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• W1968653622 abstract "Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca2+, as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, 45Ca overlay, and mass spectrometry, although there is no known motif for Ca2+ binding. LigBCen2 binds four Ca2+ as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, KD, for Ca2+ binding is 7 μm, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca2+-binding site in LigB is possibly similar to that seen in the βγ-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca2+ binding as shown by far- and near-UV CD and by fluorescence spectroscopy. In the apo form, the protein appears to be partially unfolded, as seen in the far-UV CD spectrum, and upon Ca2+ binding, the protein acquires significant β-sheet conformation. Ca2+ binding stabilizes the protein as monitored by thermal unfolding by CD (50.7–54.8 °C) and by differential scanning calorimetry (50.0–55.7 °C). Ca2+ significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca2+ in adhesion. We demonstrate that LigB is a novel bacterial Ca2+-binding protein and suggest that Ca2+ binding plays a pivotal role in the pathogenesis of leptospirosis. Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca2+, as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, 45Ca overlay, and mass spectrometry, although there is no known motif for Ca2+ binding. LigBCen2 binds four Ca2+ as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, KD, for Ca2+ binding is 7 μm, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca2+-binding site in LigB is possibly similar to that seen in the βγ-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca2+ binding as shown by far- and near-UV CD and by fluorescence spectroscopy. In the apo form, the protein appears to be partially unfolded, as seen in the far-UV CD spectrum, and upon Ca2+ binding, the protein acquires significant β-sheet conformation. Ca2+ binding stabilizes the protein as monitored by thermal unfolding by CD (50.7–54.8 °C) and by differential scanning calorimetry (50.0–55.7 °C). Ca2+ significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca2+ in adhesion. We demonstrate that LigB is a novel bacterial Ca2+-binding protein and suggest that Ca2+ binding plays a pivotal role in the pathogenesis of leptospirosis. Leptospira spp. are spirochetes, including the pathogenic species Leptospira interrogans as well as the saprophytic species Leptospira biflexa. Leptospirosis, a zoonotic disease caused by Leptospira spp., is widely distributed in developing countries and has reemerged in the United States (1Levett P.N. Clin. Microbiol. Rev. 2001; 14: 296-326Crossref PubMed Scopus (2231) Google Scholar). The severe form of leptospirosis, Weil's syndrome, includes an acute febrile illness associated with multiorgan damage, such as liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis (2Faine S.B. Adher B. Bolin C. Perolat P. Leptospira and Leptospirosis. MedSci, Melbourne, Australia1999Google Scholar), with a mortality rate up to 15% if not treated (3Segura E.R. Ganoza C.A. Campos K. Ricaldi J.N. Torres S. Silva H. Cespedes M.J. Matthias M.A. Swancutt M.A. Lopez Linan R. Gotuzzo E. Guerra H. Gilman R.H. Vinetz J.M. Clin. Infect. Dis. 2005; 40: 343-351Crossref PubMed Scopus (163) Google Scholar). Several virulence factors of this organism have been identified, including the sphingomyelinases, serine proteases, zinc-dependent proteases, collagenase (4Bulach D.M. Zuerner R.L. Wilson P. Seemann T. McGrath A. Cullen P.A. Davis J. Johnson M. Kuczek E. Alt D.P. Peterson-Burch B. Coppel R.L. Rood J.I. Davies J.K. Adler B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14560-14565Crossref PubMed Scopus (272) Google Scholar), LipL32 (5Yang C.W. Wu M.S. Pan M.J. Hsieh W.J. Vandewalle A. Huang C.C. J. Am. Soc. Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (100) Google Scholar), lipopolysaccharide (6Werts C. Tapping R.I. Mathison J.C. Chuang T.H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (573) Google Scholar), a novel factor H, laminin- and fibronectin-binding protein (Lsa24 or Len) (7Barbosa A.S. Abreu P.A. Neves F.O. Atzingen M.V. Watanabe M.M. Vieira M.L. Morais Z.M. Vasconcellos S.A. Nascimento A.L. Infect. Immun. 