Enhancement of Endoplasmic Reticulum (ER) Degradation of Misfolded Null Hong Kong α1-Antitrypsin by Human ER Mannosidase I

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong α1-antitrypsin is enhanced by overexpression of the ER processing α1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded α1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415–422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded α1-antitrypsin. We also show that misfolded α1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5–9GlcNAc released by endo-β-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5–7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested. Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong α1-antitrypsin is enhanced by overexpression of the ER processing α1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded α1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415–422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded α1-antitrypsin. We also show that misfolded α1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5–9GlcNAc released by endo-β-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5–7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested. The synthesis of glycoproteins containing N-glycans begins in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; A1AT NHK, α1-antitrypsin-null Hong Kong; ER ManI, ER α1,2-mannosidase I; EDEM, ER degradation enhancing α-mannosidase-like protein; Man8B, Man8GlcNAc2 isomer B; HEK 293, human embryonic kidney 293; Endo-H, endo-β-N-acetylglucosaminidase H; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; PVDF, polyvinylidene difluoride.1The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; A1AT NHK, α1-antitrypsin-null Hong Kong; ER ManI, ER α1,2-mannosidase I; EDEM, ER degradation enhancing α-mannosidase-like protein; Man8B, Man8GlcNAc2 isomer B; HEK 293, human embryonic kidney 293; Endo-H, endo-β-N-acetylglucosaminidase H; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; PVDF, polyvinylidene difluoride. with the addition of the Glc3Man9GlcNAc2 precursor of N-linked glycans to nascent polypeptide chains. Subsequently, the ER lectin chaperones, calnexin or calreticulin, specifically bind to monoglucosylated N-glycans, promoting proper glycoprotein folding. This ER quality control process is mediated by glucosidases I and II that remove the glucose residues from Glc3Man9GlcNAc2 and by UDP-glucose:glycoprotein glucosyltransferase that reglucosylates incompletely folded glycoproteins. Correctly folded glycoproteins exit the ER to their final destinations, whereas misfolded glycoproteins are readily degraded (for reviews see Refs. 1Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1062) Google Scholar, 2Herscovics A. Biochim. Biophys. Acta. 1999; 1473: 96-107Crossref PubMed Scopus (239) Google Scholar, 3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1965) Google Scholar). Many terminally misfolded proteins in the ER are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (for reviews see Refs. 5Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (533) Google Scholar, 6Plemper R.K. Wolf D.H. Trends Biochem. Sci. 1999; 24: 266-270Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 7Cabral C.M. Liu Y. Sifers R. Trends Biochem. Sci. 2001; 26: 619-624Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Experimental evidence in yeast and mammalian cells suggests that ER α1,2-mannosidase I (ER ManI) that primarily removes the middle branch mannose from Man9GlcNAc2 to form Man8GlcNAc2 isomer B (Man8B) (8Gonzalez D.S. Karaveg K. Vandersall-Nairn A.S. Lal A. Moremen K.W. J. Biol. Chem. 1999; 274: 21375-21386Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Tremblay L.O. Herscovics A. Glycobiology. 1999; 9: 1073-1078Crossref PubMed Scopus (99) Google Scholar) acts as a signal triggering ERAD of glycoproteins (10Knop M. Hauser N. Wolf D.H. Yeast. 1996; 12: 1229-1238Crossref PubMed Scopus (120) Google Scholar, 11Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (301) Google Scholar, 12Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Disruption of the ER α-mannosidase gene in yeast (10Knop M. Hauser N. Wolf D.H. Yeast. 