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An Essential Function of Tapasin in Quality Control of HLA-G Molecules

2003; Elsevier BV; Volume: 278; Issue: 16 Linguagem: Inglês

10.1074/jbc.m212882200

1083-351X

Boyoun Park, Kwangseog Ahn,

Reproductive Biology and Fertility

Tapasin plays an important role in the quality control of major histocompatibility complex (MHC) class I assembly, but its precise function in this process remains controversial. Whether tapasin participates in the assembly of HLA-G has not been studied. HLA-G, an MHC class Ib molecule that binds a more restricted set of peptides than class Ia molecules, is a particularly interesting molecule, because during assembly, it recycles between the endoplasmic reticulum (ER) and the cis-Golgi until it is loaded with a high affinity peptide. We have taken advantage of this unusual trafficking property of HLA-G and its requirement for high affinity peptides to demonstrate that a critical function of tapasin is to transform class I molecules into a high affinity, peptide-receptive form. In the absence of tapasin, HLA-G molecules cannot bind high affinity peptides, and an abundant supply of peptides cannot overcome the tapasin requirement for high affinity peptide loading. The addition of tapasin renders HLA-G molecules capable of loading high affinity peptides and of transporting to the surface, suggesting that tapasin is a prerequisite for the binding of high-affinity ligands. Interestingly, the “tapasin-dependent” HLA-G molecules are not empty in the absence of tapasin but are in fact associated with suboptimal peptides and continue to recycle between the ER and the cis-Golgi. Together with the finding that empty HLA-G heterodimers are strictly retained in the ER and degraded, our data suggest that MHC class I molecules bind any available peptides to avoid ER-mediated degradation and that the peptides are in turn replaced by higher affinity peptides with the aid of tapasin. Tapasin plays an important role in the quality control of major histocompatibility complex (MHC) class I assembly, but its precise function in this process remains controversial. Whether tapasin participates in the assembly of HLA-G has not been studied. HLA-G, an MHC class Ib molecule that binds a more restricted set of peptides than class Ia molecules, is a particularly interesting molecule, because during assembly, it recycles between the endoplasmic reticulum (ER) and the cis-Golgi until it is loaded with a high affinity peptide. We have taken advantage of this unusual trafficking property of HLA-G and its requirement for high affinity peptides to demonstrate that a critical function of tapasin is to transform class I molecules into a high affinity, peptide-receptive form. In the absence of tapasin, HLA-G molecules cannot bind high affinity peptides, and an abundant supply of peptides cannot overcome the tapasin requirement for high affinity peptide loading. The addition of tapasin renders HLA-G molecules capable of loading high affinity peptides and of transporting to the surface, suggesting that tapasin is a prerequisite for the binding of high-affinity ligands. Interestingly, the “tapasin-dependent” HLA-G molecules are not empty in the absence of tapasin but are in fact associated with suboptimal peptides and continue to recycle between the ER and the cis-Golgi. Together with the finding that empty HLA-G heterodimers are strictly retained in the ER and degraded, our data suggest that MHC class I molecules bind any available peptides to avoid ER-mediated degradation and that the peptides are in turn replaced by higher affinity peptides with the aid of tapasin. After being targeted to the endoplasmic reticulum (ER), 1The abbreviations used are: ERendoplasmic reticulumβ2mβ2-microglobulinTAPtransporter associated with antigen processingMHCmajor histocompatibility complexmAbmonoclonal antibodyPBSphosphate-buffered salineendo-Hendoglycosidase-HTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine nascent MHC class I heavy chains associate with a multiprotein complex that assists in their assembly with peptides and β2-microglobulin (β2m). This complex includes calnexin, calreticulin, ERp57, transporter associated with antigen processing (TAP), and tapasin (1Cresswell P. Bangia N. Dick T. Diedrich G. Immunol. Rev. 1999; 172: 21-28Crossref PubMed Scopus (270) Google Scholar). MHC class I molecules are divided into two types based on their polymorphism and levels of expression. The highly expressed polymorphic class Ia molecules bind a diverse set of peptides derived from the cytosol, whereas the less abundant, tissue-specific, nonpolymorphic class Ib molecules bind a more restricted set of peptides (2Shawar S.M. Vyas J.M. Rodgers J.R. Rich R.R. Annu. Rev. Immunol. 