Biochemical Characterization of the Penta-EF-hand Protein Grancalcin and Identification of L-plastin as a Binding Partnerстатья из журнала
Аннотация: Grancalcin is a recently described Ca2+-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca2+ although it has an unusual Ca2+ coordination loop and one that does not bind Ca2+ but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca2+ load. Flow dialysis showed that recombinant grancalcin binds two Ca2+ per subunit with positive cooperativity and moderate affinity ([Ca2+]0.5 of 25 and 83 μm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca2+-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca2+ and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca2+-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa. Grancalcin is a recently described Ca2+-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca2+ although it has an unusual Ca2+ coordination loop and one that does not bind Ca2+ but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca2+ load. Flow dialysis showed that recombinant grancalcin binds two Ca2+ per subunit with positive cooperativity and moderate affinity ([Ca2+]0.5 of 25 and 83 μm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca2+-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca2+ and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca2+-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa. penta-EF-hand octyl-β-glucopyranoside polyacrylamide gel electrophoresis 1,4-piperazinediethanesulfonic acid fast protein liquid chromatography high pressure liquid chromatography disuccinimidyl glutarate Grancalcin is a recently described protein present in some cells of hematopoietic origin and especially abundant in human neutrophils (1Teahan C.G. Totty N.F. Segal A.W. Biochem. J. 1992; 286: 549-554Crossref PubMed Scopus (36) Google Scholar, 2Boyhan A. Casimir C.M. French J.K. Teahan C.G. Segal A.W. J. Biol. Chem. 1992; 267: 2928-2933Abstract Full Text PDF PubMed Google Scholar, 3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar). Grancalcin belongs to the calpain subfamily of EF-hand Ca2+-binding proteins, comprising calpain light and heavy chain, sorcin, grancalcin, ALG-2, peflin, and YG25-yeast (4Maki M. Narayana S.V.L. Hitomi K Biochem. J. 2017; : 718-720Google Scholar, 5Kitaura Y. Watanabe M. Satoh H. Kawai T. Hitomi K. Maki M. Biochem. Biophys. Res. Commun. 1999; 263: 68-75Crossref PubMed Scopus (35) Google Scholar). So far, the members of this subfamily seem to have diverse functions: calpain functions as a protease and can regulate adhesion, sorcin binds to and regulates the cardiac ryanodine calcium channel, ALG-2 plays a role in apoptosis, and the function of grancalcin is unknown. From amino acid sequence comparison, it was initially deduced that these proteins possess four EF-hand motifs (6Nakayama S. Kretsinger R.H. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 473-507Crossref PubMed Scopus (180) Google Scholar). However, recent studies on the three-dimensional structure of dVI, the Ca2+-binding domain of calpain light chain (7Lin G. Chattopadhyay D. Maki M. Wang K.K.W. Carson M. Jin L. Yuen P. Takano E. Hatanaka M. DeLucas L.J. Narayana S.V.L. Nat. Struct. Biol. 1997; 4: 539-547Crossref PubMed Scopus (178) Google Scholar, 8Blanchard H. Grochulski P. Li Y. Arthur S.C. Davis P.L. Elce J.S. Cygler M. Nat. Struct. Biol. 1997; 4: 532-538Crossref PubMed Scopus (182) Google Scholar), surprisingly revealed the presence of a fifth EF-hand N-terminal to the previously identified EF-hands. This EF-hand is of a novel type and has therefore escaped detection by sequence similarity analysis. This group of proteins is thus characterized by five EF-hands and has accordingly been named the penta-EF-hand (PEF)1subfamily (4Maki M. Narayana S.V.L. Hitomi K Biochem. J. 2017; : 718-720Google Scholar). In addition, the members of the PEF subfamily contain N termini of varying length and sequence but all rich in glycine and hydrophobic residues. In calpain, EF1 (although of an unusual sequence composition) and EF2 are paired and bind two Ca2+ with high affinity. EF3 and EF4 are also paired and possess one site of high affinity and one of low affinity for Ca2+. Finally, EF5, which does not bind Ca2+, is paired with a similar EF5 of another monomer and is thus a dimerization module. Native calpain is a heterodimer of the calpain light and heavy chain (9Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. Biochem. Biophys. Res. Commun. 