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Vol. 20, Issue 21, 4552-4562, November 1, 2009
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*Department of Biological Responses, Institute for Virus Research, and Innovative Techno-Hub for Integrated Medical Bio-imaging, Department of Radiation Oncology and Image-Applied Therapy, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Submitted June 1, 2009;
Revised August 10, 2009;
Accepted August 28, 2009
Monitoring Editor: Ramanujan S. Hegde
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The formation of disulfide bonds between cysteine residues is critical for the proper folding and assembly of proteins entering the secretory pathway (Sevier and Kaiser, 2006). The ER contains several oxidoreductases classified in the thioredoxin superfamily that catalyze the formation and rearrangement of disulfide bonds. Thioredoxin has been demonstrated to catalyze the reduction of disulfide bonds and thereby participate in many thiol-dependent cellular processes (Nakamura, 2005). Most of the ER oxidoreductases contain thioredoxin-like domains with their characteristic CXXC active site motifs that are responsible for catalyzing thiol-disulfide exchange reactions (Ellgaard and Ruddock, 2005).
Although many ER oxidoreductases, such as protein disulfide isomerase (PDI) and ERp57, are soluble proteins in the ER lumen, there also exist several membrane-bound oxidoreductases. In a search for genes induced by transforming growth factor-β, we identified a member of the thioredoxin family, transmembrane thioredoxin-related protein (TMX) (Akiyama et al., 2000; Matsuo et al., 2001). TMX contains one catalytic thioredoxin-like domain with a unique active site motif, CPAC, and a single transmembrane region. TMX orthologues have been found in other animal species, including mammals, Drosophila melanogaster, and Caenorhabditis elegans (Ko and Chow, 2002) but not in plants, fungi, or prokaryotes. The thioredoxin-like domain of TMX is present in the ER lumen and shows reductase and isomerase activity in vitro (Matsuo et al., 2004). TMX2 and TMX3 have also been reported in the literature as human transmembrane oxidoreductases (Meng et al., 2003; Haugstetter et al., 2005). The physiological roles of these TMX proteins still remain unclear, but, considering their structural characteristics, these TMX proteins might preferentially act on membrane-anchored substrates.
The presence of a large variety of thioredoxin family members suggests a complex mechanism of redox regulation in the mammalian ER. Whether the family members are functionally redundant or each plays a separate and distinct role remains to be elucidated. For a better understanding of their mechanisms of action, it is necessary to determine the cellular substrates and binding partners for the individual proteins. In the present study, we applied a substrate-trapping approach based on the reaction mechanism of thiol-disulfide exchange. Using a trapping mutant of TMX that enables the stabilization of the mixed disulfide intermediates between the enzyme and substrate, we identify major histocompatibility complex (MHC) class I heavy chain (HC) as a target molecule for TMX. As shown in previous studies, ER oxidoreductases such as ERp57 and PDI associate with MHC class I molecules during biosynthesis (Hughes and Cresswell, 1998; Antoniou et al., 2003), and they play critical roles in the assembly of the peptide-loading complex (Garbi et al., 2006; Park et al., 2006; Kienast et al., 2007). Unlike these oxidoreductases in the ER lumen, TMX did not affect the surface expression of class I molecules, but we found that TMX prevents the retrotranslocation of class I heavy chain for proteasomal degradation, suggesting a specific role for TMX in the retention and refolding of misfolded proteins in the ER.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mouse monoclonal antibodies to calnexin (AF8) (David et al., 1993) and MHC class I heavy chain (HC10) (Stam et al., 1986) were generously provided by Dr. Masahiko Sugita (Kyoto University, Kyoto, Japan). Rabbit polyclonal antibodies specific for calreticulin (SPA-600), calnexin (SPA-865), and PDI (SPA-890) were from Assay Designs (Ann Arbor, MI). Peroxidase-conjugated mouse monoclonal antibodies to myc or V5 were from Nacalai Tesque (Kyoto, Japan), and anti-myc agarose (9E10) was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-BiP was from BD Biosciences (San Jose, CA), mouse monoclonal anti-
-tubulin was from Sigma-Aldrich (St. Louis, MO), and fluorescein isothiocyanate (FITC)-labeled anti-human MHC class I (W6/32) was from eBioscience (San Diego, CA). Mouse anti-thioredoxin monoclonal antibody (mAb) (ADF11) was described previously (Kogaki et al., 1996).
