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• W2018607054 abstract "CD1d is an MHC class I-like molecule comprised of a transmembrane glycoprotein (heavy chain) associated with β2-microglobulin (β2m) that presents lipid antigens to NKT cells. Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β2m to form the heterodimer. Here we show that in CRT-deficient cells CD1d heavy chains convert to β2m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected. Unlike the situation with MHC class I molecules, antigen presentation by CD1d is not impaired in the absence of CRT. Instead, there are elevated levels of stable and functional CD1d on the surface of CRT-deficient cells. Association of the heavy chains with the ER chaperones Grp94 and Bip is observed in the absence of CRT, and these may replace CRT in mediating CD1d folding and assembly. ER retention of free CD1d heavy chains is impaired in CRT-deficient cells, allowing their escape and subsequent expression on the plasma membrane. However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β2m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed. CD1d is an MHC class I-like molecule comprised of a transmembrane glycoprotein (heavy chain) associated with β2-microglobulin (β2m) that presents lipid antigens to NKT cells. Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β2m to form the heterodimer. Here we show that in CRT-deficient cells CD1d heavy chains convert to β2m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected. Unlike the situation with MHC class I molecules, antigen presentation by CD1d is not impaired in the absence of CRT. Instead, there are elevated levels of stable and functional CD1d on the surface of CRT-deficient cells. Association of the heavy chains with the ER chaperones Grp94 and Bip is observed in the absence of CRT, and these may replace CRT in mediating CD1d folding and assembly. ER retention of free CD1d heavy chains is impaired in CRT-deficient cells, allowing their escape and subsequent expression on the plasma membrane. However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β2m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed. CD1 molecules are encoded by a family of linked genes located outside the major histocompatibility complex (MHC) 2The abbreviations used are: MHCmajor histocompatibility complexBipimmunoglobulin-binding proteinCD1cluster of differentiation 1β2mβ2-microglobulinERendoplasmic reticulumCNXcalnexinCRTcalreticulinα-GalCerα-galactosyl ceramideCSTcastanospermineGalGalCergalactosyl-b(b1–2)-galactosyl ceramideNKTnatural killer T cellsEndo Hendoglycosidase HAPadaptor proteinDSPdithiobis succinimydyl propionateCHXcycloheximide. (reviewed in Ref. 1Brigl M. Brenner M.B. Annu. Rev. Immunol. 2004; 22: 817-890Crossref PubMed Scopus (889) Google Scholar). In humans, five CD1 isoforms have been identified, including CD1a, b, c, d, and e, that are divided into two groups based on amino acid sequence homology. Group 1 includes CD1a, b, c and e, and group 2 consists only of CD1d. CD1d is the sole CD1 species found in mice and rats. The overall structure of CD1 glycoproteins resembles that of MHC class I molecules (2Zeng Z. Castaño A.R. Segelke B.W. Stura E.A. Peterson P.A. Wilson I.A. Science. 1997; 277: 339-345Crossref PubMed Scopus (562) Google Scholar, 3Koch M. Stronge V.S. Shepherd D. Gadola S.D. Mathew B. Ritter G. Fersht A.R. Besra G.S. Schmidt R.R. Jones E.Y. Cerundolo V. Nat. Immunol. 