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

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

Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins.

Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.

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

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

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 Novel α4/7-Conotoxin QuIA Selectively Inhibits α3ß2 and α6/α3ß4 Nicotinic Acetylcholine Receptor Subtypes with High Efficacy.

α6ß4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6ß4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3ß2 and α6/α3ß4 nAChR subtypes with significantly high affinity (IC50 was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 µM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3ß4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6ß2ß3 and/or α3ß4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6ß4 nAChR.

Conotoxins: Chemistry and Biology.

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

Synthesis, Structural and Pharmacological Characterizations of CIC, a Novel α-Conotoxin with an Extended N-Terminal Tail.

Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3ß2 and α6/α3ß2ß3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.

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

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

Diversity of Conopeptides and Their Precursor Genes of Conus Litteratus.

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

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

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

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

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

Structural and Functional Analyses of Cone Snail Toxins.

Cone snails are marine gastropod mollusks with one of the most powerful venoms in nature. The toxins, named conotoxins, must act quickly on the cone snails´ prey due to the fact that snails are extremely slow, reducing their hunting capability. Therefore, the characteristics of conotoxins have become the object of investigation, and as a result medicines have been developed or are in the trialing process. Conotoxins interact with transmembrane proteins, showing specificity and potency. They target ion channels and ionotropic receptors with greater regularity, and when interaction occurs, there is immediate physiological decompensation. In this review we aimed to evaluate the structural features of conotoxins and the relationship with their target types.

Discovery Methodology of Novel Conotoxins from Conus Species.

Cone snail venoms provide an ideal resource for neuropharmacological tools and drug candidates discovery, which have become a research hotspot in neuroscience and new drug development. More than 1,000,000 natural peptides are produced by cone snails, but less than 0.1% of the estimated conotoxins has been characterized to date. Hence, the discovery of novel conotoxins from the huge conotoxin resources with high-throughput and sensitive methods becomes a crucial key for the conotoxin-based drug development. In this review, we introduce the discovery methodology of new conotoxins from various Conus species. It focuses on obtaining full N- to C-terminal sequences, regardless of disulfide bond connectivity through crude venom purification, conotoxin precusor gene cloning, venom duct transcriptomics, venom proteomics and multi-omic methods. The protocols, advantages, disadvantages, and developments of different approaches during the last decade are summarized and the promising prospects are discussed as well.

Effect of Methionine Oxidation and Substitution of α-Conotoxin TxID on α3ß4 Nicotinic Acetylcholine Receptor.

α-Conotoxin TxID was discovered from Conus textile by gene cloning, which has 4/6 inter-cysteine loop spacing and selectively inhibits α3β4 nicotinic acetylcholine receptor (nAChR) subtype. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID using a combination of electrophysiological recordings and molecular modelling. The results showed most TxID analogues had substantially decreased activities on α3β4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on α3β4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at α3β4 nAChR with an IC50 of 69 nM, which only exhibited 3.8-fold less compared with TxID. Molecular dynamics simulations were performed to expound the decrease in the affinity for α3β4 nAChR. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign conotoxins containing methionine residue.

Neuronal Nicotinic Acetylcholine Receptor Modulators from Cone Snails.

Marine cone snails are a large family of gastropods that have evolved highly potent venoms for predation and defense. The cone snail venom has exceptional molecular diversity in neuropharmacologically active compounds, targeting a range of receptors, ion channels, and transporters. These conotoxins have helped to dissect the structure and function of many of these therapeutically significant targets in the central and peripheral nervous systems, as well as unravelling the complex cellular mechanisms modulated by these receptors and ion channels. This review provides an overview of α-conotoxins targeting neuronal nicotinic acetylcholine receptors. The structure and activity of both classical and non-classical α-conotoxins are discussed, along with their contributions towards understanding nicotinic acetylcholine receptor (nAChR) structure and function.

Isolation and Composition Analysis of Bioactive Glycosaminoglycans from Whelk.

