Various Conotoxin Diversifications Revealed by a Venomic Study of Conus flavidus

Abstract

Conotoxins are peptide neurotoxins produced by predatory cone snails. They are mostly cysteine-rich short peptides with remarkable structural diversity. The conserved signal peptide sequences of their mRNA-encoded precursors have enabled the grouping of known conotoxins into a limited number of superfamilies. However, the conotoxins within each superfamily often present variable sequences, cysteine frameworks, and post-translational modifications. To understand better how conotoxins are diversified, we performed a venomic study with C. flavidus, an uninvestigated vermivorous Conus species, by combining transcriptomic and proteomic analyses. In order to obtain the full-length conotoxin sequences, protease digestion was not performed with the venom extraction prior to spectra acquisition via tandem mass spectrometry (MS/MS). Because conotoxins are produced from mRNA-encoded precursors by means of proteolytic cleavage, nonspecific digestion of precursors was applied during the database search. Special attention was also paid in interpreting the MS/MS spectra. All together, these analyses identified 69 nonredundant cDNA sequences and 31 conotoxin components with confident MS/MS spectra. A new Q-superfamily was also identified. More importantly, this study revealed that conotoxin-encoding transcripts are diversified by hypermutation, fragment insertion/deletion, and mutation-induced premature termination, and that a single mRNA species can produce multiple toxin products through alternative post-translational modifications and alternative cleavages of the translated precursor. These multiple diversification strategies at different levels may explain, at least in part, the diversity of conotoxins, and provide the basis for further investigation. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.028647, 105-118, 2014.Cone snails (Conus species) are predatory mollusks that inhabit tropical and subtropical shallow seawater. Their venom ducts produce a mixture of peptides, generally known as conotoxins, that have exquisite specificity for different ion channels, receptors, and transporters. These toxins are used to immobilize or paralyze prey, as well as for defense (1). Because of their specificity, conotoxin peptides are considered the ideal neurobiological tools for investigating specific receptors and channels. Furthermore, they are valuable lead compounds for clinical therapy. The conotoxin -MVIIA, isolated from Conus magus, is known commercially as Prialt (ziconotide) and is already in clinical use for the treatment of chronic pain (2, 3).Apart from their great potential for neurobiological and therapeutic applications, an emerging enigma concerning conotoxins is their striking diversity. Conotoxins are generally short disulfide-rich peptides of 10 to 40 amino acids. Every Conus species can produce more than 1000 different conotoxins (4, 5), and the number of Conus species with morphological differences is currently estimated as 800 (6). Hence, the total number of conotoxins is thought to be over 1 million. Becau...

Conotoxin Gene Superfamilies

“…A single A-superfamily precursor identified in the venom gland transcriptome of Conus victoriae (A_Vc22.1) encodes a predicted mature peptide that exhibits a remarkably different primary structure to known A-superfamily conotoxins, with eight cysteines arranged in a type XXII cysteine framework (C-C-C-C-C-C-C-C) [ 27 ]. Similarly, a single A-superfamily precursor identified from Conus flavidus encoded a predicted mature peptide with cysteine framework VI/VII (C-C-CC-C-C) [ 28 ]. A summary of pharmacological activities associated with selected A-superfamily conotoxins is presented in Table 1 .…”

Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS

“…In most cases, conopeptides have been isolated from venom obtained from the dissected venom ducts of several specimens (pooled dissected venom) [11][12][13][14]. The dissected venom contains cellular debris, unprocessed peptides, and other endogenous components in addition to conopeptides [15].…”

Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

“…Recent advances in nucleotide and protein sequencing have revealed that toxins from marine snails in the superfamily Conoidea, such as cone snails (Conus), comprise a remarkable diversity of cysteinerich polypeptides (1,2). Toxin expression and folding takes place in the endoplasmic reticulum (ER) of venom gland cells, where, at any given time, hundreds of distinct cysteine-rich peptides are properly folded and secreted in preparation for envenomation (3,4). No other tissue type has been reported to produce such a high density and diversity of cysteine-rich peptides.…”