Variable peptide processing of a Conus (Asprella) neocostatus α-conotoxin generates bioactive toxiforms that are potent against distinct nicotinic acetylcholine receptor subtypes

Abstract

Conus venoms are composed of peptides that are commonly post-translationally modified, increasing their chemical diversity beyond what is encoded in the genome and enhancing their potency and selectivity. This study describes how PTMs alter an α-conotoxin’s selectivity for specific nAChR subtypes. Venom from the cone snail Conus (Asprella) neocostatus was fractionated using high-performance liquid chromatography and tested using a behavioral intracranial mouse bioassay and a cholinergic calcium imaging assay using SH-SY5Y neuroblastoma cells. Four peptides were isolated from three HPLC fractions and found to have similar amino acid sequences using tandem mass spectrometry; they all contain C-terminal amidation. The four peptides appear to be encoded by a single gene as indicated by transcriptomic analysis. One of these, NcIA, contains no additional PTM. NcIB lacked the two glycine residues found in the N-terminus of NcIA and contained two hydroxylated prolines. Analogs of both peptides containing a ɣ-carboxylated glutamic residue (NcIA[E15γ] and NcIB[E13γ]) were also isolated. Functional assays revealed distinct receptor selectivity: NcIA inhibited nicotine-evoked responses by over 70 %, while NcIA[E15γ] did not. Conversely, NcIB[E13γ] was inhibitory (∼60 %), but NcIB was not. Against choline-evoked responses, NcIA was weakly inhibitory (∼40 %), whereas the other three were nearly fully inhibitory. The IC₅₀ values for NcIB and NcIB[E13γ] were 91.0 nM and 64.7 nM, respectively. These findings indicate that PTMs and N-terminal modifications influence peptide potency and receptor specificity, suggesting that cone snails use variable peptide processing not only to generate chemical diversity in their venom but also to fine-tune the pharmacology of its components.