinated hyperactivity. Impaired locomotion impedes escape, and a hyperactive snail will likely emerge from its shell, negating the protection the shell provides. Thus, χ-conotoxins are an evolutionary innovation that accompanied the emergence of molluscivory in Conus and provide solutions to problems posed by hunting other snails., Competing Interests: Conflict of Interest: The authors declare no conflict of interest in the conduct of this research., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Venomous marine gastropods of the superfamily Conoidea possess a rich arsenal of toxins, including neuroactive toxins. Venom adaptations might have played a fundamental role in the radiation of conoideans; nevertheless, there is still no knowledge about the venom of the most diversified family of the group: Raphitomidae Bellardi, 1875. In this study, transcriptomes were produced from the carcase, salivary glands, and proximal and distal venom ducts of the northeastern Atlantic species Raphitoma purpurea (Montagu, 1803). Using a gut barcoding approach, we were also able to report, for the first time, molecular evidence of a vermivorous diet for the genus. Transcriptomic analyses revealed over a hundred putative venom components (PVC), including 69 neurotoxins. Twenty novel toxin families, including some with high levels of expansion, were discovered. No significant difference was observed between the distal and proximal venom duct secretions. Peptides related to cone snail toxins (Cerm06, Pgam02, and turritoxin) and other venom-related proteins (disulfide isomerase and elevenin) were retrieved from the salivary glands. These salivary venom components may constitute ancestral adaptations for venom production in conoideans. Although often neglected, salivary gland secretions are of extreme importance for understanding the evolutionary history of conoidean venom.

Venom systems are complex traits that have independently emerged multiple times in diverse plant and animal phyla. Within each venomous lineage there typically exists interspecific variation in venom composition where several factors have been proposed as drivers of variation, including phylogeny and diet. Understanding these factors is of broad biological interest and has implications for the development of antivenom therapies and venom-based drug discovery. Because of their high species richness and the presence of several major evolutionary prey shifts, venomous marine cone snails (genus Conus) provide an ideal system to investigate drivers of interspecific venom variation. Here, by analyzing the venom gland expression profiles of ∼3,000 toxin genes from 42 species of cone snail, we elucidate the role of prey-specific selection pressures in shaping venom variation. By analyzing overall venom composition and individual toxin structures, we demonstrate that the shifts from vermivory to piscivory in Conus are complemented by distinct changes in venom composition independent of phylogeny. In vivo injections of venom from piscivorous cone snails in fish further showed a higher potency compared with venom of nonpiscivores demonstrating a selective advantage. Together, our findings provide compelling evidence for the role of prey shifts in directing the venom composition of cone snails and expand our understanding of the mechanisms of venom variation and diversification., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds., (© 2023 European Peptide Society and John Wiley & Sons, Ltd.)

The diversity of venomous organisms and the toxins they produce have been increasingly investigated, but taxonomic bias remains important. Neogastropods, a group of marine predators representing almost 22% of the known gastropod diversity, evolved a wide range of feeding strategies, including the production of toxins to subdue their preys. However, whether the diversity of these compounds is at the origin of the hyperdiversification of the group and how genome evolution may correlate with both the compounds and species diversities remain understudied. Among the available gastropods genomes, only eight, with uneven quality assemblies, belong to neogastropods. Here, we generated chromosome-level assemblies of two species belonging to the Tonnoidea and Muricoidea superfamilies (Monoplex corrugatus and Stramonita haemastoma). The two obtained high-quality genomes had 3 and 2.2 Gb, respectively, and 92-89% of the total assembly conformed 35 pseudochromosomes in each species. Through the analysis of syntenic blocks, Hox gene cluster duplication, and synonymous substitutions distribution pattern, we inferred the occurrence of a whole genome duplication event in both genomes. As these species are known to release venom, toxins were annotated in both genomes, but few of them were found in homologous chromosomes. A comparison of the expression of ohnolog genes (using transcriptomes from osphradium and salivary glands in S. haemastoma), where both copies were differentially expressed, showed that most of them had similar expression profiles. The high quality of these genomes makes them valuable reference in their respective taxa, facilitating the identification of genome-level processes at the origin of their evolutionary success., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Cone snail venom biodiversity reflects dietary preference and predatory and defensive envenomation strategies across the ≈900 species of Conidae . To better understand the mechanisms of adaptive radiations in closely related species, we investigated the venom of two phylogenetically and spatially related species, C. flavidus and C. frigidus of the Virgiconus clade. Transcriptomic analysis revealed that the major superfamily profiles were conserved between the two species, including 68 shared conotoxin transcripts. These shared transcripts contributed 90% of the conotoxin expression in C. frigidus and only 49% in C. flavidus , which showed greater toxin diversification in the dominant O1, I2, A, O2, O3, and M superfamilies compared to C. frigidus . On the basis of morphology, two additional sub-groups closely resembling C. flavidus were also identified from One Tree Island Reef. Despite the morphological resemblance, the venom duct proteomes of these cryptic sub-groups were distinct from C. flavidus. We suggest rapid conotoxin sequence divergence may have facilitated adaptive radiation and the establishment of new species and the regulatory mechanisms facilitating species-specific venom evolution.