1996; 12: 1229-1238Crossref PubMed Scopus (120) Google Scholar, 11Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (301) Google Scholar) and inhibition of ER ManI in mammalian cells (13Tokunaga F. Brostrom C. Koide T. Arvan P. J. Biol. Chem. 2000; 275: 40757-40764Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Fagioli C. Sitia R. J. Biol. Chem. 2001; 276: 12885-12892Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) both prevent ERAD. We recently reported the molecular cloning of mouse EDEM (ER degradation enhancing α-mannosidase-like protein) and its involvement in glycoprotein ERAD. It was suggested that EDEM is a putative lectin, which most likely binds Man8B on misfolded α1-antitrypsin and accelerates its degradation (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). Two groups also reported the yeast homolog of EDEM and its function in yeast ERAD (16Jakob C.A. Bodmer D. Spirig U. Bättig P. Marcil A. Dignard D. Bergeron J.J.M. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar, 17Nakatsukasa K. Nishikawa S.-I. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), indicating that the ERAD mechanisms involved in the degradation of misfolded glycoproteins are similar in yeast and mammals. Both ER ManI and EDEM are ER resident transmembrane proteins containing characteristic signature motifs of class I α1,2-mannosidases (glycosylhydrolase family 47). Despite this significant sequence similarity, EDEM lacks α1,2-mannosidase activity with Man9GlcNAc as substrate (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). In addition, these two proteins differ in their response to ER stress. EDEM is induced by various forms of ER stress, but ER ManI is not (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). In this report, we show that overexpression of human ER ManI accelerates the degradation of the terminally misfolded α1-antitrypsin genetic variant-null Hong Kong (A1AT NHK) (18Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1988; 263: 7330-7335Abstract Full Text PDF PubMed Google Scholar). We also show the combined effects of ER ManI and EDEM on the degradation of misfolded α1-antitrypsin. Glycan analysis on misfolded NHK shows that overexpression of ER ManI greatly increases the formation of Man8GlcNAc2 and Glc1Man8GlcNAc2 and also stimulates trimming of N-linked oligosaccharides to Man5GlcNAc2 in vivo. These experiments suggest that the misfolded glycoprotein interacts both with ER ManI and EDEM before being recognized by downstream ERAD components. Cell Culture and Transfection—Human HEK 293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, and transfection of plasmids encoding human ER ManI, mouse EDEM, and human α1-antitrypsin-null Hong Kong were performed using FuGENE 6 transfection reagents (Roche Molecular Biochemicals) according to the protocol recommended by the manufacturer. Approximately 1.5 × 105 cells were plated on poly-l-lysine-coated 3.5-cm dishes. After 24 h of plating, 0.5 μg of plasmid encoding ER ManI, EDEM, or pMH vector were mixed with 1 μg of plasmid encoding the α1-antitrypsin variant A1AT NHK for transfection, and cells were pulse-labeled and harvested 36 h post-transfection. Reagents—Kifunensine, an inhibitor of ER ManI, was kindly provided by Fujisawa Pharmaceutical Co. (Osaka, Japan) and added to the culture medium at a concentration of 5 μg/ml for 4 h prior to pulse-labeling. The proteasome inhibitor lactacystin was purchased from Kyowa Medics Co. (Tokyo, Japan) and added to the culture medium at a concentration of 1 mm for 4 h prior to pulse-labeling. The inhibitors were also present in the medium during the pulse and the chase periods. Plasmid Construction—Human ER ManI cDNA was subcloned into the pMH vector (Roche Molecular Biochemicals) by PCR. The entire open reading frame (9Tremblay L.O. Herscovics A. Glycobiology. 