Functions of ERp57 in the Folding and Assembly of Major Histocompatibility Complex Class I Molecules
2006; Elsevier BV; Volume: 281; Issue: 21 Linguagem: Inglês
10.1074/jbc.m512073200
ISSN1083-351X
AutoresYi-Nan Zhang, Ehtesham Baig, David B. Williams,
Tópico(s)Heat shock proteins research
ResumoERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain α3 domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-β2-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and β2 -microglobulin does not depend on an oxidized α3 domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process. ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain α3 domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-β2-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and β2 -microglobulin does not depend on an oxidized α3 domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process. Protein folding within the endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; β2m, β2-microglobulin; CNX, calnexin; CRT, calreticulin; endo H, endoglycosidase H; siRNA, small interfering RNA; PDI, protein-disulfide isomerase; MHC, major histocompatibility complex; H chain, heavy chain; DMEM, Dulbecco's modified Eagle's medium; Ab, antibody; mAb, monoclonal antibody; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; IFN, interferon; HPRT, hypoxanthine-guanine phosphoribosyltransferase. is assisted by a diverse array of folding catalysts and molecular chaperones. Folding catalysts include as many as 17 members of the protein-disulfide isomerase (PDI) family of proteins as well as peptidylprolyl cis-trans isomerases. ERp57 (1Bourdi M. Demady D. Martin J.L. Jabbour S.K. Martin B.M. George J.W. Pohl L.R. Arch. Biochem. Biophys. 1995; 323: 397-403Crossref PubMed Scopus (91) Google Scholar, 2Hirano N. Shibasaki F. Sakai R. Tanaka T. Nishida J. Yazaki Y. Takenawa T. Hirai H. Eur. J. Biochem. 1995; 234: 336-342Crossref PubMed Scopus (123) Google Scholar, 3Koivunen P. Helaakoski T. Annunen P. Veijola J. Raisanen S. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 316: 599-605Crossref PubMed Scopus (41) Google Scholar) is a member of the PDI family and contains four thioredoxin domains with two CXXC active sites located within the first and fourth domains (4Alanen H.I. Salo K.E. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W. Antioxid. Redox Signal. 2003; 5: 367-374Crossref PubMed Scopus (72) Google Scholar, 5Silvennoinen L. Myllyharju J. Ruoppolo M. Orru S. Caterino M. Kivirikko K.I. Koivunen P. J. Biol. Chem. 2004; 279: 13607-13615Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Like PDI (6Frand A.R. Cuozzo J.W. Kaiser C.A. Trends Cell Biol. 2000; 10: 203-210Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 7Noiva R. Semin. Cell Dev. Biol. 1999; 10: 481-493Crossref PubMed Scopus (239) Google Scholar), ERp57 has been shown to catalyze disulfide oxidation, isomerization, and reduction in vitro (8Antoniou A.N. Ford S. Alphey M. Osborne A. Elliott T. Powis S.J. EMBO J. 2002; 21: 2655-2663Crossref PubMed Scopus (91) Google Scholar, 9Frickel E.M. Frei P. Bouvier M. Stafford W.F. Helenius A. Glockshuber R. Ellgaard L. J. Biol. Chem. 2004; 279: 18277-18287Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 10Zapun A. Darby N.J. Tessier D.C. Michalak M. Bergeron J.J. Thomas D.Y. J. Biol. Chem. 1998; 273: 6009-6012Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). However, it is distinct from other PDI family members in that it associates noncovalently with the molecular chaperones calnexin (CNX) and calreticulin (CRT) (11Frickel E.M. Riek R. Jelesarov I. Helenius A. Wuthrich K. Ellgaard L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1954-1959Crossref PubMed Scopus (246) Google Scholar). These chaperones bind preferentially to nonnative glycoproteins bearing monoglucosylated Glc1Man5–9-GlcNAc2 oligosaccharides (12Spiro R.G. Zhu Q. Bhoyroo V. Soling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 13Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). It is thought that the ternary complex of ERp57, CNX/CRT, and unfolded glycoprotein enhances the rate of disulfide formation/isomerization by keeping the enzyme in proximity to the substrate. Consistent with this view, the activity of ERp57 toward monoglucosylated glycoprotein substrates is dramatically enhanced in vitro when CNX or CRT is present (10Zapun A. Darby N.J. Tessier D.C. Michalak M. Bergeron J.J. Thomas D.Y. J. Biol. Chem. 1998; 273: 6009-6012Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Furthermore, ERp57 forms mixed disulfide intermediates with model viral glycoproteins in vivo, and these complexes are abrogated when the formation of monoglucosylated oligosaccharides is blocked (14Molinari M. Helenius A. Nature. 1999; 402: 90-93Crossref PubMed Scopus (277) Google Scholar). Despite these suggestive findings, there is little evidence that ERp57 actually promotes oxidative folding of any protein in living cells. To address the functions of ERp57 in cells, we have been studying the biogenesis of class I molecules of the major histocompatibility complex (MHC), a system in which interactions with ERp57 are well established. Class I molecules bind to cytosolically derived peptide antigens and subsequently display them at the cell surface, where they are screened by cytotoxic T cells. They consist of a glycosylated transmembrane heavy chain (H chain) with three extracellular domains termed α1, α2, and α3, a soluble subunit termed β2-microglobulin (β2m), and a peptide ligand of 8–10 residues. Assembly of class I molecules begins within the ER, where the nascent H chain binds to the membrane-bound chaperone CNX and, indirectly, to ERp57 (15Paulsson K. Wang P. Biochim. Biophys. Acta. 2003; 1641: 1-12Crossref PubMed Scopus (63) Google Scholar, 16Lehner P.J. Cresswell P. Curr. Opin. Immunol. 2004; 16: 82-89Crossref PubMed Scopus (73) Google Scholar). At this early stage, H chain disulfide bond formation takes place, although the order of formation of the two disulfides within the α2 and α3 domains remains unclear (17Ribaudo R.K. Margulies D.H. J. Immunol. 1992; 149: 2935-2944PubMed Google Scholar). The efficiency of complete disulfide formation is substantially increased upon H chain association with β2m (17Ribaudo R.K. Margulies D.H. J. Immunol. 1992; 149: 2935-2944PubMed Google Scholar, 18Wang H. Capps G.G. Robinson B.E. Zuniga M.C. J. Biol. Chem. 1994; 269: 22276-22281Abstract Full Text PDF PubMed Google Scholar). Correct disulfide bond formation is crucial, since mutagenesis of cysteines comprising either disulfide results in reduced cell surface expression and defects in peptide loading (19Miyazaki J. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 757-761Crossref PubMed Scopus (50) Google Scholar, 20Warburton R.J. Matsui M. Rowland-Jones S.L. Gammon M.C. Katzenstein G.E. Wei T. Edidin M. Zweerink H.J. McMichael A.J. Frelinger J.A. Hum. Immunol. 1994; 39: 261-271Crossref PubMed Scopus (41) Google Scholar). Once H chain associates with β2m, a peptide loading complex assembles that consists of the class I heterodimer, CNX, or its soluble paralog CRT, ERp57, Bap31, the TAP peptide transporter, and another protein, tapasin. The peptides that bind to class I are primarily generated in the cytosol by the proteasome and are transported into the ER by the TAP transporter. Tapasin serves as a bridge between class I and TAP (21Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (333) Google Scholar, 22Suh W.K. Cohen-Doyle M.F. Fruh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (279) Google Scholar), thereby placing class I molecules close to the peptide source. In addition, tapasin stabilizes the peptide loading complexes and enhances the loading of high affinity peptides into the class I binding groove (23Momburg F. Tan P. Mol. Immunol. 