2005; 6: 819-826Crossref PubMed Scopus (334) Google Scholar). Each consists of a transmembrane heavy chain that non-covalently associates with β2m. The heavy chain can be further divided into three extracellular domains (α1, α2 and α3), a transmembrane domain, and a cytoplasmic region. The α1 and α2 domains fold to generate a binding pocket that, unlike MHC class I and class II glycoproteins, which associate with short peptides, binds a lipid. The lipid binding pocket is similar to the peptide binding pocket of MHC molecules, consisting of a pair of antiparallel α-helices overlaying an eight strand β-sheet. CD1 binding pockets are deep and highly hydrophobic, and can accommodate the alkyl chains of a wide variety of lipids (4Silk J.D. Salio M. Brown J. Jones E.Y. Cerundolo V. Annu. Rev. Cell Dev. Biol. 2008; 24: 369-395Crossref PubMed Scopus (48) Google Scholar). Similar to the way that MHC class I- and class II-restricted T cells can recognize peptide antigens presented by MHC glycoproteins, lipid antigens associated with CD1 molecules can also be recognized by effector T cells (5Beckman E.M. Porcelli S.A. Morita C.T. Behar S.M. Furlong S.T. Brenner M.B. Nature. 1994; 372: 691-694Crossref PubMed Scopus (885) Google Scholar, 6Barral D.C. Brenner M.B. Nat. Rev. Immunol. 2007; 7: 929-941Crossref PubMed Scopus (269) Google Scholar). In particular, CD1d molecules present lipids to a unique subpopulation of T lymphocytes, called NKT cells, which co-express an invariant T cell receptor (TCR) and surface markers also found on natural killer (NK) cells (7Bendelac A. Lantz O. Quimby M.E. Yewdell J.W. Bennink J.R. Brutkiewicz R.R. Science. 1995; 268: 863-865Crossref PubMed Scopus (847) Google Scholar). Recent data have shown that both endogenous lipids and exogenous lipids, e.g. from Sphingomonas and Borrelia burgdorferi species, can be presented to NKT cells by CD1d molecules (8Wang J. Li Y. Kinjo Y. Mac T.T. Gibson D. Painter G.F. Kronenberg M. Zajonc D.M. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 1535-1540Crossref PubMed Scopus (84) Google Scholar, 9Kinjo Y. Wu D. Kim G. Xing G.W. Poles M.A. Ho D.D. Tsuji M. Kawahara K. Wong C.H. Kronenberg M. Nature. 2005; 434: 520-525Crossref PubMed Scopus (805) Google Scholar, 10Mattner J. Debord K.L. Ismail N. Goff R.D. Cantu 3rd, C. Zhou D. Saint-Mezard P. Wang V. Gao Y. Yin N. Hoebe K. Schneewind O. Walker D. Beutler B. Teyton L. Savage P.B. Bendelac A. Nature. 2005; 434: 525-529Crossref PubMed Scopus (962) Google Scholar, 11Zhou D. Mattner J. Cantu 3rd, C. Schrantz N. Yin N. Gao Y. Sagiv Y. Hudspeth K. Wu Y.P. Yamashita T. Teneberg S. Wang D. Proia R.L. Levery S.B. Savage P.B. Teyton L. Bendelac A. Science. 2004; 306: 1786-1789Crossref PubMed Scopus (847) Google Scholar, 12Kinjo Y. Tupin E. Wu D. Fujio M. Garcia-Navarro R. Benhnia M.R. Zajonc D.M. Ben-Menachem G. Ainge G.D. Painter G.F. Khurana A. Hoebe K. Behar S.M. Beutler B. Wilson I.A. Tsuji M. Sellati T.J. Wong C.H. Kronenberg M. Nat. Immunol. 2006; 7: 978-986Crossref PubMed Scopus (531) Google Scholar). Upon activation, NKT cells secrete both T helper type 1 and type 2 cytokines, and play important roles in both innate and adaptive immunity (13Bendelac A. Savage P.B. Teyton L. Annu. Rev. Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1809) Google Scholar). major histocompatibility complex immunoglobulin-binding protein cluster of differentiation 1 β2-microglobulin endoplasmic reticulum calnexin calreticulin α-galactosyl ceramide castanospermine galactosyl-b(b1–2)-galactosyl ceramide natural killer T cells endoglycosidase H adaptor protein dithiobis succinimydyl propionate cycloheximide. The presentation of lipid antigens depends on the proper assembly and intracellular trafficking of CD1 glycoproteins. Shortly after their synthesis in the endoplasmic reticulum (ER) and assembly with β2m, CD1d molecules follow the secretory pathway to the cell surface (14Brutkiewicz R.R. Bennink J.R. Yewdell J.W. Bendelac A. J. Exp. Med. 1995; 182: 1913-1919Crossref PubMed Scopus (149) Google Scholar, 15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). From there, CD1d is routed to endosomal compartments by a tyrosine-based motif, YXXZ (where Y is tyrosine, X is any amino acid, and Z is a bulky hydrophobic amino acid), located in its cytoplasmic domain (16Jayawardena-Wolf J. Benlagha K. Chiu Y.H. Mehr R. Bendelac A. Immunity. 2001; 15: 897-908Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Adaptor proteins (AP) bind to this motif at the plasma membrane and direct the internalization of CD1d molecules via clathrin-coated pits (17Elewaut D. Lawton A.P. Nagarajan N.A. Maverakis E. Khurana A. Honing S. Benedict C.A. Sercarz E. Bakke O. Kronenberg M. Prigozy T.I. J. Exp. Med. 2003; 198: 1133-1146Crossref PubMed Scopus (92) Google Scholar, 18Gumperz J.E. Traffic. 