The long range goal of this proposal is to provide a more thorough understanding of the regulatory mechanisms involved in the biosynthesis and processing of the glycans found covalently attached to glycoproteins. Most proteins secreted from the cell or destined to become membrane components are glycoproteins. Failure to properly synthesize, traffic or catabolize the carbohydrates on glycoproteins causes numerous disease states leading to mental retardation, failure of reproductive hormonal control and immunological disorders leading to susceptibility to severe infection. Receptor targeting and transmembrane signaling cascades of many glycoproteins are mediated through their attendant carbohydrates. Influenza and AIDS virus uptake is mediated through carbohydrates, and cell surface glycans are altered in cancer. Clearly, understanding the fundamental control mechanisms that underlie correct glycoprotein metabolism is seminal to identifying and intervening in numerous glycopathologies. The current application proposes in four Aims studies in the small eukaryotes Saccharomyces cerevisiae and schizosaccharomyces pombe to determine the extent to which the substrate specificity of glycan processing enzymes or their subcompartmentation in elements of the secretory pathway govern glycan modeling in yeast.
The first Aim will characterize the enzymology of steps in S. cerevisiae required for the correct synthesis of Glc3Man9GlcNAc2-PP-dolichol, the precursor of initial N-glycan synthesis in the endoplasmic reticulum.
The second Aim will determine the aberrant N-linked oligosaccharide structures on a probe glycoprotein, secreted invertase, from S. cerevisiae pmrl mutant that, because of a defective Ca2+-ATPase, mistargets secretory components in vivo.
The third Aim will continue studies to define N- and O-. linked oligosaccharide structures in wild-type and five mutant S. pombe strains that have defects in sugartransferases.
The fourth Aim will examine the effect on normal glycoprotein metabolism of expressing a S. cerevisiae alpha1,3-mannosyltransferase in S. pombe, which does not have this enzyme activity. N-linked oligosaccharides are released from glycoproteins with endo-Beta-N-acetylglucosaminidase H and O-linked glycans by Beta- elimination and then purified by size on Bio-Gel P4. Glycan isomers are separated by Dionex high-pH anion-exchange chromatography and structurally defined by 1H-NMR spectroscopy and chemical and enzymatic methods as needed. The localization studies in Aims two and four will involve subcellular fractionation and immunofluorescence microscopy.
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