EXPRESSION OF GLYCOLIPID ANCHORED PRIONS IN TRYPANOSOMES

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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.