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Comparison of the Ca2+-binding Properties of Human Recombinant Calretinin-22k and Calretinin

1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês

10.1074/jbc.272.47.29663

1083-351X

Beat Schwaller, Isabelle Durussel, Doris Jermann, Brigitte Herrmann, Jos A. Cox,

Glycosylation and Glycoproteins Research

Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found specifically in cancer cells, and possesses four EF-hands and a differently processed C-terminal end. The Ca2+-binding properties of recombinant human calretinin CR-22k were investigated by flow dialysis and spectroscopic methods and compared with those of CR. CR possesses four Ca2+-binding sites with positive cooperativity (n H = 1.3) and a [Ca2+]0.5 of 1.5 μm, plus one low affinity site with an intrinsic dissociation constant (K′ D ) of 0.5 mm. CR-22k contains three Ca2+-binding sites with n H of 1.3 and [Ca2+]0.5 of 1.2 μm, plus a low affinity site with K′ D of 1 mm. All the sites seem to be of the Ca2+-specific type. Limited proteolysis and thiol reactivity suggest that that the C terminus of full-length CR, but not of CR-22k, is in close proximity of site I leading to mutual shielding. Circular dichroism (CD) spectra predict that the content of α-helix in CR and CR-22k is similar and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k and CR, even though CR-22k possesses one additional Trp at the C-terminal end, and revealed that the Trp residues are organized into a hydrophobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+. The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedly enhanced by the two proteins already in the absence of Ca2+ and is further increased by binding of Ca2+. The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+. Together, our data suggest the presence of an allosteric conformational unit encompassing sites I–III for CR-22k and I–IV for CR, with a very similar conformation and conformational changes for both proteins. In the allosteric unit of CR, site IV is fully active, whereas in CR-22k this site has a 80-fold decreased affinity, due to the decreased amphiphilic properties of the C-terminal helix of this site. Some very specific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the "sensor"-type family of Ca2+-binding proteins. Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found specifically in cancer cells, and possesses four EF-hands and a differently processed C-terminal end. The Ca2+-binding properties of recombinant human calretinin CR-22k were investigated by flow dialysis and spectroscopic methods and compared with those of CR. CR possesses four Ca2+-binding sites with positive cooperativity (n H = 1.3) and a [Ca2+]0.5 of 1.5 μm, plus one low affinity site with an intrinsic dissociation constant (K′ D ) of 0.5 mm. CR-22k contains three Ca2+-binding sites with n H of 1.3 and [Ca2+]0.5 of 1.2 μm, plus a low affinity site with K′ D of 1 mm. All the sites seem to be of the Ca2+-specific type. Limited proteolysis and thiol reactivity suggest that that the C terminus of full-length CR, but not of CR-22k, is in close proximity of site I leading to mutual shielding. Circular dichroism (CD) spectra predict that the content of α-helix in CR and CR-22k is similar and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k and CR, even though CR-22k possesses one additional Trp at the C-terminal end, and revealed that the Trp residues are organized into a hydrophobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+. The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedly enhanced by the two proteins already in the absence of Ca2+ and is further increased by binding of Ca2+. The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+. Together, our data suggest the presence of an allosteric conformational unit encompassing sites I–III for CR-22k and I–IV for CR, with a very similar conformation and conformational changes for both proteins. In the allosteric unit of CR, site IV is fully active, whereas in CR-22k this site has a 80-fold decreased affinity, due to the decreased amphiphilic properties of the C-terminal helix of this site. Some very specific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the "sensor"-type family of Ca2+-binding proteins. Calretinin (CR), 1The abbreviations used are: CR, calretinin; CR-22k, calretinin-22k; CaM, calmodulin; TNS, 2-p-toluidinylnaphtalene-6-sulfonate; ESI-MS, electrospray ionization-mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis. an intracellular Ca2+-binding protein containing six EF-hand motifs (1Kretsinger R.H. Cold Spring Harbor Symp. Quant. Biol. 1987; 52: 499-510Crossref PubMed Scopus (158) Google Scholar), is most closely related to calbindin-D28k(2Rogers J. J. Cell Biol. 1987; 105: 1343-1353Crossref PubMed Scopus (500) Google Scholar). It is abundant in neuronal tissues, especially in the olfactory bulb (3Parmentier M. Lefort A. Eur. J. Biochem. 