Control of glycoprotein synthesis. The in vitro synthesis by hen oviduct membrane preparations of hybrid asparagine-linked oligosaccharides containing 5 mannose residues.
Hen oviduct membranes were incubated with UDP-N-acetyl-D-[14C]glucosamine and [3H]GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6(Man alpha 1-3)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn (glycopeptide Gn(I)M5). Two double labeled products were obtained, both containing 5 Man and 4 GlcNAc residues. In order to separate these isomeric components, the mixture was treated with rat liver Golgi-rich membranes as a source of mannosidase II. One of the isomers was degraded by mannosidase action while the other was not, thereby allowing separation of two products (A and B). Product A was shown to be [3H]GlcNAc beta 1-2[( 14C] GlcNAc beta 1-3,4, or 6)Man alpha 1- 3Man beta 1-4GlcNAc beta 1-4GlcNAc, proving that hen oviduct membranes were capable of incorporating GlcNAc in beta-linkage into the Man alpha 1-3- residue of Gn(I)M5. Product B was identified as [3H]GlcNAc beta 1-2Man alpha 1-3[( 14C]GlcNAc beta 1-4)-[Man alpha 1-3)Man alpha 1-6]Man beta 1-4GlcNAc beta 1- f4GlcNAc , showing that hen oviduct membranes could incorporate a bisecting GlcNAc residue (linked beta 1-4 to the beta-linked Man) into Gn(I)M5. The ability of hen oviduct to carry out these two reactions in vitro supports the hypothesis first suggested by Harpaz and Schachter ( Harpaz , N., and Schachter , H. (1980) J. Biol. Chem. 255, 4894-4902) that the synthesis of bisected hybrid oligosaccharides is controlled by the insertion of a bisecting GlcNAc residue into Gn(I)M5. The presence of a bisecting GlcNAc residue prevents mannosidase II action and the synthetic pathway is therefore committed to hybrid oligosaccharide synthesis.
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002645:Chickens; D002846:Chromatography, Affinity; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D010057:Oviducts / Q000378:metabolism*; D051381:Rats
D009844:Oligosaccharides; D001216:Asparagine; D008358:Mannose