Caracterização de gicosilações em toxinas do veneno de três espécies de aranhas de importância médica do gênero Loxosceles

Abstract

Loxoscelism, symptomatology envenomation by Loxosceles spiders, is a public health issue in Brazil, affecting around 8,000 victims annually. Despite advances in the characterization of the toxins of these venoms, little is known about the role of post-translational modifications (PTMs) and their relationship with the pathophysiology of envenomation. Among PTMs, glycosylation is one of the most frequently present in animal proteins, and its presence is related, among others, to the stability, solubility, cellular and molecular interaction of proteins. Given their importance and the scarce experimental evidence of the presence and role of these PTMs in arthropod toxins, our group proposes, in this work, to investigate the predominance and characteristics of this modification in Loxosceles venom toxins. In order to have an overview of the presence of glycoproteins in the venom, through two-dimensional electrophoresis stained with a glycospecific dye (ProQ Emerald), we saw that from 17 to 43% of the spots in the molecular range of essential proteins with biological activity in envenomation, such as phospholipases D, metalloproteases and hyaluronidases were marked as glycosylated with different profiles in the three studied species. Furthermore, when using an Enzyme-linked lectin assay in order to verify the reactivity of the venoms by them, reactivity with ConA, PNA, DBA, and SNA was observed, suggesting the presence of N- and O-linked glycosylations. This presence was confirmed by the analysis of glycosylated peptides, obtained by bottom-up glycoproteomics, against the sequence bank, customized by our group, using the Byonic software. Considering the three species, 100 sequences without redundancy of N- and O-linked glycosylated peptides were found in proteins, such as serine and metalloproteases, and phospholipases D and A2. These glycosylation sites can contain from 1 to 22 non-redundant glycoforms. The N-linked glycosylation structures found were mostly paucimannose, however, the presence of oligomannose and complex/hybrid structures was also observed. These findings contribute to the current knowledge about these molecules, indicating the importance of glycosylations in this venom and raising questions about their role in the outcome of loxoscelism.