C-terminal Deletion of Human Tissue Transglutaminase Enhances Magnesium-dependent GTP/ATPase Activity
1996; Elsevier BV; Volume: 271; Issue: 49 Linguagem: Inglês
10.1074/jbc.271.49.31191
ISSN1083-351X
AutoresThung‐S. Lai, Thomas Slaughter, C. M. Koropchak, Zishan A. Haroon, Charles S. Greenberg,
Tópico(s)Blood properties and coagulation
ResumoTissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (ΔS538, ΔE447, ΔP345, ΔC290, ΔV228, and ΔF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent Km value for either GTP or ATP. In contrast, the Kcat value for GTP was increased by 4.6- and 3-fold for the ΔS538 and ΔE447 mutants, respectively. The ΔP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1.9-fold for the ΔC290, ΔV228, and ΔF185 mutants, respectively. The Kcat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the ΔP345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the ΔS538 and ΔE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed. Tissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (ΔS538, ΔE447, ΔP345, ΔC290, ΔV228, and ΔF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent Km value for either GTP or ATP. In contrast, the Kcat value for GTP was increased by 4.6- and 3-fold for the ΔS538 and ΔE447 mutants, respectively. The ΔP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1.9-fold for the ΔC290, ΔV228, and ΔF185 mutants, respectively. The Kcat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the ΔP345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the ΔS538 and ΔE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed. INTRODUCTIONTissue transglutaminase (tTG) 1The abbreviations used are: tTGtissue transglutaminaseGSTglutathione S-transferaseTGasethe activity of tTG to carry out the calcium-dependent incorporation of [3H]putrescine or biotinylated pentylamine into N,N-dimethylcaseinECMextracellular matrixBSAbovine serum albuminTBSTris-buffered saline. is a unique member of the transglutaminase gene family in that it exhibits two distinct enzyme activities (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar, 3Lee K.N. Birckbichler P.J. Patterson Jr., M.K. Biochem. Biophys. Res. Commun. 1989; 162: 1370-1375Crossref PubMed Scopus (93) Google Scholar). The calcium-dependent transglutaminase activity (TGase) catalyzes the covalent modification of proteins by the formation of γ-glutamyl-ϵ-lysine bonds between proteins or polyamines (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). The TGase activity is considered to be an important intracellular and extracellular reaction during apoptosis (4Fesus L. Davies P.J.A. Piacentini M. Eur. J. Cell. Biol. 1991; 56: 170-177PubMed Google Scholar, 5Fesus L. Thomazy V. Falus A. FEBS Lett. 1987; 224: 104-108Crossref PubMed Scopus (406) Google Scholar), bone ossification (6Aeschlimann D. Wetterwald A. Fleisch H. Paulsson M. J. Cell Biol. 1993; 120: 1461-1470Crossref PubMed Scopus (166) Google Scholar), tissue repair (7Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.P. J. Cell Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar), and tumor growth (8Johnson T.S. Knight C.R.L. El Alaoui S. Mian S. Rees R.C. Gentiles V. Davies P.J.A. Griffin M. Oncogene. 1994; : 2935-2942PubMed Google Scholar). TGase activity requires a calcium binding site and active site cysteine to form a thioester bond with the glutamine substrate (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). The active site of human tTG is located at Cys-277, and the putative calcium binding site is located between amino acids 446 and 453 based on sequence homology to the calcium binding site in the factor XIII A chains (9Gentile V. Saydak M. Chiocca E.A. Akande 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).The tTG will selectively modify a group of protein-bound glutamine residues that exist in proteins found in the extracellular matrix (ECM) including vitronectin, fibronectin, osteonectin, and nidogen (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). When tTG is released into plasma or ECM it binds to fibronectin and retains TGase activity (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). Fibronectin binding functions to localize tTG to sites of fibronectin expression and deposition and limits the availability of the enzyme for cross-linking other substrates. The fibronectin binding site is located in the N-terminal seven amino acid residues (10Jeong J.