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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. [ABSTRACT FROM AUTHOR]

Cone snails, as a type of marine organism, have rich species diversity. Traditionally, classifications of cone snails were based mostly on radula, shell, and anatomical characters. Because of these phenotypic features' high population variability and propensity for local adaptation and convergence, identifying species can be difficult and occasionally inaccurate. In addition, mitochondrial genomes contain high phylogenetic information, so complete mitogenomes have been increasingly employed for inferring molecular phylogeny. To enrich the mitogenomic database of cone snails (Caenogastropoda: Conidae), mitogenomes of four Conus species, i.e., C. imperialis (15,505 bp), C. literatus (15,569 bp), C. virgo (15,594 bp), and C. marmoreus (15,579 bp), were characterized and compared. All 4 of these mitogenomes included 13 protein-coding genes, 2 ribosomal RNA genes, 22 tRNA genes, and non-coding regions. All the Protein Codon Genes (PCGs) of both newly sequenced mitogenomes used TAA or TAG as a terminal codon. Most PCGs used conventional start codon ATG, but an alternative initiation codon GTG was detected in a gene (NADH dehydrogenase subunit 4 (nad4)) of C. imperialis. In addition, the phylogenetic relationships were reconstructed among 20 Conus species on the basis of PCGs, COX1, and the complete mitogenome using both Bayesian Inference (BI) and Maximum Likelihood (ML). The phylogenetic results supported that C. litteratus, C. quercinus, and C. virgo were clustered together as a sister group (PP = 1, BS = 99), but they did not support the phylogenetic relation of C. imperialis and C. tribblei (PP = 0.79, BS = 50). In addition, our study established that PCGs and complete mitogenome are the two useful markers for phylogenetic inference of Conus species. These results enriched the data of the cone snail's mitochondrion in the South China Sea and provided a reliable basis for the interpretation of the phylogenetic relationship of the cone snail based on the mitochondrial genome. [ABSTRACT FROM AUTHOR]

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

Abstract The genus Conus includes over 900 species of marine invertebrates known as cone snails, whose venoms are among the most powerful described so far. This potency is mainly due to the concerted action of hundreds of small bioactive peptides named conopeptides, which target different ion channels and membrane receptors and thus interfere with crucial physiological processes. By swiftly harpooning and injecting their prey and predators with such deadly cocktails, the slow-moving cone snails guarantee their survival in the harsh, competitive marine environment. Each cone snail species produces a unique venom, as the mature sequences of conopeptides from the venoms of different species share very little identity. This biochemical diversity, added to the numerous species and conopeptides contained in their venoms, results in an immense biotechnological and therapeutic potential, still largely unexplored. That is especially true regarding the bioprospection of the venoms of cone snail species found off the Brazilian coast - a region widely known for its biodiversity. Of the 31 species described in this region so far, only four - Conus cancellatus, Conus regius, Conus villepinii, and Conus ermineus - have had their venoms partially characterized, and, although many bioactive molecules have been identified, only a few have been actually isolated and studied. In addition to providing an overview on all the cone snail species found off the Brazilian coast to date, this review compiles the information on the structural and pharmacological features of conopeptides and other molecules identified in the venoms of the four aforementioned species, paving the way for future studies.

Marine cone snail venoms represent a vast library of bioactive peptides with proven potential as research tools, drug leads, and therapeutics. In this study, a transcriptome library of four different organs, namely radular sheath, venom duct, venom gland, and salivary gland, from piscivorous Conus striatus was constructed and sequenced using both Illumina next-generation sequencing (NGS) and PacBio third-generation sequencing (TGS) technologies. A total of 428 conotoxin precursor peptides were retrieved from these transcriptome data, of which 413 conotoxin sequences assigned to 13 gene superfamilies, and 15 conotoxin sequences were classified as unassigned families. It is worth noting that there were significant differences in the diversity of conotoxins identified from the NGS and TGS data: 82 conotoxins were identified from the NGS datasets while 366 conotoxins from the TGS datasets. Interestingly, we found point mutations in the signal peptide sequences of some conotoxins with the same mature sequence. Therefore, TGS broke the traditional view of the conservation of conotoxin signal peptides and the variability of mature peptides obtained by NGS technology. These results shed light on the integrated NGS and TGS technologies to mine diverse conotoxins in Conus species, which will greatly contribute to the discovery of novel conotoxins and the development of new marine drugs.