1999; 9: 1073-1078Crossref PubMed Scopus (99) Google Scholar) was amplified from cDNA using a sense primer containing a HindIII site and a Kozak sequence (5′-AAAAAAGCTTCCACCATGGCTGCCTGCGAGGGCAGGAG-3′) and an antisense primer containing a NotI site and omitting the stop codon (5′-AAAAAAAAGCGGCCGCGCTGCAGGGGTCCAGATAGGCAGAG-3′). The open reading frame amplicon was digested with HindIII and NotI and cloned into the HindIII/NotI sites of pMH in-frame with the C-terminal HA-tag. Mouse EDEM cDNA was tagged with HA at its C terminus in pCMV-SPORT2 vector (Invitrogen) as described previously (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). The NHK variant was constructed as described elsewhere (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). Metabolic Labeling and Immunoprecipitation—Cells were preincubated in DMEM lacking methionine (Invitrogen) for 30 min and labeled with Expre35S35S protein labeling mixture (PerkinElmer Life Sciences) for 15 min at a concentration of 8.2 MBq/ml. The cells were then incubated in DMEM during the chase period. After washing twice with phosphate-buffered saline lacking Ca2+ and Mg2+, cells were incubated on ice for 20 min in lysis buffer (1% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl, pH 8.0, supplemented with protease inhibitors) (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). For immunoprecipitation, Nonidet P-40 soluble cell lysates were mixed with various antibodies (1/50 dilution), and the immune complexes were collected using either Protein A- or Protein G-Sepharose beads (Amersham-Pharmacia, Amersham, UK) (19Satoh M. Hirayoshi K. Yokota S-i. Hosokawa N. Nagata K. J. Cell Biol. 1996; 133: 469-483Crossref PubMed Scopus (188) Google Scholar). One-half of the cell lysate was used for immunoprecipitation using antibody against A1AT, and the other half was incubated with anti-HA antibody. In cells co-transfected with NHK and ER ManI or EDEM, the radioactivity of immunoprecipitated NHK decreased compared with cells transfected with NHK and pMH. To obtain approximately equal signals for the immunoprecipitated NHK at chase 0 h, the gels were exposed 2 or 3 times longer for samples co-transfected with ER ManI or EDEM than for those co-transfected with pMH. Alternatively in some experiments, one-half or one-third of the cell lysates was used for immunoprecipitation in pMH transfected cells in comparison with ER ManI or EDEM transfected cells. The conditions are in the The immune was in buffer containing and by 10% in the was by the gels to a and the gels were exposed to Co. Japan) For labeling with cells were preincubated in DMEM containing 1 mm glucose (Invitrogen) for 30 min and labeled with for 1 were then for the times in DMEM containing mm glucose (Invitrogen) supplemented with 5 mm mannose J. Biol. Chem. Full Text PDF PubMed Google Scholar). Cell immunoprecipitation, and were performed as described for the labeling with and the samples were a in 5 mm The of the NHK on the were by the to a against α1-antitrypsin was purchased from and against the was from ER ManI antibody was by with a The was using the For the antibody against mouse EDEM, was synthesized using the was used for the cell lysates or as described Labeling and were to 1 × buffer containing and by 10% For μg of protein was For immunoprecipitation, μg of protein of Nonidet cell lysate was used as After to a complexes were by and exposed to of NHK on the were into The were with and then with pH times for 15 min at and the were The were then incubated with in the buffer at for was added at and 36 h of and the were times with The and were for 3 and The labeled oligosaccharides released by this were in mixed with and by as described previously Herscovics A. Biochem. J. PubMed Scopus Google Scholar). In some an of Glc1Man8GlcNAc was also It was by of labeled Glc1Man9GlcNAc for min at with of α1,2-mannosidase of F. Herscovics A. 1999; PubMed Scopus Google Scholar). of A1AT NHK by Overexpression of ER the effect of ER mannosidase I on ERAD of misfolded human ER ManI cDNA was transfected into HEK 293 cells with the A1AT A1AT is a protein to a protease inhibitor in α1-antitrypsin or D. Metabolic of Scholar). The NHK genetic variant of A1AT terminally in the ER and is degraded by ERAD Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). of human ER ManI greatly enhanced the degradation of misfolded A1AT NHK in comparison with transfected cells with NHK was not into the medium of cells co-transfected with ER ManI not of cells co-transfected with vector (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar, Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that the of transfected NHK in 293 cells was of ER ManI the of NHK to 50 min NHK immunoprecipitated from cell