Expression Vectors
TMX cDNA was subcloned into pEF6/Myc-His A (Invitrogen, Carlsbad, CA) for the selection of stable cell lines with blasticidin. To construct TMX/C59A, TMX/C56A·C59A, and TMX/P101T, substitution mutations were introduced by polymerase chain reaction (PCR) by using the following primers: C59A, F1 and R2; C56A·C59A, F3 and R4; and P101T, F5 and R6. PCR products encoding TMX140 (primer set: F7 and R8), TMX180 (F7 and R9), or TMX215 (F7 and R10) were amplified from the TMX cDNA and inserted into pcDNA3.1/MycHis A vector (Invitrogen). For the expression of soluble TMX, an ER retention motif (KDEL) was introduced by PCR at the C terminus of TMX140-myc with primers F11 and R12, and the PCR products were cloned into pcDNA3.1(–) (Invitrogen). For the inducible expression of TMX/P101T-myc, the cDNA was subcloned into the tetracycline-regulated expression vector pTRE2pur (Clontech, Mountain View, CA). The coding sequences of human Ero1 and Ero1β were amplified by PCR using the first-strand cDNAs prepared from A549 cells (primers used were as follows: Ero1, F13 and R14; and Ero1β, F15 and R16). V5-tagged Ero1s were generated by inserting the PCR products into pcDNA3.1/V5-His vector (Invitrogen). Human leukocyte antigen B27 (HLA-B27) cDNA in pSR-neo was a kind gift from Dr. Masahiko Sugita (Kyoto University, Kyoto, Japan) (Sugita and Brenner, 1995). The full-length HLA-B27 coding sequence was amplified by PCR and inserted into pcDNA3.1/V5-His vector to add a V5 tag at the C terminus. Plasmids were transfected into cells with FuGENE6 (Roche Diagnostics, Indianapolis, IN) or Lipofectamine 2000 (Invitrogen) for transient expression. The sequences of the primers used are given in Supplemental Table S1.
Cell Culture
Human lung adenocarcinoma cell line A549 (JCRB0076) and human embryonic kidney cell line 293 (JCRB9068) were obtained from Health Science Research Resources Bank (Osaka, Japan). A549, 293, and HeLa cells were maintained in DMEM (Sigma-Aldrich) supplemented with 10% fetal calf serum (Invitrogen), and human T cell leukemia line Jurkat was cultured in RPMI-1640 medium (Sigma-Aldrich) with 10% fetal calf serum. To induce the unfolded protein response, cells were treated with brefeldin A, thapsigargin, and tunicamycin from Nacalai Tesque for the indicated periods. To generate cells stably expressing TMX-myc, pEF6-TMX-myc was transfected into A549 cells by using the Nucleofector system (Amaxa Biosystems, Gaithersburg, MD). Cells were cultured in selective medium containing 2 µg/ml blasticidin (InvivoGen, San Diego, CA), and resistant colonies were picked and cultured. For the inducible expression of the trapping mutant, pTRE2pur-TMX/P101T-myc was transfected into T-REx 293 cells (Invitrogen) stably expressing the tetracycline repressor using FuGENE6 transfection reagent (Roche Diagnostics). A clonal cell line was established by selection with 0.5 µg/ml puromycin (InvivoGen) and 100 µg/ml G418 (Nacalai Tesque).
Determination of the Redox State
Cells were incubated for 10 min at 37°C with or without 0.5 mM 4,4'-dipyridyl disulfide (DPS; Nacalai Tesque) or 5 mM dithiothreitol (DTT; Nacalai Tesque), washed with ice-cold phosphate-buffered saline (PBS) containing 20 mM N-ethylmaleimide (NEM; Nacalai Tesque) immediately or after incubation in fresh medium to alkylate free thiols. The cells were lysed in 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 20 mM NEM, and 0.5% NP-40, and the lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. The redox state of TMX was determined by monitoring the mobility shift of oxidized versus reduced proteins in the immunoblot analysis. In an alternative method, after acid denaturation by trichloroacetic acid (TCA) proteins with free thiol groups were modified with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) (Kobayashi et al., 1997). In brief, cells were treated with 10% TCA in PBS for 15 min and then lysed in 50 mM Tris-HCl, pH 6.8, 1% SDS, and 1 mM AMS. The samples were incubated for 30 min at room temperature and subjected to nonreducing SDS-PAGE followed by immunoblotting.
