Functional analysis of EDEM which accelerates glycoprotein ERAD

在本研究中，我进一步分析了EDEM（ER降解增强α-甘露糖苷酶样蛋白）的功能，其加速糖蛋白的ERAD（ER相关降解）（EMBO Reports，2，415 -422（2001））。1. ER甘露糖苷酶I（ER ManI）是一种将N-连接的寡糖中的甘露糖残基修剪掉的酶，这种修剪是触发ERAD错误折叠的糖蛋白的关键事件。EDEM与ER ManI同源，但我们发现EDEM缺乏作为加工α-甘露糖苷酶的酶活性。通过分析错误折叠的NHK上的寡糖结构，（α1-抗胰蛋白酶变体null Hong Kong），我们阐明了ER ManI和EDEM对糖蛋白ERAd的作用机制不同（JBC，278，26287-26294，（2003））。2.ERAD底物NHK在其C-末端附近具有一个半胱氨酸残基，并且我们发现NHK形成分子内二硫桥，导致细胞内NHK二聚体形成。EDEM过表达抑制NHK二聚体的形成，而EDEM对野生型α1-抗胰蛋白酶的合成和分泌没有影响。这些结果表明，EDEM与内质网中的错误折叠糖蛋白结合，并作为错误折叠糖蛋白的分子伴侣，保持其降解能力。3.我们克隆了一个EDEM同源蛋白，命名为EDEM 3。EDEM 3是一种内质网腔蛋白，当它在293细胞中过表达时，能像EDEM 1一样增强糖蛋白ERAD。我们已经发现，这些同源蛋白对糖蛋白ERAD的机制是离散的，并且它们的表达受到ER应激和组织中的不同调节。

In the present study, I have further analyzed the function of EDEM (ER degradation enhancing α-mannosidase-like protein), which accelerates ERAD (ER associated degradation) of glycoproteins (EMBO Reports, 2,415-422 (2001)). The results which I obtained are summarized as follows :1.ER mannosidase I(ER ManI) is an enzyme which trimmes mannose residues from N-linked oligosaccharides, and this trimming is a key event that triggers a misfolded glycoprotein for ERAD. EDEM is homologous to ER ManI, but we have found that EDEM lacks enzyme activity as a processing α-mannosidase. By analyzing the oligosaccharide structures on misfolded NHK(α1-antitrypsin variant null Hong Kong), we have clarified that the mechanisms of ER ManI and EDEM on glycoprotein ERAd are different (JBC, 278, 26287-26294, (2003)).2.An ERAD substrate NHK has one cysteine residue near its C-terminus, and we have found that NHK makes an intramolecular disulfide bridge, resulting in NHK dimer formation within the cells. EDEM overexpression inhibited NHK dimer formation, whereas EDEM had no effect on the synthesis and secretion of wild type α1-antitrypsin. These results suggested that EDEM binds to misfolded glycoproteins in the ER, and that it acts like a molecular chaperone protein of misfolded glycoproteins by keeping them degradation competent.3.We have cloned an EDEM homologue protein, which we named EDEM3. EDEM3 is an ER lumemal protein, and enhanced the glycoprotein ERAD like EDEM1 when it was overexpressed in 293 cells. We have found that the mechanisms of these homologue proteins on glycoprotein ERAD are discrete and that their expressions are differently regulated by the ER stress and in tissues.