STRUCTURE AND 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的反应。 生物，但无菌免疫很少，如果曾经实现，作为寄生虫 在宿主细胞的细胞质中受到保护。一种72 kDa糖蛋白，GP 72， 是T.克鲁兹， 显然仅在昆虫阶段（外鞭毛体和 后环锥鞭毛体）。因为已经被用来 定义GP 72识别碳水化合物表位，是否 该基因在其他阶段表达，但差异后， 修改，尚未解决。这项建议的目的是研究 发育调节的表面糖蛋白的结构和功能， 特别强调GP 72的结构特征，其基因 (or基因，因为所有特征T. Cruzi表面抗原似乎 由基因家族编码），以及其不寻常的翻译后 修改.关注GP 72有几个原因：它仍然是 所有描述的T.克氏表面抗原 高亲和力单克隆抗体（WIC 29.26）可用于其 纯化;它是发展调节;它有非常不寻常的，如果 不是独特的，翻译后聚糖修饰，这可能是 参与了它的一些生物功能;它与 寄生虫的几种生物学功能;它是一个主要的受体， C3，它是非常免疫原性的，它已被证明是保护性的， 小鼠的免疫。 纯化了GP 72，并获得了部分氨基酸序列数据。这 这些信息将用于克隆该蛋白质的cDNA。本组织 将研究编码GP 72的基因的序列。我们预计多个基因 将存在，因此我们将确定这些中的哪些被表达，在什么地方被表达， 生命周期中的阶段，以及表达的副本是否在 分离、克隆特异性或阶段特异性方式。如果表达的副本 不同，我们将确定是否有重大的结构性和 因此基因产物之间存在潜在的功能差异。我们 将探索连接聚糖的性质和位置。使用 克隆的基因和针对相应蛋白质的抗体，我们 将研究生物合成和翻译后修饰的动力学 GP 72的最终，我们希望研究免疫预防剂 重组蛋白和合成糖肽的潜力。

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. A 72 kDa glycoprotein, GP72, is an important developmentally regulated surface antigen of T. cruzi, apparently expressed only in the insect stages (epimastigotes and metacyclic trypomastigotes). Because the antisera that have been used to define GP72 recognize carbohydrate epitopes, the question of whether the gene is expressed in other stages, but differentially posttranslationally modified, has not been resolved. The aim of this proposal is to study the structure and function of developmentally regulated surface glycoproteins, with special emphasis on the structural characterization of GP72, its gene (or genes, since all characterized T. cruzi surface antigens appear to be encoded by gene families), and the nature of its unusual posttranslational modifications. There are several reasons for focusing on GP72: it is still the best defined of all the described T. cruzi surface antigens; a high-affinity monoclonal antibody (WIC 29.26) is available for its purification; it is developmentally regulated; it has highly unusual, if not unique, posttranslational glycan modifications, which are probably involved in some of its biological functions; it has been associated with several biological functions of the parasite; it is a major acceptor for C3, it is very immunogenic, and it has been shown to be protective upon immunization of mice. GP72 has been purified, and partial amino acid sequence data obtained. This information will be used to clone a cDNA for the protein. The organization of gene(s) encoding GP72 will be studied. We anticipate that multiple genes will be present, so we will determine which of these are expressed, at what stage in the life cycle, and whether the expressed copy varies in an isolate, clone-specific, or stage-specific fashion. If the expressed copy varies, we will determine whether there are significant structural and therefore potential functional differences between the gene products. We will explore the nature and location of the attached glycans. Using the cloned gene and antibodies raised against the corresponding protein, we will study the kinetics of biosynthesis and posttranslational modification of GP72. Ultimately, we hope to investigate the immunoprophylactic potential of recombinant proteins, and synthetic glycopeptides.