Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.
Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.
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DOI：
10.1016/s0021-9258(18)33332-5
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发表时间：
1982-12
期刊：
影响因子：
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通讯作者：
J. Byrd;A. Tarentino;F. Maley;P. H. Atkinson;R. B. Trimble
中科院分区：
文献类型：
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作者：
J. Byrd;A. Tarentino;F. Maley;P. H. Atkinson;R. B. Trimble
通过[2-3H]甘露糖标记和h内糖苷酶释放产物的凝胶过滤分析，在体内研究了酿酒酵母糖蛋白n -连接低聚糖的合成。小的低聚糖，Man8-14GlcNAc，和较大的产物，Man大于20GlcNAc，都被标记。连续和脉冲跟踪标记的动力学表明，转移到蛋白质的初始产物Glc3Man9GlcNAc2被快速（t1/2 = 3 min）修剪为Man8GlcNAc2，然后更慢（t1/2 = 10-20 min）延长为更大的低聚糖。两种标记方法均未发现小于Man8GlcNAc2的低聚糖。为了证实在体内观察到的修饰反应，我们在体外通过破碎的酵母细胞将牛甲状腺球蛋白中3h标记的man9 - n -乙酰氨基葡萄糖醇和酵母低聚糖脂中[14C]Man9GlcNAc2转化为3h标记的man8 - n -乙酰氨基葡萄糖醇和[14C]Man8GlcNAc2。用凝胶过滤法纯化酵母转化酶和牛甲状腺球蛋白中的Man8GlcNAc和Man9GlcNAc，并用高场1H-NMR进行分析。转化酶Man8GlcNAc (B)和Man9GlcNAc (C)是均相化合物，与甲状腺球蛋白的Man9GlcNAc (A)不同，其末端缺乏特异性α 1,2-连接甘露糖残基。转化酶(C)的Man9GlcNAc末端有一个额外的α 1,6连接甘露糖，并且在结构上与从含有mnn1和mnn2突变的酵母中分离出来的甘露糖相同（Cohen， R. E., Zhang, w . j.）。（1982） J. Biol。化学，257,5730-5737)。综上所述，从Glc3Man9GlcNAc2中去除葡萄糖和一个甘露糖形成的Man8GlcNAc2是修饰的最终产物，也是酵母糖蛋白上低聚糖延伸的最小前体。结果表明，通过高度特异性的α -甘露糖苷酶从Man9GlcNAc2中去除一个特定的末端α 1,2-连接的甘露糖，暴露了初生的man - α 1,6- man主链，以获得额外的α 1,6-连接的甘露糖残基，根据以下方案：（公式，见文本）。
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).