The thiol oxidoreductase ERp57 is a component of the MHC class I peptide-loading complex Eric A. Hughes and Peter Cresswell

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1 Brief Communication 709 The thiol oxidoreductase is a component of the MHC class I peptide-loading complex Eric A. Hughes and Peter Cresswell The proper folding and assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER) is an intricate process involving a number of components. Nascent heavy chains of MHC class I molecules, translocated into the ER membrane, are rapidly glycosylated and bind the transmembrane chaperone calnexin [1]. In humans, after dissociation from calnexin, fully oxidized MHC class I heavy chains associate with b 2 -microglobulin (b 2 m) and the soluble chaperone calreticulin [2]. This complex interacts with another transmembrane protein, tapasin, which is believed to assist in MHC class I folding as well as in mediating the interaction between assembling MHC class I molecules and the transporter associated with antigen processing (TAP) [3,4]. The TAP heterodimer ( TAP2) introduces the final component of the MHC class I molecule by translocating peptides, predominately generated by the proteasome, from the cytosol into the ER where they can bind dimers of b 2 m and the MHC class I heavy chain [5]. Recently, the thiol oxidoreductase also known as GRP58, ERp61, ER60, Q2, H-70, and CPT [6] and first misidentified as phospholipase C-a [7] has been shown to bind in conjunction with calnexin or calreticulin to a number of newly synthesized ER glycoproteins when their N-linked glycans are trimmed by glucosidases I and II [8,9]. It was speculated that is a generic component of the glycan-dependent ER quality system [10]. Here, we show that is a component of the MHC class I peptide-loading complex. might influence the folding of MHC class I molecules at a critical step in peptide loading. Address: Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, necticut 06510, USA. Correspondence: Peter Cresswell peter.cresswell@qm.yale.edu Received: 4 February 1998 Revised: 26 March 1998 Accepted: 4 April 1998 Published: 25 May , 8: Ltd ISSN Results and discussion To determine if is a part of the MHC class I peptide-loading complex, anti- immunoprecipitates from lysates of [ 35 S]methionine-labeled HeLa M cells were denatured and the individual components identified by re-precipitation with specific antibodies (Figure 1). We have observed that the band previously identified as calreticulin occasionally separates into two very closely migrating species with apparent molecular weights of approximately 60. As shown in Figure 1, this doublet is easily resolved and re-precipitation clearly shows that the upper band is calreticulin and the lower band is. The reported cysteine-dependent protease activity [11,12] or the thiol-dependent oxido-reductase activity [13 15] of could be important for any one of the components of the MHC class I peptide-loading complex. To address this issue, a number of well-defined cell lines, deficient for various components of the complex, were assayed for the interaction between and TAP. did not co-immunoprecipitate with an anti- antibody in cells Figure 1 1º 2º None is a component of the MHC class I peptide-loading complex. HeLa M cells ( ) were treated with interferon-γ (200 U/ml) for 48 h then metabolically labeled with 2.0 mci [ 35 S]methionine. The cell pellet was lysed in 1% digitonin and the MHC class I peptide-loading complex was immunoprecipitated using the anti- antibody RING.4C. The primary immunoprecipitate (1 ) was denatured in SDS and DTT, 10% was separated by SDS PAGE (none) and the remainder was re-immunoprecipitated (2 ) with the following antibodies; R.RING.4C (), rabbit anti-calreticulin serum (crt), purified rabbit anti--peptide antibody (), purified rabbit anti-tapasin-peptide antibody (tapasin), monoclonal antibody HC10 for MHC class I heavy chain (MHC I) and anti- rabbit serum (). Normal rabbit serum was used as a antibody (con) for both the 1 and 2. MHC I

