EDEM3, a Soluble EDEM Homolog, Enhances Glycoprotein Endoplasmic Reticulum-associated Degradation and Mannose Trimming

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing α-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I α-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded α1-antitrypsin variant (null (Hong Kong)) and of TCRα. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded α1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent α1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of α1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has α1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM. Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing α-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I α-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded α1-antitrypsin variant (null (Hong Kong)) and of TCRα. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded α1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent α1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of α1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has α1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM. ER 3The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; ER ManI, ER α1,2-mannosidase I; HA, influenza hemagglutinin epitope; α1-AT, α1-antitrypsin; NHK, α1-AT null (Hong Kong); TCRα, T cell receptor α subunit; Ab, antibody; PNGase F, peptide-N-glycosidase F; Endo H, endoglycosidase H; HPLC, high pressure liquid chromatography. quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway (1Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 649-676Crossref PubMed Scopus (364) Google Scholar, 2Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1064) Google Scholar, 3Fewell S.W. Travers K.J. Weissman J.S. Brodsky J.L. Annu. Rev. Genet. 2001; 35: 149-191Crossref PubMed Scopus (264) Google Scholar, 4Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1676) Google Scholar). During this process, proteins that fail to attain their native conformation due to mutations of the polypeptides or to ER stress conditions adverse for protein folding as well as orphan subunits are degraded in a process known as ER-associated degradation (ERAD) (3Fewell S.W. Travers K.J. Weissman J.S. Brodsky J.L. Annu. Rev. Genet. 2001; 35: 149-191Crossref PubMed Scopus (264) Google Scholar, 5Tsai B. Ye Y. Rapoport T.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 246-255Crossref PubMed Scopus (551) Google Scholar, 6Sitia R. Braakman I. Nature. 2003; 426: 891-894Crossref PubMed Scopus (575) Google Scholar, 7McCracken A.A. Brodsky J.L. BioEssays. 2003; 25: 868-877Crossref PubMed Scopus (194) Google Scholar). The recognition of misfolded proteins for ERAD is still poorly understood, but there is increasing evidence for a role of mannose trimming in the targeting of glycoproteins for ERAD (8Cabral C.M. Liu Y. Sifers R.N. Trends Biochem. Sci. 2001; 26: 619-624Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 9Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1620) Google Scholar). In mammalian cells, overexpression of ER α-mannosidase I stimulates ERAD of misfolded glycoproteins (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Wu Y. Swulius M.T. Moremen K.W. Sifers R.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8229-8234Crossref PubMed Scopus (150) Google Scholar), whereas the α1,2-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin stabilize misfolded glycoproteins (12Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 13Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 14Chillaron J. Adan C. Haas I.G. Biol. Chem. 2000; 381: 1155-1164Crossref PubMed Scopus (20) Google Scholar, 15Wilson C.M. Farmery M.R. Bulleid N.J. J. Biol. Chem. 2000; 275: 21224-21232Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Tokunaga F. Brostrom C. Koide T. Arvan P. J. Biol. Chem. 2000; 275: 40757-40764Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). These observations suggested that Man8GlcNAc2 isomer B, the major product of the ER α1,2-mannosidase, is a recognition marker for ERAD of glycoproteins, but this view is being challenged, since there is increasing evidence that trimming to smaller oligosaccharides occurs on ERAD substrates (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 17Frenkel Z. Gregory W. Kornfeld S. Lederkremer G.Z. J. Biol. Chem. 2003; 278: 34119-34124Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Kitzmuller C. Caprini A. Moore S.E. Frenoy J.P. Schwaiger E. Kellermann O. Ivessa N.E. Ermonval M. Biochem. J. 2003; 376: 687-696Crossref PubMed Scopus (52) Google Scholar, 19Ermonval M. Kitzmuller C. Mir A.M. Cacan R. Ivessa N.E. Glycobiology. 