Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Glycobiology. 2010 Mar;20(3):310-21. doi: 10.1093/glycob/cwp175. Epub 2009 Nov 12.
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 Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man and Manalpha1,6Man. 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 alpha1,6-linked mannose in the Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)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 alpha1,6-linked mannose from the C-arm or an alpha1,3-linked mannose from the B-arm abrogated binding to OS-9(MRH). An immunoprecipitation experiment demonstrated that the alpha1-antitrypsin variant null(Hong Kong), but not wild-type alpha1-antitrypsin, selectively interacted with OS-9 in the cells in a sugar-dependent manner. These results suggest that trimming of the outermost alpha1,2-linked mannose on the C-arm is a critical process for misfolded proteins to enter ERAD.
错误折叠的糖蛋白从内质网（ER）易位到细胞质中，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 的重组生物素化甘露糖 6-磷酸受体同源（MRH）结构域（OS-9（MRH））以及六种突变 OS-9（MRH），并与藻红蛋白（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 通过与阳离子依赖性甘露糖 6-磷酸受体参与糖结合的同源氨基酸残基，以非依赖金属离子的方式与 HeLaS3 细胞结合，并且这种结合通过 swainsonine、脱氧甘露尼辛或 kifunensine 处理大大增加。N-乙酰葡糖胺基转移酶 I 缺陷型 Lec1 细胞，而不是 Lec2 或 Lec8 细胞，也被四聚体强烈结合。OS-9（MRH）-SA 与细胞的结合被 Manalpha1,6（Manalpha1,3）Manalpha1,6（Manalpha1,3）Man 和 Manalpha1,6Man 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 92 种不同的寡糖进行了前沿亲和层析。我们发现，在 Manalpha1,6（Manalpha1,3）Manalpha1,6（Manalpha1,3）Man 结构中含有末端 alpha1,6 连接的甘露糖的几种 N-聚糖是 OS-9（MRH）的良好配体，具有约 10（4）M（-1）的 K（a）值，并且从 C 臂上切除任一 alpha1,6 连接的甘露糖或从 B 臂上切除 alpha1,3 连接的甘露糖都会破坏与 OS-9（MRH）的结合。免疫沉淀实验表明，突变型 alpha1-抗胰蛋白酶变体 null（香港），而不是野生型 alpha1-抗胰蛋白酶，以糖依赖的方式选择性地与细胞中的 OS-9 相互作用。这些结果表明，C 臂上最外层 alpha1,2 连接的甘露糖的修剪是错误折叠的蛋白质进入 ERAD 的关键过程。
