FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2103808537> ?p ?o ?g. }

• W2103808537 endingPage "14938" @default.
• W2103808537 startingPage "14933" @default.
• W2103808537 abstract "We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly. We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly. endoplasmic reticulum major histocompatibility complex transporter associated with antigen presentation semipermeabilized dithiothreitol aurintricarboxylic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid polyacrylamide gel electrophoresis a homobifunctional reagent specific free sulfhydryls N-ethylmaleimide β2-microglobulin Complex and inter-related quality control mechanisms exist within the lumen of the mammalian endoplasmic reticulum (ER)1 to ensure correct protein folding. The biosynthesis of multisubunit proteins entering the secretory pathway is regulated at the ER where individual subunits are synthesized and their assembly is co-ordinated. Studies on the biogenesis of major histocompatibility complex (MHC) class I have highlighted intermediate stages during assembly of this multisubunit complex and suggested that a number of ER resident proteins, including calnexin (1.Capps G.G. Zuniga M.C. J. Biol. Chem. 1994; 269: 11634-11639Abstract Full Text PDF PubMed Google Scholar), calreticulin (2.Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar), tapasin (2.Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 5.Li S. Sjögren H.-O. Hellman U. Peterson R.F. Wang P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8708-8713Crossref PubMed Scopus (116) Google Scholar, 6.Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (433) Google Scholar), and the transporter associated with antigen presentation (TAP) peptide transporters (3.Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (325) Google Scholar, 4.Suh W.-K. Cohen-Doyle M.F. Fruh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (271) Google Scholar) play a role during the assembly process. Interactions with other proteins such as BiP (7.Noessner E. Parham P. J. Exp. Med. 1995; 181: 327-337Crossref PubMed Scopus (134) Google Scholar) and Grp94 (8.Li Z. Srivastava P.K. EMBO J. 1993; 12: 3143-3151Crossref PubMed Scopus (248) Google Scholar) have also been reported, but it is not clear if these interactions occur during the normal assembly of the MHC class I molecule. Mature MHC class I molecules consist of three subunits, a heavy chain, which assembles with β2-microglobulin within the ER, and an antigenic peptide whose assembly with the β2-microglobulin-heavy chain complex is essential for subsequent transport of a stable and functional class I complex to the cell surface (9.Germain R.N. Margulies D.H. Annu. Rev. Immunol. 1993; 11: 403-450Crossref PubMed Scopus (952) Google Scholar). The dynamics of ER chaperone-mediated interactions, the identity of the specific intermediates each ER protein interacts with, and the role these proteins play during assembly remain to be established. The sequence of events leading to transport of the fully assembled MHC class I complex from the ER is an area of intense study. As the MHC class I heavy chain is translocated into the ER, intra-chain disulfide bonds are formed within the α2 and α3domains (10.Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Only the fully disulfide-bonded form is assembled with β2-microglobulin, emphasizing that the formation of these bonds is a requirement for correct folding and assembly (11.Wang H. Capps G.G. Robinson B.E. Zúniga M. J. Biol. Chem. 1994; 269: 22276-22281Abstract Full Text PDF PubMed Google Scholar). Both nondisulfide- and disulfide-bonded forms of heavy chain have been shown to associate with calnexin, suggesting that this protein plays a role in preventing nonspecific association of heavy chains prior to disulfide bond formation and assembly with β2-microglobulin (10.Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Once a heavy chain-β2-microglobulin dimer is formed, this then dissociates from calnexin and associates with calreticulin, TAP, and tapasin to form a complex that is required for peptide loading (3.Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (325) Google Scholar, 6.Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (433) Google Scholar,7.Noessner E. Parham P. J. Exp. Med. 1995; 181: 327-337Crossref PubMed Scopus (134) Google Scholar). The order of assembly of this complex is unclear, but as soon as the heavy chain is loaded with peptide, the complex dissociates allowing the MHC class I complex to be transported from the ER to the cell surface (3.Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (325) Google Scholar, 4.Suh W.-K. Cohen-Doyle M.F. Fruh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (271) Google Scholar). Mutations within the heavy chain, which prevent this complex from forming (12.Lewis J.W. Neisig A. Neefjes J. Elliott T. Curr. Biol. 1996; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), or expression of heavy chains in cells lacking tapasin (6.Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampé R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (433) Google Scholar) or TAP (13.Cerundolo V. Alexander J. Anderson K. Lamb C. Cresswell P. McMicheal A. Gotch F. Townsend A. Nature. 1990; 345: 449-452Crossref PubMed Scopus (326) Google Scholar) lead to transport of empty unstable complexes to the cell surface. This indicates that this complex plays a role in ER retention or quality control as well as peptide loading. The role of the thiol-dependent oxidoreductase ERp57 (15.Oliver J.D. van der Wal F.J. Bulleid N.J. High S. Science. 1997; 275: 86-88Crossref PubMed Scopus (344) Google Scholar) in MHC class I assembly is less clear. It has been suggested that ERp57 may be involved in stabilization of the MHC class I peptide-loading complex, peptide trimming, or the ER-associated degradation of misfolded proteins (30.Hughes E.A. Cresswell P. Curr. Biol. 1998; 8: 709-712Abstract Full Text Full Text PDF PubMed Google Scholar). ERp57 has been shown previously to form a complex with newly synthesized proteins that, like the interaction with calnexin and calreticulin, is glycoprotein-specific. These studies used a cross-linking approach to identify ERp57 as a cross-linking partner that interacted with glycoproteins forming a complex with calnexin or calreticulin (15.Oliver J.D. van der Wal F.J. Bulleid N.J. High S. Science. 1997; 275: 86-88Crossref PubMed Scopus (344) Google Scholar, 27.Rupp K. Birnbach U. Lundström J. Van P.N. Söling H.-D. J. Biol. Chem. 1994; 269: 2501-2507Abstract Full Text PDF PubMed Google Scholar, 28.Oliver J.D. Roderick H.L. Llewellyn D.H. High S. Mol. Biol. Cell. 1999; 10: 2573-2582Crossref PubMed Scopus (272) Google Scholar). Significantly, ERp57 has also been shown to form a complex with MHC class I in intact cells (29.Morrice N.A. Powis S.J. Curr. Biol. 1998; 8: 713-716Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 30.Hughes E.A. Cresswell P. Curr. Biol. 1998; 8: 709-712Abstract Full Text Full Text PDF PubMed Google Scholar). This observation was extended in a kinetic analysis of rodent MHC class I heavy chain interaction with calnexin, ERp57, and TAP (29.Morrice N.A. Powis S.J. Curr. Biol. 1998; 8: 713-716Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), which suggested that there is a delay in the interaction of ERp57 with MHC class I in comparison with calnexin. In human cells, ERp57 has been shown to interact with MHC class I heavy chain, β2-microglobulin, calreticulin, and tapasin in a subcomplex that is dependent on the presence of β2-microglobulin and tapasin but not TAP (30.Hughes E.A. Cresswell P. Curr. Biol. 1998; 8: 709-712Abstract Full Text Full Text PDF PubMed Google Scholar). ERp57 may be recruited to this peptide-loading subcomplex to facilitate crucial structural changes by formation or rearrangement of disulfide bonds prior to association with TAP and peptide loading. To study what is clearly a complex process, one would ideally investigate folding and assembly within a functionally intact ER. To address this aim we have developed techniques that will allow us to delineate the time course of folding, assembly, and interaction of MHC class I molecules with ER resident proteins. This approach makes use of semipermeabilized cells (SP cells) that can be prepared from any cell line grown in culture and then added to an in vitrotranslation system where they act as a source of ER (14.Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J. 1995; 307: 679-687Crossref PubMed Scopus (131) Google Scholar). Thus, folding and assembly can be studied as it would occur in intact cells using a system amenable to experimental manipulation. We have shown that the synthesis of heavy chains can be synchronized allowing the sequential interaction with calnexin, calreticulin, and tapasin to be studied. We also show that the recently discovered glycoprotein-specific chaperone ERp57 (15.Oliver J.D. van der Wal F.J. Bulleid N.J. High S. Science. 