-M. Murthy S.N.P. Radek J.T. Lorand L. J. Biol. Chem. 1995; 270: 5654-5658Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).The tTG binds GTP and ATP inducing a conformational change that causes a reduction in the affinity for calcium and in TGase activity (11Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar, 12Bergamini C.M. FEBS Lett. 1988; 239: 255-258Crossref PubMed Scopus (73) Google Scholar). The binding of ATP and/or GTP to intracellular tTG could play a major role in suppressing TGase activity and preventing intracellular protein cross-linking reactions. In addition, a magnesium-dependent GTP hydrolysis activity (GTPase) was discovered to reside in the molecule and does not require the active site cysteine (13Lee K.N. Shelly A.A. Birckbichler P.J. Patterson Jr., M.K. Fraij B.M. Takeuchi Y. Carter H.A. Biochim. Biophys. Acta. 1993; 1202: 1-6Crossref PubMed Scopus (77) Google Scholar). The over-expression of a tTG mutant with the active site Cys-277 mutated to alanine only expressed GTPase activity and caused cell cycle arrest at the S to G2/M interphase (14Mian S. El Alaoui S. Lowry J. Gentile V. Davis P.J.A. Griffin M. FEBS Lett. 1995; 370: 27-31Crossref PubMed Scopus (86) Google Scholar). The GTPase activity of the tTG was also reported to function in cell receptor signaling by the α1-adrenoreceptor (15Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar). These recent studies emphasize the importance of understanding the molecular basis for regulating the TGase and GTPase activity.Takeuchi et al. (16Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar) reported three potential nucleotide binding sites located at amino acid residues 46-69, 345-367, and 520-544 of guinea pig tTG based on the ability of peptides to bind either GTP or ATP directly (16Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar). A 36-kDa N-terminal fragment was purified from rabbit liver nuclei that could bind GTP, suggesting that the N terminus of tTG plays an important role in nucleotide binding (17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar). 63- and 37-kDa N-terminal fragments of tTG were detected in a human erythroleukemia cell line, raising the possibility that an alternative splicing of mRNA could produce a protein that plays a role in leukemia cell proliferation (18Fraij B.M. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. Gonzales R.A. J. Biol. Chem. 1992; 267: 22616-22623Abstract Full Text PDF PubMed Google Scholar, 19Fraij B.M. Gonzales R.A. Biochim. Biophys. Acta. 1996; 1306: 63-74Crossref PubMed Scopus (27) Google Scholar). The C-terminal eight amino acid residues of tTG were recently reported to associate with the recognition and stimulation of phospholipase C (20Hwang K.-C. Gray C.D. Sivasubramanian N. Im M.-J. J. Biol. Chem. 1995; 270: 27058-27062Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).The purpose of this study was to perform C-terminal deletion analysis of the recombinant human tTG and determine the effect on GTP/ATP hydrolysis (nucleotide triphosphatase, NTPase) activity of human tTG. We report that C-terminal deletion causes a loss of TGase activity and a major increase in NTPase activity. All mutants retained the fibronectin binding property. The importance of the C terminus in regulating the NTPase function will be discussed.RESULTS AND DISCUSSIONWe investigated the role of the C terminus in regulating the GTP/ATPase activity of human tTG by expressing and purifying C-terminal deletion mutants as GST fusion proteins. We initially constructed mutants ΔS538, ΔE447, and ΔP345 (Fig. 1A). The ΔS538 mutant is similar in size to a tTG homologue expressed in human erythroleukemia cells and was recently reported to have increased GTPase activity (26Fraij B.M. Biochem. Biophy. Res. Commun. 