Wilkinson Barrie M, Purswani Jessica, Stirling Colin J
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
J Biol Chem. 2006 Mar 10;281(10):6325-33. doi: 10.1074/jbc.M510455200. Epub 2005 Dec 22.
Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose alpha-subunit contains the glycosyl hydrolase active site. The function of the beta-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the beta-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96-102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast alpha-subunit homologue Gls2p. Surprisingly, we found that Deltagtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.
葡萄糖苷酶II在内质网中糖蛋白生物合成过程中，对于从N-连接聚糖上顺序去除两个葡萄糖残基至关重要。该酶是一种异源二聚体，其α亚基含有糖基水解酶活性位点。β亚基的功能尚未明确，但人类基因中的突变已与常染色体显性多囊肝病相关联。在此，我们报告了酿酒酵母基因GTB1的鉴定和特征，该基因编码一种与人类葡萄糖苷酶II的β亚基具有21%序列相似性的多肽。Gtb1蛋白被证明是一种可溶性糖蛋白（96 - 102 kDa），定位于内质网腔，在那里它与酵母α亚基同源物Gls2p一起存在于一个复合物中。令人惊讶的是，我们发现缺失GTB1的突变细胞在单葡萄糖基化聚糖的加工过程中存在特异性缺陷。因此，尽管Gls2p足以切割倒数第二个葡萄糖残基，但Gtb1p对于切割最后一个葡萄糖至关重要。我们的数据表明，Gtb1p是正常糖蛋白生物合成所必需的，并揭示了N-聚糖加工中最后两个葡萄糖修剪步骤在机制上是不同的。