lysates transfected with ER ManI on than that from transfected cells the degradation of NHK in cells co-transfected with ER ManI 2, and and also in a in NHK with inhibition of ER α1,2-mannosidase activity with overexpression of ER ManI ERAD of misfolded NHK by mannose trimming from its N-linked This was by NHK described of ER ManI with NHK was in cells transfected with ER ManI open the transfected ER ManI was degraded and see protein of was with ER ManI using anti-HA antibody This decreased in with the ER ManI during The degradation of co-transfected ER ManI was not by the addition of with in with the that human ER ManI is not a glycoprotein (8Gonzalez D.S. Karaveg K. Vandersall-Nairn A.S. Lal A. Moremen K.W. J. Biol. Chem. 1999; 274: 21375-21386Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Tremblay L.O. Herscovics A. Glycobiology. 1999; 9: 1073-1078Crossref PubMed Scopus (99) Google Scholar). also decreased the of the protein 1 and that the is of NHK by effect of the proteasome inhibitor lactacystin on NHK degradation was then The degradation of NHK in cells transfected with ER ManI was by lactacystin with indicating that the enhanced NHK degradation induced by ER ManI overexpression the ERAD is that the transfected ER ManI is degraded open see also and protein synthesis by the of into the was not by transfection of ER The degradation of ER ManI is of with The degradation of ER ManI was not by other protease inhibitors or protease protease I. Wada, and K. Nagata, of ER ManI and EDEM on NHK we reported that mouse EDEM, which sequence to α1,2-mannosidases but lacks activity with Man9GlcNAc as accelerates ERAD of misfolded NHK transfected into 293 cells (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). EDEM was to with NHK in experiments and was to misfolded glycoproteins to for We compared the effects of transfection with ER ManI and with EDEM on ERAD of ER ManI and EDEM the of NHK with or of either ER ManI or EDEM was with NHK, using antibody against A1AT open ER indicating In the using anti-HA protein of and 150 on were in ER ManI and EDEM transfected cells, open for ER for transfected ER ManI is transfected EDEM is during the chase with of 293 cells with both ER ManI and EDEM enhanced ERAD of NHK, compared with ER ManI that is a combined effect of these two ER resident proteins on NHK degradation with of degradation of NHK was also in cells co-transfected with both ER ManI and EDEM compared with EDEM with of the degradation of NHK transfection of both ER ManI and EDEM in this cell lysates were at chase periods. The of of ER ManI, EDEM, and NHK were both ER proteins and substrate proteins were the proteasome inhibitor lactacystin was similar of labeled NHK were from the of cell lysates and whereas a of NHK was in cells both ER ManI and EDEM a chase in the of lactacystin These that the of labeled NHK immunoprecipitated from cells co-transfected with ER ManI and EDEM was to degradation by the and not to synthesis of We then ER ManI and EDEM form a with We against from both ER ManI and recognized its protein by immunoprecipitation and analysis but of the we co-transfected ER ManI with EDEM or ER ManI with We performed immunoprecipitation of labeled cell as as immunoprecipitation of cell lysates by using either or We not of ER ManI with EDEM not The in cells transfected with ER ManI was than that in EDEM transfected cells open and indicating that the proteins of on the of the oligosaccharides present on NHK, cells were labeled with and the oligosaccharides released by from immunoprecipitated NHK were by In transfected cells the labeled oligosaccharides on NHK were Glc1Man9GlcNAc and Man9GlcNAc with of The of the oligosaccharides was by with α1,2-mannosidase by of the Glc1Man9GlcNAc labeled and mannose whereas Man9GlcNAc was to mannose not times of trimming of NHK glycans to B and and to 4 h of chase not was the oligosaccharides on misfolded NHK are Man8GlcNAc2 by α1,2-mannosidase Overexpression of ER ManI a in Man8GlcNAc, and an of Glc1Man8GlcNAc and and the was a in the of Glc1Man9GlcNAc and Man9GlcNAc2 as as an in compared with transfected cells. of the from to with an of Glc1Man8GlcNAc the of the of Glc1Man8GlcNAc and the of Man9GlcNAc in cells transfected with ER ManI compared with transfected cells. The Glc1Man8GlcNAc may have by the of ER ManI on Glc1Man9GlcNAc or by the addition of glucose to the by UDP-glucose:glycoprotein These show that ER ManI is of trimming Man8GlcNAc2 in as was recently reported for ER ManI in A. Tremblay L.O. Glycobiology. 