Venoms are complex mixtures of proteins that have evolved repeatedly in the animal kingdom. Cone snail venoms represent one of the best studied venom systems. In nature, this venom can be dynamically adjusted depending on its final purpose, whether to deter predators or hunt prey. Here, the transcriptome of the venom gland and the proteomes of the predation-evoked and defensive venoms of the molluscivorous cone snail Cylinder ammiralis were catalogued. A total of 242 venom-related transcripts were annotated. The conotoxin superfamilies presenting more different peptides were O1, O2, T, and M, which also showed high expression levels (except T). The three precursors of the J superfamily were also highly expressed. The predation-evoked and defensive venoms showed a markedly distinct profile. A total of 217 different peptides were identified, with half of them being unique to one venom. A total of 59 peptides ascribed to 23 different protein families were found to be exclusive to the predatory venom, including the cono-insulin, which was, for the first time, identified in an injected venom. A total of 43 peptides from 20 protein families were exclusive to the defensive venom. Finally, comparisons of the relative abundance (in terms of number of peptides) of the different conotoxin precursor superfamilies showed that most of them present similar abundance regardless of the diet.

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.

Predator-prey interactions are thought to play a driving role in animal evolution, especially for groups that have developed venom as their predatory strategy. However, how the diet of venomous animals influences the composition of venom arsenals remains uncertain. Two prevailing hypotheses to explain the relationship between diet and venom composition focus on prey preference and the types of compounds in venom, and a positive correlation between dietary breadth and the number of compounds in venom. Here, we examined venom complexity, phylogenetic relationship, collection depth, and biogeography of the Terebridae (auger snails) to determine if repeated innovations in terebrid foregut anatomy and venom composition correspond to diet variation. We performed the first molecular study of the diet of terebrid marine snails by metabarcoding the gut content of 71 terebrid specimens from 17 species. Our results suggest that the presence or absence of a venom gland is strongly correlated with dietary breadth. Specifically, terebrid species without a venom gland displayed greater diversity in their diet. Additionally, we propose a revision of the definition of venom complexity in conoidean snails to more accurately capture the breadth of ecological influences. These findings suggest that prey diet is an important factor in terebrid venom evolution and diversification and further investigations of other understudied organisms, like terebrids, are needed to develop robust hypotheses in this area.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa.