2006; 7: 2-13Crossref PubMed Scopus (47) Google Scholar). Binding of lipid antigens to CD1d molecules occurs mainly in the endocytic system and is catalyzed by endosomal lipid transfer proteins, predominantly the saposins (19Zhou D. Cantu 3rd, C. Sagiv Y. Schrantz N. Kulkarni A.B. Qi X. Mahuran D.J. Morales C.R. Grabowski G.A. Benlagha K. Savage P. Bendelac A. Teyton L. Science. 2004; 303: 523-527Crossref PubMed Scopus (281) Google Scholar, 20Kang S.J. Cresswell P. Nat. Immunol. 2004; 5: 175-181Crossref PubMed Scopus (181) Google Scholar, 21Yuan W. Qi X. Tsang P. Kang S.J. Illarionov P.A. Besra G.S. Gumperz J. Cresswell P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5551-5556Crossref PubMed Scopus (88) Google Scholar). Abolishing endosomal targeting of CD1d, by mutating the endocytic motif, or disrupting lysosomal acidification, which affects saposin function, significantly impairs antigen presentation by CD1d (22Chiu Y.H. Park S.H. Benlagha K. Forestier C. Jayawardena-Wolf J. Savage P.B. Teyton L. Bendelac A. Nat. Immunol. 2002; 3: 55-60Crossref PubMed Scopus (171) Google Scholar, 23Roberts T.J. Sriram V. Spence P.M. Gui M. Hayakawa K. Bacik I. Bennink J.R. Yewdell J.W. Brutkiewicz R.R. J. Immunol. 2002; 168: 5409-5414Crossref PubMed Scopus (102) Google Scholar). In addition to accessing the endocytic pathway by AP-dependent endocytosis, CD1d molecules can also be directed there through an interaction with the invariant chain, normally responsible for the endocytic localization of MHC class II molecules, or by an association with MHC class II-invariant chain complexes (16Jayawardena-Wolf J. Benlagha K. Chiu Y.H. Mehr R. Bendelac A. Immunity. 2001; 15: 897-908Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 24Kang S.J. Cresswell P. EMBO J. 2002; 21: 1650-1660Crossref PubMed Scopus (110) Google Scholar). The functional significance of this alternative route for endosomal assessment is unclear. Nevertheless, there is evidence that CD1d molecules can undergo multiple rounds of recycling between the cell surface and endosomal compartments to extensively survey changes in lipid composition (16Jayawardena-Wolf J. Benlagha K. Chiu Y.H. Mehr R. Bendelac A. Immunity. 2001; 15: 897-908Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 22Chiu Y.H. Park S.H. Benlagha K. Forestier C. Jayawardena-Wolf J. Savage P.B. Teyton L. Bendelac A. Nat. Immunol. 2002; 3: 55-60Crossref PubMed Scopus (171) Google Scholar). Associated lipid antigens are presented at the plasma membrane for NKT cell recognition. Previous studies have identified accessory molecules involved in the early biogenesis of CD1d molecules inside the ER (14Brutkiewicz R.R. Bennink J.R. Yewdell J.W. Bendelac A. J. Exp. Med. 1995; 182: 1913-1919Crossref PubMed Scopus (149) Google Scholar, 15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 25Paduraru C. Spiridon L. Yuan W. Bricard G. Valencia X. Porcelli S.A. Illarionov P.A. Besra G.S. Petrescu S.M. Petrescu A.J. Cresswell P. J. Biol. Chem. 2006; 281: 40369-40378Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Like other glycoproteins, the correct folding of CD1d involves the lectin chaperones (15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). After translocation into the ER, newly synthesized CD1d heavy chains are rapidly glycosylated and bind caltreticulin (CRT) and calnexin (CNX), which recognize monoglucosylated N-linked glycans. These ER chaperones recruit the thiol oxidoreductase ERp57, which catalyzes disulfide bond formation in CD1d heavy chains. Once released from the CNX/CRT cycle, fully oxidized CD1d heavy chains associate with β2m and enter the Golgi where the glycans are further processed before transport to the plasma membrane. Unlike MHC class I and CD1b molecules, association with β2m is not strictly required for CD1d to exit the ER and small amounts of free CD1d heavy chains expressed on the cell surface may still be functional (26Balk S.P. Burke S. Polischuk J.E. Frantz M.E. Yang L. Porcelli S. Colgan S.P. Blumberg R.S. Science. 