2001; 11: 565-676Crossref PubMed Scopus (60) Google Scholar). We previously cloned mouse EDEM (ER degradation enhancing α-mannosidase-like protein) as a cDNA whose expression is up-regulated by ER stress and showed that EDEM accelerates glycoprotein ERAD (20Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (387) Google Scholar). EDEM is an integral ER membrane protein that has all the signature motifs of Class I α1,2-mannosidases (glycosylhydrolase family 47) but no detectable enzyme activity as a processing α-mannosidase in vivo or in vitro. Recently, it was found that EDEM extracts terminally misfolded glycoproteins from the calnexin cycle (21Molinari M. Calanca V. Galli C. Lucca P. Paganetti P. Science. 2003; 299: 1397-1400Crossref PubMed Scopus (389) Google Scholar, 22Oda Y. Hosokawa N. Wada I. Nagata K. Science. 2003; 299: 1394-1397Crossref PubMed Scopus (391) Google Scholar). In S. cerevisiae, the ER α1,2-mannosidase as well as Htm1p/Mnl1p belonging to the same protein family are also involved in ERAD, since disruption of the genes delays the ERAD of glycoproteins (23Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar, 24Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Although EDEM and Htm1p/Mnl1p were postulated to be lectins involved in targeting misfolded glycoproteins for ERAD, the precise mechanisms whereby EDEM and Htm1p/Mnl1p recognize and sort misfolded glycoproteins for degradation are still unclear, and their role as lectins has not been established directly. While this manuscript was in preparation, EDEM2 was reported to stimulate ERAD of misfolded glycoproteins without affecting mannose trimming (25Mast S.W. Diekman K. Karaveg K. Davis A. Sifers R.N. Moremen K.W. Glycobiology. 2005; 15: 421-436Crossref PubMed Scopus (131) Google Scholar, 26Olivari S. Galli C. Alanen H. Ruddock L. Molinari M. J. Biol. Chem. 2005; 280: 2424-2428Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Here, we show that EDEM3 is a soluble EDEM homolog located in the ER of transfected mammalian cells that accelerates ERAD of misfolded glycoproteins through a mechanism likely to be different from that of EDEM or EDEM2, since EDEM3 greatly stimulates mannose trimming in vivo. Cloning of Mouse EDEM Homolog—Five expressed sequence tag clones were sequenced, and one clone (G431003D06), which was kindly provided by Dr. Y. Hayashizaki (RIKEN, Japan) (27Carninci P. Waki K. Shiraki T. Konno H. Shibata K. Itoh M. Aizawa K. Arakawa T. Ishii Y. Sasaki D. Bono H. Kondo S. Sugahara Y. Saito R. Osato N. Fukuda S. Sato K. Watahiki A. Hirozane-Kishikawa T. Nakamura M. Shibata Y. Yasunishi A. Kikuchi N. Yoshiki A. Kusakabe M. Gustincich S. Beisel K. Pavan W. Aidinis V. Nakagawara A. Held W.A. Iwata H. Kono T. Nakauchi H. Lyons P. Wells C. Hume D.A. Fagiolini M. Hensch T.K. Brinkmeier M. Camper S. Hirota J. Mombaerts P. Muramatsu M. Okazaki Y. Kawai J. Hayashizaki Y. Genome Res. 2003; 13: 1273-1289Crossref PubMed Scopus (150) Google Scholar), contained the entire EDEM3 cDNA. Sequencing was performed by the PCR-based dideoxy termination method using BigDye version 3 (ABI) and PCR 9700 (PerkinElmer Life Sciences) and then analyzed with the ABI 3100 capillary sequencer. Plasmid Construction—The coding region of EDEM3 cDNA was subcloned into pcDNA3.1+ by PCR, and the HA tag was introduced prior to the -KDEL ER retrieval signal (EDEM3-HA). The null Hong Kong (NHK)-QQQ mutant was created by replacing Asn residues of NHK glycosylation sites with Gln, using the QuikChange™ site-directed