Background: Hematophagy arose independently multiple times during metazoan evolution, with several lineages of vampire animals particularly diversified in invertebrates. However, the biochemistry of hematophagy has been studied in a few species of direct medical interest and is still underdeveloped in most invertebrates, as in general is the study of venom toxins. In cone snails, leeches, arthropods and snakes, the strong target specificity of venom toxins uniquely aligns them to industrial and academic pursuits (pharmacological applications, pest control etc.) and provides a biochemical tool for studying biological activities including cell signalling and immunological response. Neogastropod snails (cones, oyster drills etc.) are carnivorous and include active predators, scavengers, grazers on sessile invertebrates and hematophagous parasites; most of them use venoms to efficiently feed. It has been hypothesized that trophic innovations were the main drivers of rapid radiation of Neogastropoda in the late Cretaceous. We present here the first molecular characterization of the alimentary secretion of a non-conoidean neogastropod, Colubraria reticulata. Colubrariids successfully feed on the blood of fishes, throughout the secretion into the host of a complex mixture of anaesthetics and anticoagulants. We used a NGS RNA-Seq approach, integrated with differential expression analyses and custom searches for putative secreted feeding-related proteins, to describe in detail the salivary and mid-oesophageal transcriptomes of this Mediterranean vampire snail, with functional and evolutionary insights on major families of bioactive molecules., Results: A remarkably low level of overlap was observed between the gene expression in the two target tissues, which also contained a high percentage of putatively secreted proteins when compared to the whole body. At least 12 families of feeding-related proteins were identified, including: 1) anaesthetics, such as ShK Toxin-containing proteins and turripeptides (ion-channel blockers), Cysteine-rich secretory proteins (CRISPs), Adenosine Deaminase (ADA); 2) inhibitors of primary haemostasis, such as novel vWFA domain-containing proteins, the Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5) and the wasp Antigen-5; 3) anticoagulants, such as TFPI-like multiple Kunitz-type protease inhibitors, Peptidases S1 (PS1), CAP/ShKT domain-containing proteins, Astacin metalloproteases and Astacin/ShKT domain-containing proteins; 4) additional proteins, such the Angiotensin-Converting Enzyme (ACE: vasopressive) and the cytolytic Porins., Conclusions: Colubraria feeding physiology seems to involve inhibitors of both primary and secondary haemostasis, anaesthetics, a vasoconstrictive enzyme to reduce feeding time and tissue-degrading proteins such as Porins and Astacins. The complexity of Colubraria venomous cocktail and the divergence from the arsenal of the few neogastropods studied to date (mostly conoideans) suggest that biochemical diversification of neogastropods might be largely underestimated and worth of extensive investigation.

Venom peptides from predatory organisms are a resource for investigating evolutionary processes such as adaptive radiation or diversification, and exemplify promising targets for biomedical drug development. Terebridae are an understudied lineage of conoidean snails, which also includes cone snails and turrids. Characterization of cone snail venom peptides, conotoxins, has revealed a cocktail of bioactive compounds used to investigate physiological cellular function, predator-prey interactions, and to develop novel therapeutics. However, venom diversity of other conoidean snails remains poorly understood. The present research applies a venomics approach to characterize novel terebrid venom peptides, teretoxins, from the venom gland transcriptomes of Triplostephanus anilis and Terebra subulata. Next-generation sequencing and de novo assembly identified 139 putative teretoxins that were analyzed for the presence of canonical peptide features as identified in conotoxins. To meet the challenges of de novo assembly, multiple approaches for cross validation of findings were performed to achieve reliable assemblies of venom duct transcriptomes and to obtain a robust portrait of Terebridae venom. Phylogenetic methodology was used to identify 14 teretoxin gene superfamilies for the first time, 13 of which are unique to the Terebridae. Additionally, basic local algorithm search tool homology-based searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, such as actinoporin, commonly found in venoms. This research provides novel insights into venom evolution and recruitment in Conoidean predatory marine snails and identifies a plethora of terebrid venom peptides that can be used to investigate fundamental questions pertaining to gene evolution., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells., Biological Significance: Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, we report for the first time a detailed evaluation of the phylogenetic history and molecular evolution of the major coleoid toxins: CAP, carboxypeptidase, chitinase, metalloprotease GON-domain, hyaluronidase, pacifastin, PLA2, SE-cephalotoxin and serine proteases, with the carboxypeptidase and GON-domain documented for the first time in the coleoid venom arsenal. We show that although a majority of sites in these coleoid venom-encoding genes have evolved under the regime of negative selection, a very small proportion of sites are influenced by the transient selection pressures. Moreover, nearly 70 % of these episodically adapted sites are confined to the molecular surface, highlighting the importance of variation of the toxin surface chemistry. Coleoid venoms were revealed to be as complex as other venoms that have traditionally been the recipient of the bulk of research efforts. The presence of multiple peptide/protein types in coleoids similar to those present in other animal venoms identifies a convergent strategy, revealing new information as to what characteristics make a peptide/protein type amenable for recruitment into chemical arsenals. Coleoid venoms have significant potential not only for understanding fundamental aspects of venom evolution but also as an untapped source of novel toxins for use in drug design and discovery.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The venomous marine gastropods, cone snails (genus Conus), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. Conus bullatus belongs to a clade of Conus species called Textilia, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic Textilia clade., Results: We have carried out a sequencing survey of the Conus bullatus genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an in silico pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities., Conclusions: Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of Conus bullatus venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of C. bullatus transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.

Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

Cone snail venoms have yielded pharmacologically active natural products of exceptional scientific interest. However, cone snails are a small minority of venomous molluscan biodiversity, the vast majority being tiny venomous morphospecies in the family Turridae. A novel method called lumun-lumun opens access to these micromolluscs and their venoms. Old fishing nets are anchored to the sea bottom for a period of 1-6months and marine biotas rich in small molluscs are established. In a single lumun-lumun community, we found a remarkable gastropod biodiversity (155 morphospecies). Venomous predators belonging to the superfamily Conoidea (36 morphospecies) were the largest group, the majority being micromolluscs in the family Turridae. We carried out an initial analysis of the most abundant of the turrid morphospecies recovered, Clathurella (Lienardia) cincta (Dunker, 1871). In contrast to all cDNA clones characterized from cone snail venom ducts, one of the C. cincta clones identified encoded two different peptide precursors presumably translated from a single mRNA. The prospect of easily accessing so many different morphospecies of venomous marine snails raises intriguing toxinological possibilities: the 36 conoidean morphospecies in this one net alone have the potential to yield thousands of novel pharmacologically active compounds., (Copyright © 2009. Published by Elsevier Ltd.)

Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.

The species-rich Cone snails (Conus sp.) are predatory, marine gastropods known for small venom peptides that are valuable for pharmacological research applications. Phylogenetic analyses with mitochondrial rRNA sequences have facilitated peptide discovery. However, these relatively conserved genes leave unresolved the closer relationships among many species. We sequenced 26 internal transcribed spacer 2 (ITS2) sequences from genomic ribosomal DNA to elucidate the evolutionary relationships among molluscivorous species and to piscivorous and vermivorous species. We show that ITS2 sequences are well conserved within species but are sufficiently variable among species to resolve recent divergences. Using Bayesian, maximum likelihood and log-determinant methods, we use the ITS sequences to resolve portions of the tree that could not be resolved using the more conventional mt rRNA sequences. When the ITS2 sequences are added to existing COI and to the more conserved rRNA sequences and then properly modeled, support throughout the tree is increased. This enables us to show finer relationships among the molluscivorous species that reveal three well-supported clades (Conus, Cylinder, and Darioconus) and renders the ITS2 sequences an essential component in advancing the discovery and pharmacological characterization of novel peptides from the venoms of these molluscs.