1991; 196: 79-85Crossref PubMed Scopus (76) Google Scholar) and auditory pathways. In the rat ventral cochlear nucleus, the CR content was found to be on the order of 6.4 μg/mg of protein; however, in other regions (paraventricular nucleus of the thalamus), CR contents of approximately 4 μg/mg of protein were measured (4Jacobowitz D.M. Winsky L. J. Comp. Neurol. 1991; 310: 198-218Crossref Scopus (348) Google Scholar, 5Strauss K.I. Jacobowitz D.M. Mol. Brain Res. 1993; 20: 229-239Crossref PubMed Scopus (27) Google Scholar). For calbindin-D28k, protein levels in auditory neurons were estimated to reach concentrations of up to 2 mm (6Oberholtzer J.C. Buettger C. Summers M.C. Matchinsky F.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3387-3390Crossref PubMed Scopus (75) Google Scholar) and the concentrations of CR are expected to be of the same order of magnitude. Its physiological function is not known, but seems to be related to Ca2+ buffering and diffusion (7Rogers J.H. Heizmann C.W. Novel Calcium-binding Proteins. Springer-Verlag, Berlin1991: 251-276Crossref Google Scholar). In the cochlea, CR could provide a strong, fast, and mobile buffer to cope with the unusually high, localized Ca2+ spikes (8Lenzi D. Roberts W.M. Curr. Opin. Neurobiol. 1994; 4: 496-502Crossref PubMed Scopus (53) Google Scholar). For both CR and calbindin-D28k, a role in neuroprotection from excitotoxic insults has been postulated (9Lukas W. Jones K.A. Neuroscience. 1994; 61: 307-316Crossref PubMed Scopus (144) Google Scholar), but the results remain quite controversial (for a review, see Ref. 10Andressen C. Blümcke I. Celio M.R. Cell Tissue Res. 1993; 271: 181-208Crossref PubMed Scopus (661) Google Scholar). Two recent studies have shown that important amounts of CR are associated with cell membranes (11Hubbard M. McHugh N.J. FEBS Lett. 1995; 374: 333-337Crossref PubMed Scopus (46) Google Scholar, 12Winsky L. Kuznicki J. J. Neurochem. 1995; 65: 381-388Crossref PubMed Scopus (74) Google Scholar), suggesting that besides the proposed role as Ca2+buffer, CR could also have specific target ligands in the membrane. Normal human colon epithelial cells do not express CR, but several colon carcinoma cell lines (e.g. WiDr) express this protein to various amounts (13Gotzos V. Schwaller B. Hetzel N. Bustos-Castillo M. Celio M.R. Exp. Cell Res. 1992; 202: 292-302Crossref PubMed Scopus (70) Google Scholar, 14Gander J.-C. Bustos-Castillo M. Stüber D. Hunziker W. Celio M.R. Schwaller B. Cell Calcium. 1996; 20: 63-72Crossref PubMed Scopus (18) Google Scholar). In several cell lines, alternatively spliced CR mRNA leading to C-terminally truncated proteins have been detected (15Schwaller B. Celio M.R. Hunziker W. Eur. J. Biochem. 1995; 230: 424-430Crossref PubMed Scopus (30) Google Scholar) and the presence of one of the splice products, calretinin-22k, has been demonstrated. To date, the alternatively spliced mRNAs have been detected only in cancer cells but not in the cells normally expressing CR. It has been hypothesized that the splice products could, in part, be responsible for the phenotype of transformed cells. One of the splice products derived from CR mRNA lacking exons 8 and 9 (Δ8,9) and coding for a protein named calretinin-22k (CR-22k), was found in several different colon carcinoma cell lines, indicative of a particular "physiological" function (14Gander J.-C. Bustos-Castillo M. Stüber D. Hunziker W. Celio M.R. Schwaller B. Cell Calcium. 1996; 20: 63-72Crossref PubMed Scopus (18) Google Scholar). Human CR-22k comprises the first four EF-hands of full-length calretinin, including the first 3 amino acid residues of the C-terminal helix of site IV followed by a segment of 14 residues which, due to a frameshift, are completely different from those present in bona fide calretinin. Thus, four potentially functional EF-hands are present in CR-22k, but the fourth site is different from the one in CR. In the frame of the function of CR and CR-22k as buffers, it is important to know how sensitive and selective they are toward Ca2+, especially since the alternative splicing caused quite dramatic effects in the C-terminal sites. If they are also activators, it is also important to monitor the type of the conformational changes upon binding of Ca2+ and establish the differences between CR and CR-22k. In the present report, human CR-22k was expressed and its Ca2+-binding properties and conformational changes are described. These properties were systematically compared with those of the full-length recombinant CR. The expression vectors coding for the fusion proteins named 6xHis-CR-22k and 6xHis-CR, which contain the affinity tag MRGS(H)6GS at the N terminus, have been described previously (14Gander J.