2006; 74: 6356-6364Crossref PubMed Scopus (151) Google Scholar, 8Stevenson B. Choy H.A. Pinne M. Rotondi M.L. Miller M.C. Demoll E. Kraiczy P. Cooley A.E. Creamer T.P. Suchard M.A. Brissette C.A. Verma A. Haake D.A. PLoS ONE. 2007; 2: e1188Crossref PubMed Scopus (163) Google Scholar, 9Verma A. Hellwage J. Artiushin S. Zipfel P.F. Kraiczy P. Timoney J.F. Stevenson B. Infect. Immun. 2006; 74: 2659-2666Crossref PubMed Scopus (124) Google Scholar), Loa22 (10Ristow P. Bourhy P. McBride F.W. Figueira C.P. Huerre M. Ave P. Girons I.S. Ko A.I. Picardeau M. PLoS Pathog. 2007; 3: e97Crossref PubMed Scopus (181) Google Scholar), and Lig (leptospira immunoglobulin-like) proteins (11Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 12Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 13Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar). Lig proteins, which include LigA and LigB, possess bacterial immunoglobulin-like (BIg) 3The abbreviations used are: BIgbacterial immunoglobulin-likeMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightEDSenergy-dispersive spectrometryICP-OESinductively coupled plasma optical emission spectrometryFnfibronectinNTDN-terminal domainITCisothermal titration calorimetryDSCdifferential scanning calorimetryMOPS4-morpholinepropanesulfonic acidELISAenzyme-linked immunosorbent assayGSTglutathione S-transferase. domains with 90-amino acid tandem repeats. Both proteins have identical N-terminal sequences of 630 amino acids, but their C termini are variable (11Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 12Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 13Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar). LigB also encodes a C-terminal, nonrepeat domain with 771 amino acid residues (11Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 12Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar). LigA and LigB may serve as microbial surface components recognizing adhesive matrix molecules that allow pathogenic Leptospira to bind to host extracellular matrix components, such as fibronectin (Fn), fibrinogen, laminin, and collagen (14Choy H.A. Kelley M.M. Chen T.L. Moller A.K. Matsunaga J. Haake D.A. Infect. Immun. 2007; 75: 2441-2450Crossref PubMed Scopus (210) Google Scholar, 15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar, 16Lin Y.P. Chang Y.F. J. Vet. Sci. 2008; 9: 133-144Crossref PubMed Scopus (42) Google Scholar). Lig proteins may also serve as possible vaccine candidates and/or as diagnostic antigens (12Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 17Faisal S.M. Yan W. Chen C.S. Palaniappan R.U. McDonough S.P. Chang Y.F. Vaccine. 2008; 26: 277-287Crossref PubMed Scopus (82) Google Scholar, 18Palaniappan R.U. McDonough S.P. Divers T.J. Chen C.S. Pan M.J. Matsumoto M. Chang Y.F. Infect. Immun. 2006; 74: 1745-1750Crossref PubMed Scopus (108) Google Scholar), and their expression is regulated by osmolarity (19Matsunaga J. Lo M. Bulach D.M. Zuerner R.L. Adler B. Haake D.A. Infect. Immun. 2007; 75: 2864-2874Crossref PubMed Scopus (100) Google Scholar). A high affinity Fn binding region of LigB, designated LigBCen2, contains 152 amino acids that include part of an immunoglobulin-like domain and a nonrepeated region (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar) (Fig. 1A). bacterial immunoglobulin-like matrix-assisted laser desorption ionization time-of-flight energy-dispersive spectrometry inductively coupled plasma optical emission spectrometry fibronectin N-terminal domain isothermal titration calorimetry differential scanning calorimetry 4-morpholinepropanesulfonic acid enzyme-linked immunosorbent assay glutathione S-transferase. Calcium plays a pivotal role in bacterial physiological activities, such as cell cycle, cell division (20Yu X.C. Margolin W. EMBO J. 1997; 16: 5455-5463Crossref PubMed Scopus (219) Google Scholar), competence (21Trombe M.C. Rieux V. Baille F. J. Bacteriol. 1994; 176: 1992-1996Crossref PubMed Google Scholar), pathogenesis (22Straley S.C. Plano G.V. Skrzypek E. Haddix P.L. Fields K.A. Mol. Microbiol. 1993; 8: 1005-1010Crossref PubMed Scopus (162) Google Scholar), signal transduction (23Werthen M. Lundgren T. J. Biol. Chem. 2001; 276: 6468-6472Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and motility and chemotaxis (24Tisa L.S. Adler J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11804-11808Crossref PubMed Scopus (58) Google Scholar, 25Tisa L.S. Adler J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10777-10781Crossref PubMed Scopus (55) Google Scholar). Apart from these functions, it is also known that host-pathogen interactions of some bacteria are affected by calcium (26Dardanelli M. Angelini J. Fabra A. Can. J. Microbiol. 