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• W2039153066 abstract "The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa β2-microglobulin (β2m), and an ∼8-9-residue antigenic peptide. Without β2m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a β2m with a Cys-to-Trp substitution at position 25 (β2mC25W). This substitution causes misfolding and degradation of β2mC25W but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type β2m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional β2m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases. The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa β2-microglobulin (β2m), and an ∼8-9-residue antigenic peptide. Without β2m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a β2m with a Cys-to-Trp substitution at position 25 (β2mC25W). This substitution causes misfolding and degradation of β2mC25W but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type β2m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional β2m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases. The human leukocyte antigen (HLA) 4The abbreviations used are: HLA, human leukocyte antigen; APM, antigen processing machinery; β2m, β2-microglobulin; CTL, cytotoxic T lymphocyte; ER, endoplasmic reticulum; HC, HLA class I heavy chain; IFN-γ, interferon-γ; LMP, low molecular mass polypeptide; LOH, loss of heterozygosity; mAb, monoclonal antibody; MFI, mean fluorescence intensity; TA, tumor antigen; TAP, transporter associated with antigen processing; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TyrVac, tyrosinase-expressing vaccinia strain. class I molecules, encoded by the genes located in the major histocompatibility complex, are composed of three subunits including a 45-kDa HLA class I heavy chain (HC), a 12-kDa β2-microglobulin (β2m), and an ∼8-9-residue peptide (1Saper M.A. Bjorkman P.J. Wiley D.C. J. Mol. Biol. 1991; 219: 277-319Crossref PubMed Scopus (971) Google Scholar). Expression of these molecules on the cell surface requires the stepwise assembly of HCs, β2m, and peptides in the endoplasmic reticulum (ER) followed by the transport of the trimeric molecule to the plasma membrane. These processes are dependent on a functional antigen processing machinery (APM), which includes the proteasome subunits, the peptide transporters TAP1 and TAP2, and a number of ER-resident chaperons such as calnexin, calreticulin, ERp57, and tapasin (2Cresswell P. Bangia N. Dick T. Diedrich G. Immunol. Rev. 1999; 172: 21-28Crossref PubMed Scopus (269) Google Scholar, 3Yewdell J. Mol. Immunol. 2002; 39: 125-259Crossref PubMed Scopus (2) Google Scholar). β2m plays an integral part in the assembly and transport of HLA class I molecules because it stabilizes the HC-β2m heterodimer through non-covalent protein-protein interactions, thereby allowing binding of endogenous antigenic peptides with the help of TAP and tapasin (4Hansen T.H. Lee D.R. Adv. Immunol. 1997; 64: 105-137Crossref PubMed Google Scholar). As a result, the assembled HC-β2m-peptide trimeric complexes can travel to the cell surface, where they are recognized by HLA class I-restricted, antigen-specific cytotoxic T lymphocytes (CTLs). The lack of HLA class I molecule expression on the cell surface usually reflects defects in β2m protein synthesis caused by mutations in the β2m gene, as has been found mostly in malignant cells thus far (5Ferrone S. Semin. Cancer Biol. 2002; 12: 1-86Crossref Scopus (6) Google Scholar). This abnormality renders tumor cells resistant to tumor antigen-specific CTLs and may have a negative impact on the elimination of tumor cells by host CTLs. The defects underlying β2m loss have thus far been found to be structural in nature, involving lack of translation because of small deletions or point mutations in most cases and lack of transcription because of large deletions in a few cases (5Ferrone S. Semin. Cancer Biol. 2002; 12: 1-86Crossref Scopus (6) Google Scholar). Because of a lack of β2m expression, the resulting HLA class I loss cannot be corrected by interferon (IFN-γ), a cytokine that up-regulates the expression of most of the molecules participating in the assembly and transport of HLA class I molecules. On the other hand, a low level of HLA class I expression on cells usually reflects nonstructural defects in the APM components because this abnormality can be corrected by IFN-γ (6Seliger B. Maeurer M.J. Ferrone S. Immunol. Today. 