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• W2013685938 abstract "Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp438, Ala457, Glu485, and Glu490. We mutated two glutamic acid residues (Glu485 and Glu490) and three aspartic acid residues (Asp472, Asp476, and Asp479) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The Kactvalues for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the Kact values were not changed by mutagenesis. However, Asp476 and Asp479 are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu485 and Glu490 are Ca2+-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp476 and Asp479 play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule. Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp438, Ala457, Glu485, and Glu490. We mutated two glutamic acid residues (Glu485 and Glu490) and three aspartic acid residues (Asp472, Asp476, and Asp479) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The Kactvalues for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the Kact values were not changed by mutagenesis. However, Asp476 and Asp479 are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu485 and Glu490 are Ca2+-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp476 and Asp479 play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule. factor XIII transglutaminase glutathione S-transferase The plasma factor XIII (FXIII)1 molecule is a tetrameric zymogen (A2B2) that circulates in human plasma and that is composed of two A-chains and two glycosylated B-chains (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar, 2McDonagh J.A. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis. J. B. Lippincott Co., Philadelphia1987: 289-300Google Scholar, 3Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar). In contrast, monocytes and platelet FXIII exist as an intracellular dimer composed of two A-chains (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar, 2McDonagh J.A. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis. J. B. Lippincott Co., Philadelphia1987: 289-300Google Scholar, 3Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar). X-ray crystallography studies revealed that the FXIII A-chain is composed of four distinct structural domains. Starting from the N terminus, there is a β-sandwich domain (residues 43–184) followed by a catalytic core (residues 185–515) and two β-barrels (residues 516–628 and 629–727) at the C terminus (4Yee V.C. Pederson L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar). The activation peptide (residues 1–37) of each A-subunit crosses the dimer interface and partially occludes the opening to the active site in the catalytic core of the other subunit. Thrombin cleavage of the A-chains at the Arg37–Gly38 bond is required for the A-chains to express FXIIIa (the FXIII A-chain with the activation peptide (Met1–Arg37) removed) activity in vivo (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar, 2McDonagh J.A. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis. J. B. Lippincott Co., Philadelphia1987: 289-300Google Scholar, 3Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar). Thrombin cleavage of plasma FXIII is a calcium-independent reaction (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar, 2McDonagh J.A. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis. J. B. Lippincott Co., Philadelphia1987: 289-300Google Scholar, 3Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar) that is accelerated by fibrin polymers (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar, 2McDonagh J.A. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis. J. B. Lippincott Co., Philadelphia1987: 289-300Google Scholar, 3Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar). After proteolysis, all subsequent steps in the formation and function of FXIIIa are calcium-dependent (5Folk J.E. Adv. Enzymol. Related Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar, 6Chung S.I. Folk J.E. J. Biol. Chem. 1972; 247: 2798-2807Abstract Full Text PDF PubMed Google Scholar, 7Folk J.E. Mullooly J.P. Cole P.W. J. Biol. Chem. 1967; 242: 1838-1844Abstract Full Text PDF PubMed Google Scholar, 8Folk J.E. Cole P.W. Mullooly J.P. J. Biol. Chem. 1967; 242: 2615-2621Abstract Full Text PDF PubMed Google Scholar). Detailed biochemical studies of purified plasma XIII established that calcium ions dissociate B-chains from the thrombin-cleaved A-chains of plasma FXIII (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar). Then, a calcium-dependent conformational change in the thrombin-cleaved A-chain is required to expose the active-site Cys314 (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar). Calcium ions are essential for FXIIIa to catalyze intermolecular isopeptide bonds between protein molecules (1Lorand L. Losowsky M.S. Miloszewski K.J.M. Prog. Hemostasis Thromb. 