1994; 12: 839-880Crossref PubMed Scopus (226) Google Scholar). Tapasin is indispensable for the proper function of the class Ia antigen presentation pathway. In tapasin mutant mice, the expression and stability of surface class I molecules are strongly reduced. In tapasin double-negative cells, the presentation of cytosolic antigens is markedly impaired (3Garbi N. Tan P. Diehl A.D. Chambers B.J. Ljunggren H.G. Momburg F. Hammerling G.J. Nat. Immunol. 2000; 1: 234-238Crossref PubMed Scopus (168) Google Scholar). Defects in the development of CD8+ T cells and immune responses against some viruses were also noted in tapasin double-negative mice (4Grandea III, A.G. Golovina T.N. Hamilton S.E. Sriram V. Spies T. Brutkiewicz R.R. Harty J.T. Eisenlohr L.C. Van Kaer L. Immunity. 2000; 13: 213-222Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Like class Ia molecules, tapasin plays an important role in the assembly and surface expression of certain class Ib molecules such as HLA-E (5Braud V.M. Allan D.S. Wilson D. McMichael A.J. Curr. Biol. 1998; 8: 1-10Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and H2-M3 (6Lybarger L. Yu Y.Y. Chun T. Wang C.R. Grandea III, A.G. Van Kaer L. Hansen T.H. J. Immunol. 2001; 167: 2097-2105Crossref PubMed Scopus (36) Google Scholar, 7Chun T. Grandea 3rd, A.G. Lybarger L. Forman J. Van Kaer L. Wang C.R. J. Immunol. 2001; 167: 1507-1514Crossref PubMed Scopus (31) Google Scholar). endoplasmic reticulum β2-microglobulin transporter associated with antigen processing major histocompatibility complex monoclonal antibody phosphate-buffered saline endoglycosidase-H N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Recent studies have implied that tapasin serves several functions in the assembly of class I molecule-peptide complexes. It functions to bridge the heavy chain complexes to TAP (8Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). Tapasin expression also enhances the stability of TAP heterodimers, increasing overall peptide transport into the ER (9Li S. Paulsson K.M. Chen S. Sjogren H.O. Wang P. J. Biol. Chem. 2000; 275: 1581-1586Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Lehner P.J. Surman M.J. Cresswell P. Immunity. 1998; 8: 221-231Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In insect cells, tapasin retains empty Kb molecules in the ER (11Schoenhals G.J. Krishna R.M. Grandea III, A.G. Spies T. Peterson P.A. Yang Y. Fruh K. EMBO J. 1999; 18: 743-753Crossref PubMed Scopus (100) Google Scholar). Likewise, in tapasin-deficient human 721.220 cells, tapasin prevents premature release of Kb molecules from the ER, suggesting that tapasin might participate in retaining class I molecules in the ER until an optimal peptide is loaded (12Barnden M.J. Purcell A.W. Gorman J.J. McCluskey J. J. Immunol. 2000; 165: 322-330Crossref PubMed Scopus (84) Google Scholar). Finally, other recent studies have suggested that tapasin might be involved in the peptide editing of class I molecules (13Lewis J.W. Elliott T. Curr. Biol. 1998; 8: 717-720Abstract Full Text Full Text PDF PubMed Google Scholar, 14Purcell A.W. Gorman J.J. Garcia-Peydro M. Paradela A. Burrows S.R. Talbo G.H. Laham N. Peh C.A. Reynolds E.C. Lopez De Castro J.A. McCluskey J. J. Immunol. 2001; 166: 1016-1027Crossref PubMed Scopus (136) Google Scholar, 15Williams A.P. Peh C.A. Purcell A.W. McCluskey J. Elliott T. Immunity. 