2 710, Vol 8 No 12 Figure 2 (a) TAP + TAP (b) β 2 m + (c) + does not associate with the TAP heterodimer in the absence of or tapasin. was immunoprecipitated (), using monoclonal antibody (9E10 was used as a ; con), from (a) wild-type cell line 45.1 (TAP + ) and its TAP-negative derivative.174 (TAP ), (b) the -negative Daudi cell line ( ) and Daudi cells transfected with the gene ( + ), and (c) the tapasin-negative cell line.220-b8 (tapasin ) and.220-b8 cells transfected with the tapasin gene (tapasin + ). In each case, cells were lysed in 1% digitonin and co-purifying proteins were eluted from the immunoprecipitate with 1% deoxycholic acid (DOC) [2], separated by SDS PAGE, and detected by immunoblot analysis using purified rabbit anti--peptide antibody. As a loading, a portion of each whole cell lysate used for immunoprecipitation was also separated by SDS PAGE and blotted for the total amount of present. deficient for, or tapasin (Figure 2). Therefore, associates with only in the presence of and tapasin and does not associate specifically with TAP2 or tapasin, as these molecules are known to remain associated with in the absence of. MHC class I heavy chain and calreticulin also do not interact with TAP in the absence of [2,16], and the presence of in the complex is thus more probably because it has a function in MHC class I assembly rather than in the assembly of the TAP dimer or in tapasin folding. Calreticulin and both associate with a variety of molecules in the ER [8,9,17]. Their association with each other has also been reported following cross-linking. To determine if and calreticulin associate with the MHC class I component of the TAP complex, anti- immunoprecipitates from the wild-type cell line 45.1 were eluted with 1% Triton X-100, which releases a subcomplex containing MHC class I heavy chain,, tapasin, and calreticulin from TAP [2]. As expected, no or class I heavy chains could be detected upon reimmunoprecipitation with a -specific antibody; however, both and MHC class I heavy chain were co-precipitated by anti-calreticulin or anti- antibodies (Figure 3a). This suggested that might also be a component of the sub-complex associated with the TAP heterodimer. Because antibodies specific for MHC class I molecules do not effectively precipitate TAP-associated or tapasin-associated class I molecules, we used a cross-linking approach to look for a direct interaction between and class I molecules. Cross-linking of and MHC class I heavy chains was easily detectable (Figure 3b). The class I heavy chains bound to were sensitive to endoglycosidase H, indicating that they are probably in the ER. In the TAP-deficient cell line.174, antibodies to calreticulin co-precipitate a sub-complex containing MHC class I heavy chain,, and tapasin [2]. We re-immunoprecipitated proteins from anti- immunoprecipitations from these cells and show that is also in this subcomplex (Figure 3c). The same result was obtained when the antibody used for the primary immunoprecipitation was directed against tapasin (data not shown). Immunoblot analysis also indicated that coimmunoprecipitated from.174 cell lysates with tapasin,, and calreticulin (Figure 3d). Thus, the sub-complex formed in the absence of TAP also contains. To determine if tapasin is required for the association of or calreticulin with dimers of and the class I heavy chain, we analyzed anti- immunoprecipitates from tapasin-negative as well as TAP-negative cells by immunoblot analysis. Less was recovered from TAP-negative cells than from their wild-type counterparts (Figure 4a). It was shown previously that the amount of recovered complex containing MHC class I molecules, tapasin, and calreticulin is reduced by about fourfold in the absence of TAP [2]. This was ascribed to stabilization of the sub-complex by TAP, and it seems likely that the reduced recovery of reflects this. More striking, however, was the loss of associated with class I molecules in tapasin-negative cells (Figure 4a). Similarly, no calreticulin co-precipitated with in the absence of tapasin (Figure 4b). Thus, in the absence of TAP, both and calreticulin associate with dimers of and MHC class I molecules, whereas in the absence of tapasin neither do. To determine if an interaction between calreticulin and could occur independently of MHC class I assembly, we looked for in anti-calreticulin immunoprecipitates from a number of mutant cell lines by immunoblot analysis. and calreticulin associated in wild-type and TAP-negative cells but, surprisingly, not in the absence of either or tapasin (Figure 4c e). Detectable association of calreticulin and therefore appears to depend on cooperative interactions within the sub-complex containing MHC class I heavy chain,, calreticulin,, and tapasin. may be recruited to the MHC class I peptideloading complex to rearrange disulfide bonds. Our