Cone snails, as a type of marine organism, have rich species diversity. Traditionally, classifications of cone snails were based mostly on radula, shell, and anatomical characters. Because of these phenotypic features’ high population variability and propensity for local adaptation and convergence, identifying species can be difficult and occasionally inaccurate. In addition, mitochondrial genomes contain high phylogenetic information, so complete mitogenomes have been increasingly employed for inferring molecular phylogeny. To enrich the mitogenomic database of cone snails (Caenogastropoda: Conidae), mitogenomes of four Conus species, i.e., C. imperialis (15,505 bp), C. literatus (15,569 bp), C. virgo (15,594 bp), and C. marmoreus (15,579 bp), were characterized and compared. All 4 of these mitogenomes included 13 protein-coding genes, 2 ribosomal RNA genes, 22 tRNA genes, and non-coding regions. All the Protein Codon Genes (PCGs) of both newly sequenced mitogenomes used TAA or TAG as a terminal codon. Most PCGs used conventional start codon ATG, but an alternative initiation codon GTG was detected in a gene (NADH dehydrogenase subunit 4 (nad4)) of C. imperialis. In addition, the phylogenetic relationships were reconstructed among 20 Conus species on the basis of PCGs, COX1, and the complete mitogenome using both Bayesian Inference (BI) and Maximum Likelihood (ML). The phylogenetic results supported that C. litteratus, C. quercinus, and C. virgo were clustered together as a sister group (PP = 1, BS = 99), but they did not support the phylogenetic relation of C. imperialis and C. tribblei (PP = 0.79, BS = 50). In addition, our study established that PCGs and complete mitogenome are the two useful markers for phylogenetic inference of Conus species. These results enriched the data of the cone snail’s mitochondrion in the South China Sea and provided a reliable basis for the interpretation of the phylogenetic relationship of the cone snail based on the mitochondrial genome.

The venomous insulin‐like peptides released by certain cone snails stimulate hypoglycemic shock to immobilize fish and catch the prey. Compared to human insulin (hIns), the cone snail insulins (Con‐Ins) are typically monomeric and shorter in sequence, yet they exhibit moderate hIns‐like biological activity. We have modeled six variants of Con‐Ins (G3, K1, K2, T1A, T1B, and T2) and carried out explicit‐solvent molecular dynamics (MD) simulations of eight types of insulins, two with known structures (hIns and Con‐Ins‐G1) and six Con‐Ins with modeled structures, to characterize key residues of each insulin that interact with the truncated human insulin receptor (μIR). We show that each insulin/μIR complex is stable during explicit‐solvent MD simulations and hIns interactions indicate the highest affinity for the "site 1" of IR. The residue contact maps reveal that each insulin preferably interacts with the αCT peptide than the L1 domain of IR. Through analysis of the average nonbonded interaction energy contribution of every residue of each insulin for the μIR, we probe the residues establishing favorable interactions with the receptor. We compared the interaction energy of each residue of every Con‐Ins to the μIR and observed that γ‐carboxylated glutamate (Gla), His, Thr, Tyr, Tyr/His, and Asn in Con‐Ins are favorable substitutions for GluA4, AsnA21, ValB12, LeuB15, GlyB20, and ArgB22 in hIns, respectively. The identified insulin analogs, although lacking the last eight residues of the B‐chain of hIns, bind strongly to μIR. Our findings are potentially useful in designing potent fast‐acting therapeutic insulin. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the tubular cone snail Conus betulinus is presented in this study. The C. betulinus mitochondrial genome was 16,240 bp with 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding AT-rich region (D-loop). The overall base composition was estimated to be 25.67% for A, 38.26% for T, 21.38% for G, and 14.69% for C, with a high A + T content of 63.93%. Phylogenetic analyses based on 13 PCGs showed the close relationship of vermivorous C. betulinus with the common ancestor of molluscivorous Conus textile and Conus gloriamaris, providing a basis for further studies on the phylogenetics of cone snails according to their dietary type.

Conic snails are slow-moving carnivorous that use a very powerful venom to hunt. This venom is a combination of different toxins from the family of conototoxins. This research was carried out to identify a marine Cone snail, Conus pennaceus quasimagnificus in Shibderaz Cost, South of Qeshm Island. After sampling the venom was isolated using a buffer and centrifuge. Then the lethal dose (LD50) on mice was analyzed and various conotoxins were identified. For the first time, the presence of a conotoxin called “alpha-conotoxin-like” of the A-superfamily was found in the venom of the collected specimens. This peptide was tested for its anti-bacterial activity after being synthesized. Analysis of the isolated DNA showed that the species studied was C. pennaceus and phylogenetical studies showed the closest distance to the C. pennaceus quasimagnificus subspecies. The venom of this cone snake showed the LD50 of 500 mg kg-1. This peptide showed no effect on different strains of bacteria, however, given the strong inhibitory effect of venom on nicotine acetylcholine receptors (nAChR), it can be further studied for its pain relief effect.