RNA Interference
For suppression of the endogenous TMX gene, the following synthetic oligonucleotides containing a 19-nt target sequence of human TMX (GGAGACTGGATGATAGAAT) were annealed and inserted into pSUPER RNAi vector (Oligoengine, Seattle, WA): 5'-GATCCCCGGAGACTGGATGATAGAATTTCAAGAGAATTCTATCATCCAGTCTCCTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAGGAGACTGGATGATAGAATTCTCTTGAAATTCTATCATCCAGTCTCCGGG-3'. Either the resulting plasmid pSUPER-TMX or control pSUPER vector was transfected into HeLa cells together with pUCDV-BSD (Kaken Pharmaceutical, Tokyo, Japan)) containing the blasticidin resistance marker. Transfection was performed using the Nucleofector system (Amaxa). The cells were cultured in selective medium containing 2 µg/ml blasticidin, and resistant colonies were isolated. The level of TMX gene knockdown was evaluated by immunoblot analysis with anti-TMX antibody.
Measurement of Cell Viability
Cell viability was measured using cell count reagent SF containing the tetrazolium salt WST-8 (Nacalai Tesque) following the manufacturer's instructions. Each sample was assayed in triplicate, and the experiments were repeated twice.
Immunoprecipitation and Immunoblotting
Cells expressing TMX-myc were incubated in PBS containing 20 mM NEM for 10 min on ice to preserve mixed disulfides. Cells were then lysed in 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 20 mM NEM, and 0.5% NP-40, and insoluble materials were precipitated by centrifugation at 20,000 x g for 10 min. The resulting supernatants were subjected to immunoprecipitation with anti-myc agarose. Immunoprecipitates were washed with lysis buffer and eluted in buffer containing 150 µg/ml c-myc peptide (Sigma-Aldrich). For myc/calnexin sequential immunoprecipitation, the myc peptide eluates were diluted in lysis buffer and further immunoprecipitated using anti-calnexin antibody. In immunoprecipitation experiments using anti-calnexin or anti-MHC class I heavy chain, immune complexes were recovered with protein A-Sepharose and eluted with SDS-PAGE sample buffer. Samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane Immobilon-P (Millipore, Billerica, MA) followed by blocking in Tris-buffered saline/Tween 20 containing 0.5% skim milk (Nacalai Tesque). Immunoblots were probed with the indicated antibodies, and immunodetection was carried out using enhanced chemiluminescence reagents (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom).
Two-dimensional Gel Electrophoresis
Immunoprecipitates were separated by SDS-PAGE under nonreducing conditions. The excised gel strip was incubated in SDS sample buffer containing 50 mM DTT for 30 min and placed horizontally on the top of a new SDS-PAGE gel. After separation on the second gel, proteins were transferred to the membrane and analyzed by immunoblotting.
Flow Cytometry
Cells were detached with enzyme-free cell dissociation buffer (Invitrogen) and stained with FITC-labeled mAb W6/32 for 20 min at 4°C. Cells were analyzed using a FACSCalibur (BD Biosciences) and Flowjo software (TreeStar, Ashland, OR).
Subcellular Fractionation
Cells were fractionated into cytosolic and membrane fractions using ProteoExtract subcellular proteome extraction kit (EMD Biosciences, San Diego, CA). For assessing the translocation of MHC class I heavy chain, cells were transfected with HLA-B27-V5. Forty-eight hours after transfection, cells were incubated with or without 10 µM MG-132 (Peptide Institute, Osaka, Japan) for 4 h at 37°C and then fractionated into cytosolic and membrane fractions. The resulting samples were separated by SDS-PAGE followed by immunoblotting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 293 cells were treated with trichloroacetic acid to denature and precipitate whole cell proteins, and the precipitates were dissolved in buffer containing alkylating agent AMS, which modifies free thiol groups. Covalent modification with AMS increases the molecular mass by 
0.54 kDa per thiol group, thereby causing a shift in mobility upon separation by SDS-PAGE, and reduced (AMS-modified) proteins migrate slower than oxidized forms. As shown in Figure 1B, the majority of TMX migrated with the mobility of the reduced protein. TMX contains seven cysteines, two of which constitute the active site. To test whether the mobility shift after modification with AMS reflects alkylation of the active site cysteines, we examined the redox state of exogenously expressed wild-type TMX and its mutant, in which two cysteines within the active site were replaced with serines. In cells transfected with wild-type TMX, two bands corresponding to reduced and oxidized proteins were detected (Figure 1C, lane 1), which was consistent with the redox properties of endogenous TMX. In the case of the cysteine mutant, however, we could only see a single band of the protein irrespective of DTT pretreatment (Figure 1C, lanes 3 and 4). Thus, the decreased mobility after AMS modification truly indicated that the active site was in the reduced state.