1994; 265: 259-262Crossref PubMed Scopus (147) Google Scholar, 27Kim H.S. Garcia J. Exley M. Johnson K.W. Balk S.P. Blumberg R.S. J. Biol. Chem. 1999; 274: 9289-9295Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Brossay L. Jullien D. Cardell S. Sydora B.C. Burdin N. Modlin R.L. Kronenberg M. J. Immunol. 1997; 159: 1216-1224PubMed Google Scholar, 29Amano M. Baumgarth N. Dick M.D. Brossay L. Kronenberg M. Herzenberg L.A. Strober S. J. Immunol. 1998; 161: 1710-1717PubMed Google Scholar). Consistent with the hypothesis that CD1 molecules are loaded with endogenous lipids during assembly inside the ER, multiple groups have found the association of ER-derived lipids with ER-retained or -secreted soluble CD1d (30Yuan W. Kang S.J. Evans J.E. Cresswell P. J. Immunol. 2009; 182: 4784-4791Crossref PubMed Scopus (78) Google Scholar, 31Joyce 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: 1541-1544Crossref PubMed Scopus (364) Google Scholar, 32Cox D. Fox L. Tian R. Bardet W. Skaley M. Mojsilovic D. Gumperz J. Hildebrand W. PLoS One. 2009; 4: e5325Crossref PubMed Scopus (118) Google Scholar). Loading of ER-derived lipids to nascent CD1d molecules may be mediated by microsomal triglyceride transfer protein (MTP), the only known lipid transfer protein found in the ER (33Dougan S.K. Salas A. Rava P. Agyemang A. Kaser A. Morrison J. Khurana A. Kronenberg M. Johnson C. Exley M. Hussain M.M. Blumberg R.S. J. Exp. Med. 2005; 202: 529-539Crossref PubMed Scopus (134) Google Scholar, 34Zeissig S. Dougan S.K. Barral D.C. Junker Y. Chen Z. Kaser A. Ho M. Mandel H. McIntyre A. Kennedy S.M. Painter G.F. Veerapen N. Besra G.S. Cerundolo V. Yue S. Beladi S. Behar S.M. Chen X. Gumperz J.E. Breckpot K. Raper A. Baer A. Exley M.A. Hegele R.A. Cuchel M. Rader D.J. Davidson N.O. Blumberg R.S. J. Clin. Invest. 2010; 120: 2889-2899Crossref PubMed Scopus (68) Google Scholar). However the role of lipids in the quality control of CD1d assembly is poorly understood. The assembly of MHC class I molecules also requires lectin chaperones, including CRT. The role of CRT in MHC class I antigen presentation has been studied in CRT-deficient murine embryonic fibroblasts (MEFs) (35Gao B. Adhikari R. Howarth M. Nakamura K. Gold M.C. Hill A.B. Knee R. Michalak M. Elliott T. Immunity. 2002; 16: 99-109Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 36Howe C. Garstka M. Al-Balushi M. Ghanem E. Antoniou A.N. Fritzsche S. Jankevicius G. Kontouli N. Schneeweiss C. Williams A. Elliott T. Springer S. EMBO J. 2009; 28: 3730-3744Crossref PubMed Scopus (69) Google Scholar). In such cells, MHC class I molecules assemble with β2m normally and move to the cell surface with accelerated kinetics. However, loading with optimal peptides within the peptide loading complex inside the ER is defective. Consequently a large fraction of MHC class I associates with low-affinity peptides and is unstable, and T cell recognition is impaired. These results demonstrated a key role for CRT in the quality control of class I-peptide complexes, and inspired us to examine the role of CRT in the assembly and function of CD1d molecules. In this study, we have examined the assembly and antigen presentation functions of CD1d in CRT-deficient MEFs. We find that neither of these functions is impaired. Instead, we observed accelerated conversion of CD1d free heavy chains to β2m-associated dimers and the accumulation of elevated levels of stable and functional dimers on the cell surface. However, ER retention of free CD1d heavy chains is not efficient in the absence of CRT, resulting in the expression of free heavy chains on the plasma membrane. Wild-type (K41) and CRT-deficient (K42) fibroblasts were gifts from Dr. David Williams, University of Toronto. The fibroblasts and their transfectants were maintained in Iscove's modified Dulbecco's medium (Sigma) supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in 5% CO2 atmosphere. The mouse Vα14Jα18 invariant TCR-positive cell hybridoma, DN32.D3, was kindly provided by Dr. Albert Bendelac, University of Chicago. The RetroMax Retroviral System (Imgenex) was used to express CD1d in K41 and K42 cells. Briefly, human CD1d cDNA was subcloned into the MSCV2.2 IRES-GFP vector to generate a CD1d-IRES-GFP construct. The vector was co-transfected into the packaging cell line 293T with pCL-Eco packing vector. 