The Hemochromatosis Founder Mutation in HLA-H Disrupts β2-Microglobulin Interaction and Cell Surface Expression
1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês
10.1074/jbc.272.22.14025
ISSN1083-351X
AutoresJohn N. Feder, Zenta Tsuchihashi, Alivelu Irrinki, Vince Lee, Felipa Mapa, Ebenezer Morikang, Cynthia Prass, Steven M. Starnes, Roger K. Wolff, Seppo Parkkila, William S. Sly, Randall C. Schatzman,
Tópico(s)Trace Elements in Health
ResumoWe recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), calledHLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 → tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 → aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds β2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease. We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), calledHLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 → tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 → aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds β2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease. Hereditary hemochromatosis (HH) 1The abbreviations used are:HHhereditary hemochromatosisMHCmajor histocompatibility complexPCRpolymerase chain reactionβ-COPβ-coatomer proteinERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresisPVDFpolyvinyl difluoride. is an autosomal recessive disorder of iron metabolism and represents one of the most common inherited disorders in individuals of Northern European descent with an estimated carrier frequency between 1 in 8 and 1 in 10 (1Dadone M.M. Kushner J.P. Edwards C.Q. Bishop D.T. Skolnick M.H. Am. J. Clin. Pathol. 1982; 78: 196-207Crossref PubMed Scopus (157) Google Scholar, (2Edwards C.Q. Griffen L.M. Goldgar D. Drummond C. Skolnick M.H. Kushner J.P. N. Engl. J. Med. 1988; 318: 1355-1362Crossref PubMed Scopus (567) Google Scholar). In patients with HH, excessive iron deposition in a variety of organs leads to multi-organ dysfunction. Recently, we reported a mutation in a novel MHC class I-like gene, called HLA-H (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). Eighty-three percent of HH patient DNAs were found to be homozygous for this mutation, which consists of a single base transition of G to A and results in a change of cysteine 282 → tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4Jaswinska E.C. Cullen L.M. Busfield F. Pyper W.R. Webb S.I. Powell L.W. Morris C.P. Walsh T.P. Nat. Gen. 1996; 14: 250-251Google Scholar, 5Jouanolle A.M. Gandon G. Jezequel P. Blayau M. Campion M.L. Yaouang J. Mosser J. Fergelot P. Chauvel B. Bouric P. Carn G. Andrieux N. Gicquel I. Gall J.-Y.L. David V. Nat. Gen. 1996; 14: 251-252Crossref PubMed Scopus (346) Google Scholar, 6Beutler E. Gelbart T. West C. Lee P. Adams M. Blackstone R. Pockros P. Kosty M. Vendetti C.P. Phatak P.D. Seese N.K. Chorney K.A. Elshof A.E.T. Gerhard G.S. Chorney M. Blood Cells Mol. Dis. 1996; 22: 187-194Crossref PubMed Scopus (382) Google Scholar), providing further support thatHLA-H is the primary HH locus. A second missense mutation, histidine 63 → aspartate (H63D), was also reported that was enriched in heterozygotes with the C282Y mutation (eight of nine cases) (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). The specific role that either of these mutations in HLA-H play in the etiology of HH disease has not been elucidated.The HLA-H protein is similar to MHC class I family molecules including HLA-A2, nonclassical class I molecules such as HLA-G, and the human neonatal Fc receptor (FcRn). All four of the invariant cysteine residues that form disulfide bridges in the α2 and α3 domains of MHC class I family members are present in the HLA-H protein. One of these conserved cysteine residues is altered in the C282Y mutation. The integrity of the conserved disulfide linkages has been suggested to be critical for proper maintenance of the secondary and tertiary structure of the protein allowing interactions with accessory molecules such as β2-microglobulin (7Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (612) Google Scholar). Importantly, the functional significance of an interaction between β2-microglobulin and an unknown class I-like molecule in HH disease was suggested by β2-microglobulin-deficient mice; these mice display a progressive hepatic iron overload similar to that observed in human HH (8de Souza M. Reimao R. Lacerda R. Hugo P. Kaufmann S.H.E. Porto G. Immunol. Lett. 1994; 39: 105-111Crossref PubMed Scopus (178) Google Scholar, 9Rothenberg B.E. Voland J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1529-1534Crossref PubMed Scopus (196) Google Scholar, 10Santos M. Schilham M.W. Rademakers L.H. Marx J.J. de Souza M. Clevers H. J. Exp. Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar). Other studies have demonstrated that mutation of cysteine 203 in the α3 domain of the mouse MHC class I family member H-2Ld prevented intracellular transport of the molecule from the endoplasmic reticulum to the plasma membrane (11Miyazaki J. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 757-761Crossref PubMed Scopus (49) Google Scholar).As a step toward understanding the role of HLA-H in HH disease, we examined the effects of the C282Y and H63D mutations on HLA-H cellular processing. In this report we demonstrate that wild-type HLA-H binds to β2-microglobulin and that the C282Y mutation completely abrogates this interaction and disrupts intracellular protein trafficking. The data provide support for the hypothesis that the C282Y mutation results in intracellular sequestration of the HLA-H protein, which leads to HH disease.RESULTS AND DISCUSSIONWe first sought to demonstrate an interaction of the HLA-H protein with β2-microglobulin and to examine the effects of the C282Y and H63D mutations on that interaction. Human embryonic kidney cells (293 cells) were transfected with vectors containing the wild-type HLA-H cDNA or the cDNA with either the C282Y or H63D mutation. The FLAG octapeptide sequence was fused onto the carboxyl terminus of each, providing a specific tag for detection of the expressed proteins (13Ford C.F. Suominen I. Glatz C.E. Protein Expression Purif. 1991; 2: 95-107Crossref PubMed Scopus (133) Google Scholar). We established individual stable cell lines expressing the three proteins. Immunoprecipitation of cell lysates with monoclonal antibodies directed to the FLAG sequences (M2 antibodies), to precipitate the HLA-H/FLAG fusion protein, followed by Western blotting with β2-microglobulin polyclonal antibodies demonstrated a clear interaction between the HLA-H protein and β2-microglobulin (Fig. 1 A, left panel, Wild type lane). Significantly, β2-microglobulin was not detected in immune complexes from cell lines expressing the HLA-H protein with the C282Y mutation (Fig. 1 A, left panel). This failure to detect β2-microglobulin was not due to lack of HLA-H protein in the mutant cell lines, since stripping the blots and immunodetection with rabbit polyclonal antibodies directed to the COOH-terminal 17 amino acids of HLA-H (CT1 antibodies) demonstrated that the amount of HLA-H protein in the three cell lines was similar (Fig. 1 A, right panel). The results with the H63D mutant were similar to the wild-type HLA-H protein; β2-microglobulin was co-immunoprecipitated along with that mutant protein (Fig. 1 A, left panel, compare H63D and Wild type lanes). It is of interest to note that the wild-type or H63D HLA-H proteins detected in the right panel appeared to migrate as a doublet of 49 and 46 kDa in lighter exposures, whereas the C282Y appeared as only a single band of approximately 46 kDa.The β2-microglobulin/HLA-H interaction results were corroborated by performing the inverse experiment in which cell lysates were initially immunoprecipitated with β2-microglobulin antibodies followed by Western blotting with antibodies directed toward the COOH-terminal sequence of HLA-H (CT1 antibodies). In this experiment, the β2-microglobulin antibodies co-immunoprecipitated HLA-H protein from the wild-type and H63D mutant expressor cell lines, but failed to do so in the C282Y mutant expressor cell line (Fig. 1 B, left panel). Stripping the blots and reprobing with β2-microglobulin antibodies demonstrated that similar amounts of β2-microglobulin protein were immunoprecipitated from each cell line (Fig. 1 B, right panel). These results further confirm an interaction between wild-type HLA-H protein and β2-microglobulin and demonstrate that the C282Y, but not the H63D, mutation disrupts this association.Previous reports have suggested that association of the MHC class I heavy chain with β2-microglobulin is critical for cell-surface expression (14Arce-Gomez B. Jones E.A. Barnstable C.J. Solomon E. Bodmer W.F. Tissue Antigens. 