Glycosaminoglycans (GAGs) are found covalently attached to proteins, which create conjugates known as proteoglycans. GAGs have remarkable biological activity as co-receptors for a variety of growth factors, cytokines, and chemokines. The present study identifies the key compositional differences between the GAGs isolated from whelk and mammalian GAGs. This polysaccharide represents a new, previously undescribed GAG with cytotoxic activity on cancer cells. Disaccharides were obtained by sample digestion with heparinases I, II, and III and chondroitinase ABC. The resistant oligosaccharides from whelk GAGs treated with heparinase I, II, and III and chondroitinase ABC were retained by the filter due to their larger size. Disaccharide analysis was performed using Glycan Reduction Isotope Labeling (GRIL LCQ-MS). The amounts of filter-retained fragments, as assessed by monosaccharides analysis, suggested that a proportion of the whelk GAG chains remained resistant to the enzymes used in the disaccharide analysis. Thus, the proportions of individual disaccharide produced in this analysis may not truly represent the overall proportions of disaccharide types within the intact whelk GAGs chain. However, they do serve as important descriptors for the classification and make-up of the anti-cancer GAGs chains. Furthermore, these data represent clear evidence of the compositional differences between whelk GAGs and commercial mammalian GAGs.

High Throughput Identification of Novel Conotoxins from the Vermivorous Oak Cone Snail (Conus quercinus) by Transcriptome Sequencing.

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.

Venom Insulins of Cone Snails Diversify Rapidly and Track Prey Taxa.

A specialized insulin was recently found in the venom of a fish-hunting cone snail, Conus geographus Here we show that many worm-hunting and snail-hunting cones also express venom insulins, and that this novel gene family has diversified explosively. Cone snails express a highly conserved insulin in their nerve ring; presumably this conventional signaling insulin is finely tuned to the Conus insulin receptor, which also evolves very slowly. By contrast, the venom insulins diverge rapidly, apparently in response to biotic interactions with prey and also possibly the cones' own predators and competitors. Thus, the inwardly directed signaling insulins appear to experience predominantly purifying sele\ction to target an internal receptor that seldom changes, while the outwardly directed venom insulins frequently experience directional selection to target heterospecific insulin receptors in a changing mix of prey, predators and competitors. Prey insulin receptors may often be constrained in ways that prevent their evolutionary escape from targeted venom insulins, if amino-acid substitutions that result in escape also degrade the receptor's signaling functions.

Molecular Engineering of Conus Peptides as Therapeutic Leads.

The venom from carnivorous marine snails of the Conus genus is a cocktail of peptides, proteins and small molecules that is used by the snail to capture prey. The peptides within this venom have been the focus of many drug design efforts as they exhibit potent and selective targeting of therapeutically important receptors, transporters and channels, particularly in relation to the treatment of chronic pain. The most well studied class of Conus peptides are the conotoxins, which are disulfide-rich and typically have well-defined three dimensional structures that are important for both biological activity and stability. In this chapter we discuss the molecular engineering approaches that have been used to modify these conotoxins to improve their pharmacological properties, including potency, selectivity, stability, and minimisation of the bioactive pharmacophore. These engineering strategies include sidechain modifications, disulfide substitution and deletion, backbone cyclisation, and truncations. Several of these re-engineered conotoxins have progressed to pre-clinical or clinical studies, which demonstrates the promise of using these molecular engineering techniques for the development of therapeutic leads.

Identification of a Novel O-Conotoxin Reveals an Unusual and Potent Inhibitor of the Human α9α10 Nicotinic Acetylcholine Receptor.

Conotoxins are a pool of disulfide-rich peptide neurotoxins produced by cone snails for predation and defense. They are a rich reservoir of novel ligands for ion channels, neurotransmitter receptors and transporters in the nervous system. In this study, we identified a novel conotoxin component, O-conotoxin GeXXVIIA, from the venom of Conus generalis. The native form of this component is a disulfide-linked homodimer of a 5-Cys-containing peptide. Surprisingly, our electrophysiological studies showed that, in comparison to the folded monomers, the linear peptide of this toxin had the highest inhibitory activity at the human α9α10 nicotinic acetylcholine receptor (nAChR), with an IC50 of 16.2 ± 1.4 nM. The activities of the N-terminal and C-terminal halves of the linear toxin are markedly reduced compared with the full-length toxin, suggesting that the intact sequence is required to potently inhibit the hα9α10 nAChR. α9α10 nAChRs are expressed not only in the nervous system, but also in a variety of non-neuronal cells, such as cochlear hair cells, keratinocytes, epithelial and immune cells. A potent inhibitor of human α9α10 nAChRs, such as GeXXVIIA, would facilitate unraveling the functions of this nAChR subtype. Furthermore, this unusual nAChR inhibitor may lead to the development of novel α9α10 nAChR-targeting drugs.