Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.蛇毒是由分泌型蛋白质与多肽构成的生物武器系统，其功能为麻痹或击杀猎物。尽管翻译后修饰（post-translational modifications）因在诸多生物学现象中发挥关键作用而被广泛研究，但目前学界对蛋白质糖基化（glycosylation）如何影响毒蛋白质组的变异与稳定性仍缺乏深入认知。为解决这一科学问题，本研究采用鸟枪法蛋白质组学（shotgun proteomics）策略，对7种矛头蝮属（Bothrops）蛇的毒蛋白质组开展了系统表征。此外，我们对比了天然蛇毒与去糖基化蛇毒的电泳图谱；为解析其糖蛋白亚蛋白质组，我们通过与3种具有不同糖特异性的凝集素（lectins）亲和结合的方式，鉴定了其中的糖蛋白及其潜在糖基化位点。鉴于蛋白酶是丰度较高的糖基化毒素，我们考察了N-去糖基化（N-deglycosylation）对其催化活性的影响，结果显示7种蛇毒中的蛋白酶均会因N-聚糖的移除而受到相似的影响。此外，我们对源自巴西矛头蝮（B. jararaca）毒腺数据集的转录本的潜在糖基化位点进行了预测，并检测到与毒素家族相关的特异性糖基化模式。基于本研究的全局分析，我们发现矛头蝮属蛇毒的蛋白质组与糖蛋白质组包含一组核心组分，这些组分显著决定了其组成特征，且在进化过程中保持保守，与其他用于确定其系统发育分类的分子标记呈平行演化态势。