Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology
Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology
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DOI：
10.1371/journal.ppat.1005559
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发表时间：
2016-04-01
期刊：
影响因子：
6.7
通讯作者：
Campetella, Oscar
中科院分区：
文献类型：
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作者：
Lantos, Andres B.;Carlevaro, Giannina;Campetella, Oscar
克氏锥虫是查加斯病或美洲锥虫病的鞭毛状原生动物病原体，不能从头合成唾液酸。粘蛋白和反式唾液酸酶(TS)分别是从寄主清除唾液酸的糖生物系统的底物和酶，在毛滴虫生活史中起着至关重要的作用。唾液酸残基的获得使寄生虫能够避免血清因子的裂解，并与宿主细胞相互作用。研究唾液酸化动力学和锥虫糖偶联物周转的一个主要缺点是很难识别和跟踪最近获得的唾液酸基残基。为了解决这个问题，我们采用了一种非天然糖的方法作为生物正交化学记者，在这种方法中，使用叠氮唾液酰基残基可以识别获得的糖。利用先进的显微技术和生物化学方法，从糖生物学的角度研究了类鞭毛虫膜。通过生化程序和蛋白质标志物鉴定主要唾液酸受体为粘蛋白。除了测定它们的脱落和周转速度外，我们还报道了几种膜蛋白，包括TS及其底物，以及糖基磷脂酰肌醇锚定蛋白，分别分布在寄生虫表面，并包含在不同的高度稳定的膜微区中。值得注意的是，α(1，3)半乳糖残基的标记只与唾液酸化的粘蛋白部分共定位，表明确实存在两种糖基化的粘蛋白，它们是在寄生虫表面分离的。此外，唾液酸化的粘蛋白包含在脂筏结构域中，而TS分子不包括在内。表面锚定的TS的位置太远，不能分析粘蛋白，这是由脱落的TS所起的作用。磷脂酰肌醇-磷脂酶-C活性实际上在Try-pomastis中不存在。因此，TS的脱落是通过微泡而不是完全溶解的形式发生的。
Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for a(1,3) Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in try-pomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.