Chen Y , Hu D , Yabe R , Tateno H , Qin SY , Matsumoto N , Hirabayashi J , Yamamoto K .

	FIGURE 1:. Purification of recombinant malectin and malectin mutants. (A) Construction of E. coli expression vectors for recombinant malectin and malectin mutants. (B) Recombinant malectin and malectin mutants were refolded from inclusion bodies and purified. The proteins were analyzed by SDS–PAGE and visualized with Coomassie brilliant blue. (C) CD spectra of wild-type and mutant malectins were obtained as described in Materials and Methods.
	FIGURE 2:. Malectin binds to N-glycans on the surface of DNJ-treated cells. (A) HeLaS3 cells treated with 1 mM CST, 1 mM DNJ, 8.6 μM KIF, or 58 μM SW for 20 h were incubated with 0.5 μg/ml PE-labeled sMAL-SA (filled histogram) or PE-SA as a control (thin line) and then analyzed by flow cytometry. (B) HeLaS3 cells cultured in the presence of 1 mM DNJ for 20 h were treated with or without 104 U/ml endo H and then incubated with PE-labeled sMAL-SA (filled histogram) or PE-SA as a control (thin line), followed by flow cytometry. (C) DNJ-treated HeLaS3 cells were incubated with 0.5 μg/ml PE-labeled sMAL-SA in the presence of 10 mM lactose, nigerose, or α1,2-mannobiose and then analyzed by flow cytometry. (D) DNJ-treated HeLaS3 cells were incubated with 0.5 μg/ml PE-labeled sMAL-SA or the indicated malectin mutant and then analyzed by flow cytometry. The numbers indicate the mean fluorescence intensity.
	FIGURE 3:. Malectin specifically binds to diglucosylated high mannose–type glycans. (A) Structure of the N-glycan precursor G3M9. The mannose residues are organized into three branches, with a triglucosylated A-arm and the 6′-pentamannosyl branch subdivided into B- and C-arms. GI hydrolyzes α1,2-linked glucose and GII cleavages α1,3-linked glucoses at two sites. (B) Structures of the PA-labeled N-glycans used for FAC. (C) The affinity of each PA-labeled oligosaccharide for human malectin was determined by FAC. The Ka values represent the results of three independent experiments.
	FIGURE 4:. Significant interaction between malectin and ATNHK. (A) HeLa cells were transfected with expression vectors for human AT, ATNHK , and the N-glycosylation-deficient variant of ATNHK, ATNHK-Q3, along with FLAG-tagged malectin for 24 h. Cells were starved and then metabolically labeled with 35S-methionine and 35S-cysteine for 3 h. For DNJ treatment, the cells were cultured in the presence of 1 mM DNJ during the starvation and radiolabeling procedures. Cell lysates were subjected to immunoprecipitation using anti–human AT antibody (A) or anti-FLAG antibody (B), and then immune complexes were separated by SDS–PAGE under nonreducing conditions. Asterisk represents dimeric form. (C) HeLa cells were transfected with expression vectors for ATNHK and FLAG-tagged malectin, radiolabeled, and then treated with 1 mM DNJ or CST. Radiolabeled cell lysates were subjected to immunoprecipitation using anti-FLAG antibody or anti–human AT antibody, and then immune complexes were analyzed by SDS–PAGE under nonreducing conditions. (D) HeLa cells were transfected with expression vectors for FLAG-tagged wild-type malectin or the indicated malectin mutants (Y104A, Y131A, F132A, or D201A) along with an expression vector for ATNHK. Cells were radiolabeled and cultured in the presence of 1 mM DNJ. Radiolabeled cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody; immune complexes were analyzed by SDS–PAGE under nonreducing conditions. (E) Quantification of the results in D.
	FIGURE 5:. Overexpression of malectin reduces the association of CNX with ATNHK. HeLa cells were cotransfected with pcDNA3.1-AT or pcDNA3.1-ATNHK along with p3xFLAG-CMV9-malectin or an empty vector as a control. Cells were cultured in the presence or absence of 1 mM DNJ. Cell lysates were subjected to immunoprecipitation using anti-CNX antibody, and cell lysates (A) and immune complexes (B) were analyzed by SDS–PAGE under nonreducing conditions, followed by immunoblot using anti–human AT antibody. (C) Data of B were quantified by scanning densitometry. The data shown are mean ± SD from four independent experiments. NS, not significant.
	FIGURE 6:. Malectin overexpression abrogates the secretion of ATNHK. HeLa cells were transfected with expression vectors for FLAG-tagged malectin and AT, ATNHK, or ATNHK-Q3 for 24 h. Cells were cultured in serum-free medium for 48 h, and then cell lysate and medium were collected. (A) To confirm the expression levels of FLAG-tagged malectin, immunoblot was performed using anti-FLAG and anti–β-actin antibodies, respectively. The presence of AT (B) ATNHK and ATNHK-Q3 (C) in the cell lysate or culture medium was analyzed by immunoblot using an anti–human AT antibody. (D) HeLa cells were transfected with expression vectors for FLAG-tagged malectin or the indicated malectin mutants along with ATNHK. Secreted and intracellular ATNHK, FLAG-tagged malectin, and β-actin were analyzed by immunoblot. (E) Data were quantified by scanning densitometry from fluorographs. Secreted ATNHK in the culture medium was detected by immunoblot using an anti–human AT antibody. Data are representative of two independent experiments.
	FIGURE 7:. Downregulation of ATNHK secretion is due to enhancement of ERAD. (A) HeLa cells were transfected with expression vectors for FLAG-tagged malectin, or empty vector, and ATNHK. After 24 h, cells were cultured in complete medium containing 2 μM MG132. The culture medium and cell lysates were collected 48 h later. Secreted and intracellular ATNHK, FLAG-tagged malectin, and β-actin were analyzed by immunoblot using the indicated antibodies. (B) Data were quantified as described for Figure 6E. The data shown are mean ± SD from three independent experiments. NS, not significant. (C) HeLa cells were transfected with expression vectors for malectin and FLAG-tagged OS-9 along with expression vectors for ATNHK or ATNHK-Q3. After 24 h, cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody, and then the presence of coimmunoprecipitated ATNHK or ATNHK-Q3 (right) was analyzed by SDS–PAGE under nonreducing conditions. Protein expression was monitored using an anti–human AT antibody, anti–human malectin antibody, or anti-FLAG antibody (left).

