African trypanosomiasis is caused by the parasitic protozoan Trypanosoma brucei. Currently, drugs used to treat the disease are toxic and vaccine development is hampered by """"""""antigenic variation"""""""", whereby the parasite acquires a new variant surface glycoprotein (VSG) coat which is antigenically distinct and enables the infection to be sustained by evasion of the immune system. All VSGs attach to the plasma membrane by a glycosyl phosphatidylinositol (GPI) protein anchor which when cleave renders trypanosomes sensitive to lysis by the complement-mediated system. It is our aim to provide basic biochemical information on GPI catabolism which could be exploited chemotherapeutically. T. brucei glycosyl phosphatidylinositol-specific phospholipase C (GPI- PLC) can cleave dimyristoyl glycerol from the GPI anchor, releasing VSG from membranes. GPI-PLC activity is """"""""activated"""""""" in bloodstream trypanosomes in response to the initiation of cell lysis or an increase in intracellular calcium. We have identified two forms GPI-PLC protein which differ in molecular weight by 4 kDa, the larger of which (39 Kda) might be the form present when cells are activated for VSG GPI cleavage. By metabolic labeling to detect posttranslational modifications, and in vitro treatment with enzymes capable of posttranslationally modifying proteins we shall characterize the modifications, and in vitro treatment with enzymes capable of posttranslationally modifying proteins we shall characterize the modifications associated with GPI-PLC before and after activation (Specific aim 1). We have expressed GPI-PLC in E. coli: biochemical studies will be performed on the recombinant enzyme to relate the structure to function. Deletion analysis and site-specific mutagenesis of the GPI-PLC gene expressed in E. coli in combination with chemical modification and photoaffinity labeling (with purified glycan from the VSG GPI) of the purified protein will identify amino acid residues involved in catalysis or maintenance of enzyme structural integrity (Specific Aim 2). To study the effect of GPI depletion on T. brucei infection of rodents we shall overexpress a GPI-PLC specifically designed to localize in the endoplasmic reticulum of T. brucei, where GPIs are transferred to nascent VSG (Specific Aim 3). The recombinant gene will be stably integrated int the genome of T. brucei by homologous recombination. Clones will be analyzed for glycosylation of the recombinant GPI-PLC and subsequently studied for the effect presence of GPI-PLC in the endoplasmic reticulum on the availability of membrane associated VSG. Ability of these clones to infect rodents will also be examined.
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| Mensa-Wilmot, K; LeBowitz, J H; Chang, K P et al. (1994) A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways. J Cell Biol 124:935-47 |