1997; 275: 86-88Crossref PubMed Scopus (344) Google Scholar) interacts with heavy chain during disulfide bond formation. The cDNA coding for HLA-B2705 heavy chain was a gift from Dr. Linda Tussey (John Radcliffe Hospital, Oxford, UK) and was subcloned as an EcoRI-BamHI fragment into pBluescript SK+ (Stratagene, Cambridge, UK). The recombinant plasmid was designated pAJM2. Transcription reactions were carried out as described by Gurevich et al. (16.Gurevich V.V. Pokrovskaya I.D. Obukhova T.A. Zozulya S.A. Anal. Biochem. 1991; 195: 207-213Crossref PubMed Scopus (131) Google Scholar). Recombinant plasmid was linearized with BamHI and transcribed using T7 RNA polymerase (Promega, Southampton, UK). Reactions (100 μl) were incubated at 37 °C for 2 h. Following purification over RNeasy columns (Qiagen, Dorking, UK), the RNA was resuspended in 100 μl of RNase-free water containing 1 mmdithiothreitol (DTT) and 40 units of RNasin (Promega). The human T-lymphoblastoid cell line CEM (17.Howell D.N. Andreotti P.E. Dawson J.E. Cresswell P. J. Immunol. 1985; 134: 971-976PubMed Google Scholar) was maintained in Iscoves' modified Dulbecco's medium supplemented with 10% fetal calf serum. SP cells from this cell line were prepared as described previously (18.Allen S. Bulleid N.J. Biochem. J. 1997; 328: 113-119Crossref PubMed Scopus (23) Google Scholar). RNA was translated using a rabbit reticulocyte lysate (FlexiLysate, Promega) at 30 °C. The translation reaction (per 25 μl) contained 17.5 μl of reticulocyte lysate, 0.5 μl of 1 mm amino acids (minus methionine), 0.5 μl of 100 mm KCl, 15 μCi ofl-[35S]methionine (NEN, Dreiech, Germany), 1 μl of transcribed RNA, and 5.5 μl (approximately 2 × 105) of SP cells. Translations were carried out for 5 min prior to the addition of aurintricarboxylic acid (ATCA) (75 μm) to prevent re-initiation of translation (19.Stewart M.L. Grollman A.P. Huang M.-T. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 97-101Crossref PubMed Scopus (117) Google Scholar). Translation reactions were incubated for various lengths of time after ATCA addition as described in individual experiments. Translation was terminated by the addition of N-ethylmaleimide (NEM) to a final concentration of 20 mm and by the isolation of the SP cells by centrifugation at 12,000 × g for 2 min. Cell pellets were resuspended in KHM buffer (110 mm KOAc, 20 mm Hepes (pH 7.2), 2 mm MgOAc) and re-isolated by centrifugation as above. Cell pellets were processed for immunoprecipitation as described below. Cross-linking was performed using BMH, a homobifunctional reagent specific free sulfhydryls. Following in vitro translation, the membrane fraction was incubated for 20 min on ice in the presence of 50 μm BMH added from a 10 mm stock in Me2SO4. Control reactions were carried out in the presence of Me2SO4 alone. The reaction was quenched by the addition of 0.1 volume of 100 mm DTT, and the samples were incubated for 10 min on ice. After cross-linking, samples were subjected to denaturation in immunoprecipitation buffer containing 1% SDS for 5 min at 100 °C. This was followed by cooling on ice, removal of 10% of the sample to provide a total cross-linked sample, and the addition of 10 volumes of immunoprecipitation buffer without added SDS. Samples were subjected to immunoprecipitation with specific antibodies as described below. Isolated cell pellets were made up to a final volume of 1 ml with ice-cold CHAPS immunoprecipitation buffer (50 mm HEPES (pH 7.4), containing 2% CHAPS, 200 mmNaCl, 0.02% (w/v) sodium azide) and pre-incubated with 50 μl of protein A-Sepharose (10% (w/v) in phosphate-buffered saline) for 60 min at 4 °C to preclear the samples of protein A binding components. Insoluble cell debris was removed by centrifugation at 12,000 ×g for 5 min after preclearing. Precleared samples were incubated for 2 h with 50 μl of protein A-Sepharose and either 10 μl of monoclonal antibody HC10 (Dr. Hidde Ploegh, MIT, Cambridge, MA), 1 μl of W6/32 (Serotec Ltd., Oxford, UK), or 1 μl of polyclonal antibody to β2-microglobulin (AMS Biotechnology UK, Oxon, UK), 5 μl of calnexin (12.Lewis J.W. Neisig A. Neefjes J. Elliott T. Curr. Biol. 