Human and animal-infective trypanosomes have major medical and veterinary consequences for equatorial Africa. Failure of the immune system to eliminate trypanosome infections is largely attributable to the Salivarian trypanosomes' unique mechanism for antigenic variations. Each trypanosome is covered by a surface coat consisting of a closely packed monolayer of about ten million molecules of a single member of a large family of variant surface glycoproteins (VSGs).
The aim of this proposal is to study the carboxy-terminal post-translational modification of the VSGs of Trypanosoma brucei, whereby a hydrophobic peptide tail, present on the primary VSG mRNA translation product, is cleaved and replaced by a complex glycan inositol phospholipid, which anchors VSG to the surface membrane. The question of variable galactosylation of the VSG-linked glycolipid will be resolved by extending structural at a glycolipid having several of teh characteristic expected of an immediate precursor of the VSG- linked glycolipid will be determined, suing the previously characterized VSG-linked as a model for experimental design and methodology. There are indications that peptide as a model for experimental design and methodology. There are indications that peptide cleavage and glycolipid attachment occur in a co- ordinated reaction at the endoplasmic reticulum, but that further processing of the glycan side-chain may occur in the Golgi apparatus. Cell-free systems will be developed, using trypanosome microsomal fractions, in which details of the modification reaction can be investigated. A further aim of this proposal is to investigate the role of the glycolipid moiety in VSG secretion, function, turnover and release. Conceptual and experimental contributions to these aspects can be expected from the expanding awareness of similar post-translational modification of several mammalian proteins. Since trypanosomes attach glycolipid to 10% of their protein (the VSG), they may represent the ideal model in which to explore some aspects of a modification pathway that may be important to all eukaryotic cells. The VSG-releasing enzyme, a glycan-phosphatidylinositol- specific phospholipase C (GPI-PLC), will be further characterized. Further specificity studies will be performed, its subcellular location will be determined, and the regulation of its activity by effectors and inhibitors will be determined. The GPI-PLC gene will be cloned and sequenced, for studies of its biosynthesis, regulation, and to facilitate characterization of the corresponding mammalian enzyme.
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