2002; 16: 509-520Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Although there is little doubt that tapasin is a class I-dedicated chaperone, the actual mechanism for each proposed function of tapasin during its interactions with class I alleles remains to be elucidated. Tapasin dependence could reflect a direct role for tapasin in ER retention of heavy chains, TAP stabilization, peptide editing, or any combination of these functions. Determining which of these interrelated functions are primary rather than secondary manifestations of tapasin's interactions with class I molecules has proven to be difficult. In addition to the uncertainty regarding the precise functions of tapasin, various class Ia molecules differ in their dependence on tapasin for both efficient surface expression and presentation of antigenic determinants to CTL, as observed in studies of 721.220 transfectants (13Lewis J.W. Elliott T. Curr. Biol. 1998; 8: 717-720Abstract Full Text Full Text PDF PubMed Google Scholar, 16Peh C.A. Burrows S.R. Barnden M. Khanna R. Cresswell P. Moss D.J. McCluskey J. Immunity. 1998; 8: 531-542Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Grandea III, A.G. Lehner P.J. Cresswell P. Spies T. Immunogenetics. 1997; 46: 477-483Crossref PubMed Scopus (77) Google Scholar, 18Greenwood R. Shimizu Y. Sekhon G.S. DeMars R. J. Immunol. 1994; 153: 5525-5536PubMed Google Scholar). In 721.220 cells, tapasin is not required for high levels of surface expression of the HLA-B2705 allele or for presentation of viral determinants to cytotoxic T lymphocytes (16Peh C.A. Burrows S.R. Barnden M. Khanna R. Cresswell P. Moss D.J. McCluskey J. Immunity. 1998; 8: 531-542Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In contrast, for the HLA-B4402 allele, functional antigen presentation and surface expression are highly dependent on tapasin; HLA-B0801 falls between the B2705 and B4402 alleles in the spectrum of tapasin dependence (16Peh C.A. Burrows S.R. Barnden M. Khanna R. Cresswell P. Moss D.J. McCluskey J. Immunity. 1998; 8: 531-542Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). These relationships reflect the complexity of tapasin function. In fact, the aforementioned studies regarding the function of tapasin took place before the concept was established that the tapasin dependence of class I molecules is allele-specific. Thus, the conclusions of these studies might be biased, depending on which class I alleles were analyzed. HLA-G, an MHC class Ib molecule, is expressed primarily in trophoblast cells and has limited polymorphism (2Shawar S.M. Vyas J.M. Rodgers J.R. Rich R.R. Annu. Rev. Immunol. 1994; 12: 839-880Crossref PubMed Scopus (226) Google Scholar). Due to a scarcity of natural endogenous peptide ligands in most cells, the supply of peptides is the rate-limiting factor for the intracellular transport kinetics of HLA-G (19Park B. Lee S. Kim E. Chang S. Jin M. Ahn K. Immunity. 2001; 15: 213-224Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Recent evidence shows that HLA-G protects trophoblast cells from recognition by NK cells (20Munz C. Holmes N. King A. Loke Y.W. Colonna M. Schild H. Rammensee H.G. J. Exp. Med. 1997; 185: 385-391Crossref PubMed Scopus (129) Google Scholar, 21Rajagopalan S. Long E.O. J. Exp. Med. 1999; 189: 1093-1100Crossref PubMed Scopus (621) Google Scholar); expression of HLA-G on melanoma cells protects them from lysis by NK cells (22Paul P. Rouas-Freiss N. Khalil-Daher I. Moreau P. Riteau B. Le Gal F.A. Avril M.F. Dausset J. Guillet J.G. Carosella E.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4510-4515Crossref PubMed Scopus (388) Google Scholar). HLA-G is a particularly interesting molecule in protein trafficking, because it recycles between the ER and the cis-Golgi until it is loaded with a high affinity peptide (19Park B. Lee S. Kim E. Chang S. Jin M. Ahn K. Immunity. 2001; 15: 213-224Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This feature makes it possible to estimate whether HLA-G molecules are loaded with high affinity or low affinity peptides. Whether the process involved in the assembly of MHC class Ia molecule-peptide complexes applies to the assembly of HLA-G-peptide complexes has not been studied. Because HLA-G binds a restricted set of peptides (23Diehl M. Munz C. Keilholz W. Stevanovic S. Holmes N. Loke Y.W. Rammensee H.G. Curr. Biol. 1996; 6: 305-314Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 24Lee N. Malacko A.R. Ishitani A. Chen M.C. Bajorath J. Marquardt H. Geraghty D.E. Immunity. 