<i>N</i> -Glycoproteomic Portraits of <i>Bothrops</i> 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 <i>Bothrops</i> venoms, including neutral sugar quantification, glycoprotein profiling by lectin blot, and determination of <i>N</i>-glycosylation sites in proteins and their <i>N</i>-glycan compositions, by direct, intact glycopeptide analysis by mass spectrometry. Interestingly, all identified <i>N</i>-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 <i>N</i>-glycosylated toxins identified. The results indicated that in <i>Bothrops</i> venoms, there is an important level of variation in protein primary structure that is not restricted to regions containing <i>N</i>-sequons. Overall, the signatures of <i>N</i>-glycosylated and nonglycosylated peptide backbones and of <i>N</i>-glycan site occupation by different <i>N-</i>glycans revealed conservation of venom phenotype framework and diversification of <i>N</i>-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 <i>Bothrops</i> venoms according to the species phylogenetic classification.