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Article |
Address correspondence to Patricia Camacho, Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: (210) 567-6558. Fax: (210) 5674410. email: camacho{at}uthscsa.edu
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Abstract |
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Key Words: calreticulin; calcium ATPases; endoplasmic reticulum; calcium oscillations; glycoprotein folding
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Introduction |
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The lumen of the ER is a specialized protein-folding environment. It contains molecular chaperones such as calreticulin (CRT), calnexin (CLNX), and ER protein 57 (also known as ER-60, GRP58; ERp57; High et al., 2000; Ellgaard and Helenius, 2003; Kostova and Wolf, 2003; Schrag et al., 2003). Optimal [Ca2+]ER is necessary for protein folding (Ashby and Tepikin, 2001). ER Ca2+ depletion inhibits protein folding and maturation (Hardman and Wetmore, 1996; Chen et al., 1997) and facilitates protein degradation (Ramsden et al., 2000). Ca2+ can also regulate the formation of chaperone complexes in the ER (Corbett et al., 1999).
ERp57 is a ubiquitous ER thiol-dependent oxidoreductase that promotes the formation of intra- or intermolecular disulfide bonds during glycoprotein folding (Marcus et al., 1996; High et al., 2000; Ellgaard and Helenius, 2003; Kostova and Wolf, 2003; Schrag et al., 2003). ERp57 has also been identified as a key component in the assembly of class I major histocompatibility complexes (Hughes and Cresswell, 1998; Lindquist et al., 1998; Morrice and Powis, 1998; Antoniou et al., 2002; Dick et al., 2002; Bouvier, 2003). Critical to our paper is the demonstration, firmly established in the literature, of a specific interaction between either CLNX or CRT with ERp57 (Oliver et al., 1997, 1999; Van der Wal et al., 1998; Zapun et al., 1998; High et al., 2000; Frickel et al., 2002).
Our group discovered that CRT, as well as CLNX, inhibited Ca2+ oscillations using the Xenopus oocyte system (Camacho and Lechleiter, 1995; John et al., 1998; Roderick et al., 2000). The precise molecular mechanism responsible for this inhibitory effect is not completely known, however, CRT and CLNX may directly regulate SERCA 2b or they may recruit other enzymes such as ERp57 to cause the effect. Two conserved cysteines in the longest ER facing loop 4 (L4) of SERCA 2b are potential targets of ERp57. In this paper, we address the issues of whether ERp57 modulates Ca2+ oscillations through an interaction with L4 thiols; whether this interaction requires the enzymatic activity of ERp57; whether the interaction is specific and Ca2+ dependent; and whether CRT is required to recruit ERp57 to L4.
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Results |
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ERp57 and PDI catalytic activity lack Ca2+ dependence
Because the association of ERp57 with the L4 substrate is stronger at higher Ca2+ concentrations, we tested whether the intrinsic activity of ERp57 is Ca2+ dependent. We measured in vitro the catalytic activity of purified GST-ERp57 at the same range of [Ca2+] used in the GST pull-down assay (300, 150, 50, and 10 µM). The activity of ERp57 was only mildly but not significantly dependent on Ca2+ at the concentrations measured (Fig. 6 A). A similar assay was also performed for GST-PDI and in this case we found no Ca2+ dependence of its activity (Fig. 6 B).
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Discussion |
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ERp57 and its homologue PDI have different substrate specificities (Freedman et al., 2002; Clissold and Bicknell, 2003). ERp57 specifically associates with N-linked glycoproteins, whereas PDI does not. This specificity is mainly mediated by an interaction with CRT or CLNX (Oliver et al., 1997, 1999; Van der Wal et al., 1998; Zapun et al., 1998; High et al., 2000; Frickel et al., 2002). Using in vitro GST pull-down experiments, we also demonstrated that ERp57 specifically interacts with the L4 of SERCA 2b, whereas PDI did not. It is possible that there is a direct interaction between ERp57 and L4, but it is equally likely that an indirect protein interaction is occurring in a pull-down assay. For example, microsomes contain CRT and glycoproteins (e.g., SERCA 2b), which could then link ERp57 to L4. In fact, the bulk of our results favor the requirement of CRT to recruit ERp57 to the L4.