3 Brief Communication 711 Figure 3 (a) (b) (c) (d) 1º +DSP DSP 1º GP96 2º 2º Endo H MHC 69 class I MHC 28 class I MHC I MHC class I heavy chain,,, calreticulin, and tapasin form a sub-complex. (a) -associated proteins were immunoprecipitated (1 ) from cells lysed in 1% digitonin using the monoclonal antibody The complex was partially disrupted with 1% Triton X-100 and the released material re-immunoprecipitated with antibodies to TAP.1 (148.3), calreticulin (crt; rabbit anti-calreticulin serum), and (monoclonal antibody BBM.1). Secondary immunoprecipitates (2 ) were eluted with 1% DOC, separated by SDS PAGE, and and MHC class I heavy chain detected by immunoblot analysis using purified rabbit anti--peptide antibody and rat monoclonal antibody 3B10.7, respectively. trol antibodies (con) were the monoclonal antibody 9E10 (1 ) and normal rabbit serum (2 ). (b) Cells ( ) from the wild-type 45.1 line were lysed in 1% digitonin with or without 800 µm of the protein cross-linker dithiobis (N-succinimidyl propionate; DSP) for 30 min at 4 C. The lysate was denatured with SDS and diluted with 1% Triton X-100. and, as a, an abundant soluble ER-retained chaperone GP96 were immunoprecipitated (using monoclonal antibodies and 9G10, respectively), treated with or without endoglycosidase H (endo H), separated by SDS PAGE, and MHC class I heavy chains detected by immunoblot analysis using 3B10.7. A portion of each cell lysate was blotted for total MHC class I heavy chain as a loading. (c) Cells ( ) from the TAP-negative.174 line were metabolically labeled with 2.0 mci [ 35 S]methionine for 3 h and chased with an excess of cold methionine for 30 min. The cell pellet was lysed in 1% digitonin and the MHC class I sub-complex was immunoprecipitated with rabbit anti- serum (1 ). After denaturation with SDS and DTT, the indicated proteins were reimmunoprecipitated (2 ) as for Figure 1. (d) Cells ( ) from the.174 line were lysed in 1% digitonin., tapasin,, and calreticulin () were immunoprecipitated and associated proteins were eluted with 1% DOC, separated by SDS PAGE, and detected by immunoblot analysis; antibodies and s were as for Figure 2. analysis of the complexes formed in the various mutant cell lines is consistent with the idea that associates with assembled dimers of and the MHC class I heavy chain at the same time as both calreticulin and tapasin. As class I molecules in the complex have completely oxidized disulfide bonds while calnexin-associated heavy chains do not [2,18 20], perhaps is responsible for their formation. It has been suggested that has cysteine protease activity as degradation of calreticulin and protein disulfide isomerase (PDI) by purified in vitro is sensitive to Figure 4 (a) TAP (b) (c) (d) (e) TAP + + TAP TAP + + TAP TAP and calreticulin do not associate with the heterodimer of and the MHC class I heavy chain in the absence of tapasin, and do not detectably interact with each other in the absence of or tapasin. (a,b) and (c,d) calreticulin (crt) were immunoprecipitated from the indicated cell lines as described in the legend to Figure 2 using the monoclonal antibody BBM.1 and rabbit antiserum, respectively. Monoclonal antibody 9E10 was used as a (con). Associated proteins were eluted with 1% DOC, separated by SDS PAGE, and (a,c e) or (b) calreticulin were detected by immunoblot analysis. As a loading a portion of each whole cell lysate used for immunoprecipitation was also separated by SDS PAGE and blotted for the total amount of either (a,b) MHC class I heavy chain or (c e).