1996; 218: 45-49Crossref PubMed Scopus (21) Google Scholar). The ΔE447 mutant lacks the putative calcium binding domain (amino acids 446-453). The ΔP345 was designed to remove additional charged residues that could play a role in GTP and ATP binding (16Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar). Based on the sequence alignment with the structural domains of factor XIII A chain (27Yee 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 (320) Google Scholar), the truncation site Ser-538 was located in the barrel 1 region, and Glu-447 and Pro-345 were located in the catalytic core region (Fig. 1A). Since the GTP/ATP hydrolysis activity was increased by 34-fold in the ΔP345 mutant (Fig. 1, C and D, see below), additional C-terminal truncation mutants were constructed. The ΔC290, ΔV228, and ΔF185 mutants stopped at intron-exon boundaries for exon 5, 4, and 3, respectively. These truncation sites were located in the catalytic core region when aligned with factor XIII A chain (Fig. 1A). All mutants were expressed and purified as GST-fusion proteins and migrated on SDS-polyacrylamide gel electrophoresis with their expected molecular weights (Fig. 1A). All mutants except ΔC290 were shown to be greater than 90% pure by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis separation. The ΔC290 was approximately 50% pure and consistently had an E. coli protein copurify with the mutant. In preliminary studies, we established that cleavage of the GST was not necessary to obtain reproducible TGase activity measurements, GTPase/ATPase hydrolysis, and fibronectin binding results. The results obtained with the GST fusion protein were comparable with those obtained from cleaving the GST and purifying the tTG. Therefore, the affinity-purified fusion proteins were used directly in all investigations to facilitate the rapid purification of the mutants by the same method.We found there was a substantial decrease in the calcium-dependent TGase activity when the 149 amino acid residues were deleted from the C terminus (Fig. 1B). From 1 to 5% of the original TGase activity was detected for the ΔS538 and ΔE447 mutants. The ΔP345, ΔC290, ΔV228, and ΔF185 mutants had no detectable TGase activity even when assayed at 50-fold higher protein concentration. When the calcium chloride concentration was increased to 100 mM, there was no further increase in the TGase activity for the inactive tTG mutants. All of the truncation mutants displayed specific binding to the fibronectin-coated plate, demonstrating that the N terminus was exposed and could bind to fibronectin.We found that 1 mM MgCl2 was the optimal concentration required for hydrolysis of both GTP and ATP for all the C-terminal truncation mutants, and we used this concentration to characterize all the mutants. The apparent Km value for GTP varied from 110 to 181.9 μM and from 30.9 to 43 μM for ATP. These relatively minor changes demonstrate all mutants retained comparable affinity for ATP and GTP. In contrast, the Kcat for GTP was increased by 4.6- and 3-fold for the ΔS538 and ΔE447 mutants (Fig. 1C). There was a 34-fold increase in hydrolysis for the ΔP345 mutant and a subsequent reduction in activity to only an 8.1-, 8.7-, and 1.9-fold increase in hydrolysis for the ΔC290, ΔV228, and ΔF185 mutants, respectively (Fig. 1C). The change in Kcat for ATP increased in parallel with the GTP results, and the highest Kcat was detected for the ΔP345 mutant (Fig. 1D). The removal of the C terminus apparently allows the GTP/ATPase catalytic domain to fold in a manner that increases NTP hydrolysis activity. These data are consistent with published data demonstrating that an N-terminal 36-kDa fragment purified from rabbit liver retained GTP binding properties (17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar) and that a human tTG homologue found in erythroleukemia cells had higher GTP hydrolysis activity (26Fraij B.M. Biochem. Biophy. Res. Commun. 1996; 218: 45-49Crossref PubMed Scopus (21) Google Scholar). These data localize a GTP/ATPase hydrolytic domain to the N-terminal 185 amino acids of tTG with a predicted Mr of 20,825. It is possible that selective proteolysis of the C terminus of tTG could occur at some intracellular or extracellular compartment to produce a N-terminal fragment with increased NTPase activity. In preliminary studies, we have been able to detect an increase in the GTP/ATP hydrolysis activity of tTG by treating it with several different proteases. This finding demonstrated that the activity obtained with our deletion mutants was not an artifact of expressing C-terminal deletion mutants. 