JBC, Vol. 251, Issue 20, 6420-6425, Oct, 1976

Lipid-saccharide intermediates in glycoprotein biosynthesis. III. Comparison of oligosaccharide-lipids formed by slices from several tissues

M. J. Spiro, R. G. Spiro and V. D. Bhoyroo

The synthesis of oligosaccharide-lipids thought to play a role in the attachment of carbohydrate to protein has been studied in incubations of slices from calf kidney, pancreas, thymus, and liver, as well as from hen oviduct. These compounds were characterized after radiolabeling of their saccharide moiety by incubation with [14C]glucose or [14C]mannose and a comparison was made with the oligosaccharide-lipid produced by thyroid slices. Furthermore, the unlabeled glycolipid was prepared from hen oviduct for the purpose of quantitating its sugar constituents. Purification of the oligosaccharide-lipids extracted with chloroform/methanol/water (10/10/3) was achieved by DEAE-cellulose chromatography and their carbohydrate moieties were released by mild acid hydrolysis. On the basis of gel filtration it was determined that the lipid-bound oligosaccharides formed by oviduct, thymus, kidney, and liver had molecular weights comparable to that from thyroid (about 2400). The saccharide moiety of the glycolipid from pancreas was however distinctly smaller in size with a molecular weight of approximately 1800. Analyses of the radiolabeled oligosaccharide-lipids from oviduct, kidney, and thymus indicated that they, like the compound from thyroid slices, but unlike those believed to be formed by cell-free systems from various tissues, contained glucose in addition to mannose and N-acetylglucosamine as their monosaccharide constituents. This compositional data was supported by the finding that the unlabeled oligosaccharide from oviduct consists of 10 mannose, 1 glucose, and 2 N-acetylglucosamine residues. Sodium borohydride reduction of this oviduct saccharide moiety indicated that 1 of the 2 glucosamines was situated in a reducing terminal position. The radiolabeled oligosaccharide from the glycolipid produced by pancreas differed from the others analyzed in that it contained only trace amounts of glucose. Upon treatment with alpha-mannosidase this glucose-deficient pancreatic oligosaccharide was extensively digested (85% of the mannose released). In contrast, the carbohydrate moieties of oviduct, kidney, and thymus, like that of thyroid, underwent a more limited digestion with the alpha-mannosidase (55% or less of the mannose released) suggesting that the presence of glucose may serve to block a more complete degradation of these oligosaccharides by this enzyme.
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