4 712, Vol 8 No 12 the cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal (LLnL) and N-acetyl-leucyl-leucyl-methionine [12]. Thus, associated with MHC class I heavy chains could be involved in the trimming of long peptides translocated by TAP which can occur in the ER [21]. sistent with this idea is our finding that trimming of signalsequence-derived peptides associated with HLA-A2 molecules in TAP-negative cells is also dependent on an ER protease sensitive to LLnL [22]. Finally, it has also been suggested that might be a component of the proteolytic machinery involved in the degradation of misfolded ER proteins as it binds an ERretained mutant form of lysozyme in vivo and degrades denatured lysozyme in vitro [12]. Degradation of the mutant lysozyme was sensitive to LLnL, but LLnL has also been found to inhibit proteosome-mediated degradation of misfolded ER proteins [23] after they have been retro-translocated into the cytosol [24,25]. Association of with misfolded proteins might reflect a requirement for reduction and denaturation before they are shunted to the cytosol. Misfolded MHC class I heavy chains can be extruded into the cytosol when they have bound but have failed to bind peptide [23]. As the -associated class I heavy chains are fully oxidized, their degradation could require an interaction with and their reduction before they can be retro-translocated into the cytosol. Materials and methods Immunoprecipitations in digitonin, elution of co-precipitated proteins in DOC and Triton X-100, dithiobis cross-linking and the various cells and antibodies used have been described previously [2,4,16]. Supplementary material Additional methodological details are published with this paper on the internet. Acknowledgements We thank R. Salter and T. Spies for supplying cell lines, and S. High and J.L. Holtzman for antibodies. We also thank Nancy Dometios for her yeoman service in preparation of this manuscript. This work was supported by National Institutes of Health (NIH) grant AI23081 and by the Howard Hughes Medical Institute. E.A.H. is supported by the NIH Medical Scientist Training Program. References 1. Williams DB, Watts TH: Molecular chaperones in antigen presentation. Curr Opin Immunol 1995, 7: Sadasivan B, Lehner PJ, Ortmann B, Spies T, Cresswell P: Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. Immunity 1996, 5: Ortmann B, Copeman J, Lehner PJ, Sadasivan B, Herbert JA, Grandea AG, et al.: A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes. Science 1997, 277: Lehner PJ, Surman MJ, Cresswell P: Soluble tapasin restores MHC class I expression and function in the tapasin negative cell line.220. Immunity 1998, 8: Androlewicz MJ, Cresswell P: How selective is the transporter associated with antigen processing? Immunity 1996, 5: Mobbs CV, Kaplitt M, Pfaff D: H-70/GRP58/ERp61/PLC-a/CPT: a single gene product and member of the protein disulfide isomerase gene family with thiol oxidoreductase activity subject to neuroendocrine regulation. In Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other Structurally Related Proteins. Edited by Guzman NA. New York: Marcel Dekker Inc; 1997: Bennett CF, Balcarek JM, Varrichio A, Crooke ST: Molecular cloning and complete amino-acid sequence of form-i phosphoinositidespecific phospholipase C. Nature 1988, 334: Oliver JD, van der Wal FJ, Bulleid NJ, High S: Interaction of the thioldependent reductase with nascant glycoproteins. Science 1997, 275: Elliott JG, Oliver SD, High S: The thiol-dependent reductase interacts specifically with N-glycosylated integral membrane proteins. J Biol Chem 1997, 272: Helenius A, Trombetta ES, Hebert DN, Simons JF: Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol 1997, 7: Urade R, Nasu M, Moriyama T, Wada K, Kito M: Protein degradation by the phosphoinositide-specific phospholipase c-a family from rat liver endoplasmic reticulum. J Biol Chem 1992, 267: Otsu M, Urade R, Kito M, Omura F, Kikuchi M: A possible role of ER- 60 protease in the degradation of misfolded proteins in the endoplasmic