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• W2007067293 abstract "Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the γ chains of fibrin(ogen) more efficiently than the Aα chains. On the other hand, tTG is thrombin-independent and forms predominantly Aα and Aα-γ chain complexes. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269, 28309–28313). To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension. Other work from this laboratory (Achyuthan, K. E., Slaughter, T. F., Santiago, M. A., Enghild, J. J., and Greenberg, C. S. (1993) J. Biol. Chem.268, 21284–21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5). Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged. FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis. FXIII preferentially formed the γ-γ dimer, whereas tTG formed Aα-γ complexes. Chimera 7 formed Aα-γ complexes that resembled the cross-linking pattern of tTG. This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aα and Aα-γ cross-link pattern characteristic of tTG. Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the γ-γ dimer, whereas chimera 3 was not active. In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aα and Aα-γ complexes) instead of FXIII (γ-γ dimers). Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the γ chains of fibrin(ogen) more efficiently than the Aα chains. On the other hand, tTG is thrombin-independent and forms predominantly Aα and Aα-γ chain complexes. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269, 28309–28313). To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension. Other work from this laboratory (Achyuthan, K. E., Slaughter, T. F., Santiago, M. A., Enghild, J. J., and Greenberg, C. S. (1993) J. Biol. Chem.268, 21284–21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5). Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged. FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis. FXIII preferentially formed the γ-γ dimer, whereas tTG formed Aα-γ complexes. Chimera 7 formed Aα-γ complexes that resembled the cross-linking pattern of tTG. This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aα and Aα-γ cross-link pattern characteristic of tTG. Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the γ-γ dimer, whereas chimera 3 was not active. In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aα and Aα-γ complexes) instead of FXIII (γ-γ dimers). During the process of blood coagulation, plasma transglutaminase, also known as factor XIII (FXIII), 1The abbreviations used are: FXIII, factor XIII; tTG, tissue transglutaminase; BP, 5-(biotinamido)pentylamine; GST, glutathione S-transferase.1The abbreviations used are: FXIII, factor XIII; tTG, tissue transglutaminase; BP, 5-(biotinamido)pentylamine; GST, glutathione S-transferase. is converted to FXIIIa by the action of thrombin (1Lorand L. Konishi K. Arch. Biochem. Biophys. 1964; 105: 58-67Crossref PubMed Scopus (167) Google Scholar). FXIIIa then acts within the fibrin clot to stabilize the clot by increasing the mechanical strength (2Shen L. Lorand L. J. Clin. Invest. 1983; 71: 1336-1341Crossref PubMed Scopus (120) Google Scholar) and reducing the susceptibility of the clot to proteolytic degradation by plasmin (3Gaffney P.J. Whitaker A.N. Thromb. Res. 1979; 14: 85-94Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 4Sakata Y. Aoki N. J. Clin. Invest. 1982; 69: 536-542Crossref PubMed Scopus (233) Google Scholar, 5Sakata Y. Mimuro J. Aoki N. Blood. 1984; 63: 1393-1401Crossref PubMed Google Scholar, 6Francis C.W. Marder V.J. Blood. 1988; 71: 1361-1365Crossref PubMed Google Scholar). The stabilization of the clot occurs when FXIIIa catalyzes the formation of covalent ε-(γ-glutamyl)lysine cross-links between fibrin molecules (7McKee P.A. Mattock P. Hill R.L. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 738-744Crossref PubMed Scopus (259) Google Scholar, 8Lorand L. Ann. N. Y. Acad. Sci. 1972; 202: 6-30Crossref PubMed Scopus (124) Google Scholar, 9Folk J.E. Finlayson J.S. Adv. Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (783) Google Scholar, 10Mosher D.F. Ann. N. Y. Acad. Sci. 1978; 312: 38-42Crossref PubMed Scopus (31) Google Scholar, 11Sakata Y. Aoki N. J. Clin. Invest. 1980; 65: 290-297Crossref PubMed Scopus (306) Google Scholar, 12Tamaki T. Aoki N. Biochim. Biophys. Acta. 1981; 661: 280-286Crossref PubMed Scopus (76) Google Scholar). Fibrinogen is a large (340 kDa) trinodular glycoprotein composed of three pairs of different polypeptide chains (Aα, Bβ, γ) linked by disulfide bonds (13McKee P.A. Rogers L.A. Marler E. Hill R.L. Arch. Biochem. Biophys. 1966; 116: 271-279Crossref PubMed Scopus (91) Google Scholar, 14Hermans J. McDonagh J. Semin. Thromb Hemostasis. 1982; 8: 11-24Crossref PubMed Scopus (98) Google Scholar, 15Doolittle R.F. Ann. N. Y. Acad. Sci. 1983; 408: 13-27Crossref PubMed Scopus (86) Google Scholar, 16Henschen A. Lottspeich F. Kehl M. Southan C. Ann. N. Y. Acad. Sci. 1983; 408: 28-43Crossref PubMed Scopus (206) Google Scholar). The rapid cross-linking of the γ chains into the γ-γ dimer (17Pisano J.J. Finlayson J.S. Peyton M.P. Science. 1968; 160: 892-893Crossref PubMed Scopus (245) Google Scholar, 18Doolittle R.F. Chen R. Lau F. Biochem. Biophys. Res. Commun. 1971; 44: 94-100Crossref PubMed Scopus (54) Google Scholar) occurs when fibrinogen is converted to fibrin by the action of thrombin (19Blomback B. Vestermark A. Ark. Kemi. 1958; 12: 173Google Scholar). Formation of the γ-γ dimer is then followed by intermolecular cross-linking between the γ chains and Aα chains to form Aα-γ2 hybrids (20Shainoff J.R. Urbanic D.A. DiBello P.M. J. Biol. Chem. 1991; 266: 6429-6437Abstract Full Text PDF PubMed Google Scholar). It has also been shown that FXIIIa cross-links the γ chains into γ trimers and tetramers (21Siebenlist K.R. Mosesson M.W. Biochemistry. 1992; 31: 936-941Crossref PubMed Scopus (31) Google Scholar, 22Mosesson M.W. Siebenlist K.R. Amrani D.L. DiOrio J.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1113-1117Crossref PubMed Scopus (80) Google Scholar). In contrast, the tissue transglutaminase (tTG) preferentially cross-links the Aα chains and an Aα-γ complex of fibrin (20Shainoff J.R. Urbanic D.A. DiBello P.M. J. Biol. Chem. 1991; 266: 6429-6437Abstract Full Text PDF PubMed Google Scholar, 23Chung S.I. Ann. N. Y. Acad. Sci. 1972; 202: 240-255Crossref PubMed Scopus (121) Google Scholar,24Murthy S.N. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9679-9682Crossref PubMed Scopus (31) Google Scholar). Immunoelectrophoretic analysis of cross-linked fibrin(ogen) can readily distinguish whether the cross-linked products resulted from the action of tTG or FXIII (20Shainoff J.R. Urbanic D.A. DiBello P.M. J. Biol. Chem. 1991; 266: 6429-6437Abstract Full Text PDF PubMed Google Scholar, 23Chung S.I. Ann. N. Y. Acad. Sci. 1972; 202: 240-255Crossref PubMed Scopus (121) Google Scholar). This methodology has been used successfully to distinguish tTG and FXIII cross-linked fibrin(ogen) complexes in atherosclerotic aortic intimas and vascular graft pseudo-intimas (25Shainoff J.R. Valenzuela R. Urbanic D.A. DiBello P.M. Lucas F.V. Graor R. Blood Coagul. Fibrinolysis. 1990; 1: 499-503Crossref PubMed Scopus (12) Google Scholar, 26Valenzuela R. Shainoff J.R. DiBello P.M. Urbanic D.A. Anderson J.M. Matsueda G.R. Kudryk B.J. Am. J. Pathol. 1992; 141: 861-880PubMed Google Scholar). The purpose of the present study was to localize the substrate recognition domains between these two homologous transglutaminases using recombinant chimeric FXIII/tTG molecules. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (27Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar). In addition, using synthetic peptides, we also identified two other exons (exons 3 and 5) that might play a role in substrate recognition (28Achyuthan K.E. Slaughter T.F. Santiago M.A. Enghild J.J. Greenberg C.S. J. Biol. Chem. 1993; 268: 21284-21292Abstract Full Text PDF PubMed Google Scholar). To determine whether the primary amino acid sequence in these exons was responsible for defining the characteristic cross-link pattern between FXIII and tTG, recombinant FXIII/tTG chimeras were created in which exons 3, 5, and 7 of FXIII were replaced with the corresponding amino acids from tTG. Replacing exon 7 (the exon that contains the active site) of FXIII with that of tTG resulted in a FXIII chimera that produced fibrin cross-link products characteristic of the tTG. This finding suggests that some of the specificity for the Aα and Aα-γ cross-link pattern characteristic of tTG resides in the primary amino acid sequence of exon 7. All restriction enzymes, T4 DNA ligase, Taq polymerase, and streptavidin alkaline phosphatase were purchased from Life Technologies, Inc. FXIII-free human fibrinogen was obtained from American Diagnostica, Inc. (Greenwich, CT). Human α-thrombin was the generous gift of Dr. J. W. Fenton II (New York State Department of Health, Albany). Oligonucleotides were synthesized by Biosynthesis, Inc. (Lewisville, TX). 5-(Biotinamido)pentylamine (BP) was obtained from Pierce. SDS-polyacrylamide gel electrophoresis molecular weight markers were obtained from Bio-Rad. The monoclonal antibodies specific for the fibrinogen γ chain (4A5) and the Aα chain (F103) were the generous gifts of Dr. Gary Matsueda (Bristol Myers Squibb, Princeton, NJ) and Dr. Joan Sobel (Columbia University, New York), respectively. All other reagents were purchased from Sigma unless otherwise stated. The polyclonal antibody to the FXIII A chain was purchased from Calbiochem. The construction of the plasmids for FXIII (pG-FXIII) and tTG (pG-tTG) have been described previously (29Hettasch J.M. Bandarenko N. Burchette J.L. Lai T.S. Marks J.R. Haroon Z.A. Peters K. Dewhirst M.W. Iglehart J.D. Greenberg C.S. Lab. Invest. 1996; 75: 637-645PubMed Google Scholar). The plasmids containing the cDNA for wild type FXIII and tTG were created using the pGEX-3X vector (Pharmacia Biotech Inc.). This vector provides a means for expressing these two transglutaminases as glutathione S-transferase (GST) fusion proteins to simplify purification. The cDNAs for three different FXIII/tTG chimeras were generated using a polymerase chain reaction-based method known as gene splicing by overlap extension (30Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 61-68Crossref PubMed Scopus (2634) Google Scholar, 31Horton R.M. Pease L.R. McPherson M.J. Directed Mutagenesis. IRL Press, New York1991: 217-247Google Scholar). The plasmids pG-tTG and pG-FXIII, synthetic oligonucleotides, and the polymerase chain reaction were used to exchange precisely segments of FXIII with corresponding regions of tTG. These three exons were postulated to be important for substrate specificity, and the amino acid substitutions were based on sequence alignment of tTG and FXIII. Three different FXIII/tTG chimeras were generated: (i) an exon 3 swap (designated chimera 3), where amino acid residues 43–105 of FXIII were replaced with residues 1–62 of tTG; (ii) an exon 5 swap (designated chimera 5), where amino acid residues 190–229 of FXIII were replaced with residues 144–183 of tTG; and (iii) an exon 7 swap (designated chimera 7), where amino acid residues 266–323 of FXIII were replaced with residues 227–285 of tTG. The aligned sequences for these three exons are illustrated in Fig.1 A, and schematics of the FXIII/tTG chimeras are represented in Fig. 1 B. After the chimeric regions were generated by splice overlap extension, theNcoI-SacI fragment from pG-FXIII was replaced with the NcoI-SacI fragment from chimera 3 and chimera 5 to generate pG-chimera 3 and pG-chimera 5, respectively. The pG-chimera 7 construct was created by replacing theNcoI-StuI fragment of pG-FXIII with theNcoI-StuI fragment of chimera 7. DNA sequencing (32Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar, 33Biggin M.D. Gibson T.J. Hong G.