2T.-S. Lai and C. S. Greenberg, unpublished findings. Further studies are in progress to test the hypothesis that proteases activate the GTP/ATPase activity.To investigate whether protein folding is required for GTP/ATPase activity, we selected full-length tTG and ΔP345 mutant for this experiment. Since the conditions for protein renaturation vary between different proteins, we adapted the conditions that have been established for a homologous protein, plasma transglutaminase (i.e. coagulation factor XIII A chains) (25Rinas U. Risse B. Jaenicke R. Abel K.J. Zettlmeissl G. Biol. Chem. Hoppe-Seyler. 1990; 371: 49-56Crossref PubMed Scopus (25) Google Scholar). The results demonstrated that 90-95% of the protein remained soluble after renaturation. However, only 8-13% and 15% of the original GTPase activity was detected for the full-length tTG and ΔP345 mutant, respectively. There was no detectable TGase activity after renaturation of either the full-length tTG or the ΔP345 mutant. These results indicate that proper protein folding is required for displaying the GTP/ATPase and TGase activities. Furthermore, TGase activity is more sensitive to denaturation than the hydrolysis activity of the tTG.Although other investigators reported that tTG had ATPase activity (28Takeuchi Y. Birckbichler P.J. Patterson M.K. Lee K.N. Carter H.A. Z. Naturforsch. 1994; 49: 453-457Crossref PubMed Scopus (9) Google Scholar), none of the earlier investigations determined the nature of the reaction products generated by incubating ATP with tTG. The reaction products of ATP hydrolysis for both full-length tTG and the ΔP345 mutant were studied by TLC as described under "Materials and Methods." Degradation of ATP occurred in two consecutive steps; there was very rapid formation of ADP followed by a much slower conversion of ADP to AMP. More than 50% of the ATP was converted to ADP during the first 10 min, and 85% was converted to ADP after 30 min of incubation. There was a substantial lag phase for the formation of AMP during the first 40 min of incubation, and less than 5% of the ADP was converted to AMP after 60 min of incubation (results not shown). Control experiments performed with either no enzyme or only the GST protein did not produce any ADP or AMP. These results indicate that the ΔP345 mutant hydrolyzes ATP to ADP at a much faster rate than the conversion of ADP to AMP. We found a similar pattern of hydrolysis for wild type tTG with more than 90% of the ATP converted to ADP, and <5% of ADP converted to AMP after 60 min of incubation. The inability of tTG to convert ADP to AMP suggests that the ADP could play a role in modulating the intracellular activity of the enzyme. We recently found that ADP can bind and inhibit the ATPase activity of the tTG.2. Therefore, once ADP is formed it would prevent ATP hydrolysis. In addition, ATP-tTG is inactive while ADP-tTG retains calcium-dependent TGase function.2The majority of tTG is found in the cytoplasm of the cell while 4-20% is associated with the particulate fraction (29Korner G. Schneider D.E. Purdon M.A. Bjornsson T.D. Biochem. J. 1989; 262: 633-641Crossref PubMed Scopus (39) Google Scholar). The recent finding that the full-length and the 36-kDa cleaved form of tTG associates with the nuclear pore of rat liver nuclei suggests that the tTG could play a role in modifying the structure of the nuclear pore or in trafficking of molecules through the pore (17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar). Specific processing by proteases in the nuclei could regulate the TGase and NTPase activity in this cellular compartment and warrants further analysis.The ECM surrounding endothelial cells and fibroblasts contain tTG (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar) and are exposed to ATP and other NTPs at sites of cell and tissue injury. The tTG could play a role in converting ATP to ADP and altering the response of cells to these vasoactive molecules (30Chen Z.-P. Levy A. Lightman S. J. Neuroendocrinol. 