Peptide-bound Major Histocompatibility Complex Class I Molecules Associate with Tapasin before Dissociation from Transporter Associated with Antigen Processing
1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês
10.1074/jbc.274.13.8649
ISSN1083-351X
AutoresSuling Li, Kajsa Paulsson, Hans‐Olov Sjögren, Ping Wang,
Tópico(s)Immunotherapy and Immune Responses
ResumoMajor histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum. Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum. Major histocompatibility complex (MHC) 1The abbreviations used are:MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; ER, endoplasmic reticulum; β2m, β2-microglobulin; tapasin, TAP-associated glycoprotein; HC, heavy chain; OVA, ovalbumin.1The abbreviations used are:MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; ER, endoplasmic reticulum; β2m, β2-microglobulin; tapasin, TAP-associated glycoprotein; HC, heavy chain; OVA, ovalbumin. class I molecules present antigenic peptides to CD8+ T cells (1Townsend A. Ohlen C. Bastin J. Ljunggren H.-G. Foster L. Karre K. Nature. 1989; 340: 443-448Crossref PubMed Scopus (877) Google Scholar, 2Rammensee H.-G. Falk K. Rotzschke O. Annu. Rev. Immunol. 1993; 11: 213-244Crossref PubMed Scopus (708) Google Scholar). The majority of peptides found associated with class I molecules are derived from nuclear and cytosolic proteins, and they are generated largely by the proteasome complex (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar, 4Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (869) Google Scholar). Peptides are transported from the cytosol into the lumen of the endoplasmic reticulum (ER) by a peptide transporter, which is known as the transporter associated with antigen processing (TAP) (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar, 5Howard J.C. Curr. Opin. Immunol. 1995; 7: 69-76Crossref PubMed Scopus (76) Google Scholar). Both proteins contain a hydrophobic multimembrane spanning domain and a cytosolic nucleotide binding domain. Comparing with other members of the ABC-transporter proteins, which have 2 ATP binding sites and 12–16 transmembrane segments (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar), it was predicted that TAP1/2 functions as a plausible heterodimeric complex. The requirement of TAP1/2 for peptide transport across the ER membrane was elucidated by different assay systems (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar, 4Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (869) Google Scholar, 5Howard J.C. Curr. Opin. Immunol. 1995; 7: 69-76Crossref PubMed Scopus (76) Google Scholar, 6Momburg F. Neefjes J.J. Hämmerling G.J. Curr. Opin. Immunol. 1994; 6: 32-37Crossref PubMed Scopus (93) Google Scholar). These studies have shown that TAP preferentially transports peptides of 8–15 residues in an ATP-dependent fashion. Peptide translocation occurs in two steps involving ATP-independent peptide binding to TAP and peptide translocation across the ER membrane, which is ATP dependent (7Androlewicz M.J. Cresswell P. Immunity. 1994; 1: 7-14Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 8Van Endert P.M. Riganelli D. Greco G. Fleischhauer K. Sidney J. Sette A. Bach J.-F. J. Exp. Med. 1995; 182: 1883-1895Crossref PubMed Scopus (169) Google Scholar, 9Uebel S. Meyer T.H. Kraas W. Kienle S. Jung G. Wiesmuller K.-H. Tampe R. J. Biol. Chem. 1995; 270: 18512-18516Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 10Wang P. Gyllner G. Kvist S. J. Immunol. 1996; 157: 213-220PubMed Google Scholar, 11Wang P. Raynoschek C. Svensson K. Ljunggren H.-G. J. Biol. Chem. 1996; 271: 24830-24835Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). In addition to functioning as a peptide transporter, a physical association between TAP1 and class I heavy chain (HC)/β2-microglobulin (β2m) dimer has been demonstrated (12Such W.-K. Cohen-Doyle M.F. Früh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (272) Google Scholar, 13Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (328) Google Scholar). Because the binding of class I HC/β2m to TAP1 is not required for the peptide translocation, the binding of peptides to class I molecules is thought to be facilitated by association of assembled MHC class I HC/β2m heterodimers with the TAP complex (12Such W.-K. Cohen-Doyle M.F. Früh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (272) Google Scholar, 13Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (328) Google Scholar, 14Androlewicz M.J. Ortmann B. van Endert P.M. Spies T. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12716-12720Crossref PubMed Scopus (138) Google Scholar).Two findings suggested that TAP is not only required for peptide transport across the ER membrane but is also required for the assembly of peptide and MHC class I HC/β2m complex. A first finding came from the study of mutant HLA-A2.1 with a point mutation of threonine 134 to lysine (T134K) (15Peace-Brewer A.L. Tussey L.G. Matsui M. Li G. Quinn D.G. Frelinger J.A. Immunity. 1996; 4: 505-514Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Lewis J.W. Neisig A. Neefjes J. Elliott T. Curr. Biol. 1996; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). This mutation makes the heavy chain incapable of interacting with the TAP complex. This results in decreased cell surface expression of HLA-A2.1, as well as the loss of capacity of newly synthesized class I HC/β2m complex to load peptide in a TAP-dependent manner (15Peace-Brewer A.L. Tussey L.G. Matsui M. Li G. Quinn D.G. Frelinger J.A. Immunity. 1996; 4: 505-514Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Lewis J.W. Neisig A. Neefjes J. Elliott T. Curr. Biol. 1996; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Moreover, direct delivery of peptide to the ER in a TAP-independent manner restores the ability of T134K, HLA-A2.1 to present antigenic peptide to peptide-specific CTL (15Peace-Brewer A.L. Tussey L.G. Matsui M. Li G. Quinn D.G. Frelinger J.A. Immunity. 1996; 4: 505-514Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In a human mutant cell line, .220, it has been found that MHC class I fails to associate with TAP (17Grandea A.G. Androlewicz M.J. Athwal R.S. Geraghty D.E. Spies T. Science. 1995; 270: 105-108Crossref PubMed Scopus (175) Google Scholar). In this cell line, TAP1/2 are normally expressed and peptide transport into the ER is as effective as wild type cells. The MHC class I HC-β2m dimer in the cell appeared to lack associated peptides (17Grandea A.G. Androlewicz M.J. Athwal R.S. Geraghty D.E. Spies T. Science. 1995; 270: 105-108Crossref PubMed Scopus (175) Google Scholar). This finding suggests that interaction of MHC class I HC-β2m dimer with TAP is required for the association of peptides to class I (17Grandea A.G. Androlewicz M.J. Athwal R.S. Geraghty D.E. Spies T. Science. 1995; 270: 105-108Crossref PubMed Scopus (175) Google Scholar) indicating the involvement of TAP in the assembly of peptide and class I HC/β2m.With anti-TAP1 antiserum, several proteins were coprecipitated with TAP1/2 (13Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (328) Google Scholar, 18Früh K. Ahn K. Djaballah H. Samppé P. van Endert P.M. Tampé R. Peterson P.A. Yang Y. Nature. 1995; 375: 415-418Crossref PubMed Scopus (520) Google Scholar). One of the coprecipitates was identified as a 48-kDa glycoprotein (tapasin) and was found in complex with TAP1/2, calreticulin, and MHC class I (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar). Tapasin is a type I transmembrane glycoprotein with a double lysine motif that mediates the retrieval of proteins back from the cis-Golgi and thus maintains membrane proteins in the ER (20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar, 21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herbert J.A. Grandea A.G. Riddell S.R. Tampe R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar). Analysis of .220 cells showed that they lacked the expression of tapasin (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar). Transfection of tapasin into .220 cells restored class I-TAP association and association of peptide with MHC class I (21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herbert J.A. Grandea A.G. Riddell S.R. Tampe R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar). These pieces of evidence indicate that tapasin mediates the interaction of MHC class I HC-β2m with TAP, and this interaction is essential for peptide loading onto MHC class I HC-β2m. Moreover, the stability of the complex of tapasin and TAP1/2 was analyzed in comparison with the association of MHC class I and TAP in both normal and β2m-deficient cells (20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar). Tapasin was stably present in immunoprecipitates in roughly stoichiometric amounts with TAP1/2. However, MHC class I was rapidly dissociated from TAP (20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar). A similar stable association of tapasin with TAP1/2 was also obtained in β2m-deficient cells (20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar). This indicates that TAP1, -2, and tapasin form a trimeric complex. Tapasin serves as a docking site on the TAP complex specific for interaction with MHC class I HC-β2m. Interaction of tapasin with peptide in the presence of ATP suggested the function of tapasin in the loading of peptide onto MHC class I (20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar). More recently, studies with transfection of soluble tapasin into .220 cells indicated that association of tapasin with MHC class I is sufficient to facilitate peptide loading and assembly of MHC class I molecules (22Lehner P.J. Surman M.J. Cresswell P. Immunity. 