1995; 208: 376-383Crossref PubMed Scopus (80) Google Scholar). The recent crystallization of apograncalcin (10Han Q. Jia J. Li Y. Lollike K. Cygler M. Acta Crystallogr. Sec. D. 2000; 56: 772-774Crossref PubMed Scopus (6) Google Scholar, 11Jia J. Han Q. Borregaard N. Lollike K. Cygler M. J. Mol. Biol. 2000; 300: 1271-1281Crossref PubMed Scopus (42) Google Scholar) has shown great overall similarity to the crystal structure of calpain. Grancalcin was also found to form dimers by paring of cognate EF5s. Unfortunately, grancalcin precipitated in the presence of Ca2+ and the topology of active Ca2+-binding sites could not be determined, except for binding of Ca2+ to EF3 from one monomer. Moreover, the N terminus of grancalcin is disordered in the crystals, and the first well defined residue is Ser53. In molecular sieve chromatography, purified grancalcin migrates as a homodimer (1Teahan C.G. Totty N.F. Segal A.W. Biochem. J. 1992; 286: 549-554Crossref PubMed Scopus (36) Google Scholar) and ALG-2 as a monomer (12Maki M. Yamaguchi K. Kitaura Y. Satoh H. Hitomi K. J. Biochem. 1998; 124: 1170-1177Crossref PubMed Scopus (52) Google Scholar, 13Lo K.W.H. Zhang Q. Li M. Zhang M. Biochemistry. 1999; 38: 7498-7508Crossref PubMed Scopus (71) Google Scholar). However, when using a chemical cross-linker, ALG-2 could be shown to exist also as a dimer (13Lo K.W.H. Zhang Q. Li M. Zhang M. Biochemistry. 1999; 38: 7498-7508Crossref PubMed Scopus (71) Google Scholar). The cDNA for grancalcin is 1.65 kilobase pairs long and contains an open reading frame for 217 amino acids; the first 14 amino acids have been reported to be removed posttranslationally to give rise to a 203-residue-long functional protein (2Boyhan A. Casimir C.M. French J.K. Teahan C.G. Segal A.W. J. Biol. Chem. 1992; 267: 2928-2933Abstract Full Text PDF PubMed Google Scholar). This presumably functional grancalcin has a calculated molecular mass of 22.4 kDa, while grancalcin migrates as a 28-kDa protein in SDS-PAGE (1Teahan C.G. Totty N.F. Segal A.W. Biochem. J. 1992; 286: 549-554Crossref PubMed Scopus (36) Google Scholar, 2Boyhan A. Casimir C.M. French J.K. Teahan C.G. Segal A.W. J. Biol. Chem. 1992; 267: 2928-2933Abstract Full Text PDF PubMed Google Scholar). The reason for this discrepancy is unknown, but it is not due to glycosylation (1Teahan C.G. Totty N.F. Segal A.W. Biochem. J. 1992; 286: 549-554Crossref PubMed Scopus (36) Google Scholar). Grancalcin translocates to membranes upon binding of Ca2+ (2Boyhan A. Casimir C.M. French J.K. Teahan C.G. Segal A.W. J. Biol. Chem. 1992; 267: 2928-2933Abstract Full Text PDF PubMed Google Scholar, 3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar, 14Borregaard N. Kjeldsen L. Lollike K. Sengeløv H. FEBS Lett. 1992; 304: 195-197Crossref PubMed Scopus (27) Google Scholar). This is a common feature of several EF-hand proteins. Translocation is often mediated by exposure of hydrophobic patches of amino acids subsequent to Ca2+binding, as is the case for calpain (15Takeyama Y. Nakizishi H. Uratsuji Y. Kishimoto A. Nishizuka Y. FEBS Lett. 1986; 194: 110-114Crossref PubMed Scopus (37) Google Scholar) and calretinin (16Winsky L. Kuznicki J. J. Neurochem. 