STRUCTURE AND PROCESSING OF TRYPANOSOME VARIANT ANTIGENS

锥虫变异抗原的结构和加工

• 批准号: 2061552
• 负责人: GEORGE ALAN MARTIN CROSS
• 金额: $ 18.08万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2061552
• 关键词: Trypanosoma; acylation; electroporation; galactosyltransferases; gene mutation; glycolipids; glycoprotein biosynthesis; glycoprotein structure; glycosylation; hydrolase; laboratory mouse; laboratory rabbit; laboratory rat; molecular cloning; phosphatidylinositols; phospholipase C; polymerase chain reaction; posttranslational modifications; protein sequence; protein signal sequence; protozoal antigen; surface antigens; trypanosomiasis

Human and animal-infective trypanosomes have major medical and veterinary consequences for equatorial Africa and Latin America. Failure of the immune system to eliminate infections with the Salivarian trypanosomes is largely attributable to the phenomenon of antigenic variation. Each trypanosome is covered by a surface coat consisting of a closely packed monolayer of about 10 million molecules of a single member of a large family of variant surface glycoproteins (VSGs). VSGs are anchored to the trypanosome surface membrane via a covalently attached glycosylphosphatidylinositol (GPI) moiety. The integrity of the VSG coat is essential for trypanosome survival. Trypanosoma brucei also contains a GPI-specific phospholipase-C (GPIPLC). This situation suggests that VSGs are GPI anchored for very specific but so far elusive reasons, perhaps related to VSG turnover. Our previous studies led to the elucidation of the VSG-linked GPI structure, the structural characterization of GPI precursors, and an outline knowledge of the pathway of GPI biosynthesis. These studies are of general scientific interest since all eukaryotic cells have subsequently been found to express GPI-anchored cellsurface glycoproteins with diverse functions. The core structure of the GPI anchor appears to have been conserved during evolution. In trypanosomes, the major surface glycoproteins of bloodstream-form and procyclic T. brucei and of the infective stages of Leishmania and T. cruzi are GPI anchored. All of these glycoproteins probably have critical roles in infectivity and virulence. This continuation proposal will focus on the possible function of trypanosome GPI anchors through an exploration of the roles of the GPIPLC, the pathways that determine whether GPI anchors will be PIPLC-sensitive or resistant, the signals for GPI addition to proteins in trypanosomes, and GPI galactosylation. A primary objective of these studies is to identify aspects of GPI structure and function that may be distinct from host pathways. So far, GPIPLC has only been convincingly purified from T. brucei and its gene has been cloned. Its intracellular location is uncertain and its role in VSG metabolism is unknown. We propose genetic and organellar fractionation approaches to obtain more information on the role and subcellular location of GPIPLC in trypanosomes. Now that procyclic and bloodstream-form trypanosomes can routinely be transfected and cultured in vitro, we propose to use genetic approaches to further explore the carboxy-terminal amino acid sequence requirements for GPI anchoring of VSGs and PARP and determine whether the remarkably conserved VSG carboxy-terminal sequences indicate a very specific sequence requirement for GPI anchoring in trypanosomes. These studies may open up new in vitro approaches to identifying components of the GPI transfer machinery.

感染人类和动物的锥虫有主要的医疗和兽医 对赤道非洲和拉丁美洲的影响。失灵了 免疫系统消除对卵巢锥体的感染是 很大程度上归因于抗原变异现象。每个人 锥虫被一层表面涂层覆盖，该涂层由紧密堆积的 由大约1000万个大分子的单个成员组成的单层 变异型表面糖蛋白家族(VSG)。VSG被锚定到 通过共价连接的锥虫表面膜 糖基磷脂酰肌醇(GPI)部分。VSG涂层的完整性 对锥虫的生存至关重要。布氏锥虫还含有一种 GPI特异性磷脂酶C(GPIPLC)。这种情况表明，VSG GPI是出于非常具体但到目前为止难以捉摸的原因吗？ 与VSG的营业额相关。我们之前的研究导致了对 VSG连接的GPI结构，GPI的结构特征 前体，并概述了GPI生物合成途径的知识。 这些研究具有普遍的科学意义，因为所有真核生物 随后发现细胞表达GPI锚定的细胞表面 具有多种功能的糖蛋白。GPI的核心结构 锚似乎在进化过程中是保守的。在锥虫体内， 血液型和原环型主要表面糖蛋白。 布氏杆菌、利什曼原虫和克鲁兹旋毛虫的感染期是GPI 抛锚了。所有这些糖蛋白可能在 传染性和致命性。 这份延续提案将重点放在可能的功能上 通过对锥虫GPI锚的作用的探索 GPIPLC，决定GPI锚点是否将被 PIPLC敏感或抗性，GPI加入蛋白质的信号 锥体和GPI半乳糖化。这些活动的一个主要目标是 研究是确定GPI结构和功能的可能方面 与宿主途径不同。到目前为止，GPIPLC只是令人信服 从布氏毛滴虫中分离纯化并克隆了其基因。它的细胞内 定位尚不确定，其在VSG代谢中的作用也尚不清楚。我们 提出遗传和细胞器分级方法，以获得更多 关于GPIPLC的作用和亚细胞位置的信息 锥虫。现在，原环状和血流形式的锥虫可以 常规地在体外进行转基因和培养，我们建议使用基因 进一步探索羧基末端氨基酸序列的途径 VSG和PARP的GPI锚定要求，并确定是否 非常保守的VSG羧基末端序列表明 锥体中GPI锚定的特定序列要求。这些 研究可能会开辟新的体外方法来鉴定黄连的成分 GPI传输机器。