FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061344384> ?p ?o ?g. }

• W2061344384 endingPage "44844" @default.
• W2061344384 startingPage "44838" @default.
• W2061344384 abstract "Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and β2-microglobulin (β2m) in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need β2m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with β2m. These differences led us to investigate early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. Here we show that CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin. Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and β2-microglobulin (β2m) in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need β2m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with β2m. These differences led us to investigate early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. Here we show that CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin. CD1 molecules are transmembrane glycoproteins encoded by linked genes outside the major histocompatibility complex (MHC) 1The abbreviations used are: MHC, major histocompatibility complex; β2m, β2-microglobulin; B-LCL, B-lymphoblastoid line; ER, endoplasmic reticulum; DTT, dithiothreitol; DSP, dithiobis(succinimidylpropionate); CST, castanospermine; mAb, monoclonal antibody; Endo H, endoglycosidase H; TAP, transporter-associated with antigen processing; TBS, Tris-buffered saline. (reviewed in Ref. 1Porcelli S.A. Modlin R.L. Annu. Rev. Immunol. 1999; 17: 297-329Google Scholar). The human CD1a, -b, and -c molecules constitute group I, whereas human and mouse CD1d molecules form group II. Like classical MHC class I and class II molecules, CD1 molecules are recognized by T cells. Certain αβ+ and γδ+ CD4-CD8− T cells were initially shown to lyse tumor cells expressing CD1a or CD1c (2Porcelli S. Brenner M.B. Greenstein J.L. Balk S.P. Terhorst C. Bleicher P.A. Nature. 1989; 341: 447-450Google Scholar). Subsequent studies showed that mycobacterial lipids, such as mycolic acids, lipoarabinomannan, phosphatidylinositol mannosides, and hexosyl-1-phosphoisoprenoids could be presented to cytotoxic T cells by CD1b and CD1c molecules (3Porcelli S. Morita C.T. Brenner M.B. Nature. 1992; 360: 593-597Google Scholar, 4Beckman E.M. Porcelli S.A. Morita C.T. Behar S.M. Furlong S.T. Brenner M.B. Nature. 1994; 372: 691-694Google Scholar, 5Sieling P.A. Chatterjee D. Porcelli S.A. Prigozy T.I. Mazzaccaro R.J. Soriano T. Bloom B.R. Brenner M.B. Kronenberg M. Brennan P.J. Modlin R.L. Science. 1995; 269: 227-230Google Scholar, 6Moody D.B. Ulrichs T. Muhlecker W. Young D.C. Gurcha S.S. Grant E. Rosat J.P. Brenner M.B. Costello C.E. Besra G.S. Porcelli S.A. Nature. 2000; 404: 884-888Google Scholar). Recently, gangliosides and sulfatides have been identified as natural host antigens presented by CD1b and CD1a, respectively (7Shamshiev A. Donda A. Carena I. Mori L. Kappos L. De Libero G. Eur. J. Immunol. 1999; 29: 1667-1675Google Scholar, 8Shamshiev A. Donda A. Prigozy T.I. Mori L. Chigorno V. Benedict C.A. Kappos L. Sonnino S. Kronenberg M. De Libero G. Immunity. 2000; 13: 255-264Google Scholar, 9Shamshiev A. Gober H.J. Donda A. Mazorra Z. Mori L. De Libero G. J. Exp. Med. 2002; 195: 1013-1021Google Scholar). Although no bacterial antigens presented by CD1d molecules have been identified, they present a marine sponge-derived lipid, α-galactosylceramide, to both mouse and human NKT cells (10Kawano T. Cui J. Koezuka Y. Toura I. Kaneko Y. Motoki K. Ueno H. Nakagawa R. Sato H. Kondo E. Koseki H. Taniguchi M. Science. 1997; 278: 1626-1629Google Scholar, 11Brossay L. Naidenko O. Burdin N. Matsuda J. Sakai T. Kronenberg M. J. Immunol. 1998; 161: 5124-5128Google Scholar, 12Brossay L. Chioda M. Burdin N. Koezuka Y. Casorati G. Dellabona P. Kronenberg M. J. Exp. Med. 1998; 188: 1521-1528Google Scholar, 13Brossay L. Tangri S. Bix M. Cardell S. Locksley R. Kronenberg M. J. Immunol. 1998; 160: 3681-3688Google Scholar, 14Burdin N. Brossay L. Koezuka Y. Smiley S.T. Grusby M.J. Gui M. Taniguchi M. Hayakawa K. Kronenberg M. J. Immunol. 1998; 161: 3271-3281Google Scholar, 15Spada F.M. Koezuka Y. Porcelli S.A. J. Exp. Med. 1998; 188: 1529-1534Google Scholar). Recently, natural lipid ligands, including phosphatidylinositol and phosphatidylethanolamine, recognizable by autoreactive CD1d-restricted T cells, have been identified (16Gumperz J.E. Roy C. Makowska A. Lum D. Sugita M. Podrebarac T. Koezuka Y. Porcelli S.A. Cardell S. Brenner M.B. Behar S.M. Immunity. 