2002; 39: 217-233Crossref PubMed Scopus (92) Google Scholar, 24Ortmann B. Copeman J. Lehner P.J. Sadasivan B. Herberg J.A. Grandea A.G. Riddell S.R. Tampe R. Spies T. Trowsdale J. Cresswell P. Science. 1997; 277: 1306-1309Crossref PubMed Scopus (449) Google Scholar). Upon peptide binding to class I, the loading complex dissociates, thereby permitting fully assembled class I molecules to be exported from the ER to the cell surface, a process that is facilitated in the ER by Bap31 (25Paquet M.E. Cohen-Doyle M. Shore G.C. Williams D.B. J. Immunol. 2004; 172: 7548-7555Crossref PubMed Scopus (84) Google Scholar). Relative to other members of the PDI family, ERp57 appears to enjoy a privileged position in class I biogenesis. ERp57, but not PDI, has been detected in association with both free and β2m-associated H chains and as a component of the peptide loading complex (8Antoniou A.N. Ford S. Alphey M. Osborne A. Elliott T. Powis S.J. EMBO J. 2002; 21: 2655-2663Crossref PubMed Scopus (91) Google Scholar, 26Farmery M.R. Allen S. Allen A.J. Bulleid N.J. J. Biol. Chem. 2000; 275: 14933-14938Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Lindquist J.A. Hammerling G.J. Trowsdale J. FASEB J. 2001; 15: 1448-1450Crossref PubMed Scopus (68) Google Scholar). Furthermore, Cresswell and co-workers have demonstrated that between 15 and 80% of the total ERp57 pool can be associated with class I peptide loading complexes and that ERp57 is present exclusively in disulfide linkage to tapasin (28Peaper D.R. Wearsch P.A. Cresswell P. EMBO J. 2005; 24: 3613-3623Crossref PubMed Scopus (149) Google Scholar). The introduction of cysteine mutants in tapasin not only abolishes the formation of the ERp57-tapasin complex but also prevents full oxidation of the class I H chain and impairs the loading of high affinity peptides (29Dick T.P. Bangia N. Peaper D.R. Cresswell P. Immunity. 2002; 16: 87-98Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Whether these phenotypes are due to the lack of ERp57 or a secondary consequence of the tapasin mutations remains to be determined. These studies have led to suggestions that ERp57 may participate in the oxidative folding of the class I H chain, and, together with tapasin, may make class I molecules more receptive to peptide loading through isomerization of the disulfide bond within the peptide binding groove. In this study, we used RNA interference to reduce the expression of ERp57 as a means to assess its functions in class I biogenesis. We show that in the absence of ERp57, H chain disulfide bond formation and α3 domain folding are substantially delayed, whereas the rate of ER to Golgi transport is slowed only slightly. No effect of ERp57 depletion was observed on the kinetics of H chain-β2m association, assembly of the peptide loading complex, or the loading of peptides onto class I. These findings support a role for ERp57 in H chain disulfide formation but suggest that the ERp57-tapasin disulfide conjugate may not be required for peptide loading. Interestingly, reducing the expression of another ER thiol oxidoreductase, ERp72, was completely without effect on class I biogenesis, consistent with a specialized role for ERp57 in this process and the existence of substrate preferences among different thiol oxidoreductases. Cells and Antibodies—Mouse L cells stably expressing the class I molecules, H-2Kb or H-2Dd, were maintained in high glucose DMEM supplemented with 10% fetal bovine serum, glutamine, and antibiotics. Mouse EL4 cells expressing a truncated version of ovalbumin (residues 253–386; a kind gift of Dr. N. Shastri, University of California Berkeley) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, glutamine, and antibiotics. The following antibodies (Abs) were used in this study: anti-8, a rabbit polyclonal antiserum, which recognizes the carboxyl terminus of the Kb molecule encoded by exon-8 (30Smith M.H. Parker J.M. Hodges R.S. Barber B.H. Mol. Immunol. 