12: Google Scholar). In cells EDEM the labeled oligosaccharides were Glc1Man9GlcNAc and as in transfected cells with of and a similar to that in was in the of the Man9GlcNAc and a of Glc1Man8GlcNAc were cells were with the formation of was that ER α1,2-mannosidase II is not for trimming NHK oligosaccharides in of on the oligosaccharides of misfolded were transfected with NHK and as described in were harvested a The cells were preincubated in either the or the of 5 μg/ml for 4 h prior to labeling with The of the oligosaccharides is as in In the present study, we have that overexpression of human ER ManI in HEK 293 cells ERAD of the terminally misfolded glycoprotein degradation of NHK in cells ER ManI was by lactacystin and by indicating that misfolded NHK was degraded the ERAD and that overexpression of ER ManI enhanced ERAD by trimming of its It that the processing of N-linked oligosaccharides to Man8B by ER ManI acts as a signal for degradation of misfolded glycoproteins L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1062) Google Scholar, 7Cabral C.M. Liu Y. Sifers R. Trends Biochem. Sci. 2001; 26: 619-624Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, F. Brostrom C. Koide T. Arvan P. J. Biol. Chem. 2000; 275: 40757-40764Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Fagioli C. Sitia R. J. Biol. Chem. 2001; 276: 12885-12892Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). studies to were on the that Man8B is the on misfolded glycoproteins to ER ManI activity and on of carbohydrate trimming from misfolded glycoproteins by in their In this analysis of the glycans on terminally misfolded NHK, the show that a mixture of oligosaccharides is on NHK and that the that Man8B is by ER ManI to be In transfected cells, and were the oligosaccharides on NHK, but of and were also It is that oligosaccharides other than Man8B, or in addition to Man8B, may as recognition for ERAD. The of and on NHK be the of ERAD of NHK these The of Glc1Man9GlcNAc on NHK is with previous studies NHK with calnexin A. Sifers R.N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The also show that the trimming is not to ER α-mannosidase II was to Overexpression of ER ManI, which ERAD of NHK, also greatly trimming of mannose residues to and Man8GlcNAc, as as the formation of the of the the oligosaccharides were in transfected cells, that the of NHK degradation by overexpression of ER ManI is not likely by the formation of The trimming of N-glycans in cells ER ManI is with previous experiments with ER ManI in indicating that ER ManI is than previously A. Tremblay L.O. Glycobiology. 12: Google Scholar). The of oligosaccharides in EDEM and transfected cells is but is a of the Man9GlcNAc that to a of that EDEM either trimming of Glc1Man9GlcNAc to or of by glycoprotein We combined effects of ER ManI and of EDEM on ERAD. We previously reported that EDEM interacts with and accelerates the degradation of misfolded NHK, that EDEM may be a putative lectin, which misfolded glycoproteins for ERAD (15Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). NHK degradation is in cells co-transfected with both ER ManI and EDEM than in cells transfected with ER ManI or EDEM We also evidence indicating that ER ManI and EDEM are of complexes proteins were with of and evidence that ER ManI and EDEM form a with other was We propose a model whereby misfolded glycoproteins are recognized by ER ManI and by The of the proteins with ER ManI and EDEM to be which may in the of glycoprotein and in the to and degradation by ER ManI transfected into 293 cells was degraded a mechanism of cytoplasmic Despite its we the of transfected ER ManI by analysis using antibody against or human ER ManI The of of ER ManI in transfected cells is than that of ER ManI we not the by analysis using antibody. the molecular mechanism and the for the degradation of ER ManI the suggest that the of ER ManI is We for in and for on the

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