The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.