Puccia R, Grondin B, Herscovics A
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Biochem J. 1993 Feb 15;290 ( Pt 1)(Pt 1):21-6. doi: 10.1042/bj2900021.
Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis.
酿酒酵母中N-连接寡糖的加工起始于从Glc3Man9GlcNAc2去除葡萄糖和甘露糖残基，以形成Man8GlcNAc2的单一异构体。在编码负责Man8GlcNAc2合成的特定α1,2-甘露糖苷酶的MNS1基因被破坏的酿酒酵母菌株中，研究了甘露糖去除对于后续外链合成的重要性[Camirand、Heysen、Grondin和Herscovics（1991年）《生物化学杂志》266卷，15120 - 15127页]。在mns1细胞（即含有被破坏基因的细胞）的Northern印迹中未观察到1.85 kb和1.7 kb的MNS1转录本。在用[2-³H]甘露糖脉冲10分钟后，对内切β-N-乙酰葡糖胺酶-H敏感的寡糖进行Bio-Gel P-6分析，结果显示在对照细胞和mns1细胞中，从凝胶中排除的标记外链的量相似。对包含的寡糖进行高效液相色谱分析表明，mns1细胞中形成的中间体是Man9GlcNAc，而不是Man8GlcNAc。通过高效液相色谱分析从免疫沉淀的羧肽酶Y和蔗糖酶中得到的[³H]甘露糖标记的核心寡糖，结果显示mns1细胞和对照细胞中的大小分布相似。从[³⁵S]甲硫氨酸标记的mns1细胞中免疫沉淀的蔗糖酶高度糖基化，但在变性聚丙烯酰胺凝胶电泳上的迁移速度比对照细胞中的蔗糖酶略快，表明糖基化存在微小差异。在非变性凝胶电泳后的蔗糖酶活性染色中也观察到了类似的迁移差异。得出的结论是，由MNS1编码的α-甘露糖苷酶是体内唯一负责去除甘露糖的酶，并且这一加工步骤对于外链合成不是必需的。