Peroxiredoxins (Prxs) play an important role against various oxidative stresses and intra-cellular signal transduction. Peroxiredoxin 6 (PrxVI) was identified from the disk abalone Haliotis discus discus cDNA library and named HdPrxVI. The full length cDNA of HdPrxVI was 1457 bp with a 654 bp open reading frame (ORF) encoding 218 amino acids. The predicted molecular mass and estimated isoelectric point (pI) of HdPrxVI were 24 kDa and 7.3, respectively. The deduced amino acid sequence demonstrated the greatest degree (72.4%) of identity with Crassostrea gigas PrxVI. The conserved peroxidase catalytic center (42PVCTTE47) with a conserved cysteine residue (Cys44) and a catalytic center for PLA2 activity (27GGSWA31) were observed in the sequence, indicating that it is a member of 1-Cys Prx. Real time PCR results revealed that HdPrxVI mRNA is constitutively expressed in all tissues in a tissue-specific manner. During exposure to haemorrhagic septicaemia virus (VHSV), HdPrxVI mRNA transcription was down-regulated in the gill, suggesting that the abalone responded to the viral infection quickly, and HdPrxVI played a physiological role against virus-induced oxidative stress. The purified recombinant HdPrxVI, together with dithiothreitol (DTT), was shown to scavenge H2O2 in human leukemia THP-1 cells and provided protection against H2O2-induced apoptosis.

Background: Members of the predatory gastropod genus Conus use a venom comprised of a cocktail of peptide neurotoxins, termed conotoxins or conopeptides, to paralyze prey and conotoxin gene family members diversify via strong positive selection. Because Conus venoms are used primarily to subdue prey, the evolution of venoms is likely affected by predator-prey interactions., Methodology/principal Findings: To identify the selective forces that drive the differentiation of venoms within species of Conus, we examined the distribution of alleles of a polymorphic O-superfamily conotoxin locus of Conus ebraeus at Okinawa, Guam and Hawaii. Previous analyses of mitochondrial cytochrome oxidase I gene sequences suggest that populations of C. ebraeus, a worm-eating Conus, are not structured genetically in the western and central Pacific. Nonetheless, because the sample size from Guam was relatively low, we obtained additional data from this location and reexamined patterns of genetic variation at the mitochondrial gene at Okinawa, Guam and Hawaii. We also utilized a DNA-based approach to identify prey items of individuals of C. ebraeus from Guam and compared this information to published data on diets at Okinawa and Hawaii. Our results show that conotoxin allelic frequencies differ significantly among all three locations, with strongest differentiation at Hawaii. We also confirm previous inferences that C. ebraeus exhibits no genetic differentiation between Okinawa, Guam and Hawaii at the mitochondrial locus. Finally, DNA-based analyses show that eunicid polychaetes comprise the majority of the prey items of C. ebraeus at Guam; while this results compares well with observed diet of this species at Okinawa, C. ebraeus preys predominantly on nereid polychaetes at Hawaii., Conclusions/significance: These results imply that strong selection pressures affect conotoxin allelic frequencies. Based on the dietary information, the selection may derive from geographic variation in dietary specialization and local coevolutionary arms races between Conus and their prey.

The origin and evolution of venom in many animal orders remain controversial or almost entirely uninvestigated. Here we use cDNA studies of cephalopod posterior and anterior glands to reveal a single early origin of the associated secreted proteins. Protein types recovered were CAP (CRISP, Antigen 5 [Ag5] and Pathogenesis-related [PR-1]), chitinase, peptidase S1, PLA(2) (phospholipase A(2)), and six novel peptide types. CAP, chitinase, and PLA(2) were each recovered from a single species (Hapalochlaena maculosa, Octopus kaurna, and Sepia latimanus, respectively), while peptidase S1 transcripts were found in large numbers in all three posterior gland libraries. In addition, peptidase S1 transcripts were recovered from the anterior gland of H. maculata. We compare their molecular evolution to that of related proteins found in invertebrate and vertebrate venoms, revealing striking similarities in the types of proteins selected for toxic mutation and thus shedding light on what makes a protein amenable for use as a toxin.