2003; 49: 399-405Crossref PubMed Scopus (29) Google Scholar, 27Izutsu K.T. Belton C.M. Chan A. Fatherazi S. Kanter J.P. Park Y. Lamont R.J. FEMS Microbiol. Lett. 1996; 144: 145-150Crossref PubMed Google Scholar). Several types of Ca2+-binding motifs in bacterial proteins have been identified, which include EF-hand motif (28Michiels J. Xi C. Verhaert J. Vanderleyden J. Trends Microbiol. 2002; 10: 87-93Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), leukotoxin (29Chang Y.F. Shi J. Ma D.P. Shin S.J. Lein D.H. DNA Cell Biol. 1993; 12: 351-362Crossref PubMed Scopus (38) Google Scholar) or hemolysin-type calcium-binding domain (30Cruz W.T. Young R. Chang Y.F. Struck D.K. Mol. Microbiol. 1990; 4: 1933-1939Crossref PubMed Scopus (39) Google Scholar), and orphan motifs in which oxygen atoms provided by several charged glutamate or aspartate residues are used in ligation (28Michiels J. Xi C. Verhaert J. Vanderleyden J. Trends Microbiol. 2002; 10: 87-93Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). It appears that Lig proteins do not have any of these known Ca2+-binding motifs. LigBCen2 shows sequence similarity to the c-type lectin domain of other adhesins, including invasin of Yersinia pseudotuberculosis and intimin of Escherichia coli (12Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar) (Fig. 1B). Although Lig proteins are likely to play a significant role in pathogenicity, little is known about the mechanisms of action of these proteins. We undertook this study to identify novel properties of Lig proteins. Based on the structural homology of immunoglobulin-like fold with lens βγ-crystallin type Greek key motif (31Goode D. Crabbe M.J. Comput. Chem. 1995; 19: 65-74Crossref PubMed Scopus (11) Google Scholar), we wondered if Lig proteins would bind Ca2+ (32Caday C.G. Steiner R.F. J. Biol. Chem. 1985; 260: 5985-5990Abstract Full Text PDF PubMed Google Scholar). We therefore performed these studies and report that Ca2+ binds to the high affinity Fn binding region of LigB, LigBCen2. Ca2+ binding increases the stability of LigBCen2 and significantly influences its conformation. Further, we demonstrate that Ca2+ modulates the binding of LigB to N-terminal domain (NTD) of Fn, suggesting that it plays a major role in bacterial infection. Reagents and Antibodies—Calcium Green™-1 was obtained from Molecular Probe (Eugene, OR). Fibronectin (human plasma fibronectin), NTD of Fn, EGTA, Stains-all, sodium chloride, Tris, and calcium chloride were from Sigma. Plasmid Construction, Protein Purification, and Decalcification—The construct for the expression of histidine tag fused with a LigBCen2 DNA fragment (amino acids 1014–1165) was generated using the vector pQE30 (Qiagen, Alencia, CA). Construction, expression, and purification procedures were as previously described (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar, 18Palaniappan R.U. McDonough S.P. Divers T.J. Chen C.S. Pan M.J. Matsumoto M. Chang Y.F. Infect. Immun. 2006; 74: 1745-1750Crossref PubMed Scopus (108) Google Scholar). Protein was decalcified with 3 mm EDTA incubation for 45–60 min followed by buffer exchange with Chelex-100 resin-treated Tris buffer. All buffer solutions used for Ca2+-binding studies were passed through Chelex-100 resin and stored in plasticware. Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES)—Standard analysis procedures were performed by the Soil and Plant Laboratory (Cornell University) to analyze the total mineral and heavy metal content by ICP-OES (Varian, Inc., Palo Alto, CA). Protein samples included 25 mm Tris buffer, pH 7.0, which contained 150 mm NaCl and 70 μm of LigBCen2 either untreated or treated with 1 mm CaCl2 or 5 mm EGTA, for 1 h at room temperature. Unbound calcium and EGTA were removed to trace levels (<0.1 μg/ml) via extensive dialysis against Chelex-100-treated Tris buffer. MALDI-TOF Mass Spectrometry—The molecular weight of purified LigBCen2 protein was analyzed using an Applied Biosystem 4700 mass spectrometer (Applied Biosystems, Foster City, CA). Protein samples (70 μm LigBCen2) were incubated with 1 mm CaCl2, pH7, for 1 h at room temperature. Unbound excess calcium was removed by dialysis against deionized water. Energy-dispersive Spectrometry (EDS)—EDS analyses were