1995; 3: 591-600Abstract Full Text PDF PubMed Scopus (297) Google Scholar) and recycles between the ER andcis-Golgi, it is an attractive model for investigating the roles of individual components of the ER peptide-loading complex in the assembly of functional MHC class I molecules. In this study, we examined the tapasin dependence of HLA-G in terms of tapasin's critical role in the assembly and intracellular transport of HLA-G. Our findings demonstrate that in the absence of tapasin, HLA-G molecules are not able to bind high affinity peptides despite an abundant supply of peptides, suggesting that tapasin is a prerequisite for the binding of high affinity peptides. Interestingly, immediately after synthesis, these tapasin-dependent HLA-G molecules associate constitutively with low affinity endogenous peptides and exit the ER, thereby avoiding ER-mediated degradation. We propose that a critical function of tapasin is to transform the peptide-binding groove of HLA-G into a high affinity, peptide-receptive form, which could promote the replacement of low affinity peptides with high affinity peptides. All HLA cDNAs and their mutagenized derivatives were inserted into the mammalian expression vector pcDNA3.1 (Invitrogen). The cDNA encoding human tapasin was kindly provided by Dr. Cresswell (Yale University, New Haven, CT) and was subcloned into the pcDNA3.1/Hygro vector (Invitrogen). The cDNA encoding human β2m was contained in the pcDNA3.1/Neomycin vector (Invitrogen). Point mutations of HLA-G were made by changing the codons by PCR with Pfu DNA polymerase (Stratagene). The sequences for all mutations were confirmed by sequencing. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), penicillin (50 units/ml), and streptomycin (50 μg/ml). NIH3T3 cells were transfected with either human β2m cDNA or human β2m and human tapasin cDNAs. Stable transfectants expressing human β2m (NIH3T3.hβ2m) were selected with 1 mg/ml G418 (Sigma). Stable transfectants expressing both human β2m and human tapasin (NIH3T3.hβ2m.hTpn) were selected with 1 mg/ml G418 and 0.35 mg/ml hygromycin (Invitrogen). Tapasin expression was restored in 721.220 cells by transfection with the cDNA encoding human tapasin, and transfectants were selected with 0.35 mg/ml hygromycin, giving rise to the 721.220.Tpn cell line. The 721.220 and 721.220.Tpn cells were cultured in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum, 2 mmglutamine, penicillin (50 units/ml), and streptomycin (50 μg/ml). The HLA-G-specific monoclonal antibody (mAb) G233 was a gift from Dr. Loke (University of Cambridge). The mAb W6/32 recognizes only MHC class I heavy chains associated with β2m. Polyclonal rabbit K455 antibody reacts with MHC class I heavy chains and β2m in both assembled and nonassembled forms. The rabbit polyclonal antibody to human tapasin (R.gp48N) was a gift from Dr. Cresswell. The rabbit polyclonal antibody to PDI (SPA-890) and the anti-β-COP mAb M3A5 were purchased from Stressgen (Victoria, Canada) and Sigma, respectively. The mAb to the cis-Golgi marker GM130 was purchased from BD Transduction Laboratories (Franklin Lakes, NJ.). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG and Texas Red-conjugated goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated streptavidin was purchased from Pierce. Cells (5 × 106) were transfected by electroporation, starved for 40 min in medium lacking methionine, labeled for 20 min with 0.1 mCi/ml [35S]methionine (TranS-label; PerkinElmer Life Sciences), and chased for the indicated times in normal medium. Cells were lysed by using 1% Nonidet P-40 (Sigma) in phosphate-buffered saline (PBS) with protease inhibitor mixture (Sigma) for 30 min at 4 °C. After preclearing lysates with protein G-Sepharose (Amersham Biosciences), primary antibodies and protein G-Sepharose were added to the supernatant and incubated at 4 °C with rotation for 2 h. The beads were washed three times with 0.1% Nonidet P-40 in PBS. Proteins were eluted from the beads by boiling in SDS sample buffer and separated by 12% SDS-PAGE. The gels were dried, exposed to BAS film for 14 h, and analyzed with the phosphorimaging system BAS-2500 (Fuji Film, Tokyo, Japan). For endoglycosidase-H (endo-H) treatment, immunoprecipitates were digested with 3 milliunits of endo-H (Roche Molecular Biochemicals) at 37 °C overnight in 50 mm sodium acetate (pH 5.6), 0.3% SDS, and 150 mm β-mercaptoethanol (Sigma). The surface expression of HLA-G molecules was determined by flow cytometry (FACScalibur, Becton Dickinson Biosciences, Mountain View, CA). Cells (1 × 106) were washed twice with cold PBS containing 1% bovine serum albumin and incubated for 1 h at 4 °C with a saturating concentration of mAb G233. Normal mouse IgG was used as a negative control for each test. The cells were washed twice with cold PBS containing 1% bovine serum albumin and then stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG for 30 min. A total of 10,000 gated events were collected by the FACScalibur cytometer and analyzed with CellQuest software (Becton Dickinson Biosciences). For immunofluorescence staining of permeabilized cells, NIH3T3 cells were fixed in 3.7% formaldehyde, made permeable with 0.1% Triton X-100, and incubated with the appropriate primary antibody for 1 h. MRC-1024 confocal microscopy was used for confocal imaging (Bio-Rad). Cells were lysed in 1% digitonin in digitonin buffer containing 25 mmHEPES, 100 mm NaCl, 10 mm CaCl2, and 5 mm MgCl2 (pH 7.6) supplemented with protease inhibitors. Lysates were precleared with protein G-Sepharose (Amersham Biosciences) for 1 h at 4 °C. For immunoprecipitation, samples were incubated with the appropriate antibodies for 2 h at 4 °C before protein G-Sepharose beads were added. Beads were washed four times with 0.1% digitonin, and bound proteins were eluted by boiling in SDS sample buffer. Proteins were separated by 12% SDS-PAGE, transferred onto a nitrocellulose membrane, blocked with 5% skim milk in PBS with 0.1% Tween 20 for 2 h, and probed with the appropriate antibodies for 4 h. Membranes were washed three times in PBS with 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated streptavidin (Pierce) for 1 h. The immunoblots were visualized with ECL detection reagent (Pierce). Microsomes from 721.220 and 721.220.Tpn cells expressing HLA-G or HLA-G/E114H heavy chains were prepared and purified as previously described (25Saraste J. Palade G.E. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6425-6429Crossref PubMed Scopus (177) Google Scholar). Biotinylated peptide KIPAQFYIL was conjugated to the photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS; Pierce) as described (9Li S. Paulsson K.M. Chen S. Sjogren H.O. Wang P. J. Biol. Chem. 2000; 275: 1581-1586Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). For the peptide-loading assay, reporter peptides, with or without various concentrations of the unlabeled peptide (KIPAQFYIL), were mixed with 15 μl of microsomes (concentration of 60 A280/ml) in a total volume of 50 μl of RM buffer (250 mm sucrose, 50 mmtriethanolamine-HCl, 50 mm potassium acetate, 2 mm magnesium acetate, 1 mm dithiothreitol, and 10 mm ATP). The mixture was incubated for 30 min at 26 °C in a flat-bottomed 96-well tissue culture plate. The samples were maintained on ice during a 3-min exposure to shortwave (365-nm) ultraviolet irradiation. After centrifugation, the membranes were washed once with cold RM buffer and lysed with 1% digitonin, and the cross-linked proteins were immunoprecipitated with mAb G233. The precipitates were separated by 12% SDS-PAGE and transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was incubated with horseradish peroxidase-conjugated streptavidin for 1 h, and biotinylated proteins were visualized by using ECL Western blotting reagent (Pierce). Peptide translocation was determined after incubating microsomes with biotin-conjugated reporter peptides in the absence of competitor for 30 min at 26 °C with or without 1 mm ATP. Microsomal membranes were recovered by centrifugation at 75,000 × g for 10 min through a 0.5m sucrose cushion in cold RM buffer. After washing with cold RM buffer twice, the membrane pellet was directly dissolved in sample buffer. The samples were analyzed on Tricine/SDS-PAGE, appropriate for resolution of low mass polypeptides as described (26Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar), and probed with horseradish