Thrombin Hydrolysis of Human Osteopontin Is Dependent on Thrombin Anion-binding Exosites
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m708629200
ISSN1083-351X
AutoresTimothy Myles, Lawrence Leung,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic α4β1/α9β1 integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a kcat/Km value of 1.14 × 105 m–1 s–1. Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC50 = 1.2 ± 0.2 μm), unfractionated heparin (IC50 = 56.6 ± 8.4 μg/ml) and low molecular weight (5 kDa) heparin (IC50 = 31.0 ± 7.9 μg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162–197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162–197) with a specificity constant of kcat/Km = 1.64 × 104 m–1 s–1. Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (–4.1 ± 1.0) and OPN-(162–197) (–2.4 ± 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation. The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic α4β1/α9β1 integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a kcat/Km value of 1.14 × 105 m–1 s–1. Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC50 = 1.2 ± 0.2 μm), unfractionated heparin (IC50 = 56.6 ± 8.4 μg/ml) and low molecular weight (5 kDa) heparin (IC50 = 31.0 ± 7.9 μg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162–197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162–197) with a specificity constant of kcat/Km = 1.64 × 104 m–1 s–1. Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (–4.1 ± 1.0) and OPN-(162–197) (–2.4 ± 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation. Osteopontin (OPN) 2The abbreviations used are: OPN, osteopontin; WT, wild type; LMW, low molecular weight; PPACK, d-Phe-Pro-Arg-chloromethylketone; DTT, dithiothreitol; ABE, anion-binding exosite; HPLC, high pressure liquid chromatography; GFC, gel filtration chromatography; CTF, C-terminal fragment. is a secreted, acidic, phosphorylated glycoprotein that can act as either an extracellular matrix protein important in bone resorption or as a cytokine (1Denhardt D.T. Noda M. J. Cell. Biochem. Suppl. 1998; 30/31: 92-102Crossref Google Scholar, 2Mazzali M. Kipari T. Ophascharoensuk V. Wesson J.A. Johnson R. 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Studies with OPN-null mice show that OPN has roles in a broad range of homeostatic (e.g. bone remodeling, cellular immunity, wound healing), and pathologic (e.g. tumor metastasis) processes (1Denhardt D.T. Noda M. J. Cell. Biochem. Suppl. 1998; 30/31: 92-102Crossref Google Scholar, 4Ishijima M. Rittling S.R. Yamashita T. Tsuji K. Kurosawa H. Nifuji A. Denhardt D.T. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (199) Google Scholar, 5Liaw L. Birk D.E. Ballas C.B. Whitsitt J.S. Davidson J.M. Hogan B.L. J. Clin. Investig. 1998; 101: 1468-2478Crossref PubMed Google Scholar, 6Ashkar S. Weber G.F. Panoutsakopoulou V. Sanchirico M.E. Jansson M. Zawaideh S. Rittling S.R. Denhardt D.T. Glimcher M.J. Cantor H. Science. 2000; 287: 860-864Crossref PubMed Scopus (969) Google Scholar, 7Chabas D. Baranzini S.E. Mitchell D. Bernard C.C.A. Rittling S.R. Denhardt D.T. Sobel R.A. Lock C. Karpuj M. Pedotti R. Heller R. Oksenberg J.R. Steinman L. Science. 2001; 294: 1731-1735Crossref PubMed Scopus (766) Google Scholar, 8Koh A. da Silva A.P. Bansal A.K. Bansal M. Sun C. Lee H. Glogauer M. Sodek J. Zohar R. Immunology. 2007; 122: 446-475Crossref Scopus (112) Google Scholar). OPN is chemotactic for several cell types, in particular monocytes and macrophages, and stimulates cell motility and cell survival. These functions are in part dependent on the RGD sequence in OPN, which interacts with a number of integrins, including αvβ1, αvβ3, αvβ5 (9Liaw L. Skinner M.P. Raines E.W. Ross R. Cherish D.A. Schwartz S.M. Giachelli C. J. Clin. Investig. 