Quaternary and domain structure of glycoprotein processing glucosidase II
Quaternary and domain structure of glycoprotein processing glucosidase II
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DOI：
10.1021/bi010629u
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发表时间：
2001-09-04
期刊：
影响因子：
2.9
通讯作者：
Helenius, A
中科院分区：
文献类型：
--
作者：
Trombetta, ES;Fleming, KG;Helenius, A
新合成的糖蛋白中的葡萄糖修剪调节它们与钙联蛋白/钙网蛋白伴侣系统的相互作用。我们最近提出葡萄糖苷酶 II 由两个不同的亚基 α 和 β 组成。 α 亚基是催化成分，酵母中其同源物的缺失会消除葡萄糖苷酶 II 的活性。粟酒裂殖糖中非催化β亚基同源物的缺失会显着降低葡萄糖苷酶II活性，但β亚基在葡萄糖苷酶11活性中的作用尚未确定。此外，OL 和 fi 亚基之间的直接相互作用尚未得到证实。通过化学交联和分析超速离心的流体动力学分析，我们发现这两个亚基形成了一个确定的复合物，由一个催化亚基和一个辅助亚基（α（1）β（1））组成，分子量为161 kDa。该复合物的 s 值为 6.3 S，表明其形状高度非球状。 α (1)β (1) 复合物的不对称形状通过其对蛋白酶的高敏感性得到证实。 β 亚基可以通过蛋白水解从 α (1)β (1) 复合物中去除，而不影响催化作用，这表明它不是体外葡萄糖苷酶 II 活性所必需的。此外，我们分离了α亚基的单体C端片段，它保留了完整的葡萄糖苷酶活性。我们得出结论，葡萄糖苷酶 11 的催化核心位于 α 亚基的球状结构域中。它可以独立于β亚基发挥作用，而完整的α和β亚基组装成具有高度延伸构象的确定的异二聚体复合物，这可能有利于与内质网（ER）中的其他蛋白质相互作用。通过其C端HDEL信号，β亚基可以在ER中保留完整的α(1)β(1)复合物。
Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta. The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase 11 activity has not been established. Furthermore, a direct interaction between OL and fi subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha (1)beta (1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the alpha (1)beta (1) complex was confirmed by its high susceptibility to proteases. The beta subunit could be proteolytically removed from the alpha (1)beta (1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase 11 resides in a globular domain of the alpha subunit. which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha (1)beta (1) complex in the ER.