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3963-3965Crossref PubMed Scopus (1411) Google Scholar) was performed on the regions that were generated by the polymerase chain reaction to verify that the nucleotide sequence was not altered during this manipulation. The carboxyl-terminal truncated forms of the FXIII/tTG chimeras were constructed by replacing the NcoI-Sst1 fragment of pG-K513 with theNcoI-Sst1 fragment from each of the full-length chimeras (pG-chimera 3, pG-chimera 5, or pG-chimera 7) to produce pG-chimera 3-K513, pG-chimera 5-K513, and pG-chimera 7-K513. The construction of pG-K513 has been described previously (34Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg G.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar). The generation of pG-chimera 3, pG-chimera 5, and pG-chimera 7 described above creates a system for expressing the FXIII/tTG chimeras as GST fusion proteins. These GST fusion proteins were purified using a glutathione-agarose affinity resin as described previously (34Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg G.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar). Briefly, the Escherichia coli strain JM105 containing the plasmid for FXIII, tTG, or the 3 FXIII/tTG chimeras was grown in 2 liters of enriched medium. The E. coli were chilled on ice, harvested by centrifugation, and resuspended in 200 ml of cold buffer A (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 15% glycerol, 1 mm EDTA/dithiothreitol, 10 μg/ml aprotinin, 0.2 mm phenylmethylsulfonyl fluoride). The resuspended cells were then sonicated seven times for 10 s with 20-s rest between bursts. The lysates were centrifuged at 22,000 ×g for 20 min at 4 °C. After centrifugation, the supernatant was applied to a glutathione-agarose column (8 × 2.6 cm) that was equilibrated with buffer A. The column was then washed with 20 bed volumes of buffer A containing 0.5% Triton X-100 followed by 20 bed volumes of buffer A without detergent. The GST fusion proteins were eluted with 50 mm Tris-HCl, pH 7.5, 15% glycerol, 10 mm glutathione. The fractions containing the recombinant protein were concentrated in an Amicon ultrafiltration cell and then dialyzed extensively against 20 mm Tris acetate, pH 8.0, 1 mm dithiothreitol, 1 mm EDTA, 20% glycerol. Protein concentration was determined using the Bio-Rad protein assay with bovine serum albumin as the standard. The recombinant proteins were aliquoted, frozen, and stored at −80 °C until assayed for activity. FXIIIa activity was assayed using a microtiter plate assay described previously (35Slaughter T.F. Achyuthan K.E. Lai T.S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (154) Google Scholar). Briefly, microtiter plates (Costar Corp., Cambridge MA) were coated with 200 μl ofN,N-dimethylcasein (20 mg/ml) for 1 h at room temperature followed by blocking with 3% bovine serum albumin in TBST (100 mm Tris, pH 8.5, 150 mm NaCl, 0.05% Tween 20). FXIII, chimera 7, chimera 5, or chimera 3 (0.3–32 μg/ml) was added to the wells followed by a solution (50 μl) containing 100 mm Tris, pH 8.5, 20 mm CaCl2, and 40 units/ml thrombin. The microtiter plate was incubated at 37 °C for 30 min to activate FXIII. After activation, 50 μl of a solution containing 100 mm Tris, 40 mm dithiothreitol, and 2 mm BP was added to the wells of the microtiter plate. After the addition of BP, the plate was incubated at 37 °C for 40 min to allow FXIIIa to cross-link BP into the dimethylcasein. The microtiter plates were washed once with TBST containing 1 mm EDTA, followed by three washes with TBST. The plates were then incubated for 1 h at room temperature with 100 μl of streptavidin-alkaline phosphatase diluted 1:500 in 3% bovine serum albumin/TBST. Plates were then washed with TBST, and 200 μl of the substrate p-nitrophenyl phosphate (1 mg/ml in 100 mm Tris, pH 9.8, 100 mm NaCl, 5 mmMgCl2) was added. The rate of color development was monitored for 15 min at 15-s intervals at 405 nm in aV max kinetic microplate reader (Molecular Devices, Menlo Park, CA). In experiments where fibrin was used as the