In the ER, there exist cellular mechanisms controlling the redox state of the oxidoreductases. In mammalian cells, glutathione, a low-molecular-weight thiol compound, has been shown to directly reduce an oxidoreductase and maintain the protein in a reduced form (Jessop and Bulleid, 2004). In comparison, Ero1 has been shown to reoxidize PDI for further catalytic cycles in an oxidative folding pathway (Frand and Kaiser, 1999; Mezghrani et al., 2001). Considering that TMX is partially oxidized in cultured human cells, we investigated the redox state of TMX in cells overexpressing either human Ero1 (Cabibbo et al., 2000) or Ero1β (Pagani et al., 2000). Unlike the Ero1-PDI pathway, both Ero1 and Ero1 β could not alter the redox state of TMX (Figure 1D), suggesting that TMX seemed not to be a substrate for Ero1. Recovery from protein oxidation by DPS also was examined. In Figure 1E, DPS-treated Jurkat cells were further incubated in the absence of DPS, and the shift of the redox state was monitored. Three hours after removal of the oxidizing agent, we observed that some proteins were restored to the reduced forms, which were not detectable immediately after DPS treatment (Figure 1E, lane 4). Thus, TMX seemed to be maintained in a reduced state with exposed cysteine residues, which may be involved in the reduction/isomerization of the target molecules.
TMX Expression Is Not Induced by the Unfolded Protein Response
Cells respond to the accumulation of unfolded proteins in the ER by activating an intracellular signaling pathway called the unfolded protein response (UPR) (Ron and Walter, 2007). Activation of the UPR promotes the expression of molecular chaperones and folding catalysts such as BiP and PDI. We tested whether the expression of TMX was up-regulated by cellular stresses that induce the UPR. A549 cells were treated with brefeldin A, thapsigargin, and tunicamycin. These agents induced the UPR, as indicated by the up-regulation of the UPR marker BiP (Figure 2). Under these stress conditions, we observed no significant increase in TMX protein expression, suggesting that TMX gene may not be directly regulated by the UPR. Similar results were obtained in 293 cells (data not shown).
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-tubulin (Figure 3A, third panel). TMX down-regulation did not affect the expression of BiP and another oxidoreductase PDI (Figure 3A, top and second panels, respectively). The unaltered expression of these UPR markers indicated that, under normal culture conditions, TMX knockdown did not cause severe ER dysfunction, leading to the activation of the UPR. When cells were treated with ER stress-inducing reagent tunicamycin, the early stress response of the TMX knockdown cells seemed to be normal, because the induction of BiP after tunicamycin treatment was unaffected (Figure 3B). After longer incubation, however, down-regulation of TMX rendered cells more sensitive to tunicamycin, resulting in decreased cell viability (Figure 3C), suggesting the protective role of TMX under stress conditions in these cells.
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). The interaction was rather specific for calnexin, as calreticulin, a soluble homologue of calnexin in the ER lumen, was not coprecipitated with TMX-myc (Figure 4A, middle).
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). The KDEL sequence prevented the secretion of the protein, and after subcellular fractionation TMX140-mycKDEL was found in the membrane fraction containing the ER (Figure 4D, left) exactly like endogenous TMX. The purity of the membrane fraction was confirmed by the absence of a cytosolic marker thioredoxin (Figure 4D, right). Using these TMX variants, we tested each for binding to calnexin. Although TMX without its cytoplasmic segments (TMX215-myc) was still capable of binding calnexin (Figure 4E, lane 3), no interaction could be detected between soluble TMX and calnexin (Figure 4E, lane 4), suggesting that the transmembrane domain is responsible for their association. We also examined whether the CXXC motif of TMX was involved in calnexin binding. Substitution of the two cysteines within the active site with alanine residues (C56A·C59A) did not alter the interaction with calnexin (Figure 4E, lane 6), demonstrating that an intact CXXC motif was not required for TMX to associate with calnexin. Although the C56A·C59A mutant retained the ability to interact with calnexin, cysteine-to-serine mutation (C56S·C59S) significantly reduced the binding affinity for calnexin (Figure 4E, lane 5). Potentially, the substitution to polar serine residues with hydroxyl groups induced conformational change in the protein and affected the stability of TMX-calnexin complex. Thus, TMX specifically interacts with calnexin despite the lack of N-linked glycans suggesting their cooperative behavior in the protein folding process in the ER.