Previous studies have indicated that each conotoxin precursor has a hyperconserved signal region, a rather conserved pro region and a hypervariable mature region, and nucleotide mutations are the main driven factor. However, in this study, we made an in-depth analysis on the M-superfamily conotoxin precursors and found that the diversity of the signal, pro and mature regions are more complicated than previous findings. Different conotoxin precursors can have same signal, pro and/or mature regions, especially different conotoxin precursors with same mature region but different signal and pro regions. In addition, insertions and deletions (indels) were detected in conotoxin precursors. Indels are infrequent in the signal region but frequent in the pro and mature regions. In contrast to deletions that dominate in the pro region, insertions dominate in the mature region. The number of amino acids is crucial for the physiological functions of mature conotoxins, therefore indels, especially insertions in the mature region, play an important role in the sequence and function diversity of conotoxins. [ABSTRACT FROM AUTHOR]

Background: For the past two decades, few conotoxins entered in clinical trial and some are available for pain relieving as well as for treatment of neurological disorders. Aim: The present investigation was made to elucidate the antiproliferative effect of crude venom from Conus virgo against human lung adenocarcinoma cells. Acute toxicity was determined on adult Zebra fish. Methods: Cytotoxicity was assessed by MTT assay where the DNA damage was carried out by DNA fragmentation method and the in vivo toxicity was assessed by OECD guidelines. Results: The cell viability was found to decrease and the cell lost their morphological integrity with increasing concentration (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/ml) with an IC50 concentration of 74.69 µg/ml. The nuclear integrity and DNA fragmentation assay revealed the induction of DNA damage which was observed at 5 h. Further acute toxicity study was made, the LD50 was found at a concentration of 96.393µg/ml. The crude venom affects the locomotory activity of adult Zebra fish and induces the paralytic activity at 50, 75, 100 and 125 µg/ml concentrations. Histology of brain tissue indicated slight variations in the pallium. Conclusion: From the investigation, it was inferred that C. virgo venom could be a potential candidate for cancer chemotherapy. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the tubular cone snail Conus betulinus is presented in this study. The C. betulinus mitochondrial genome was 16,240 bp with 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding AT-rich region (D-loop). The overall base composition was estimated to be 25.67% for A, 38.26% for T, 21.38% for G, and 14.69% for C, with a high A + T content of 63.93%. Phylogenetic analyses based on 13 PCGs showed the close relationship of vermivorous C. betulinus with the common ancestor of molluscivorous Conus textile and Conus gloriamaris, providing a basis for further studies on the phylogenetics of cone snails according to their dietary type. [ABSTRACT FROM AUTHOR]

Cone snails have evolved a variety of insulin-like molecules that may help with the development of better treatments for diabetes.

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Conotoxins form a diverse group of peptide toxins found in the venom of predatory marine cone snails. Decades of conotoxin research have provided numerous measurable scientific and societal benefits. These include their use as a drug, diagnostic agent, drug leads, and research tools in neuroscience, pharmacology, biochemistry, structural biology, and molecular evolution. Human envenomations by cone snails are rare but can be fatal. Death by envenomation is likely caused by a small set of toxins that induce muscle paralysis of the diaphragm, resulting in respiratory arrest. The potency of these toxins led to concerns regarding the potential development and use of conotoxins as biological weapons. To address this, various regulatory measures have been introduced that limit the use and access of conotoxins within the research community. Some of these regulations apply to all of the ≈200,000 conotoxins predicted to exist in nature of which less than 0.05% are estimated to have any significant toxicity in humans. In this review we provide an overview of the many benefits of conotoxin research, and contrast these to the perceived biosecurity concerns of conotoxins and research thereof.

Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.