2000; 12: 211-221Google Scholar). The structure of CD1 is similar to that of MHC class I molecules in that CD1 molecules form heterodimers of 43–49-kDa heavy chains noncovalently associated with β2-microglobulin (β2m). However, unlike class I molecules, which bind peptide antigens in the ER, CD1 glycoproteins, like class II molecules, survey antigens in the endocytic pathway. Peptide binding by class I molecules is an essential requirement for their egress from the ER. CD1d molecules may bind autologous lipids in the ER, such as phosphatidylinositol or glycosylphosphatidylinositol (17Joyce S. Woods A.S. Yewdell J.W. Bennink J.R. De Silva A.D. Boesteanu A. Balk S.P. Cotter R.J. Brutkiewicz R.R. Science. 1998; 279: 1541-1544Google Scholar, 18De Silva A.D. Park J.J. Matsuki N. Stanic A.K. Brutkiewicz R.R. Medof M.E. Joyce S. J. Immunol. 2002; 168: 723-733Google Scholar), to satisfy quality control mechanisms and allow transport, before exchanging them in the endocytic pathway for other autologous or foreign lipids. Previous studies have examined the assembly of CD1 molecules. CD1b heavy chain was observed to bind calnexin and calreticulin in the ER and to require β2m for subsequent transport to the cell surface (19Huttinger R. Staffler G. Majdic O. Stockinger H. Int. Immunol. 1999; 11: 1615-1623Google Scholar, 20Sugita M. Porcelli S.A. Brenner M.B. J. Immunol. 1997; 159: 2358-2365Google Scholar). In contrast, mouse and human CD1d molecules are expressed both as free heavy chains and as complexes with β2m. This was observed not only in transfectants but also in primary tissue cells (21Balk S.P. Burke S. Polischuk J.E. Frantz M.E. Yang L. Porcelli S. Colgan S.P. Blumberg R.S. Science. 1994; 265: 259-262Google Scholar, 22Kim H.S. Garcia J. Exley M. Johnson K.W. Balk S.P. Blumberg R.S. J. Biol. Chem. 1999; 274: 9289-9295Google Scholar, 23Amano M. Baumgarth N. Dick M.D. Brossay L. Kronenberg M. Herzenberg L.A. Strober S. J. Immunol. 1998; 161: 1710-1717Google Scholar). CD1d expressed in β2m-negative cells is fully capable of activating cognate T cells (23Amano M. Baumgarth N. Dick M.D. Brossay L. Kronenberg M. Herzenberg L.A. Strober S. J. Immunol. 1998; 161: 1710-1717Google Scholar, 24Cardell S. Tangri S. Chan S. Kronenberg M. Benoist C. Mathis D. J. Exp. Med. 1995; 182: 993-1004Google Scholar), indicating that heterodimer formation may not be critical for CD1d function. Although a recent study showed that β2m seems to modulate the structure of the heavy chain carbohydrates (22Kim H.S. Garcia J. Exley M. Johnson K.W. Balk S.P. Blumberg R.S. J. Biol. Chem. 1999; 274: 9289-9295Google Scholar), the underlying mechanisms regulating CD1d expression with or without β2m remain to be elucidated. Here we present a detailed analysis of CD1d biosynthesis and an examination of the roles of ER chaperones in CD1d assembly. We show that CD1d heavy chains associate with calnexin, calreticulin, and the thiol oxidoreductase ERp57 and demonstrate a role for this association in the formation of CD1d heavy chain disulfide bonds. The B-LCL C1R and .221, and their CD1d transfectants, were maintained in Iscove's medium (Invitrogen) containing 5% bovine calf serum or 10% fetal bovine serum at 37 °C. The mouse mAbs to human CD1d, 51.1.3 (22Kim H.S. Garcia J. Exley M. Johnson K.W. Balk S.P. Blumberg R.S. J. Biol. Chem. 1999; 274: 9289-9295Google Scholar, 25Exley M. Garcia J. Balk S.P. Porcelli S. J. Exp. Med. 1997; 186: 109-120Google Scholar, 26Exley M. Garcia J. Wilson S.B. Spada F. Gerdes D. Tahir S.M. Patton K.T. Blumberg R.S. Porcelli S. Chott A. Balk S.P. Immunology. 2000; 100: 37-47Google Scholar) and D5 (22Kim H.S. Garcia J. Exley M. Johnson K.W. Balk S.P. Blumberg R.S. J. Biol. Chem. 1999; 274: 9289-9295Google Scholar, 26Exley M. Garcia J. Wilson S.B. Spada F. Gerdes D. Tahir S.M. Patton K.T. Blumberg R.S. Porcelli S. Chott A. Balk S.P. Immunology. 2000; 100: 37-47Google Scholar, 27Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495Google Scholar), were gifts from Dr. S. Porcelli (Albert Einstein College of Medicine) and Dr. S. Balk (Harvard Medical School), respectively. The mAbs GAP.A3 (anti-HLA-A3 (28Berger A.E. Davis J.E. Cresswell P. Hybridoma. 1982; 1: 87-90Google Scholar)), MaP.ERp57 (anti-ERp57 (29Diedrich G. Bangia N. Pan M. Cresswell P. J. Immunol. 2001; 166: 1703-1709Google Scholar)), and HC10 (anti-class I (30Stam N.J. Spits H. Ploegh H.L. J. Immunol. 