1978; 11: 96-112Crossref PubMed Scopus (156) Google Scholar, 15Seong R.H. Clayberger C.A. Krensky A.M. Parnes J.R. J. Exp. Med. 1988; 167: 288-299Crossref PubMed Scopus (73) Google Scholar). Because of the failure of the HLA-H protein containing the C282Y mutation to interact with β2-microglobulin, we next investigated whether this mutation would also affect cell-surface presentation of the HLA-H protein. Parental 293 cell lines and those expressing the wild-type HLA-H protein or the C282Y mutant were examined for cell-surface protein expression by immunostaining with rabbit polyclonal antibodies specific to sequences residing in the predicted external domain of the HLA-H protein (EX1 and EX2 antibodies) followed by detection with immunofluorescence. Parental 293 cells displayed no detectable surface labeling by these antibodies (Fig. 2 A), consistent with the undetectable levels of HLA-H protein observed in the Western blotting experiments (Fig. 1 A, right panel). Wild-type HLA-H-expressing cells demonstrated a distinct pattern of surface labeling as evidenced by a punctate pattern of labeling that was much more intense at the edges of the cells (Fig. 2 B). By contrast, cells expressing the C282Y mutation displayed no surface labeling and were indistinguishable from the parental controls (Fig. 2, compare C and A). The specificity of the antibody labeling was demonstrated by preincubating the EX1 and EX2 antibodies with their respective peptides; in these experiments the punctate surface labeling observed in the wild-type HLA-H expressor cells was completely abolished (data not shown).Figure 2The C282Y mutation in HLA-H prevents cell surface expression. To examine HLA-H determinants exposed on the cell surface, parental 293 cell lines, and 293 cells stably transfected with wild-type HLA-H (Wild type) or HLA-H containing the C282Y or H63D mutations were first fixed in paraformaldehyde and then labeled with a 1:1 mixture of antibodies EX1 and EX2 at 50 μg/ml followed by fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies (A–C, Non-permeablized). For a comparison with surface staining, cells were fixed and then permeablized with saponin and labeled as in A–C(D–F, Permeablized), both internal and to a lesser extent, surface determinants are labeled with this procedure. Magnification, × 630.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We examined the possibility that the C282Y mutant protein was expressed in the transfected cells but remained intracellularly localized. Immunostaining was performed following treatment of the cells with saponin to permeablize them. Staining of these cells for the FLAG-tagged C282Y mutant HLA-H protein demonstrated strong perinuclear fluorescence, which was absent in the parental control cells (Fig. 2, compare D and F). Permeablized wild-type HLA-H protein expressor cells showed similar intracellular staining with the FLAG-M2 antibody, suggesting that not all of the wild-type protein in these transfected cells reaches the cell surface (E). Experiments utilizing the EX1, EX2, or CT1 antibodies yielded the same results (data not shown). These results clearly demonstrate that the C282Y mutation specifically disrupts cell-surface presentation of the HLA-H protein.To examine the distribution of wild-type and mutant HLA-H proteins within the cell in more detail, we performed subcellular fractionations on stepwise sucrose gradients to separate the various membrane components. Three separate postnuclear membrane fractions were obtained; the 20/35% interface contained the lightest density membranes (L); dense membranes (D) partitioned at the 40/50% interface, whereas the 35/40% medium-density (M) interface contained a mixture of light and dense membrane-derived components. We initially characterized the efficacy of our subcellular membrane fractionation scheme by assaying these fractions for marker proteins. Antibodies to Na+/K+-ATPase were utilized as markers for plasma membrane, β-coatomer protein (β-COP) for Golgi, and calnexin for ER membrane identification. Samples representing membranes from equal numbers of cells from