Synthesis of the N-linked oligosaccharides of glycoproteins. Assembly of the lipid-linked precursor oligosaccharide and its relation to protein synthesis in vivo.
The asparagine-linked oligosaccharides of chick embryo fibroblast glycoproteins were previously shown to derive from a common lipid-linked precursor, Glc3Man9GlcNAc2. The formation of this precursor oligosaccharide was examined in intact chick embryo fibroblasts, NIL-8 cells, and Chinese hamster ovary cells. The labeling kinetics and compositions of the lipid-linked oligosaccharides were examined, and the results indicate that lipid-linked Man5GlcNAc2 is rapidly assembled (< 1.5 min) and then extended (< 2.5 min) to Glc3Man9GlcNAc2 via the intermediate Man8GlcNAc2. Chain elongation from Man5GlcNAc2- to Man8GlcNAc2-lipid probably occurs by addition of single mannose residues. The pool of lipid-linked Glc3Man9GlcNAc2 turns over with a half-time of 3.5 to 6 min; since there is little if any degradation (the mannose residues do not turn over), this reflects the rate at which completed chains are transferred to acceptor proteins. The same intermediates and similar kinetics were observed in all three cell types. Oligosaccharide-lipid assembly was also examined in cells in which protein synthesis was decreased (using actinomycin D to depress levels of mRNA) or abolished (using cycloheximide). The results indicate that the rate of oligosaccharide-lipid synthesis is proportional to the rate of protein synthesis. The regulated step is prior to the Man5GlcNAc2 stage, and we suggest that the most likely control mechanism is limitation of available oligosaccharide carrier lipid.
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D010053:Ovary
D006017:Glycolipids; D006023:Glycoproteins; D009844:Oligosaccharides; D008358:Mannose