2000; 21: 455-464Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). In the present study, we have elucidated the mechanism underlying HLA class I down-regulation identified in a melanoma cell line and in the metastasis from which the cell line was derived (7Yamshchikov G.V. Mullins D.W. Chang C.C. Ogino T. Thompson L. Presley J. Galavotti H. Aquila W. Deacon D. Ross W. Patterson J.W. Engelhard V.H. Ferrone S. Slingluff Jr. C.L. J. Immunol. 2005; 174: 6863-6871Crossref PubMed Scopus (85) Google Scholar). This HLA class I down-regulation phenotype cannot be corrected by IFN-γ and was unexpectedly found to be caused by an abnormal full-length β2m protein that can neither form stable complexes with HCs nor assist in loading peptides onto the HLA class I peptide binding groove. Cell Lines—The VMM5A and VMM5B melanoma cell lines were established from two sequential metastatic melanoma deposits surgically removed from patient VMM5 at two time points (7Yamshchikov G.V. Mullins D.W. Chang C.C. Ogino T. Thompson L. Presley J. Galavotti H. Aquila W. Deacon D. Ross W. Patterson J.W. Engelhard V.H. Ferrone S. Slingluff Jr. C.L. J. Immunol. 2005; 174: 6863-6871Crossref PubMed Scopus (85) Google Scholar). Other melanoma cell lines included DM6 cells (a gift from Dr. T. L. Darrow, Duke University, Durham, NC), which express HLA-A2 and multiple melanocytic differentiation antigens including gp100, melanoma antigen recognized by T cells (MART-1) and tyrosinase, and FO-1 cells, which do not express HLA class I molecules because of β2m loss (8D'Urso C.M. Wang Z.G. Cao Y. Tatake R. Zeff R.A. Ferrone S. J. Clin. Investig. 1991; 87: 284-292Crossref PubMed Scopus (295) Google Scholar). LG-2 is a human B lymphoblastoid cell line. All of these cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum in a 5% CO2 atmosphere at 37 °C. A tyrosinase369-377D-specific 5D means a post-translational modification from N to D at position 371 when the protein tyrosinase is processed inside the cells. CTL line was derived from patient VMM119 who had been vaccinated with a mixture of four peptides including tyrosinase369-377D (YMDGTMSQV) (9Yamshchikov G.V. Barnd D.L. Eastham S. Galavotti H. Patterson J.W. Deacon D.H. Teates D. Neese P. Grosh W.W. Petroni G. Engelhard V.H. Slingluff Jr. C.L. Int. J. Cancer. 2001; 92: 703-712Crossref PubMed Scopus (96) Google Scholar). Monoclonal and Polyclonal Antibodies—The following mAbs were developed and characterized as described: the mAb W6/32, which recognizes a monomorphic determinant expressed on β2m-associated HLA-A, -B, and -C heavy chains (10Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Cell. 1978; 14: 9-19Abstract Full Text PDF PubMed Scopus (1598) Google Scholar); the mAb LG III-147.4.1, which recognizes a determinant expressed on β2m-associated HLA-A heavy chains except A23, A24, A25, and A32 (11Wang X. Liang B. Rebmann V. Lu J. Celis E. Kageshita T. Grosse-Wilde H. Ferrone S. Tissue Antigens. 2003; 62: 139-147Crossref PubMed Scopus (17) Google Scholar); the mAb B1.23.2, which recognizes a determinant restricted to HLA-B and -C antigens (12Rebai N. Malissen B. Tissue Antigens. 1983; 22: 107-114Crossref PubMed Scopus (158) Google Scholar); the mAb HC-10, which recognizes a determinant expressed on all β2m-free HLA-B and -C heavy chains and on β2m-free HLA-A10, -A28, -A29, -A30, -A31, -A32, and -A33 heavy chains (13Stam N.J. Vroom T.M. Peters P.J. Pastoors E.B. Ploegh H.L. Int. Immunol. 1990; 2: 113-122Crossref PubMed Scopus (236) Google Scholar, 14Sernee M.F. Ploegh H.L. Schust D.J. Mol. Immunol. 