The vast diversity of neuronal nicotinic acetylcholine subunits expressed in the central and peripheral nervous systems, as well as in non-neuronal tissues, constitutes a formidable challenge for researchers and clinicians to decipher the role of particular subtypes, including complex subunit associations, in physiological and pathophysiological functions. Many natural products target the nAChRs, but there is no richer source of nicotinic ligands than the venom of predatory gastropods known as cone snails. Indeed, every single species of cone snail was shown to produce at least one type of such α-conotoxins. These tiny peptides (10–25 amino acids), constrained by disulfide bridges, proved to be unvaluable tools to investigate the structure and function of nAChRs, some of them having also therapeutic potential. In this review, we provide a recent update on the pharmacology and subtype specificity of several major α-conotoxins. [ABSTRACT FROM AUTHOR]

Marine drugs have developed rapidly in recent decades. Cone snails, a group of more than 700 species, have always been one of the focuses for new drug discovery. These venomous snails capture prey using a diverse array of unique bioactive neurotoxins, usually named as conotoxins or conopeptides. These conotoxins have proven to be valuable pharmacological probes and potential drugs due to their high specificity and affinity to ion channels, receptors, and transporters in the nervous systems of target prey and humans. Several research groups, including ours, have examined the venom gland of cone snails using a combination of transcriptomic and proteomic sequencing, and revealed the existence of hundreds of conotoxin transcripts and thousands of conopeptides in each Conus species. Over 2000 nucleotide and 8000 peptide sequences of conotoxins have been published, and the number is still increasing quickly. However, more than 98% of these sequences still lack 3D structural and functional information. With the rapid development of genomics and bioinformatics in recent years, functional predictions and investigations on conotoxins are making great progress in promoting the discovery of novel drugs. For example, ω-MVIIA was approved by the U.S. Food and Drug Administration in 2004 to treat chronic pain, and nine more conotoxins are at various stages of preclinical or clinical evaluation. In short, the genus Conus, the big family of cone snails, has become an important genetic resource for conotoxin identification and drug development. [ABSTRACT FROM AUTHOR]

Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to Stenotrophomonas bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized. [ABSTRACT FROM AUTHOR]

A novel anti-mollusk conopeptide pn4c was isolated from theConus pennaceusvenom by repeated HPLC fractionation based on the activity against freshwater snails. The primary structure of pn4c was determined by the mass spectrometricde novosequencing analysis. In addition, pn3a was isolated from the same fraction containing pn4c, as a peptide with unknown functions. [ABSTRACT FROM AUTHOR]

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

Abstract During evolution, nature has embraced different strategies for species to survive. One strategy, applied by predators as diverse as snakes, scorpions, sea anemones and cone snails, is using venom to immobilize or kill a prey. This venom offers a unique and extensive source of chemical diversity as it is driven by the evolutionary pressure to improve prey capture and/or to protect their species. Cone snail venom is an example of the remarkable diversity in pharmacologically active small peptides that venoms can consist of. These venom peptides, called conopeptides, are classified into two main groups based on the number of cysteine residues, namely disulfide-rich and disulfide-poor conopeptides. Since disulfide-poor conotoxins are minor components of this venom cocktail, the number of identified peptides and the characterization of these peptides is far outclassed by its cysteine-rich equivalents. This review provides an overview of 12 families of disulfide-poor peptides identified to date as well as the state of affairs.

Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.

Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 μM, and it does not affect the hα3β2 subtype at 6.5 μM.

The primary objective of this study was to realize the large-scale discovery of conotoxin sequences from different organs (including the venom duct, venom bulb and salivary gland) of the vermivorous Oak cone snail, Conus quercinus. Using high-throughput transcriptome sequencing, we identified 133 putative conotoxins that belong to 34 known superfamilies, of which nine were previously reported while the remaining 124 were novel conotoxins, with 17 in new and unassigned conotoxin groups. A-, O1-, M-, and I2- superfamilies were the most abundant, and the cysteine frameworks XIII and VIII were observed for the first time in the A- and I2-superfamilies. The transcriptome data from the venom duct, venom bulb and salivary gland showed considerable inter-organizational variations. Each organ had many exclusive conotoxins, and only seven of all the inferred mature peptides were common in the three organs. As expected, most of the identified conotoxins were synthesized in the venom duct at relatively high levels; however, a number of conotoxins were also identified in the venom bulb and the salivary gland with very low transcription levels. Therefore, various organs have different conotoxins with high diversity, suggesting greater contributions from several organs to the high-throughput discovery of new conotoxins for future drug development.

