Formation of lipid-linked saccharide intermediates of glycoprotein biosynthesis in lactating bovine mammary explants from an organ culture.

Abstract

During an organ culture of lactating bovine mammary explants in the presence of lactogenic hormones, the radioactivity from D-[1-14C]mannose (Man) and N-acetyl-D-[1-14C]glucosamine (GlcNAc) was incorporated rapidly into the lipid-saccharide (C/M), lipid-oligosaccharide (C/M/W) and protein fractions, which were endogenous acceptors in the N-linked glycoprotein fromation. The distribution of radioactivity in the C/M, C/M/W and protein fractions at 6 hr incubation was 5, 2 and 93% for [14C]GlcNAc, but 34, 1 and 65% for [14C]Man, respectively. The labelled sugars in the C/M and C/M/W fractions were identified to be Man and GlcNAc. Man in the C/M fraction was mannosyl phosphoryl dolichol, and both Man and GlcNAc in the C/M/W fraction were incorporated into lipid-linked oligosaccharide, which was composed of trisaccharide through dodecasaccharide. Polyacrylamide gel electrophoretic analysis of the protein fraction indicated that six polypeptides were labelled. Of the major peaks, one was caseins and the other was glycoproteins corresponding to the glycoproteins of the plasma membrane. The results suggest that the molecular size of lipid-linked oligosaccharide and the acceptor proteins differed from those of the bovine mammary membranes and rabbit mammary explants.