1986; 137: 2299-2306Google Scholar)) have been previously described. The mAb AF8 (anti-calnexin (31Hochstenbach F. David V. Watkins S. Brenner M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4734-4738Google Scholar)) was a gift from Dr. M. Brenner (Harvard Medical School). The rabbit anti-calreticulin serum and rat anti-Grp94 mAb were purchased from StressGen Biotechnologies Corp. (Victoria, BC, Canada) and anti-β2m serum from Roche Applied Science (Indianapolis, IN). Dithiobis(succinimidylpropionate) (DSP) was purchased from Pierce and castanospermine (CST) from Roche Applied Science.N-Butyldeoxynojirimycin was a gift of Dr. K. Cannon (32Cannon K.S. Cresswell P. EMBO J. 2001; 20: 2443-2453Google Scholar). The vector CD1d/pSRα-neo was a gift of Dr. S. Porcelli. C1R and .221 cells were transfected by electroporation at 230 and 210 V/960 microfarads, respectively. C1R.CD1d and .221.CD1d were selected for neomycin resistance at 1.8 and 0.6 mg/ml G418, respectively. They were screened for CD1d expression by flow cytometry. Labeling with [35S]methionine and cysteine (ICN, Costa Mesa, CA), immunoprecipitations, and endoglycosidase H (Endo H) digestion were performed as previously described (33Denzin L.K. Hammond C. Cresswell P. J. Exp. Med. 1996; 184: 2153-2165Google Scholar). Reimmunoprecipitation experiments were performed as described (34Kang S.J. Cresswell P. EMBO J. 2002; 21: 1650-1660Google Scholar). For elution in reducing conditions, washed primary immunoprecipitates were boiled for 5 min in 100 μl of 1% SDS, 5 mm dithiothreitol (DTT), TBS (10 mm Tris, 150 mm NaCl, pH 7.4) before diluting with 1 ml of 1% Triton X-100, 10 mmiodoacetamide, TBS. For nonreducing elution, DTT and iodoacetamide were omitted. The beads were centrifuged and the supernatants were used for the second immunoprecipitations. The samples were boiled with Laemmli SDS sample buffer and separated by reducing, unless indicated otherwise, SDS-PAGE prior to autoradiography. All quantitation was performed using Bio-Rad GS-525 phosphorimaging and Molecular Analyst Software. Cells were metabolically labeled for 45 min and lysed on ice in 1% digitonin in Dulbecco's phosphate-buffered saline (Invitrogen) with or without 1 mmDSP. Lysates were diluted in 1% digitonin or 1.2% Triton X-100 before immunoprecipitation. CD1d biosynthesis was examined in the B-LCL, C1R.CD1d, which is capable of activating CD1d-specific T cells (15Spada F.M. Koezuka Y. Porcelli S.A. J. Exp. Med. 1998; 188: 1529-1534Google Scholar). Cells were labeled with [35S]methionine/cysteine for 15 min and chased up to 20 h. They were lysed in 1% Triton X-100 and immunoprecipitated with the anti-CD1d antibodies, 51.1.3 or D5, which recognize heavy chain-β2m dimers and free heavy chains, respectively. Exit from the ER was monitored by loss of susceptibility to Endo H. Fig. 1 shows that the majority of Endo H-sensitive free heavy chains were converted to heavy chain-β2m complexes within 2 h (Fig. 1 B). Both β2m-associated CD1d heavy chains and the residual free heavy chains underwent complex carbohydrate modification, manifested as smeared bands, starting at about 1 h for the heterodimer and 2–4 h for the free heavy chains. Concomitant loss of Endo H-sensitive bands indicates transport from the ER, and these results indicate that β2m facilitates, but is not essential for, CD1d exit from the ER. That 51.1.3-recognizable CD1d is associated with β2m was confirmed by comparing kinetics of maturation by immunoprecipitation with 51.1.3 and with anti-β2m serum. The immunoprecipitates were eluted from the beads by boiling in 1% SDS/DTT/TBS, diluted in 1% Triton X-100/iodoacetamide/TBS, and the supernatants were reimmunoprecipitated with D5. Immunoprecipitation with either antibody showed identical CD1d transport kinetics in C1R.CD1d and in a second CD1d-expressing cell line, .221.CD1d (Fig. 1 C). To examine how fast CD1d heterodimers arrive at the cell surface, C1R.CD1d cells were radiolabeled for 15 min and chased up to 8 h. At each chase point, surface CD1d was captured by incubating the cells with the 51.1.3 mAb. Unbound 51.1.3 was removed by washing and the surface CD1d was isolated by adding protein G-Sepharose after lysis. The residual, mostly intracellular, CD1d was immunoprecipitated by adding additional 51.1.3 and protein G-Sepharose (Fig.2 A). The immunoprecipitates were eluted from the beads, and released CD1d heavy chains were reimmunoprecipitated with the D5 mAb. The results showed that CD1d was detectable at the cell surface after 1 h and reached a plateau after ∼2.5 h (Fig. 2, B, upper panel, andC). The rate