Top-down venomics of the East African green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals

We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K⁺ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution.Dendroaspis angusticeps (eastern green mamba) and D. polylepis (black mamba) are African snake species of medical concern. Their venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Studies on the venoms of D. polylepis and D. angusticeps have focused on the biochemical and pharmacological characterization of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species. Only very recently an overview of the composition of the venom of a Dendroaspis species, D. polylepis, has been reported through a bottom-up venomics strategy. Peptide-centric approaches provide incomplete sequence coverage, and in general do not allow to distinguish between different proteoforms or closely related toxin isoforms. To overcome this shortcoming we have now applied top-down venomics to unveil the complexity of the toxin arsenals of the black mamba and the eastern green mamba at locus resolution. Our data show that the green and the black mamba venom contain, respectively, ≥232 and ≥268 protein species, highlighting that D. angusticeps and D. polylepis venom comprise a much higher complexity than the 20 and the 27 toxin sequences available, respectively for these snake species, in the non-redundant NCBI database. On the other hand, 36 (D. angusticeps) and 3 (D. polylepis) minor venom proteins showed mass shifts of +42Da modifications, suggesting the presence of monoacetyl lysine residues. Noteworthy, although both venoms have highly similar global molecular compositions, the identity of the major proteins and their relative abundances vary between D. angusticeps and D. polylepis. Our data lay the foundation for rationalizing the notably different venom toxicity profiles of the green and the black mamba.