1986; 23: 1077-1092Crossref PubMed Scopus (44) Google Scholar); monoclonal antibody (mAb) 34-2-12S, which recognizes the foldedα3 domain of Dd molecules with an intact disulfide bond (17Ribaudo R.K. Margulies D.H. J. Immunol. 1992; 149: 2935-2944PubMed Google Scholar, 31Ozato K. Mayer N.M. Sachs D.H. Transplantation. 1982; 34: 113-120Crossref PubMed Scopus (481) Google Scholar); mAb 34-5-8S, specific for β2m-associated Dd molecules (31Ozato K. Mayer N.M. Sachs D.H. Transplantation. 1982; 34: 113-120Crossref PubMed Scopus (481) Google Scholar); mAbs Y3 and 20-8-4S, which recognize β2m-associated Kb molecules (32Ozato K. Sachs D.H. J. Immunol. 1981; 126: 317-321PubMed Google Scholar, 33Jones B. Janeway Jr., C.A. Nature. 1981; 292: 547-549Crossref PubMed Scopus (174) Google Scholar); and mAb 25-D1-16, which is specific for Kb molecules complexed with the ovalbumin-derived peptide SIINFEKL (34Porgador A. Yewdell J.W. Deng Y. Bennink J.R. Germain R.N. Immunity. 1997; 6: 715-726Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Rabbit anti-tapasin antiserum directed against the carboxyl-terminal 20 amino acids of murine tapasin and anti-CNX antiserum raised against the ER luminal domain of dog CNX have been described previously (35Danilczyk U.G. Williams D.B. J. Biol. Chem. 2001; 276: 25532-25540Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 36Suh W.K. Derby M.A. Cohen-Doyle M.F. Schoenhals G.J. Fruh K. Berzofsky J.A. Williams D.B. J. Immunol. 1999; 162: 1530-1540PubMed Google Scholar). Anti-ERp57 antiserum was raised against glutathione S-transferase-fused mouse ERp57. The anti-glutathione S-transferase antibodies were removed by adsorption to glutathione S-transferase-agarose prior to use. Anti-CRT antiserum (SPA-600), anti-ERp72 antiserum (SPA-720), and anti-PDI antiserum (SPA-891) were purchased from StressGen Biotechnologies (Victoria, Canada). Rabbit anti-actin antiserum was purchased from Sigma. Anti-CaBP1 antiserum was the generous gift of Dr. D. Ferrari (Max Planck Institute for Biophysical Chemistry, Goettingen, Germany). RNA Interference and Transfections—Two double-stranded small interfering RNAs (siRNAs) corresponding to mouse ERp57 DNA sequences AGCCAGCAACTTGAGAGATAA (siRNA 1) and AAGAGGCTTGCCCCTGAGTAT (siRNA 2) were synthesized and annealed by Qiagen (Valencia, CA). siRNA 2 was fluorescein isothiocyanate-labeled to facilitate measurement of transfection efficiency. siRNA targeting mouse ERp72 (AAGCAGTTTGCTCCAGAATAT) and nontargeting control siRNA (AATTCTCCGAACGTGTCACGT) were also purchased from Qiagen. Eighteen hours before transfection, 1 × 105 mouse L cells expressing H-2Kb or H-2Dd were seeded into 6-well dishes. siRNAs were transfected into the cells using oligofectamine (Invitrogen) according to the manufacturer's protocol with a final siRNA concentration of 40 nm. Cells were transfected for 4 days before assays were performed. For mouse EL4 TO cells, ERp57 siRNA was premixed with solution T included in the Nucleofector kit T (final siRNA concentration of 240 nm), and 6 × 105 cells were transfected with the Nucleofector device using parameter O17 (Amaxa GmbH, Cologne, Germany). Cells were maintained in RPMI 1640 in the presence of siRNA for 3 days before being assessed for surface expression of Kb-SIINFEKL peptide complexes. For all RNA interference experiments, ∼1 × 105 siRNA-transfected cells were used to assess the efficiency of protein knockdown by Western blot. Cell lysates were separated by SDS-PAGE, transferred onto an Immobilon membrane (Millipore Corp., Bedford, MA), and probed with the appropriate antiserum. Detection was performed by ECL (Amersham Biosciences). Metabolic Radiolabeling and Immunoisolation—Pulse-chase