substrate, fibrinogen (10 μg/ml) was used to coat the microtiter plate wells (1 h at room temperature), and then 20 units/ml thrombin was added to convert the fibrinogen to fibrin. The thrombin was then washed out with TBST, and the assay was performed as described above for dimethylcasein. FXIII, chimera 7, chimera 5, or chimera 3 (9 nm) was incubated with FXIII-free fibrinogen (1 mg/ml) in the presence of 20 mm Tris-HCl, pH 7.4, 100 mmNaCl, 10 mm CaCl2, and 20 units/ml α-thrombin. Clots were allowed to form for 1 h and then squeezed mechanically to release the clot liquor. Gel electrophoresis of clot supernatants was performed on a 4–10% polyacrylamide gradient using the buffer system of Laemmli (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). After electrophoresis, the proteins were transferred to nitrocellulose (0.2 μm) as described by Towbin et al.(37Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44843) Google Scholar). After the transfer was complete, the nitrocellulose membrane was blocked for 1 h with 3% nonfat milk dissolved in 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% Tween 20. The FXIIIa or FXIIIa/tTG chimeric antigen was detected by incubation for 1 h with a polyclonal antibody against the human FXIII A chain subunit (Calbiochem), followed by incubation for 1 h with goat anti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad). The FXIIIa or FXIIIa/tTG chimeric antigen was visualized by precipitation of the chromogenic substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. The binding of wild type FXIII and the FXIII/tTG chimeras to fibrin clots was measured by quantitating the disappearance of FXIIIa or FXIIIa/tTG chimeric antigen from the clot supernatants using a Hoefer GS 300 densitometer. Previously established assay conditions designed to determine the rate of fibrin cross-linking by FXIIIa were used to analyze the cross-linking activity of the FXIII/tTG chimeras (28Achyuthan K.E. Slaughter T.F. Santiago M.A. Enghild J.J. Greenberg C.S. J. Biol. Chem. 1993; 268: 21284-21292Abstract Full Text PDF PubMed Google Scholar). In these experiments, FXIII (30 nm), tTG (325 nm), or chimera 3, 5, or 7 (0.03–1.2 μm) was incubated with FXIII-free fibrinogen (0.5 mg/ml) in the presence of 0.1 m Tris acetate, pH 7.4, 0.15 m NaCl, 0.1% polyethylene glycol 8000, 10 mm CaCl2, and 20 units/ml α-thrombin for 1 h at room temperature. The total assay volume was 300 μl, and the reactions were stopped by the addition of SDS-polyacrylamide gel electrophoresis sample buffer containing 6 m urea, followed by boiling for 5 min. Gel electrophoresis of these samples (3.75 μg of fibrinogen was loaded/well) was performed on a 4–10% polyacrylamide gradient gel, and the proteins were transferred to nitrocellulose (0.2 μm) as described above. After transfer was complete, the nitrocellulose was blocked with 3% nonfat dry milk dissolved in 20 mm Tris, pH 7.4, 150 mm NaCl, 0.5% Tween 20 for 1 h. To detect the cross-linked γ chain products, the nitrocellulose was incubated for 1 h with a monoclonal antibody to the human fibrinogen γ chain (4A5) followed by incubation for 1 h with goat anti-mouse IgG conjugated to alkaline phosphatase (Bio-Rad). Fibrinogen on the nitrocellulose was visualized by precipitation of the chromogenic substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. To detect the cross-linked Aα chain products, a monoclonal antibody (F103) to the human fibrinogen Aα chain was used in place of 4A5. To gain insight into the molecular basis of transglutaminase substrate specificity, three recombinant chimeras of FXIII and tTG were expressed, purified, and biochemically characterized. We demonstrated previously that several amino acid residues around the active site cysteine in exon 7 were important for FXIII catalysis (27Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar). In addition, our laboratory has identified two regions (one in exon 3 and the other in exon 5) as potential substrate