1995; 7: 83-96Crossref PubMed Scopus (85) Google Scholar). The tTG in the ECM could be cleaved at the C terminus by a protease released in response to tissue injury, and the cleaved form of tTG could modify local nucleotide concentrations. Cleaved forms of tTG were detected in human atherosclerotic aortic tissues and human breast cancer tumor tissue.2 If the proteolysis of tTG leaves the N terminus intact, fragments would remain bound to ECM and be localized to stimulate platelet activation by converting ATP to ADP. In preliminary studies, we have confirmed these findings by stimulating platelet aggregation by incubating platelet-rich plasma with a solution of ATP previously incubated with proteases and recombinant tTG.2 Since ATP and ADP are potent bioactive molecules in vascular tissues where tTG is expressed at high levels, the regulation of latent NTPase activity could be an important function for this ECM protein.Recently, the erythrocyte membrane protein band 4.2 (Pallidin) was shown to bind ATP but not GTP, and the ATP binding consensus sequence was located at amino acids 340-352 (31Azim A.C. Marfatia S.M. Korsgren C. Dotimas E. Cohen C.M. Chishti A. Biochemistry. 1996; 35: 3001-3006Crossref PubMed Scopus (22) Google Scholar), which corresponds to amino acids 321-329 of human tTG. An ATP binding consensus sequence does not exist in tTG or any of the other published TG sequence. Since the ΔC290, ΔV228, and ΔF185 mutants retained significant GTP/ATP hydrolysis, amino acids 321-329 are not essential for hydrolysis, and the GTP/ATP binding capacity must reside in the first 185 amino acid residues.The C terminus also plays an important role in regulating the TGase activity since the activity of ΔS538 and ΔE447 mutants were decreased to 1-5% of the original value. The residual TGase activity displayed by the ΔE447 mutant suggests that additional calcium binding site(s) could substitute for the putative calcium binding domain located between amino acid residues 446 and 453. In addition, Ikura et al. (32Ikura K. Yu C. Nagao M. Sasaki R. Furuyoshi S. Kawabata N. Arch. Biochem. Biophys. 1995; 318: 307-313Crossref PubMed Scopus (11) Google Scholar) also reported that the anionic amino acid residues (Glu-445 to Glu-452) in this domain were not essential for guinea pig liver TG to display calcium-dependent TGase activity. Further work is needed to localize the calcium binding site(s) in tTG.In conclusion, we have localized the ATP and GTP hydrolytic domain to amino acid residues 1-185 in the N terminus of tTG. The predicted structure of this fragment contains β-sandwich domain and part of the catalytic core region when it aligns with the structural domains of factor XIII A chain (27Yee 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 (320) Google Scholar). The C terminus of tTG functions to inhibit the expression of this endogenous GTP/ATPase activity in the intact protein. Studies are in progress to define the role of the NTPase activity of tTG in healthy and injured tissues and define the importance of this reaction in regulating the cell cycle and cell receptor signaling. INTRODUCTIONTissue transglutaminase (tTG) 1The abbreviations used are: tTGtissue transglutaminaseGSTglutathione S-transferaseTGasethe activity of tTG to carry out the calcium-dependent incorporation of [3H]putrescine or biotinylated pentylamine into N,N-dimethylcaseinECMextracellular matrixBSAbovine serum albuminTBSTris-buffered saline. is a unique member of the transglutaminase gene family in that it exhibits two distinct enzyme activities (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar, 3Lee K.N. Birckbichler P.J. Patterson Jr., M.K. Biochem. Biophys. Res. Commun. 1989; 162: 1370-1375Crossref PubMed Scopus (93) Google Scholar). The calcium-dependent transglutaminase activity (TGase) catalyzes the covalent modification of proteins by the formation of γ-glutamyl-ϵ-lysine bonds between proteins or polyamines (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). The TGase activity is considered to be an important intracellular and extracellular reaction during apoptosis (4Fesus L. Davies P.J.A. Piacentini M. Eur. J. Cell. Biol. 1991; 56: 170-177PubMed Google Scholar, 5Fesus L. Thomazy V. Falus A. FEBS Lett. 1987; 224: 104-108Crossref PubMed Scopus (406) Google Scholar), bone ossification (6Aeschlimann D. Wetterwald A. Fleisch H. Paulsson M. J. Cell Biol. 1993; 120: 1461-1470Crossref PubMed Scopus (166) Google Scholar), tissue repair (7Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.P. J. Cell Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar), and tumor growth (8Johnson T.S. Knight C.R.L. El Alaoui S. Mian S. Rees R.C. Gentiles V. Davies P.J.A. Griffin M. Oncogene. 