The sugar-binding ability of human OS-9 and its involvement in ER-associated degradation
The sugar-binding ability of human OS-9 and its involvement in ER-associated degradation
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DOI：
10.1093/glycob/cwp175
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发表时间：
2010-03-01
期刊：
影响因子：
4.3
通讯作者：
Yamamoto, Kazuo
中科院分区：
文献类型：
--
作者：
Mikami, Kaoru;Yamaguchi, Daisuke;Yamamoto, Kazuo
错误折叠的糖蛋白从内质网（ER）转位到细胞质中进行蛋白酶体介导的降解。OS-9蛋白被认为参与ER相关糖蛋白降解（ERAD）。制备了人OS-9甘露糖6-磷酸受体同源结构域（OS-9（MRH））和6种突变的OS-9（MRH），并与R-藻红蛋白（PE）标记的链霉亲和素混合形成四聚体（OS-9（MRH）-SA）。PE标记的OS-9（MRH）-SA结合到HeLaS 3细胞中的金属离子不依赖的方式，通过氨基酸残基同源的那些参与糖结合的阳离子依赖性甘露糖6-磷酸受体，这种结合大大增加了苦马豆素，脱氧mannojirimycin，或kifunensine治疗。N-乙酰葡糖胺转移酶I缺陷的Lec 1细胞，但不是Lec 2或Lec 8细胞，也强烈结合的四聚体。OS-9（MRH）-SA与细胞的结合被Man α 1，6（Man α 1，3）Man α 1，6（Man α 1，3）Man和Man α 1，6 Man强烈抑制。为了进一步确定OS-9（MRH）的天然配体的特异性，使用各种各样的92种不同的寡糖进行正面亲和层析。我们发现，在Man α 1，6-甘露糖链中含有几个末端α 1，6-甘露糖的N-聚糖，（Man alpha 1，3）Man alpha 1，6（Man α 1，3）Man结构-是OS-9（MRH）的良好配体，具有约10（4）M-1的K-a值，并且从C-臂或α 1，来自B臂的3-连接的甘露糖消除了与OS-9（MRH）的结合。免疫沉淀实验表明，α 1-抗胰蛋白酶变体null（香港），但不是野生型α 1-抗胰蛋白酶，选择性地相互作用与OS-9在细胞中的糖依赖性方式。这些结果表明，修剪C臂上最外面的α 1，2-连接的甘露糖是错误折叠的蛋白质进入ERAD的关键过程。
Misfolded glycoproteins are translocated from the endoplasmic reticulum (ER) into the cytoplasm for proteasome-mediated degradation. OS-9 protein is thought to participate in ER-associated glycoprotein degradation (ERAD). The recombinant biotinylated mannose 6-phosphate receptor homology (MRH) domain of human OS-9 (OS-9(MRH)) together with six kinds of mutated OS-9(MRH) were prepared and mixed with R-phycoerythrin (PE)-labeled streptavidin to form tetramers (OS-9(MRH)-SA). The PE-labeled OS-9(MRH)-SA bound to HeLaS3 cells in a metal ion-independent manner through amino acid residues homologous to those participating in sugar binding of the cation-dependent mannose 6-phosphate receptor, and this binding was greatly increased by swainsonine, deoxymannojirimycin, or kifunensine treatment. N-Acetylglucosaminyltransferase I-deficient Lec1 cells, but not Lec2 or Lec8 cells, were also strongly bound by the tetramer. OS-9(MRH)-SA binding to the cells was strongly inhibited by Man alpha 1,6(Man alpha 1,3) Man alpha 1,6(Man alpha 1,3) Man and Man alpha 1,6Man. To further determine the specificity of native ligands for OS-9(MRH), frontal affinity chromatography was performed using a wide variety of 92 different oligosaccharides. We found that several N-glycans containing terminal alpha 1,6-linked mannose in the Man alpha 1,6(Man alpha 1,3) Man alpha 1,6(Man alpha 1,3) Man structure-were good ligands for OS-9(MRH), having K-a values of approximately 10(4) M-1 and that trimming of either an alpha 1,6-linked mannose from the C-arm or an alpha 1,3-linked mannose from the B-arm abrogated binding to OS-9(MRH). An immunoprecipitation experiment demonstrated that the alpha 1-antitrypsin variant null(Hong Kong), but not wild-type alpha 1-antitrypsin, selectively interacted with OS-9 in the cells in a sugar-dependent manner. These results suggest that trimming of the outermost alpha 1,2-linked mannose on the C-arm is a critical process for misfolded proteins to enter ERAD.