NR2B-selective inhibitors show lower side-effects in preclinical pain models than non-selective NMDA receptor (NMDAR) antagonists, but it is unclear whether the improved safety of NR2B-selective inhibitors is due to their subtype selectivity or to a unique mode of inhibition of the receptor. In this study, the analgesic effects of intracerebral bolus injections of conantokin peptides with different NMDAR subunit selectivity were determined in mice by the standard hot-plate test, and following stimuli with acetic acid, formalin and complete Freund's adjuvant (CFA). In the standard hot-plate model, con-G[S16Y], a NR2B-selective inhibitor, showed the highest analgesic activity among conantokin peptides tested. In the acetic acid- and CFA-induced pain models, con-G[S16Y] and, to a lesser extent, con-G exhibited higher analgesic activity compared to non-selective inhibitors, such as con-R[1-17]. In the formalin test, while all conantokin peptides could partially suppress the first phase response, only con-G[S16Y] and con-G significantly inhibited the second phase response and suppressed paw edema. Our results suggest that the antinociceptive action of the conantokins may be related to their NR2B-selectivity and that these peptides may be useful as both neurobiological tools for probing mechanisms of nociception and as therapeutic agents for pain relief.

Understanding the evolution of ecological specialization is important for making inferences about the origins of biodiversity. Members of the predatory, marine gastropod genus Conus exhibit a variety of diets and the ability to capture prey is linked to a venom comprised of peptide neurotoxins, termed conotoxins. We identified conotoxin transcripts from Conus leopardus, a species of Conus that uniquely preys exclusively on hemichordates, and compared its venom duct transcriptome to that of four other Conus species to determine whether a shift to a specialized diet is associated with changes in the venom composition of this species. We also examined the secondary structure of predicted amino acid sequences of conotoxin transcripts of C. leopardus to identify substitutions that may be linked to specialization on hemichordates. We identified seven distinct conotoxin sequences from C. leopardus that appear to represent transcripts of seven distinct loci. Expression levels and the diversity of conotoxins expressed by C. leopardus are considerably less than those of other Conus. Moreover, gene products of two transcripts exhibited unique secondary structures that have not been previously observed from other Conus. These results suggest that transition to a specialist diet is associated with reduction in the number of components expressed in venoms of Conus and that diverse venoms of Conus are maintained in species with a broad dietary width.

The >10,000 living venomous marine snail species [superfamily Conoidea (Fleming, 1822)] include cone snails (Conus), the overwhelming focus of research. Hastula hectica (Linnaeus, 1758), a venomous snail in the family Terebridae (Mörch, 1852) was comprehensively investigated. The Terebridae comprise a major monophyletic group within Conoidea. H. hectica has a striking radular tooth to inject venom that looks like a perforated spear; in Conus, the tooth looks like a hypodermic needle. H. hectica venom contains a large complement of small disulfide-rich peptides, but with no apparent overlap with Conus in gene superfamilies expressed. Although Conus peptide toxins are densely post-translationally modified, no post-translationally modified amino acids were found in any Hastula venom peptide. The results suggest that different major lineages of venomous molluscs have strikingly divergent toxinological and venom-delivery strategies., (2007 Wiley-Liss, Inc)

Mammals envenomed by either the Eastern diamondback rattlesnake (Crotalus adamanteus) or the cottonmouth (Agkistrodon piscivorus piscivorus) exhibit an immediate but transitory pupillar contraction, a parasympathomimetic effect mediated through the ciliary ganglion that can be prevented by a retrobulbar injection of anesthetic. The venom of the cottonmouth injected into the lymph spaces of the frog (Rana pipiens) produces an immediate and total collapse of the lung sacs. Applied locally to the surface, it produces a constriction that eventually collapses the entire sac. Tests of venoms and toxins from both anterior and posterior parts of the venom apparatus indicate that the lung-collapsing moiety originates in the accessory, not the main portion of the venom gland. This is the first example of a functional specialization within the whole structure. It seems that this factor is elaborated primarily in snakes that prey upon frogs, although insufficient data are available from this study to confirm this. In both reptile species, the predatory strike is accompanied by an immediate effect, perhaps mediated by the parasympathetic nervous system, designed to incapacitate the prey and facilitate capture. These effects cannot now be attributed to neurotoxins because the effect of the former is transitory (and not lethal) and neither has been purified sufficiently to determine potency or structure. Both take part in securing, but not killing, the prey, and both directly oppose the sympathetic nervous system "fright-fight/flight" response. Evidence is presented to support the possibility that known epigenetic mechanisms are capable of effecting heritable changes in gene expression that could allow for the development of factors that facilitate prey acquisition and promote rapid adaptation to environmental change., ((c) 2007 Wiley-Liss, Inc.)

Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin-related peptides that have been found in the venom of cone snails, a genus of marine predatory molluscs that envenom their prey with a complex mixture of neuroactive peptides. In the present paper, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. This novel peptide, designated gamma-conopressin-vil, has the sequence CLIQDCPgammaG* (gamma is gamma-carboxyglutamate and * is C-terminal amidation). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is gamma-carboxyglutamate instead of a neutral or basic residue; therefore it could not be directly classified into either the vasopressin or the oxytocin peptide families. Nano-NMR spectroscopy of the peptide isolated directly from the cone snails revealed that the native gamma-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the gamma-carboxyglutamate residue. However, the negatively charged residues in the sequence of gamma-conopressin-vil may mediate calcium binding by a novel mechanism not observed in other peptides of this family.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.

One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel gamma-carboxyglutamic acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five gamma-carboxyglutamic acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two gamma-carboxyglutamic acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal acid.

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds., (Copyright 2004 John Wiley & Sons, Ltd.)

The objective of this investigation was to purify and characterize polypeptides from the venom ducts of the turrid snails Polystira albida and Gemmula periscelida (superfamily: Conoidea, family: Turridae), collected in Mexican waters. Venoms of other groups in the superfamily (family: Conidae, genus: Conus) have peptide toxins ('conotoxins'), but no venom components have been characterized from any turrid species. Crude venoms were fractionated using reversed-phase high performance liquid chromatography, and one major component from each venom was characterized. In contrast to most conotoxins, the polypeptides characterized contain a high proportion of Met, Tyr and Arg residues, and few, if any, Cys residues. The two peptides had some regions of homology, but were not significantly similar to other peptides. Both peptides are predicted to contain alpha-helical structures, and the peptide from P. albida is predicted to form a coiled-coil motif. This structural motif could provide conformational stability for these turrid venom components ("turritoxins"), which in the case of conotoxins is primarily achieved by disulfide bonds. Thus, the first turritoxins characterized are strikingly different from the conotoxins, suggesting divergent biochemical strategies in the venoms of different major groups included in the superfamily Conoidea.

The 500 different species of venomous cone snails (genus Conus) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides (approximately 50,000-100,000), most of which are 12-30 aa in length with two to three disulfide crosslinks. In vitro, forming the biologically relevant disulfide configuration is often problematic, suggesting that in vivo mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a Conus peptide is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in Conus venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of "microproteins" that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.

The full-length cDNAs of two A-superfamily conotoxins, kappaA-SIVA and alpha-SII, were respectively cloned and sequenced from Conus striatus using 3' RACE and 5' RACE. The cDNA of kappaA-SIVA encodes a precursor of 68 residues, including a signal peptide of 21 residues, a pro-peptide of 17 residues, and a mature peptide of 30 residues with an additional residue Gly which is prerequisite for the amidation of the preceding C-terminal Cys. The cDNA-deduced sequence of alpha-SII is composed of a signal peptide of 21 residues, a pro-peptide of 29 residues, a mature peptide of 19 residues and three additional residues Arg-Thr-Ile at the C-terminus. This tripeptide might be cleaved off by proteolytic processing. Although these two conotoxins belong to different families and target voltage-gated potassium channel and nicotinic acetylcholine receptor, respectively, they share the same signal sequence, and both are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The length of 3' untranslational region of alpha-conotoxin SII was extraordinarily large about 10 times longer than that of kappaA-SIVA with 770 and 75 bp, respectively. The elucidated cDNAs of these two toxins will facilitate a better understanding of the process of their post-translational modifications.

Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related alpha6 and alpha3 subunits. nAChRs containing alpha3, alpha4, or alpha6 subunits in combination with beta2, occasionally beta4, and sometimes beta3 or alpha5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, alpha-conotoxin PIA, potently blocks the chimeric alpha6/alpha3beta2beta3 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR alpha4beta2. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing alpha6 and alpha3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.