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis-trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis-trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression. [ABSTRACT FROM AUTHOR]

The pharmacological relevance of peptides isolated from cone snails is gaining interest, particularly for pain management. Conotoxins are small well-structured peptides with specific functions over a number of specific physiological targets. Despite the large number and variety of toxins that these organisms can produce, only a handful of three-dimensional structures has been experimentally determined. Theoretical models of toxins, developed with bioinformatics method, contribute to the understanding of the structure and function of these peptides. RsXXVIA is a conotoxin previously isolated from the Conus regularis venom that has been shown to block N-type calcium channels. In this work, we modeled 12 theoretical cysteine frameworks (disulfide bonds) to elucidate the 3D structure of RsXXIVA to explain its activity. We used, as a template, the ω-conotoxin MVIIA (ziconotide), a prototype conotoxin with high sequence similarity to RsXXVIA. Particularly, the spatial arrangement of two amino acid residues, Lys2 and Tyr13 (in ziconotide), responsible for the pharmacological activity was taken into account. Remarkably, 3D models rendered a particularly suitable spatial disposition of key amino acids responsible for the activity on the N-type calcium channel. Additionally, this work explains, through computational models, how the conotoxin might be acting on the channel, thus, paving the way to find the principal RsXXVIA's physiological target. [ABSTRACT FROM AUTHOR]

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top candidates for the development of innovative drugs. Instead of relying on the classical, low-throughput bioassay-guided approach to identify innovative bioactive peptides, this work exploits a recent paradigm to access to venom diversity. This strategy bypasses the classical approach by combining high-throughput transcriptomics, proteomics and bioinformatics cutting-edge technologies to generate reliable peptide sequences. The strategy employed to generate hundreds of reliable sequences from Conus venoms is deeply described. The study led to the discovery of (i) conotoxins that belong to known pharmacological families targeting various GPCRs or ion-gated channels, and (ii) new families of conotoxins, never described to date. It also focusses on the diversity of genes, sequences, folds, and PTM's provided by such species. [ABSTRACT FROM AUTHOR]

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels that are expressed throughout the nervous system. ASICs have been implicated in several neuronal disorders, like ischemic stroke, neuronal inflammation, and pathological pain. Several toxins from venomous animals have been identified that target ASICs with high specificity and potency. These toxins are extremely useful in providing protein pharmacophores and to characterize function and structure of ASICs. Marine cone snails contain a high diversity of toxins in their venom such as conotoxins, which are short polypeptides stabilized by disulfide bonds, and conopeptides, which have no or only one disulfide bond. Whereas conotoxins selectively target specific neuronal proteins, mainly ion channels, the targets of conopeptides are less well known. Here, we perform an in vitro screen of venoms from 18 cone snail species to identify toxins targeting ASICs. We identified a small conopeptide of only four amino acids from the venom of Conus textile that strongly potentiated currents of ASIC3, which has a specific role in the pain pathway. This peptide, RPRFamide, belongs to the subgroup of cono-RFamides. Electrophysiological characterization of isolated dorsal root ganglion (DRG) neurons revealed that RPRFamide increases their excitability. Moreover, injection of the peptide into the gastrocnemius muscle strongly enhanced acid-induced muscle pain in mice that was abolished by genetic inactivation of ASIC3. In summary, we identified a conopeptide that targets the nociceptorspecific ion channel ASIC3. [ABSTRACT FROM AUTHOR]

Background: The venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides or conotoxins. These peptides have proven to be valuable pharmacological probes and potential drugs because of their high specificity and affinity to important ion channels, receptors and transporters of the nervous system. Most previous studies have focused specifically on the conopeptides from piscivorous and molluscivorous cone snails, but little attention has been devoted to the dominant vermivorous species. Results: The vermivorous Chinese tubular cone snail, Conus betulinus, is the dominant Conus species inhabiting the South China Sea. The transcriptomes of venom ducts and venom bulbs from a variety of specimens of this species were sequenced using both next-generation sequencing and traditional Sanger sequencing technologies, resulting in the identification of a total of 215 distinct conopeptides. Among these, 183 were novel conopeptides, including nine new superfamilies. It appeared that most of the identified conopeptides were synthesized in the venom duct, while a handful of conopeptides were identified only in the venom bulb and at very low levels. Conclusions: We identified 215 unique putative conopeptide transcripts from the combination of five transcriptomes and one EST sequencing dataset. Variation in conopeptides from different specimens of C. betulinus was observed, which suggested the presence of intraspecific variability in toxin production at the genetic level. These novel conopeptides provide a potentially fertile resource for the development of new pharmaceuticals, and a pathway for the discovery of new conotoxins. [ABSTRACT FROM AUTHOR]

Peptide toxins typically bind to their target ion channels or receptors with high potency and selectivity, making them attractive leads for therapeutic development. In some cases the native peptide as it is found in the venom from which it originates can be used directly, but in many instances it is desirable to truncate and/or stabilize the peptide to improve its therapeutic properties. A complementary strategy is to display the key residues that make up the pharmacophore of the peptide toxin on a non-peptidic scaffold, thereby creating a peptidomimetic. This review exemplifies these approaches with peptide toxins from marine organisms, with a particular focus on conotoxins.