peroxidase-conjugated streptavidin. The relative densities of the peptide bands were determined by use of an imaging densitometer (GS-700; Bio-Rad) and MultiAnalyst densitometer software (Bio-Rad). To determine the function of tapasin for the assembly of HLA-G-peptide complexes, we first examined the tapasin dependence of HLA-G for cell surface expression. The genes encoding HLA-G heavy chains were independently transfected into both 721.220 and 721.220.Tpn cell lines. In the absence of tapasin, low levels of surface expression were observed (Fig.1A). Significantly, upon expression of tapasin, surface expression increased more than 5-fold, indicating the dependence of HLA-G on tapasin for its surface expression. In recent efforts to identify the factors that determine the relative tapasin dependence of various class Ia alleles, we have found that the nature of the amino acid residues present at the naturally polymorphic position 114 determines tapasin dependence. 2B. Park and K. Ahn, unpublished data. To test whether this is also the case for the HLA-G molecules, we constructed substitution mutants and examined their dependence on tapasin for surface expression (Fig. 1A). Surprisingly, a glutamic acid to histidine substitution at position 114 (HLA-G/E114H) allows the otherwise tapasin-dependent HLA-G to have levels of surface expression comparable with the levels seen in the presence of tapasin. The HLA-G/E114Q mutant, in which residue 114 was replaced by the neutrally charged glutamine, fell between HLA-G wild type and HLA-G/E114H in the spectrum of tapasin dependence. These results indicate that, like HLA class Ia molecules, the tapasin dependence of HLA-G is also influenced by the nature of the amino acid at position 114. Since it is unlikely that the point mutation causes a gross conformational change in class I molecules, we used the HLA-G/E114H mutant in parallel with wild-type HLA-G as a control for the tapasin-independent phenotype. To investigate the mechanism by which tapasin affects surface expression of HLA-G, we compared the intracellular maturation and transport of HLA-G in the 721.220 and 721.220.Tpn cells. In the absence of tapasin, 50% of HLA-G heavy chains remained sensitive to endo-H digestion, even after 8 h (Fig. 1B), reflecting retention of these molecules in the ER. Conversely, in the presence of tapasin, most HLA-G molecules had become endo-H-resistant by this time (Fig. 1B). These findings suggest that the impaired intracellular transport of HLA-G molecules in the absence of tapasin accounts for their low levels of surface expression. Pulse-chase experiments from tapasin-negative and -positive cells revealed comparable acquisition of endo-H resistance by HLA-G/E114H (Fig.1C), indicating its tapasin independence for normal intracellular transport. We have shown that the availability of high affinity peptides dictates the transport kinetics of HLA-G (19Park B. Lee S. Kim E. Chang S. Jin M. Ahn K. Immunity. 2001; 15: 213-224Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To determine whether the inefficient transport of HLA-G in the absence of tapasin can be overcome by supplying high affinity peptides, we examined the intracellular trafficking of HLA-G and HLA-G/E114H upon expression of high affinity peptides by the minigene expression system. The minigene encoding MIPAQFYIL, a high affinity peptide ligand for HLA-G (23Diehl M. Munz C. Keilholz W. Stevanovic S. Holmes N. Loke Y.W. Rammensee H.G. Curr. Biol. 1996; 6: 305-314Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), was co-expressed with the gene encoding either HLA-G or HLA-G/E114H in 721.220 and 721.220.Tpn cells. In the presence of tapasin, no discernible difference in the transport rate between HLA-G and HLA-G/E114H was seen upon expression of high affinity peptides (Fig.1D), but both HLA-G and HLA-G/E114H molecules were transported with much faster kinetics when compared with the kinetics of molecules without the supply of high affinity peptides (Fig. 1,B and C, right). In the absence of tapasin, the supply of high affinity peptides increased the transport kinetics of HLA-G/E114H (Fig. 1, compare C (left) and E (right)), whereas this supply did not influence the