-C. Bustos-Castillo M. Stüber D. Hunziker W. Celio M.R. Schwaller B. Cell Calcium. 1996; 20: 63-72Crossref PubMed Scopus (18) Google Scholar). Briefly, the cDNA of clone CR16.17 coding for CR-22k was cut with SspI (this site is located 35 nucleotides downstream of the stop codon of CR-22k), the site was filled by Klenow enzyme, and the plasmid further digested withNcoI. The NcoI site (CCATGG) contains the start codon ATG for CR-22k. The vector pDS56.1/RBSII 6xHis,NcoI-(HindIII, filled) was used to ligate the isolated fragment. For the 6xHis-CR vector, the cDNA clone CR3.9 was digested with NcoI and HindIII and the isolated fragment containing the entire reading frame of CR and including the 3′-nontranslating region of the CR cDNA was cloned into the same expression vector as above (pDS56.1/RBSII 6xHis,NcoI-HindIII). To remove the affinity tag, a fragment coding for the recognition site for protease Xa was cloned in between the 6xHis tag and the CR or CR-22k cDNAs (Fig.1). For this reason, two complementary oligodeoxynucleotides with NcoI half-sites were synthesized that code for the peptide Ile-Glu-Gly-Arg-Ser, and this linker was inserted into the NcoI site. After transformation and plating, positive clones from each ligation were chosen according to several restriction enzyme digests. The correctness of the sequence of the introduced linker region was verified by dedeoxy sequencing.Escherichia coli cultures were grown in TB medium, induced by isopropyl-1-thio-β-d-galactopyranoside, and further grown for 4 h at 37 °C. After pelleting the E. colicultures (3000 × g, 30 min, 4 °C), the pellets were solubilized in 8 m guanidine HCl and the overexpressed proteins purified on a nickel chelate column as described (16Stüber D. Matile H. Garotta G. Lefkovitz I. Parnis B. System for High-level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies and Structure-Function Analysis. Immunological Methods. IV. Academic Press, New York1990: 121-152Google Scholar). Fractions containing the fusion proteins were analyzed by SDS-PAGE (12.5%). For the removal of the affinity tag, the purified fusion proteins were dialyzed in 20 mm Tris, pH 8.0, 100 mm NaCl, 2 mm CaCl2 (TNC buffer). MRGS(H)6GSIEGRS-CR was digested with protease Xa (New England Biolabs) at a ratio of 1:2000 for 4–6 h at 37 °C and MRGS(H)6GSIEGRS-CR-22k at a protease ratio of 1:200 for 30 min at 37 °C. Samples were loaded onto the nickel chelate column, and the flow-through was collected. Undigested protein and the affinity tag were eluted from the column by lowering the pH to 4.0. The purity of the eluted proteins CR-22k and CR was checked by SDS-PAGE. For the kinetics experiments, the proteins containing the affinity tag were incubated in TNC-buffer, protease Xa was added (1:200, w/w), and after different times small aliquots were removed, boiled in SDS sample buffer, and separated on a 12.5% gel. The procedure was similar to the protocol described for rat CR (27Kuznicki J. Wand T.-C. Martin B.M. Winsky L. Jacobowitz D.M. Biochem. J. 1995; 308: 607-612Crossref PubMed Scopus (32) Google Scholar). Briefly, purified CR and CR-22k (0.25 mg/ml) were incubated at 37 °C with trypsin (sequencing grade, Boehringer Mannheim) at a ratio of 1:100 (w/w) in buffer A (50 mm Tris-HCl, 150 mm KCl, pH 7.5) containing either 1 mm CaCl2 or 1 mm EGTA. Samples were digested for 0.5–50 min, and the digestion was blocked by the addition of soybean trypsin inhibitor (Boehringer Mannheim) at a ratio 1:2 (enzyme:inhibitor). The digested fragments were separated by SDS-PAGE (15%) and stained by Coomassie Blue. Protein samples were desalted using capillary reverse phase-HPLC (Hewlett Packard, Waldbronn, Germany, model 1090, LC packings POROS R2/H, cartridge 5 mm × 0.8 mm) and an acetonitrile, 0.1% trifluoroacetic acid gradient for elution. The desalted fractions were lyophilized, taken up in 10 μl of acetonitrile, 1 m acetic acid (1:1), and flow-injected with