GPI-PHOSPHOLIPASE C OF TRYPANOSOMA BRUCEI

非洲锥虫病是由寄生原生动物锥虫引起的 布鲁塞。 目前，用于治疗该病的药物是有毒的， 发展受到“抗原变异”的阻碍， 获得一种新的变异表面糖蛋白（VSG）外壳， 抗原性不同，并使感染持续， 免疫系统的逃避。 所有VSG都通过以下方式附着在质膜上 糖基磷脂酰肌醇（GPI）蛋白质锚，其在切割时 使得锥虫对补体介导的裂解敏感 系统 我们的目标是提供关于GPI的基本生化信息 可以用于化学治疗的催化剂。 T.布鲁氏菌糖基磷脂酰肌醇特异性磷脂酶C（GPI- PLC）可以从GPI锚上裂解二肉豆蔻酰甘油，释放VSG from membrane膜. GPI-PLC活性在血流中被“激活” 锥虫对细胞裂解起始或增加的反应 细胞内钙离子 我们鉴定了两种GPI-PLC蛋白 其分子量相差4 kDa，其中较大的（39 kDa） 可能是当细胞被激活进行VSG GPI切割时存在的形式。 通过代谢标记检测翻译后修饰， 用能够后修饰的酶进行体外处理 蛋白质，我们将表征的修改，并在体外治疗 利用能够对蛋白质进行后期修饰的酶， 表征GPI-PLC前后相关的修改 具体目标1）。 在E.大肠杆菌：生化研究将是 在重组酶上进行，以将结构与功能联系起来。 GPI-PLC基因的缺失分析和定点突变 在大肠杆菌中表达大肠杆菌与化学修饰的组合， 光亲和标记（用来自VSG GPI的纯化聚糖） 纯化的蛋白质将鉴定参与催化的氨基酸残基 或维持酶的结构完整性（具体目标2）。 研究GPI缺失对T.啮齿动物布氏杆菌感染 我们将过度表达一个GPI-PLC，专门设计用于定位在 T.布氏杆菌，其中GPIs被转移到新生 VSG（Specific Aim 3）。 重组基因将稳定整合到 T.通过同源重组获得布氏杆菌。 克隆将 分析重组GPI-PLC的糖基化，随后 研究GPI-PLC在内质网中的作用 膜相关VSG的可用性。 这些克隆人的能力 感染啮齿类动物的方法。

African trypanosomiasis is caused by the parasitic protozoan Trypanosoma brucei. Currently, drugs used to treat the disease are toxic and vaccine development is hampered by "antigenic variation", whereby the parasite acquires a new variant surface glycoprotein (VSG) coat which is antigenically distinct and enables the infection to be sustained by evasion of the immune system. All VSGs attach to the plasma membrane by a glycosyl phosphatidylinositol (GPI) protein anchor which when cleave renders trypanosomes sensitive to lysis by the complement-mediated system. It is our aim to provide basic biochemical information on GPI catabolism which could be exploited chemotherapeutically. T. brucei glycosyl phosphatidylinositol-specific phospholipase C (GPI- PLC) can cleave dimyristoyl glycerol from the GPI anchor, releasing VSG from membranes. GPI-PLC activity is "activated" in bloodstream trypanosomes in response to the initiation of cell lysis or an increase in intracellular calcium. We have identified two forms GPI-PLC protein which differ in molecular weight by 4 kDa, the larger of which (39 Kda) might be the form present when cells are activated for VSG GPI cleavage. By metabolic labeling to detect posttranslational modifications, and in vitro treatment with enzymes capable of posttranslationally modifying proteins we shall characterize the modifications, and in vitro treatment with enzymes capable of posttranslationally modifying proteins we shall characterize the modifications associated with GPI-PLC before and after activation (Specific aim 1). We have expressed GPI-PLC in E. coli: biochemical studies will be performed on the recombinant enzyme to relate the structure to function. Deletion analysis and site-specific mutagenesis of the GPI-PLC gene expressed in E. coli in combination with chemical modification and photoaffinity labeling (with purified glycan from the VSG GPI) of the purified protein will identify amino acid residues involved in catalysis or maintenance of enzyme structural integrity (Specific Aim 2). To study the effect of GPI depletion on T. brucei infection of rodents we shall overexpress a GPI-PLC specifically designed to localize in the endoplasmic reticulum of T. brucei, where GPIs are transferred to nascent VSG (Specific Aim 3). The recombinant gene will be stably integrated int the genome of T. brucei by homologous recombination. Clones will be analyzed for glycosylation of the recombinant GPI-PLC and subsequently studied for the effect presence of GPI-PLC in the endoplasmic reticulum on the availability of membrane associated VSG. Ability of these clones to infect rodents will also be examined.