transport kinetics of HLA-G (Fig. 1, compare B(right) and E (left)). These results indicate that for efficient intracellular transport of HLA-G molecules, an abundance of high affinity peptides cannot compensate for the lack of tapasin. In the absence of tapasin, the impaired intracellular transport and the low surface expression of HLA-G might be due to its inability to load high affinity peptides. To test this possibility, we examined peptide loading into HLA-G or HLA-G/E114H molecules as a function of tapasin by using the reporter peptide, KIPAQFYIL, which is known to be a high affinity ligand for HLA-G (23Diehl M. Munz C. Keilholz W. Stevanovic S. Holmes N. Loke Y.W. Rammensee H.G. Curr. Biol. 1996; 6: 305-314Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), in the presence of the competitors at different concentrations. We were surprised at the marked differences in the ability of HLA-G and HLA-G/E114H to load peptides in the absence of tapasin. In the case of no competitor added, the level of binding of the reporter peptide by HLA-G was only 25% of the binding observed in the HLA-G/E114H substitution mutant (Fig.2A). Furthermore, the reporter peptides loaded into HLA-G were completely outcompeted by a lower concentration (1.6 μm) of unlabeled peptide, whereas as much as 6.4 μm of unlabeled peptide was not sufficient for completely outcompeting reporter peptide binding to HLA-G/E114H. However, in the presence of tapasin, no discernible difference in the peptide binding ability of HLA-G and HLA-G/E114H was seen (Fig.2B). These results indicate that HLA-G molecules are highly dependent on tapasin for efficient loading with high affinity peptides, but the substitution of glutamic acid to histidine at position 114 renders the molecules independent of tapasin for high affinity peptide loading. Accordingly, the impaired intracellular transport and reduced surface expression of HLA-G in the absence of tapasin are probably the result of the inadequacy of HLA-G for high affinity peptide loading. To test whether the differential peptide loading observed in the absence of tapasin might be due to differences in the luminal availability of the peptide, we quantitated the amount of reporter peptides that were translocated into the ER lumen. Comparable amounts of peptides were recovered between HLA-G and HLA-G/E114H (Fig.2C). Therefore, we exclude the notion that luminal availability of the peptide plays an important role in dictating class I loading in tapasin-deficient cells. In control experiments in which ATP was omitted, little peptide was recovered. Since peptide translocation via TAP requires ATP (27Jun Y. Kim E. Jin M. Sung H.C. Han H. Geraghty D.E. Ahn K. J. Immunol. 2000; 164: 805-811Crossref PubMed Scopus (98) Google Scholar), we conclude that the modified reporter peptides entered the ER lumen in a TAP-dependent manner. HLA-G molecules that are loaded with peptides of suboptimal affinity are retrieved back to the ER. Loading of HLA-G with high affinity peptides abrogates this retrieval and allows HLA-G-peptide complexes to transport forward to the cell surface (19Park B. Lee S. Kim E. Chang S. Jin M. Ahn K. Immunity. 2001; 15: 213-224Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To examine whether tapasin affects the recycling behavior of HLA-G as a function of the availability of high affinity peptides, we stably expressed minigenes encoding MIPAQFYIL in NIH3T3.hβ2m cells and NIH3T3.hβ2m.hTpn cells and then treated cells with nocodazole. Nocodazole disrupts microtubules, leading to disintegration of the Golgi and interruption of traffic between the Golgi and the ER (28Lippincott-Schwartz J. Donaldson J.G. Schweizer A. Berger E.G. Hauri H.P. Yuan L.C. Klausner R.D. Cell. 1990; 60: 821-836Abstract Full Text PDF PubMed Scopus (734) Google Scholar). On the basis of cell size and ease of manipulation, we used NIH3T3 cells for the immunofluorescence and confocal microscopy experiments throughout the study. In the absence of high affinity peptides, in nocodazole-treated cells, both HLA-G and HLA-G/E114H exhibited the punctate staining pattern around the perinuclear region regardless of tapasin (Fig.3, A and B), an indication of recycling. In the presence