Human OS-9, a lectin required for glycoprotein endoplasmic reticulum-associated degradation, recognizes mannose-trimmed N-glycans.
In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of alpha1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER alpha1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.
From the Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan. Electronic address: nobuko@frontier.kyoto-u.ac.jp.
Nobuko
N
Hosokawa
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, Japan; Graduate School of Pharmaceutical Sciences, Nagaya City University, Nagoya 467-8063, Japan.
Yukiko
Y
Kamiya
Graduate School of Pharmaceutical Sciences, Nagaya City University, Nagoya 467-8063, Japan.
Daiki
D
Kamiya
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, Japan; Graduate School of Pharmaceutical Sciences, Nagaya City University, Nagoya 467-8063, Japan; The Glycoscience Institute, Ochanomizu University, Tokyo 112-8610, Japan. Electronic address: kkatonmr@ims.ac.jp.
Koichi
K
Kato
From the Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan.
Kazuhiro
K
Nagata
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D037102:Lectins; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D006023:Glycoproteins; D037102:Lectins; D037241:Mannose-Binding Lectins; D009363:Neoplasm Proteins; C098898:OS9 protein, human; D011134:Polysaccharides; D011506:Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C117384:SEL1L protein, human; D000515:alpha 1-Antitrypsin; D008358:Mannose