radiolabeling experiments with siRNA-transfected L cells expressing either Kb or Dd molecules were performed in 35-mm plates. Cells were starved for 30 min with methionine-free RPMI 1640 and pulse-labeled for 2 min with 0.5 ml of medium containing 0.1 mCi of [35S]Met (>1000 Ci/mmol; Amersham Biosciences). Cells were then washed with Met-free RPMI 1640 and chased for various periods in DMEM containing 1 mm Met and 500 μm cycloheximide. After washing for 3 min in cold PBS containing 20 mm N-ethylmaleimide (NEM), lysis was conducted in 500 μl of PBS, pH 6.8, containing 1% Nonidet P-40, 20 mm NEM, and protease inhibitors (60 μg/ml 2-aminoethyl-benzenesulfonylfluoride and 10 μg/ml each leupeptin, antipain, and pepstatin (BioShop, Burlington, Canada)). Following centrifugation at 10,000 × g to remove nuclei and cell debris, the supernatant fraction was incubated on ice for 2 h with either anti-8 or 20-8-4S Ab for Kb and either 34-2-12S or 34-5-8S Ab for Dd. Immune complexes were recovered by shaking for 1 h with 30 μl of packed protein A-agarose beads. Proteins were eluted with SDS-PAGE sample buffer from the beads and analyzed either by nonreducing or reducing SDS-PAGE (10% gel) followed by fluorography. Films were scanned using an EPSON 1680 scanner and were quantified using NIH Image software (National Institutes of Health). In all cases, backgrounds were subtracted by quantifying a blank area of the film corresponding in size to that of the gel band of interest. In experiments to assess the formation of the peptide loading complex, cells were radiolabeled for 45 min with [35S]Met, washed in cold PBS containing 20 mm NEM, and lysed in digitonin lysis buffer (1% digitonin in PBS, pH 6.8, 20 mm N-ethylmaleimide, and protease inhibitors). The lysate was incubated with anti-tapasin antiserum for 2 h. Immune complexes were recovered with protein A-agarose and then washed twice with 0.2% digitonin in PBS, pH 6.8, before elution and analysis by SDS-PAGE. For class I ER to Golgi transport assays, Kb and Dd molecules were immunoisolated with anti-8 and 34-2-12S Ab, respectively, as described above and then were eluted from protein A beads with 0.1 m citrate buffer, pH 6, containing 0.1% SDS and digested with 2 units of endo-β-N-acetylglucosaminidase H (endo H; New England Biolabs, Beverly, MA) at 37 °C for 2 h prior to analysis by SDS-PAGE. Flow Cytometry Analysis—To assess the cell surface levels of class I molecules, 3–5 × 105 cells were trypsinized from culture dishes and incubated with mouse anti-class I mAbs (1.5 μg of mAb Y3 for Kb or mAb 34-5-8S for Dd) in 100 μl of culture medium for 20 min on ice. After incubation, cells were washed once with fluorescence-activated cell sorting buffer (Hanks' balanced salt solution, 1% bovine serum albumin, and 0.01% NaN3) and then incubated with 0.4 μg of phycoerythrin-conjugated goat anti-mouse IgG (H+L chain-specific, Cedarlane, Hornby, Canada) in 100 μl of fluorescence-activated cell sorting buffer for 20 min on ice. Cells were washed twice with fluorescence-activated cell sorting buffer and then fixed in 0.5% paraformaldehyde in PBS, pH 7.4. Subsequent flow cytometry was performed using an EPICS Elite flow cytometer (Beckman Coulter, Fullerton, CA). For analysis of the turnover kinetics of cell surface class I molecules, Kb -or Dd-expressing L cells were incubated for 18 h at 26 °C in DMEM containing 10 μg/ml human β2m (Sigma). Human β2m-containing DMEM was then replaced with prewarmed DMEM containing 10 μg/ml brefeldin A (Sigma), and cells were transferred to a 37 °C incubator. Cells (3–5 × 105) were removed at the indicated time points and analyzed by flow cytometry as described above. Kb-SIINFEKL complexes on the surface of EL4 TO cells were measured by flow cytometry using mAb 25-D1-16. The SIINFEKL peptide loading efficiency was expressed as the Kb-SIINFEKL