Mutation of Cys59 or Pro101 of TMX Leads to the Accumulation of Disulfide-linked Complexes
During the thiol-disulfide exchange reactions, it is thought that a transient mixed disulfide bond is formed between an oxidoreductase and its substrate. To investigate whether TMX can participate in the formation of mixed disulfides with other proteins, cells transfected with myc-tagged TMX were treated with NEM to trap disulfide-linked intermediates and analyzed by immunoblotting under nonreducing conditions. Only the monomeric TMX-myc was observed, and we failed to detect any mixed disulfides (Figure 5A, lane 1). Because such transient mixed disulfides tend to be unstable, we attempted to stabilize the complex by mutating a conserved amino acid residue within the thioredoxin-like domain. We found that mutating the C-terminal cysteine of the CXXC motif to alanine (C59A) successfully led to the accumulation of multiple bands reacting with the myc antibody under nonreducing conditions (Figure 5A, lane 2). These bands disappeared when the samples were treated with reducing agents (Figure 5A, lane 5), indicating that they represent disulfide-linked complexes containing TMX-myc. It has been reported that conversion of the cis-proline of DsbA, an Escherichia coli oxidase in the periplasm, resulted in the accumulation of disulfide-linked DsbA-substrate complexes (Kadokura et al., 2004). The corresponding proline residue is also conserved among mammalian thioredoxin family members including TMX (Supplemental Figure S1), and mutation of Pro101 of TMX to threonine (P101T) had an effect in detecting mixed disulfides (Figure 5A, lane 3). Thus, these mutants could be useful for stabilizing mixed disulfides formed during the thiol-disulfide exchange, leading to identification of the substrates of TMX.
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). We established 293 cells expressing the TMX/P101T trapping mutant under the control of a tetracycline-inducible promoter. After treatment with doxycycline at 0.4 µg/ml, TMX/P101T-myc was expressed at levels similar to those of endogenous TMX (Figure 5B, top). The cells were pretreated with increasing doses of CST and then cultured in the presence of doxycycline to induce the expression of TMX/P101T-myc. Cell lysates were separated by SDS-PAGE under reducing and nonreducing conditions and immunoblotted with anti-myc antibodies. Treatment with CST reduced the formation of mixed disulfides (Figure 5B, bottom), suggesting that some substrates of TMX contain monoglucosylated N-glycans and they might be recognized by TMX through the cooperative interaction with calnexin. The presence of TMX–substrate complexes in anti-calnexin immunoprecipitates supported this possibility. As shown in Figure 5C, several high molecular weight complexes containing TMX/P101T-myc were observed in anti-calnexin immunoprecipitates under nonreducing conditions (lane 5). These were disrupted upon treatment with DTT (lane 2), or by mutating both of the two active site cysteines of TMX (lane 6), indicating that they are TMX-substrate conjugates. Similar results were obtained in the experiments using C59A trapping mutant (Supplemental Figure S2). The only exception was a band of 75 kDa. The band was susceptible to reduction by DTT, but under nonreducing conditions it was also present with TMX/C56A·C59A mutant containing no active site cysteines (lane 6, asterisk). Although its identity is still unclear, the 75 kDa band, judging from the size, most likely represents a disulfide-linked dimer of TMX-myc, and noncatalytic cysteines might contribute to dimerization. It should be noted that even in the presence of CST, mixed disulfide formation was not completely blocked, and we could still detect TMX–substrate complexes (Figure 5B), indicating that the association of TMX with several substrates was not dependent on glucose trimming.
