Journal
GLYCOBIOLOGY
Volume 20, Issue 3, Pages 310-321Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwp175
Keywords
ERAD; lectin; N-glycan; OS-9; sugar-binding specificity
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Funding
- Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency
- Ministry of Education, Culture, Sports, Science and Technology, Japan [21390173]
- Grants-in-Aid for Scientific Research [21390173] Funding Source: KAKEN
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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.
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