performed with a JEOL 8900 electron probe microanalyzer (JEOL, Ltd. Tokyo, Japan). The operating conditions were 10-kV acceleration voltages. Sample preparation is described under “MALDI-TOF Mass Spectrometry.” Aqueous solutions of proteins were lyophilized, and a powder form of each sample was analyzed by EDS. Isothermal Titration Calorimetry (ITC)—The reaction enthalpy measurements were carried out with a VP-ITC calorimeter (MicroCal Inc.) at 30 °C. Before the ITC experiment, residual Ca2+ was removed from the protein by EDTA incubation, followed by taking EDTA off with Chelex-100 resin-treated Tris pH 7 buffer containing 50 mm KCl. In a typical ITC experiment, the cell contained 1.39 ml of thoroughly degassed 75 μm protein in 50 mm Tris (pH 7) containing 50 mm KCl, titrated against the same buffer containing 7 mm CaCl2. Following thermal equilibration, titrant was added to the 1.39-ml sample at an initial delay of 60 s, and sample in the cell was stirred at 300 rpm by a syringe. After 50 injections, protein was saturated with Ca2+ until there was no further heat change. In another experiment with the same parameters, Mg2+ versus LigBCen2 titration was performed, and after 50 injections the same protein was used for Ca2+ titration. Titration was performed as follows: 50 injections of 3 μl of ligand delivered over 4 s with a 220-s spacing between injections to allow complete equilibrium. Titration of Tris buffer with ligand was performed with the same parameters as mentioned above, and these reference data (heat of dilution) were subtracted with standard. Data were analyzed using MicroCal LLC ITC software (MicroCal), fitting them to an independent binding model. Differential Scanning Calorimetry (DSC)—Excess heat capacity Cp(T) of the apo and holo form of LigBCen2 was measured using a DSC Q1000 microcalorimeter (Waters, New Castle, DE). Degassed samples containing 3 μm LigBCen2 with and without 1 mm CaCl2 in Tris buffer (pH 7.0) were heated at a 10 K/h scan rate. Cp(T) data were recorded, corrected for buffer base line, and normalized to the amount of the sample. The TA Universal Analysis software (Waters, New Castle, DE) was used for the data analysis and display. CD Spectrometry—CD spectra were recorded on a Jasco J-815 spectropolarimeter under N2 atmosphere at room temperature (25 °C) in a 0.02- and 0.5-cm path length quartz cell for far- and near-UV CD spectra, respectively, with eight accumulations. Aliquots of calcium chloride solution were added to the protein solution and incubated for 5 min. All spectra were recorded in 50 mm Tris buffer, pH 7, containing 50 mm KCl. In thermal unfolding experiments, 2 μm LigBCen2 in the absence and presence of 1 mm CaCl2 was subjected to thermal unfolding, and data were collected at 1 °C/min increments from 25 to 70 °C recording the ellipticity at 215 nm, with 30-s temperature equilibrations, followed by 30-s data averaging. In order to measure the melting point, a first order derivative was applied to the results from the melting experiment. In all CD experiments, the background spectrum of Tris buffer (pH 7.0) without protein was subtracted from the protein spectra. The Stains-all binding assay was performed essentially as described earlier (32Caday C.G. Steiner R.F. J. Biol. Chem. 1985; 260: 5985-5990Abstract Full Text PDF PubMed Google Scholar, 33Sharma Y. Rao C.M. Rao S.C. Krishna A.G. Somasundaram T. Balasubramanian D. J. Biol. Chem. 1989; 264: 20923-20927Abstract Full Text PDF PubMed Google Scholar). LigBCen2 (5 μm) was mixed with the dye solution (60–100 μm) in 2 mm MOPS, pH 7.2, containing 30% ethylene glycol, and incubated for 5 min, and CD spectra were recorded from 400–700 nm. For studying the interaction, aliquots of NTD of Fn were mixed to the LigBCen2-Stains-all complex in the presence or absence of Ca2+ and CD spectra recorded. Fluorescence Spectrometry—Fluorescence emission spectra were measured on a Hitachi F-4500 spectrofluorometer (Hitachi, Tokyo, Japan). All spectra were recorded in correct spectrum mode of the instrument with excitation and emission band passes of 5 nm each. The intrinsic Trp fluorescence of the protein was recorded by exciting the solution at 295 nm and measuring the emission in the 300–400 nm regions. For calcium or magnesium titration, 0.1, 0.3, 0.5, 0.8, or 1.0 mm of calcium chloride was mixed with 10 μm of LigBCen2 in 50 mm Tris buffer, pH 7.2, containing 