| Conotoxins are small, disulfide-rich peptides (10-40aa) derived from the venom of snail conus, with divergent sequences and different specific biological activity. So far the conotoxins can be classified into several superfamilies, A-, T-, O-, M-, P-, I-and S-superfamily, pharmacologically targeting the sodium, potassium and calcium channels or receptors. Thus, conotoxins are great potential rools for neuroscience, and several are being developed as diagnostic and therapeutic agents. Here we isolated and characterized eight novel M-superfamily conotoxins from a molluscivorous species C. marmoreus, then the genes of mr3.4 and mr3.8 were cloned via RACE approach. Partial reduction of mr3.4 was carried out by TCEP to determine the disulfide connectivity. All the novel conotoxins can be divided into two groups according to the sequence alignments and phylogenetic tree analysis. Although these conotoxins share the same Cys pattern and similar amino acid spacing in their loops, they remarkably differ in the protein motif, disulfide linkage, conserved signal peptide sequences, 5'and 3'UTR and Cys codon usage, etc. Then it is presumed that the two group conotoxins rooted in the same ancestor and diverged by duplication or mutation in the evolution. This observation not only reclassifies the M-superfamily conotoxins, but also improves and complements the superfamily definition of conotoxins. That a superfamily conotoxin can hold different conserved signal sequences will facilitate the definition of a conotoxin superfamily. 34 novel conotoxins were cloned by RACE from C. marmoreus, C. quercinus, C. leopardus, C. miles and C. achatinus, belonging to A-, T-, O-superfamily and conantokin, respectively. Phylogenetic trees were constructed based on the sequence alignments of these novel conotoxins.
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