1996; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), 1 μl of calreticulin (Cambridge Biosciences, Cambridge, UK), 1 μl of tapasin Rgp48C (Dr. Peter Cresswell, Howard Hughes Medical Institute, Yale University Medical School), or 1 μl of ERp57 (Dr. T. Wileman, IAH, Pirbright, UK). The complexes were washed three times with immunoprecipitation buffer then prepared for analysis by SDS-polyacrylamide gel electrophoresis (PAGE) as described below. Samples were prepared for electrophoresis by the addition of SDS-PAGE loading buffer (0.0625 m Tris/HCl (pH 6.8), SDS (2% w/v), glycerol (10% v/v), and bromphenol blue) in the presence or absence of 50 mm DTT and boiled for 5 min. After electrophoresis, the gels were dried, processed for autoradiography, and exposed to Kodak X-Omat AR film or image intensities were quantified by phosphorimage analysis. We have previously demonstrated that, when HLA-A2 heavy chain was translated in the presence of SP cells derived from the human cell line HT1080, a heavy chain/β2-microglobulin heterodimer could be formed as judged by immunoprecipitation with the conformation-specific antibody W6/32 (14.Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J. 1995; 307: 679-687Crossref PubMed Scopus (131) Google Scholar). This assembly was inefficient, probably due to a limited amount of β2-microglobulin or peptide in this cell line. To improve efficiency of assembly we use here the human cell line CEM, which is lymphoblastoid in origin and has been used previously to study MHC class I assembly (20.Sadasivan B.K. Cariappa A. Waneck G.L. Cresswell P. Cold Spring Harbor Symp. Quant. Biol. 1995; LX: 267-275Crossref Scopus (64) Google Scholar). For these studies we translated HLA-B27 heavy chain, but it should be noted that HLA-A2 also assembled efficiently with SP cells prepared from this cell line (results not shown). Our initial experiments were designed to demonstrate that the translated heavy chain was targeted to, translocated across, and integrated into the ER membrane of SP cells. In the absence of added cells, HLA-B27 heavy chain is translated (Fig.1 A, lane 1) but remains sensitive to proteinase K treatment (Fig. 1A, lane 2). Translocation could be demonstrated by protection of the translation product synthesized in SP cells from proteolysis by added proteinase K (Fig. 1 A, lane 3). The increase in mobility observed after proteinase K treatment is caused by the removal of the cytoplasmic tail, demonstrating that the translated heavy chain was translocated and integrated into the ER membrane (Fig.1 A, compare lanes 3 and 5). We also investigated whether the translocated heavy chain becameN-link-glycosylated. Such a post-translational modification was suggested by the decrease in mobility of the translation product synthesized in the presence of cells as compared with the translation product synthesized in the absence of cells. Glycosylation was confirmed by sensitivity of the translation product to digestion with endoglycosidase H (Fig. 1 A, lanes 6). We also demonstrate that, when heavy chain was translated in the presence of SP cells derived from CEM cells, assembly occurs as judged by immunoprecipitation with the conformation-specific antibody W6/32 (Fig.1 B). The HLA-B27 complex has been shown to be relatively stable in the presence of peptide (22.Tan L. Anderson M.H. Elliott T. Haurum J.S. J. Immunol. Methods. 1997; 209: 25-36Crossref PubMed Scopus (15) Google Scholar), and we confirmed these results by showing that the complex formed in our system was stable up to 55 °C (Fig. 1 B). This would suggest that the majority of complexes were loaded with peptide. These experiments show that when B27 heavy chain RNA was translated in the presence of SP cells derived from CEM cells, assembly of the translocated, glycosylated product with endogenous β2-microglobulin and peptide resulted in the formation of a stable MHC class I complex. In these initial experiments, the HLA-B27 mRNA was translated for 60 min, which resulted in a heterologous mixture of polypeptide chains at various stages of synthesis, folding, and assembly. To follow the folding and assembly of a more homogenous population of molecules, we attempted to synchronize the synthesis of polypeptide chains by only allowing a single wave of translational initiation. We incubated our translation reactions for 5 min to allow initiation to occur and then added aurintricarboxylic acid (ATCA), a compound that inhibits initiation but does not inhibit chain extension or cause dissociation of mRNA-ribosome complexes (19.Stewart M.L. Grollman A.P. Huang M.