the HPLC system into a Perkin Elmer/Sciex (Concord, Canada) model API-300 mass spectrometer. Spectra were obtained at a flow rate of 10 μl/min in positive ion mode. Matrix-assisted laser desorption ionization mass spectrometry was performed on a Perseptive Biosystems Voyager Elite instrument (Framingham, MA) equipped with a time-of-flight mass analyzer (MALDI-TOF). For removal of contaminating metals and for complete equilibration of the protein in the assay buffer, the proteins were either extensively dialyzed against 1 mm EDTA-containing buffer or precipitated with 3% trichloroacetic acid, redissolved in 50 mm Tris-HCl, pH 7.5, 1 mm β-mercaptoethanol, and then passed through a 40 × 0.8-cm Sephadex G-25 column equilibrated in buffer A. Total Ca2+ and Mg2+concentrations were determined with a Perkin-Elmer 2380 atomic absorption spectrophotometer. The protein concentration was determined spectrophotometrically using a molar extinction coefficient at 276 nm of 26,100 and 26,900 m−1 cm−1 for CR-22k and CR, respectively. Ca2+ binding was measured at 25 °C by the flow dialysis method (17Colowick S.P. Womack F.C. J. Biol. Chem. 1969; 244: 774-777Abstract Full Text PDF PubMed Google Scholar) in 50 mmTris-HCl, pH 7.5, 150 mm KCl. Protein concentrations were 25–35 μm. Treatment of the raw data and evaluation of the intrinsic metal-binding constants was as described (18Cox J.A. Celio M. Guidebook to the Calcium Binding Proteins. Oxford University Press, Oxford1996: 1-14Google Scholar). The accuracy of the flow dialysis method is estimated to be in the order of 100 ± 10%. Since the binding isotherms show positive and negative cooperativity, the data were analyzed with the equation of Adair. ν={K1[Ca2+]+2K1K2[Ca2+]2+nK1K2…Kn[Ca2+]n}/{1+K1[Ca2+]+K1K2[Ca2+]2+K1K2…Kn[Ca2+]n}Equation 1 K 1, K 2, andK n are the stoichiometric association constants for the binding of the first, second, and nth Ca2+. The intrinsic constants were calculated from the stoichiometric ones using the appropriate statistical factors (18Cox J.A. Celio M. Guidebook to the Calcium Binding Proteins. Oxford University Press, Oxford1996: 1-14Google Scholar). Punctuate binding experiments were carried out with the equilibrium gel filtration method of Hummel and Dryer (19Hummel J.P. Dryer W.J. Biochim. Biophys. Acta. 1962; 63: 530-532Crossref PubMed Scopus (934) Google Scholar). Emission fluorescence spectra were taken with a Perkin-Elmer LS-5B spectrofluorimeter on 1 μmsolutions of the metal-free proteins in buffer A at room temperature with excitation wavelength at 278 nm and slits of 5 nm. 20 μm EGTA, 5 mm MgCl2, 1 mm CaCl2, and 4 m guanidine HCl were added subsequently to obtain the metal-free, Mg2+, Ca2+, and denatured forms, respectively. Buffer and guanidine HCl contribution were subtracted. Stability curves were obtained by monitoring the Trp fluorescence changes upon titration of the metal-free proteins with guanidine HCl. UV-absorption spectra and difference spectra were measured with a Perkin-Elmer λ 5 spectrophotometer at room temperature. The metal-free protein solution (40 μm) was equilibrated in buffer A and 20 μm EGTA, 5 mmMgCl2, 1 mm CaCl2, and 4m guanidine HCl were added subsequently to obtain the metal-free, Mg2+, Ca2+, and fully denatured forms, respectively. Difference spectra were taken on solutions with an optical density at 278 nm of 1 and normalized to an optical density of 1.0 at 278 nm. Circular dichroism (CD) spectra were acquired with a Jasco J-715 spectropolarimeter on solutions of 0.25 mg/ml protein in 5 mm phosphate buffer, pH 7.5, in a cell of 1 mm optical path. Ellipticities were normalized to residue concentration using the relationship [θ] = θob/(lcN), where [θ] is the mean residue ellipticity, θob the observed ellipticity, l the path length (in mm), c the molar concentration of the protein, and N the total number of residues in the protein. The Ca2+- and Mg2+-dependent changes in hydrophobic surface of CR-22k and CR were followed by monitoring the fluorescence properties of TNS as described (20McClure W.O. Edelman G.M. Biochemistry. 