EXPRESSION OF GLYCOLIPID ANCHORED PRIONS IN TRYPANOSOMES

布鲁氏锥虫引起动物和人类的一种疾病，通常被称为非洲锥虫病，Nagana或睡眠病。锥虫可以在被感染的宿主体内无限期地存活，通过抗原变异过程逃避免疫系统，这种变异是由变异的表面糖蛋白(VSG)的顺序表达所介导的，这些变异的表面糖蛋白在锥体表面形成了一层坚不可摧的‘外套’。每个锥虫表达1000万个糖基磷脂酰肌醇(GPI)锚定的VSG分子，占细胞总蛋白的10%。布氏毛滴虫的表面积与红细胞的表面积差不多，而在大鼠或小鼠感染的高峰期，锥虫的产量约为红细胞数量的三分之一。对VSGs的研究提供了明确的化学证据，随后提供了第一个完整的GPI膜锚定结构，并阐明了GPI合成的复杂途径的许多细节。随后在广泛的真核细胞中的数百种蛋白质中发现的糖脂锚定共享一个共同的核心结构。布氏锥虫是内源性GPI锚定蛋白表面表达的工厂：VSG和原环酸性重复蛋白(PARP)分别占血液和昆虫(Glossina，Tesetse)“原环”形式锥虫总蛋白的10%。大多数哺乳动物的GPI锚定蛋白占细胞总蛋白的比例不到0.1%。尽管积累了与几种脑病有关的普恩或突变的普恩基因的知识，但核心理论--普恩传播涉及内源性细胞蛋白(PrPC)到传染性瘙痒病(PrPSc)的自催化转化--由于无法从自然来源或通过重组技术大量产生感染性PrPSc或天然PrPC，因此不容易被检验。尽管最近发表的核磁共振研究是在复性的非糖基化非GPI锚定的重组蛋白上进行的，但关于PrPC结构的一些问题仍然存在。锥体产生的大量GPI锚定的VSG，以及允许锥体稳定转化表达异位基因的方法的发展，为生产高兴趣、高价值的GPI锚定的哺乳动物蛋白，特别是PrP，提供了可靠的前景，可以快速评估。这项提案将侧重于在布氏锥虫中生产不同形式的PrP。

Trypanosoma brucei causes a disease of animals and humans that is commonly called African Trypanosomiasis, Nagana or Sleeping Sickness. Trypanosomes can persist indefinitely in the infected host, evading the immune system through a process of antigenic variation, mediated by the sequential expression of variant surface glycoproteins (VSGs) that form an impregnable 'coat' on the trypanosome surface. Each trypanosome expresses 10 million molecules of a single glycosylphosphatidylinositol- (GPI) anchored VSG, representing 10 percent of the total cell protein. The surface area of T. brucei is about the same as that of an erythrocyte and the yield of trypanosomes, at the peak of infection in a rat or mouse, is about one third of the number of erythrocytes. Studies of VSGs provided the definitive chemical evidence and, subsequently, the first complete structure of a GPI membrane anchor and the elucidation of many details of a complex pathway for GPI synthesis. The glycolipid anchors subsequently found in hundreds of proteins in a wide range of eukaryotic cells share a common core structure. T. brucei is a 'factory' for the surface expression of endogenous GPI-anchored proteins: VSG and the procyclic acidic repetitive protein (PARP) each comprise 10 percent of the total trypanosome protein, in the bloodstream and insect (Glossina, the Tesetse) 'procyclic' forms, respectively. Most mammalian GPI-anchored proteins comprise less than 0.1 percent of the total cellular protein. Despite the accumulated knowledge implicating prions or mutant prion genes in several encephalopathies, the central theory - that prion propagation involves a self-catalysed conversion of the endogenous cellular protein (PrPC) to the infectious scrapie prion (PrPSc) - cannot readily be tested because of the inability to produce infective PrPSc or native PrPC in large amounts from natural sources or by recombinant techniques. Several questions about the structure of PrPC remain, despite recently published NMR studies that were necessarily performed on renatured non-glycosylated non-GPI-anchored recombinant protein. The vast amount of GPI-anchored VSG that is made by trypanosomes and the development of methods that allow trypanosomes to be stably transformed to express ectopic genes, offer credible prospects, which can be rapidly evaluated, for producing high-interest high-value GPI-anchored mammalian proteins in general and PrP in particular. This proposal will focus on producing different forms of PrP in T. brucei.