Whole Genome Duplication and Gene Evolution in the Hyperdiverse Venomous Gastropods.

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.

Comparative Venomics of C. flavidus and C. frigidus and Closely Related Vermivorous Cone Snails.

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.

A Combined Transcriptomics and Proteomics Approach Reveals the Differences in the Predatory and Defensive Venoms of the Molluscivorous Cone Snail Cylinder ammiralis (Caenogastropoda: Conidae).

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.

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.

Diet Diversity in Carnivorous Terebrid Snails Is Tied to the Presence and Absence of a Venom Gland.

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.

A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

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 Profiling Reveals Extraordinary Diversity of Venom Peptides in Unexplored Predatory Gastropods of the Genus Clavus.

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.

Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins.

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.

Shotgun Proteomics Analysis of Saliva and Salivary Gland Tissue from the Common Octopus Octopus vulgaris.

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.

Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals.

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.

From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins.

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.

The venomous cocktail of the vampire snail Colubraria reticulata (Mollusca, Gastropoda).

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.

Molecular Diversity and Gene Evolution of the Venom Arsenal of Terebridae Predatory Marine Snails.

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.

Discovery by proteogenomics and characterization of an RF-amide neuropeptide from cone snail venom.

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.

Molecular phylogeny and evolution of the proteins encoded by coleoid (cuttlefish, octopus, and squid) posterior venom glands.

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.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

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.

Characterization of the Conus bullatus genome and its venom-duct transcriptome.

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.

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus.

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.

Functional hypervariability and gene diversity of cardioactive neuropeptides.

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.

Conopeptide characterization and classifications: an analysis using ConoServer.

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.