1995; 95: 713-724Crossref PubMed Google Scholar), α5β1 (10Barry S.T. Ludbrook S.B. Murrison E. Horgan C.M.T. Biochem. Biophys. Res. Commun. 2000; 267: 764-769Crossref PubMed Scopus (75) Google Scholar), and α8β1 (11Denda S. Reichardt L.F. Müller U. Mol. Biol. Cell. 1998; 9: 1425-1435Crossref PubMed Scopus (162) Google Scholar), or binds to either of two variant forms of CD44 (12Weber G.F. Ashkar S. Glimcher M.J. Cantor H. Science. 1996; 71: 509-512Crossref Scopus (812) Google Scholar). OPN also has a conserved thrombin cleavage site downstream of and contiguous to the RGD domain. Cleavage of OPN by thrombin increases the adhesion, spreading, and migration of a variety of cultured cells in vitro (13Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van De Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar, 14Senger D.R. Perruzzi C.A. Biochim. Biophys. Acta. 1996; 1314: 13-24Crossref PubMed Scopus (145) Google Scholar). Melanoma cells and HT1080 fibrosarcoma cells will only bind thrombin-cleaved OPN, suggesting that thrombin cleavage is critical in the pathology of certain cancers (13Senger D.R. Perruzzi C.A. Papadopoulos-Sergiou A. Van De Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (180) Google Scholar, 14Senger D.R. Perruzzi C.A. Biochim. Biophys. Acta. 1996; 1314: 13-24Crossref PubMed Scopus (145) Google Scholar, 15Smith L.L. Giachelli C.M. Exp. Cell Res. 1998; 242: 351-360Crossref PubMed Scopus (91) Google Scholar). Thrombin cleavage of human OPN (Arg168–Ser169) exposes a cryptic integrin binding motif, 162SVVYGLR168, that specifically binds integrins α4β1, α4β7, and α9β1 (16Barry S.T. Ludbrook S.B. Murison E. Horgan C.M.T. Exp. Cell Res. 2000; 258: 342-351Crossref PubMed Scopus (96) Google Scholar, 17Bayless K.J. Davis G.E. J. Biol. Chem. 2001; 276: 13483-13489Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Green P.M. Ludbrook S.B. Millar D.D. Horgan C.M. Barry S.T. FEBS Lett. 2001; 503: 75-79Crossref PubMed Scopus (90) Google Scholar, 19Yokosaki Y. Matsuura N. Sasaki T. Murakami I. Schneider H. Higashiyama S. Saitoh Y. Yamakido M. Taooka Y. Sheppard D. J. Biol. Chem. 1999; 274: 36328-36334Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar); this sequence is adjacent to the RGD domain. The thrombin-cleaved N-terminal fragment of OPN, where RGD has been mutated to RAA, still binds to α9-transfected cells (19Yokosaki Y. Matsuura N. Sasaki T. Murakami I. Schneider H. Higashiyama S. Saitoh Y. Yamakido M. Taooka Y. Sheppard D. J. Biol. Chem. 1999; 274: 36328-36334Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Cell adhesion can be inhibited by alanine substitution of Tyr165 or by the double deletion of Leu167/Arg168 in the SVVYGLR motif. The integrin α4β1 also recognizes the SVVYGLR motif and is functionally distinct from the RGD domain (16Barry S.T. Ludbrook S.B. Murison E. Horgan C.M.T. Exp. Cell Res. 2000; 258: 342-351Crossref PubMed Scopus (96) Google Scholar, 17Bayless K.J. Davis G.E. J. Biol. Chem. 2001; 276: 13483-13489Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Green P.M. Ludbrook S.B. Millar D.D. Horgan C.M. Barry S.T. FEBS Lett. 2001; 503: 75-79Crossref PubMed Scopus (90) Google Scholar). As thrombin and OPN are both present at sites of tissue injury and inflammation, thrombin cleavage of OPN has been postulated to be important in enhancing the pro-inflammatory effect of OPN in vivo. However, although thrombin cleavage of OPN appears to be important in regulating its biological role, its biochemistry has not been studied in detail. This paper establishes OPN as a bona fide substrate of thrombin and defines the domains in that are thrombin important for its recognition and hydrolysis. Materials—Human wild-type (WT) and alanine-substituted mutant thrombins were expressed, purified, and titrated with PPACK (d-Phe-Pro-Arg-chloromethylketone) as described previously (20Tsiang M. Paborsky L.R. Li W.X. Jain A.K. Mao C.T. Dunn K.E. Lee O.W. Matsumura S.Y. Matteucci M.D. Coutré S.E. Leung L.L. Gibbs C.S. Biochemistry. 