1980; 5: 245-290PubMed Google Scholar, 3Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (932) Google Scholar, 5Folk J.E. Adv. Enzymol. Related Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar). There are two distinct biochemical steps that occur during FXIIIa catalysis. In the first step, the active-site Cys314 of FXIIIa binds the peptide-bound glutamine substrate, forming a thioester bond intermediate and releasing ammonia (5Folk J.E. Adv. Enzymol. Related Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar). Then, the enzyme-substrate complex interacts with either a primary amine or a peptide-bound lysine residue, producing an isopeptide bond (5Folk J.E. Adv. Enzymol. Related Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar). Calcium ions are required for both steps of catalysis (6Chung S.I. Folk J.E. J. Biol. Chem. 1972; 247: 2798-2807Abstract Full Text PDF PubMed Google Scholar, 7Folk J.E. Mullooly J.P. Cole P.W. J. Biol. Chem. 1967; 242: 1838-1844Abstract Full Text PDF PubMed Google Scholar, 8Folk J.E. Cole P.W. Mullooly J.P. J. Biol. Chem. 1967; 242: 2615-2621Abstract Full Text PDF PubMed Google Scholar). Calcium ions also protect FXIIIa from proteolysis at the Lys513-Ser514 site (9McDonagh J. McDonagh R.P. Br. J. Haematol. 1975; 30: 465-477Crossref PubMed Scopus (18) Google Scholar, 10Mary A. Achyuthan K.E. Greenberg C.S. Arch. Biochem. Biophys. 1988; 261: 112-121Crossref PubMed Scopus (18) Google Scholar, 11Takahashi N. Takahashi Y. Putnam F.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8019-8023Crossref PubMed Scopus (130) Google Scholar). The precise location and number of calcium-binding sites in factor XIII A-chains are hampered by the relative low affinity of the calcium binding (12Lewis B.A. Freyssinet J.-M. Holbrook J.J. Biochem. J. 1978; 169: 397-402Crossref PubMed Scopus (22) Google Scholar). Studies performed by Lewis et al. (12Lewis B.A. Freyssinet J.-M. Holbrook J.J. Biochem. J. 1978; 169: 397-402Crossref PubMed Scopus (22) Google Scholar) demonstrated that the FXIII A-chain binds 1.2–1.5 calcium ions with low affinity (Kd ∼ 10−4m) and up to 8 calcium ions/molecule of plasma FXIII at higher calcium concentrations. A putative calcium-binding site was first proposed by Takahashi et al. (11Takahashi N. Takahashi Y. Putnam F.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8019-8023Crossref PubMed Scopus (130) Google Scholar) as an EF-hand-like structure located in a fragment between Gln467 and Asp479. This area is rich in the negative charged amino acid residues that are postulated to bind calcium ions. Recent x-ray crystallography studies of factor XIII A-chain crystals grown in the presence of Sr2+ or Yb3+ demonstrated there is no EF-hand-like motif (13Pedersen L.C. X-ray Structure Determination of Factor XIII.Doctoral dissertation. University of Washington, 1994Google Scholar, 14Yee V.C. Le Trong I. Bishop P.D. Pedersen L.C. Stenkamp R.E. Teller D.C. Semin. Thromb. Hemostasis. 1996; 22: 377-384Crossref PubMed Scopus (48) Google Scholar). X-ray crystallography identified a major cation-binding site containing Asp438, Ala457, Glu485, and Glu490 and a minor site containing Asp270 and Glu272 (13Pedersen L.C. X-ray Structure Determination of Factor XIII.Doctoral dissertation. University of Washington, 1994Google Scholar, 14Yee V.C. Le Trong I. Bishop P.D. Pedersen L.C. Stenkamp R.E. Teller D.C. Semin. Thromb. Hemostasis. 1996; 22: 377-384Crossref PubMed Scopus (48) Google Scholar, 15Fox B.A. Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. J. Biol. Chem. 1999; 274: 4917-4923Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Interestingly, there were no major conformational changes between the cation-bound factor XIIIa and the zymogen structures (13Pedersen L.C. X-ray Structure Determination of Factor XIII.Doctoral dissertation. University of Washington, 1994Google Scholar, 14Yee V.C. Le Trong I. Bishop P.D. Pedersen L.C. Stenkamp R.E. Teller D.C. Semin. Thromb. Hemostasis. 1996; 22: 377-384Crossref PubMed Scopus (48) Google Scholar, 15Fox B.A. Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. J. Biol. Chem. 1999; 274: 4917-4923Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The role of these cation-binding sites in expression of FXIIIa activity and regulation of the conformation of the protein in solution remains unclear. In addition, there were differences in coordinating ligands between the Sr2+- and Yb3+-bound structures, and fewer than the ideal number of coordinating ligands were reported (14Yee V.C. Le Trong I. Bishop P.D. Pedersen L.C. Stenkamp R.E. Teller D.C. Semin. Thromb. Hemostasis. 1996; 22: 377-384Crossref PubMed Scopus (48) Google Scholar, 15Fox B.A. Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. J. Biol. Chem. 1999; 274: 4917-4923Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). These data suggest that other amino acids are also involved in the binding of cations. Since high calcium concentrations (>50 mm) enable the platelet factor XIII zymogen to express enzymatic activity in the absence of thrombin cleavage, there may be other lower affinity calcium-binding sites that play a role in regulating catalysis and protein conformation. Therefore, site-directed mutagenesis of amino acid residues identified by x-ray crystallography studies and other potential calcium-binding residues is necessary to confirm their role in regulating catalysis and protein conformation. In this study, we performed site-directed mutagenesis analysis of two calcium-binding ligands (Glu485 and Glu490) identified by x-ray crystallography. These amino acid sequences are conserved in other transglutaminases such as human tissue transglutaminase (TGase), bovine endothelial TGase, and guinea pig TGase (16Ikura K. Nasu T. Yokota H. Tsuchiya Y. Sasaki R. Chiba H. Biochemistry. 1998; 27: 2898-2905Crossref Scopus (149) Google Scholar, 17Gentile V. Saydak M. Chiocca E.A. Akanda O. Birckbichler P.J. Lee K.N. Stein J.P. Davies P.J.A. J. Biol. Chem. 1991; 266: 478-483Abstract Full Text PDF PubMed Google Scholar, 18Nakanishi K. Nara K. Hagiwara H. Aoyama Y. Ueno H. Hirose S. Eur. J. Biochem. 1991; 202: 15-21Crossref PubMed Scopus (47) Google Scholar). In addition, three aspartic residues (Asp472, Asp476, and Asp479) located in close proximity to Glu485 and Glu490were mutated. The substitution mutants were expressed, purified, and analyzed for their catalytic properties and sensitivity to thrombin proteolysis. The effects of these mutations on the structure and function of FXIIIa will be discussed. All restriction enzymes, T4 DNA ligase, bacteria alkaline phosphatase, LB medium, and yeast extract were obtained from Life Technologies, Inc. Human α-thrombin was supplied by Dr. J. W. Fenton II (New York State Department of Health, Albany, NY). Oligonucleotides were synthesized by Biosynthesis, Inc. (Lewisville, TX) or Life Technologies, Inc. All other reagents used in this study were purchased from Sigma unless stated otherwise. All factor XIII A-chain mutants were constructed using oligonucleotide-mediated mutagenesis (T7-Gen mutagenesis kit, Amersham Pharmacia Biotech) as described previously (19Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar, 20Lai T.-S. Santiago M.A. Achyuthan K.E. Greenberg C.S. Protein Expression Purif. 1994; 5: 125-132Crossref PubMed Scopus (15) Google Scholar). The single point mutations E485A, E490A, D472A, D476A, and D479A were constructed using oligonucleotides 1 (GATA CTTACA AATTC CAAGC CGGTC AAGAA GAAGA GAG), 2 (GAAGG TCAAG AAGAA GCGAG ATTGG CCCTA GAAAC TG), 3 (AATTG GTGGT GCGGG CATGA TGG), 4 (GGCAT GATGG CGATT ACTGA TAC), and 5 (GGATA TTACT GCGAC TTACA AATT), respectively. In addition to the single point mutations, four double mutants (E485A,E490A, D472A,D476A, D472A,D479A, and D476A,D479A) and one triple mutant (D472A,D476A,D479A) were also constructed. The DNA sequences for each mutant were confirmed by DNA sequencing. The Nco I-Pst I fragments of these constructs were then subcloned into the Nco I and Pst I sites of pKK233-2 as described previously (20Lai T.-S. Santiago M.A. Achyuthan K.E. Greenberg C.S. Protein Expression Purif. 1994; 5: 125-132Crossref PubMed Scopus (15) Google Scholar). The E485A, E490A, E485A,E490A, D476A,D479A, and D472A,D476A,D479A mutants were also constructed in the pGEX-MCS vector and purified as glutathione S-transferase fusion proteins as described previously (19Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar). The expression and preparation of Escherichia coli lysates were as described (19Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar, 20Lai T.