of transport to the cell surface was approximately the same as the rate of complex carbohydrate modification (Figs. 1 and 2 B, lower panel). This suggests that the majority of newly synthesized CD1d is transported along the secretory pathway through the Golgi to the cell surface without detouring through endosomal compartments like class II molecules. Transport to the cell surface is not as rapid as seen for MHC class I molecules, but is faster than for MHC class II, a pattern very similar to that observed for CD1b (35Briken V. Jackman R.M. Dasgupta S. Hoening S. Porcelli S.A. EMBO J. 2002; 21: 825-834Google Scholar). We observed similar surface transport kinetics by using a surface biotinylation technique (data not shown). To determine whether the ER-resident proteins involved with the assembly of MHC class I molecules associate with CD1d, we performed co-immunoprecipitations in the detergent digitonin, conditions where their interactions with class I are maintained. Class I heavy chains bound calnexin, calreticulin, ERp57, the transporter associated with antigen processing (TAP), and tapasin, whereas CD1d associated only with the chaperones calnexin and calreticulin (data not shown). This is consistent with previous studies of TAP-deficient cells that showed that the class I peptide loading complex is not involved with CD1d assembly (36Brutkiewicz R.R. Bennink J.R. Yewdell J.W. Bendelac A. J. Exp. Med. 1995; 182: 1913-1919Google Scholar, 37Teitell M. Holcombe H.R. Brossay L. Hagenbaugh A. Jackson M.J. Pond L. Balk S.P. Terhorst C. Peterson P.A. Kronenberg M. J. Immunol. 1997; 158: 2143-2149Google Scholar). We also observed normal surface expression of CD1d in the tapasin-negative .220 B-LCL (38Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampe R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Google Scholar) transfected with CD1d (data not shown). Calnexin and calreticulin are homologous lectin domain-containing ER chaperones that regulate glycoprotein folding in the ER by interacting with their N-linked glycans (reviewed in Ref. 39Ellgaard L. Helenius A. Curr. Opin. Cell Biol. 2001; 13: 431-437Google Scholar). After transfer to a protein two terminal glucose residues are removed from the glycan by glucosidase I. Glycoproteins bearing monoglucosylatedN-linked glycans bind calnexin and calreticulin through their lectin domains and undergo folding and assembly. After release by the action of glucosidase II, which removes the terminal glucose, proper folding is monitored by the enzyme UDP-glucose:glycoprotein glucosyltransferase, which reglucosylates the N-linked glycans of nonnative structures. A correct conformation allows the glycoprotein to avoid reglucosylation and further interactions with calnexin and calreticulin. To determine whether calnexin and calreticulin association with CD1d is dependent on glucose trimming, radiolabeled C1R.CD1d cells were incubated with the glucosidase inhibitor CST for various times. Calnexin and calreticulin dissociation from CD1d was inhibited by CST, indicating that both interactions are glycan-dependent (data not shown). Calnexin and calreticulin cooperate with the thiol oxidoreductase ERp57 to facilitate the formation or isomerization of disulfide bonds during glycoprotein folding (40Elliott J.G. Oliver J.D. High S. J. Biol. Chem. 1997; 272: 13849-13855Google Scholar, 41Zapun A. Darby N.J. Tessier D.C. Michalak M. Bergeron J.J. Thomas D.Y. J. Biol. Chem. 1998; 273: 6009-6912Google Scholar, 42Oliver J.D. Roderick H.L. Llewellyn D.H. High S. Mol. Biol. Cell. 1999; 10: 2573-2582Google Scholar, 43Molinari M. Helenius A. Nature. 1999; 402: 90-93Google Scholar). To investigate this for CD1d we first examined the redox state of CD1d heavy chains at various stages; the forms associated with calnexin or calreticulin, the β2m-free, D5-reactive immature form, and the β2m-associated, 51.1.3-reactive mature form. C1R.CD1d cells were labeled for 45 min and extracts were immunoprecipitated with 51.1.3, D5, anti-calnexin, or anti-calreticulin antibodies. Anti-chaperone immunoprecipitates were eluted under nonreducing conditions (1% SDS and boiling), diluted in Triton X-100, and CD1d heavy chains were reimmunoprecipitated with D5. After treatment with Endo H to remove N-linked glycans and improve resolution, SDS-PAGE analysis showed that, whereas all forms of CD1d had identical mobilities under reducing conditions, there were differences under nonreducing conditions, reflecting differences in oxidation states (Fig. 3). β2m-associated CD1d heavy chains had the greatest mobility, i.e. were the most oxidized, whereas the calnexin- and calreticulin-associated forms and the D5-recognized form showed the same two bands. One is identical in mobility to the putatively fully oxidized, 51.1.3-recognized form, whereas the second is intermediate between that form and that seen upon complete reduction, consistent with only one of the internal disulfide bonds being oxidized. We did not detect ERp57 association with CD1d under conditions that maintain the association between ERp57 and the class I loading complex (data not shown). However, detergents disrupt the interaction of ERp57 with most glycoproteins. We therefore used a cross-linking agent to preserve the interaction. C1R.CD1d cells were labeled for 45 min and lysed in 1% digitonin in the absence or presence of the reducible cross-linking agent, DSP (1 mm). The extracts were diluted in 1% Triton X-100 and immunoprecipitated with antibodies to ERp57, calnexin, calreticulin, and, as a negative control, Grp94. After elution in SDS, CD1d heavy chains were reimmunoprecipitated with the mAb D5 followed by reducing SDS-PAGE. The results show that CD1d remains associated with ERp57 only if cross-linker is added (Fig.4 A). The association of CD1d with calnexin and calreticulin was maintained in Triton X-100 without the cross-linker, and no interaction with Grp94 was observed regardless of the presence of the cross-linker. By serial immunoprecipitation and SDS stripping, first with anti-ERp57 followed by anti-calnexin or anti-calreticulin and then by anti-CD1d, we also observed ternary complexes of ERp57, CD1d, and calnexin or calreticulin when the DSP cross-linker was used (Fig. 4 B). To ask whether ERp57 association with CD1d is glucose trimming-dependent, C1R.CD1d cells were labeled for 45 min in the absence or presence of glucosidase inhibitors CST orN-butyldeoxynojirimycin. DSP cross-linked lysates were immunoprecipitated with antibodies against ERp57, calnexin, or calreticulin, SDS-eluted, and reimmunoprecipitated with the anti-CD1d antibody, D5. Analysis by SDS-PAGE showed that ERp57, together with calnexin and calreticulin, failed to associate with CD1d in the presence of the inhibitors (Fig. 4 C). Previous reports indicate that ERp57 does not have lectin-like domains (41Zapun A. Darby N.J. Tessier D.C. Michalak M. Bergeron J.J. Thomas D.Y. J. Biol. Chem. 1998; 273: 6009-6912Google Scholar). These data, together with other reports (40Elliott J.G. Oliver J.D. High S. J. Biol. Chem. 1997; 272: 13849-13855Google Scholar, 44Van der Wal F.J. Oliver J.D. High S. Eur. J. Biochem. 1998; 256: 51-59Google Scholar), indicate that CD1d associates with ERp57 indirectly via the lectin domains of calnexin or calreticulin. During assembly of class I molecules newly synthesized free heavy chains associate with calnexin, whereas class I-β2m heterodimers associate with calreticulin (45Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Google Scholar). To determine whether this is the case for CD1d, C1R.CD1d cells were radiolabeled for 45 min, lysed in 1% digitonin, and the extract was immunoprecipitated with anti-β2m serum. β2m-associated molecules were competitively eluted with human β2m and reimmunoprecipitated with antibodies against calnexin and calreticulin. The associated CD1d was detected by immunoprecipitation with D5. The data show that, whereas class I-β2m dimers (HLA-C molecules in this case) were co-precipitated with calreticulin, CD1d-β2m dimers were co-precipitated with neither calnexin nor calreticulin (Fig.4 D). This indicates that both calnexin and calreticulin interact only with free CD1d heavy chains. Because both calnexin and calreticulin associate with β2m-free CD1d heavy chain, and CD1d has fourN-glycans, 2S.-J. Kang and P. Cresswell, unpublished observation. it seemed possible that CD1d heavy chain could form a ternary complex with both calnexin and calreticulin. This was tested by additional serial immunoprecipitation/stripping experiments on DSP cross-linked extracts. The results clearly show the existence of such a ternary complex (Fig.4 E). Given that both calnexin- and calreticulin-associated CD1d heavy chains are also associated with ERp57, the existence of a quaternary complex of CD1d, calnexin, calreticulin, and ERp57 seems likely. The data suggest that disulfide bond formation in CD1d heavy chains is regulated by ERp57. Given that D5-reactive, calnexin-associated, and calreticulin-associated