Gañán S, Cazzulo J J, Parodi A J
Instituto de Investigaciones Bioquímicas, Fundación Campomar, Buenos Aires, Argentina.
Biochemistry. 1991 Mar 26;30(12):3098-104. doi: 10.1021/bi00226a017.
N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.
缺乏葡萄糖残基的N-连接高甘露糖型寡糖可能会在哺乳动物、植物、真菌和原生动物细胞的内质网中直接从UDP-葡萄糖进行瞬时糖基化。形成的产物已被鉴定为N-连接的Glc1Man5-9GlcNAc2，葡糖苷酶II显然是负责这些化合物在体内去糖基化的酶。由于新糖基化的糖蛋白会立即去糖基化，目前尚不清楚瞬时糖基化是涉及所有或几乎所有N-连接的糖蛋白，还是相反，仅影响其中一小部分。为了评估被糖基化的N-连接寡糖的摩尔比例，锥虫原生动物克氏锥虫（一种在蛋白质N-糖基化中转移Man9GlcNAc2的寄生虫）的细胞在[14C]葡萄糖以及葡糖苷酶II抑制剂脱氧野尻霉素和栗精胺存在的情况下培养，其浓度比产生50%克氏锥虫酶抑制所需的浓度高1000倍以上。约52 - 53%的总N-连接寡糖似乎含有葡萄糖残基。这些化合物被鉴定为Glc1Man7-9GlcNAc2。当细胞在脱氧野尻霉素存在下用[14C]葡萄糖脉冲追踪60分钟时，得到了相同的百分比。未获得从糖基化化合物产生GlcMan的内切甘露糖苷酶存在的证据。由于糖蛋白中每个分子的N-连接寡糖平均数量高于一个，这些结果表明超过52 - 53%的总糖蛋白被糖基化，并且瞬时糖基化是糖蛋白正常加工过程中的一个主要事件。
