Studies on the Biosynthesis of Glycoproteins

Spiro, Robert

Abstract

Most proteins destined for secretion or incorporation into cell surfaces or basement membranes become glycosylated during their formation. The biosynthesis of glycoprotains requires an elaborate enzymatic machinery which functions at pre-, co- and post-translational stages to yield a variety of carbohydrate units which are well suited to serve as recognition signals in diverse biological interactions. Numerous sites for regulation are afforded by saccharide unit assembly and the possibilities for malfunction are many and may be operative in diseases like diabetes, neoplasia and cystic fibrosis. It is the objective of this research proposal to undertake a multifaceted investigation involving several aspects of the biosynthesis of asparagine-linked carbohydrate units, the sulfation and recycling of cell surface glycoproteins and the biogenesis of the glycoprotein components of basement membranes and regulation thereof. Studies on N-linked saccharides in thyroid and liver will seek to accomplish the following: isolation and characterization with defined exogenous acceptors of the glucosyltransferases involved in the formation of the dolichyl pyrophosphoryl-linked precursor oligosaccharide; delineation of the mechanism and energetics involved in the transmembrane (endoplasmic reticulum) passage of the activated glucose needed for these reactions; purification of the oligosaccharyltransferase responsible for N-glycosylation and evaluation of the hypothesis that this enzyme has an oligosaccharide-lipid hydrolysis function of potential regulatory importance; exploration, through studies with microsomal vesicles and isolation of enzymes, of the alternate routes of carbohydrate unit processing which occur in the endoplasmic reticulum; definition of the physiological role of endo-Beta-N-acetylglucosaminidase by determining with pulse-chase experiments the origin of the released glucosylated and unglucosylated polymannose-GlcNAc oligosaccharides. Furthermore information about the mechanism and subcellular site of sulfation steps will be sought from an investigation of the biosynthesis of the well-defined sulfated thyroid cell surface glycoproteins. These glycoproteins will also be the subject of studies dealing with the internalization and recycling of cell surface molecules and for this purpose their strong reactivity with B. Simplicifolia lectin will be utilized. Biosynthesis of basement membrane glycoproteins will be studied by immunochemical and radiolabeling procedures in cultured lens epithelial cells and in a kidney mRNA translation system. The effect of diabetes and the influence of glucose concentration and various hormones on the production of the basement membrane components will be evaluated.