1998; 8: 221-231Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). The lack of evidence for a tapasin analogue in mouse has suggested a direct interaction of mouse MHC class I with TAP (23Koopmann J.-O. Hämmerling G.J. Momburg F. Curr. Opin. Immunol. 1997; 9: 80-88Crossref PubMed Scopus (94) Google Scholar).We have now cloned the mouse homologue of tapasin and analyzed the interaction of mouse tapasin and TAP1/2 as well as peptide binding in both wild type and TAP2 mutant cells. Mouse tapasin is similar in both function and amino acid sequence to the human homologue. Interaction of tapasin with TAP1 and MHC class I is independent of TAP2. Tapasin associates with peptide-bound TAP1/2 and MHC class I, indicative of a functional difference between tapasin and calreticulin.DISCUSSIONAs a peptide transporter, TAP functions to translocate peptides from the cytosol into the lumen of the ER (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar, 4Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (869) Google Scholar, 5Howard J.C. Curr. Opin. Immunol. 1995; 7: 69-76Crossref PubMed Scopus (76) Google Scholar). In addition to translocating peptides, interaction of MHC class I with TAP has been found to be important in the assembly of peptide and MHC class I (12Such W.-K. Cohen-Doyle M.F. Früh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (272) Google Scholar,13Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (328) Google Scholar). Recently, it has been discovered that tapasin, a subunit of the TAP complex, mediates the association of MHC class I and TAP. Cells lacking tapasin have a deficient expression of surface MHC class I as a result of reduced assembly of MHC class I and peptides in the ER (17Grandea A.G. Androlewicz M.J. Athwal R.S. Geraghty D.E. Spies T. Science. 1995; 270: 105-108Crossref PubMed Scopus (175) Google Scholar,19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar). Transfection of tapasin restored the assembly and surface expression of class I (21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herbert J.A. Grandea A.G. Riddell S.R. Tampe R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar). Human tapasin has been cloned and the amino acid sequence showed a type I transmembrane glycoprotein with a strong ER retention signal at the C terminus (20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar, 21Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herbert J.A. Grandea A.G. Riddell S.R. Tampe R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (434) Google Scholar). Because a mouse analogue of tapasin had not been identified from coprecipitated TAP complex, it was suggested that mouse MHC class I may not need tapasin for interaction with TAP (23Koopmann J.-O. Hämmerling G.J. Momburg F. Curr. Opin. Immunol. 1997; 9: 80-88Crossref PubMed Scopus (94) Google Scholar). We have now cloned mouse tapasin. The predicted amino acid sequence reveals 78% identity to human tapasin with identical signal peptide, N-glycosylation site, transmembrane domain, and double lysine motif at the C-terminal end, indicative of a similar function of human and mouse tapasin. An interesting feature of the sequence of mouse tapasin is the predicted cytosolic domain, which showed less than 50% homology with the human protein. In addition, mouse tapasin has 14 extra amino acids at the C terminus compared with human tapasin, suggestive of species specificity of the tapasin function. In line with the sequence results, it was recently found that the intracellular maturation and surface expression of HLA-B*4402 in murine cells required co-expression of human tapasin but not human TAP1/2 (37Chen A.P. 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 (226) Google Scholar). The cytosolic domain of tapasin may determine its interaction with MHC class I or TAP1.In studies of the interaction between MHC class I and TAP, it was found that TAP1, but not TAP2, is required for the association of TAP with class I molecules (12Such W.