50 mm KCl, and spectra were recorded after 3 min of incubation. ANS fluorescence was measured by adding ANS to a final concentration of 100 μm to the protein solution (18 μm) and incubated for 5 min, and spectra were recorded between 400 and 600 nm at an excitation wavelength of 365 nm. Fibronectin binding to LigBCen2 was assayed by measuring the change in Trp fluorescence upon the addition of aliquots of NTD of Fn in the presence of 100 μm CaCl2 or in the absence of calcium chloride (in the presence of 100 μm EGTA). The mixture was incubated for 5 min before recording the emission spectra at the excitation of 295 nm. In a calcium competition experiment, 20 μm Calcium Green™-1 was mixed with or without 30 μm LigBCen2, and 100 μl/well was dispensed into a microtiter plate and incubated with 0.046, 0.093, 0.187, 0.375, 0.75, 1.5, or 3 μm CaCl2 in Tris buffer, pH 7.0, for 5 min. Enhanced fluorescence due to binding of free calcium and Calcium Green™-1 was monitored at 528 nm with excitation at 485 nm using a Synergy™ HT multidetection microplate reader (Bio-TEK Instruments, Inc. Winooski, VT). The association constant, Ka, was deduced using a Scatchard plot, and rearrangement of the Chang-Prusoff equation was used to calculate the dissociation constant (KD) of LigBCen2 and calcium (34Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12294) Google Scholar). Kapp=KD(dye)(1+[LigBCen2]KD(LigBCen2))(Eq. 1) Kapp is the apparent dissociation constant measured in the presence of a concentration of LigBCen2 in Calcium Green™-1 solution with calcium; KD(dye) or KD(LigBCen2) is the KD of calcium with Calcium Green™-1 or LigBCen2, respectively. All spectra were recorded in the correct spectrum mode excitation and emission band passes of 5 nm each and corrected for volume changes before further analysis. All measurements were performed at 25 °C. Fibronectin Binding Assay by ELISA—To determine the binding of LigBCen2 to NTD of Fn in the presence and absence of Ca2+, serial concentrations of NTD or bovine serum albumin (negative reference and data not shown) were coated on microtiter plate wells and blocked subsequently as previously described (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). 10 nm in the absence of or 1 mm in the presence of CaCl2, GST-LigBCen2, or GST in 100 μl of Tris buffer (pH 7.0) was added onto microtiter plate wells and incubated for 1 h at 37 °C. To detect the binding of GST-LigBCen2 or GST, rabbit anti-GST (1:200) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000) were used as primary and secondary antibody, respectively (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). After washing the plates three times with TBST, (0.05% Tween 20 in Tris buffer) 100 μl of 3, 3′, 5, 5′-tetramethylbenzidine (KPL, Gaithersburg, MD) was added to each well and incubated for 5 min. The reaction was stopped by adding 100 μl of 0.5% hydrofluoric acid to each well. Each plate was read at 630 nm using an ELISA plate reader (Bioteck EL-312; Bio-TEK Instruments). Each value represents the mean ± S.E. of three trials in triplicate samples. Statistically significant (p < 0.05) differences are indicated by an asterisk. Statistical Analysis—Each data point represents the mean S.E. of sample tested in triplicate. Data were analyzed by Student's t test, and statistically significant differences were claimed at p values of <0.05. Previously, Lig proteins, including LigA and LigB, have been shown to bind to extracellular matrix (14Choy H.A. Kelley M.M. Chen T.L. Moller A.K. Matsunaga J. Haake D.A. Infect. Immun. 2007; 75: 2441-2450Crossref PubMed Scopus (210) Google Scholar, 15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar, 16Lin Y.P. Chang Y.F. J. Vet. Sci. 2008; 9: 133-144Crossref PubMed Scopus (42) Google Scholar). the central region of LigB, annotated as LigBCen2 (Fig. 1A) was selected for further functional studies (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). The sequence was selected from amino acids 1014–1165 of LigB, as seen in Fig. 1B. Interestingly, this region contains the BIg domain and the nonrepeat region of 46 amino acids (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). Proteins that contain the BIg domain are found in a variety of bacterial and phage surface proteins, such as intimins or invasins, which are bacterial cell adhesion molecules that mediate intimate bacterial host-cell interaction (35Isberg