each interface were analyzed by Western blotting and quantitated on a Molecular Dynamics scanning densitometer. Plasma membranes were found primarily in the light-density interface and to a lesser extent in the medium-density layer: L, 90%; M, 10%; D, 0%. Golgi membranes were distributed nearly equally throughout the three interfaces: L, 30%; M, 40%; D, 30%. ER membranes were found mostly in the dense membrane interface: L, 0%; M, 20%; D, 80%. The fractionations from each of the three cell lines gave equivalent results.We determined the specific distribution of HLA-H proteins in the sucrose gradient interfaces by Western blotting and probing with HLA-H antibodies. As with the co-immunoprecipitation results (Fig. 1), the immunostaining suggested that the wild-type HLA-H protein migrated as a doublet of the 49 and 46 kDa forms (Fig. 3, left panel). The slower migrating 49-kDa form was found principally in those fractions containing plasma membranes, whereas the lower molecular mass 46-kDa form was distributed in a pattern similar to that of the Golgi marker, β-COP. By contrast, the C282Y mutant HLA-H protein consisted only of the faster migrating 46-kDa species. Like the wild-type 46-kDa protein, the mutant 46-kDa protein was distributed in a pattern that most closely resembled that of the Golgi marker protein, suggesting the possibility of incomplete posttranslational processing or modification (Fig. 3, middle panel). The H63D mutant proteins migrated in a pattern resembling that of the wild-type protein, implying that this mutation had little or no effect on intracellular HLA-H protein trafficking (Fig. 3, right panel). In other studies, no HLA-H protein was detected in the cytosolic fractions of any of the wild-type or mutant cell lines, suggesting that neither the C282Y nor the H63D mutation cause a redistribution of the protein to the cytoplasm (data not shown).Figure 3The C282Y mutation in HLA-H alters the subcellular distribution of the protein. Membranes from 293 cells stably expressing the wild-type HLA-H or the C282Y or H63D mutant proteins were fractionated by isopycnic stepwise sucrose gradient centrifugation as described under "Experimental Procedures." Membranes from each interface were then resolved on SDS-PAGE, transferred to PVDF membrane, and the HLA-H protein detected with a 1:1 mixture of EX1 and EX2 HLA-H antibodies followed by ECL. L, 20/35% sucrose light-density interface; M, 35/40% sucrose medium-density interface; D, 40/50% sucrose high-density interface; WT, wild-type HLA-H expressor cell line;C282Y, C282Y HLA-H mutant expressor cell line;H63D, H63D HLA-H mutant expressor cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Taken together these results demonstrate that the C282Y mutation prevents the HLA-H molecule from interacting with β2-microglobulin and eliminates cell-surface presentation. Cysteine 282 is one of four cysteine residues that are invariant in both classical and nonclassical MHC class I molecules and forms a critical disulfide bridge in the α3-immunoglobulin-like domain (7Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (612) Google Scholar). Thus, the integrity of this structure is critical to the formation of the heterodimer of β2-microglobulin and HLA-H and also for proper intracellular processing of the protein. Class I MHC molecules are noncovalently linked heterodimers between an α heavy chain and β2-microglobulin (light chain) (7Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (612) Google Scholar). The role of the β2-microglobulin/heavy chain interaction is to facilitate and stabilize the folding of the heavy chain during biosynthesis through interactions with the α1-α2platform and the α3 domain (16Hansen T.H. Myers N.B. Lee D.R. J. Immunol. 1988; 140: 3522-3527PubMed Google Scholar) (7Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (612) Google Scholar). Previous work demonstrated that mutating the reciprocal cysteine residue (cysteine 203) in mouse H-2Ld protein abolished cell-surface presentation (11Miyazaki J. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 757-761Crossref PubMed Scopus (49) Google Scholar). Interestingly, the mutant H-2Ld molecule retained the ability to associate with β2-microglobulin. Mouse H-2Ld belongs to the family of classical antigen-presenting molecules, whereas HLA-H is nonpolymorphic and, therefore, resembles nonclassical molecules such as HLA-G or the human Fc receptor (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). It is conceivable that the integrity of the α3 domain may be less stringent for β2-microglobulin association with classical antigen-presenting proteins (such as H-2Ld) than for nonclassical molecules (such as HLA-H).The biogenesis of MHC class I molecules is well documented. The heavy chain of a MHC class I molecule is synthesized on membrane-bound polysomes, and N-linked glycosylation occurs co-translationally in the ER (17Ploegh H.L. Cannon L.E. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2273-2277Crossref PubMed Scopus (146) Google Scholar). Subsequently, the modified heavy chain associates with chaperone proteins and β2-microglobulin in the ER and is then transported through the cis-Golgi network, to the middle and trans-Golgi cisternae where the glycosyl side chain is modified to a more complex form en route to the plasma membrane (18Krangel M.S. Orr H.T. Strominger J.L. Cell. 1979; 18: 979-991Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 19Owen M.J. Kissonerghis A.-M. Lodish H.L. J. Biol. Chem. 1980; 255: 9678-9684Abstract Full Text PDF PubMed Google Scholar, 20Williams D.B. Watts T.H. Curr. Opin. Immunol. 1995; 7: 77-84Crossref PubMed Scopus (96) Google Scholar). Class I molecules that fail to assemble properly are recycled between the ER and Golgi, rather than being retained exclusively in the ER (21Hsu V.W. Yuan L.C. Nuchtern J.G. Lippincott-Schwartz J. Hammerling G.J. Klausner R.D. Nature. 1991; 352: 441-444Crossref PubMed Scopus (129) Google Scholar). In our studies the C282Y mutant of HLA-H is retained on intracellular membranes in a pattern that would be consistent with these earlier observations. The mutant protein migrates similar to the Golgi marker protein β-COP in subcellular fractionations, but because of the limited resolution of the step-gradient, we cannot rule out that some protein may also be in the ER. The perinuclear pattern of staining noted in the immunofluorescence studies does not definitively resolve this. More detailed studies will be necessary to ascertain the specific point at which intracellular transport of the C282Y mutant is disrupted.In contrast to our results with the C282Y mutation, we found no detectable changes in the β2-microglobulin interaction or intracellular processing of the H63D mutant form of HLA-H, which is enriched in C282Y heterozygous patients (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). Other studies have demonstrated alterations in intracellular transport of class I molecules by mutations in the peptide-binding groove of HLA-A (22Salter R.D. Immunogenetics. 1994; 39: 266-271Crossref PubMed Scopus (2) Google Scholar). The H63D mutation is localized in the α1 domain between the third and fourth β strands of the external peptide-binding region. It is possible that the effect of this mutation is to alter the affinity of the HLA-H protein for an as yet unidentified ligand or to alter the manner that the mutant protein interacts with other proteins in the cell membrane. Alternatively, this mutation may represent a common polymorphism with little or no effect on the biological functioning of the protein. The definitive answer to this question will await further investigation as we elucidate how the HLA-H molecule regulates iron metabolism in the body. Hereditary hemochromatosis (HH) 1The abbreviations used are:HHhereditary hemochromatosisMHCmajor histocompatibility complexPCRpolymerase chain reactionβ-COPβ-coatomer proteinERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresisPVDFpolyvinyl difluoride. is an autosomal recessive disorder of iron metabolism and represents one of the most common inherited disorders in individuals of Northern European descent with an estimated carrier frequency between 1 in 8 and 1 in 10 (1Dadone M.M. Kushner J.P. Edwards C.Q. Bishop D.T. Skolnick M.H. Am. J. Clin. Pathol. 