1996; 35: 16449-16457Crossref PubMed Scopus (76) Google Scholar). Low molecular weight (LMW) heparin (5 kDa), porcine intestinal mucosal unfractionated heparin, PPACK, and unsulfated hirulog were from Sigma. The peptides SVVYGLR and OPN-(162–197) (SVVYGLR/SKSKKFQRPDIQYPDATDEDITSHMESEE) were synthesized, purified, and quantitated by the peptide synthesis facility at Stanford University School of Medicine. Purification of Human OPN from Milk—OPN was purified from human milk (Mothers Milk Bank, San Jose, CA) using a previously published procedure with modifications (21Bayless K.J. Davis G.E. Meininger G.A. Protein Expression Purif. 1997; 9: 309-314Crossref PubMed Scopus (69) Google Scholar). Briefly, 1 liter of human milk pooled from several donors was allowed to separate on ice. The whey was carefully decanted from the curd and clarified by centrifugation at 23,000 × g for 60 min at 4 °C and then clarified further over a 0.2-μm pore size filtration unit. Benzamidine (2 mm) and DTT (2 mm) were added, and the clarified whey fraction was batch-adsorbed to Q-Sepharose (GE Healthcare) for 2 h at 4°C. The resin was washed with 10 mm sodium phosphate/0.2 m NaCl, 2 mm DTT, 2 mm benzamidine, pH 7.4, and then eluted with 10 mm sodium phosphate/1.0 m NaCl, 2 mm DTT, pH 7.4. The eluant was dialyzed overnight in 10 mm sodium phosphate/4 m NaCl, 2 mm DTT, pH 7.4. The dialyzed protein was then loaded onto a 5-ml HiTrap phenyl-Sepharose HP column (GE Healthcare) at 1 ml/min and washed to base line. A linear gradient was developed from 4 to 1 m NaCl at 1 ml/min with 1-ml fractions collected. Pure fractions were pooled and dialyzed extensively against phosphate-buffered saline, pH 7.4. Partially pure fractions were diluted in 10 mm sodium phosphate/4 m NaCl, 2 mm DTT, pH 7.4, and reapplied over the HiTrap phenyl-Sepharose HP column. The concentration of purified protein was estimated at 280 nm using an extinction coefficient (1 cm path length) of 22920. Hydrolysis of Human Milk OPN by Thrombin and the Quantitation of the Cleaved C-terminal Fragment—OPN (4–31 μm) was reacted with thrombin (≈5–100 nm) in assay buffer containing 20 mm HEPES, 5 mm CaCl2, 0.1% polyethylene glycol 8000, pH 7.5, and at various salt concentrations (≈20–200 mm NaCl) at 37 °C. Reactions were terminated with the addition of PPACK (5 μm) followed by DTT (5 mm). HPLC-gel filtration chromatography (HPLC-GFC) was employed to quantitate the generation of the 15-kDa C-terminal fragment (CTF) liberated by thrombin cleavage. Various amounts (10–40 μl) of the reaction mixture were injected over a Shodex 8 × 300 mm KW-803 column (Showa Denko America Inc, New York), and proteins were resolved in 20 mm sodium phosphate, 300 mm NaCl, 5 mm DTT, pH 7.6, at 1 ml/min. The peak area was detected at 254 nm and was converted to nmoles of product using a calibration curve. Inhibition of Thrombin Hydrolysis of Human Milk OPN or OPN-(162–197) Peptide by LMW Heparin, Unfractionated Heparin, and Unsulfated Hirulog—Human milk OPN (5 μm) or OPN-(162–197) (10 μm) was preincubated with 5 kDa of LMW heparin, porcine intestinal unfractionated heparin (0.25–1000 μg/ml), or unsulfated hirugen (10 nm–10 μm) at 37°C for 10 min. Thrombin was added to milk OPN (50 nm) and OPN-(162–197) (100 nm) and incubated for 3 and 5 min, respectively, at 37 °C. Reactions containing human milk OPN were terminated with 5 μm PPACK followed by 5 mm DTT. Thrombin hydrolysis of OPN-(162–197) was terminated by the addition of 10% perchloric acid. The concentration of thrombin used and the incubation time were determined empirically to give no more than 30% substrate hydrolysis. The amount of product