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 ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.
Highlights
To cite this version: Sébastien Dutertre, Ai-Hua Jin, Quentin Kaas, Alun Jones, Paul Alewood, et al
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 Matrix-assisted laser desorption ionization (MALDI) and electrospray Ionization (ESI)-mass range (MS), respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument
Conopeptides belonging to gene superfamily N and H show typical mature conotoxins, while gene superfamily B, E, and F are represented by only one sequence and appear to be quite divergent
Summary
To cite this version: Sébastien Dutertre, Ai-Hua Jin, Quentin Kaas, Alun Jones, Paul Alewood, et al. Cone snails are slow-moving predatory marine gastropods that hunt a variety of preys including fish [1] using venom optimized through more than 33 million years of evolution [2] The success of this strategy relies on the deployment of potent toxins targeted to the nervous system and musculature of the prey using a specialized radula tooth [3]. Cone snail venoms are regarded as pharmacological treasures, and significant research efforts are being made to uncover the therapeutic potential of these molecules [8] One such molecule, the N-type channel selective blocker -conotoxin MVIIA, is an FDA-approved drug to treat unmanageable chronic pain [9], and an optimized version of the norepinephrine transporter inhibitor -conotoxin MrIA (Xen2174) is in Phase IIa trials for cancer and post-surgical pain [10]. Several other cone snail compounds are being investigated for the treatment of neuropathic pain, epilepsy, cardiac infarction, and neurological diseases [11]
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