mutagenesis kit (Stratagene). The EDEM3 E147Q mutant was constructed by the same method. FLAG-tagged TCRα was kindly provided by Dr. F. Tokunaga (Osaka City University Graduate School of Medicine). In Vitro Translation and Translocation—EDEM3-HA cDNA was linearized with EcoRI, and transcribed in vitro by T7 RNA polymerase (Promega). The transcript was then translated in vitro in the reticulocyte lysate (Flexi-lysate; Promega) supplemented with [35S]methionine with and without canine pancreas microsomes. The localization of the EDEM3 product was by the method U. E. B. Rapoport T.A. EMBO J. PubMed Scopus Google Scholar), by Mouse EDEM with HA was used as a control ER membrane protein (20Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (387) Google Scholar), and was used as a control ER protein K. Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). Cell and and cells by were in supplemented with and and in cells were in supplemented with and cells the EDEM3 were established using the which has a T. T. K. H. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), and in the of were with a Plasmid and transfected using the as previously (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). RNA from cells was using on were a membrane Life Sciences) and then were with for in Japan) membrane was used to mouse for EDEM, and were by the method EDEM3 were by PCR, using the of the EDEM3 cDNA its termination to the activity of the and to with EDEM. and cell and were as previously (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), that and was used of α1-AT were from the HA tag were from was from and mouse was from was from and kifunensine was a from Endo and were from or cells were on a in a prior to the cells were with for and with tag and for and then with an and an for were by of NHK and of NHK by was performed as previously (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, B. Herscovics A. Biochem. J. PubMed Scopus Google Scholar). of total glycoproteins in and cell EDEM3, were by using and were by on an Japan) as previously S. PubMed Scopus Google Scholar). were the and the oligosaccharides were by a of and from to for in were by Cloning of EDEM3, a of transcript was in a region to R. I. D.A. C.M. K. H. C. K.W. 2001; 73: PubMed Scopus Google Scholar). The of has a sequence to Class I α1,2-mannosidases (glycosylhydrolase family 47) and to the product T. N. K. M. Y. O. A. H. N. N. Res. 3 PubMed Scopus Google Scholar). is the of the mouse EDEM (20Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (387) Google Scholar), we the mouse expressed sequence tag for the Sequencing of expressed sequence tag clones a mouse cDNA of a protein of 931 amino acids consists of a region acids to Class I α1,2-mannosidases (glycosylhydrolase family by a C-terminal region a protease-associated motif acids K. Trends Biochem. Sci. 2001; 26: Full Text Full Text PDF PubMed Scopus Google that is in EDEM and in all Class I α1,2-mannosidases has a signal sequence the as well as an signal its The mouse EDEM3 cDNA sequence with its in the coding region and The mouse and are in amino The mouse EDEM3 has amino acids the with the The α-mannosidase domain and amino EDEM3 and EDEM and EDEM3 and ER α-mannosidase I (ER acidic amino acids that are essential for activity F. Herscovics A. 1999; PubMed Scopus Google are conserved proteins the residues for activity of the yeast ER α1,2-mannosidase F. Herscovics A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google are not conserved in EDEM3 or EDEM. The protease-associated motif is a sequence found in K. Trends Biochem. Sci. 2001; 26: Full Text Full Text PDF PubMed Scopus Google Scholar), the of which in EDEM3 is of EDEM3 and of ER a major EDEM3 transcript of in all mouse as from the cloned with high in and as reported for the homolog R. I. D.A. C.M. K. H. C. K.W. 2001; 73: PubMed Scopus Google Scholar). EDEM is expressed in and in whereas the expression is in and We EDEM3 expression is by ER as previously shown for EDEM (20Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (387) Google Scholar, H. T. Hosokawa