1995; 65: 381-388Crossref PubMed Scopus (73) Google Scholar). In other instances, such as in the recoverin family, a covalently bound fatty acid becomes exposed after Ca2+-induced conformational changes, and this leads to translocation of the protein to membranes, a mechanism also called the myristoyl switch (17Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (293) Google Scholar). Which of these molecular schemes prevails for grancalcin is at present unknown. EF-hand proteins have affinities for Ca2+ varying from 108 to 103m−1. Moreover, some show great selectivity toward Ca2+, such as calmodulin, S100 proteins, and calretinin, whereas others also bind Mg2+, such as parvalbumin and recoverin (for a review, see Ref. 18Cox J.A. Celio M.R. Pauls T. Schwaller B. Guidebook to the calcium-binding Proteins. Oxford University Press, Oxford1996: 1-12Google Scholar). Neither the affinity nor the selectivity can be deduced from the sequence of the EF-hands. Except for a positive 45Ca2+ overlay, no information on the ion-binding and conformational changes of grancalcin is available. Because the biochemical characterization of grancalcin has been very sparse and nothing is known about the function of grancalcin, we decided to address these issues. In this report, we have identified a N-terminal posttranslational modification of wild-type grancalcin by mass spectroscopy and sequencing. Because grancalcin precipitates in the presence of Ca2+ in most experiments, we have generated three mutants of grancalcin with varying N-terminal deletions. We show that these mutants do not precipitate to the same degree as recombinant grancalcin. We have also compared critical properties of wild type and recombinant grancalcin, determined the binding characteristics of grancalcin for Ca2+ by flow dialysis, and probed cation induced conformational changes by fluorometry of the intrinsic Trp residues. Finally, using affinity chromatography, we identify the actin-bundling protein L-plastin as binding partner of grancalcin and document the Ca2+ dependence of this interaction. Human neutrophils were isolated from volunteer donors as described (19Boyum A. Scand. J. Clin. Lab. Invest. 1968; 21: 77-90Crossref PubMed Scopus (990) Google Scholar). In short, erythrocytes were sedimented by 2% dextran (Amersham Pharmacia Biotech) in saline, and the leukocyte-rich supernatant was submitted to 400 × g density centrifugation for 30 min on Lymphoprep (Nycomed, Oslo, Norway). The pellet was submitted to hypotonic lysis of contaminating erythrocytes for 30 s in pure water, after which tonicity was restored by the addition of NaCl. The neutrophils were washed once, counted, and resuspended in saline. SDS-PAGE (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar) and immunoblotting (21Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44841) Google Scholar) were performed on Bio-Rad systems according to the instructions given by the manufacturer (Bio-Rad) and as described (3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar), except for visualization of immunoblots, which was by metal-enhanced diaminobenzidine tetrahydrochloride (Pierce). Crude protein concentration was determined by the method of Bradford according to the instructions given by the manufacturer (Bio-Rad), and catalase ranging from 0.05 to 0.5 mg/ml was used as a standard. Grancalcin was quantified by enzyme-linked immunosorbent assay as previously described (3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar). Purified grancalcin was quantified by the ultraviolet absorption spectrum using a molar extinction coefficient ε278 nm of 28,000m−1cm−1. Isolated neutrophils at 3 × 107 cells/ml were incubated in saline with 5 mm diisopropyl fluorophosphate (Sigma) for 5 min on ice and centrifuged at 200 × g for 6 min. Cell pellets were resuspended at 2 × 108 cells/ml in binding buffer (100 mm KCl, 3 mm NaCl, 10 mm PIPES (pH 7.2)) containing 0.5 mmphenylmethylsulfonyl fluoride, 200 units/ml aprotinin, and 100 μg/ml leupeptin (all three Sigma-Aldrich) and disrupted by nitrogen cavitation at 400 p.s.i. for 5 min at 4 °C as described (22Borregaard N. Heiple J.M. Simons E.R. Clark R.A. J. Cell Biol. 1983; 97: 52-61Crossref PubMed Scopus (652) Google Scholar). The cavitate was ultracentrifuged at 100,000 × g for 45 min, and the supernatant was kept as cytosol. Human neutrophil cytosol (prepared as above) in binding buffer plus 0.5 mm CaCl2 was purified on a self-packaged phenyl-Sepharose (Amersham Pharmacia Biotech) column at room temperature. After binding of cytosolic proteins, the column was washed thoroughly in binding buffer plus 0.5 mm CaCl2. Bound proteins were eluted in fractions of 2 ml with binding buffer plus 5 mm EGTA. Fractions containing grancalcin (as determined by SDS-PAGE) were submitted to a buffer change to 20 mm Trizma (pH 7.4) using a Centriprep column (Amicon, Beverly, MA) and passed through a Mono-Q (Amersham Pharmacia Biotech) column coupled to an FPLC instrument (Amersham Pharmacia Biotech). Bound proteins were eluted with a 0–1 m NaCl nonlinear gradient, and 0.5-ml fractions were evaluated by SDS-PAGE and immunoblotting. Recombinant grancalcin was produced and purified as previously described (3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar). N-terminal deletion mutants were cloned from the grancalcin clone using the following primers for the N terminus: Δ42-grancalcin, 5′-CGC GGA TCC GCA TAT TCA GAC ACT TAT TCC-3′; Δ50-grancalcin, 5′-CGC GGA TCC GCT GGT GAC TCC GTG TAT AC-3′; and Δ53-grancalcin, 5′-CGC GGA TCC TCC GTG TAT ACT TTC AGT G-3′. For all three mutants, the following primer was used for the C terminus: 5′-CCG GAA TTC TCA AAT TGC CAT AGT GCC CTG C-3′. The obtained clones and the vector (pGEX2T (Pharmacia Amersham Biotech)) were cut with BamHI and EcoRI and, following ligation, transformed into Escherichia coli. The expressed clones were checked by sequencing (model 377 DNASequencer (PE Biosystems, Foster City, CA)), and only clones with correct sequences were used for protein expression. Procedures for expression and purification of N-terminal deletion mutants were as described for recombinant grancalcin (3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar). For the identification of the N-terminal modification in wild type grancalcin, 20 μg of both wild type and recombinant grancalcin was incubated with 0.5 μg of endoproteinase Glu-C (Roche Molecular Biochemicals) in 500 μl 50 mm NH4HCO3 (pH 7.8) including 10% acetonitrile for 20 h at room temperature. The reaction was stopped by the addition of 1 ml 1% trifluoroacetic acid. The resulting fragments were purified by HPLC on a 2.1 × 150-mm C-4 column (Vydac, Hesperia, CA) eluted with a linear gradient (1% per min) from solvent A (0.1% trifluoroacetic acid) to solvent B (0.1% trifluoroacetic acid in acetonitrile). Peak fractions were collected manually. The putative N-terminal fragment was localized by mass spectrometry, dried, and submitted to cleavage with 0.1 μg of thermolysin (Roche Molecular Biochemicals) in 100 μl of buffer (100 mm NH4HCO3, 5 mmCaCl2 (pH 7.8) including 10% actonitrile) for 3 h at 37 °C. The reaction was stopped by the addition of 400 μl of 1% trifluoroacetic acid. The resulting fragments were purified by HPLC on a 2.1 × 150-mm C-8 column (Vydac, Hesperia, CA) as described above. The purified peptides were analyzed in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Biflex, Bruker-Franzen, Bremen, Germany). For analysis, 0.5 μl of the sample was mixed with 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid in acetonitrile/methanol; Hewlett Packard), and 0.5 μl of the mixture was applied to the probe and allowed to dry. The purified proteins (wild-type grancalcin and recombinant grancalcin) were diluted 4–10 times with a 20 mm octyl-β-glucopyranoside (OG) (Roche Molecular Biochemicals) solution in formic acid/H2O/acetonitrile (1:3:2, by volume) before mixing with the matrix. Most samples were analyzed in the positive mode. Spectra were recorded for 20–100 laser shots. The method has an accuracy of 0.1%. The amino acid sequences of the purified peptides were determined employing an automatic protein sequencer (model 494A; PerkinElmer Life Sciences) equipped with an online HPLC system for detection of the amino acid-phenylthiohydantoin derivatives. All chemicals and solvents were sequence or HPLC grade supplied by PerkinElmer Life Sciences. 