We describe the purification and biochemical characterization of three components from the venom of the toxoglossate gastropod Terebra subulata. The three polypeptide venom components, augertoxins s6a, s7a and s11a, are 40-41AA in length with 3-4 disulfide linkages. The arrangement of Cys residues is reminiscent of certain conopeptide superfamilies, but molecular cloning failed to show the highly conserved sequence features diagnostic of the conopeptide gene superfamily with a similar arrangement of Cys residues. One of the purified peptides, s7a, elicited an uncoordinated twisting syndrome when injected into the nematode Caenorhabditis elegans, but had no effect on mice. T. subulata belongs to the family Terebridae, one of four major groups of toxoglossate gastropods in the superfamily Conacea. The results reveal that some features of the augertoxins and conotoxins are generally similar, such as the organization of prepropeptide precursors and their proteolytic processing into mature toxins; however, Terebra may have evolved generally larger venom components that are less highly post-translationally modified. The results suggest that Conus peptide gene superfamilies probably do not extend to the Terebridae, suggesting that distinctive venom gene superfamilies may be expressed in each major division of Conacean gastropods.

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

mu-Conotoxins (mu-CTX) are potent oligopeptide blockers of sodium channels. The best characterized forms of mu-CTX, GIIIA and GIIIB, have similar primary and three-dimensional structures and comparable potencies (IC(50) approximately 30 nM) for block of wild-type skeletal muscle Na(+) channels. The two toxins are thus considered to be indistinguishable by their target channels. We have found mutations in the domain II pore region (D762K and E765K) that decrease GIIIB blocking affinity approximately 200-fold, but reduce GIIIA affinity by only approximately 4-fold, compared with wild-type channels. Synthetic mu-CTX GIIIA mutants reveal that the critical residue for differential recognition is at position 14, the site of the only charge difference between the two toxin isoforms. Therefore, engineered Na(+) channels, but not wild-type channels, can discriminate between two highly homologous conotoxins. Latent specificity of toxin-channel interactions, such as that revealed here, is a principle worthy of exploitation in the design and construction of improved biosensors.

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.

alpha-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different alpha-conotoxins are able to discriminate among different subtypes of mammalian nAChRs. In this report, the activity of two peptides from the venom of Conus pennaceus, alpha-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks alpha3beta2 nAChRs, whereas PnIB prefers the alpha7 subtype. Point mutation chimeras of these alpha-conotoxins were synthesized and their activities assessed on Xenopus oocytes expressing specific nAChRs. Change of a single residue, Ala10 to Leu, in PnIA (to form PnIA [A10L]) converts the parent peptide from alpha3beta2-preferring to alpha7-preferring; furthermore, PnIA [A10L] blocks the alpha7 receptor with an IC(50) (12.6 nM) that is lower than that of either parent peptide. Kinetic analysis indicates that differences in affinity among the analogues are primarily due to differences in off-rate, with PnIA [A10L]'s interaction with alpha7 having the smallest off-rate (k(off) = 0.17 min(-)(1)). Thermodynamic analysis indicates that Leu10 enhances the peptide's interaction with alpha7, but not alpha3beta2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both alpha7 and alpha3beta2 nAChRs.

Predatory snails in the marine gastropod genus Conus stun prey by injecting a complex mixture of peptide neurotoxins. These conotoxins are associated with trophic diversification and block a diverse array of ion channels and neuronal receptors in prey species, but the evolutionary genesis of this functional diversity is unknown. Here we show that conotoxins with little amino acid similarity are in fact products of recently diverged loci that are rapidly evolving by strong positive selection in the vermivorous cone, Conus abbreviatus, and that the rate of conotoxin evolution is higher than that of most other known proteins. Gene duplication and diversifying selection result in the formation of functionally variable conotoxins that are linked to ecological diversification and evolutionary success of this genus.

All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50-200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10-35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro-region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment-specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.