Immunochemical, biochemical and evolutionary studies on Micrurus nigrocinctus nigrocinctus venom toxins

Abstract: Immunochemical, biochemical and evolutionary studies on Micrurus nigrocinctus nigrocinctus venom toxins Monoclonal antibodies (MAbs) were produced against venom proteins of M. nigrocinctus nigrocinctus, the medically most important elapid of Central America. Affinity purified polyclonal antibodies and some of the MAbs were used to develop immunoassays for the quantitative deterrnination of M. n. nigrocinctus venom antigens in biological fluids. It was shown that these assays readily detect venoms from the the most important coral snakes of North and Central America at the level of nanograms. An ELISA-based non-radioactive assay was used to detect the nicotinic acetylcholine receptor (AchR) binding activity of M. n. nigrocinctus venom alpha-neurotoxins. Four alpha-neurotoxins were isolated and found to be antigenically and structurally related to short- and long-chain alpha-neurotoxins from cobras. A therapeutic antivenom was found to contain antibodies which readily inhibited the binding of the M n. nigrocinctus alpha-neurotoxins to AchR and also antibodies which reverse the binding of toxin(s) already complexed with the receptor. One MAb was used for the immunoaffinity isolation of a group of closely related PLA2 isozymes, called nigroxins A, B, Cl, C2, and C3, which are antigenically similar and constitute a new subclass of PLA2s. The myotoxic activities of nigroxins A and B were characterized, their primary structures were established and a molecular model of nigroxin A was built. The structures of nigroxins A and B were compared with those of 19 other PLA2s, whose myotoxic activity has been previously studied. The combination of amino acid residues Arg-17, Ala-100, Asn-108 and a hydrophobic residue at position 109 was found to be present exclusively among class I myotoxic PLA2s. These residues cluster on the protein surface and have been conserved for a long time of divergent evolution (> 25 million years) in venom PLA2s from different elapid subfamilies. Based on structural and phylogenetic analysis, we reconstructed the evolutionary pathway in which elapid PLA2s gained the myotoxic activity in the scaffold of their pancreatic PLA2-like precursor. In addition, we reconstructed the evolution of potassium channel blockers in the scaffold of the pancreatic trypsin inhibitor. Thus, the isolation and study of the nigroxins allowed to clarify that during the evolution of elapid venom glands, existing structures were repeatedly recruited and adapted to serve toxic functions. Key words: Monoclonal antibody, Phospholipase A2, neurotoxin, myotoxin, protein evolution