STRUCTURE AND PROCESSING OF TRYPANOSOME VARIANT ANTIGENS

锥虫变异抗原的结构和加工

• 批准号: 2061553
• 负责人: GEORGE ALAN MARTIN CROSS
• 金额: $ 18.82万
• 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/2061553
• 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具有重要的医学和兽医学意义。 对赤道非洲和拉丁美洲的影响。 失败 免疫系统，以消除感染与唾液锥虫， 主要归因于抗原变异现象。 每个 锥虫表面覆盖着一层紧密排列的 一个大分子的单个成员的约1000万个分子的单层 变异表面糖蛋白家族（VSG）。 VSG锚定到 锥虫表面膜通过共价连接 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（GPPLC）。 这种情况表明， GPI的锚定是出于非常具体但迄今为止难以捉摸的原因， 与VSG营业额有关。 我们先前的研究阐明了 VSG连接的GPI结构，GPI的结构表征 前体，以及GPI生物合成途径的概述知识。 这些研究具有普遍的科学意义，因为所有的真核生物都是 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被保存下来。在锥虫中， 血流型和原环型T. 布氏利什曼原虫和T. cruzi是GPI 锚定。 所有这些糖蛋白可能都在 传染性和毒性。 这一续设建议将侧重于 锥虫GPI锚通过探索的作用， GPPLC，决定GPI锚是否将被 PIPLC敏感或耐药，GPI添加到蛋白质中的信号， 锥虫和GPI半乳糖基化。 其中的一个主要目标是 研究的目的是确定GPI结构和功能的各个方面， 与宿主途径不同。 到目前为止，GPPLC只是令人信服地 从T.并克隆了其基因。 其细胞内 位置不确定，其在VSG代谢中的作用未知。 我们 提出遗传和细胞器分馏方法，以获得更多的 GPPLC的作用和亚细胞定位的信息， 锥虫 既然前循环和血流形式的锥虫可以 常规转染和体外培养，我们建议使用遗传 进一步探索羧基末端氨基酸序列的方法 VSG和PARP的GPI锚定要求，并确定 非常保守的VSG羧基末端序列表明， GPI锚定在锥虫中的特定序列要求。 这些 这些研究可能会开辟新的体外方法来识别 GPI转移机械。