reticulum. J Biol Chem 1995, 270: Srivastava SP, Fuchs JA, Holtzman JL: The reported cdna sequence for phospholipase Ca encodes protein disulfide isomerase, isozyme Q-2 and not phospholipase-c. Biochem Biophys Res Comm 1993, 193: Hirano N, Shibasaki F, Sakai R, Tanaka T, Nishida J, Yazaki Y, et al.: Molecular cloning of the human glucose-regulated protein /GRP58, a thiol-dependent reductase. Identification of its secretory form and inducible expression by the oncogenic transformation. Eur J Biochem 1995, 234: Srivastava SP, Chen N-Q, Liu Y-X, Holtzman JL: Purification and characterization of a new isozyme of thiol:protein-disulfide oxidoreductase from rat hepatic microsomes. J Biol Chem 1991, 266: Ortmann B, Androlewicz M, Cresswell P: MHC class I/b 2 - microglobulin complexes associate with TAP transporters before peptide binding. Nature 1994, 368: Peterson JR, Ora A, Van PN, Helenius A: Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins. Mol Biol Cell 1995, 6: Tector M, Salter RD: Calnexin influences folding of human class I histocompatibility proteins but not their assembly with b 2 - microglobulin. J Biol Chem 1995, 270: Tector M, Zhang Q, Salter RD: b 2 -microglobulin and calnexin can independently promote folding and disulfide bond formation in class I histocompatibility proteins. Mol Immunol 1997, 34: Van Leeuwen JEM, Kearse KP: Deglucosylation of N-linked glycans is an important step in the dissociation of calreticulin-class I-TAP complexes. Proc Natl Acad Sci USA 1996, 93: Snyder HL, Yewdell JW, Bennink JR: Trimming of antigenic peptides in an early secretory compartment. J Exp Med 1994, 180: Hughes EA, Ortmann B, Surman M, Cresswell P: The protease inhibitor, N-actyl-L-leucyl-L-leucyl-L-norleucinal, decreases the pool of major histocompatibility complex class I-binding peptides and inhibits peptide trimming in the endoplasmic reticulum. J Exp Med 1996, 183: Hughes EA, Hammond C, Cresswell P: Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome. Proc Natl Acad Sci USA 1997, 94: Cresswell P, Hughes EA: Protein degradation: the ins and outs of the matter. Curr Biol 1997, 7: Huppa JB, Ploegh HL: The a chain of the T cell antigen receptor is degraded in the cytosol. Immunity 1997, 7:

5 Supplementary material S1 The thiol oxidoreductase is a component of the MHC class I peptide-loading complex Eric A. Hughes and Peter Cresswell 25 May 1998, 8: Materials and methods Cell lines The highly γ-interferon-inducible HeLa derivative HeLa M was a gift from P. Lengyel. The B-cell line 45.1 and its TAP-negative derivative,.174, have previously been described [S1]. The Daudi cell line [S2] transfected with β 2 m and Daudi cells mock-transfected with vector only were kindly supplied by R. Salter. The tapasin-deficient cell line.220-b8, was generously provided by T. Spies and the subsequent reconstitution of this cell with tapasin has previously been described [3]. Antibodies Rabbit anti-calreticulin serum and the anti-grp94 (GP96) rat monoclonal antibody (clone 9G10) were purchased from Affinity Bioreagents. Peptide-specific anti-, anti-calreticulin and anti-tapasin rabbit sera were generated by immunization with the synthetic peptides (in single-letter amino-acid code) CATNPPVIQEEKPKKKKKAQ, CDKEEDEEEDVPGQAKDEL and CLGWAAVYLSTCKDSKKKAE, respectively, conjugated to KLH and purified on peptide affinity columns. The monoclonal antibody and the purified rabbit serum R.RING.4C, (anti-), the mouse monoclonal antibody HC10 and rat monoclonal antibody 3B10.7 (anti-hla class I heavy chain) and the mouse monoclonal antibody BBM.1 (anti-) have previously been described [16,S3 S6]. Rabbit anti- serum was purchased from Boehringer Mannheim. Metabolic labeling HeLa M cells ( ) treated with 200 U/ml of interferon-γ for 48 h or untreated.174 cells were incubated in methionine-free medium containing 5% dialyzed FCS (Hyclone) for 1 h at 37 C. HeLa M was labeled with 2.0 mci [ 35 S]methionine (ICN Biochemicals) for 2.5 h in fresh methionine-free medium, while.174 was labeled with 2.0 mci for 3.0 h and chased with