-T. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 97-101Crossref PubMed Scopus (117) Google Scholar). Using this approach, in the absence of added SP cells, we observed the first full length chains 8 min after initiation of synthesis with no further increase of translation product 12 min after synthesis (Fig.2, A and B). Hence, we could follow a wave of translation products with the time difference between synthesis of the first and last chains being 3–4 min. Such an approach has previously been used to study the sequential interaction of cytosolic chaperones with newly synthesized proteins (24.Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (559) Google Scholar). Having established this synchronized translation system, we next investigated the time course of assembly of heavy chains with β2-microglobulin in our SP cells. Translation reactions were incubated for 5 min prior to the addition of ATCA and allowed to proceed for a further 5, 10, 15, 25, or 40 min. Cells were isolated from the translations and solubilized, and immunoprecipitation was carried out with antibodies to β2-microglobulin, or antibodies to heavy chain, W6/32 and HC10. In the presence of cells the first full-length chains were observed 10 min after initiation of synthesis (Fig. 3 A,bottom panel) as judged by immunoprecipitation with HC10 that recognizes unassembled chains. The time difference of appearance of full length chains between translations carried out in the presence and absence of cells probably reflects the time taken for targeting to the ER and translocation across and integration into the ER membrane. The first appearance of β2-microglobulin-heavy chain (HC-β2M) dimers occurred 5 min later (Fig. 3 B,middle panel), which compares well with the time taken for assembly in intact cells (25.Hill A. Takiguchi M. McMicheal A. Immunogenetics. 1993; 37: 95-101Crossref PubMed Scopus (45) Google Scholar, 26.Neefjes J.J. Hämmerling G.J. Momburg F. J. Exp. Med. 1993; 178: 1971-1980Crossref PubMed Scopus (70) Google Scholar). The precipitation of translocated heavy chain with the W6/32 antibody followed a similar time course confirming the appearance of HC-β2M dimers (Fig.3 A, top panel). The ratio of heavy chains present as part of a complex with β2-mircoglobulin increased dramatically over the time course (ratio unassembled:assembled was 1:0.5 at 15 min and 1:2 at 40 min) and indicated that the unassembled full length chains were assembling post-translationally with β2-microglobulin. The amount of heavy chain assembling with β2-microglobulin was still increasing after 40 min, whereas the amount of heavy chain immunoprecipitated by W6/32 did not increase after 30 min (Fig. 3 B), indicating that the supply of peptide to these cells is limiting. The results demonstrate that a population of heavy chain molecules synthesized within SP cells can assemble with endogenous β2-microglobulin and peptide and that we can follow the assembly of these complexes within a synchronized translation system. When the same translation products were separated without prior reduction, the disulfide status could be evaluated. The rabbit reticulocyte lysate used in these experiments contains no added DTT and supports the formation of disulfide bonds in proteins translocated across ER membranes (14.Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J. 1995; 307: 679-687Crossref PubMed Scopus (131) Google Scholar). All the heavy chain precipitated with W6/32 and β2-microglobulin antibodies migrated with a faster mobility than the reduced samples, thus indicating the formation of intra-chain disulfide bonds as would be expected for assembled chains (Fig. 3 A, top and middle panels). The unassembled chains precipitated by the HC10 antibody were mostly nondisulfide-bonded at early time points but became progressively more oxidized (Fig. 3 B, bottom panel). These disulfide-bonded chains migrated with a diffuse pattern that could reflect partial oxidation or the formation of non-native disulfides (compare samples precipitated with W6/32 and HC10 run under nonreducing conditions). Thus it would appear that in our SP cell system we have a population of heavy chains, which form disulfide bonds and associate with β2-microglobulin, and a population of heavy chains, which remain nondisulfide-bonded or form non-native disulfides and remain unassembled. This indicates that for heavy chain the process of disulfide bond formation and assembly with β2-microglobulin is connected as has been suggested previously (10.Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To investigate the interaction of the translocated heavy chains with the glycoprotein-specific chaperones calnexin and calreticulin we carried out a similar time course and immunoprecipitated the products of translation with antibodies specific for these proteins. The results are shown in Fig. 4 A(top and middle panels) and clearly demonstrate that both these antibodies precipitate heavy chains and, therefore, interact during assembly, which confirms previous results (1.Capps G.G. Zuniga M.C. J. Biol. Chem. 1994; 269: 11634-11639Abstract Full Text PDF PubMed Google Scholar, 10.Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The time course and the disulfide status of the heavy chains interacting with calnexin were quite distinct from those interacting with calreticulin. Hence, calnexin interacted predominantly with heavy chains soon after synthesis and with nondisulfide-bonded forms at early time points. The oxidized form of heavy chain that calnexin interacted with at later time points showed a similar diffuse mobility to the material immunoprecipitated with the HC10 antibody, suggesting that calnexin interacts mainly with unassembled chains. In contrast, calreticulin interacted with heavy chain at later time points and almost exclusively with disulfide-bonded forms, which migrated as a sharp band similar to the material immunoprecipitated with W6/32 and anti-β2-microglobulin, suggesting that calreticulin interacts with assembled chains (Fig. 4 A, middle panel). This is in agreement with previous results that suggest calreticulin and not calnexin forms a complex with HC-β2M dimers (2.Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). We were not able to determine whether the material immunoprecipitated with calreticulin was indeed assembled, because the conditions required to elute the heavy chains from calreticulin disrupt the heavy chain-β2-microglobulin complex (data not shown). The difference in the timing of interaction (Fig.4 B) and the mobility of the heavy chains when separated under nonreducing conditions are highly suggestive that calreticulin associates with assembled dimers, whereas calnexin associates with unassembled chains. We also investigated the interaction of ERp57 with heavy chains during assembly using the synchronized SP cell translation system. Here we show that ERp57 interacts with heavy chains during assembly using a co-immunoprecipitation approach (Fig. 4 A, lower panel). This co-immunoprecipitation was specific because no heavy chains were immunoprecipitated with antibodies to protein disulfide isomerase (results not shown). The disulfide status of the heavy chains interacting with ERp57 indicated that there is an association with both native and oxidized heavy chains, particularly at early time points. This is in contrast with the exclusively disulfide-bonded material interacting with calreticulin and is more similar to the material associating with calnexin. Thus, these results suggest that ERp57 interacts with a separate population of heavy chain polypeptides than calreticulin, at least during early stages of assembly. The interaction of ERp57 with a nondisulfide-bonded population at earlier time points may suggest a role for ERp57, in association with calnexin, in the folding and disulfide bond formation of heavy chain prior to an interaction with calreticulin and the TAP-associated peptide loading complex. At later time points, ERp57 interacts with fully oxidized heavy chain that is likely to be complexed with β2-microglobulin, calreticulin, and tapasin. Recruitment of a thiol-dependent reductase to assembled heavy chains would not suggest a role directly in disulfide bond formation because the intra-chain disulfide bonds have formed prior to assembly but could indicate a role in preventing disulfide reduction. Alternatively, the co-immunoprecipitation of heavy chains with antibodies to ERp57 could simply reflect a stable interaction with calreticulin. A role for ERp57 also has been suggested in the degradation of misfolded proteins (31.Bangia N. Lehner P.J. Hughes E.A. Surman M. Cresswell P. Eur. J. Immunol. 