Fujimoto K, Kornfeld R
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1991 Feb 25;266(6):3571-8.
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.
在正常的亲代BW5147小鼠淋巴瘤细胞和α-葡萄糖苷酶II缺陷型PHAR2.7突变细胞中，研究了N-连接寡糖加工的动力学以及加工中间体的结构。突变细胞积累了糖基化中间体，但能够将约40%的寡糖去糖基化并加工成复合型。这种加工并非由于残留的α-葡萄糖苷酶II活性，因为α-葡萄糖苷酶抑制剂1-脱氧野尻霉素（DNJ）和N-丁基-DNJ并不能阻止它。在存在DNJ和N-丁基-DNJ的情况下，亲代细胞也表现出与α-葡萄糖苷酶II无关的加工。来自亲代和突变细胞的膜制剂都具有内切α-甘露糖苷酶活性，即能将Glc1,2Man9GlcNAc裂解为Glc1,2Man加Man8GlcNAc，这表明这是突变细胞中形成复合寡糖的替代途径的一个候选者。一项平衡研究中，从[2-³H]甘露糖标记的突变细胞中分离并分析了细胞糖蛋白、细胞内水溶性糖类和分泌到培养基中的糖类，结果表明细胞形成的二糖和三糖Glc1Man和Glc2Man的量与细胞糖蛋白中发现的去糖基化寡糖相当。这一结果明确表明，α-葡萄糖苷酶II缺陷型突变细胞利用内切α-甘露糖苷酶作为N-连接寡糖加工的旁路途径。