24–48h after transfection the virus supernatant was harvested to infect K41 and K42 cells and stable transfectants were sorted by flow cytometry using GFP expression. The pQCXIH vector containing wild-type mouse CRT cDNA was provided by Dr. D. Williams and was transduced into K42.CD1d cells as described (37Ireland B.S. Brockmeier U. Howe C.M. Elliott T. Williams D.B. Mol. Biol. Cell. 2008; 19: 2413-2423Crossref PubMed Scopus (51) Google Scholar). The monoclonal mouse antibodies (mAbs) to human CD1d, CD1d51, and D5, have been previously described (15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The polyclonal rabbit anti-CRT antibody, the rat anti-Grp94 mAb, and the polyclonal rabbit anti-Bip (Grp78) antibody were from Stressgen. The mouse anti-GAPDH mAb was from RDI. The mouse anti-GFP mAb was from Clontech. The rat anti-Lamp1 mAb was from the Developmental Studies Hybridoma Bank. The glycosidases Endo H and PNGase F were from New England Biolabs. Brefeldin A and cycloheximide were from Sigma-Aldrich. α-GalCer and galactosyl-(α1–2)-galactosyl ceramide (GalGalCer) were synthesized as described (38van den Elzen P. Garg S. León L. Brigl M. Leadbetter E.A. Gumperz J.E. Dascher C.C. Cheng T.Y. Sacks F.M. Illarionov P.A. Besra G.S. Kent S.C. Moody D.B. Brenner M.B. Nature. 2005; 437: 906-910Crossref PubMed Scopus (306) Google Scholar). Western blot analyses were performed as previously described (25Paduraru C. Spiridon L. Yuan W. Bricard G. Valencia X. Porcelli S.A. Illarionov P.A. Besra G.S. Petrescu S.M. Petrescu A.J. Cresswell P. J. Biol. Chem. 2006; 281: 40369-40378Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For quantitative Western blots, the protein content of the post-nuclear supernatant was quantified by Bradford assay (BioRad) and 3-fold serial dilutions were resolved by SDS-PAGE. Following primary antibody incubation, membranes were probed with alkaline phosphatase-coupled secondary antibody (1:5000) (Jackson Labs) and imaged using a fluorimager with ECF substrate (Amersham Biosciences). MEF transfectants were harvested, washed, starved for 1 h in medium without methionine/cysteine, pulse-labeled with 35S labeling mix (PerkinElmer Life Sciences), chased in an excess of unlabeled methionine/cysteine for the indicated times and washed with cold PBS. For experiments examining the lectin-mediated retention of CD1d within the ER, cells were starved and labeled without castanospermine (CST) (Sigma) and chased in the presence of 2 mm CST as indicated. For all immunoprecipitation experiments except as otherwise indicated, cells were solubilized in 1% digitonin in TBS with a proteinase inhibitor mixture (Roche). Post-nuclear supernatants were precleared with normal rabbit or mouse serum and protein G-Sepharose (GE healthcare) prior to specific immunoprecipitation with the indicated antibody and protein G-Sepharose. For re-precipitation, immunoprecipitated proteins were eluted by heating to 95 °C in 1% SDS, 5 mm dithiothreitol. The eluted material was then diluted into 1% Triton X-100 in TBS and free CD1d heavy chains precipitated with D5 mAb and protein G-Sepharose prior to SDS-PAGE. When Endo H digestion was performed, precipitated proteins were eluted using Endo H buffer, and incubated overnight with Endo H, as suggested by the manufacturer. Cells (0.5 × 106) were stained as previously described (25Paduraru C. Spiridon L. Yuan W. Bricard G. Valencia X. Porcelli S.A. Illarionov P.A. Besra G.S. Petrescu S.M. Petrescu A.J. Cresswell P. J. Biol. Chem. 