1999; 29: 1858-1870Crossref PubMed Scopus (140) Google Scholar). It is possible that the population of heavy chain molecules that do not assemble with β2-microglobulin or peptide remain associated with ERp57 to facilitate disulfide reduction prior to unfolding and dislocation from the lumen of the ER for proteasome-mediated degradation. The kinetics of MHC class I interaction with tapasin was also analyzed. The anti-tapasin antibody Rgp48C was utilized to precipitate tapasin-heavy chain complexes. This antibody is only functional under denaturing conditions, and, therefore, it was necessary to perform a cross-linking reaction prior to immunoprecipitation. Conditions for BMH-mediated cross-linking were determined (Fig.5 A), and these were used to isolate heavy chain complexes in the synchronized MHC class I assembly assay (Fig. 5 B). Tapasin-specific heavy chain cross-links were clearly isolated after 25 min and suggest a 1:1 stoichiometry between tapasin and heavy chain, because the size of the cross-link was approximately 90 kDa (heavy chain has a molecular mass of 43 kDa and that of tapasin is 48 kDa). The interaction of tapasin with MHC class I at later time points is in agreement with the suggestion that the heterodimeric complex of heavy chain-β2-microglobulin must form prior to the assembly of a peptide-loading subcomplex which also comprises calreticulin. The discrete nature of the tapasin-specific cross-linking product is also suggestive of a direct interaction between heavy chain and tapasin and emphasizes the crucial role that this protein plays in the association between heavy chains and the TAP peptide transporter (31.Bangia N. Lehner P.J. Hughes E.A. Surman M. Cresswell P. Eur. J. Immunol. 1999; 29: 1858-1870Crossref PubMed Scopus (140) Google Scholar). In summary, we have developed a synchronized SP cell translation system that has allowed us to evaluate the timing of interaction of calnexin, calreticulin, ERp57, and tapasin with unassembled and assembled chains. The ability to add any cell line to this system will give us the opportunity to determine the folding, assembly, and interaction of MHC class I molecules with ER resident proteins in cells deficient in specific proteins thought to be involved in this process. Using this approach, we have clearly demonstrated distinct roles for calnexin and calreticulin during the assembly of the MHC class I complex. In addition, we have confirmed that tapasin interacts directly with heavy chain and that this interaction is probably dependent on the formation of a heavy chain-β2-microglobulin heterodimer. We have also shown that ERp57 interacts with both unassembled and assembled heavy chains and suggest that this protein plays a role in initial MHC class I heavy folding, the final stages of assembly, and possibly, the degradation of misfolded heavy chains. We are grateful to all our colleagues who have provided us with reagents for this work (see “Experimental Procedures”)." @default.
• W2103808537 created "2016-06-24" @default.
• W2103808537 creator A5006100200 @default.
• W2103808537 creator A5008290085 @default.
• W2103808537 creator A5056463927 @default.
• W2103808537 creator A5087826692 @default.
• W2103808537 date "2000-05-01" @default.
• W2103808537 modified "2023-10-17" @default.
• W2103808537 title "The Role of ERp57 in Disulfide Bond Formation during the Assembly of Major Histocompatibility Complex Class I in a Synchronized Semipermeabilized Cell Translation System" @default.
• W2103808537 cites W1483092865 @default.
• W2103808537 cites W1528375303 @default.
• W2103808537 cites W1536570090 @default.
• W2103808537 cites W1569077405 @default.
• W2103808537 cites W1852053696 @default.
• W2103808537 cites W1887538332 @default.
• W2103808537 cites W1969965954 @default.
• W2103808537 cites W1975088706 @default.
• W2103808537 cites W1987752224 @default.
• W2103808537 cites W1997215036 @default.
• W2103808537 cites W2000617551 @default.
• W2103808537 cites W2024023167 @default.
• W2103808537 cites W2026970642 @default.
• W2103808537 cites W2037488340 @default.
• W2103808537 cites W2045600868 @default.
• W2103808537 cites W2047474039 @default.
• W2103808537 cites W2052504607 @default.
• W2103808537 cites W2068523436 @default.
• W2103808537 cites W2075018537 @default.
• W2103808537 cites W2079057068 @default.