recognition sites for transglutaminases using synthetic peptides (28Achyuthan K.E. Slaughter T.F. Santiago M.A. Enghild J.J. Greenberg C.S. J. Biol. Chem. 1993; 268: 21284-21292Abstract Full Text PDF PubMed Google Scholar). Fig. 1 Aillustrates the aligned amino acid sequence of these three exons from FXIII and tTG. The sequence identity between tTG and FXIII in the exon 3, exon 5, and exon 7 domains is 29, 53, and 55%, respectively, and is highlighted by the boxed regions. In this study, these three exons in FXIII were exchanged with the corresponding exons of tTG using a polymerase chain reaction technique known as gene splicing by overlap extension (for details of the exchange, see “Experimental Procedures”). Fig. 1 B is a schematic diagram of the structure of the three recombinant FXIII/tTG chimeras that were created. Chimera 3 has amino acid residues 43–105 of FXIII replaced with residues 1–62 of tTG, chimera 5 has amino acid residues 190–229 of FXIII exchanged with residues 144–183 of tTG, and chimera 7 has amino acid residues 266–323 of FXIII interchanged with residues 227–285 of tTG. The cDNAs for FXIII and tTG were subcloned into a commercially available vector, pGEX-3X as described (34Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg G.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar, 38Lai T.-S. Slaughter T.F. Koropchak C.M. Haroon Z.A. Greenberg C.S. J. Biol. Chem. 1996; 271: 31191-31195Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The cDNAs for the chimera proteins were created using gene splicing by overlap extension and subcloned into the pGEX-3X vector as described under “Experimental Procedures.” This vector provides a means for expressing transglutaminases as GST fusion proteins, thereby simplifying the purification procedure. A representative Coomassie Blue-stained gel of these GST-transglutaminases is illustrated in Fig.2 A. From the left, the first lane shows the location of molecular weight markers, and the second, third,fourth, fifth, and sixth lanes show GST-FXIII, GST-chimera 3, GST-chimera 5, GST-chimera 7, and GST-tTG, respectively, after elution from the glutathione affinity column. There are some minor lower molecular weight bands in the chimera 3, 5, and 7 lanes; immunoblot analysis (Fig. 2 B) with a polyclonal antibody to FXIII indicates that these bands are proteolytic fragments of the recombinant proteins. The exchange of amino acid residues between the tTG and FXIII may create a molecule that is more susceptible to proteolytic degradation. Densitometric scanning of the Coomassie Blue-stained gel indicates that the GST-transglutaminase preparations contain at least 85% full-length protein. The activity of the purified recombinant FXIII chimeras was first evaluated by examining the incorporation of a small biotinylated primary amine (BP) into dimethylcasein. Fig.3 A compares the activities of FXIIIa and chimeras 7, 5, and 3. The incorporation of BP into dimethylcasein was rapid as the protein concentration was increased from 1.8–12 nm for FXIII and chimera 7. In contrast, the incorporation of BP into dimethylcasein by chimera 5 proceeded at a slower rate and plateaued at a rate of milli-optical density units/min that was 35% that of the wild type enzyme. In addition, the protein concentration of chimera 5 required to achieve this activity was 5-fold (60 nm) greater than that of FXIIIa (12 nm). The activity of chimera 5 was not enhanced even when the concentration of this enzyme was increased 16-fold (to 192 nm). Chimera 3 (1.8–192 nm) was not active in this assay. Similar results were obtained when the incorporation of BP into fibrin was examined (Fig. 3 B). Chimera 7 had the same degree of activity as FXIIIa. The enzymatic activity of chimera 5 was reduced by 70%, and chimera 3 was not active in this assay. Quantitative immunoblot analysis revealed that FXIII and the three chimeras were cleaved 75–85% by 20 units/ml α-thrombin, demonstrating that the differences in activity were not