-S. Santiago M.A. Achyuthan K.E. Greenberg C.S. Protein Expression Purif. 1994; 5: 125-132Crossref PubMed Scopus (15) Google Scholar). Briefly, after resuspending E. coli pellets in 20 mm Tris-Cl, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol/EDTA, and 15% glycerol, the cells were lysed by lysozyme and sonication as described. Cell debris was removed by centrifugation at 22,000 × g for 20 min, and the supernatants were aliquoted and stored at −70 °C. Fresh aliquots were used for each experiment, and unused samples were discarded. The glutathione S-transferase (GST)-factor XIII A-chain fusion proteins were purified using glutathione-agarose affinity resins as described previously (19Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar). The amount of protein was determined by the Bradford method (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215560) Google Scholar) using a commercial reagent (Bio-Rad). Bovine serum albumin was used as the protein standard. The concentrations of affinity-purified GST-factor XIII A-chains, GST-E485A, GST-E490A, GST-E485A,E490A, GST-D476A,D479A, and GST-D472A,D476A,D479A were further normalized by scanning densitometry of the Coomassie Blue-stained gel of each protein band. The transglutaminase activities of E. coli lysates containing wild-type factor XIII A-chains or mutants were quantitated by measuring the incorporation of [3H]putrescine (NEN Life Science Products) or 5-(biotinamido)pentylamine into N, N′-dimethylcasein essentially as described (22Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar,23Slaughter T.F. Achyuthan K.E. Lai T.-S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (156) Google Scholar). Increasing concentrations (2.5–150 μg/ml) of the thrombin-activated materials were used to estimate the specific activity, and all assays were performed in triplicate. Aliquots were taken to analyze FXIIIa formation by quantitative immunoblotting and were used to normalize the specific activity. The Michaelis-Menten kinetic constant for the glutamine substrate (N, N′-dimethylcasein) was measured by the [3H]putrescine incorporation assay, and the kinetic constant for the primary amine substrate (5-(biotinamido)pentylamine) was determined using an assay as described earlier (22Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar, 23Slaughter T.F. Achyuthan K.E. Lai T.-S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (156) Google Scholar, 24Miraglia C.C. Greenberg C.S. Anal. Biochem. 1985; 144: 165-171Crossref PubMed Scopus (28) Google Scholar). The transglutaminase activity data were transformed by an Eadie-Hofstee plot to determine the Km. The enzyme concentration used in the assays expressed <5% of the total thrombin-independent FXIIIa activity. The measurement of the activation constant (Kact) for calcium ions was carried out by incubating FXIII with thrombin in the absence of calcium chloride at 37 °C for 15 min, followed by exposure to 0.04–5 mmcalcium chloride, and then measuring the calcium-dependent activity by the 3H incorporation assay. The Kact was determined by an Eadie-Hofstee plot of the calcium-dependent FXIIIa activity. The assay was performed essentially as described previously (19Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar, 25Fickenscher K. Aab A. Stuber W. Thromb. Haemostasis. 1991; 65: 535-540Crossref PubMed Scopus (115) Google Scholar). Affinity-purified GST-factor XIII A-chains, GST-E485A, GST-E490A, GST-E485A,E490A, GST-D476A,D479A, and GST-D472A,D476A,D479A (20–100 ng/ml) were thrombin-activated at 37 °C for 15 min. Aliquots were taken to measure the thrombin cleavage efficiency by quantitative immunoblotting and were used to normalize the FXIIIa concentration in the reaction (19Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar). The thrombin-activated mixtures were added to the reaction mixture containing glycine ethyl ester, synthetic peptide substrate, NADH, α-ketoglutarate, and bovine glutamate dehydrogenase provided by the manufacturer (Behring Diagnostics, Inc., Somerville, NJ) and incubated at 37 °C for 10 min. The release of ammonia was measured by the decrease in NADH and was quantified by measuring the decrease in absorbance (λ340 nm, milli-absorbance units/min) using a Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA). The data were then averaged for two duplicate experiments and are expressed as