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

Abstract

Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

Keywords: bioinformatic; conotoxin; cysteine-rich peptides; proteomic; transcriptomic.

Fig. 1.

Macroscopic anatomy of a cone snail (A), its venom apparatus (B), and a radula tooth (C).

Fig. 2.

Fractionation methods used to purify protein samples. Reverse-phase HPLC traces (214 nm) of VD [<30 kDa (A); >30 kDa (B)], as well as RS (C) and SG (D) protein extracts are shown. Raw and reduced protein extracts have also been separated by 1D-PAGE and revealed with Coomassie blue (E). Total VD proteins have been separated by 2D-PAGE and revealed with Coomassie blue (F). The fraction (HPLC) and spot (gels) numbers that refer to MS fragments (Dataset S1) are also mentioned.

Fig. S1.

New precursor forms of known mature conotoxins. Alignments of known (framed) and novel sequences are shown in the figure (low to high amino acid conservation are represented from light to darker colors; names with a “-2” suffix designate novel toxins containing an unknown signal peptide along with known cone snail sequence signatures in their proregion and mature regions).

Fig. 3.

Number of new precursor conopeptides per gene superfamily and compartment. These toxin precursors all contain a signal peptide and have been classified with ConoSorter at the highest score (matching Conus signal, pro, and mature signatures without conflicts).

Fig. S2.

Average number of parent and variant precursors (n = 3,303) as a function of their similarity rate in the venom gland. The line plot in red shows the variation of the number of parent conopeptide precursors when different similarity thresholds were applied. The bar chart in green represents the average number of variants per parent sequence (and the corresponding SEM represented by gray vertical bars) for different similarity thresholds.

Fig. 4.

Distribution of mature toxins per superfamily and known cysteine pattern (A). (Bottom) The number of mature toxins containing cysteine framework VI/VII (C-C-CC-C-C) and I (CC-C-C) classified per loop formula in (B) and (C), respectively.

Fig. S3.

New mature conopeptides containing the cysteine pattern VI/VII (C-C-CC-C-C). Sequences are grouped according to their internal loop formula and were aligned using the BLOSUM62 cost matrix. Conserved cysteine amino acids are framed in red.

Fig. S4.

New mature conopeptides containing the cysteine pattern I (CC-C-C). Sequences are grouped according to their internal loop formula and were aligned using the BLOSUM62 cost matrix. Conserved cysteine amino acids are framed in red.

Fig. S5.

Speculation on the origin of mature conotoxin scaffolds containing an odd number of cysteine residues. The mature regions of 208 new conotoxin sequences containing an odd number of cysteines were aligned with known mature conopeptides (n = 2,258 sequences obtained from the ConoServer website) using the CD-HIT algorithm. Clusters of new (name in regular font) and known (name in italic) sequences sharing >70% identity show the position of the insertion (bold red) or deletion (bold blue) of cysteines compared with the conserved residues (green frame).

Fig. S6.

New conopeptide gene superfamilies. Sequences were aligned using the BLOSUM62 cost matrix. The number of sequences present in the original cDNA library (second column), their maximal identity percentage (third column) compared with the closest known conopeptide (name and superfamily between parentheses), as well as their signal region (delimited by red lines), are mentioned.

Fig. 5.

Relative synonymous codon usage (RSCU) of cone snail cysteine frameworks (A) and profiles of point nucleotide substitutions or indels in the signal (red), proregion (green), and mature (blue) conotoxin regions (B).