The biosynthesis of mammalian asparagine-linked carbohydrate chains involves the participation of a dolichol-linked G1c3MangGlcNac2 intermediate. After transfer of the oligosaccharide portion from the lipid carrier to nascent proteins, a processing sequence occurs that involves excision of the glucose residues and most of the mannose residues, followed by maturation of the carbohydrate chain with the attachment of peripheral sugars. We are focusing on the role of glucose in the assembly of the dolichol-linked oligosaccharide intermediates, and consequently, on the glycosylation of proteins. We have already shown that vesicular stomatitis virus-infected fibroblasts deprived of glucose cannot efficiently complete the assembly of Glc3MangGlcNAc2-P-P-Dol, resulting in an abnormally glycosylated G-protein. In the first year of this proposal our efforts will be directed toward a structural determination of the [3H]mannose-labeled oligosaccharides produced by glucose starvation. Methodologies such as lectin affinity chromatography, glycosidase digestion, and methylation analysis will be used for this purpose. We will then concentrate on elucidating the molecular events that lead to the abnormal protein glycosylation during glucose deprivation. It is possible that this glucose-dependent glycosylation of protein is due to the sensitivity of the dolichol intermediate pathway to either the cellular concentrations of glucose or to the participation of an effector molecule, such as glucose or a metabolite of glucose. This aspect will be examined by determining the cellular pools of selected metabolites (glucose, glucose-6-phosphate, UDP-glucose, and GDP-mannose) and by assaying the glycosylating enzymes in cell-free membrane preparations. We will also undertake an investigation of the assembly of the dolichol-linked oligosaccharides and protein glycosylation in 3T3-L1 adipocytes and in diabetic adipocytes isolated from hyperinsulinemic mice to examine the possible implications of glucose-dependent glycosylation of proteins in diabetes.
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