1982; 78: 196-207Crossref PubMed Scopus (157) Google Scholar, (2Edwards C.Q. Griffen L.M. Goldgar D. Drummond C. Skolnick M.H. Kushner J.P. N. Engl. J. Med. 1988; 318: 1355-1362Crossref PubMed Scopus (567) Google Scholar). In patients with HH, excessive iron deposition in a variety of organs leads to multi-organ dysfunction. Recently, we reported a mutation in a novel MHC class I-like gene, called HLA-H (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). Eighty-three percent of HH patient DNAs were found to be homozygous for this mutation, which consists of a single base transition of G to A and results in a change of cysteine 282 → tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4Jaswinska E.C. Cullen L.M. Busfield F. Pyper W.R. Webb S.I. Powell L.W. Morris C.P. Walsh T.P. Nat. Gen. 1996; 14: 250-251Google Scholar, 5Jouanolle A.M. Gandon G. Jezequel P. Blayau M. Campion M.L. Yaouang J. Mosser J. Fergelot P. Chauvel B. Bouric P. Carn G. Andrieux N. Gicquel I. Gall J.-Y.L. David V. Nat. Gen. 1996; 14: 251-252Crossref PubMed Scopus (346) Google Scholar, 6Beutler E. Gelbart T. West C. Lee P. Adams M. Blackstone R. Pockros P. Kosty M. Vendetti C.P. Phatak P.D. Seese N.K. Chorney K.A. Elshof A.E.T. Gerhard G.S. Chorney M. Blood Cells Mol. Dis. 1996; 22: 187-194Crossref PubMed Scopus (382) Google Scholar), providing further support thatHLA-H is the primary HH locus. A second missense mutation, histidine 63 → aspartate (H63D), was also reported that was enriched in heterozygotes with the C282Y mutation (eight of nine cases) (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E.E. Meyer N.C. Mintier G.A. Moeller N.N. Moore T. Morikang E. Prass C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3327) Google Scholar). The specific role that either of these mutations in HLA-H play in the etiology of HH disease has not been elucidated. hereditary hemochromatosis major histocompatibility complex polymerase chain reaction β-coatomer protein endoplasmic reticulum polyacrylamide gel electrophoresis polyvinyl difluoride. The HLA-H protein is similar to MHC class I family molecules including HLA-A2, nonclassical class I molecules such as HLA-G, and the human neonatal Fc receptor (FcRn). All four of the invariant cysteine residues that form disulfide bridges in the α2 and α3 domains of MHC class I family members are present in the HLA-H protein. One of these conserved cysteine residues is altered in the C282Y mutation. The integrity of the conserved disulfide linkages has been suggested to be critical for proper maintenance of the secondary and tertiary structure of the protein allowing interactions with accessory molecules such as β2-microglobulin (7Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (612) Google Scholar). Importantly, the functional significance of an interaction between β2-microglobulin and an unknown class I-like molecule in HH disease was suggested by β2-microglobulin-deficient mice; these mice display a progressive hepatic iron overload similar to that observed in human HH (8de Souza M. Reimao R. Lacerda R. Hugo P. Kaufmann S.H.E. Porto G. Immunol. Lett. 1994; 39: 105-111Crossref PubMed Scopus (178) Google Scholar, 9Rothenberg B.E. Voland J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1529-1534Crossref PubMed Scopus (196) Google Scholar, 10Santos M. Schilham M.W. Rademakers L.H. Marx J.J. de Souza M. Clevers H. J. Exp. Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar). Other studies have demonstrated that mutation of cysteine 203 in the α3 domain of the mouse MHC class I family member H-2Ld prevented intracellular transport of the molecule from the endoplasmic reticulum to the plasma membrane (11Miyazaki J. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 757-761Crossref PubMed Scopus (49) Google Scholar). As a step toward understanding the role of HLA-H in HH disease, we examined the effects of the C282Y and H63D mutations on HLA-H cellular processing. In this report we demonstrate that wild-type HLA-H binds to β2-microglobulin and that the C282Y mutation completely abrogates this interaction and disrupts intracellular protein trafficking. The data provide support for the hypothesis that the C282Y mutation results in intracellular sequestration of the HLA-H protein, which leads to HH disease. RESULTS AND DISCUSSIONWe first sought to demonstrate an interaction of the HLA-H protein with β2-microglobulin and to examine the effects of the C282Y and H6
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