2006; 281: 40369-40378Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and analyzed using a Becton Dickinson FACScalibur (Mountain View, CA). MEF cells transfected with CD1d were incubated with α-GalCer for 4 h or GalGalCer overnight. After three washes in PBS, the cells were fixed in 0.05% glutaraldehyde for 30 s, quenched with an equal volume of 0.4 m glycine and washed in medium three times. The fixed cells (1 × 105 cells/well) were then plated in triplicate in 96-well plates and co-cultured with 1 × 105 DN32.D3 cells/well for 24 h. Secreted mIL-2 was detected by an Opti-ELISA kit from BD Pharmingen following the manufacturer's instructions. Cells were lysed on ice in bicine buffer (0.13 m NaCl, 0.02 m Bicine, pH 8.2) containing 1% Triton X-100, 200 μg/ml dithiobis (succinimydyl propionate) (DSP) (Thermo Scientific), and the proteinase inhibitor mixture. Lysates were quenched with 10 mm glycine before immunoprecipitation. Cells growing on cover slips were fixed at room temperature for 15 min in 3% formaldehyde, quenched with 10 mm glycine for 10 min and permeabilized in PBS containing 0.5% saponin (Sigma-Aldrich) and 0.5% BSA. For co-staining, cells were incubated with D5 mouse mAb and rat anti-Lamp1 mAb, followed by Alexa Fluor 546-conjugated highly cross-absorbed goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rat IgG antibodies (Molecular Probes). All images were acquired by using a Leica TCS SP2 confocal system and processed with Leica software. To investigate the role of CRT in the assembly of CD1d molecules, we used the CRT-sufficient MEF cell line K41 and its CRT-deficient counterpart K42. Because MEF cells have no detectable CD1d expression (20Kang S.J. Cresswell P. Nat. Immunol. 2004; 5: 175-181Crossref PubMed Scopus (181) Google Scholar), we retrovirally transduced a CD1d-IRES-GFP construct into K41 and K42 cells to generate the stable transfectants K41.CD1d and K42.CD1d. The internal ribosome entry site (IRES) from encephalomyocarditis virus (ECMV) between the CD1d- and GFP-coding regions permitted both genes to be translated from a single bicistronic mRNA. We used flow cytometry to sort stably transduced cell populations that expressed similar levels of GFP, assessed by Western blotting (Fig. 1, A and B) or flow cytometry (Fig. 4A). As expected, this resulted in equal rates of CD1d protein synthesis in both transfected cell lines as determined by a short metabolic labeling experiment (Fig. 1C). However, when steady state protein levels were assessed by Western blot, CD1d levels were found to be substantially higher in the absence of CRT (Fig. 1A, compare lanes 2 and 4, and Fig. 1B). Quantitative Western blotting showed that steady state protein levels of CD1d in CRT-deficient K42.CD1d cells are ∼2-fold higher than in CRT-sufficient K41.CD1d cells. In contrast, levels of GFP and GAPDH, two non-glycoproteins, were indistinguishable in the two cell lines (Fig. 1, A and B). To confirm that the elevated CD1d level was caused by the absence of CRT, we reconstituted CRT expression in K42.CD1d, generating the cell line K42.CD1d.CRT. Introduction of CRT caused a significant decrease in CD1d steady state protein levels (Fig. 1A, compare lanes 4 and 5 and Fig. 1B), whereas levels of GFP and GAPDH remained unaffected. The CD1d level in K42.CD1d.CRT still remained somewhat above that in K41.CD1d, which may be because of the ∼2-fold higher CRT expression in the latter (Fig. 1A, compare lanes 2 and 5 and Fig. 1B).FIGURE 4Absence of CRT promotes elevated levels of functional surface CD1d. A, surface expression of β2m-associated CD1d dimers on K41.CD1d, K42.CD1d and K42.CD1d.CRT cells was examined by flow cytometry using the CD1d51 antibody (unfilled histograms, middle panel). Corresponding GFP expression in each cell type was also assessed by flow cytometry (unfilled histograms, left panel). Background staining was analyzed by incubating parental CD1d-negative MEFs with CD1d51 antibody (filled histograms, left and middle panels). Right panel, statistical analysis