1998; 35: 177-184Crossref PubMed Scopus (115) Google Scholar, 15Perosa F. Luccarelli G.M. Favoino E. Ferrone S. Dammacco F. J. Im-munol. 2003; 171: 1918-1926Crossref PubMed Scopus (143) Google Scholar); the β2m-specific mAbs L368 (16Lampson L.A. Fisher C.A. Whelan J.P. J. Immunol. 1983; 30: 2471-2478Google Scholar) and KJ-2, which recognize HLA class I heavy chain-free β2m 6S. Ferrone, unpublished results. ; the delta (Y)-specific mAb SY-5; the MB1 (X)-specific mAb SJJ-3; the Z-specific mAb NB-1; the LMP2-specific mAb SY-1; the LMP7-specific mAb HB-2; the LMP10-specific mAb TO-7 (17Bandoh N. Ogino T. Cho H.S. Hur S.Y. Shen J. Wang X. Kato S. Miyokawa N. Harabuchi Y. Ferrone S. Tissue Antigens. 2005; 66: 185-194Crossref PubMed Scopus (58) Google Scholar); the TAP1-specific mAb NOB-1 (18Wang X. Campoli M. Cho H.S. Ogino T. Bandoh N. Shen J. Hur S.Y. Kages-hita T. Ferrone S. J. Immunol. Methods. 2005; 299: 139-151Crossref PubMed Scopus (53) Google Scholar); the TAP2-specific mAb NOB-2 (18Wang X. Campoli M. Cho H.S. Ogino T. Bandoh N. Shen J. Hur S.Y. Kages-hita T. Ferrone S. J. Immunol. Methods. 2005; 299: 139-151Crossref PubMed Scopus (53) Google Scholar); the calnexin-specific mAb TO-5 (19Ogino T. Wang X. Kato S. Miyokawa N. Harabuchi Y. Ferrone S. Tissue Antigens. 2003; 62: 385-393Crossref PubMed Scopus (78) Google Scholar); the calreticulin-specific mAb TO-11 (19Ogino T. Wang X. Kato S. Miyokawa N. Harabuchi Y. Ferrone S. Tissue Antigens. 2003; 62: 385-393Crossref PubMed Scopus (78) Google Scholar); the ERp57-specific mAb TO-2 (19Ogino T. Wang X. Kato S. Miyokawa N. Harabuchi Y. Ferrone S. Tissue Antigens. 2003; 62: 385-393Crossref PubMed Scopus (78) Google Scholar); and the tapasin-specific mAb TO-3 (19Ogino T. Wang X. Kato S. Miyokawa N. Harabuchi Y. Ferrone S. Tissue Antigens. 2003; 62: 385-393Crossref PubMed Scopus (78) Google Scholar). The human actin-specific mAb (sc-8432) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-idiotypic mAb MK2-23 (20Kusama M. Kageshita T. Chen Z.J. Ferrone S. J. Immunol. 1989; 143: 3844-3852PubMed Google Scholar) and mouse IgG2a (BD Biosciences) were used as isotype controls. R-phycoerythrin-conjugated goat anti-mouse Fcγ F(ab′)2 fragments and goat anti-mouse IgG antibodies were purchased from Dako (Carpinteria, CA) and GE Healthcare, respectively. Peptides, IFN-γ, Wild-type Human β2m, and Pharmacological Inhibitors—The HLA class I-associated peptides HER2/neu369-377, KIFGSLAFL, and tyrosinase369-377D YMDGTMSQV were synthesized with a free amide N terminus and free acid C terminus by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry using a model AMS422 peptide synthesizer (Gilson Co. Inc., Middleton, WI). Peptides were purified to >98% purity by reverse-phase high pressure liquid chromatography on a C-8 column (Vydac, Hesperia, CA) at the University of Virginia biomolecular core facility. Purity and identity were confirmed using a triple quadrupole mass spectrometer (Finnigan, San Jose, CA). Recombinant human IFN-γ was purchased from Roche Applied Science. The wild-type human β2m was purchased from Sigma. The proteasome inhibitor MG132 and the trans-Golgi-to-secretory vesicles traffic inhibitor monensin were purchased from Sigma and Alexis Corp. (San Diego, CA), respectively. Low pH Treatment of Cells and Restabilization of Cell Surface HLA Class I Molecules—Low pH treatment of cells was performed as described previously (21Sugawara S. Abo T. Kumagai K. J. Immunol. Methods. 1987; 100: 83-90Crossref PubMed Scopus (118) Google Scholar). Briefly, cell pellets containing ∼1-10 × 106 cells were resuspended with 0.5 ml of stripping buffer (0.13 m citric acid, 66 mm Na2HPO4, 1% bovine serum albumin, pH 3.0) for 2 min on ice and then neutralized immediately with 50 ml of RPMI 1640 medium. After three washes, cells (1 × 106) were pulsed with 40 μg/ml peptide and 2.5 μg/ml human β2m (Sigma) for 3 h at room temperature and then washed three times with 1% bovine serum albumin/phosphate-buffered saline. Subsequently, cells were subjected to staining and flow cytometric analysis as described. Flow Cytometry—Cell surface staining and intracellular staining were performed as described (22Ogino T. Wang X. Ferrone S. J. Immunol. Methods. 