formed was determined by HPLC-GFC for human milk OPN and reverse-phase HPLC for OPN-(162–197). Determination of the Michaelis-Menten Parameters for Thrombin Hydrolysis of Milk OPN—Several concentrations of OPN (4–31 μm) were digested with thrombin (typically 5 nm) at 37 °C for various times, such that there was less than 10% hydrolysis of substrate. The reactions were terminated by the addition of 5 μm PPACK and 5 mm DTT. The amount of product formed (OPN CTF) was determined by HPLC-GFC as described above. The values for Km and kcat were determined by plotting the initial velocity of cleavage against the different substrate concentrations and then fitting to the Michaelis-Menten equation by nonlinear regression analysis. Experiments were performed in duplicate, and the data were pooled for analysis. Estimation of kcat/Km for Cleavage of OPN under First Order Rate Conditions—Values for kcat/Km were determined by full reaction progress curves at a substrate concentration well below Km for WT and representative mutants of thrombin within anion-binding exosite I (ABE-I; R20A, K21A, Q24A, R62A, K65A, H66A, R68A, T69A, R70A, Y71A, R73A, K77A, K106A, K107A), ABE-II (R89A/R93A/E94A, R98A, D122A/R123A/E124A, E169A/K174A/D175A, R178A/R180A/D183A, R245A, K248A), the 50-insertion loop (W50A), and the Na+ binding site (E229A). 5 μm OPN (0.26 × [Km]) was preincubated in assay buffer for 10 min at 37 °C. Reactions were started with the addition of thrombin (typically 50 nm) and incubated at 37 °C. Aliquots were removed at several time points (0–1800 s) and terminated with 5 μm PPACK followed by 5 mm DTT. The amount of product generated (nmoles) for each time point was determined by HPLC-GFC as detailed above. Values for kcat/Km were determined by fitting data from the time course experiments to the following equation (22Philippou H. Rance J. Myles T. Hall S.W. Ariens R.A. Grant P.J. Leung L.L. Lane D.A. J. Biol. Chem. 2003; 278: 32020-32026Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), kcat/KM=−ln(1−{[CTF]/[CTFf]}/[ET]×t(Eq. 1) where [CTF] is the concentration of the CTF at a given time, CTFf is the concentration of the CTF at full activation, and [ET] is the total enzyme concentration of thrombin at any given time (t). Curve fitting was performed using Prism 5 (GraphPad Software). Determination of Km, kcat, and kcat/Km for Hydrolysis of the Peptide OPN-(162–197)—For the estimation of Michaelis-Menten parameters, 10–300 μm OPN-(162–197) peptide was incubated with WT or mutant thrombin in assay buffer containing 20 mm HEPES, 145 mm NaCl, 5 mm CaCl2, 0.1% polyethylene glycol 8000, pH 7.5. Enzyme concentration and incubation time were varied such that there was less than 10% substrate depletion. Reactions were terminated with the addition of 10% perchloric acid. The cleaved and uncleaved peptides were separated by reverse-phase HPLC on a Waters Symmetry C18 (4.6 × 150 mm, 5 μm) column using a 0.2–35% acetonitrile/0.1% trifluoroacetic acid gradient over 30 min at a flow rate of 1.0 ml/min. Cleavage was specific with no secondary cleavage sites. The cleaved peptide peak OPN-(162–168) (SVVYGLR) eluted at 19.8 min and was well resolved from OPN-(162–197) (23.6 min) and the OPN-(169–197) cleavage product (22.3 min). Identity of the cleavage products was confirmed by mass spectrometry. The area under the SVVYGLR peak was converted to nmoles of peptide using a calibration curve constructed with purified and quantitated SVVYGLR peptide. The values for Km and kcat were determined by fitting to the Michaelis-Menten equation by non-linear regression analysis as described above. The deduced value for Km was used to determine the reaction conditions for estimating the specificity constant, kcat/Km. OPN-(162–197) peptide (10 μm, 0.04 × Km) was preincubated in assay buffer (20 mm