of the data in left and middle panels. MFI values of K42.CD1d and K42.CD1d.CRT were normalized against that of K41.CD1d cells. Data shown are the average ± S.E. of three separate experiments. B, presentation of α-GalCer and GalGalCer by K41.CD1d, K42.CD1d and K42.CD1d.CRT. Cells were incubated with different concentrations of α-GalCer for 4 h (left panel) or with GalGalCer overnight (right panel). After washing and fixing, the cells were co-cultured with the mouse NKT hybridoma, DN32.D3, in triplicate. Secretion of mIL-2 into the medium was analyzed after 24 h by sandwich ELISA. Results were expressed as the mean ± S.E. of triplicate values. The data shown represent one of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CD1d ran as a doublet upon SDS-PAGE (Figs. 1A and 2A), and we speculated that the two bands correspond to different glycosylation isoforms. Consistent with this, treatment of whole cell lysates with peptide-N-glycosidase F (PNGase F), which cleaves N-linked oligosaccharides from glycoproteins, gave rise to a single CD1d band with a substantially lower apparent molecular weight (Fig. 2A). CD1d has four N-linked glycosylation sites, one of which, at position Asn-42, fails to be processed to the complex form in the Golgi apparatus because of its proximity to the associated β2m (25Paduraru C. Spiridon L. Yuan W. Bricard G. Valencia X. Porcelli S.A. Illarionov P.A. Besra G.S. Petrescu S.M. Petrescu A.J. Cresswell P. J. Biol. Chem. 2006; 281: 40369-40378Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and remains sensitive to the enzyme endoglycosidase H (Endo H). When detergent lysates were treated with Endo H, subjected to SDS-PAGE and blotted for CD1d, two major bands were observed, which represent the partially Endo H-resistant post-ER CD1d and the Endo H-sensitive ER-localized CD1d (Fig. 2A). In K41.CD1d cells the Endo H-sensitive ER form of CD1d was present at a substantially higher level, whereas K42.CD1d cells contained more partially Endo H-resistant post-ER forms (Fig. 2A, compare lanes 2 and 5). The data indicate that in the absence of CRT ER retention of CD1d is lower despite the higher steady state level. We previously observed that the interaction of CD1d free heavy chains with CRT and CNX is glycosylation-dependent (15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). After the core Glc3Man9GlcNAc2 oligosaccharide is transferred onto newly synthesized CD1d free heavy chains, it is trimmed by glucosidases I and II to the Glc1Man9GlcNAc2 processing intermediate, which is the specific ligand for CRT and CNX (39Spiro R.G. Zhu Q. Bhoyroo V. Söling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 40Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). If the generation of this monoglucosylated folding intermediate is inhibited by the glucosidase inhibitor CST, CRT and CNX fail to interact with CD1d (15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Moreover, as for other glycoprotein substrates (41Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 42Cannon K.S. Helenius A. J. Biol. Chem. 1999; 274: 7537-7544Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), dissociation of CRT and CNX from CD1d heavy chains is dependent on trimming of the terminal glucose from the glycan (15Kang S.J. Cresswell P. J. Biol. Chem. 2002; 277: 44838-44844Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The observed increase in Endo H-sensitive CD1d when CRT is present suggested that CRT might be a critical retention factor. To examine this question, K41.CD1d and K42.CD1d cells were pulse-labeled with [35S]methionine/cysteine without CST for 15 min and chased in the presence of CST for various times to inhibit the removal of the terminal glucose. CD1d remained completely sensitive to Endo H in CST-treated CRT-sufficient K41.CD1d cells (Fig. 2, B, left panels and C, left panel), indicating a block in release from the ER. However, although the rate of transport was reduced in CR" @default.
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