Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation.

Thyroid slices were found to incorporate radioactivity from 14C-labeled sugars into the carbohydrate moiety of a polar lipid soluble in chloroform/methanol/water, 10/10/3. This radiolabeled glycolipid was purified by chromatography on DEAE-cellulose and was shown to have as its monosaccharide constituents mannose, glucose, and glucosamine. This compound cloud also be labeled by incubation of thyroid slices with [3H]mevalonic acid or [32P]phosphate as demonstrated by the coincidence of elution profiles upon DEAE-cellulose chromatography. This suggested that the lipid portion of the molecule is a polyprenol derivative and the lipid-saccharide linkage region involves a phosphate bridge. Mild acid hydrolysis of the glycolipid labeled with 14C in its carbohydrate released a neutral oligosaccharide which on the basis of Bio-Gel filtration studies was shown to have a molecular weight of approximately 2,400. This oligosaccharide contained [14C]mannose and [14C]glucose in about the same ratio as that occurring in the intact glycolipid. The oligosaccharide-lipid appeared to be distributed rather widely in thyroid particulate fractions obtained after differential and density gradient centrifugation. Its highest specific activity occurred in fractions rich in endoplasmic reticulum. By means of pulse-chase experiments in slices a relationship was demonstrated between the disappearance of radioactivity from the lipid-bound oligosaccharide and its appearance in protein-bound form. When protein synthesis was inhibited by the addition of puromycin during the chase period of the experiment transfer of oligosaccharide from the lipid to protein appeared to be blocked and the level of radiolabeled oligosaccharide-lipid increased. The observation that mannose and glucose were similarly affected during the pulse-chase studies suggests that transfer of the intact oligosaccharide unit was involved in the addition of carbohydrate to protein.