R.R. Voorhis D.L. Falkow S. Cell. 1987; 50: 769-778Abstract Full Text PDF PubMed Scopus (409) Google Scholar, 36Jerse A.E. Yu J. Tall B.D. Kaper J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7839-7843Crossref PubMed Scopus (931) Google Scholar). E. coli intimin contains three domains (two immunoglobulin-like domains and a C-type lectin-like module), implying that carbohydrate recognition may be important in intimin-mediated cell adhesion. However, the exact functions of these proteins, barring some preliminary studies on fibronectin interactions, are not known (15Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). As mentioned above, nothing is known about the function of Lig proteins except that they are thought to play a role in virulence or pathogenesis. We were interested in identifying the functions of these important proteins. Structurally, Lig proteins belong to the bacterial immunoglobulin fold or BIg fold. In one of the earlier studies, the structural and functional similarities between various proteins of the Greek key/immunoglobulin fold were assessed (31Goode D. Crabbe M.J. Comput. Chem. 1995; 19: 65-74Crossref PubMed Scopus (11) Google Scholar). Among the proteins selected for functional prediction, lens βγ-crystallins and immunoglobulin functions were chosen. Since both families of proteins possess the Greek key type fold (31Goode D. Crabbe M.J. Comput. Chem. 1995; 19: 65-74Crossref PubMed Scopus (11) Google Scholar), it prompted us to look for the function of Lig proteins in the context of lens βγ-crystallins. The exact function of lens βγ-crystallins is not known. However, we have shown earlier that βγ-crystallins belong to a different superfamily of low affinity Ca2+-binding proteins (37Jobby M.K. Sharma Y. FEBS J. 2007; 274: 4135-4147Crossref PubMed Scopus (49) Google Scholar, 38Rajini B. Graham C. Wistow G. Sharma Y. Biochemistry. 2003; 42: 4552-4559Crossref PubMed Scopus (28) Google Scholar). Based on the fold similarities, we predicted that these Lig proteins might bind Ca2+. Therefore, we assessed Ca2+ binding to LigBCen2 by a number of methods as described below. Since there is no known motif in LigBCen2 for Ca2+ binding, it was necessary to probe Ca2+ binding by a number of methods to examine the specificity of Ca2+ binding. First, we probed Ca2+ binding to LigBCen2 by ICP-OES. LigBCen2 in the presence or absence (in the presence of EGTA) of calcium chloride were applied to ICP-OES. As shown in Table 1, calcium was present only in calcium chloride-treated LigBCen2 and not in untreated or EGTA-treated LigB-Cen2. The results indicate that Ca2+ binds to LigBCen2, since there was no Ca2+ binding to EGTA-treated or untreated LigBCen2. To further confirm the Ca2+ binding activity revealed by ICP-OES, Ca2+ binding to LigBCen2 was assessed by EDS. As seen in Fig. 2, A and B, a prominent calcium signal was seen in Ca2+-bound LigBCen2 and not in the apo form of LigBCen2.TABLE 1The concentration of the metal ions detected by ICP-optical emissionCalciumManganeseCobaltCopperCadmiumMagnesiumZincμg/mlμg/mlμg/mlμg/mlμg/mlμg/mlμg/mlLigBCen2 with Ca2+4.813—a—, concentration below 0.1 μg/ml.—————LigBCen2 with EGTA———————LigBCen2———————a —, concentration below 0.1 μg/ml. Open table in a new tab We also performed 45Ca binding to LigBCen2 using a well known method of overlay (39Maruyama K. Mikawa T. Ebashi S. J. Biochem. 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar). Seen as a dark spot on the membrane, radioactive calcium 45Ca binds to LigB-Cen2, thereby confirming the specificity of Ca2+ binding to the protein (data not shown). Further, the molecular mass of the holo form of LigBCen2 is higher (18,292 Da), as indicated by MALDI-TOF peaks, than that of the apo form of LigBCen2 (18,131 Da), further indicating that Ca2+ binds to LigBCen2 (Fig. 2C). Since there was a 161-Da difference in the molecular mass between holo and apo forms, it is likely that at least four Ca2+ molecules were bound to LigBCen2. The above results of mass spectroscopy and 45Ca binding experiments demonstrate that LigBCen2 is a Ca2+-binding protein. We next assessed the affinity and stoichiometry of the Ca2+" @default.
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• W1968653622 title "Calcium Binds to Leptospiral Immunoglobulin-like Protein, LigB, and Modulates Fibronectin Binding" @default.
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