Peng et al. GigaScience (2016) 5:17 DOI 10.1186/s13742-016-0122-9 RESEARCH Open Access High-throughput identification of novel conotoxins from the Chinese tubular cone snail (Conus betulinus) by multi- transcriptome sequencing Chao Peng1†, Ge Yao2†, Bing-Miao Gao3†, Chong-Xu Fan2, Chao Bian1,4, Jintu Wang1, Ying Cao2, Bo Wen1, Yabing Zhu1, Zhiqiang Ruan1,4, Xiaofei Zhao5, Xinxin You1,4, Jie Bai1,4, Jia Li1,4, Zhilong Lin1, Shijie Zou1, Xinhui Zhang1,4, Ying Qiu1,4, Jieming Chen1,4, Steven L. Coon6, Jiaan Yang5, Ji-Sheng Chen2* and Qiong Shi1,4,7* Abstract Background: The venom of predatory marine cone snails mainlycontainsadiversearrayofuniquebioactive 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. Keywords: Cone snail, Conopeptide, Conotoxin, Conus betulinus, Transcriptome, Venom duct, Venom bulb * Correspondence: [email protected]; [email protected] †Equal contributors 2Research Institute of Pharmaceutical Chemistry, Beijing 102205, China 1BGI-Shenzhen, Shenzhen 518083, China Full list of author information is available at the end of the article © 2016 Peng et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Peng et al. GigaScience (2016) 5:17 Page 2 of 14 Background human clinical trials for intractable epilepsy [33]. In The genus Conus is classified in the Coninae subfamily addition, more and more conopeptides are undergoing de- within the Conidae family that belongs to the Conoidea velopment for the treatment of pathologies including pain, superfamily (a branch of the Neogastropoda clade) [1, 2]. Parkinson’s disease, cardiac infarction, hypertension and With an estimation of 700 species [3, 4], all cone snails are various neurological diseases [12, 28, 34–37]. classified in Conus, which is the largest genus among mar- With a few exceptions, each conopeptide precursor ine invertebrates. Venomous cone snails are carnivorous generally consists of three distinct regions: a highly con- and predatory marine gastropod mollusks that use a served N-terminal signal peptide region, a less conserved complex cocktail of venom components for many rea- intervening propeptide region, and a hypervariable C- sons, including capture and digestion of prey, defense terminal mature toxin region [38, 39]. Based on the se- against foes [5], avoidance of competitors and other quence similarities of signal peptides in the precursors biological purposes [6]. According to variation in diet, [40], conopeptides are currently classified into 26 gene cone snails are divided into three groups: piscivorous superfamilies (A, B1, B2, B3, C, D, E, F, G, H, I1, I2, I3, J, species that hunt small fish, molluscivorous species K, L, M, N, O1, O2, O3, P, S, T, V and Y) [41–47] and that feed on other marine snails including other cone 13 temporary gene superfamilies for those identified in snails, and vermivorous cone snails that prey on poly- the early divergent clade species [40, 42, 48, 49]. Al- chaetes and hemichordates [7]. though amino acid conservation in the mature pep- As slow-moving predatory marine gastropods, cone tide sequences of conopeptides within a same gene snails have developed successful strategies to subdue superfamily is much lower than in the signal and pro- quicker or stronger prey during more than 55 million peptide regions, certain characteristic cysteine frame- years of evolution [8], including a hollow, harpoon-like works within the mature conotoxins are often (but radular tooth and potent toxins targeted to the nervous not always) specific to a conotoxin superfamily. So system and musculature of the prey [9]. The modified far, 26 distinct cysteine frameworks have been de- radular tooth (along with a venom gland) can be scribed, and they may be associated with particular launched out from the snail’s mouth deep into the prey’s pharmacological families [39, 40, 50]. flesh in a harpoon-like action. The injected venom rap- Early views of the conotoxin-producing structures idly enters the victim’s circulatory system, interacts with concluded that the muscular bulbous organ (the a range of molecular targets in the nervous system, and venom bulb, located at the end of the venom duct) causes paralysis in a short time (sometimes within a few was likely to participate in venom biosynthesis [51]. seconds) [10, 11]. With the development of molecular biology, re- In contrast to the well-known large protein toxins in searchers found that the epithelial cells lining the snake venom, Conus venom mainly contains a diverse cone snail’s venom duct were rich in mature mRNAs array of unique bioactive peptides commonly referred to encoding precursor conopeptides, and the venom bulb as conopeptides or conotoxins. These small polypeptide may function to propel the venom toward the pharynx conotoxins typically range from seven to 46 amino acids while preying or defending [51–53]. in length, with many of them consisting of 12–30 amino With early estimates of an average of 100 conotox- acids [12]. They have high specificity and affinity to ins per species, recent reports proved the presence of voltage-gated ion channels, ligand-gated ion channels, 1000 to 2000 different conopeptides in a single sam- G-protein-coupled receptors and neurotransmitter ple of venom using high-sensitivity mass spectrometry transporters in the central and peripheral nervous sys- [44, 54, 55]. A single mutation in a mature sequence tems [3, 6, 12–18]. Because of their bioactive specificity, can add one more conopeptide at the DNA level and, Conus venoms have become a potent resource for subsequently, owing to the 14 different post-translational pharmacological neuroscience research [19–22] and a modifications known in addition to other undefined alter- promising source for the discovery of new drugs to treat ations, an average of 20 different toxin variants for each a wide variety of human neurological diseases [6, 23–30]. conopeptide precursor can be characterized at the protein To date, several conotoxins have already demonstrated level [44]. This raises the possibility that the total of 500 potential therapeutic effects in preclinical or clinical trials. to 700 species of cone snails, providing upwards of 50,000 The most well-known is ω-MVIIA (commercially known conopeptide genes and 1,000,000 mature conotoxins as as ziconotide), derived from the venom of C. magus, potential pharmacological targets, constitutes the largest which has been approved by the US Food and Drug Ad- single library of natural drug candidates. ministration (FDA) to treat previously unmanageable Despite the large number of potential conopeptide chronic pain in cancer and AIDS patients [30–32]. An- genes and mature conotoxins, only approximately 1400 other conotoxin, conantokin G, a specific antagonist nucleotide sequences of conotoxin genes have been re- against the NR2B subunit of the NMDA receptor, is in ported from 100 Conus species by traditional approaches Peng et al. GigaScience (2016) 5:17 Page 3 of 14 over the past decades, with as few as 210 peptides being species [42], only 53 mature conotoxins from nine validated at the protein level [40, 41]. Traditional gene superfamilies (Fig. 1b) were derived from precur- methods, which may isolate and sequence these sors or via traditional approaches (see Additional file 1). potential bioactives, are generally time-consuming, of Next-generation whole-transcriptome sequencing of the low sensitivity, and often limited by sample availability. In C. betulinus venom duct has never been attempted.