N. Nagata K. K. Dev. Cell. 2003; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). The of which ER stress by of a of EDEM3 on the cell The EDEM3 transcript is increased in and cells, whereas no is in and in EDEM3 is also up-regulated by cells with the or the (ER but the of EDEM3 not in cells to stress that greatly stimulates expression EDEM3 in the ER the region the as a signal sequence into the ER or it as a we integral membrane proteins from soluble proteins by EDEM3 RNA is translated in of the EDEM3 is in the soluble We a in the by of to EDEM3 was into not with Endo or PNGase the of high mannose from EDEM3 as from the sequence we the localization of EDEM3 by EDEM3 into a the that with the EDEM3 is not into the in of transfected HEK293 cells not we that EDEM3 is an ER EDEM3 then EDEM3 glycoprotein We used the α1-antitrypsin variant NHK as a soluble ERAD Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, R.N. S. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of EDEM3 the ERAD of NHK and of EDEM3 with NHK was using to α1-AT or that EDEM3 with NHK in the was and of the in of NHK trimming of the that the EDEM3 and NHK is with increased mannose trimming from NHK and with and and with NHK degradation is by in the of EDEM3, that EDEM3 accelerates glycoprotein ERAD by The of NHK was in cells with EDEM3 in cells with with The of NHK in cells is with that of NHK by PNGase The of the α1,2-mannosidase kifunensine greatly NHK degradation in cells EDEM3 and the of NHK on that the of NHK in cells with EDEM3 is by the mannose trimming from the different mechanisms for ERAD of soluble and proteins been K.J. A.A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (150) Google Scholar, S. W. C. J. Cell Biol. 2001; PubMed Scopus Google Scholar, Z. M. Brodsky J.L. S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), we the of EDEM3 on a FLAG-tagged TCRα, a ERAD H. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of EDEM3 the degradation of and which is by kifunensine EDEM3 not the degradation of NHK all sites its for glycoproteins and is degraded NHK that the and we that was also degraded by ERAD not These that the of glycoprotein ERAD by EDEM3 on mannose trimming from the of EDEM3 on from of NHK and of the results in suggested that overexpression of EDEM3 stimulates mannose trimming from on NHK, the oligosaccharides were cells with The from NHK by Endo were analyzed by Overexpression of EDEM3 greatly stimulates trimming of from NHK to and and but there is Man8GlcNAc2 found on NHK in cells EDEM3 with of there is only a of and on NHK from cells, whereas and are in cells transfected with EDEM3. The of and with of in cells and is the of and in Furthermore, all the of and is in cells in the In the of in Man8GlcNAc2 is in the of EDEM3. of oligosaccharides that overexpression of EDEM3 stimulates mannose trimming from the increased mannose trimming is due to activity of EDEM3, the of the E147Q mutant on NHK oligosaccharides were is a conserved that to and to in the of yeast and ER α1,2-mannosidases, F. Karaveg K. Herscovics A. Moremen K.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, F. F. P. B. Herscovics A. EMBO J. 2000; 19: PubMed Scopus Google Scholar). is essential for enzyme since mutation of this abolishes α1,2-mannosidase activity F. Herscovics A. 1999; PubMed Scopus Google Scholar). The of oligosaccharides on NHK from cells E147Q is to that of the of and are the same as in control cells and of mannose trimming by EDEM3 was also by of total glycoproteins from cells EDEM3 is a of with a of with the of results that EDEM3 has α1,2-mannosidase activity in vivo. with NHK degradation was greatly in cells the E147Q mutant with EDEM3 that the activity of EDEM3 is for its on ERAD of The that EDEM3 is a soluble homolog of EDEM that accelerates ERAD of soluble NHK and TCRα in an The of ERAD stimulation on NHK degradation is to that previously reported overexpression of EDEM and of ER (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Wu Y. Swulius M.T. Moremen K.W. Sifers R.