Functional analysis of EDEM which accelerates glycoprotein ERAD

加速糖蛋白 ERAD 的 EDEM 功能分析

基本信息

批准号：
15570157
负责人：
HOSOKAWA Nobuko
金额：
$ 1.79万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

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，该功能加速了糖蛋白的ERAD（ER相关降解）（EMBO Reports，2,415-422（2001））。我获得的结果总结如下：1。甘露糖苷酶I（ER Mani）是一种从N-连接的寡糖中修剪甘露糖残留物的酶，并且这种修剪是一个关键事件，可触发ERAD的错误折叠的糖蛋白。 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（α1-抗胰蛋白酶变体无效洪孔）的寡糖结构，我们已经澄清说，Er Mani和Edem在糖蛋白上的机制是不同的（JBC，JBC，278，278，278，26287-26294，（2003））。而且我们发现NHK建立了分子内二硫键，从而导致细胞内NHK二聚体形成。 EDEM过表达抑制了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ER中错误折叠的糖蛋白结合，并且它的作用像是通过使它们降解均能降解的分子链蛋白的分子链酮蛋白。3.我们将EDEM同源物蛋白克隆，我们命名EDEM3。 EDEM3是一种ER腔蛋白，当在293个细胞中过表达时，可以增强糖蛋白ERAD，如EDEM1。我们发现，这些同源蛋白在糖蛋白上的机制是离散的，它们的表达受到ER应激和组织中的不同调节。