Identification, expression, and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis
Identification, expression, and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis
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DOI：
10.1074/jbc.274.30.21375
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发表时间：
1999-07-23
影响因子：
4.8
通讯作者：
Moremen, KW
中科院分区：
文献类型：
--
作者：
Gonzalez, DS;Karaveg, K;Moremen, KW
我们分离了编码人 α1,2-甘露糖苷酶的全长 cDNA 克隆，该酶催化哺乳动物 Asn 连接寡糖加工中的第一个甘露糖修剪步骤。该酶被提议用于调节真核细胞内质网 (ER) 中质量控制糖蛋白降解的时间。通过与哺乳动物和酵母寡糖加工甘露糖苷酶的序列相似性来鉴定人表达序列标签克隆，并通过 cDNA 末端的 5'-快速扩增和来自人胎盘 cDNA 的直接聚合酶链式反应的组合来分离推定的甘露糖苷酶同源物的全长编码区。开放阅读框预测了一个 663 个氨基酸的 II 型跨膜多肽，具有短胞质尾（47 个氨基酸）、单个跨膜结构域（22 个氨基酸）和一个大的 COOH 末端催化结构域（594 个氨基酸）。 Northern 印迹检测到类似于 2.8 KB 对的转录本，该转录本在人体组织中普遍表达。在正常大鼠肾细胞中表位标记的全长形式的人甘露糖苷酶同源物的表达导致了 ER 定位模式。当由与人甘露糖苷酶同源物的 COOH 末端管腔结构域融合的蛋白 A 组成的重组蛋白在 COS 细胞中表达时，发现该融合蛋白仅从 Man(9)GlcNAc(2) 上裂解单个 α1,2-甘露糖残基，产生独特的 α1,2-甘露糖残基。 Man(8)GlcNAc(2) 异构体 (Man8B)。甘露糖裂解反应需要二价阳离子，如用 EDTA 或 EGTA 抑制并通过添加 Ca2+ 逆转抑制所示。该酶对脱氧甘露尻霉素和 kifunensine 的抑制也很敏感，但对苦马豆素不敏感。定位、底物特异性和抑制剂谱的结果表明，本文报道的 cDNA 编码先前指定为 ER 甘露糖苷酶 I 的酶。使用人 ER 甘露糖苷酶 I 和重组高尔基体甘露糖苷酶 IA 组合进行的酶反应表明，这两种酶在将 Man(9)GlcNAc(2) 寡糖裂解为 Man(5)GlcNAc(2) 方面是互补的。
We have isolated a full-length cDNA clone encoding a human alpha 1,2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asn-linked oligosaccharides. This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endoplasmic reticulum (ER) of eukaryotic cells. Human expressed sequence tag clones were identified by sequence similarity to mammalian and yeast oligosaccharide-processing mannosidases, and the full-length coding region of the putative mannosidase homolog was isolated by a combination of 5'-rapid amplification of cDNA ends and direct polymerase chain reaction from human placental cDNA. The open reading frame predicted a 663-amino acid type II transmembrane polypeptide with a short cytoplasmic tail (47 amino acids), a single transmembrane domain (22 amino acids), and a large COOH-terminal catalytic domain (594 amino acids). Northern blots detected a transcript of similar to 2.8 kilobase pairs that was ubiquitously expressed in human tissues. Expression of an epitope-tagged full-length form of the human mannosidase homolog in normal rat kidney cells resulted in an ER pattern of localization, When a recombinant protein, consisting of protein A fused to the COOH-terminal luminal domain of the human mannosidase homolog, was expressed in COS cells, the fusion protein was found to cleave only a single alpha 1,2-mannose residue from Man(9)GlcNAc(2) to produce a unique Man(8)GlcNAc(2) isomer (Man8B). The mannose cleavage reaction required divalent cations as indicated by inhibition with EDTA or EGTA and reversal of the inhibition by the addition of Ca2+. The enzyme was also sensitive to inhibition by deoxymannojirimycin and kifunensine, but not swainsonine. The results on the localization, substrate specificity, and inhibitor profiles indicate that the cDNA reported here encodes an enzyme previously designated ER mannosidase I. Enzyme reactions using a combination of human ER mannosidase I and recombinant Golgi mannosidase IA indicated that that these two enzymes are complementary in their cleavage of Man(9)GlcNAc(2) oligosaccharides to Man(5)GlcNAc(2).