2003; 278: 33-44Crossref PubMed Scopus (36) Google Scholar). Results are presented as -fold increase in mean fluorescence intensity over the control (-fold MFI). Reverse Transcription-PCR—Total RNA isolation from cells was performed with TRIzol (Invitrogen) according to the manufacturer's instructions. First-strand cDNA synthesis was conducted using the SuperScript™ system (Invitrogen) according to the manufacturer's instructions. The PCR was performed using β2m-specific primers, forward 261 (5′-CCTGAAGCTGACAGCATTCG-3′) and reverse 262 (5′-ACCTCCATGATGCTGCTTAC-3′); GAPDH-specific primers, GAPDH-F (5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′) and GAPDH-R (5′-CATGTGGGCCATGAGGTCCACCAC-3′). PCR products were run on a 1.2% agarose gel (Roche Applied Science) and visualized by ethidium bromide staining. The predicted sizes of PCR products were 401 bp for β2m and 983 bp for GAPDH. The PCR products were gel-purified, and their sequences were analyzed by a DNA sequencer (ABI PRISM model 377, Applied Biosystems). Western Blot Analysis—Western blot analysis of cell lysates with β2m-, HLA class I heavy chain-, and APM component-specific antibodies was performed as described (22Ogino T. Wang X. Ferrone S. J. Immunol. Methods. 2003; 278: 33-44Crossref PubMed Scopus (36) Google Scholar). Genomic DNA Extraction and PCR—Genomic DNA extraction was performed with blood and cell culture DNA midi kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. PCR amplification of β2m gene exon 2 was conducted with the primers, 491 intron 1-specific (5′-CCTGGCAATATTAATGTG-3′) and 462 intron 2-specific (5′-CATACACAACTTTCAGCAGCT-3′). The predicted PCR product size is 362 bp. The PCR products were gel-purified and their sequences analyzed by a DNA sequencer (ABI PRISM model 377, Applied Biosystems). Loss of Heterozygosity (LOH) Analysis of the β2m Gene—LOH analysis of the β2m gene was performed as described previously (23Ramal L.M. Feenstra M. van der Zwan A.W. Collado A. Lopez-Nevot M.A. Tilanus M. Garrido F. Tissue Antigens. 2000; 55: 443-448Crossref PubMed Scopus (40) Google Scholar) with minor modifications. Briefly, purified genomic DNA (100 ng) was subjected to a PCR amplification using two pairs of primers specific to the two short tandem repeat markers (D15S126 and D15S209) located near the β2m gene. The sequences of the primers are as follows: D15S126F, 5′-GTGAGCCAAGATGGCACTAC-3′; D15S126R, 5′-GCCAGCAATAATGGGAAGTT-3′; D15S209F, 5′-AAACATAGTGCTCTGGAGGC-3′; and D15S209R, 5′-GGGCTAACAACAGTGTCTGC-3′. The amplification conditions are as follows: 95 °C for 12 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s for 10 cycles; 89 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s for 20 cycles; and a final extension at 72 °C for 10 min. PCR products were then fractionated on a 4% agarose gel and visualized by ethidium bromide staining. The intensity of the staining was determined by the AlphaImager™ 2200 system (Alpha Innotech Corp., San Leandro, CA). LOH is determined by the following formula: (intensity of tumor allele one/intensity of tumor allele two)/(intensity of normal allele one/intensity of normal allele two) (referred to as the percent LOH index). An LOH index less than 50% is considered significant. Radiolabeling of Cells, Indirect Immunoprecipitation, and SDSPAGE—Radiolabeling of cells, indirect immunoprecipitation, and SDS-PAGE were performed as described (24Bangia N. Lehner P.J. Hughes E.A. Surman M. Cresswell P. Eur. J. Immunol. 