HEPES, 145 mm NaCl, 5 mm CaCl2, 0.1% polyethylene glycol 8000, pH 7.5) for 10 min. Reactions were started with the addition of WT or mutant thrombin (100–500 nm) with aliquots removed at several time points (0–1800 s) and then terminated with the addition of 10% perchloric acid. The effect of NaCl and choline chloride (ChCl) on kcat/Km was determined in the presence of assay buffer with salt concentrations ranging from 20 to 200 mm. Assays with ChCl were buffered with 5 mm Tris-HCl, pH 7.5, to reduce Na+ ions from pH adjustment. The amount of product (SVVYGLR) formed was quantitated as described above by reverse-phase HPLC analysis, and the values for kcat/Km were determined as described above. Determination of the Kinetic Parameters for Hydrolysis of Purified Human Breast Milk OPN by Thrombin—The hydrolysis of human milk derived OPN by thrombin gave estimates for Km (19.1 ± 3.9 μm), kcat (0.9 ± 0.1 s–1), and kcat/Km (0.47 × 105 m–1 s–1) under assay conditions looking at initial reaction velocities over several substrate concentrations up to 31 μm (Fig. 1A). However, significant substrate and/or product inhibition was observed at concentrations of 50 μm and higher (data not shown). To account for substrate/product inhibition effects, we estimated a value for the specificity constant kcat/Km of (1.14 ± 0.17) × 105 m–1 s–1 (Fig. 1B) by full reaction progress curves using a substrate concentration estimated to be 0.26 × Km. Under these conditions the estimate for kcat/Km is ∼2.5 times better and most likely reflects the best estimate for the specificity constant. Unfractionated Heparin, LMW Heparin, and Unsulfated Hirugen Inhibit Thrombin Hydrolysis of OPN—Osteopontin contains two putative heparin-binding domains (23Kazanecki C.C. Uzwiak D.J. Denhardt D.T. J. Cell. Biochem. 2007; 102: 912-924Crossref PubMed Scopus (214) Google Scholar), therefore we hypothesized that heparin could act as a bridge between the heparin-binding domains and the thrombin ABE-II domain, enhancing hydrolysis. OPN can be bound to HiTrap heparin-Sepharose and eluted with 0.37 m NaCl, suggesting the presence of OPN heparin-binding domains (data not shown). However, we found that LMW heparin and unfractionated heparin caused dose-dependent inhibition of thrombin hydrolysis of OPN with IC50 values of 31.0 ± 7.9 and 56.6 ± 8.4 μg/ml, respectively (Fig. 2A). Further, the inhibition curves were not bell-shaped, suggesting that heparin does not function as a macromolecular bridge binding both thrombin and OPN to enhance thrombin cleavage of OPN. Rather, both unfractionated and LMW heparins compete against OPN for binding to the thrombin ABE-II, indicating the importance of ABE-II in thrombin cleavage of OPN. Unsulfated hirugen is a tight binding inhibitor specific only for thrombin ABE-I. Hirugen also caused dose-dependent inhibition of thrombin cleavage of OPN with an IC50 of 1.2 ± 0.2 μm (Fig. 2B). The inhibition studies using LMH heparin and hirugen suggest that both exosites play a role in thrombin cleavage of OPN. Mapping of Residues in ABE-I and ABE-II Important for the Hydrolysis of OPN—A bank of more than 56 mutant thrombins has been used to map domains important in the cleavage of FV (24Myles T. Yun T.H. Hall S.W. Leung L.L. J. Biol. Chem. 2001; 276: 25143-25149Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), FVIII (25Myles T. Yun T.H. Leung L.L. Blood. 2002; 100: 2820-2826Crossref PubMed Scopus (58) Google Scholar), FXI (26Yun T.H. Baglia F.A. Myles T. Navaneetham D. Lopez J.A. Walsh P.N. Leung L.L. J. Biol. Chem. 2003; 278: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), FXIII (22Philippou H. Rance J. Myles T. Hall S.W. Ariens R.A. Grant P.J. Leung L.L. Lane D.A. J. Biol. Chem. 2003; 278: 32020-32026Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and fibrinogen (27Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L.K. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) for thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (27Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L.K. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 28Hall S.W. Nagashima M. Zhao L. Morser J. Leung L.L. J. Biol. Chem. 1999; 274: 25510-25516Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and for heparin-accelerated inhibition of antithrombin, heparin cofactor II, and protein C inhibitor (29Tsiang M. Jain A.K. Gibbs C.S. J. Biol. Chem. 1997; 272: 12024-12029Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 30Fortenberry Y.M. Whinna H.C. Gentry H.R. Myles T. Leung L.L. Church F.C. J. Biol. Chem. 2004; 279: 43237-43244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Fortenberry Y.M. Whinna H.C. Cooper S.T. Myles T. Leung L.L. Church F.C. J. Thromb. Haemost. 2007; 5: 1486-1492Crossref PubMed Scopus (7) Google Scholar). These studies have identified key residues within ABE-I and ABE-II important in defining the interaction interface for all these interactions. The specificity constant was determined for 14 mutants in ABE-I and seven mutants in ABE-II along with two thrombin mutants (W50A and E229A) known to affect the thrombin active site (23Kazanecki C.C. Uzwiak D.J. Denhardt D.T. J. Cell. Biochem. 2007; 102: 912-924Crossref PubMed Scopus (214) Google Scholar, 24Myles T. Yun T.H. Hall S.W. Leung L.L. J. Biol. Chem. 2001; 276: 25143-25149Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 25Myles T. Yun T.H. Leung L.L. Blood. 2002; 100: 2820-2826Crossref PubMed Scopus (58) Google Scholar, 26Yun T.H. Baglia F.A. Myles T. Navaneetham D. Lopez J.A. Walsh P.N. Leung L.L. J. Biol. Chem. 2003; 278: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 27Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L.K. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 28Hall S.W. Nagashima M. Zhao L. Morser J. Leung L.L. J. Biol. Chem. 1999; 274: 25510-25516Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 29Tsiang M. Jain A.K. Gibbs C.S. J. Biol. Chem. 1997; 272: 12024-12029Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 30Fortenberry Y.M. Whinna H.C. Gentry H.R. Myles T. Leung L.L. Church F.C. J. Biol. Chem. 2004; 279: 43237-43244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Fortenberry Y.M. Whinna H.C. Cooper S.T. Myles T. Leung L.L. Church F.C. J. Thromb. Haemost. 2007; 5: 1486-1492Crossref PubMed Scopus (7) Google Scholar, 32Carter W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Ten thrombin ABE-I mutants had less than 50% WT activity, with mutants K65A (29%), H66A (14%), Y71A (16%), and R73A (20%) severely affected (Table 1), which indicates a very large interaction interface within ABE-I. Three mutants in ABE-II (total of seven alanine-substituted residues) have impaired activity relative to WT thrombin, R98A (41%), R89A/R93A/E94A (31%), and R178A/R180A/D1778A (46%), confirming the role of this ABE from the heparin inhibition studies. The mutant thrombins W50A and E229A were also assessed for their ability to hydrolyze OPN. Trp50 forms part of the 50-insertion loop and is important in defining the insertion loop and the aryl binding site, and Glu229 maintains the integrity of the Na+-binding loop. The crystal structure of E229A shows the collapse of the active site with almost complete occlusion (32Carter W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Alanine substitution of both of these residues has a profound effect in the ability of thrombin to hydrolyze OPN, both with 9% WT activity. These mutations clearly show that a functional thrombin active site is needed to cleave at the P1 arginine of SVVYGLR/SKSKKFQ.TABLE 1Impaired hydrolysis of OPN by selected alanine substituted thrombin