a 15-fold excess of unlabeled methionine for 0.5 h. Immunoprecipitations Cells were pelleted and lysed in 10 mm Tris, 150 mm NaCl (TBS), ph 7.4, 1% digitonin (Wako Pure Chemical Industries), containing protease inhibitors, and post-nuclear supernatants were pre-cleared for 1 h with normal rabbit serum (NRS) or the monoclonal antibody 9E10 and protein-g Sepharose and immunoprecipitation performed as previously described [2]. To detect associated proteins by blot, the primary immunoprecipitate was washed in 0.1% digitonin/tbs three times and incubated in 1% DOC/TBS/pH 8.2 for 0.5 h. The eluted material was separated by SDS PAGE and blotted as described below. To detect -associated sub-complexes, the primary immunoprecipitate was incubated in 1% Triton X-100/TBS for 1 h at 4 C. A second immunoprecipitation was performed on the Triton X- 100 supernatant and the associated proteins eluted in 1% DOC/TBS/pH 8.2, separated by SDS PAGE and blotted as described below. To identify TAP-associated proteins or -associated proteins in labeled cells, the primary immunoprecipitates were washed three times and heated at 100 C for 5 min in 2% SDS, 2 mm DTT in TBS, diluted 10-fold in 1% Triton X-100 in TBS with 10 mm iodoacetamide, and allowed to incubate at room temperature for 30 min. The released proteins were pre-cleared again with NRS and protein-g Sepharose, followed by immunoprecipitation with the appropriate antibody and protein-a Sepharose. (N-succinimidyl propionate; DSP; Pierce) for 0.5 h at 4 C. The crosslinking was stopped with 10 µm glycine. The post-nuclear supernatant was adjusted to 2% SDS, heated to 100 C for 5 min, and diluted 10- fold in 1% Triton X-100/PBS prior to immunoprecipitation and SDS PAGE. Endoglycosidase H (Boehringer-Mannheim) digestions were performed as described previously [S7]. Immunoblots Western blots to detect protein in whole cell lysates or protein eluted in 1% DOC were performed as described [S8]. The Immobilon P membrane (Millipore) was blocked for 1 h in PBS containing 0.05% Tween 20 and 4% dehydrated milk (Blotto), rinsed in PBS, and incubated overnight at 4 C with a 2.0 µg/ml solution of purified rabbit anti-, a 2.0 µg/ml solution of purified rabbit anti-calreticulin peptide antibody, or a 1:1 dilution of 3B10.7 hybridoma supernatant in Blotto. Bands were visualized with horseradish-peroxidase-conjugated secondary goat anti-rabbit IgG (F(ab ) 2 fragment-specific antibody) or horseradish peroxidase-conjugated secondary mouse anti-rat IgG (F(ab ) 2 fragment-specific antibody; Jackson Immunoresearch Laboratories) and chemiluminescent substrate (Pierce). Exposure times were typically sec. References S1. DeMars R, Rudersdorf R, Chang C, Petersen J, Strandtmann J, Korn N, et al.: Mutations that impair a post-transcriptional step in expression of HLA-A and -B antigens. Proc Natl Acad Sci USA 1985, 82: S2. Sege K, Rask L, Peterson PA: Role of b 2 -microglobulin in the intracellular processing of HLA antigens. Biochemistry 1981, 20: S3. Meyer TH, Van Endert PM, Uebel S, Ehring B, Tampé R: Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells. FEBS Lett 1994, 351: S4. Stam NJ, Spits H, Ploegh HL: Monoclonal antibodies raised against denatured HLA-B locus H-chains permit biochemical characterization of certain HLA-C locus products. J Immunol 1986, 137: S5. Lutz PM, Cresswell P: An epitope common to HLA class I and class II antigens, Ig light chains, and b 2 microglobulin. Immunogenetics 1987, 25: S6. Brodsky FM, Bodmer WF, Parham P: Characterization of a monoclonal anti-b 2 -microglobulin antibody and its use in the genetic and biochemical analysis of major histocompatibility antigens. Eur J Immunol 1979, 9: S7. Salter RD, Cresswell P: Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J 1986, 5: S8. Anderson KS, Cresswell P: A role for calnexin (90) in the assembly of class II MHC molecules. EMBO J 1994, 13: Cross-linking and carbohydrate cleavage To demonstrate association with MHC class I, 45.1 cells were lysed in 1% digitonin/pbs/ph 7.4 containing 800 µm dithiobis