Transfer of nonglucosylated oligosaccharide from lipid to protein in a mammalian cell.
We have previously shown that the glucosidase inhibitor, N-methyl-1-deoxynojirimycin (MedJN), only partially inhibited N-linked complex oligosaccharide biosynthesis in F9 teratocarcinoma cells whereas the alpha-mannosidase I inhibitor, manno-1-deoxynojirimycin, completely prevented this synthesis (Romero, P. A. and Herscovics, A. (1986) Carbohydr. Res. 151, 21-28). In order to determine whether a pathway independent of processing glucosidases can occur, F9 cells were pulse-labeled for 2 min with D-[2-3H]mannose in the presence or absence of 2 mM MedJN. In control cells, Man7GlcNAc was identified in the protein-bound oligosaccharides released with endo-beta-N-acetylglucosaminidase H, in addition to the expected Glc1-3Man9GlcNAc and Man9GlcNAc arising from processing of Glc3Man9GlcNAc. MedJN completely prevented the removal of glucose residues from Glc3Man9GlcNAc, but did not greatly affect the appearance of Man7GlcNAc associated with protein. Labeled Man7GlcNAc was also found in the lipid-linked oligosaccharides of both control and treated cells. The 2-min pulse-labeled Man7GlcNAc obtained from both the lipid and protein fractions were shown to have identical structures by concanavalin A-Sepharose chromatography and by acetolysis and were clearly different from the Man7GlcNAc obtained from the usual processing pathway. These results demonstrate that transfer of a nonglucosylated oligosaccharide (Man7GlcNAc2) from dolichyl pyrophosphate to protein occurs in F9 cells.
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004288:Dolichol Phosphates / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D050356:Lipid Metabolism; D008358:Mannose / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*
D004288:Dolichol Phosphates; D009844:Oligosaccharides; D011506:Proteins; D017485:1-Deoxynojirimycin; C026406:dolichol pyrophosphate; D005944:Glucosamine; D008358:Mannose; C033424:N-methyldeoxynojirimycin