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Identification of an N-domain Histidine Essential for Chaperone Function in Calreticulin

2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês

10.1074/jbc.m309497200

1083-351X

Lei Guo, Jody Groenendyk, Sylvia Papp, Monika Dąbrowska, Barbara Knoblach, Cyril M. Kay, J. M. R. Parker, Michał Opas, Marek Michalak,

Biochemical and Molecular Research

Calreticulin is an endoplasmic reticulum (ER) luminal Ca2+-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt-/- cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders. Calreticulin is an endoplasmic reticulum (ER) luminal Ca2+-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt-/- cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders. The endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumMDHmalate dehydrogenaseANS8-anilino-1-naphthalene-sulfonic acidHAhemagglutininMOPS4-morpholinepropanesulfonic acid.1The abbreviations used are: ERendoplasmic reticulumMDHmalate dehydrogenaseANS8-anilino-1-naphthalene-sulfonic acidHAhemagglutininMOPS4-morpholinepropanesulfonic acid. plays an essential role in a variety of cellular processes, including Ca2+ homeostasis, protein and lipid synthesis, and post-translational modification and folding of membrane-associated and secreted proteins (1.Baumann O. Walz B. Int. Rev. Cytol. 2001; 205: 149-214Crossref PubMed Scopus (328) Google Scholar). The ER ensures that only correctly folded proteins proceed through the secretory pathway and directs misfolded proteins to ER-associated degradation (2.McCracken A.A. Brodsky J.L. BioEssays. 2003; 25: 868-877Crossref PubMed Scopus (194) Google Scholar, 3.Cabral C.M. Liu Y. Moremen K.W. Sifers R.N. Mol. Biol. Cell. 2002; 13: 2639-2650Crossref PubMed Scopus (91) Google Scholar). The lumen of the ER is a dynamic environment that contains numerous molecular chaperones and Ca2+-binding proteins that are designed for these tasks. Molecular chaperones are proteins that bind to misfolded/unfolded proteins in a transient manner to assist in their folding. endoplasmic reticulum malate dehydrogenase 8-anilino-1-naphthalene-sulfonic acid hemagglutinin 4-morpholinepropanesulfonic acid. endoplasmic reticulum malate dehydrogenase 8-anilino-1-naphthalene-sulfonic acid hemagglutinin 4-morpholinepropanesulfonic acid. Calreticulin is a Ca2+-binding chaperone that resides in the lumen of the ER and is involved in modulation of Ca2+ homeostasis and in the folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle" (4.Schrag J.D. Procopio D.O. Cygler M. Thomas D.Y. Bergeron J.J. Trends Biochem. Sci. 2003; 28: 49-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 5.Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 6.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1645) Google Scholar, 7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). Calreticulin and calnexin are both ER lectins, which bind transiently to virtually all newly synthesized glycoproteins (5.Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 6.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1645) Google Scholar, 7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). Chaperone-assisted protein folding has been studied extensively using Escherichia coli GroEL heat shock proteins, which are cytoplasmic (8.Feltham J.L. Gierasch L.M. Cell. 2000; 100: 193-196Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Numerous studies have been carried out on ER-associated chaperones (2.McCracken A.A. Brodsky J.L. BioEssays. 2003; 25: 868-877Crossref PubMed Scopus (194) Google Scholar, 3.Cabral C.M. Liu Y. Moremen K.W. Sifers R.N. Mol. Biol. Cell. 2002; 13: 2639-2650Crossref PubMed Scopus (91) Google Scholar, 4.Schrag J.D. Procopio D.O. Cygler M. Thomas D.Y. Bergeron J.J. Trends Biochem. Sci. 2003; 28: 49-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 5.Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 6.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1645) Google Scholar, 7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar); yet, the molecular features of calreticulin that confer its chaperone function have not yet been determined (7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). Three distinct structural domains have been identified in calreticulin: the amino-terminal, globular N-domain; the central P-domain; and the carboxyl-terminal C-domain (7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). NMR (9.Ellgaard L. Riek R. Herrmann T. Guntert P. Braun D. Helenius A. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3133-3138Crossref PubMed Scopus (155) Google Scholar), modeling (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar), and biochemical studies (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar) indicate that the globular N-domain and the "extended arm" P-domain of calreticulin may form a functional protein-folding unit (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar). This region of calreticulin contains a Zn2+ binding site and one disulfide bond, and it may also bind ATP (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar, 13.Andrin C. Corbett E.F. Johnson S. Dabrowska M. Campbell I.D. Eggleton P. Opas M. Michalak M. Protein Expression Purif. 2000; 20: 207-215Crossref PubMed Scopus (16) Google Scholar, 14.Corbett E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar). When calreticulin binds Zn2+, it undergoes dramatic conformational changes (15.Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar). Chemical modification of calreticulin has revealed that four histidines located in the N-domain of the protein (His25, His82, His128, and His153) are involved in the Zn2+ binding (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar). The Zn2+-dependent conformational change in calreticulin affects its ability to bind to unfolded protein/glycoprotein substrates in vitro (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar), suggesting that conformational changes in calreticulin may modify its chaperone function. The role of the Zn2+ binding histidine residues in calreticulin function is not known. Calreticulin deficiency is embryonic lethal, and cells derived from calreticulin knockout embryos have impaired Ca2+ homeostasis and compromised protein folding and quality control (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar, 17.Knee R. Ahsan I. Mesaeli N. Kaufman R.J. Michalak M. Biochem. Biophys. Res. Commun. 2003; 304: 661-666Crossref PubMed Scopus (27) Google Scholar). The availability of calreticulin-deficient cells provides an excellent tool for investigation of the molecular events associated with calreticulin function in the lumen of the ER. In this study, we created site-specific mutants of calreticulin and reconstituted them into the ER lumen of calreticulin-deficient cells. In calreticulin-deficient cells, folding of the bradykinin receptor is altered, which impairs its ability to initiate inositol 1,4,5-trisphosphate-dependent Ca2+ release (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Therefore, the reestablishment of bradykinin-dependent Ca2+ release from the ER was used as a marker for calreticulin function. Because Zn2+-dependent conformational changes in calreticulin are critical for its interaction with substrate proteins (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar), we focused on the role of histidine residues in calreticulin function. We show that of the histidine residues in the N-domain of calreticulin only His153 is essential for its function. Mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders. Materials—Trypsin, malate dehydrogenase (MDH), 8-anilino-1-naphthalene-sulfonic acid (ANS), bradykinin, and Dulbecco's modified Eagle's medium were obtained from Sigma. Fetal bovine serum was from Invitrogen. SDS-PAGE reagents and molecular weight makers were form Bio-Rad. Effectene Transfection reagent, Ni2+-nitrilotriacetic acid-agarose beads was from Qiagen. Zeocin, Pfx DNA polymerase, pBAD/glIII A, and pCDNA3.1/Zeo plasmids were from Invitrogen. EGGstarct IgY purification system was from Promega. All chemicals were of the highest grade available Plasmid and Site-directed Mutagenesis—For E. coli expression of calreticulin, wild type full-length rabbit calreticulin gene was amplified and cloned into NcoI and XbaI restriction enzyme sites of plasmid pBAD/glIII A (Invitrogen) to generate pBAD-CRT. To express calreticulin in eukaryotic cells, the rabbit calreticulin gene was amplified and cloned into EcoRI and XbaI of pcDNA3.1/Zeo. For easy detection of the recombinant protein, a hemagglutinin epitope (HA) tag was engineered to the C terminus of calreticulin to generate pcDNA-CRT-HA. Site-specific mutagenesis was carried out using a megaprimer polymerase chain reaction technique (18.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar, 19.Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar) using Gene Amp PCR system 9700 thermal cycler and Pfx DNA polymerase. For biochemical and biophysical studies, calreticulin mutant proteins were expressed in E. coli. To generate E. coli expression vector, cDNA encoding calreticulin (PstINotI restriction DNA fragment of pcDNA-CRT-HA plasmids) was cloned into PstI-NotI restriction sites of pBAD-CRT plasmid. The following histidine to alanine or histidine deletion mutants were generated: H25A, H82A, H128A, H153A, and H25Del, H82Del, H128Del, and H153Del deletion mutants. Identical results were obtained whether hisitidine to alanine or histidine deletion mutations were used. Throughout this paper wild type calreticulin and H25A, H82A, H128A, H153A mutants are designated as CRT-wt and CRT-His25, CRT-His82, CRT-His128, and CRT-His153, respectively. Cell Culture and Cytoplasmic Ca2+ Measurements—Wild type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts were used in this study (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Cells were grown at 37 °C in a 5% CO2 environment in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. crt-/- cells were transfected with pcDNA3.1/Zeo expression vectors containing cDNA encoding either wild type or mutant calreticulin. Transfections were carried out using Effectene transfection reagent, and stable transfected cell lines were selected with 350 μg/ml Zeocin. The following cell lines expressing wild type calreticulin (crt-/--wt) or specific histidine mutants (crt-/--His25, crt-/--His82, crt-/--His128, and crt-/--His153) were generated. For measurement of cytoplasmic Ca2+ concentration, 1.5 × 106 ml were loaded with the fluorescent Ca2+ indicator fura-2/AM (2 μm) (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Ca2+ release from internal store was stimulated with 200 nm bradykinin and monitored in a Ca2+-free buffer (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Immunofluorescence Microscopy—Cultured mammalian cells were washed with phosphate-buffered saline and lysed with radioimmune precipitation buffer containing 50 mm Tris, pH, 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS. Proteins were separated on SDS-PAGE (10% acrylamide) and transferred to nitrocellulose membrane. Western blots were probed with rabbit anti-HA (Roche Applied Science) at 1:300 dilution. Peroxidase-conjugated goat anti rabbit IgG was used as the secondary antibody at 1:10,000 dilution. Cells were grown on 25-mm circular cover-slips in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde. Goat anti-calreticulin antibodies (1:70 dilution) were used. Texas Redconjugated donkey anti-goat IgG (1:70 dilution) was used as secondary antibody. Cells were mounted in Vinol 205S and examined with a Bio-Rad confocal fluorescence microscope (model MRC-600) equipped with a krypton/argon laser. Expression and Purification of Recombinant Calreticulin—Proteins were expressed in Top10F′ E. coli cells in LB medium containing 100 μg of ampicillin/ml. Cultures were grown to the midlog phase followed by the induction of the expression of recombinant proteins with arabinose (final concentration of 0.002%) for 4 h. Cells were spun down at 6,000 rpm for 15 min. The pellet was resuspended in a buffer containing 50 mm Tris, pH 8.0, 300 mm NaCl, 10% glucose and lysed in the French press set at 1,000 p.s.i. followed by centrifugation at 10,000 rpm for 10 min. His-tagged proteins were purified by one-step Ni2+-nitrilotriacetic acid-agarose affinity chromatography in native condition. Samples of E. coli lysates were mixed with the Ni2+-nitrilotriacetic acid-agarose beads equilibrated with a buffer containing 50 mm Tris, pH 8.0, and 300 mm NaCl, applied onto the column, washed, and eluted with a buffer containing 50 mm Tris, pH 8.0, 300 mm NaCl, and 20 mm imidazole. Recombinant proteins were concentrated by a centrifugal filter (Biomax 30K NMWL membrane; Millipore), and proteins were dissolved in a buffer containing 10 mm Tris, pH 7.0, and 1 mm EDTA. Over 90% of the protein was purified to homogeneity by one-step Ni2+-nitrilotriacetic acid-agarose column chromatography. Protein concentration was determined by a Beckman System 6300 amino acid analyzer or by using Bio-Rad protein assay reagent using bovine serum albumin as a standard (20.