A post-translational modification perspective of Bothrops venom toxins: site-specific proteomic characterization of N-glycans and Cys residues.

Abstract

Snake venoms are important biological fluids, used for predation and defense, which appeared during the development of the order Squamata and later evolved in a variable mode in the group of advanced snakes. The variability of their compositions can be observed in all taxonomical levels, however, the factors that drove and selected such features are still under discussion. The hypothesis of recruitment of expression in the venom of genes involved with physiological functions and the neofunctionalization of these genes in the venom is corroborated by comparative transcriptomic analyses between the venom gland and other snake organs. However, these analyses are based only in the comparison of the primary structures of the proteins and the product of their paralog genes, without taking into account the post-translational modifications present in these proteins. In this context, toxin glycosylation is abundant in viperid snake venoms and previous studies of our group on its role in speciation events, in the Bothrops genus, indicate its conservation during snake evolution, in parallel with other molecular markers that defined their phylogenetic classification. Nevertheless, accurate analyses about the distribution and occurrence of sites of N-glycosylation and O-glycosylation in snake venom glycoproteins are still missing. Thus, in this project we propose to identify by mass spectrometry the N- and O-glycosylation sites in glycoproteins of B. jararaca, B. alcatraz, B. insularis, B. fonsecai, B. cotiara, B. jararacussu and B. moojeni venoms, in order to evaluate their role in directing the toxins to their targets, and to compare these results to the phylogenetic classification of these species. We also propose: i) to evaluate the role of sialic acid present in glycoproteins of B. jararaca venom in their pathological effects; ii) to evaluate the role of glycans present in the substrates of bothropasin, a hemorrhagic metalloproteinase of B. jararaca venom, on its primary specificity.