The sugar-binding ability of human OS-9 and its involvement in ER-associated degradation.
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.
Mikami
Kaoru
K
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Yamaguchi
Daisuke
D
Tateno
Hiroaki
H
Hu
Dan
D
Qin
Sheng-Ying
SY
Kawasaki
Norihito
N
Yamada
Michiyuki
M
Matsumoto
Naoki
N
Hirabayashi
Jun
J
Ito
Yukishige
Y
Yamamoto
Kazuo
K
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism
D037102:Lectins; D009363:Neoplasm Proteins; C098898:OS9 protein, human; D011134:Polysaccharides