marine drugs Article Diversity of Conopeptides and Their Precursor Genes of Conus Litteratus Xinjia Li, Wanyi Chen, Dongting Zhangsun * and Sulan Luo * Key Laboratory of Tropical Biological Resources of Ministry of Education, Key Laboratory for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China; [email protected] (X.L.); [email protected] (W.C.) * Correspondence: [email protected] (D.Z.); [email protected] (S.L.) Received: 14 August 2020; Accepted: 10 September 2020; Published: 14 September 2020 Abstract: 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. Keywords: conotoxin; diversity; superfamily; cysteine framework; gene structure 1. Introduction The genus Conus is a large family of gastropods belonging to mollusks. There are about 800 species of Conus worldwide, which are distributed in tropical seas [1,2]. According to their diet habits, cone snails can be divided into three groups, vermivorous (V), molluscivorous (M), and piscivorous (P) species [3–5]. Although they move slowly, cone snails can prey on creatures with quick movement by skillfully injecting a small amount of a complex cocktail containing potent venom peptides. These venom peptides are commonly named conotoxins or conopeptides, and are a mixture of different bioactive compounds for defense and preying. These peptides, with different pharmacological activities, are composed of a small number of amino-acid residues that are especially rich in cysteines, and have become one of the main sources of peptide medicine [6–8]. With structural stability, target specificity and high affinity, conotoxins are able to skillfully target different ion channels and receptors, displaying massive potential in the study of peptide drugs and probes [9–12]. In 1978, one conotoxin, called myotoxin, from Conus geographus venom was purified and characterized for the first time. Since then, researchers have been engaged in the systematic study Mar. Drugs 2020, 18, 464; doi:10.3390/md18090464 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 464 2 of 17 Mar. Drugs 2020, 18, x 2 of 18 of conotoxins [7,13]. As is well known, !-MVIIA (ziconotide) has already approved by the FDA forsystematic clinical study application of conotoxins in the treatment [7,13]. As ofis well severe known, pain ω‐ [14MVIIA–16]. Generally, (ziconotide) conopeptide has already precursors approved possessby the FDA a conserved for clinical topological application organization, in the treatment which of issevere composed pain [14–16]. of three Generally, regions, including conopeptide an N-terminusprecursors possess signal regiona conserved (including topological about organization, 20 hydrophobic which amino is composed acids and of highly three regions, conserved including in the samean N‐terminus gene superfamily), signal region an intervening(including about propeptide 20 hydrophobic region, and amino a mature acids toxinand highly region conserved (hypervariable in the region)same gene [17 ,superfamily),18]. an intervening propeptide region, and a mature toxin region (hypervariable region)So [17,18]. far, there are three main classification methods for conotoxins. According to the similarity of the signalSo far, peptide there are region, three various main classification conotoxins are methods mainly for divided conotoxins. into 29 According gene superfamilies, to the similarity i.e., A, B1, of B2,the B3,signal and peptide C, etc. Thereregion, are various also 15 conotoxins temporary are gene mainly superfamilies divided into identified 29 gene in superfamilies, early divergent i.e., clade A, species.B1, B2, B3, For and conotoxins C, etc. There rich inare cysteines, also 15 temporary according gene to the superfamilies latest update identified of ConoServer in early on divergent Monday, 31clade August species. 2020, Forthere conotoxins are 31 kinds rich of in confirmed cysteines, cysteineaccording frameworks, to the latest designated update of as ConoServer I, II, III, IV, etc.on OnMonday, the basis 31 ofAugust pharmacological 2020, there activityare 31 kinds and target, of confirmed 12 pharmacological cysteine frameworks, families of designated conotoxins as (α -,I, γII,-, δIII,-, "IV,-, ι-, etc.κ-, µOn-, ρ -,theσ-, basisτ-, χ- of and pharmacological!-families) have activity been defined and target, [19–21 12]. pharmacological families of conotoxinsPrevious (α‐ studies, γ‐, δ‐, ε‐ have, ι‐, shown κ‐, μ‐, ρ‐ that, σ‐ each, τ‐, χ‐ Conusand ω‐speciesfamilies) contains have thousands been defined of conopeptides, [19–21]. which meansPrevious that there studies may have be more shown than that 1,000,000 each Conus natural species conotoxins contains thousands in cone snails of conopeptides, (about 800 species) which allmeans over that the there world may [22 be–25 more]. According than 1,000,000 to the natural latest conotoxins ConoServer in cone statistics, snails less(about than 800 only species) 1% all of conopeptideover the world sequences [22–25]. According have been to reported. the latest Fewer ConoServer than 300 statistics, conopeptides less than have only been 1% of characterized conopeptide pharmacologicallysequences have been [21]; therefore,reported. a largeFewer portion than of300 conotoxins conopeptides still remain have tobeen be discovered characterized and characterized.pharmacologically Nowadays, [21]; therefore, conotoxins a large are widelyportion used of conotoxins in the research still remain and development to be discovered of marine and peptidecharacterized. drugs, Nowadays, providing conotoxins a new ligand are toolwidely for used the study in the of research receptors, and ion development channels, etc.of marine [9–12]. Traditionalpeptide drugs, methods, providing including a new isolation ligand tool and for Sanger the study sequencing, of receptors, are generally ion channels, considered etc. [9–12]. to be time-consuming,Traditional methods, of lower including efficiency, isolation and and often Sanger limited sequencing, by sample are availability. generally considered to be time‐ consuming,With the of development lower efficiency, of high-throughput and often limited sequencing by sample platforms, availability. the combination of transcriptomic or proteomicWith the sequencing development with bioinformaticalof high‐throughput screening sequencing is expected platforms, to accelerate the ligandcombination and target of discoverytranscriptomic [26–28 or]. Inproteomic the present sequencing work, the with Hiseq bioinformatical Xten sequencing screening approach is wasexpected applied to to accelerate uncover theligand venom and ducttarget transcriptome discovery [26–28]. of a worm-hunting In the present species work, ofthe cone Hiseq snail, XtenC. sequencing litteratus, of approach different sizes was (Smallapplied 6–7cm, to uncover Middle the 8–10cm, venom Big duct 10–12cm; transcriptome Figure1). Weof furthera worm characterized‐hunting species its conotoxin of cone diversitysnail, C. oflitteratus cysteine, of framework, different sizes pharmacological (Small 6–7cm, activity Middle and gene8–10cm, superfamily. Big 10–12cm; The simple Figure sequence 1). We repeatsfurther (SSRs),characterized codon usageits conotoxin bias, and diversity gene structure of cysteine of the conotoxinsframework, were pharmacological also analyzed. activity This research and gene will supportsuperfamily. novel The bioactive simple peptide sequence discovery repeats and (SSRs), genomic codon feature usage studies bias, and for thegene exploitation structure ofof thisthe speciesconotoxins in the were future. also analyzed. This research will support novel bioactive peptide discovery and genomic feature studies for the exploitation of this species in the future. Figure 1. TheThe shell shell and and venom venom duct duct of of C.C. litteratus litteratus. (.(A)A Small) Small conus conus (SC), (SC), (B) (middleB) middle