CD1d heavy chains show the same two bands on SDS-PAGE under nonreducing conditions (Fig. 3), we hypothesized that the D5-reactive partially oxidized form initially associates with calnexin, calreticulin, and ERp57 and that formation of the second disulfide bond occurs during association. If this is correct, inhibition of the CD1d-chaperone interaction should result in the accumulation of D5-reactive partially oxidized CD1d heavy chains. To test this, C1R.CD1d cells were radiolabeled for 5 min and chased in the absence or presence of CST. CD1d heavy chains were immunoprecipitated with D5, treated with Endo H, and subjected to nonreducing SDS-PAGE. The ratio of partially oxidized to fully oxidized heavy chains was indeed higher throughout the chase period in the presence of CST (Fig. 5, Aand B). A model of the early biogenesis of CD1d molecules based on the data presented here is depicted in Fig.6. For simplicity only two of theN-linked glycans are shown. Calnexin and calreticulin simultaneously associate via monoglucosylated N-linked glycans with newly synthesized CD1d heavy chains in which one disulfide bond is already formed. Here we show the nonoxidized one as that in the α2 domain although there is no evidence to support this. Calnexin and/or calreticulin bring ERp57 to the β2m-free CD1d heavy chain and this mediates the formation of the second disulfide bond. The terminal glucose residues of theN-linked glycans are trimmed and the fully oxidized CD1d heavy chain then dissociates from the chaperones. The majority of the CD1d binds β2m but it can leave the ER even without it. Useful comparisons can be made between the assembly pathway illustrated in Fig. 6 and that of classical MHC class I molecules and even class II molecules. Both of these pathways involve calnexin, and class I assembly also involves calreticulin and ERp57. Class II molecules consist of heterodimers of transmembrane glycoproteins that associate with trimers of a third glycoprotein, the invariant chain, in the ER. Intermediates containing one or two αβ dimers bound to an invariant chain trimer remain associated with calnexin until the ultimate nonameric complex with three αβ dimers is complete (46Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Google Scholar). Whether ERp57 plays a role in this process is unknown. In class I assembly, newly synthesized heavy chains first bind calnexin. Calreticulin replaces calnexin when β2m binds to the heavy chain, and, together with associated ERp57, this assembly is recruited to become part of a multisubunit “peptide loading complex” that also contains the heterodimeric TAP transporter and transmembrane glycoprotein tapasin (reviewed in Ref. 47Cresswell P. Bangia N. Dick T. Diedrich G. Immunol. Rev. 1999; 172: 21-28Google Scholar). Recruitment of class I molecules to the loading complex requires calreticulin (48Gao B. Adhikari R. Howarth M. Nakamura K. Gold M.C. Hill A.B. Knee R. Michalak M. Elliott T. Immunity. 2002; 16: 99-109Google Scholar) and depends upon the generation of a monoglucosylated N-linked glycan in the class I heavy chain (40Elliott J.G. Oliver J.D. High S. J. Biol. Chem. 1997; 272: 13849-13855Google Scholar). 3Radcliffe, C. M., Diedrich, G., Harvey, D. J., Dwek, R. A., Cresswell, P., and Rudd, P. M. (2002) J. Biol. Chem. 277, 46415–46423.Recently it has been shown that tapasin binds to ERp57 via an interchain disulfide bond, and cooperates with ERp57 in forming the correct disulfide bonds in the class I heavy chain (49Dick T.P. Bangia N. Peaper D.R. Cresswell P. Immunity. 2002; 16: 87-98Google Scholar). Formation of the loading complex, including calreticulin association (48Gao B. Adhikari R. Howarth M. Nakamura K. Gold M.C. Hill A.B. Knee R. Michalak M. Elliott T. Immunity. 2002; 16: 99-109Google Scholar) and tapasin-mediated ERp57 association, is required for proper peptide binding to class I molecules, and peptide binding is required for the class I-β2m dimer to leave the ER (50Degen E. Cohen-Doyle M.F. Williams D.B. J. Exp. Med. 1992; 175: 1653-1661Google Scholar, 51Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Google Scholar). Thus, this whole process can be understood as a unique quality control mechanism in which all the components, i.e. β2m, calnexin, calreticulin, ERp57, TAP, tapasin, and even peptides, participate. There are obviously significant differences between the CD1d and MHC class I assembly and transport pathways. First, β2m association is not as critical for CD1d folding as it is for conventional class I molecules. Complete disulfide bond formation is achieved before β2m associates with CD1d and β2m-free CD1d heavy chain can leave the ER. This is further supported by previous studies, which showed that, even in vivo, β2m is not critical for the function of CD1d. β2m-free CD1d expressed in splenic cells from β2m-deficient mice is fully capable of activating cognate T cells (23Amano M. Baumgarth N. Dick M.D. Brossay L. Kronenberg M. Herzenberg L.A. Strober S. J. Immunol. 