milli-absorbance units/min/mg. The fibrin binding assay was performed as described previously (22Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar). E. coli lysates of wild-type or mutant FXIII A-chains (0.75 μg/ml) were incubated with factor XIII-free fibrinogen (2.5 mg/ml; (American Diagnostica Inc.) in the presence of 20 mm Tris-Cl, pH 7.4, 100 mm NaCl, 2 mm CaCl2, and 0.73 μmα-thrombin. Fibrin clots were squeezed to remove the liquor. Gel electrophoresis and immunoblotting were performed on clot supernatants of wild-type and mutant factor XIII A-chains in parallel with control samples containing no fibrin. The binding of wild-type and mutant FXIIIa to fibrin was measured by quantitating the disappearance of FXIIIa antigen in the supernatants using scanning densitometry. The data are presented as percentage bound to fibrin relative to the wild-type FXIII A-chain. Fibrin cross-linking was performed at room temperature for 30 min in a reaction volume of 60 μl containing 50 mm Tris-Cl, pH 7.5, 50 μg of E. coli lysates containing wild-type FXIII A-chains or mutants, 10 mm CaCl2, 2 μm FXIII-free fibrinogen, and 0.73 μm human α-thrombin. The reactions were stopped by the addition of SDS-polyacrylamide gel electrophoresis loading buffer, separated by 6–15% SDS-polyacrylamide gel electrophoresis, and stained with Coomassie Blue. The proteolysis experiments were performed using the E. coli lysates (50 μg) containing either wild-type FXIII A-chains (or mutants) or 4 μg of affinity-purified GST-FXIII A-chains (or GST-E485A, GST-E490A, GST-E485A,E490A, GST-D476A,D479A, or GST-D472A,D476A,D479A) in a reaction volume of 50 μl containing 100 mm Tris acetate, pH 7.5, 100 mm NaCl, 0.1% polyethylene glycol 8000, increasing concentrations of α-thrombin (0–9.1 μm), and 5 mm CaCl2 or EDTA at 37 °C for 15 min. After adding SDS-polyacrylamide gel loading buffer to stop the reaction, the reaction mixtures were separated by 8.5% SDS-polyacrylamide gel electrophoresis and subjected to Western blotting. The factor XIII A-chain-related antigen was visualized using rabbit polyclonal antibody against factor XIII A-chains (Calbiochem). For experiments with affinity-purified GST fusion proteins, FXIIIa and the 51-kDa fragment were easily visualized after Coomassie Blue staining. The three-dimensional coordinates of the blood coagulation factor XIII A-chain were downloaded from the Protein Data Bank (Research Collaboratory for Structural Bioinformatics, Rutgers University) with access code 1fie (4Yee V.C. Pederson L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar). The molecular modeling of the FXIII A-chain was performed using the Composer module of the Sybyl Version 6.4 molecular modeling program (Tripos Associates, St. Louis, MO). The locations of the amino acid residues selected for mutagenesis are shown in relationship to the domain structure of the factor XIII A-chain (Fig. 1 A). In this model, the calcium-binding ligands Glu485 and Glu490 are located at the junction between the catalytic core domain and the first β-barrel domain (Fig. 1 A). These calcium-binding residues are also in close proximity (15 Å) to the secondary thrombin cleavage site (Lys513-Ser514) that is surface-exposed (Fig.1 A). Asp472 is located the farthest from the major calcium-binding coordinates (Fig. 1 B), although it is closer to the catalytic triad (Cys314, His373, and Asp396) than any of the other residues mutated in this study (Fig. 1 A). Asp476 is located in a β-sheet and Asp479 in a helical turn that are in close proximity to the reported calcium-binding site (Fig. 1 B). All three side chains of Asp472, Asp476, and Asp479 are solvent-accessible. The Asp479 side chains do not participate in any hydrogen bonding with other amino acid residues. The side chains of Asp472 participate in hydrogen bonding with Lys704. The side chains of Asp476also participate in hydrogen bonding with Thr478. Asp476 and Asp479 are <5 Å apart. The shortest distances between Asp472, Asp476, or Asp479 and Glu485 and Glu490 are 29.5, 16, and 10.6 Å, respectively (Fig. 1 B). The shortest distance between Asp472, Asp476, or Asp479 and the catalytic triad residues is ∼13 Å. All FXIII mutants were expressed in E. coli to the same level as wild-type FXIII A-chains (20 mg/liter). When analyzed by SDS-polyacrylamide gel electrophoresis, they migrated with the same mobility as the wild-type FXIII A-chains. The summary of the biochemical data derived from the study of