1999; 29: 1858-1870Crossref PubMed Scopus (140) Google Scholar). Transfection—Cells were transfected with a plasmid encoding the wild-type β2m or the mutant β2m (β2mC25W) utilizing Lipofectamine-mediated gene transfer (Invitrogen) according to the manufacturer's instructions. Briefly, pcDNA3.1-b2m, pcDNA3.1-b2mC25W, or the empty plasmid pcDNA3.1-neo (Invitrogen) was mixed with Lipofectamine™ 2000 before being added to melanoma cells grown in monolayers with a 90% confluence. Cells were selected 2 days after transfection in medium containing G418 sulfate (1 mg/ml) (Calbiochem). After ∼2-3 weeks of selection, G418-resistant clones were picked, expanded, and screened by flow cytometry for HLA class I expression. Positive clones were then further expanded in complete media supplemented with a maintenance dose (0.3 mg/ml) of G418. Recombinant Vaccinia Virus—Virus encoding full-length human tyrosinase (TyrVac) was constructed as described previously (25Mosse C.A. Meadows L. Luckey C.J. Kittlesen D.J. Huczko E.L. Slingluff C.L. Shabanowitz J. Hunt D.F. Engelhard V.H. J. Exp. Med. 1998; 187: 37-48Crossref PubMed Scopus (107) Google Scholar). Purified recombinant vaccinia virus stock was titered and tested for proper expression of tyrosinase using specific HLA-A2-restricted CTL (data not shown). Cytotoxicity Assay in Vitro—Standard 51Cr release assays were performed to determine CTL recognition of the HLA-A2-restricted epitope from the melanocyte differentiation protein tyrosinase (tyrosinase369-377D). Target cells were prepared by either loading with tyrosinase369-377D peptide (40 μg/ml) for 1 h at room temperature or by infecting with TyrVac (10 plaque-forming units/cell, 106 target cells) in 1 ml of Hanks' balanced salt solution supplemented with 0.1% bovine serum albumin, 1.6 mm MgSO4, and 1.8 mm CaCl2 for 1 h and then 4 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum for 8 h to allow for expression followed by labeling with 100 μCi of Na51CrO4 for 1 h for a standard 4 h 51Cr release assay. Percent cytotoxicity was calculated by the formula: % specific lysis = ((experimental release - spontaneous release)/(total release - experimental release)) × 100. Marked HLA Class I Down-regulation on VMM5B Melanoma Cells— Fluorescence-activated cell sorting analysis of cells stained with mAb W6/32 showed that HLA class I molecules were barely detectable on melanoma cells VMM5B as compared with autologous VMM5A melanoma cells (Fig. 1A). The low level of staining of VMM5B cells with mAb W6/32 does not represent nonspecific background staining because no staining was detected when the β2m-deficient FO-1 cells (8D'Urso C.M. Wang Z.G. Cao Y. Tatake R. Zeff R.A. Ferrone S. J. Clin. Investig. 1991; 87: 284-292Crossref PubMed Scopus (295) Google Scholar) were stained with mAb W6/32. Incubation of VMM5B cells with IFN-γ (300 units/ml) for 48 h at 37 °C had no detectable effect on the staining of VMM5B cells, as well as FO-1 cells, by mAb W6/32. To corroborate the specificity of the staining of VMM5B cells by mAb W6/32, cells were treated with low pH (pH 3.0) and then stained with mAb W6/32, a method used to disintegrate the trimeric HLA class I complex on the cell surface (21Sugawara S. Abo T. Kumagai K. J. Immunol. Methods. 1987; 100: 83-90Crossref PubMed Scopus (118) Google Scholar). As shown in Fig. 1B, following treatment with pH 3.0, VMM5B cells were not stained by mAb W6/32. These results imply the dissociation of peptides and β2m from the HCs because the antigenic determinant recognized by mAb W6/32 requires the association of HCs with β2m for its expression (10Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Cell. 1978; 14: 9-19Abstract Full Text PDF PubMed Scopus (1598) Google Scholar). This possibility is supported by the restoration of the staining with mAb W6/32 following loading of low pH-treated VMM5B cells with wild-type β2m along with a HLA-A2-binding peptide (HER-2/neu369-397, KIFGSLAFL, t½ = 481.2 min). This result reflects the reassembly of the trimeric HLA class I complex on the cell surface. It is noteworthy that the level of the restored HLA class I expression is similar to that on untreated