Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom

Abstract

Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50 -200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N-and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ϳ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail. Molecular & Cellular

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

“…Despite a weak correlation between gene superfamilies and pharmacological properties, some functional redundancy among members of a same superfamily exists (56). To date, 16 empirical gene superfamilies (designated as A, D, I1, I2, I3, J, L, M, O1, O2, O3, P, S, T, V, Y) have been annotated (57), plus 31 novel superfamilies have been discovered during the past two years (38,39,46,(57)(58)(59)(60).…”

δ-Conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails

“…R. Soc. B 282: 20150817 in ancestral cone snails [4]. The vermivorous species studied here belong to the Tesselliconus (C. suturatus), Virroconus (C. ebraeus and C. miliaris), Virgiconus (C. frigidus), Lividoconus (C. sanguinolentus) and Strategoconus (C. varius) phylogenetic clades.…”

“…Recent estimates suggest that each species of cone snail produces hundreds to thousands of conotoxins, the majority of which remain pharmacologically uncharacterized [4,17]. Not surprisingly, many conotoxins affect action potential initiation and propagation, including the m-and mO-conotoxins that block the pore or alter Na V channel gating, [1,[18][19][20], the d-conotoxins that delay Na V inactivation by binding to site 6, and the i-conotoxins that enhance Na V channel opening [21][22][23].…”

“…Total RNA extraction, mRNA purification and 454 pyrosequencing were carried out as described previously [4]. The raw reads were uploaded in our in-house searchable database and d-conotoxinlike sequences were retrieved using CONOSORTER [13], aligned with MULTALIN program [14] and edited in JALVIEW [15].…”

“…Through a cladistics approach and cDNA cloning, it was shown that all fishhunting Conus species analysed produced at least one d-conotoxin-like sequence, supporting an essential biological role for these secreted peptides [3]. In addition, d-conotoxins belong to the widely distributed and ancestral plesiotypic gene superfamily O1, suggesting their early evolution in Conidae [4]. While ancestral cone snails preyed upon marine worms, the evolutionary path allowing a slowmoving, worm-eating (vermivorous) snail to switch to fish-hunting (piscivorous) behaviour has not been unravelled [5].…”