Cloning and expression of a specific human α1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis
Cloning and expression of a specific human α1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis
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DOI：
10.1093/glycob/9.10.1073
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发表时间：
1999-10-01
期刊：
影响因子：
4.3
通讯作者：
Herscovics, A
中科院分区：
文献类型：
--
作者：
Tremblay, LO;Herscovics, A
我们报道了一种新型人类 cDNA 的分离，该 cDNA 编码 79.5 kDa 的 II 型膜蛋白，其氨基酸序列与 I 类 α1,2-甘露糖苷酶相似。该酶的催化结构域在毕赤酵母中表达为分泌蛋白。重组酶从 Man(9)GlcNAc 中去除单个甘露糖残基，[H-1]-NMR 分析表明唯一的产物是 Man(8)GlcNAc 异构体 B，即缺少中臂末端 α 1,2-甘露糖的形式。酶活性需要钙，1-deoxymannojirimycin 和 kifunensine 都会抑制人 α1,2-甘露糖苷酶。这种人 α 1,2-甘露糖苷酶的特性和特异性与来自酿酒酵母的内质网 α 1,2-甘露糖苷酶相同，但与之前克隆的高尔基体 α 1d,2-甘露糖苷酶不同，后者在 N-聚糖成熟过程中从 Man(9)GlcNAc(2) 中去除最多四个甘露糖残基。 Northern 印迹分析表明，所有检查的人体组织都表达不同数量的 3 kb 转录物。这种高度特异性的 α 1,2-甘露糖苷酶可能参与糖蛋白质量控制，因为越来越多的证据表明，酵母细胞中将 Man(9)GlcNAc(2) 修剪为 Man(9)GlcNAc(2) 异构体 B 对于靶向错误折叠的糖蛋白进行降解非常重要。
We report the isolation of a novel human cDNA encoding a type II membrane protein of 79.5 kDa with amino acid sequence similarity to Class I alpha 1,2-mannosidases. The catalytic domain of the enzyme was expressed as a secreted protein in Pichia pastoris. The recombinant enzyme removes a single mannose residue from Man(9)GlcNAc and [H-1]-NMR analysis indicates that the only product is Man(8)GlcNAc isomer B, the form lacking the middle-arm terminal alpha 1,2-mannose. Calcium is required for enzyme activity and both 1-deoxymannojirimycin and kifunensine inhibit the human alpha 1,2-mannosidase. The properties and specificity of this human alpha 1,2-mannosidase are identical to the endoplasmic reticulum alpha 1,2-mannosidase from Saccharomyces cerevisiae and differ from those of previously cloned Golgi alpha 1d,2-mannosidases that remove up to four mannose residues from Man(9)GlcNAc(2) during N-glycan maturation. Northern blot analysis showed that all human tissues examined express variable amounts of a 3 kb transcript. This highly specific alpha 1,2-mannosidase is likely to be involved in glycoprotein quality control since there is increasing evidence that trimming of Man(9)GlcNAc(2) to Man(9)GlcNAc(2) isomer B in yeast cells is important to target misfolded glycoproteins for degradation.