1998; 161: 1710-1717Google Scholar, 24Cardell S. Tangri S. Chan S. Kronenberg M. Benoist C. Mathis D. J. Exp. Med. 1995; 182: 993-1004Google Scholar). Second, the point in the assembly process where calreticulin interacts with MHC class I is different from that with CD1d. Class I associates with calreticulin only when complexed with β2m, whereas CD1d associates with calreticulin as a free heavy chain. Moreover, CD1d can form a quaternary complex with calnexin, calreticulin, and ERp57. Calnexin and calreticulin may bind to different glycans of the CD1d heavy chain, similar to the situation with influenza virus hemagglutinin (52Hebert D.N. Zhang J.X. Chen W. Foellmer B. Helenius A. J. Cell Biol. 1997; 139: 613-623Google Scholar). How calnexin and calreticulin mediate the folding of CD1d heavy chain and which one is responsible for recruiting ERp57 remains to be elucidated. Surface expression of CD1d in the calnexin-negative cell CEM.NKR is not impaired 4S.-J. Kang and P. Cresswell, unpublished data. so calreticulin may be more critical for CD1d folding and assembly. Third, class I assembly may need to be tightly coupled to peptide loading because class I must survey the available peptides in the ER and optimize the associated repertoire. Quality control of CD1d may not need to be tightly coupled to optimal lipid binding in the ER, although lipids may bind there (see below), because it samples antigenic lipids in the endocytic pathway. In other words, optimization of antigen binding may be separable from quality control, being restricted to the place where antigen presenting molecules survey antigens. This is further substantiated by studies of class Ib molecules, H2-M3, HLA-E, and Qa-1. These are structurally similar to class I molecules but bind hydrophobic peptides,N-formylated peptides, or signal peptides of class Ia molecules, respectively. When their cognate peptides are not available, they are retained in the ER (53Chiu N.M. Chun T. Fay M. Mandal M. Wang C.R. J. Exp. Med. 1999; 190: 423-434Google Scholar, 54Chun T. Grandea 3rd, A.G. Lybarger L. Forman J. Van Kaer L. Wang C.R. J. Immunol. 2001; 167: 1507-1514Google Scholar, 55Lybarger L. Yu Y.Y. Chun T. Wang C.R. Grandea 3rd, A.G. Van Kaer L. Hansen T.H. J. Immunol. 2001; 167: 2097-2105Google Scholar, 56Lee N. Goodlett D.R. Ishitani A. Marquardt H. Geraghty D.E. J. Immunol. 1998; 160: 4951-4960Google Scholar, 57Bai A. Broen J. Forman J. Immunity. 1998; 9: 413-421Google Scholar, 58Bai A. Aldrich C.J. Forman J. J. Immunol. 2000; 165: 7025-7034Google Scholar). Similarly, the stability of class II molecules is determined by peptide exchange mediated in the endocytic pathway by DM molecules. Given the hydrophobic nature of the CD1d binding groove it seems unlikely that the CD1d molecule would satisfy ER quality control mechanisms, and avoid reglucosylation of one or more of itsN-linked glycans by UDP-glucose:glycoprotein glucosyltransferase, unless the groove is occupied. As already discussed, the binding site of class I molecules must be occupied by peptides, and the peptide binding site of MHC class II molecules also must be occupied for efficient transport from the ER, in this case by the CLIP region of the invariant chain. For the CD1d family, ER-derived lipids, such as the phosphatidylinositol and glycosylphosphatidylinositol associated with soluble, secreted mouse CD1d, are the likely occupants of the binding groove in the ER (17Joyce S. Woods A.S. Yewdell J.W. Bennink J.R. De Silva A.D. Boesteanu A. Balk S.P. Cotter R.J. Brutkiewicz R.R. Science. 1998; 279: 1541-1544Google Scholar,18De Silva A.D. Park J.J. Matsuki N. Stanic A.K. Brutkiewicz R.R. Medof M.E. Joyce S. J. Immunol. 2002; 168: 723-733Google Scholar). We thank Nancy Dometios for help with preparation of this manuscript." @default.
• W2061344384 created "2016-06-24" @default.
• W2061344384 creator A5055658138 @default.
• W2061344384 creator A5065397632 @default.
• W2061344384 date "2002-11-01" @default.
• W2061344384 modified "2023-10-16" @default.