TMX Forms a Mixed Disulfide with MHC Class I Heavy Chain
Given the association of TMX with calnexin, we next tested the possibility that a membrane protein recognized by calnexin could be a target of TMX. We found that endogenous MHC class I HC was coprecipitated by anti-myc antibodies from cells expressing myc-tagged TMX/C59A (Figure 6A, lane 2). Integrin β1, another substrate for calnexin (Lenter and Vestweber, 1994), was not coprecipitated with TMX (data not shown), indicating the specific interaction between TMX and class I HC. It is noted that under nonreducing conditions bands of 80 kDa were detected with anti-class I HC in immunoprecipitates from C59A-expressing cells (Figure 6A, lane 6). The molecular weight of the bands was consistent with a complex of TMX-myc (37 kDa) and class I HC (43 kDa), and they were susceptible to reduction with DTT, suggesting that the trapping mutant forms a disulfide-linked dimer with endogenous class I HC. TMX/P101T trapping mutant could also form a mixed disulfide with class I HC (Supplemental Figure S3A), but wild-type TMX-myc and C56A·C59A mutant did not capture any mixed disulfides (Figure 6A, lanes 5 and 7). Similar results were obtained when the immunoprecipitation was performed with anti-class I HC followed by immunoblotting with anti-myc (Supplemental Figure S3B).
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Oxidative Folding of Class I Heavy Chain Is Not Affected by TMX Gene Knockdown
To elucidate the functional significance of the interaction between TMX and MHC class I HC, we first examined the effects of TMX down-regulation on the expression of class I HC. Cell lysates prepared from control or TMX knockdown cells were analyzed by immunoblotting to quantify the total amount of class I HC. As shown in Figure 7A, there was no significant difference in the expression level of the class I HC between these cells. Furthermore, in the steady state, TMX gene knockdown did not substantially affect the surface expression of MHC class I molecules as quantified by flow cytometry (Figure 7B).
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). During maturation, some disulfides are buried in the folded structure and become insensitive to reducing agents unless the proteins are denatured (Braakman et al., 1992; Tatu et al., 1993). To analyze oxidative folding of class I HC, we assessed DTT sensitivity of HLA-B27 expressed in control and TMX knockdown cells (Figure 7C). Cells transfected with V5-tagged HLA-B27 were incubated in medium containing 5 mM DTT for 5 min. After washing out DTT, cell extracts were prepared, separated by nonreducing SDS-PAGE, and analyzed by immunoblotting with anti-V5 antibody. Although a small fraction of HLA-B27-V5 was reduced by DTT treatment (indicated by closed arrowhead), most of the proteins acquired a conformation resistant to DTT reduction both in the control and TMX knockdown cells (Figure 7C, lanes 2 and 4, open arrowhead). Thus, down-regulation of TMX did not affect the conversion of class I HC from DTT-sensitive to DTT-resistant form, and oxidative folding of class I HC seemed to proceed normally.
Tunicamycin Treatment Promotes the Formation of TMX–Class I Heavy Chain Complex
Next, we examined the requirement for N-linked glycans of MHC class I HC in its association with TMX. A549 cells stably expressing TMX-myc were cultured in the presence or absence of tunicamycin, a potent inhibitor of protein glycosylation. In tunicamycin-treated cells, nonglycosylated class I HC additionally appeared as a faster migrating species (Figure 8A, lane 2, open arrowhead). We noted that class I HC was not obviously detectable in the anti-myc immunoprecipitates from unstressed A549 cells (Figure 8A, lane 3). Interestingly, the amount of coprecipitated class I HC apparently increased in tunicamycin-treated cells (Figure 8A, lane 4). Furthermore, only the nonglycosylated forms of class I HC were coprecipitated, as judged by their mobility on SDS-PAGE, whereas no interaction was observed with the glycosylated proteins. Because TMX trapping mutants could recognize glycosylated class I HC (Figure 6), TMX seemed not to monitor the glycosylation status of class I HC. This led us to suspect that TMX might preferentially bind the incompletely folded proteins produced under conditions of ER stress.
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TMX Prevents the Degradation of HLA-B27
During conditions of stress, proteins are unable to adopt their native conformation, and terminally misfolded proteins are degraded by a process referred to as ER-associated degradation (Meusser et al., 2005). Misfolded MHC class I molecules are retrotranslocated from the ER to the cytosol where they are deglycosylated and degraded by proteasomes. Previous studies have shown that when proteasomal proteolysis is inhibited characteristic deglycosylated forms of class I HC accumulate in the cytosol (Wiertz et al., 1996; Hughes et al., 1997). To study the degradation process of class I HC, 293 cells transfected with V5-tagged HLA-B27, which is prone to misfold in the ER (Colbert, 2000), were fractionated into cytosolic and membrane fractions. We observed the accumulation of cytosolic HLA-B27 derivatives only in the presence of proteasome inhibitor MG-132, which seemed to be the deglycosylated degradation intermediates as judged by their smaller size (Figure 8C, top, lane 3). Calnexin was absent from the cytosolic fraction, which demonstrates the efficient separation of cytosolic and membrane components (Figure 8C, bottom). These results indicate that a portion of the exogenous HLA-B27 was targeted for proteasomal degradation and transported to the cytosol.
