TRANS-SIALIDASE OF TRYPANOSOMA CRUZI

克鲁兹锥虫的转唾液酸酶

• 批准号: 3148059
• 负责人: Victor Nussenzweig
• 金额: $ 25.07万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3148059
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; Trypanosoma cruzi; affinity chromatography; carbohydrate structure; cell migration; enzyme activity; enzyme inhibitors; enzyme linked immunosorbent assay; exo alpha sialidase; flow cytometry; gene expression; high performance liquid chromatography; host organism interaction; intracellular parasitism; ion exchange chromatography; laboratory mouse; laboratory rabbit; membrane proteins; molecular cloning; nucleic acid structure; oligosaccharides; protein structure function; sialate

T. cruzi does not synthesize sialic acid, but the infective trypomastigotes contain an unusual trans-sialidase (TS) on their surface membranes. Within seconds after leaving host cells to enter the blood stream, the trypomastigote surface glycoproteins are sialylated. This reaction leads to the assembly of the Ssp-3 epitope, which plays a essential role in the adhesion and subsequent invasion of other host cells. The TS does not employ CMP-NeuAc, the normally used donor for sialic acid transfer reactions. Such a TS has not been described in mammalian cells, making the enzyme a potential target for chemotherapy. Independently of the possible role of TS in the biology of T. cruzi and pathology of Chagas' disease, the Ts is of particular interest to carbohydrate biochemists. In this proposal we describe experimental approaches: 1) to clarify the structure and function of the TS, and its relationship to a previously described T. cruzi sialidase. We will: a) clone and express the DNA coding for the TS gene(s) and assay the activity of the product(s) for TS and sialidase activities; b) titrate TS and sialidade in extracts of various strains of T. cruzi, to determine whether the two enzymatic activities are closely correlated, or vary independently of each other; c) isolate the two activities from crude trypomastigote extracts, and verify whether they co-purify; d) study some properties of the isolated enzyme(s). 2) to determine the role of the sialic acid-containing molecules in target cell invasion, and in the protecting trypomastigotes from lysis by complement. 3) to verify whether the sialylated molecules can be ligands for members of the LECCAM family of host receptors, and play a role in the parasite migration in the mammalian host's tissues. 4) to characterize the structure of the oligosaccharides which are sialylated by the TS, and specifically, of L)those oligosaccharides of the Ssp-3-bearing surface molecules.

T. cruzi不合成唾液酸，而是感染性锥虫 在其表面膜上包含一个不寻常的跨硅化酶（TS）。 之内 离开宿主细胞进入血流后的几秒钟， 锥虫表面糖蛋白被溶解。 该反应导致 SSP-3表位的组装，在 粘附和随后侵袭其他宿主细胞。 TS没有 采用CMP-NEUAC，这是通常用于唾液酸转移的供体 反应。 这种TS在哺乳动物细胞中尚未描述，使得 酶是化学疗法的潜在靶标。 独立于 TS在Cruzi和Chagas的病理学生物学中的可能作用 疾病，碳水化合物生物化学家特别感兴趣。 在 我们描述了实验方法：1）澄清 TS的结构和功能及其与以前的关系 描述了克鲁齐唾液酸酶。 我们将：a）克隆并表达DNA编码 对于TS基因并测定TS和TS产品的活性 唾液酸酶活性； b）滴定ts和sialidade在各种提取物中 克鲁齐的菌株，以确定两种酶活性是否是 密切相关或彼此独立的相关性； c）隔离两者 来自粗锥虫提取物的活动，并验证它们是否 共纯化； d）研究分离的酶的一些特性。 2）到 确定含唾液酸分子在靶细胞中的作用 入侵，并在保护锥虫中免受补体裂解。 3）验证溶解分子是否可以是成员的配体 宿主受体家族，并在寄生虫中发挥作用 在哺乳动物宿主的组织中迁移。 4）表征 由TS溶解的寡糖的结构， 具体而言，l）SSP-3表面的那些寡糖 分子。