The trypanosomatid Leishmania donovani is the etiological agent of kala azar, a chronic and often fatal form of visceral leishmaniasis in humans. In its digenetic life cycle, this protozoan parasite resides in two hydrolytic environments: 1) extracellularly in a promastigote form in the alimentary tract of its sandfly vector and 2) intracellularly in an amastigote form within the lysosomes of mammalian macrophages. Cell surface macromolecules, such as complex carbohydrates, are likely to be responsible for the parasite's survival and for host-parasite interactions. Our long-term objective is to provide a complete understanding of the structure, biosynthesis, and function of complex carbohydrates of the parasite. In this research proposal our immediate objective will be focused on two aspects: 1) The characterization of the cellular location, structure, and biosynthesis of an unusual acidic glycoconjugate expressed by the organism. Localization of the glycoconjugate will be accomplished by either an indirect immunofluorescence technique or cell surface radiolabeling protocols. Sufficient quantities of the substance, which contains galactose, mannose, and phosphate in an approximate equimolar ratio of 1:1:1, can be obtained to perform most of the structural work on chemical amounts. Standard procedures that will be used to determine the structure include: oligosaccharide size determination by HPLC, methylation linkage analysis, glycosidase digestions, and acetolysis. 2) The involvement of lipid-linked oligosaccharide intermediates in the biosynthesis of the parasite's glycoproteins. The structure of the lipid, carbohydrate, and phosphate linkage region of the metabolically-labeled intermediates, as well as the protein-bound carbohydrate chains, will be determined using the standard procedures. The actual participation of oligosaccharide-lipid intermediates in glycoprotein biosynthesis will be evaluated by experiments that determine the effect of tunicamycin on [H3]mannose incorporation in intact cells and by in vitro experiments in which radiolabeled oligosaccharide-lipids are added to membrane preparations from the parasite. From these studies, we hope to contribute to the understanding of the role glycoconjugates play in the pathogenesis of leishmaniasis and to provide a biochemical rationale for the design of chemotherapeutic regimens that exploit complex carbohydrate differences between those of the mammalian host and of the parasite.
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