Using this assay system, we investigated whether TMX is involved in the mechanism that regulates the degradation of class I HC. We first tested whether TMX can interact with V5-tagged HLA-B27. Endogenous TMX was coprecipitated by anti-V5 antibody from cells expressing HLA-B27-V5 (Figure 8D, lane 4, arrowhead), but not from mock-transfected cells (lane 3), demonstrating the interaction between TMX and HLA-B27. In Figure 8E, HLA-B27-V5 was cotransfected either with TMX-myc or an empty vector into 293 cells, and after incubation with MG-132, cytosolic and membrane fractions were prepared. We found a decreased level of cytosolic deglycosylated HLA-B27 in cells expressing TMX-myc compared with vector control cells (Figure 8E, top, compare lane 1 to lane 2; the protein band intensities were quantified as shown in the graph), indicating that coexpression of TMX prevented the translocation of HLA-B27. TMX-myc was found only in the membrane fraction, and the ER-to-cytosol translocation of TMX itself was not detectable (Figure 8E, middle). Equal protein loading was confirmed by Coomassie blue staining of the gel (Figure 8E, bottom).
Next we assessed the effect of TMX down-regulation on the degradation process of class I HC. HLA-B27-V5 was transfected into control or TMX knockdown cells, and the translocation of HLA-B27 was examined. In the presence of MG-132, deglycosylated forms of HLA-B27 in the cytosol were increased in the two independent TMX knockdown clones compared with control cells expressing endogenous TMX (Figure 8F, compare lane 4 with lanes 5 and 6). Thus, the depletion of TMX enhanced the rate of degradation of HLA-B27, suggesting that TMX acts to retain MHC class I heavy chain in the ER and thereby protects the protein from degradation.
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DISCUSSION |
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; Jessop and Bulleid, 2004). We also found that a significant fraction of TMX was oxidized in some cell lines. Although it is uncertain whether the oxidized form is biologically active, TMX may have a physiological role as an oxidase like PDI. Alternatively, the oxidized TMX might be a reaction intermediate, which would subsequently be reduced. ERp57 is maintained in a reduced state, and after treatment with oxidizing agent it was restored to the reduced form very rapidly (Jessop and Bulleid, 2004). In contrast, the recovery of reduced TMX was relatively slow, and oxidized TMX was still present three hours after removal of the oxidizing agent (Figure 1E). Therefore, the delay in the recovery from protein oxidation may provide an explanation for TMX seeming to be partially oxidized.
The diversity among thioredoxin family members in the ER may reflect their functional specialization. To determine whether these redox proteins share overlapping biological activities or they have separate and distinct functions, it is required to identify binding partners and substrate proteins for each oxidoreductase. PDI can interact directly with substrate proteins, and its b' domain is known to be the primary peptide binding site (Klappa et al., 1998). ERp57, the closest homologue of PDI, has been shown to act as a glycoprotein-specific oxidoreductase through the interaction with the lectin-like chaperones calnexin and calreticulin (Molinari and Helenius, 1999; Oliver et al., 1999; Jessop et al., 2009). We found that TMX bound to calnexin. Given that the luminal part of TMX lacks a b'-like peptide binding domain and CST treatment partially inhibited the formation of mixed disulfides, TMX might require calnexin to recruit a portion of its substrates. Another issue to be considered is how TMX is retained in the ER. TMX lacks known ER retention signal like C-terminal dilysine motif (Jackson et al., 1990), and no specific residues or sequence motifs required for its proper localization have been identified. The association with calnexin may be important for retaining TMX on the ER membrane.
Considering the instability of the intermediate mixed disulfides, experimental approaches have been used to trap substrates by mutating the C-terminal cysteine in the CXXC active site (Motohashi et al., 2001; Jessop et al., 2007; Schwertassek et al., 2007). An alternative approach to mutate the conserved cis proline has been developed to identify substrates for E. coli oxidase DsbA (Kadokura et al., 2004). We showed that the TMX mutant harboring a P101T substitution was capable of capturing the disulfide-linked complexes as seen with the C59A mutant, demonstrating that the mutation of the corresponding proline residue is also useful in mammalian systems for the detection of mixed disulfide intermediates.