• W2061344384 title "Calnexin, Calreticulin, and ERp57 Cooperate in Disulfide Bond Formation in Human CD1d Heavy Chain" @default.
• W2061344384 cites W1491069734 @default.
• W2061344384 cites W1491748015 @default.
• W2061344384 cites W1508427423 @default.
• W2061344384 cites W1551389252 @default.
• W2061344384 cites W1558024223 @default.
• W2061344384 cites W1564678593 @default.
• W2061344384 cites W1607176255 @default.
• W2061344384 cites W1813398788 @default.
• W2061344384 cites W1886629964 @default.
• W2061344384 cites W1906987668 @default.
• W2061344384 cites W1920719136 @default.
• W2061344384 cites W1972054911 @default.
• W2061344384 cites W1972085567 @default.
• W2061344384 cites W1986645576 @default.
• W2061344384 cites W1987824008 @default.
• W2061344384 cites W2003625969 @default.
• W2061344384 cites W2015569988 @default.
• W2061344384 cites W2018302186 @default.
• W2061344384 cites W2020746301 @default.
• W2061344384 cites W2022284744 @default.
• W2061344384 cites W2024624695 @default.
• W2061344384 cites W2027679419 @default.
• W2061344384 cites W2031192254 @default.
• W2061344384 cites W2034273045 @default.
• W2061344384 cites W2046549183 @default.
• W2061344384 cites W2053088462 @default.
• W2061344384 cites W2054871814 @default.
• W2061344384 cites W2058910688 @default.
• W2061344384 cites W2064507821 @default.
• W2061344384 cites W2068920418 @default.
• W2061344384 cites W2069102137 @default.
• W2061344384 cites W2075018537 @default.
• W2061344384 cites W2075908603 @default.
• W2061344384 cites W2084416493 @default.
• W2061344384 cites W2087113665 @default.
• W2061344384 cites W2093497258 @default.
• W2061344384 cites W2105433002 @default.
• W2061344384 cites W2105857780 @default.
• W2061344384 cites W2110693976 @default.
• W2061344384 cites W2115183373 @default.
• W2061344384 cites W2119347540 @default.
• W2061344384 cites W2122029067 @default.
• W2061344384 cites W2130875567 @default.
• W2061344384 cites W2131641718 @default.
• W2061344384 cites W2138205101 @default.
• W2061344384 cites W2147402069 @default.
• W2061344384 cites W2151292780 @default.
• W2061344384 cites W2154859242 @default.
• W2061344384 cites W2156460811 @default.
• W2061344384 cites W2158117877 @default.
• W2061344384 cites W2161307276 @default.
• W2061344384 cites W2163164309 @default.
• W2061344384 cites W2167688505 @default.
• W2061344384 cites W2186755194 @default.
• W2061344384 cites W2405228935 @default.
• W2061344384 cites W4313310004 @default.
• W2061344384 cites W4313331714 @default.
• W2061344384 cites W4313333799 @default.
• W2061344384 doi "https://doi.org/10.1074/jbc.m207831200" @default.
• W2061344384 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12239218" @default.
• W2061344384 hasPublicationYear "2002" @default.
• W2061344384 type Work @default.
• W2061344384 sameAs 2061344384 @default.
• W2061344384 citedByCount "109" @default.
• W2061344384 countsByYear W20613443842012 @default.
• W2061344384 countsByYear W20613443842013 @default.
• W2061344384 countsByYear W20613443842014 @default.
• W2061344384 countsByYear W20613443842015 @default.
• W2061344384 countsByYear W20613443842016 @default.
• W2061344384 countsByYear W20613443842017 @default.
• W2061344384 countsByYear W20613443842018 @default.
• W2061344384 countsByYear W20613443842020 @default.
• W2061344384 countsByYear W20613443842022 @default.
• W2061344384 countsByYear W20613443842023 @default.
• W2061344384 crossrefType "journal-article" @default.
• W2061344384 hasAuthorship W2061344384A5055658138 @default.
• W2061344384 hasAuthorship W2061344384A5065397632 @default.
• W2061344384 hasBestOaLocation W20613443841 @default.
• W2061344384 hasConcept C104317684 @default.
• W2061344384 hasConcept C121332964 @default.
• W2061344384 hasConcept C1276947 @default.
• W2061344384 hasConcept C130361206 @default.
• W2061344384 hasConcept C158617107 @default.
• W2061344384 hasConcept C185592680 @default.
• W2061344384 hasConcept C199185054 @default.
• W2061344384 hasConcept C27256138 @default.
• W2061344384 hasConcept C47450691 @default.
• W2061344384 hasConcept C55493867 @default.
• W2061344384 hasConcept C84613547 @default.
• W2061344384 hasConcept C86803240 @default.
• W2061344384 hasConcept C95444343 @default.

• next