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8229-8234Crossref PubMed Scopus (150) Google Scholar, N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (387) Google Scholar). the role of EDEM3 in ERAD of NHK is likely to be different from that of EDEM, since EDEM3 overexpression greatly stimulates mannose trimming of from NHK, whereas overexpression of EDEM not (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Furthermore, EDEM3 is the of oligosaccharides from NHK is different from that on NHK from ER In cells EDEM3, there is trimming of to with Man8GlcNAc2 and whereas in cells ER ManI, there is increased of Man8GlcNAc2 and with increased trimming to smaller oligosaccharides (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). the of EDEM3 on the trimming of is by the essential acidic to and B, and the increased ERAD of NHK due to EDEM3 overexpression is greatly by this mutation is found in the of processing Class I α-mannosidases by F. Karaveg K. Herscovics A. Moremen K.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, F. F. P. B. Herscovics A. EMBO J. 2000; 19: PubMed Scopus Google Scholar, F. A. T. P. Herscovics A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, F. H. R. J. J. Mol. Biol. 2001; PubMed Scopus Google Scholar, W. Karaveg K. Liu J. Moremen K.W. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and is essential for enzyme activity F. Herscovics A. 1999; PubMed Scopus Google Scholar). Although results that EDEM3 stimulation of NHK ERAD is by its α1,2-mannosidase an likely is that the mutation of to the conformation of EDEM3 and abolishes the on NHK degradation of enzyme activity. overexpression of EDEM3 stimulates mannose trimming from total glycoproteins as well as from the misfolded glycoprotein NHK EDEM3 is likely as a processing α1,2-mannosidase in vivo, trimming of Man8GlcNAc2 oligosaccharides to to be different from that of ER that greatly stimulates trimming to Man8GlcNAc2 and (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The results that Man8GlcNAc2 is not an targeting signal for ERAD of glycoproteins and that smaller oligosaccharides to misfolded glycoproteins in this in with (10Hosokawa N. Tremblay L.O. You Z. Herscovics A. Wada I. Nagata K. J. Biol. Chem. 2003; 278: 26287-26294Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 17Frenkel Z. Gregory W. Kornfeld S. Lederkremer G.Z. J. Biol. Chem. 2003; 278: 34119-34124Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Kitzmuller C. Caprini A. Moore S.E. Frenoy J.P. Schwaiger E. Kellermann O. Ivessa N.E. Ermonval M. Biochem. J. 2003; 376: 687-696Crossref PubMed Scopus (52) Google Scholar, 19Ermonval M. Kitzmuller C. Mir A.M. Cacan R. Ivessa N.E. Glycobiology. 2001; 11: 565-676Crossref PubMed Scopus (60) Google Scholar, F. Parodi A.J. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). While this manuscript was in preparation, reported on EDEM which EDEM2 (25Mast S.W. Diekman K. Karaveg K. Davis A. Sifers R.N. Moremen K.W. Glycobiology. 2005; 15: 421-436Crossref PubMed Scopus (131) Google Scholar, 26Olivari S. Galli C. Alanen H. Ruddock L. Molinari M. J. Biol. Chem. 2005; 280: 2424-2428Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Although EDEM2 stimulates glycoprotein ERAD, it has no on mannose trimming from misfolded glycoproteins, that its mechanism of is different from that of EDEM3 in the In the of EDEM3 is but its is not further The mechanisms involved in ERAD is an of the not only for cell but also for ERAD is in the of a of by protein the mechanisms whereby the cell misfolded proteins and to ERAD are not showed that ER and EDEM stimulate ERAD of glycoproteins, a mechanism has been whereby targeting of misfolded glycoproteins on by which is then by EDEM. it is from and from the that the targeting for ERAD is since there are EDEM proteins and trimming of oligosaccharides on misfolded glycoproteins to smaller the and of EDEM3 a to the of the quality control of misfolded We Dr. Y. Hayashizaki (RIKEN, Japan) for the cDNA Dr. F. Tokunaga (Osaka City University Graduate School of for the FLAG-tagged TCRα and K. for

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