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar). Aggregation Assay—One μm MDH was mixed with varying amounts of wild type and mutant calreticulin at room temperature, and samples were incubated at 45 °C in 50 mm sodium phosphate, pH 7.5 (total volume, 1.2 ml) and monitored for light scattering for 2 h. IgY was isolated from chicken egg yolk according to the protocol of the EGG-stract IgY purification system. IgY was dialyzed overnight against denaturing buffer containing 100 mm Tris, pH 7.0, 6 m guanidinium hydrochloride, and 40 mm dithiothreitol. The denatured IgY protein (0.25 μm) was suspended in a buffer containing 10 mm Tris-HCl, pH 7.0, 150 mm NaCl, 5 mm CaCl2 (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar) followed by the addition of wild type or mutant calreticulin (0.25 μm). Protein aggregation was induced by increasing sample temperature to 44 °C. Light scattering was measured using a spectrofluorometer system C43/2000 (PTI) equipped with a temperature-controlled cell holder; the excitation and emission wavelengths were set to 320 and 360 nm, respectively. Intrinsic Fluorescence Measurement and Circular Dichroism—Intrinsic fluorescence measurements were performed at 25 °C in a spectrofluorometer system C43/2000 (Photon Technology International Inc.). Three μm calreticulin wild type and mutant proteins was used for fluorescence measurement in a buffer containing 10 mm MOPS, pH 7.1, 3 mm MgCl2, and 150 mm KCl (15.Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar). The excitation wavelength was set to 286 nm, and the range of emission wavelength was set to 295-450 mm. The effect of Zn2+ on the intrinsic fluorescence of protein was evaluated at a wavelength of 334 nm. CD analysis was performed at 25 °C using a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD), interfaced to an Epson Equity 386/25 and controlled by Jasco software as described previously (21.Corbett E.F. Oikawa K. Francois P. Tessier D.C. Kay C. Bergeron J.J.M. Thomas D.Y. Krause K.-H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The CD spectra were analyzed for secondary structure elements by the Contin ridge regression analysis program of Provencher and Glöckner (22.Provencher S.W. Glöckner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1857) Google Scholar). Proteolytic Digestions—Ten μg of purified, recombinant wild type and mutant calreticulin expressed in E. coli were incubated with trypsin at 1:100 (trypsin/protein; w/w) (14.Corbett E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar). Aliquots were taken at indicated time points, and the reaction was stopped by the addition of the Laemmli sample buffer (23.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). The proteins were separated in SDS-PAGE (10% acrylamide) and stained with Coomassie Blue. Expression of Histidine Mutants in Calreticulin-deficient Cells—The N-domain of calreticulin contains 5 histidine residues, which are involved in Zn2+ binding to the protein (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar). Zn2+ binding to calreticulin causes significant conformational changes, which promote the interaction of calreticulin with substrates (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar, 14.Corbett E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar, 15.Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar, 16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar, 24.Baksh S. Burns K. Andrin C. Michalak M. J. Biol. Chem. 1995; 270: 31338-31344Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 25.Li Z. Stafford W.F. Bouvier M. Biochemistry. 2001; 40: 11193-11201Crossref PubMed Scopus (57) Google Scholar). To assess the role of the N-domain histidines in calreticulin function, we mutated residues His25, His82, His128, and His153 to create a series of plasmids containing cDNA encoding these mutants and designated as CRT-His25, CRT-His82, CRT-His128, and CRT-His153, respectively. Calreticulin-deficient cells were stably transfected with these expression vectors to create cell lines expressing wild type calreticulin (crt-/--wt) or the specific histidine mutants crt-/--His25, crt-/--His82, crt-/--His128, and crt-/--His153. For easy identification of recombinant proteins, the HA epitope was introduced at the C terminus. We have shown previously that introduction of a C-terminal HA epitope does not affect calreticulin function (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar, 26.Arnaudeau S. Frieden M. Nakamura K. Castelbou C. Michalak M. Demaurex N. J. Biol. Chem. 2002; 277: 46696-46705Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 27.Gao B. Adhikari R. Howarth M. Nakamura K. Gold M.C. Hill A.B. Knee R. Michalak M. Elliott T. Immunity. 