200 μl of recombinant protein (10 μm) or cytosol in binding buffer with or without 0.5 mm Ca2+ was applied to a Superdex 75 column (FPLC system; Amersham Pharmacia Biotech) equilibrated in binding buffer with or without 0.5 mm Ca2+, and eluted fractions were analyzed by absorbance and/or grancalcin enzyme-linked immunosorbent assay. The following markers were used to calibrate the column: blue dextran 2000 (>2,000,000), ribonuclease A (13,700), chymotrypsinogen A (25,000), and ovalbumin (43,000). Proteins were cross-linked with the chemical cross-linker DSG (Pierce) essentially as described (13Lo K.W.H. Zhang Q. Li M. Zhang M. Biochemistry. 1999; 38: 7498-7508Crossref PubMed Scopus (71) Google Scholar). In short, proteins were incubated for 30 min at room temperature in 100 μl of cross-linking buffer (20 mm HEPES (pH 8.0), 50 mm NaCl, and 1 ml of dithiothreitol) to which 5 μl of DSG was added (from 10 mm stock in N, N-dimethylformamide). The reaction was quenched by the addition of 125 μl of 1 m Tris-HCl, pH 7.5, and cross-linking was evaluated by immunoblotting. Recombinant grancalcin at 2 or 10 μm and Δ42-grancalcin and Δ53-grancalcin at 10 μm in binding buffer (with or without 25 mm OG) were supplemented with varying concentrations of Ca2+ and rotated end over end for 30 min at room temperature. The tubes were centrifuged at 14,800 × gfor 5 min. at 4 °C, and the supernatants were assayed by absorption to determine protein concentrations. Since the method of trichloroacetic acid precipitation (14Borregaard N. Kjeldsen L. Lollike K. Sengeløv H. FEBS Lett. 1992; 304: 195-197Crossref PubMed Scopus (27) Google Scholar) irreversibly modified the Trp fluorescence spectra of grancalcin, the concentrated protein sample was supplemented with 1 mm EGTA, dialyzed overnight against buffer A containing 0.1 mm EGTA, and passed through a Sephadex G25 column (0.8 × 40 cm) equilibrated in buffer A (50 mm Tris-HCl buffer (pH 7.5), 150 mm KCl). The protein contained less than 0.1 mol of Ca2+/mol. Ca2+ binding was measured on the recombinant protein at 25 °C by the flow dialysis method (23Colowick S.P. Womack F.C. J. Biol. Chem. 1969; 244: 774-777Abstract Full Text PDF PubMed Google Scholar) in buffer A. Protein concentrations were 25 μm. Processing of data and evaluation of the binding constants were as described (18Cox J.A. Celio M.R. Pauls T. Schwaller B. Guidebook to the calcium-binding Proteins. Oxford University Press, Oxford1996: 1-12Google Scholar). Emission fluorescence spectra were obtained in a PerkinElmer Life Sciences LS-5B spectrofluorometer. The measurements were carried out at 25 °C on 4 μm wild-type or recombinant grancalcin in buffer A in the presence of 37.5 mm OG with an excitation wavelength at 278 nm and both slits at 5 nm. Measurements were performed in the presence of either 50 μm EGTA, 0.5 mmCaCl2 or 4 m guanidine-HCl, respectively. Recombinant grancalcin at 1 mg/ml in the presence of 0.5 mm CaCl2 or 0.5 mm EGTA was coupled to CNBr-activated Sepharose™ according to the manufacturer's instructions (Amersham Pharmacia Biotech). Three columns were packaged: one containing recombinant grancalcin in the presence of Ca2+, one containing recombinant grancalcin without calcium, and one without any protein present. Purified human neutrophils at 3 × 107 cells/ml were solubilized in solubilization buffer (10 mm HEPES (pH 7.4), 100 mm KCl, 25 mm OG, 0.2% cetyltrimethylammonium bromide, 1 mm phenylmethylsulfonyl fluoride, 200 units/ml aprotinin, 100 μg/ml leupeptin, and 100 μm EGTA) and incubated overnight at 4 °C. Unsolubilized material were spun down (5000 × g for 10 min), and 10 ml of supernatant, supplemented with 0.5 mm EGTA, were applied to each to each of the three columns. The columns were washed extensively in binding buffer plus 0.5 mm EGTA, and bound proteins were eluted with binding buffer plus 5 mm CaCl2. Fractions of 1 ml were collected and evaluated by SDS-PAGE. Fractions with a visible protein band were pooled and concentrated by centrifugation and subjected to SDS-PAGE. The identified band was cut out of the SDS gel and digested with trypsin essentially as described by Wilm et al. (24Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1505) Google Scholar). In short, the gel piece was cut into small cubes and washed for 1 h in 100 mmNH4HCO3 (AB), and excess liquid was removed. Then the proteins were reduced for 30 min at 60 °C in 100 μl of 4 mm dithiothreitol in 100 mm AB, cooled, and alkylated by the addition of 10 μl of 100 mmiodoacetamide followed by a 30-min incubation at room temperature in the dark. Excess liquid was removed, and the gel pieces were washed for 1 h in 50% acetonitrile in 100 mm AB followed by shrinkage in acetonitrile and vacuum centrifugation. The tube with the dry gel pieces was placed in an ice bath and allowed to swell in 25 mm AB including 2 μg/100 μl modified trypsin (Promega). After 45 min, the excess liquid was removed, and buffer was added to cover the gel pieces during overnight incubation at 37 °C. The next day, the liquid was removed and combined with two consecutive extractions with 50 μl of 0.1% trifluoroacetic acid in 60% acetonitrile. The extract was dried in a vacuum centrifuge to near dryness followed by the addition of 5 μl of 0.1% trifluoroacetic acid in 30% acetonitrile. For analysis, 0.4 μl of the sample was mixed with 0.1 μl of internal standard mixture (angiotensin II and dynorphin, 0.06 and 1 pmol/μl, respectively) and 0.5 μl matrix solution (α-cyano-4 hydroxycinnamic acid) and measured by mass spectrometry as described above, except the instrument was upgraded with the time lag focusing option (delayed extraction). The peptide molecular mass fingerprint was used for a search of the SWISS-PROT and TrEMBL data base using the "PeptIdent" tool on the ExPASy Molecular Biology Server of the Swiss Institute of Bioinformatics (25Appel R.D. Bairoch A. Hochstrasser D.F. Trends Biochem. Sci. 1994; 19: 258-260Abstract Full Text PDF PubMed Scopus (512) Google Scholar). Cytosol from human neutrophils (prepared as described above) was diluted with binding buffer to a final concentration of 1 mg/ml (as determined by Bio-Rad). 5 μl of anti-grancalcin or anti-L-plastin antibodies (two different antibodies; LPL4A1, which binds to L-plastin irrespective of Ca2+ load, and LPL7,2, which only binds to the Ca2+-loaded form of L-plastin (described in Ref. 26Jones S.L. Brown E.J. J. Biol. Chem. 1996; 271: 14623-14630Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), a kind gift from Dr. Eric J. Brown (Division of Infectious Disease, Howard Hughes Medical Institute, St. Louis, MO)) were added to 1 ml of the diluted cytosol in the presence of 0.5 mm CaCl2 or 5 mm EGTA, respectively. The solution were rotated end over end overnight at 4 °C. Next, 100 μl of protein A-Sepharose® beads (Amersham Pharmacia Biotech) (prepared in binding buffer as described by the manufacturer) were added, and the slurry was rotated end over end for 2 h at 4 °C. The tubes were centrifuged to pellet beads, and the supernatant was gently removed. The pellets were washed three times in binding buffer with either 0.5 mm CaCl2 or 5 mm EGTA present before reconstitution into the initial volume. Samples were run on SDS-PAGE and immunoblotted as described above. For immunoblotting with L-plastin and MRP14 antibodies, the following dilutions were used: LPL4A1 primary antibody, 1:1000; secondary antibody (goat anti-mouse (D0486) (Dako A/S, Glostrup, Denmark), 1:1000; LPL7,2 primary