-K. Cohen-Doyle M.F. Früh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (272) Google Scholar, 13Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (328) Google Scholar). Because tapasin is essential for the association of MHC class I to TAP (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar), tapasin may directly interact with TAP1. We have now shown that tapasin interacts with TAP1 and MHC class I in RMA-S cells, which have a mutation that causes premature termination of the TAP2 protein. Thus, the interaction between TAP1 and MHC class I appears to be mediated by the tapasin. From these results, the predicted order of interaction between different molecules in the TAP complex is TAP2 to TAP1, TAP1 to tapasin, and tapasin to MHC class I. By these linked molecules, the translocation and loading of peptides are rapidly and efficiently processed in the same microenviroment.Previous reports have indicated the existence of several ER chaperones, which interact with MHC class I (4Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (869) Google Scholar, 19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 27Lindquist J.A. Jensen O.N. Mann M. Hammerling G.J. EMBO J. 1998; 17: 2186-2195Crossref PubMed Scopus (195) Google Scholar, 28Jackson M.R. Cohen-Doyle M.F. Peterson P.A. Williams D.B. Science. 1994; 263: 384-387Crossref PubMed Scopus (220) Google Scholar, 29Noessner E. Parham P. J. Exp. Med. 1995; 181: 327-337Crossref PubMed Scopus (135) Google Scholar, 30Li Z. Srivastava P.K. EMBO J. 1993; 12: 3143-3151Crossref PubMed Scopus (248) Google Scholar). Among them, calnexin and calreticulin were best characterized (4Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (869) Google Scholar, 31Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar). By using conformation specific antibodies and a β2m mutant cell line, a distinct difference between calreticulin and calnexin in their mode of association with MHC class I was found (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 29Noessner E. Parham P. J. Exp. Med. 1995; 181: 327-337Crossref PubMed Scopus (135) Google Scholar, 32van Leeuwen J.E. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 24: 13997-14001Crossref Scopus (75) Google Scholar). Calnexin binds only to β2m-free heavy chain in human cells (29Noessner E. Parham P. J. Exp. Med. 1995; 181: 327-337Crossref PubMed Scopus (135) Google Scholar, 33David V. Hochstenbach F. Rajagopalan S. Brenner M.B. J. Biol. Chem. 1993; 268: 9585-9592Abstract Full Text PDF PubMed Google Scholar), whereas, calreticulin binds only to the MHC class I HC-β2m dimer (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 32van Leeuwen J.E. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 24: 13997-14001Crossref Scopus (75) Google Scholar). Similarly to calreticulin, interaction of tapasin with MHC class I is β2m-dependent (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar), despite the fact that calreticulin and tapasin are quite different molecules. Like calnexin, calreticulin is an ER chaperone with lectin-like activity, and it binds to several other glycoproteins in the ER besides MHC class I (34Helenius A. Mol. Biol. Cell. 1994; 5: 253-255Crossref PubMed Scopus (558) Google Scholar, 35Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-525Crossref PubMed Scopus (587) Google Scholar). The binding is regulated by glucose trimming of nascentN-linked oligosaccharides (34Helenius A. Mol. Biol. Cell. 1994; 5: 253-255Crossref PubMed Scopus (558) Google Scholar, 35Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-525Crossref PubMed Scopus (587) Google Scholar). Tapasin so far was found only in the complexes where MHC class I and/or TAP1/2 are present (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar,20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar). Because tapasin is also an N-linked glycoprotein, it has not yet been proven whether tapasin can directly bind to calreticulin. In addition, tapasin is a subunit of the TAP complex, but calreticulin does not directly associate with TAP (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 20Li S. Sjögren H.