2002; 16: 99-109Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Western blot analysis showed that all of the transfected cells expressed recombinant calreticulin (Fig. 1). A relatively lower level of expression of recombinant protein was consistently observed in cells expressing the His25 and His128 mutants (Fig. 1A), and although several different stably transfected cell lines were generated, we were unable to obtain cells with a higher level of expression. Next we carried out immunofluorescence analysis of the cell lines to confirm that the recombinant calreticulin was localized to the ER. All of the cell lines (crt-/--wt, crt-/--His25, crt-/--His82, crt-/--His128, and crt-/--His153) expressed recombinant calreticulin, and the protein was localized to an ER-like network (Fig. 1). Morphologically, at a light microscope level, the ER appeared intact in all cell lines as judged by staining with antibodies against PDI, Grp94, and calnexin (data not shown). Bradykinin-induced Ca2+ Release in Cells Expressing Calreticulin Mutants—We have shown previously that calreticulin-deficient cells have inhibited bradykinin-dependent Ca2+ release, which is restored by expression of full-length recombinant calreticulin (Fig. 2) (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). This is because, in the absence of calreticulin, the bradykinin receptor does not fold properly, so it is unable to bind bradykinin to generate inositol 1,4,5-trisphosphate-dependent signals (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Consequently, measurement of bradykinin-dependent Ca2+ release from the ER makes an excellent model system to study the function of calreticulin and calreticulin mutants in crt-/- cells. Therefore, we assessed bradykinin-dependent Ca2+ release in cells expressing the calreticulin mutants we had generated. We performed these experiments with the Ca2+-sensitive fluorescent dye fura-2. Fig. 2 shows that, as expected, bradykinin caused a rapid and transient increase in the cytoplasmic Ca2+ concentration in wild type cells but not in crt-/- cells (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Also, the expression of recombinant calreticulin in crt-/- cells restored bradykinin-dependent Ca2+ release (Fig. 2, crt-/- + CRT). Bradykinin-induced Ca2+ release was then measured in the crt-/--His25, crt-/--His82, crt-/--His128, and crt-/--His153 cell lines. Expression of the His25, His82, and His128 mutants fully restored bradykinin-dependent Ca2+ release (Fig. 2), whereas expression of the His153 mutant did not. This indicates that His153 must play an essential role in calreticulin's structure and chaperone function. His153 Mutant Does Not Prevent Thermal Aggregation of MDH and IgY—In order to examine the role of His153 in the chaperone activity of calreticulin, we exploited an in vitro assay used previously by Williams's group (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar). This assay makes use of MDH, a nonglycosylated substrate, and IgY, a glycosylated substrate (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar). MDH is susceptible to heat-induced aggregation at 45 °C, as measured by light scattering, and it has been widely used as a model substrate in aggregation and refolding assays with many other molecular chaperones (28.Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (651) Google Scholar, 29.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 30.Manna T. Sarkar T. Poddar A. Roychowdhury M. Das K.P. Bhattacharyya B. J. Biol. Chem. 2001; 276: 39742-39747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Williams's group has shown recently that full-length recombinant calreticulin efficiently prevents MDH or IgY thermal-induced aggregation in vitro (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar). To further investigate the role of histidine residues in calreticulin function, we examined the effectiveness of the His25, His82, His128, and His153 mutants in prevention of thermal aggregation of MDH and IgY, in vitro. In order to do this, we expressed the recombinant proteins in E. coli and purified them. Fig. 3 shows that one-step purification of the recombinant proteins on a nickel column was sufficient. As previously reported, when MDH was heated to 45 °C it began to form insoluble aggregates that could be detected by light scattering (Fig. 3) (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar, 28.Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (651) Google Scholar, 29.Veinger L. Diamant S. Buchner J. Goloubinoff P. J. Biol. Chem. 1998; 273: 11032-11037Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 30.Manna T. Sarkar T. Poddar A. Roychowdhury M. Das K.P. Bhattacharyya B. J. Biol. Chem. 2001; 276: 39742-39747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). As expected, aggregation was reduced in the presence of different concentrations of wild type calreticulin (16.Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar). However, the addition of the His153 calreticulin mutant to 0.1 μm (calreticulin/MDH = 0.1:1) or 0.2 μm (calreticulin/MDH = 0.2:1) did not prevent MDH aggregation (Fig. 3B).