mutants from ABE-I, ABE-II, and the active siteThrombinaThrombin-based numbering.ChymotrypsinbChymotrypsinogen-based numbering.Locationkcat/Km × 104WT activitym-1s-1%WTWT11.36 ± 1.72100R20AR35AABE-I14.22 ± 1.18125K21AK36A3.56 ± 0.0731Q24AQ38A10.42 ± 1.2592R62AR67A4.65 ± 0.2841K65AK70A3.31 ± 0.2029H66AH71A1.58 ± 0.1914R68AR73A3.80 ± 0.4934T69AT74A7.90 ± 0.9570R70AR75A4.65 ± 0.5141Y71AY76A1.84 ± 0.1716R73AR77AA2.31 ± 0.1520K77AK81A4.73 ± 0.3842K106AK109A6.25 ± 0.2555K107AK110A5.10 ± 0.3645R89A/R93A/E94AR93A/R97A/E97AAABE-II3.10 ± 0.1931R98AR101A4.69 ± 0.2841D122A/R123A/E124AD125A/R126A/E127A7.60 ± 0.3867E169A/K174A/D175AE164A/K169A/D170A7.02 ± 0.4262R178A/R180A/D183AR173A/R175A/D178A5.24 ± 0.4746R245AR233A7.28 ± 0.4464K248AK236A7.24 ± 0.2964W50AW60DAActive site0.96 ± 0.148E229AE217A1.02 ± 0.149a Thrombin-based numbering.b Chymotrypsinogen-based numbering. Open table in a new tab Identification of an OPN Domain That Interacts with ABE-I—OPN has a hirudin-like domain, DIQYPDATDEDITSHMESEE (33Rydel T.J. Tulinsky A. Bode W. Huber R. J. Mol. Biol. 1991; 221: 583-601Crossref PubMed Scopus (322) Google Scholar), adjacent and contiguous to the thrombin cleavage site, that could potentially interact with ABE-I. To address this potential interaction, a peptide was made (OPN-(162–197)) that spanned the cleavage site and hirudin-like ABE-I-binding domain. Values for Km (296.6 ± 10.9 μm), kcat (3.3 ± 0.1 s–1), and kcat/Km (1.11 × 104 m–1 s–1) were initially determined over a range of substrate concentrations; the value derived for Km was used to define experimental conditions to estimate kcat/Km under first order rate conditions (Fig. 3). The specificity constant kcat/Km was determined for the cleavage of this OPN-(162–197) by WT thrombin and was found not to be a good substrate (kcat/Km 1.64 ± 0.06 × 104 m–1 s–1) when compared with milk OPN (kcat/Km 1.14 ± 0.17 × 105 m–1 s–1). Hirugen was found to inhibit thrombin cleavage of OPN-(162–197) (Fig. 2B) with an IC50 = 1.1 ± 0.1 μm showing a requirement for ABE-I. The ability of selected ABE-I and ABE-II mutants to cleave OPN-(162–197) was compared with that of WT thrombin (Table 2). The ABE mutants K65A, H66A, R68A, Y71A, and R73A in general showed similar decreases in activity relative to WT thrombin when compared with the cleavage of milk OPN. Heparin had no effect, excluding the participation of ABE-II (Fig. 2A). Consistent with this finding, ABE-II mutants R98A, E160A/K174A/D175A, and R178A/R180A/D183A in general had no effect on cleavage of the peptide, suggesting that the OPN hirudin-like domain interacts principally with the thrombin ABE-I. The ABE-II R89A/R93A/E94A mutant showed 44% WT activity. The residue Glu94 lies on the rim between ABE-II and the active site, so it is conceivable that mutation of this residue could influence cleavage within the active site.TABLE 2ABE-I alanine-substituted thrombin mutants have impaired catalysis for the OPN-(162–197) peptideThrombinLocationkcat/KmWT activitymm-1s-1%WT16.35 ± 0.58100K65AABE-I5.38 ± 1.2533H66A2.87 ± 0.4118R68A5.80 ± 0.3235Y71A3.76 ± 0.0123R73A7.41 ± 0.1945R89A/R93A/E94AABE-II7.13 ± 0.4044R98A15.10 ± 0.8392E169A/K174A/D175A14.79 ± 1.2390R178A/R180A/D183A16.33 ± 0.35100 Open table in a new tab Electrostatic Interactions Are Important for Hydrolysis of OPN by Thrombin—Studies between thrombin and hirudin show the importance of complementary electrostatic fields in enhancing the rate of complex formation by "electrostatic steering" (34Wade R.C. Gabdoulline R.R. Lüdemann S.K. Lounnas V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5942-5949Crossref PubMed Scopus (178) Google Scholar, 35Karshikov A. Bode W. Tulinsky A. Stone S.R. Protein Sci. 1992; 1: 727-735Crossref PubMed Scopus (90) Google Scholar). To study t
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