Evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin.
To obtain information on the mechanism of glycosylation of ovalbumin, three types of experiments were performed with either hen oviduct membrane preparations or tissue slices and the antibiotic tunicamycin. First, experiments involving the addition of tunicamycin to oviduct membranes demonstrated that this antibiotic inhibited the synthesis of a N-acetylglucosaminyl-lipid with the properties of N-acetyl-glucosaminylpyrophosphorylpolyisoprenol. No inhibitory effects on the other steps in the synthesis of oligosaccharide-lipid were observed. Thus, tunicamycin inhibits the lipid-linked pathway for membrane protein glycosylation by blocking the first step in the synthesis of oligosaccharide-lipid, namely, the synthesis of N-acetylglucosaminylpyrophosphorylpolyisoprenol. Second, in experiments using membranes prepared from oviduct tissue slices preincubated with tunicamycin, it was found that mannosylphosphoryldolichol was the only saccharide-lipid synthesized. This result indicates that tunicamycin administered in vivo depletes endogenous pools of N-acetylglucosamine-lipid precursors to oligosaccharide-lipid in a manner consistent with its activity in vitro. Finally, it was found that tissue slices incubated in the presence of tunicamycin synthesized the polypeptide chain of ovalbumin at almost normal rates. However, the protein newly synthesized in vivo did not contain labeled N-acetylglucosamine or mannose and had the properties of unglycosylated ovalbumin. These results indicate that saccharide-lipids participate in the assembly of the core oligosaccharide of the secretory glycoprotein, ovalbumin.
D000118:Acetylglucosaminidase; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D005260:Female; D006017:Glycolipids / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009844:Oligosaccharides / Q000096:biosynthesis*; D010047:Ovalbumin / Q000096:biosynthesis*; D010057:Oviducts / Q000187:drug effects / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D000998:Antiviral Agents; D006017:Glycolipids; D009844:Oligosaccharides; D006155:Guanosine Diphosphate Mannose; D014537:Uridine Diphosphate N-Acetylglucosamine; D010047:Ovalbumin; D000118:Acetylglucosaminidase