antibody, 1:1000; secondary antibody (rabbit anti-rat (P0450) (Dako A/S), 1:1000; MRP14 primary antibody (Bachem, Bubendorf, Switzerland), 1:2000 (biotinylated); secondary layer (avidine horseradish peroxidase (P0347) (Dako A/S)), 1:1000. One aim of this study was to determine if native grancalcin has a lipid anchor or other posttranslational modifications. Furthermore, we wanted to verify the reported N terminus of grancalcin. Since many EF-hand proteins expose hydrophobic areas upon binding of Ca2+, we tested if grancalcin also becomes hydrophobic in the presence of high concentrations of Ca2+. Human neutrophil cytosol was passed through a phenyl-Sepharose column in the presence of 0.5 mm Ca2+, and the column was washed with Ca2+ buffer. Replacement of Ca2+ by an excess of EGTA led to elution of a limited number of proteins (Fig. 1 A). By Coomassie staining, several bands can be seen, which all are expected to be Ca2+-binding proteins or proteins that interact with Ca2+-binding proteins. Indeed, several well known Ca2+-binding proteins could be identified (data not shown). The band at 28 kDa, showing retarded EGTA elution, was identified as grancalcin by immunoblotting. Fractions containing grancalcin were further purified by anion-exchange chromatography as described under "Experimental Procedures." This resulted in pure grancalcin as evaluated by SDS-PAGE and immunoblotting (Fig.1 B). Thus, we here present a novel and fast protocol for purification of grancalcin from the cytosol of human neutrophils, based on its Ca2+-dependent binding to a hydrophobic matrix. Recombinant grancalcin was produced and purified as described (3Lollike K. Sørensen O. Bundgaard J.R. Segal A.W. Boyhan A. Borregaard N. J. Immunol. Methods. 1995; 185: 1-8Crossref PubMed Scopus (11) Google Scholar). The grancalcin clone was checked by conventional sequencing techniques and found to be identical with the published (2Boyhan A. Casimir C.M. French J.K. Teahan C.G. Segal A.W. J. Biol. Chem. 1992; 267: 2928-2933Abstract Full Text PDF PubMed Google Scholar) sequence (data not shown). Recombinant grancalcin is synthesized as a glutathione S-transferase fusion protein linked by a cleavage site for thrombin and differs from prograncalcin (we use the term prograncalcin for the hypothetical protein corresponding to the coding sequence, including the N-terminal Met) by an additional Gly-Ser dipeptide at the N terminus, as was confirmed by N-terminal sequence analysis (data not shown). The molecular mass of purified recombinant grancalcin was determined to be 24,124 Da, in good agreement with the theoretical mass of 24,154 Da. In order to solve solubility problems (see below), we also generated three mutants with varying deletions in the N terminus, named after the starting amino acid according to the nomenclature for prograncalcin: Δ42-grancalcin, Δ50-grancalcin, and Δ53-grancalcin, respectively. All three mutants were easily expressed and purified to electrophoretic homogeneity (Fig. 2) following the same procedure as for recombinant grancalcin. Our polyclonal (rabbit) anti-grancalcin antibodies reacted with all three mutants (data not shown). The purified wild-type grancalcin gave no signal in protein sequence analysis, indicating that the N terminus is blocked. Moreover, by mass spectrometry wild-type grancalcin was found to have a molecular mass of 23,856 Da as compared with 24,010 Da calculated for prograncalcin (hypothetical protein, corresponding to the coding sequence), suggesting that the N terminus of wild-type grancalcin is modified. Cleavage of wild-type grancalcin with endoproteina
Год издания: 2001
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Calpain Protease Function and Regulation, Cell Adhesion Molecules Research, Protease and Inhibitor Mechanisms
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Том: 276
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Страницы: 17762–17769