Byrd J C, Tarentino A L, Maley F, Atkinson P H, Trimble R B
J Biol Chem. 1982 Dec 25;257(24):14657-66.
Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).
通过用[2-³H]甘露糖标记并对内切糖苷酶H释放的产物进行凝胶过滤分析，在体内研究了酿酒酵母糖蛋白N-连接寡糖的合成。小寡糖（Man8-14GlcNAc）和较大的产物（Man大于20GlcNAc）都被标记。连续标记和脉冲追踪标记的动力学表明，转移到蛋白质上的初始产物Glc3Man9GlcNAc2迅速（半衰期约为3分钟）被修剪为Man8GlcNAc2，然后更缓慢地（半衰期 = 10 - 20分钟）延长为更大的寡糖。两种标记方法都未发现明显小于Man8GlcNAc2的寡糖。为了证实体内观察到的修剪反应，来自牛甲状腺球蛋白的³H标记的Man9-N-乙酰葡糖胺醇和来自酵母寡糖-脂质的[¹⁴C]Man9GlcNAc2在体外被破碎的酵母细胞转化为³H标记的Man8-N-乙酰葡糖胺醇和[¹⁴C]Man8GlcNAc2。通过凝胶过滤纯化来自酵母转化酶和牛甲状腺球蛋白的Man8GlcNAc和Man9GlcNAc，并通过高场¹H-NMR分析进行检测。转化酶Man8GlcNAc（B）和Man9GlcNAc（C）是均一的化合物，它们与甲状腺球蛋白的Man9GlcNAc（A）不同，缺少一个特定的末端α1,2-连接的甘露糖残基。转化酶的Man9GlcNAc（C）有一个额外的末端α1,6-连接的甘露糖，其结构与从含有mnn1和mnn2突变的酵母中分离得到的甘露糖相同（科恩，R.E.，张，W.-j.，和巴卢，C.E.（1982年）《生物化学杂志》257，5730 - 5737）。得出的结论是，通过从Glc3Man9GlcNAc2中去除葡萄糖和一个甘露糖形成的Man8GlcNAc2是修剪的最终产物，也是酵母糖蛋白上寡糖延长的最小前体。结果表明，一种高度特异性的α-甘露糖苷酶从Man9GlcNAc2中去除特定的末端α1,2-连接的甘露糖，暴露出新生的Man-α1,6-Man主链，以便根据以下方案用额外的α1,6-连接的甘露糖残基进行延长：（公式，见原文）。
