Conotoxin truncation as a post-translational modification to increase the pharmacological diversity within the milked venom of Conus magus

Milked venoms of Conus demonstrate direct lineage to US Food and Drug Administration approved and present in-trial drug leads. Yet the complexity of the milked venom has not been adequately investigated or characterized, in a sustainable manner. In this study we determine the extent of molecular mass differentiation in milked venom from captive Conus magus and confirm the expression of known conotoxin constituents. We demonstrate the presence of post-translational N-terminal peptide truncation, which differentiates the milked venom constituent α-conotoxin MI from the novel α-conotoxin MIC. This truncation has a direct effect on peptide bioactivity - K_i of 89.1±9.1 and 248.7±10.9nM (α-conotoxin MI and MIC respectively) toward the muscle-type nAChR (Torpedo). These milked venom conotoxins demonstrated acute lethality in fish, with a LD₅₀ of 12.24 and 23.29μgkg^-1 for α-conotoxin MI and MIC respectively. By synthesizing and investigating the synthetic intermediate variant des[Gly]¹α-conotoxin MI, it was demonstrated that retention of the N-terminal arginine residue increased affinity at the muscle-type nAChR site (binding K_i of 73.3±5.8nM and lethal toxicity level LD₅₀ of 8.19μgkg^-1). This post-translational modification event within the milked venom of C. magus represents a unique mechanism by which cone snails are able to increase the chemical and pharmacological diversity of their venoms.