STRUCTURE/FUNCTION OF T CRUZI SURFACE GLYCOPROTEINS

克氏锥虫是一种粪生锥虫，引起恰加斯病， 慢性病影响到中部和南部2 000多万人 美国参考这种专性细胞内寄生虫具有复杂的生命周期， 在昆虫载体和哺乳动物中具有多个发育阶段 主持人宿主的免疫系统对病毒产生强烈反应 生物体，但无菌免疫很少，如果曾经实现，因为 寄生虫在宿主细胞的细胞质中受到保护。GP72是一个72 kDa的 免疫显性表面糖蛋白，据推测只在 昆虫期（外鞭毛体和后环锥鞭毛体）的T.克鲁兹 因为定义GP72的单克隆抗体（WIC 29.26）识别 碳水化合物表位，基因是否表达的问题， 其他阶段，但不同的后期修改，还没有被 解决了GP72是目前已知的T. cruzi表面抗原。有一些可能的功能的迹象 GP72的它是C3的主要受体，具有很强的免疫原性，GP72- 免疫的小鼠对后循环锥鞭毛体的攻击具有抗性。 然而，先前用于免疫研究的GP72的纯度是 可疑的。纯化了GP72，克隆了其基因， 测序t. cruzi Y株含有一对GP72基因和一个 通过连续缺失GP72基因构建了Bull突变体。 这些研究表明，WIC 29.26单克隆抗体识别 除了GP72之外的糖缀合物。零突变体有一个惊人的 形态表型，涉及鞭毛脱离。形态 异常持续到感染性锥鞭毛体阶段， 这是迄今为止GP72在这些阶段正常表达的最好证据。 本研究的目的是进一步研究其结构、表达、 GP72及其基因的细胞定位和生物学功能， 连接的聚糖。调查将确定结构和位置 连接的聚糖。GP72和WIC 29.26聚糖在 昆虫载体的定殖，其在无效突变体中受损， 将进一步研究。所使用的方法将利用我们最近的 发展出了进行复杂的基因操作的能力， 锥虫原生动物。例如，表位标记将提供一个表位标记。 标记，以澄清细胞位置和阶段特异性 GP72多肽的表达，这不能通过现有的 方法.使用克隆的基因和针对 相应的蛋白质，或标记的变体，我们将研究 GP72的生物合成和翻译后修饰。从 生命周期期间从GP72结构研究中获得的知识， 重组蛋白和合成的免疫预防潜力 将评价糖肽。

Trypanosoma cruzi, a Stercorarian trypanosome, causes Chagas' disease, a chronic illness affecting over 20 million people in Central and South America. This obligate intracellular parasite has a complex life cycle, with multiple developmental stages in the insect vector and the mammalian host. The immune system of the host mounts a strong response to the organisms, but sterile immunity is rarely if ever achieved, as the parasite lies protected in the cytoplasm of host cells. GP72 is a 72 kDa immunodominant surface glycoprotein, supposedly expressed only in the insect stages (epimastigotes and metacyclic trypomastigotes) of T. cruzi. Because the monoclonal antibody (WIC 29.26) that defined GP72 recognizes carbohydrate epitopes, the question of whether the gene is expressed in other stages, but differently posttranslationally modified, has not been resolved. GP72 is now one of the best defined of all the described T. cruzi surface antigens. There are some indications of possible functions of GP72. It is a major acceptor for C3, it is very immunogenic, and GP72- immunized mice are resistant to challenge by metacyclic trypomastigotes. However, the purity of GP72 previously used for immunization studies is questionable. GP72 has been purified and its gene has been cloned and sequenced. T. cruzi Y strain contains a single pair of GP72 genes and a Bull mutant has been constructed by sequential deletion of GP72 genes. These studies showed that the WIC 29.26 monoclonal antibody recognizes glycoconjugates other than GP72. The null mutant has a striking morphological phenotype, involving flagellar detachment. Morphological abnormalities persist into the infective trypomastigote stages, providing the best evidence so far that GP72 is normally expressed in these stages. The aim of this proposal is to further study the structure, expression, cellular location and biological function of GP72, its gene, and its novel attached glycans. Investigations will determine the structure and location of the attached glycans. The role of GP72 and the WIC 29.26 glycans in colonization of the insect vector, which is impaired in the null mutant, will be further studied. The approaches used will utilize our recently developed ability to perform sophisticated genetic manipulations of trypanosomatid protozoa. For example, epitope tagging will provide a marker to clarify both the cellular location and the stage-specific expression of the GP72 polypeptide, which could not be proven by existing methods. Using the cloned gene and antibodies raised against the corresponding protein, or a tagged variant, we will study the kinetics of biosynthesis and posttranslational modification of GP72. From the knowledge gained from structural studies of GP72 during the life cycle, the immunoprophylactic potential of recombinant proteins and synthetic glycopeptides will be evaluated.