Characterization of lipid-linked octa-, nona-, and decasaccharides formed during in vitro synthesis of mammary glycoproteins

The lipid-linked octa-, nona-, and decasaccharides, isolated from incubations of a membrane preparation from the lactating bovine mammary tissue with GDP-[14C]mannose and UDP-N-acetylglucosamine were subjected to mild acid hydrolysis and purified extensively by repeated gel filtration and paper chromatography. Structural characterization of the oligosaccharides containing six to eight mannose residues linked to an N,N'-diacetylchitobiose unit utilizing digestions with alpha-mannosidase, beta-mannosidase, endo-beta-N-acetylglucosaminidase, D, H, and L, acetolysis, and methylation analysis revealed the presence of several isomers within each size species. Supplementation of the incubations with 0.1 mM dolichol phosphate reduces the number of isomers within these oligosaccharides; the predominant isomers of saccharides from these incubations appear to be similar to the saccharides isolated from in vivo preparations of Chinese hamster ovary cells.