GPI-PHOSPHOLIPASE C OF TRYPANOSOMA BRUCEI

非洲锥虫病是由寄生原虫锥虫引起的 布鲁塞。目前，用于治疗这种疾病的药物是有毒的和疫苗 发育受阻于“抗原变异”，即寄生虫 获得了一种新的变异型表面糖蛋白(VSG)涂层 抗原性不同，使感染能够通过 逃避免疫系统。所有VSG通过以下方式附着在质膜上 糖基磷脂酰肌醇(GPI)蛋白锚点，当切割时 通过补体介导使锥虫对裂解敏感 系统。我们的目标是提供有关GPI的基本生化信息 分解代谢，可用于化疗。 布氏支原体糖基磷脂酰肌醇特异性磷脂酶C(GPI- PLC)可以从GPI锚点上裂解二肉豆蔻基甘油，释放VSG 从膜上。GPI-PLC活性在血液中被“激活” 锥体对细胞裂解的启动或增加的反应 细胞内的钙离子。我们已经鉴定出两种形式的GPI-PLC蛋白 它们的相对分子质量相差4 kDa，其中较大的(39 KDa) 可能是当细胞被激活以进行VSG GPI切割时出现的形式。 通过代谢标记来检测翻译后修饰，并在 翻译后修饰酶的体外治疗 我们将对修饰和体外处理的蛋白质进行表征 有了能够翻译后修饰蛋白质的酶，我们将 描述与GPI-PLC相关的修改前后的特征 激活(特定目标1)。 我们已经在大肠杆菌中表达了GPI-PLC：生化研究将在 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，结合化学修饰和 光亲和标记(从VSG GPI中纯化的多糖) 纯化的蛋白质将识别与催化有关的氨基酸残基 或维持酶结构的完整性(具体目标2)。 GPI耗竭对啮齿动物布氏毛滴虫感染的影响 我们将过度表达专门设计用于本地化的GPI-PLC 布氏毛滴虫的内质网，在那里GPI被转移到新生 VSG(特定目标3)。重组基因将稳定整合到 布氏毛滴虫基因组的同源重组。克隆人将成为 对重组GPI-PLC进行糖基化分析 GPI-PLC在内质网中作用的研究 膜结合型VSG的可用性研究。这些克隆人的能力 还将检查感染啮齿动物的情况。

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.