each mutant is shown in Table I. The specific activity of the D472A mutant was unchanged, whereas the specific activities of the point mutations D476A and D479A were reduced by only ∼15%. An additional Ala substitution at Asp472 to the D476A and D479A mutants did not further modify the activity. In contrast, there was a significant inhibition of activity if both aspartic acids 476 and 479 were converted to alanine. The activity of the D476A,D479A double mutant was reduced by 69%, and an additional Ala substitution at Asp472 reduced activity by 86%. The specific activity of E485A was similar to that of E490A and was reduced by 47 and 49%. The activity of the E485A,E490A double mutant was reduced by 79%. The mutants that had reduced activity had the activation peptide cleaved by thrombin when analyzed by immunoblotting (data not shown). Therefore, the loss of FXIIIa activity displayed by these mutants was not due to a defect in cleaving the activation peptide or in cleavage at another site within the catalytic core that would inactivate FXIIIa.Table ISummary of biochemical data for each mutantKm(BP)1-aBP, 5-(biotinamido)pentylamine; mAU, mill-absorbance units; ND, not determined.Km(casein)Kact(Ca2+)Specific activity1-bThe specific activity was determined in reactions containing 1 mm Ca2+.Fibrin bindingAmmonia releaseμmμmμm%% boundmAU/min/mgWild-type75 ± 133 ± 1337 ± 401008717.8 ± 1.6E485A99 ± 192.7 ± 1.5934 ± 8053 ± 69110.1 ± 1.1E490A90 ± 152.5 ± 22688 ± 38151 ± 8929.3 ± 0.8E485A,E490A77 ± 223.5 ± 1.67170 ± 56021 ± 10899.2 ± 1.5D472A89 ± 276.1 ± 1451 ± 64106 ± 1684NDD476A75 ± 14.1 ± 1.5307 ± 8684 ± 288NDD479A77 ± 25 ± 2.7268 ± 18284 ± 688NDD472A,D476A73 ± 25.5 ± 2579 ± 13677 ± 987NDD472A,D479A70 ± 74.8 ± 1.5424 ± 1882 ± 0.491NDD476A,D479A107 ± 14.3 ± 0.7159 ± 4031 ± 5904.0 ± 1.3D472A,D476A,D479A103 ± 89 ± 2213 ± 16514 ± 2881.1 ± 0.51-a BP, 5-(biotinamido)pentylamine; mAU, mill-absorbance units; ND, not determined.1-b The specific activity was determined in reactions containing 1 mm Ca2+. Open table in a new tab The apparent Km was unchanged when the glutamine substrate (N, N′-dimethylcasein) was studied for the single and double mutants E485A, E490A, E485A,E490A, D472A, D476A, D479A, D472A,D476A, D472A,D479A, and D476A,D479A. The Km for the triple mutant D472A,D476A,D479A was only ∼3-fold higher than that for wild-type FXIIIa (Table I). The apparent Km for the primary amine substrate (5-(biotinamido)pentylamine) was similar for all the thrombin-activated FXIII A-chain mutant molecules studied (75–107 μm). We examined whether the loss of activity was due to a defect in the formation of the thioester bond or in the catalysis of the amide transfer reaction for the E485A, E490A, E485A,E490A, D476A,D479A, and D472A,D476A,D479A mutants. The formation of the thioester bond was measured by quantitating ammonia release using a glutamine peptide substrate as described previously (16Ikura K. Nasu T. Yokota H. Tsuchiya Y. Sasaki R. Chiba H. Biochemistry. 1998; 27: 2898-2905Crossref Scopus (149) Google Scholar, 22Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar). The affinity-purified E485A, E490A, E485A,E490A, D476A,D479A, and D472A,D476A,D479A mutants were thrombin-activated and found to have thioester bond formation reduced by 1.8-, 1.9-, 1.9-, 4.5-, and 16-fold, respectively, when compared with wild-type FXIIIa. The Kact values for calcium for the E485A, E490A, and E485A,E490A mutants increased by 2.8-, 8-, and 21-fold, respectively, whereas the D476A and D479A mutants had similar Kact values (∼337 μm) for calcium ions compared with wild-type FXIIIa. The Kact values for the D472A, D472,479A, and D472,479A mutants were only 1.3–1.7-fold higher than that for wild-type FXIIIa. The Kact values for the D476A,D479A and D472A,D476A,D479A mutants were slightly reduced by 2.1- and 1.6-fold, respectively (Table I). All mutants bound to a fibrin clot to the same extent as wild-type FXIIIa (84–92%) (Table I). The FXIIIa mutant molecules also displayed the same pattern of fibrin cross-linking as wild-type FXIIIa, with γ-chain dimer formation occurring prior to α-chain polymer formation. However, the rate of cross-linking was reduced for mutants that had reduced activity as measur" @default.
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• W2013685938 title "Site-directed Mutagenesis of the Calcium-binding Site of Blood Coagulation Factor XIIIa" @default.
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