The trapping mutants enabled us to identify MHC class I HC as a candidate substrate for TMX. Previous studies have shown that class I HC associates with ERp57 during the early events in its assembly (Lindquist et al., 1998; Morrice and Powis, 1998). At a later stage, ERp57 has also been detected as a component of the peptide-loading complex (Hughes and Cresswell, 1998; Antoniou et al., 2003), and B cell-specific deletion of ERp57 in mice resulted in reduced surface levels and stability of class I molecules (Garbi et al., 2006). More recently, another ER oxidoreductase, PDI, has been identified as a component of the peptide-loading complex, and it stabilizes the peptide-receptive site of the class I molecule (Park et al., 2006). Unlike these classical ER oxidoreductases, TMX was found not to be essential for the oxidative protein folding of class I HC under normal conditions. In exploring their functional relationship, however, we demonstrated that TMX prevented the degradation of class I HC expressed in excess in human cell lines. Furthermore, we see an increased interaction of TMX with nonglycosylated class I HC in tunicamycin-treated cells. Considering that protein overload or tunicamycin treatment should promote aberrant folding of proteins, TMX might act on proteins with malfolded or incompletely folded structures that need to be refolded or degraded. Our data also suggest that calnexin is involved in the TMX–class I HC complex formation under stress conditions. Their interaction might be mediated by the glycan-independent chaperone function of calnexin, which contributes to the quality control of misfolded proteins (Arunachalam and Cresswell, 1995; Ihara et al., 1999; Swanton et al., 2003).
The accumulation of misfolded species in the ER can interfere with cellular processes, and stressed cells cope with protein overload in the ER by increasing the folding capacity or activating the ER-associated protein degradation pathway. Several studies have shown that the retrotranslocation and degradation of misfolded proteins require a sequence of events, including unfolding, disulfide bond reduction, and deglycosylation (Kopito, 1997; Fagioli et al., 2001). However, it is not well understood how cells discriminate between correctly-folded and misfolded proteins or regulate the decision of either repairing the protein or degrading it. It has been suggested that one possible mechanism involves regulation of the redox state of proteins. For example, ER-to-cytosol translocation is sensitive to agents that modify free thiols and alter the redox state of the cell (Tortorella et al., 1998). PDI has been shown to act as a redox-dependent chaperone to unfold cholera toxin and facilitate retrograde transport of the A1 chain into the cytosol where it exerts toxic effects (Tsai et al., 2001). On the contrary, ERp72 and ERp44, both of which belong to the thioredoxin family, act to retain their substrates in the ER (Anelli et al., 2003; Forster et al., 2006). It is possible that TMX mediates the ER retention of membrane proteins including class I HC through a mechanism analogous to that observed in the ER lumen. Reversible thiol-disulfide exchange reactions catalyzed by oxidoreductases must involve a large conformational change that may enable the cellular triage system to distinguish proteins to be refolded and proteins destined for degradation. Further investigation is required to clarify the underlying molecular mechanisms of this redox-based quality control system.
Alterations in the ER environment affect the protein folding, and the resulting accumulation of misfolded proteins is known to contribute to a variety of diseases (Kaufman, 2002). MHC class I is critically important as an antigen-presenting molecule in host defense against intracellular infections, but hyperexpression and misfolding of MHC class I could trigger the onset of autoimmune diseases (Singer et al., 1997; Colbert, 2000). Increased interaction of TMX with class I HC under stress conditions raises the possibility that TMX plays a protective role in pathological conditions such as inflammation and infectious diseases under which large amounts of class I molecules are synthesized. Future development of knockout animal models will enable us to investigate the function of TMX in vivo and its relevance to pathophysiology associated with protein misfolding in the ER.
In this study, we have identified the physical and functional interaction between TMX and MHC class I HC. At present, it remains unclear whether TMX is involved in the redox regulation of other glycoproteins sharing common structures with MHC class I. The classification of substrate proteins for TMX will provide a better understanding of the precise function of this transmembrane oxidoreductase.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Junji Yodoi (yodoi{at}virus.kyoto-u.ac.jp).
Abbreviations used: AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; CST, castanospermine; DPS, 4, 4'-dipyridyl disulfide; DTT, dithiothreitol; ER, endoplasmic reticulum; HC, heavy chain; HLA, human leukocyte antigen; MHC, major histocompatibility complex; NEM, N-ethylmaleimide; PDI, protein disulfide isomerase; UPR, unfolded protein response.
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