The long-term goal of this research is to understand the regulation of N- linked oligosaccharide processing during glycoprotein transport and secretion. Yeast glycoprotein synthesis has many parallels with that in animal cells, and oligosaccharide structural studies imply tight control of the numerous sugar transferases involved in yeast glycan maturation. However, little is known about the substrate specificity or compartmentalization of these enzymes, or about the structure of oligosaccharide intermediates. In the proposed work, Saccharomyces, Pichia and Schizosaccharomyces yeasts will be studied in four complementary Aims to provide new information regarding the structural biochemistry, cell biology and enzymology of early events in the complex process of glycan maturation. First, the alpha1,3-mannosyl will be purified, characterized and its gene cloned to learn more about oligosaccharide-lipid metabolism. This enzyme is defective in the Saccharomyces alg3 mutant. Second, invertase oligosaccharides conditionally truncated in the cis- to medial- Golgi of Saccharomyces mutants pmr1, ypt1-ts and sec7-ts will be assigned structures using high-field 1H NMR coupled as needed with chemical and enzymatic methods, to elucidate Golgi processing intermediates.
The third aim i s the purification, characterization and cloning of the Saccharomyces alpha1,3-mannosyl transferase, which adds terminal alpha1,3-linked mannose to Man10GlcNAc to yield Man11-14GlcNAc. This gene will be expressed in Pichia, unable to add alpha1,3-linked mannose to oligosaccharides, to study expression and targeting of this enzyme. In the fourth aim, Schizosaccharomyces invertase oligosaccharides will be structurally defined by the above methodology to determine the specificity of alpha-linked galactose addition in the Golgi. Most secreted and integral membrane components and receptors in eucaryotic cells are glycoproteins. N-linked carbohydrate has been implicated in numerous cellular functions, including receptor-mediated events such as clearance, signal transduction, virus uptake and maturation and the intracellular targeting of critical cellular components. Many human diseases result from a failure to properly synthesize, process or degrade glycoprotein glycans, and apart from direct causal relationships, glycoprotein metabolism abnormalities are associated with pathological states such as metastatic cancer, cystic fibrosis and Wiscott-Aldrich syndrome. Understanding how glycoproteins are made, transported, and function within the organism is seminal to developing a rational basis for curing identifiable disease states.
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