TRANS-SIALIDASE OF TRYPANOSOMA CRUZI

T. Cruzi不合成唾液酸，但感染性锥鞭毛体 它们的表面膜上含有一种不寻常的转唾液酸酶（TS）。 内 在离开宿主细胞进入血流后的几秒钟， 锥鞭毛体表面糖蛋白被唾液酸化。 这种反应导致 到Ssp-3表位的组装，其在免疫应答中起重要作用。 粘附和随后的侵入其他宿主细胞。 TS不 使用CMP-NeuAc，通常用于唾液酸转移的供体 反应. 这种TS尚未在哺乳动物细胞中描述，使得 这种酶是化疗的潜在靶点。 独立于 TS在T. cruzi和南美锥虫病的病理学 疾病时，Ts对碳水化合物生物化学家特别感兴趣。 在 本建议我们描述实验方法：1）澄清 TS的结构和功能，以及它与先前 描述了T. cruzi唾液酸酶 我们将：a）克隆并表达编码 并测定产物的TS活性， 唾液酸酶活性; B）滴定各种植物提取物中的TS和唾液酸酶 木霉菌株cruzi，以确定两种酶活性是否 紧密相关，或彼此独立地变化; c）将两者分离 活性的粗锥鞭毛体提取物，并验证它们是否 共纯化; d）研究分离的酶的一些性质。2)到 确定含唾液酸分子在靶细胞中的作用 入侵，并保护锥鞭毛体免受补体溶解。 3)为了验证唾液酸化的分子是否可以是 宿主受体的LECCAM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 被TS唾液酸化的寡糖的结构，和 具体地，L）具有Ssp-3的表面的那些寡糖 分子。

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.