We have described that protein-linked, high mannose-type oligosaccharides lacking glucose units are glucosylated directly from UDP-Glc (that is, without dolichol derivatives as intermediates) in the endoplasmic reticulum (ER) of mammalian, plant, fungal and protozoan cells and that the glucose units are subsequently removed, probably by glucosidase II. The ultimate aim of this proposal is to find a role for these reactions. For this purpose the UDP-Glc: Glycoprotein Glucosyltransferase (an enzyme apparently loosely attached to the lumen of the ER) will be purified, antibodies against it will be raised an d its localization will be established by immunocytochemistry. Double localization of glucosidase II and the glucosyltransferase by the immunogold technique will indicate if both enzymes share the same ER compartments. Purification of the enzyme and availability of monospecific antibodies will allow, in the long term, to isolate the corresponding gene. Its disruption may provide clues on the role of the reaction. We also propose to study the influence of the oligosaccharide and peptide moieties of glycoproteins on the glycosylation rate. For these propose glycopeptides containing variable oligosaccharide moieties (Man7, 8, 9GlcNAc2) and the same peptide or alternatively, the same oligosaccharide (Man9 GlcNAc2) but variable peptide moieties will be prepared and incubated with the enzyme. If the glucosyltransferase shows a different specificity with respect to the oligosaccharides, this will indicate that processing by ER mannosidases influences the glycosylation of glycoproteins. We already have evidence that some unknown feature in a protein domain close to the oligosaccharide strongly affects the rate of glycosylation. Identification of amino acids interacting with the glucosyltransferase will allow in the long term, to affect glycosylation of known glycoproteins by site-directed mutagenesis and thus obtain clues on the role of the glycosylation reaction. Finally, we propose to incubate intact cells with the appropriate precursor in such a way that only the glucose units transferred from UDP-Glc to glycoproteins will become labeled, to inhibit glucosidase II with certain drugs and to investigate, using antibodies against known glycoproteins, if glycosylation is restricted to certain types of glycoproteins (membrane bound, secreted, malfolded, lysosomal, etc.) or if it forms part of the machinery by which cells transport different glycoproteins from the ER to the Golgi at different rates.