The mechanisms that regulate SERCA activity from the ER have been until now somewhat unclear. Decreased lumenal Ca2+ induces a strong stimulation of SERCA activity in isolated pancreatic acinar cells (Mogami et al., 1998). We demonstrate that the association of ERp57 with the L4 is Ca2+ dependent in the physiological range of ER lumenal Ca2+ (10, 50, 150, and 300 µM). The Ca2+ dependency of the association between ERp57 and L4 is likely to be mediated by CRT, because Ca2+ does not regulate the intrinsic enzyme activity of the ERp57. It has been reported that Ca2+ is required for CRT binding to oligosaccharides, although these experiments were conducted beyond physiological [Ca2+] (10 mM Ca2+; Vassilakos et al., 1998). Ca2+ has also been shown to modulate the interaction between CRT and ERp57 (Corbett et al., 1999). Together, our data suggest the following role of ER Ca2+ in the feedback mechanism that regulates SERCA 2b activity (Fig. 10). When ER Ca2+ stores are full (300 µM Ca2+), ERp57 binds to L4 promoting disulfide bond formation in the loop, which inhibits pump activity. When Ca2+ stores become depleted (
10 µM Ca2+), ERp57 dissociates from L4, resulting in the reduced form of SERCA 2b that is more active. The mechanism by which thiol groups in the L4 are reduced after ERp57 dissociates from the pump is unknown. The ER lumen maintains a neutral to slightly oxidized environment (GSHGSSG
13; Hwang et al., 1992). Our results suggest that ERp57 must be bound to L4 in order to maintain it in an oxidized state. It is possible that additional ER proteins play a role here. Regardless of the precise underlying mechanism, our data clearly indicate that the reduced form of L4 supports higher pump activity to rapidly refill the ER.
The CLNXCRT cycle has a well-established role in productive glycoprotein folding and quality control of nascent glycoproteins undergoing folding (High et al., 2000; Ellgaard and Helenius, 2003; Kostova and Wolf, 2003; Schrag et al., 2003). Our observations indicate that these ER chaperones interact with a mature ER resident glycoprotein (SERCA 2b). Association/dissociation of other ER chaperones with mature proteins has also been reported during the unfolded protein response (Bertolotti et al., 2000). We speculate that the classical CLNXCRT cycle and the system we have characterized here exist in parallel and that the action of CRTCLNX on SERCA 2b provides the homeostatic feedback necessary to maintain Ca2+ conditions that promote productive glycoprotein folding. Without it, Ca2+ depletion could result in the well-known unfolded protein response, which can bring about cellular apoptosis under certain stress conditions (Kaufman, 1999; Welihinda et al., 1999; Patil and Walter, 2001; Harding et al., 2002). Maintaining optimal Ca2+ concentrations in the ER is clearly important for nascent protein folding (Hardman and Wetmore, 1996; Chen et al., 1997; Stevens and Argon, 1999; Ramsden et al., 2000). The ability of the lectin chaperones to "talk" to a mature protein in the Ca2+ signaling machinery provides the mechanism by which the optimal Ca2+ environment is maintained for protein folding.
Oxidant treatment inactivates SERCA 2b to a greater extent than other pump isoforms (Grover et al., 1997; Barnes et al., 2000). Our observations imply that SERCA 2b is in fact a redox sensor. In the oxidized state of L4, this pump exhibits lower activity. On the other hand, the pump favors rapid Ca2+ uptake in the L4 mutants that are constitutively reduced by cysteine mutagenesis. Interestingly, the ryanodine receptor, a Ca2+ release channel located on the SRER is also a redox sensor (Zable et al., 1997; Xia et al., 2000; Sun et al., 2001). The oxidized channel correlates with the open state, whereas the reduced form correlates with channel closure. The IP3R may also sense the redox potential because it contains cysteines in the ER lumenal facing loop that line the channel pore. Consequently, modulation of redox potential of reactive thiols in the ER might be a general mechanism by which SERCA 2b, IP3R, and ryanodine receptor control ER Ca2+. In terms of Ca2+ homeostasis, our observations with ERp57 fit the view that ER redox modulates Ca2+ release and uptake in coordinate and opposite directions. In particular, oxidization appears to favor Ca2+ release by opening Ca2+ channels and inhibiting Ca2+ pumping. On the other hand, reduction seems to favor Ca2+ uptake by channel closure and increasing Ca2+ pumping. The combined effect of these two actions helps to minimize the loss of ER Ca2+ and maximize refilling of the stores, thereby protecting the physiological functions of the ER such as glycoprotein folding.
