N‑Glycoproteomic Portraits of Bothrops Snake Venoms Reveal Evolutionarily Conserved and Divergent Phenotypes

Glycosylation is a major protein post-translational modification in snake venom proteins and contributes to the diversification of proteomes. In this study, we carried out an in-depth analysis of the glycosylation profile of seven Bothrops venoms, including neutral sugar quantification, glycoprotein profiling by lectin blot, and determination of N-glycosylation sites in proteins and their N-glycan compositions, by direct, intact glycopeptide analysis by mass spectrometry. Interestingly, all identified N-glycosylated peptides were from enzymatic venom components, mainly proteolytic enzymes that are key in envenomation. All venoms revealed a prominent occurrence of fucose and sialic acid in all N-glycosylated toxins identified. The results indicated that in Bothrops venoms, there is an important level of variation in protein primary structure that is not restricted to regions containing N-sequons. Overall, the signatures of N-glycosylated and nonglycosylated peptide backbones and of N-glycan site occupation by different N-glycans revealed conservation of venom phenotype framework and diversification of N-glycan usage. Hence, the molecular mechanisms of toxin structure and function evolution are at the same time dynamic in that they involve a fine-tuning for the presence of distinct glycans as an evolutionary novelty and are subjected to some conservation that results in the clustering of Bothrops venoms according to the species phylogenetic classification.