Gonzalez D S, Karaveg K, Vandersall-Nairn A S, Lal A, Moremen K W
Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
J Biol Chem. 1999 Jul 23;274(30):21375-86. doi: 10.1074/jbc.274.30.21375.
We have isolated a full-length cDNA clone encoding a human alpha1, 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 approximately 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 alpha1,2-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 Ca(2+). 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).
我们分离出了一个编码人α1,2-甘露糖苷酶的全长cDNA克隆，该酶催化哺乳动物N-连接寡糖加工过程中的第一步甘露糖修剪反应。有人提出这种酶可调控真核细胞内质网（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鉴定出人类表达序列标签克隆，并通过5'-cDNA末端快速扩增和从人胎盘cDNA进行直接聚合酶链反应相结合的方法分离出假定甘露糖苷酶同源物的全长编码区。开放阅读框预测为一个663个氨基酸的II型跨膜多肽，带有短的胞质尾巴（47个氨基酸）、单个跨膜结构域（22个氨基酸）和大的COOH末端催化结构域（594个氨基酸）。Northern印迹检测到在人类组织中普遍表达的约2.8千碱基对的转录本。在正常大鼠肾细胞中表达人甘露糖苷酶同源物的表位标签全长形式，其定位呈现内质网模式。当在COS细胞中表达由与人甘露糖苷酶同源物的COOH末端腔内结构域融合的蛋白A组成的重组蛋白时，发现该融合蛋白仅从Man(9)GlcNAc(2)上切割单个α1,2-甘露糖残基，产生独特的Man(8)GlcNAc(2)异构体（Man8B）。如用EDTA或EGTA抑制并通过添加Ca(2+)逆转抑制所示，甘露糖切割反应需要二价阳离子。该酶对脱氧甘露基野尻霉素和基夫内新敏感，但对苦马豆素不敏感。关于定位、底物特异性和抑制剂谱的结果表明，此处报道的cDNA编码一种先前称为内质网甘露糖苷酶I的酶。使用人内质网甘露糖苷酶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)寡糖切割为Man(5)GlcNAc(2)方面具有互补性。
