STRUCTURE AND FUNCTION OF T. CRUZI SURFACE GLYCOPROTEINS

克氏锥虫是一种变态锥虫，会引起查加斯病。 中南部2000多万人罹患慢性病 美国。这种专性的细胞内寄生虫有一个复杂的生命周期， 在昆虫媒介和哺乳动物中有多个发育阶段 主持人。宿主的免疫系统会对病毒产生强烈的反应 生物体，但无菌免疫力很少，就像寄生虫 被保护在宿主细胞的细胞质中。一个72 kDa的糖蛋白，GP72， 是克鲁兹毛滴虫重要的发育调节表面抗原， 显然只在昆虫阶段表达(表鞭毛体和 半环类鞭毛虫)。因为已经被用于治疗的抗血清 定义GP72识别的碳水化合物表位，问题是 基因在其他阶段表达，但在翻译后差异表达 已修改，但尚未解决。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， 重点研究了其基因GP72的结构特征 (或基因，因为所有特化的克氏锥虫表面抗原似乎都是 由基因家族编码)，以及其不寻常的翻译后的性质 修改。关注GP72有几个原因：它仍然是 所有已描述的克氏锥虫表面抗原中定义最好的； 高亲和力单抗(WIC 29.26)可用于ITS 净化；它是发育调节的；它有极不寻常的，如果 不是唯一的，翻译后的多糖修饰，可能是 参与了它的一些生物学功能；它与 寄生虫的几种生物学功能；它是 C3，它具有很强的免疫原性，并已被证明对 小鼠的免疫。 对GP72进行了纯化，获得了部分氨基酸序列数据。这 这些信息将被用来克隆该蛋白质的cdna。该组织 对编码gp72的基因(S)进行了研究。我们预计有多个基因 将会出现，所以我们将确定其中哪些被表达，在什么地方表达 生命周期中的阶段，以及表达的副本是否在 隔离、克隆特定或阶段特定的方式。如果表达的副本 不同，我们将确定是否存在显著的结构性和 因此基因产物之间存在潜在的功能差异。我们 将探索附着的多聚糖的性质和位置。使用 克隆的基因和针对相应蛋白的抗体 将研究生物合成和翻译后修饰的动力学 对GP72进行了分类。最终，我们希望研究免疫预防 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.