Venomics and biochemical analysis of the black-tailed horned pitviper, Mixcoatlus melanurus, and characterization of Melanurutoxin, a novel crotoxin homolog.

Abstract We report a structural and functional venomics characterization of the black-tailed horned pitviper, Mixcoatlus melanurus. The venom phenotype of this small and elusive pitviper endemic to Mexico comprise peptides and proteins of 16 toxin families whose relative abundance mirror those of neurotoxic (type II) venoms described for some species within genera distributed in Central Asia (Gloydius) and the Americas (Sistrurus, Crotalus, Ophryacus, and Bothriechis). A novel β-neurotoxic heterodimeric PLA2, termed Melanurutoxin was characterized. With a relative abundance of 14.8% of the total M. melanurus venom proteome and a median lethal dose of 0.31 μg/g mouse body weight, Melanurutoxin accounted for 37.8% of the lethality of the whole venom (0.82 μg/g). The low percentage (1.1%) of snake venom metalloproteinases (PIII-SVMPs) and the high content of Melanurutoxin and bradykinin-potentiating peptides (BPP, 16%) found in the type-II venom proteome of M. melanurus correlate with the severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest observed in ex vivo neuromuscular junction experiments and in vivo experimental murine envenoming. Mexican antivenoms manufactured by Birmex and Bioclon showed low neutralization potency per vial (95 LD50s, Birmex; 114 LD50s, Antivipmyn®), and failed to reverse completely the paralysis and the hypotensive effect induced by the black-tailed horned pitviper, Mixcoatlus melanurus. We suggest that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom may be attributed to the use of immunization mixtures that include venom of taxa, C. basiliscus (Birmex) and C. simus (Antivipmyn®), that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s. Biological significance This study represents the first proteomics and funcional investigations conducted on the venom of the black-tailed horned, Mixcoatlus melanurus, a pitviper species endemic to Mexico. The venom's features unveiled through combination of bottom-up venomics and ex vivo and in vivo functional assays provided complementary evidence pointing to severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest as the predominant mechanism of murine prey immobilization and death caused by M. melanurus. A novel β-neurotoxic heterodimeric PLA2, coined Melanurutoxin, was identified as a major contributor to the lethality of the whole venom. Our study also showed the inefficacy of two commercial Mexican antivenoms to reverse competely the paralytic and hypotensive effects induced by M. melanurus venom in the murine model. We hypothesize that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom should be ascribed to the use as immunogens of venoms that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s.