Biosynthesis of glycoproteins in human placenta: differential labeling of mannose and heterogeneity of oligosaccharide lipid intermediates.

The biosynthesis of lipid-linked oligosaccharides has been studied in first trimester human placentas. Tissue was pulsed with [2-3H]mannose, [1-3H]glucosamine, and [1-3H]galactose (for [3H]glucose incorporation). The lipid-linked oligosaccharides (OSL) were purified on DEAE-cellulose. After cleaving the lipid from OSL the oligosaccharides were purified by paper chromatography and borate high-voltage electrophoresis. Four major oligosaccharides with the composition Glc(0-3)Man9GlcNAc2 thus obtained were characterized enzymatically by digestion with endo-beta-N-acetylglucosaminidase H and alpha-mannosidase, and chemically by methylation, acetolysis, and Smith degradation. While identical Glc(1-3)Man9GlcNAc2 oligosaccharides have been isolated from other in vivo systems, the presence of Man9Glc2 is novel for human placenta. Furthermore, Man9GlcNAc2 is present in appreciable amounts in placenta. Second, one mannose in Man9GlcNAc2 had a significantly higher specific radioactivity than the other mannosyl residues of the Glc(0-3)Man9GlcNAc2 oligosaccharides. Third, the differential labeling in Man9GlcNAc2 and our pulse-chase studies indicate that Man9GlcNAc2 is not a major precursor of Glc3Man9GlcNAc2. The data also suggest that the ninth mannose, which has the highest radioactivity, may be incorporated in a different subcellular compartment and may serve as a regulatory step in the biosynthesis of lipid-linked oligosaccharides. A model is proposed which outlines possible multiple pathways of lipid-linked oligosaccharide biosynthesis in human placenta.