level divided by the surface Kb level (as detected with mAb Y3). Quantification of Interferon α and β mRNAs—L cells expressing Kb molecules were either untransfected or transfected with control siRNA, siRNA targeting ERp57, and siRNA targeting ERp72. Cells were harvested after 12 h, and total RNA was extracted with the RNeasy Mini Kit (Qiagen). Digestion of contaminating DNA was performed using Fermentas DNaseI according to the manufacturer's protocol. cDNA was synthesized using 1 μg of RNA in the presence of random primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer's protocol. For quantitative PCR, reaction components were obtained from the LightCycler® FastStart DNA Master SYBR GreenPLUS I kit (Roche Applied Science). The LightCycler® instrument (Roche Applied Science) and corresponding software was used for all reactions. The following reaction conditions were used: preincubation at 95 °C for 10 min, followed by 45 amplification cycles of 95 °C for 10 s, 60 °C for interferon (IFN)-β, 65 °C for IFN-α2, and 60 °C for hypoxanthine-guanine phosphoribosyltransferase (HPRT) for 5 s, 72 °C for 10 s, melting curve analysis at 95 °C for 0 s, 65 °C for 15 s, and a continuous acquisition mode of 95 °C with a temperature transition rate of 0.1 °C/s. The PCR was performed in a final volume of 20 μl containing 1× Master SYBR GreenPLUS I buffer, 20 pmol of HPRT and IFN-β primers, 10 pmol of IFN-α2 primers, and 5 μl of template cDNA (concentration 100 ng/μl). The following primer sets were used: for IFN-α2, 5′-AAAGGGGAGCCTCCTCAT-3′ (forward) and 5′-TGCTTTCCTCGTGATGCTGA-3′ (reverse); for IFN-β, 5′-ACACAAGCTTAACCACCATGAACAACAGGTGGATCCTCCACGC-3′ (forward) and 5′-GTTAGGAATTCTCAGTTTTGGAAGTTTCTGGTAAGTCTTCG-3′ (reverse); and for HPRT, 5′-CAAGCTTGCTGGTGAAAAGGA-3′ (forward) and 5′-TGAAGTACTCATTATAGTCAAGGGCATATC-3′ (reverse). Standard curves for IFN-α2, IFN-β, and HPRT were established using serial dilutions of template cDNA in triplicate in the RelQuant software (Roche Applied Science). Levels of IFN-α2, IFN-β, and HPRT mRNA were calculated based on these established standard curves, and the amounts of IFN-α2 and IFN-β mRNA were normalized to the amount of HPRT in each sample. All samples were analyzed in triplicate. Delayed H Chain Disulfide Bond Formation Is Observed upon Depletion of ERp57 but Not ERp72—To study the function of ERp57 in the process of class I biogenesis, we reduced the expression of ERp57 in L cells expressing the class I H-2Kb molecule by transfecting with 21-bp siRNAs. Following a 4-day transfection with siRNA 1, greater than 90% of ERp57 was depleted (Fig. 1A). No expression differences were observed for several other proteins, including CNX or the thiol oxidoreductases ERp72, PDI, and CaBP1. As a control to assess the effect of reducing the expression of a thiol oxidoreductase not previously implicated in class I biogenesis, we performed a similar experiment using siRNA directed against ERp72. Again, the level of ERp72 was reduced by >90% with no effects on the expression of ERp57 or several other proteins (Fig. 1B). Based on previous studies suggesting an interaction of ERp57 with free class I H chains (8Antoniou A.N. Ford S. Alphey M. Osborne A. Elliott T. Powis S.J. EMBO J. 2002; 21: 2655-2663Crossref PubMed Scopus (91) Google Scholar, 27Lindquist J.A. Hammerling G.J. Trowsdale J. FASEB J. 2001; 15: 1448-1450Crossref PubMed Scopus (68) Google Scholar), we anticipated that this enzyme might participate in H chain disulfide formation. Consequently, the effect of ERp57 depletion on H chain disulfide bond formation was monitored in a pulse-chase experiment. After transfecting with ERp57 siRNA 1 or with control siRNA, L cells expressing the class I H-2Kb molecule were radiolabeled for 2 min with [35S]Met and chased in medium containing unlabeled Met and cycloheximide for periods of up to 30 min. Kb