• W2103808537 cites W2084416493 @default.
• W2103808537 cites W2085868042 @default.
• W2103808537 cites W2089256829 @default.
• W2103808537 cites W2089473305 @default.
• W2103808537 cites W2107857115 @default.
• W2103808537 cites W2118471528 @default.
• W2103808537 cites W2129596312 @default.
• W2103808537 cites W2141988411 @default.
• W2103808537 cites W2147402069 @default.
• W2103808537 doi "https://doi.org/10.1074/jbc.275.20.14933" @default.
• W2103808537 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10809738" @default.
• W2103808537 hasPublicationYear "2000" @default.
• W2103808537 type Work @default.
• W2103808537 sameAs 2103808537 @default.
• W2103808537 citedByCount "82" @default.
• W2103808537 countsByYear W21038085372012 @default.
• W2103808537 countsByYear W21038085372014 @default.
• W2103808537 countsByYear W21038085372016 @default.
• W2103808537 countsByYear W21038085372019 @default.
• W2103808537 countsByYear W21038085372020 @default.
• W2103808537 countsByYear W21038085372021 @default.
• W2103808537 crossrefType "journal-article" @default.
• W2103808537 hasAuthorship W2103808537A5006100200 @default.
• W2103808537 hasAuthorship W2103808537A5008290085 @default.
• W2103808537 hasAuthorship W2103808537A5056463927 @default.
• W2103808537 hasAuthorship W2103808537A5087826692 @default.
• W2103808537 hasBestOaLocation W21038085371 @default.
• W2103808537 hasConcept C104317684 @default.
• W2103808537 hasConcept C105580179 @default.
• W2103808537 hasConcept C12554922 @default.
• W2103808537 hasConcept C149364088 @default.
• W2103808537 hasConcept C154945302 @default.
• W2103808537 hasConcept C185592680 @default.
• W2103808537 hasConcept C207936829 @default.
• W2103808537 hasConcept C27256138 @default.
• W2103808537 hasConcept C2777212361 @default.
• W2103808537 hasConcept C41008148 @default.
• W2103808537 hasConcept C55493867 @default.
• W2103808537 hasConcept C86803240 @default.
• W2103808537 hasConcept C95444343 @default.
• W2103808537 hasConceptScore W2103808537C104317684 @default.
• W2103808537 hasConceptScore W2103808537C105580179 @default.
• W2103808537 hasConceptScore W2103808537C12554922 @default.
• W2103808537 hasConceptScore W2103808537C149364088 @default.
• W2103808537 hasConceptScore W2103808537C154945302 @default.
• W2103808537 hasConceptScore W2103808537C185592680 @default.
• W2103808537 hasConceptScore W2103808537C207936829 @default.
• W2103808537 hasConceptScore W2103808537C27256138 @default.
• W2103808537 hasConceptScore W2103808537C2777212361 @default.
• W2103808537 hasConceptScore W2103808537C41008148 @default.
• W2103808537 hasConceptScore W2103808537C55493867 @default.
• W2103808537 hasConceptScore W2103808537C86803240 @default.
• W2103808537 hasConceptScore W2103808537C95444343 @default.
• W2103808537 hasIssue "20" @default.
• W2103808537 hasLocation W21038085371 @default.
• W2103808537 hasOpenAccess W2103808537 @default.
• W2103808537 hasPrimaryLocation W21038085371 @default.
• W2103808537 hasRelatedWork W1988515303 @default.
• W2103808537 hasRelatedWork W2045355728 @default.
• W2103808537 hasRelatedWork W2060268288 @default.
• W2103808537 hasRelatedWork W2064876270 @default.
• W2103808537 hasRelatedWork W2329655268 @default.
• W2103808537 hasRelatedWork W2333726361 @default.
• W2103808537 hasRelatedWork W2341499342 @default.
• W2103808537 hasRelatedWork W2950785785 @default.
• W2103808537 hasRelatedWork W2951715315 @default.
• W2103808537 hasRelatedWork W644041026 @default.
• W2103808537 hasVolume "275" @default.
• W2103808537 isParatext "false" @default.

• next