TRANS-SIALIDASE OF TRYPANOSOMA CRUZI

克氏毛滴虫不会合成唾液酸，但具有感染性的类鞭毛虫 它们的表面膜上含有一种不寻常的反式唾液酸酶(TS)。在 在离开宿主细胞进入血流的几秒钟后， 锥虫表面糖蛋白是唾液酸化的。这种反应导致了 SSP-3表位的组装，它在 其他宿主细胞的黏附和随后的入侵。TS不会 使用CMP-NeuAc，通常用于唾液酸转移的供体 反应。这样的TS还没有在哺乳动物细胞中被描述，使 这种酶是化疗的潜在靶点.独立于 TS在克氏毛滴虫生物学和查加斯病病理中的可能作用 对于疾病，碳水化合物生物化学家特别感兴趣的是TS。在……里面 这一建议我们描述了实验方法：1)澄清 TS的结构和功能，以及它与以前 对克氏锥虫唾液酸酶进行了描述。我们将：a)克隆和表达DNA编码 为TS基因(S)，并测定产物(S)对TS和 唾液酸酶活性；b)滴定TS和唾液酸度 克氏锥虫菌株，以确定这两种酶活性是否 密切相关的，相互独立的；c)将两者分开 从类鞭毛虫粗提物中提取活性，并验证它们是否 共提纯；d)对分离出的酶(S)的一些性质进行研究。2)至 确定含唾液酸分子在靶细胞中的作用 侵袭，以及通过补体保护类鞭毛虫免受裂解。 3)验证唾液酸化的分子是否可以作为以下成员的配体 LECCAM家族的宿主受体，并在寄生虫中发挥作用 在哺乳动物宿主组织中的迁徙。4)描述 被TS唾液酸化的寡糖的结构，以及 具体地说，L)那些含SSP-3表面的低聚糖 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.