cells, remaining at a low level, unlike the full restoration of HLA class I expression on low pH-treated autologous VMM5A cells following loading with exogenous peptide and β2m (data not shown). These results suggest that the amount of HCs transported to the plasma membrane is the limiting factor in VMM5B cells. This finding is different from the efficient transport to the cell surface of open-form (peptide-free or low affinity peptide-bound) HCs, which can subsequently be refolded to their native, bioactive conformation following loading with a high affinity peptide, as has been observed in the TAP-deficient T2 cells (3Yewdell J. Mol. Immunol. 2002; 39: 125-259Crossref PubMed Scopus (2) Google Scholar). To determine whether the low level of HLA class I molecules on the membrane of VMM5B cells is caused by a reduced level of all HCs and/or by a defect in β2m, HC and β2m expression in VMM5A and VMM5B cell lysates were analyzed by Western blotting. As shown in Fig. 2A, HCs were detected in both cell lysates and were markedly up-regulated by IFN-γ. In contrast, β2m was barely detectable with β2m-specific rabbit polyclonal antibodies in a VMM5B cell lysate following IFN-γ treatment. β2m was not detected in a VMM5B cell lysate with a panel of β2m-specific mouse mAb (Fig. 2A and data not shown). The latter results may reflect the insufficient sensitivity of the Western blotting technique because β2m was detected in VMM5B cells by intracellular staining with the β2m-specific mAb L368 (Fig. 2B). These results in conjunction with the lack of up-regulation of HLA class I molecules by IFN-γ suggest that the abnormal HLA class I phenotype of VMM5B cells is caused by a defect in β2m. Cys-to-Trp substitution in β2m Caused by a Point Mutation in the β2m Gene in VMM5B Cells—To investigate whether structural mutation(s) underlie the low β2m level and its functional abnormalities, we amplified the full-length β2m cDNA fragments (Fig. 3A) and performed nucleotide sequencing of the open reading frame of the β2m gene in VMM5B cells. As shown in Fig. 3, B and C, a cytosine-to-guanine transversion mutation at position 135 in exon 2 was detected. This mutation changes codon 25 from Cys to Trp (β2mC25W) (Fig. 4A), abolishing the disulfide bond between residue 25 (Cys-25) and 80 (Cys-80) of the full-length β2m protein (B). It is noteworthy that this mutation was not acquired during in vitro culture of the VMM5B cell line because the identical mutated nucleotide was detected in the genomic DNA extracted from the melanoma metastasis from which the cell line had been derived (Fig. 5A). Moreover, the wild-type sequence of the β2m gene in VMM5B cells was not detected suggesting that one β2m gene copy was lost. This possibility is supported by the LOH identified at two chromosome 15 short tandem repeat sites (D15S126 and D15S209) (23Ramal L.M. Feenstra M. van der Zwan A.W. Collado A. Lopez-Nevot M.A. Tilanus M. Garrido F. Tissue Antigens. 2000; 55: 443-448Crossref PubMed Scopus (40) Google Scholar) flanking the β2m gene in genomic DNA extracted from VMM5B cells and the corresponding melanoma metastasis (Fig. 5, B and C).FIGURE 4Nucleotide and deduced amino acid sequences of β2m cDNA synthesized from VMM5A and VMM5B cells. A, sequence conservation is indicated by dashes. Downward triangles indicate the exon-exon junctions. Bold underlined nucleotide letters indicate the mutated codon. Bold nucleotide letters indicate the stop codon. Italic amino acid letters indicate the signal peptide sequence. Bold amino acid letters indicate the residues where the substitution occurs. B, the lack of the disulfide bond in the β2m protein in VMM5B cells is schematically shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Detection of C-to-G point mutation and LOH of the β2m locus in genomic DNA extracted from VMM5B metastasis. A, the nucleotide sequence analysis shows a mix ofC(blue)andG(black) at position 135 in genomic DNA prepared from VMM5B metastasis. B, the heterozygosity status of the β2m locus in VMM5A cells, VMM5B cells, cryopreserved VMM5B tumor (VMM5B-T), and cryopreserved peripheral blood lymphocytes (PBL) of the patient was subjected to LOH analysis. C, the degree of LOH of the β2m locus in VMM5B cells and VMM5B metastasis is quantitatively presented.