The sugar-binding ability of human OS-9 and its involvement in ER-associated degradation

Abstract

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-9MRH) together with six kinds of mutated OS-9MRH were prepared and mixed with R-phycoerythrin (PE)-labeled streptavidin to form tetramers (OS-9MRH-SA). The PE-labeled OS-9MRH-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-9MRH-SA binding to the cells was strongly inhibited by Manα1,6(Manα1,3)Manα1,6(Manα1,3)Man and Manα1,6Man. To further determine the specificity of native ligands for OS-9MRH, frontal affinity chromatography was performed using a wide variety of 92 different oligosaccharides. We found that several N-glycans containing terminal α1,6-linked mannose in the Manα1,6(Manα1,3)Manα1,6(Manα1,3)Man structure were good ligands for OS-9MRH,havingKa values of approximately 104 M-1 and that trimming of either an α1,6-linked mannose from the C-arm or an α1,3-linked mannose from the B-arm abrogated binding to OS-9MRH. An immunopre-cipitation experiment demonstrated that the α1-antitrypsin variant nullHongKong, but not wild-type α1-antitrypsin, selectively interacted with OS-9 in the cells in a sugar-dependent manner. These results suggest that trimming of the outer-most α1,2-linked mannose on the C-arm is a critical process for misfolded proteins to enter ERAD. © The Author 2009. Published by Oxford University Press.