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Mol Biol Cell
2011 Oct 01;2219:3559-70. doi: 10.1091/mbc.E11-03-0201.
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Role of malectin in Glc(2)Man(9)GlcNAc(2)-dependent quality control of α1-antitrypsin.
Chen Y
,
Hu D
,
Yabe R
,
Tateno H
,
Qin SY
,
Matsumoto N
,
Hirabayashi J
,
Yamamoto K
.
???displayArticle.abstract??? Malectin was first discovered as a novel endoplasmic reticulum (ER)-resident lectin from Xenopus laevis that exhibits structural similarity to bacterial glycosylhydrolases. Like other intracellular lectins involved in glycoprotein quality control, malectin is highly conserved in animals. Here results from in vitro membrane-based binding assays and frontal affinity chromatography confirm that human malectin binds specifically to Glc(2)Man(9)GlcNAc(2) (G2M9) N-glycan, with a K(a) of 1.97 × 10(5) M(-1), whereas binding to Glc(1)Man(9)GlcNAc(2) (G1M9), Glc(3)Man(9)GlcNAc(2) (G3M9), and other N-glycans is barely detectable. Metabolic labeling and immunoprecipitation experiments demonstrate that before entering the calnexin cycle, the folding-defective human α1-antitrypsin variant null Hong Kong (AT(NHK)) stably associates with malectin, whereas wild-type α1-antitrypsin (AT) or N-glycan-truncated variant of AT(NHK) (AT(NHK)-Q3) dose not. Moreover, malectin overexpression dramatically inhibits the secretion of AT(NHK) through a mechanism that involves enhanced ER-associated protein degradation; by comparison, the secretion of AT and AT(NHK)-Q3 is only slightly affected by malectin overexpression. ER-stress induced by tunicamycin results in significantly elevated mRNA transcription of malectin. These observations suggest a possible role of malectin in regulating newly synthesized glycoproteins via G2M9 recognition.
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).
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