1966; 5: 1908-1919Crossref PubMed Scopus (431) Google Scholar). Solutions of 30 μmTNS and 2 μm metal-free protein were excited at 328 nm and the emission spectra recorded with slits of 5 nm. EGTA (20 μm), MgCl2 (5 mm), or CaCl2 (1 mm) were added to obtain the metal-free, Mg2+, or Ca2+ forms, respectively. The thiol reactivity was assayed on CR-22k and CR that were previously reduced by overnight incubation with 100 mm dithiothreitol at pH 8.5 and passed on a Sephadex G-25 column equilibrated in nitrogen-saturated buffer A. The thiol reactivity was monitored by measuring the kinetics of the reduction of Ellman's reagent by spectrophotometry at 412 nm according to Riddles et al. (21Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; 91: 49-60Crossref PubMed Scopus (1073) Google Scholar). The protein 6xHis-CR-22k was overexpressed and purified to homogeneity on a nickel chelate column as described previously (14Gander J.-C. Bustos-Castillo M. Stüber D. Hunziker W. Celio M.R. Schwaller B. Cell Calcium. 1996; 20: 63-72Crossref PubMed Scopus (18) Google Scholar) and additionally characterized by MALDI-TOF and ESI-MS. The molecular mass estimated by MALDI-TOF was 23,572, which was slightly too high due to a small amount of dimer M22+ not separated from the peak of M+. The M r of 6xHis-CR-22k was additionally measured by ESI-MS, and the calculated mass of 23,555 ± 1.5 Da was as expected from the calculated molecular weight. Since an interference by the 6xHis affinity tag on the cation binding properties cannot be ruled out, an expression system was developed, with a recognition sequence for protease Xa between the 6xHis affinity tag and the CR or CR-22k protein. After cleavage with protease Xa, CR-22k and CR were identical to the human recombinant proteins except for one additional serine residue at the N terminus. The two proteins were produced in E. coli and purified as described under "Materials and Methods," and SDS gels of the purified proteins CR and CR-22k are shown in Fig. 2. The size difference between CR-22k and CR before and after cleavage at the protease Xa cleavage site is clearly visible and is a result of the removal of the peptide MRGS(H)6GSMIEGR from the N termini of both proteins. In preliminary proteolytic experiments during the purification of CR-22k, a protein band appeared of approximately 17–18 kDa, significantly smaller than the expected size of CR-22k (Fig. 2). Amino acid sequence comparison revealed that segment 36–39 2The numbering used is that of the native protein. (Ile-Glu-Gly-Lys) in the loop of EF-hand site I is almost identical to the sequence recognized by protease Xa (Ile-Glu-Gly-Arg). Since this cleavage was not detected during the purification of CR, we determined the time course of the digestion of both proteins. Whereas for CR-22k the cleavage was complete after 20 min, in the case of CR undigested precursor protein was still present after 40 min (Fig. 2). Prolonged incubation of CR with protease Xa did not lead to the internal cleavage presumably after amino acid Lys-39, whereas the cleavage of CR-22k at this site was complete under identical conditions (Fig. 2). The different kinetics of cleavage at the introduced specific site and the different sensitivity toward the internal cleavage site demonstrated that the accessibility for the protease Xa was different for the two proteins in the region of Ca2+-binding site I. Trypsin digestion experiments in the presence or absence of Ca2+ with CR-22k containing the affinity tag are shown in Fig. 3 (A and B). In the presence of 1 mm Ca2+, the protein was cleaved at Lys-60, giving rise to fragments of 15 and 7 kDa. The 15-kDa fragment was then further cleaved yielding a fragment of 11 kDa. In the absence of Ca2+, several fragments were already visible after 0.5 min and also the 15-kDa fragment was rapidly cleaved into very short fragments. When the digestion experiment was repeated on CR-22k from which the affinity tag had been previously removed, the pattern (not shown) was identical to that shown in Fig. 3 (Aand B). Since in the latter experiment time points were chosen so that many different fragments accumulated, these fragments could be analyzed by ESI-MS; the results are listed in TableI and Fig. 3 C. In the presence of Ca2+, CR-22k was preferentially cleaved at Lys-60, while in the apo form the protein was additionally cleaved at residues Lys-141 and Lys-170, as well as at other sites, which could not be unambiguously identified.Table ITryptic fragments of CR-22k and CR identified by ESI-MSProtein fragmentsAmino acid1-apositions corresponding to the published sequence; the first Met (ATG) corresponds to residue 1; −1 denotes the additional Ser residue at the amino terminus of the purified protein. Calculated sizes >1000 Da are average molecular weights, while for the fragment 1000 Da are average molecular weights, while for the fragment 0.2) at 236 nm (not shown). The Phe environment does not seem to be sensitive to Ca2+binding, although the fo