-O. Hellman U. Pettersson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar). Assembly of MHC class I and peptides was defective in the absence of tapasin, and the presence of calreticulin (19Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar), indicating that either calreticulin is not required for the loading of peptides to MHC class I or both tapasin and calreticulin are essential for the association of peptide with MHC class I. In this study, a distinct difference in association of these two molecules with peptide-bound MHC class I molecules was found. Tapasin, but not calreticulin, bound to peptide-loaded MHC class I before this complex dissociated from TAP. This may indicate that tapasin directly catalyzes the loading of peptides onto MHC class I, and calreticulin controls the conformation of β2m-heavy chain dimer before it interacts with TAP. Lehner et al. (22Lehner P.J. Surman M.J. Cresswell P. Immunity. 1998; 8: 221-231Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) have shown that TAP1/2-free soluble tapasin can restore MHC class I expression in tapasin-negative cell line .220. This finding, together with the demonstrated interaction between tapasin and peptide-loaded MHC class I, suggests that tapasin functions not only to form a bridge between TAP1/2 and MHC class I but also directly to facilitate the assembly of MHC class I with peptides.The interaction of TAP with MHC class I molecules was found to be polymorphic (36Neisig A. Wubbolts R. Zang X. Melief C. Neefjes J. J. Immunol. 1996; 156: 3196-3206PubMed Google Scholar). Some HLA-B alleles associate less or not at all with TAP (36Neisig A. Wubbolts R. Zang X. Melief C. Neefjes J. J. Immunol. 1996; 156: 3196-3206PubMed Google Scholar). The binding of different peptides to TAP-free MHC class I may differ from that of TAP associated MHC class I molecules. Polymorphism in mechanisms of peptide loading has been found recently in studies of HLA-B*2705, which can present antigenic peptides in the absence of tapasin (37Chen A.P. 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 (226) Google Scholar). In comparison, expression of B*4402 was tapasin-dependent (37Chen A.P. 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 (226) Google Scholar), although tapasin mediated interaction of both B*2705 and B*4402 with TAP1/2. The question is whether the difference in peptide loading is dependent on the peptides selected by these alleles or on the native structure of different alleles resulting in different stability of the MHC class I HC-β2m dimer in the ER. It is important now to determine the molecular structure of the binding between MHC class I and tapasin as well as between tapasin and TAP1. This will form a basis for understanding both the function of tapasin and the selectivity of peptide assembly with different MHC class I.Based on our results and the data previously published, we conclude that mouse tapasin is similar to the human analogue both in amino acid sequence and function. The difference between the mouse and human molecules in the cytosolic domain may indicate a species specificity of tapasin. Tapasin interacts with TAP1 and MHC class I in the absence of TAP2, raising the possibility of sequential interaction of TAP2-TAP1-tapasin-MHC class I in the TAP complex. Interaction of peptide-bound MHC class I with tapasin, but not calreticulin suggests that tapasin may directly influence the loading of peptides onto MHC class I. Major histocompatibility complex (MHC) 1The abbreviations used are:MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; ER, endoplasmic reticulum; β2m, β2-microglobulin; tapasin, TAP-associated glycoprotein; HC, heavy chain; OVA, ovalbumin.1The abbreviations used are:MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; ER, endoplasmic reticulum; β2m, β2-microglobulin; tapasin, TAP-associated glycoprotein; HC, heavy chain; OVA, ovalbumin. class I molecules present antigenic peptides to CD8+ T cells (1Townsend A. Ohlen C. Bastin J. Ljunggren H.-G. Foster L. Karre K. Nature. 1989; 340: 443-448Crossref PubMed Scopus (877) Google Scholar, 2Rammensee H.-G. Falk K. Rotzschke O. Annu. Rev. Immunol. 1993; 11: 213-244Crossref PubMed Scopus (708) Google Scholar). The majority of peptides found associated with class I molecules are derived from nuclear and cytosolic proteins, and they are generated largely by the proteasome complex (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar, 4Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (869) Google Scholar). Peptides are transported from the cytosol into the lumen of the endoplasmic reticulum (ER) by a peptide transporter, which is known as the transporter associated with antigen processing (TAP) (3Heemels M.-T. Ploegh H.L. Annu. Rev. Biochem. 1995; 64: 463-491Crossref PubMed Scopus (415) Google Scholar, 5Howard J.C. Curr.
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