molecules were recovered from cell lysates with anti-8, an antiserum that reacts with free or β2m-associated Kb H chains. Isolated Kb H chains were analyzed by SDS-PAGE under nonreducing conditions to allow resolution of species containing zero, one, or two disulfide bonds. The results show that disulfide bond formation was substantially slowed in the absence of ERp57 (Fig. 2A, top; quantified in Fig. 2B, siRNA 1). In control cells, 50% of H chains acquired both disulfide bonds by ∼2 min of chase, whereas in ERp57-depleted cells, the rate was 9-fold slower, with 50% of H chains acquiring both disulfide bonds by 18 min of chase. In this experiment, no zero-disulfide bond species was detected, possibly because the first disulfide bond formed co-translationally and therefore was already present following the 2-min pulse. To confirm these results, the same experiment was performed using siRNA 2, which targets a different region of the ERp57 mRNA (Fig. 2A, middle). In this case, the rate of H chain disulfide formation was slowed 11-fold upon depletion of ERp57 (quantified in Fig. 2B, siRNA 2). The half-time for acquisition of both disulfide bonds was ∼2 min in control cells versus 22 min in ERp57-depleted cells. In experiments testing the effect of ERp57 siRNA 2 on various control proteins, it was noted that this siRNA caused a ∼50% reduction of ERp72 expression in addition to the depletion of ERp57 (data not shown). However, this "off-target" depletion of ERp72 by siRNA 2 did not contribute to the observed slower H chain oxidation, since knockdown of ERp72 expression alone by >90% had no significant effect on H chain oxidation (Fig. 2A, bottom; quantified in Fig. 2B). The lack of effect due to ERp72 depletion, coupled with previous studies showing a lack of PDI interaction during class I biogenesis (26Farmery M.R. Allen S. Allen A.J. Bulleid N.J. J. Biol. Chem. 2000; 275: 14933-14938Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), suggests that ERp57 may be the preferred thiol oxidoreductase promoting oxidative class I H chain folding. ERp57 Is Required for α3 Domain Folding but Not for H Chain-β2m Association—As an independent means to assess the involvement of ERp57 in H chain disulfide formation, the folding rate of the H chain α3 domain of class I H-2Dd molecules was assessed using mAb 34-2-12S. This antibody only recognizes Dd molecules that have a folded α3 domain with an intact disulfide bond (17Ribaudo R.K. Margulies D.H. J. Immunol. 1992; 149: 2935-2944PubMed Google Scholar). In this experiment, L cells expressing D molecules were transfected with ERp57 siRNA 1 or control siRNA and subjected to pulse-chase radiolabeling, and cell lysates were immunoisolated with mAb 34-2-12S. In contrast to cells treated with control siRNA, the α3 domain epitope formed more slowly when ERp57 expression was reduced by RNA interference (Fig. 3A, top). Whereas the t½ for epitope formation was ∼2 min in control cells, it was 8-fold slower in ERp57-depleted cells (t½ siRNA 1 = 16 min) (Fig. 3B). This finding was confirmed using ERp57 siRNA 2, for which a 9-fold slower rate of epitope formation was observed relative to control cells (Fig. 3A, middle; quantified in Fig. 3B). The observed slower α3 domain folding was not due to simultaneous partial depletion of ERp72 by siRNA 2, since ERp72 depletion alone had no effect (Fig. 3A, bottom). In contrast to H chain disulfide formation, all efforts to detect effects of ERp57 depletion on the kinetics of H chain-β2m association were unsuccessful. As shown in Fig. 4A (top), isolation of β2m-associated Dd H chains with mAb 34-5-8s at various times during a pulse-chase experiment showed no differences in either the kinetics or extent of H chain-β2m formation in the absence or presence of ERp57. Similar results were obtained for Kb using mAb 20-8-4S, which reacts
Referência(s)