caused by alterations in the thrombin cleavage pattern of these recombinant proteins. To evaluate the ability of recombinant chimera 7 and chimera 5 to recognize soluble fibrinogen as a substrate, the capacity of fibrinogen to inhibit the incorporation of BP into dimethylcasein was examined. In these experiments, preactivated FXIIIa (3 nm), chimera 7 (3 nm), or chimera 5 (30 nm) was incubated with 0.5 mm BP in the absence or presence of increasing concentrations of soluble fibrinogen (0.02–1.25 mg/ml). Fig.4 illustrates the inhibition curves obtained for FXIII and these two recombinant FXIII/tTG chimeras. Fibrinogen blocked BP incorporation into dimethylcasein with an IC50 of 0.4 mg/ml for all three recombinant proteins. These findings demonstrate that chimera 7 and chimera 5 are capable of binding and interacting with soluble fibrinogen with the same affinity as FXIIIa. The next series of experiments examined the capacity of chimera 3 to bind substrate. Because chimera 3 was inactive, this study was designed to examine whether chimera 3 could act as a competitive inhibitor of FXIIIa. In these experiments, FXIIIa (3 nm) was incubated with 0.5 mm BP in the absence or presence of 7.5–240 nm chimera 3. Fig. 5illustrates a concentration-dependent inhibition of FXIIIa activity by chimera 3. When the concentration of chimera 3 (240 nm) was increased 80-fold over FXIIIa (3 nm), there was a 60% decrease in the activity of the wild type enzyme. Similar results were obtained with a catalytically inactive mutant of FXIII. This inactive form of FXIII has been characterized previously (27Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar) and was created by converting the active site cysteine (residue 314) to alanine. This point mutation produces a catalytically inactive molecule that still has the capacity to bind fibrin. The FXIII C314A mutant produced a dose-dependent inhibition of FXIIIa (3 nm) over the same concentration range (7.5–240 nm) as chimera 3. When the concentration of FXIII C314A (240 nm) was increased 80-fold over the wild type enzyme (3 nm), FXIIIa activity was reduced by 50%. These findings demonstrate that chimera 3 is capable of recognizing and binding substrate and suggest that even though chimera 3 is catalytically inactive the molecular structure of the protein is still intact. To evaluate the ability of these chimeras to recognize large macromolecular substrates, the extent to which these recombinant chimeras bound fibrin was examined. TableI summarizes the fibrin binding data and illustrates that chimera 7, chimera 5, and chimera 3 bind fibrin to the same extent as FXIIIa. Therefore, even though the exchange of exon 5 and exon 3 of tTG into the corresponding region of FXIII has altered the catalytic activity of FXIIIa, the ability of these chimeras to recognize and bind fibrin substrate has not changed; this implies that the overall structure of the molecule has not been altered by the exchange of these amino acid residues.Table IBinding of FXIII and FXIII chimeras to fibrinProteinBinding to fibrin%Wild type FXIII91Chimera 791Chimera 589Chimera 388The binding data represent the average of three independent experiments. Open table in a new tab The binding data represent the average of three independent experiments. Fibrin cross-linking analysis of several FXIII mutants (34Lai T.-S. Achyuthan K.E. Santiago M.A. Greenberg G.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar) as well as information available from the x-ray crystal structure (39Pedersen L.C. Yee V.C. Bishop P.D. Le Trong I. Teller D.C. Stenkamp R.E. Pr" @default.
• W2007067293 created "2016-06-24" @default.
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• W2007067293 date "1997-10-01" @default.
• W2007067293 modified "2023-10-16" @default.
• W2007067293 title "Analysis of Factor XIII Substrate Specificity Using Recombinant Human Factor XIII and Tissue Transglutaminase Chimeras" @default.
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