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Materials and methods |
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A construct encoding the L4 of SERCA 2b was generated by PCR amplification spanning methionine 814 and arginine 922 with primers 5'-ACTGGGATCCATGAACAAACCCCCACGGAACCCA-3' and 5'-ACTGAAGCTTTTACCTCAGCAGGGACTGGTTTTC-3'. The PCR product was ligated into the BamHI and HindIII sites of the similarly digested vector pHN (plasmid pHN-SERCA 2b-L4). To generate plasmid pHN-GFP-L4, a BamHI fragment from pHN-GFP-S65T-TAA, as described previously (John et al., 1998), was subcloned into plasmid pHN-SERCA 2b-L4 that was digested with BamHI and treated with calf intestinal phosphatase. Plasmid pHN-GFT S65T was generated previously (John et al., 1998). The Xenopus expression vector of DsRed-IP3R was generated by a three-step PCR protocol to create a fusion of DsRed to the NH2-terminal sequence of the IP3R. First, the full-length DsRed was amplified by PCR from the template of pDSRed 1-N1 (CLONTECH Laboratories, Inc.). The forward and reverse primers were 5'-ACTGGAATTCATGGTGCGCTCCTCCAAGAACGTC-3' and 5'-AAGAAAGCTGGACATTTCATTCATCAGGAACAGGTGGTGGCGGCC-3'. Second, the NH2 terminus of IP3R was amplified by PCR from template pHN-IP3R with the forward and reverse primers as 5'-GGCCGCCACCACCTGTTCCTGATGAATGAAATGTCCAGCTTTCTT-3' and 5'-GTGCACATGTAGCATCAGGTGGCAGAATGA-3'. The purified PCR products served as templates to create the final product DsRe-IP3R. In this PCR reaction, the forward and reverse primers were 5'-ACTGGAATTCATGGTGCGCTCCTCCAAGAACGTC-3' and 5'-GTGCACATGTAGCATCAGGTGGCAGAATGA-3'. The final PCR fusion product was digested with EcoRI and NsiI and subcloned into the EcoRI and NsiI sites of PHN-IP3R.
The Xenopus expression vector of ERp57 (plasmid pHN-ERp57) was generated in a three-step PCR protocol. First, the mature human ERp57 was amplified from the template pET9-ERp613 (a gift from D.Y. Thomas, Biotechnology Research Institute, Montréal, Canada) with primers 5'-CTGCTCGGCCTGGCCGCCGCCTCCGACGTGCTAGAACTCACG-3' and 5'-ACGTAAGCTTTTAGAGATCCTCCTGTGCCTTCTT-3'. A second PCR generated the signal peptide of CRT from the template pHN-CRT (Camacho and Lechleiter, 1995) using primers 5'-TAATACGACTCACTATAGGG-3' and 5'-CGTGAGTTCTAGCACGTCGGAGGCGGCGGCCAGGCCGAGCAG-3'. The purified PCR products served as templates to generate a fusion of the CRT signal peptide and mature ERp57. In this PCR reaction, the forward and reverse primers were 5'-TAATACGACTCACTATAGGG-3' and 5'-ACGTAAGCTTTTAGAGATCCTCCTGTGCCTTCTT-3'. The final PCR fusion product was subcloned into the HindIII and SSTI sites of the Xenopus expression vector pHN. Three mutants of ERp57 were generated. The first mutant had cysteines 57 and 60 mutated to serines in the NH2-terminal thioredoxin motif (mutant ERp57-T1) and was generated by PCR using primer 5'-CGAGTTCTTCGCTCCCTGGTCTGGACACTCCAAGAGACTTGCACC-3' and its reverse complement. The second construct had cysteines 406 and 409 in the COOH-terminal thioredoxin motif mutated to serines (mutant ERp57-T2) and was generated using primer 5'-GAATTTTATGCCCCTTGGTCTGGTCATTCTAAGAACCTGGAGCCC-3' and its reverse complement. The third construct was a double mutant generated in two PCR steps with the above primers (mutant ERp57-T1T2).
Rat PDI cDNA was cloned from a rat liver cDNA library (Invitrogen) by PCR using primers 5'-ACTGGGATCCATGCTGAGCCGTGCTTTGCTGTGC-3' and 5'-ACTGTCTAGACTACAGTTCATCCTTCACGGC-3' based on the GenBank/EMBL/DDBJ accession no. X02918. The PCR product was subcloned into the BamHI and XbaI sites of pHN and fully sequenced.