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Instability of β2mC25W in VMM5B Cells—To test whether loss of a disulfide bond in the mutant β2m identified in VMM5B cells caused conformational instability of the full-length protein, we examined the effect of Cys-to-Trp substitution at residue 25 on the stability of β2m structure using molecular modeling. The wild-type β2m is a β-sandwich structure composed of two anti-parallel β-pleated sheets connected by a disulfide bond between Cys-25 and Cys-80 (Fig. 6A). Each of the β-pleated sheets contains three β strands. In the wild-type Cys-25-Cys-80 hydrophobic core, the linked sulfur atoms exhibit favorable van der Waals contacts with respect to the surrounding atoms 4.43, 4.81, and 3.88 Å for Cys25SG-Val27CG1, Cys25SG-Gln8Cα, and Cys80SG-Val82CG2, respectively (Fig. 6B). However, when Cys-25 is replaced with Trp-25, the bulky indole ring of Trp side chain displays drastic steric clashes with the side chains of the neighboring residues. These clashes occur with all 14 possible Trp-25 side chain rotameric conformations analyzed. Fig. 6C shows one representative conformation in which the ring carbon and nitrogen members are within the unfavorable van der Waals distances with the neighboring atoms 2.63, 1.62, 2.74, and 1.95Å for Trp25CZ3-Tyr66CD1, Trp25CZ2-Val27CG1, Trp25NE1-Val82CG2, and Trp25CD1-Cys80SG, respectively. Because all of these distances are below 3.0Å, the minimum distance between two nonbonded carbon atoms, the free energy is drastically increased, leading to a thermodynamically unstable state of the mutantβ2m (β2mC25W). Degradation of β2mC25W and Lack of Stable HC-β2mC25W Association in VMM5B Cells—Next we tested whether β2mC25W was degraded by the proteasome in VMM5B cells, especially because it was present at a very low level intracellularly, approximately at a level 29-fold and 41-fold lower than that in VMM5A cells under basal conditions and following incubation with IFN-γ (300 units/ml), respectively (Fig. 2B). To this end, VMM5B cells were sequentially incubated with IFN-γ (300 units/ml) for 24 h at 37 °C and with the proteasome inhibitor MG132 (5 μm) for 12 h at 37 °C. The intracellular levels of the steady-state free β2mC25W, free HCs, and HC-β2mC25W complexes in IFN-γ/MG132-treated and in IFN-γ-treated VMM5B cells were compared utilizing fluorescence-activated cell sorting analysis of cells intracellularly stained with mAbs. As shown in Fig. 7, A and B, both the β2mC25W and HCs were increased ∼2-fold following incubation of cells with IFN-γ, but the level of HC-β2mC25W complexes remained unchanged. In the presence of IFN-γ and MG132, the level of β2mC25W increased markedly (5-fold above that in untreated cells) along with an ∼3-fold increase in HCs, but the level of HC-β2mC25W complexes still remained unchanged. On the other hand, increased secretion of β2mC25W did not appear to play a role in its low intracellular accumulation because the level of β2mC25W, HCs, and HC-β2mC25W complex expression was not increased by the addition, 4 h before harvest, of monensin, an inhibitor of trans-Golgi-to-secretory vesicles traffic, to MG132-treated cells cultured in the presence of IFN-γ. Therefore, β2mC25W was removed through proteasome-mediated degradation. Even when it accumulates, the mutant β2m cannot form stable complexes with HCs. This conclusion is corroborated by the lack of immunoprecipitation of HC-β2mC25W complexes with mAb W6/32 from MG132 and IFN-γ-treated VMM5B cell lysates (Fig. 7C). Attemp" @default.
• W2039153066 created "2016-06-24" @default.
• W2039153066 creator A5023454962 @default.
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