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Sislic acid recognition proteins of Trypanosoma species

锥虫物种的硅酸识别蛋白

基本信息

批准号：
11694291
负责人：
UEMURA Haruki
金额：
$ 2.11万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The unique enzyme of some infective Trypanosoma species, trans-sialidase catalyzes the sialic acid transfer reactions from host derived glycoconjugates to the parasite surface mucinlike acceptor molecules and vice versa. Several lines of evidence have suggested that trans-sialidase and sialylated molecules on the parasite and host cell surface are important for parasite invasion to the host cells and escape from host defense systems. We have investigated the functional analysis of trans-sialidase in molecular level.In this study, we have successfully purified and characterized trans-sialidase obtained from the culture medium of Trypanosome brucei, which causes African sleeping sickness. This enzyme showed several biochemical similarities to that of Trypanosoma cruzi, which is the etiological agent of Chagas' disease. We also have expressed and purified homogeneously the recombinant trans-sialidase of T. cruzi using bacterial expression system. These enzymes are used for the recognition studies of the parasite protein by host cells. The comparison of enzymatically active and inactive forms of purified recombinant proteins were important for understanding the function of trans-sialidase activitis.We have obtained and analyzed several T. cruzi trans-sialidase genes expressed in trypomastigote and epimastigote stages of this parasite. The enzyme expressed in infective trypomastigote stage (T-TS) is located on the parasite surface through a glycosyl phosphatididylinositol (GPI) anchor, and readily shed into culture supernatant in vitro and into the bloodstream of animals in vivo. On the other hands, trans-sialidase of noninfective epimastigote stage is not released into the medium nor recognized by antibodies against the carboxyl terminal repeats of T-TS.We have sequences these family genes to understand functions, molecular mechanisms of expression and localization of these two different types of trans-sialidases.

转唾液酸酶是某些感染性锥虫属物种的独特酶，催化唾液酸从宿主衍生的糖缀合物转移到寄生虫表面粘蛋白样受体分子的反应，反之亦然。一些证据表明，寄生虫和宿主细胞表面的转唾液酸酶和唾液酸化分子对于寄生虫入侵宿主细胞和逃离宿主防御系统是重要的。本研究从分子水平上对转唾液酸酶的功能进行了研究，成功地从引起非洲昏睡病的布氏锥虫（Trypanosomebrucei）的培养基中分离纯化了转唾液酸酶，并对其进行了结构鉴定。这种酶与克氏锥虫（锥虫是南美锥虫病的病原体）的酶显示出几种生化相似性。我们还表达并纯化了T. cruzi，使用细菌表达系统。这些酶用于宿主细胞对寄生虫蛋白的识别研究。比较纯化的重组蛋白的酶活性和失活形式对于理解转唾液酸酶活性的功能是重要的。cruzi转唾液酸酶基因在该寄生虫的锥鞭毛体和上鞭毛体阶段表达。在感染性锥鞭毛体阶段（T-TS）表达的酶通过糖基磷脂酰肌醇（GPI）锚位于寄生虫表面，并且在体外容易地脱落到培养上清液中，在体内容易地脱落到动物的血流中。另一方面，非感染性外鞭毛体阶段的trans-sialidase不释放到培养基中，也不被针对T-TS的羧基末端重复序列的抗体识别。我们对这两种不同类型的trans-sialidase家族基因进行了测序，以了解其功能、表达和定位的分子机制。