To obtain GST-ERp57 fusion proteins including wild type and mutants as well as GST-PDI, we amplified the corresponding mature cDNA fragments by PCR and subcloned them into pGEX-4T-2 vector (Amersham Biosciences). For ERp57 and its mutants, the primers were 5'-TCGAGGATCCATGTCCGACGTGCTAGAACTCACG-3' and 5'-ACTGCTCGAGTTAGAGATCCTCCTGTGCCTTCTT-3', whereas for PDI, the primers were 5'-ACTGGGATCCATGGACGCTCTGGAGGAGGAGGAC-3' and 5'-ACTGCTCGAGCTACAGTTAATCCTTCACGGC-3'. The PCR products were ligated into the BamHI and XhoI sites of pGEX-4T-2.
The Xenopus vectors encoding CRT, CRT-NP, CRT-NC, and CRT-N domains have been published previously (Camacho and Lechleiter, 1995). The construct for expression of the P domain (plasmid pHN-CRT-P) was generated by PCR mutagenesis designed to remove the N domain from template pHN-CRT-NP (Camacho and Lechleiter, 1995). The primers used had sequence 5'-GCCGCCGAGCCCGATGACTGGGACTTCCTACCCCCCAAGAAGATAAAGGACCCA-3' and 5'-TGGGTCCTTTATCTTCTTGGGGGGTAGGAAGTCCCAGTCATCGGGCTCGGCGGC-3'. The resulting construct contains the CRT signal peptide followed by the P domain (amino acids 181260) and a KDEL sequence for ER retention.
Automatic sequencing of all cDNA constructs was performed at the UTHSCSA Advance Nucleic Acid Core Facility. All primers were purchased from Operon Technologies (QIAGEN). Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich and all restriction enzymes were purchased from Invitrogen Life Technologies.
In vitro transcriptions and translations
Synthetic mRNA was prepared as described previously (Camacho and Lechleiter, 1995). In vitro translations were performed as described previously (Roderick et al., 2000).
Western blots
Oocytes extracts were prepared as described previously (Camacho and Lechleiter, 1995). In brief, 510 oocytes were pooled and homogenized in buffer containing 20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 250 mM sucrose, and protease inhibitors in a mix containing 0.2 mM AEBSF, 10 µM leupeptin, 1 µM pepstatin A, and 0.8 mM benzamidine (Calbiochem). After centrifugation at 4,500 g for 15 min at 4°C, the supernatant was ultracentrifuged at 125,000 g for 20 min at 4°C. Microsomal pellets were resuspended in solubilization buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1% Triton X-100, and protease inhibitors). A second centrifugation (15,000 g for 10 min) was performed to discard the insoluble material. SDS-PAGE was performed by loading 12 oocyte equivalents per lane and transferred to nitrocellulose. A polyclonal rabbit anti-SERCA antibody ( NI, 1:8,000 dilution, a gift from J. Lytton, University of Calgary, Calgary, Canada) was used to detect SERCA 2b. A rabbit anti-ERp57 polyclonal antibody (1:4,000 dilution, a gift from S. High, University of Manchester, Manchester, UK) was used to detect ERp57. HRP-conjugated donkey antirabbit IgG secondary antibodies (Jackson ImmunoResearch Laboratories) in a dilution of 1:10,000 were used in all Western blots and visualized by ECL (PerkinElmer Life Sciences, Inc.).
GST fusion protein purification
BL21 bacteria transformed with pGEX-4T-2, pGEX-4T-2-PDI or pGEX-4T-2-ERp57 (and mutants) were grown to OD 600 nm 0.6. Isopropyl-ß-D-thiogalactopyranoside (Research Products International Corp.) was added to a final concentration of 1 mM to induce protein expression for 4 h at 37°C. Bacteria were lysed by sonication in 1x PBS containing 100 mM EDTA and a mix of protease inhibitors including (200 µM AEBSF, 10 µM leupeptin, and 1.5 µM pepstatin A) and 0.8 mM benzamidine. Bacterial lysates were centrifuged at 22,000 g and the supernatant collected. Binding of GST or GST fusion proteins to glutathione-Sepharose 4B (Amersham Biosciences) was performed at 4°C for 1 h followed by three washes with equilibration buffer (0.5 M Tris-HCl, pH 8.0, 4 mM EDTA, 0.1% ß-ME, 5% glycerol). Elution of bound protein was performed in this buffer supplemented with 15 mM glutathione. Proteins were dialysed against 0.05 M Tris-HCl, pH 7.5, containing 1 mM EDTA, 0.2% ß-ME, and stored at 80°C.