Arnold, Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity. 2000, Pubmed

Arnold, Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity. 2000, Pubmed
Barile, Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct. 2005, Pubmed
Bernasconi, A dual task for the Xbp1-responsive OS-9 variants in the mammalian endoplasmic reticulum: inhibiting secretion of misfolded protein conformers and enhancing their disposal. 2008, Pubmed
Bukau, Getting newly synthesized proteins into shape. 2000, Pubmed
Christianson, OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD. 2008, Pubmed
Clerc, Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. 2009, Pubmed
Deprez, More than one glycan is needed for ER glucosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle. 2005, Pubmed
Elbein, Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I. 1990, Pubmed
Galli, Malectin participates in a backup glycoprotein quality control pathway in the mammalian ER. 2011, Pubmed
Hammond, Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. 1994, Pubmed
Hartl, Molecular chaperones in cellular protein folding. 1996, Pubmed
Helenius, Roles of N-linked glycans in the endoplasmic reticulum. 2004, Pubmed
Hirao, EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. 2006, Pubmed
Hosokawa, EDEM accelerates ERAD by preventing aberrant dimer formation of misfolded alpha1-antitrypsin. 2006, Pubmed
Hosokawa, Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP. 2008, Pubmed
Hosokawa, Human OS-9, a lectin required for glycoprotein endoplasmic reticulum-associated degradation, recognizes mannose-trimmed N-glycans. 2009, Pubmed
Hosokawa, A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation. 2001, Pubmed
Hosokawa, Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I. 2003, Pubmed
Hu, Sugar-binding activity of the MRH domain in the ER alpha-glucosidase II beta subunit is important for efficient glucose trimming. 2009, Pubmed
Kawasaki, Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains. 2007, Pubmed
Kawasaki, The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2. 2008, Pubmed
Le, Association between calnexin and a secretion-incompetent variant of human alpha 1-antitrypsin. 1994, Pubmed
Lederkremer, Glycoprotein folding, quality control and ER-associated degradation. 2009, Pubmed
Lee, Proteasome inhibitors: valuable new tools for cell biologists. 1998, Pubmed
Liu, Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome. 1999, Pubmed
Maley, Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. 1989, Pubmed
Mikami, The sugar-binding ability of human OS-9 and its involvement in ER-associated degradation. 2010, Pubmed
Ni, ER chaperones in mammalian development and human diseases. 2007, Pubmed
Niwa, Efficient selection for high-expression transfectants with a novel eukaryotic vector. 1991, Pubmed
Nyfeler, Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin. 2008, Pubmed
Olivari, A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation. 2005, Pubmed
Ou, Association of folding intermediates of glycoproteins with calnexin during protein maturation. 1993, Pubmed
Palma, Multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin. 2010, Pubmed
Sasak, Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells. 1985, Pubmed
Satoh, Structural basis for oligosaccharide recognition of misfolded glycoproteins by OS-9 in ER-associated degradation. 2010, Pubmed
Saunier, Inhibition of N-linked complex oligosaccharide formation by 1-deoxynojirimycin, an inhibitor of processing glucosidases. 1982, Pubmed
Schallus, Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation. 2008, Pubmed , Xenbase
Schallus, Analysis of the specific interactions between the lectin domain of malectin and diglucosides. 2010, Pubmed , Xenbase
Sifers, A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum. 1988, Pubmed
Stigliano, Glucosidase II beta subunit modulates N-glycan trimming in fission yeasts and mammals. 2009, Pubmed
Tateno, Frontal affinity chromatography: sugar-protein interactions. 2007, Pubmed
Taylor, The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. 2004, Pubmed
Totani, The recognition motif of the glycoprotein-folding sensor enzyme UDP-Glc:glycoprotein glucosyltransferase. 2009, Pubmed
Travis, Human plasma proteinase inhibitors. 1983, Pubmed
Tulsiani, Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. 1982, Pubmed
Tulsiani, Swainsonine induces the production of hybrid glycoproteins and accumulation of oligosaccharides in male reproductive tissues of the rat. 1990, Pubmed
Ware, The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. 1995, Pubmed
Yamaguchi, VIPL has sugar-binding activity specific for high-mannose-type N-glycans, and glucosylation of the alpha1,2 mannotriosyl branch blocks its binding. 2007, Pubmed
Yamaguchi, Human XTP3-B binds to alpha1-antitrypsin variant null(Hong Kong) via the C-terminal MRH domain in a glycan-dependent manner. 2010, Pubmed
Yamamoto, Detection of weak-binding sugar activity using membrane-based carbohydrates. 2010, Pubmed
Zapun, Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57. 1998, Pubmed