alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.
The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.
Fujimoto
K
K
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
R
R
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives; D000818:Animals; D002846:Chromatography, Affinity; D002854:Chromatography, Paper; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D065089:Glycoside Hydrolase Inhibitors; D007700:Kinetics; D008223:Lymphoma / Q000235:genetics; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000520:alpha-Glucosidases / Q000172:deficiency*; D043323:alpha-Mannosidase
D065089:Glycoside Hydrolase Inhibitors; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; C059896:miglustat; D008361:Mannosidases; D000520:alpha-Glucosidases; D043323:alpha-Mannosidase; D005944:Glucosamine