De novo Transcriptome Assembly of the Venom Gland of Conus inscriptus Provides Insights into its Conotoxin Repertoire

Abstract

Cone snails (Conus spp.) produce complex venoms rich in conotoxins, a diverse group of cysteine-rich peptides with high specificity toward ion channels, receptors, and transporters, making them valuable candidates for drug discovery. Despite the pharmacological potential of cone snail venoms, the venom composition of Conus inscriptus remains largely unexplored. In this study, we present the first comprehensive venom gland transcriptome of C. inscriptus collected from the southwest coast of India. High-throughput Illumina sequencing generated 179.6 million paired-end reads, which were assembled into 259,828 transcripts and 75,366 predicted coding sequences (CDS). Functional annotation revealed enrichment of genes involved in cellular processes, metabolism, protein processing, and signal transduction, reflecting the active biosynthetic nature of the venom gland. A total of 6,066 putative conotoxin genes were identified, of which 4,921 were classified into 23 recognised conotoxin superfamilies. The A, O1, and M superfamilies were the most abundant. Additionally, 1,145 transcripts were assigned to conflict groups due to overlapping superfamily characteristics. Analysis of conflict-associated transcripts revealed remarkable cystine framework diversity, including several previously unreported cysteine-rich architectures containing up to 20 cysteine residues. Structural characterisation using AlphaFold and FoldSeek identified both conserved proteins and a large proportion of highly novel proteins lacking recognisable structural homologs. Many of these proteins exhibited high intrinsic disorder, suggesting the presence of previously undescribed peptide scaffolds and lineage-specific venom components. Overall, the transcriptome of C. inscriptus reveals an extensive and previously undocumented repertoire of conotoxins and structurally unique proteins. These findings provide new insights into cone snail venom evolution and establish C. inscriptus as a promising source of novel bioactive peptides with potential applications in marine biotechnology, neuropharmacology, and peptide-based drug development.