GST pull-down assays
In vitro translation of L4 was accomplished in a rabbit reticulocyte lysates supplement with canine pancreatic microsomes (both from Promega) and L-[35S]methionine (PerkinElmer Life Sciences). Microsomes were isolated and resuspended in solubilization buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1% Triton X-100 and a mixture of the protease inhibitors described in Western blots). Binding of in vitrotranslated L4 to GST-ERp57 fusion protein(s) was performed at 4°C overnight in the presence of glutathione Sepharose 4B (Amersham Biosciences) in binding solution containing in mM 10 Tris-HCl, pH 8.0, 70 KCl, 2 MgCl2, 50 µM EGTA, 5% BSA, and protease inhibitors supplemented with experimental CaCl2 concentrations (60, 100, 200, and 350 µM). Proteins bound to glutathione Sepharose beads were washed three times in buffer containing 0.2 M Tris-HCl, pH 8.0, 0.1% Triton X-100, 70 mM KCl, 2 mM MgCl2, 50 µM EGTA, protease inhibitors, and corresponding CaCl2 concentrations. Proteins were resolved by 15% SDS-PAGE and visualized by autoradiography. Ca2+ concentrations were calculated according to existing algorithms (Fabiato and Fabiato, 1979).
Insulin turbidity assay
The insulin turbidity assay was performed as described previously (Holmgren, 1979; Hirano et al., 1995). In this assay, oxidized insulin was used as substrate to measure thiol-dependent reductase activity of ERp57. Under reducing conditions, the two interchain disulphide bonds of insulin are cleaved, resulting in the formation of a white insoluble precipitate. 10 mg/ml of insulin (Sigma-Aldrich) stock solution was prepared as described previously (Holmgren, 1979). In brief, 100 mg of insulin was first resuspended in 8 ml 0.05 M Tris-HCl, pH 8.0. Subsequently, the pH was adjusted between 2.0 and 3.0 by adding 1 M HCl and rapidly titrated to pH 8.0 with 1 M NaOH. The final volume of the solution was adjusted to 10 ml with H2O. The clear stock solution was stored at 20°C. On the experimental day, the insulin stock solution was diluted to 1 mg/ml with a buffer containing in 100 mM KAC, pH 7.5, and 2 mM EDTA, supplemented with CaCl2 concentrations (2.44, 2.28, 2.17, and 2.12 mM) to yield free [Ca2+] of 300, 150, 50, and 10 µM. The final reaction volume was adjusted to 1 ml after purified ERp57 or its mutants were added. The reaction was initiated by adding 3 µl of 100 mM DTT. Abs650 nm was measured every 5 min in a BioSpec-1601 spectrophotometer (Shimadzu). Enzyme activity was defined by measuring the slope of the linear portion of the absorbance curve. Ca2+ concentrations were calculated according to known algorithms (Fabiato and Fabiato, 1979).
Oocyte methods and confocal imaging
Stage VIdefolliculated oocytes were injected a bolus of 50 nl of 1 µg mRNA using a standard positive pressure injector (Drummond Scientific). Oocytes were cultured in 50% L-15 media (Invitrogen) for 57 d at 18°C. Intracellular Ca2+ was imaged as described previously (Roderick et al., 2000). Oocytes were injected with fluorescent Ca2+ indicator Oregon green II (12.5 µM final; Molecular Probes) 3060 min before imaging. Ca2+ release was initiated by injecting a 50 nl bolus of 6 µM IP3 (Calbiochem) to yield 300 nM final concentration. Imaging was performed in ND96 buffer containing 1 mM EGTA. Images were acquired at a rate of 0.75 s /frame on a confocal laser scanning microscope (model PCM2000; Nikon) using a 10x objective (NA = 0.45) at zoom 1.
To characterize the ER localization of L4, GFP-L4 was coexpressed in oocytes with DsRed-IP3R. Fluorescence was monitored using a confocal microscope (model Fluoview 500; Olympus). GFP-L4 as well as GFP (as a negative control) were detected using a 488-nm laser line for excitation and a combination of a 510-nm long pass and a 550-nm short pass barrier filter for signal emission. DsRed fluorescence was obtained with a 568-nm laser line for excitation and a 585-nm long pass barrier filter for emission. Emission signals were collected using a 60x oil (NA = 1.4) objective (Olympus) at zoom 5.
Imaging analysis
The analysis of Ca2+ images was performed using the public domain NIH ImageJ program (developed at the National Institutes of Health and available at http://rsb.info.nih.gov/ij/).
Statistical analysis
Statistical significance was determined by t test or one-way ANOVA as appropriate and accepted at P < 0.05.
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Acknowledgments |
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This work was funded by National Institutes of Health grant R01 GM55372 to P. Camacho.
Submitted: 2 July 2003
Accepted: 25 November 2003
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References |
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