1994; : 2935-2942PubMed Google Scholar). TGase activity requires a calcium binding site and active site cysteine to form a thioester bond with the glutamine substrate (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). The active site of human tTG is located at Cys-277, and the putative calcium binding site is located between amino acids 446 and 453 based on sequence homology to the calcium binding site in the factor XIII A chains (9Gentile V. Saydak M. Chiocca E.A. Akande 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).The tTG will selectively modify a group of protein-bound glutamine residues that exist in proteins found in the extracellular matrix (ECM) including vitronectin, fibronectin, osteonectin, and nidogen (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). When tTG is released into plasma or ECM it binds to fibronectin and retains TGase activity (1Greenberg C.S. Birckbichler P. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (928) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar). Fibronectin binding functions to localize tTG to sites of fibronectin expression and deposition and limits the availability of the enzyme for cross-linking other substrates. The fibronectin binding site is located in the N-terminal seven amino acid residues (10Jeong J.-M. Murthy S.N.P. Radek J.T. Lorand L. J. Biol. Chem. 1995; 270: 5654-5658Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).The tTG binds GTP and ATP inducing a conformational change that causes a reduction in the affinity for calcium and in TGase activity (11Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar, 12Bergamini C.M. FEBS Lett. 1988; 239: 255-258Crossref PubMed Scopus (73) Google Scholar). The binding of ATP and/or GTP to intracellular tTG could play a major role in suppressing TGase activity and preventing intracellular protein cross-linking reactions. In addition, a magnesium-dependent GTP hydrolysis activity (GTPase) was discovered to reside in the molecule and does not require the active site cysteine (13Lee K.N. Shelly A.A. Birckbichler P.J. Patterson Jr., M.K. Fraij B.M. Takeuchi Y. Carter H.A. Biochim. Biophys. Acta. 1993; 1202: 1-6Crossref PubMed Scopus (77) Google Scholar). The over-expression of a tTG mutant with the active site Cys-277 mutated to alanine only expressed GTPase activity and caused cell cycle arrest at the S to G2/M interphase (14Mian S. El Alaoui S. Lowry J. Gentile V. Davis P.J.A. Griffin M. FEBS Lett. 1995; 370: 27-31Crossref PubMed Scopus (86) Google Scholar). The GTPase activity of the tTG was also reported to function in cell receptor signaling by the α1-adrenoreceptor (15Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar). These recent studies emphasize the importance of understanding the molecular basis for regulating the TGase and GTPase activity.Takeuchi et al. (16Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar) reported three potential nucleotide binding sites located at amino acid residues 46-69, 345-367, and 520-544 of guinea pig tTG based on the ability of peptides to bind either GTP or ATP directly (16Takeuchi Y. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. FEBS Lett. 1992; 307: 177-180Crossref PubMed Scopus (32) Google Scholar). A 36-kDa N-terminal fragment was purified from rabbit liver nuclei that could bind GTP, suggesting that the N terminus of tTG plays an important role in nucleotide binding (17Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar). 63- and 37-kDa N-terminal fragments of tTG were detected in a human erythroleukemia cell line, raising the possibility that an alternative splicing of mRNA could produce a protein that plays a role in leukemia cell proliferation (18Fraij B.M. Birckbichler P.J. Patterson Jr., M.K. Lee K.N. Gonzales R.A. J. Biol. Chem. 1992; 267: 22616-22623Abstract Full Text PDF PubMed Google Scholar, 19Fraij B.M. Gonzales R.A. Biochim. Biophys. Acta. 1996; 1306: 63-74Crossref PubMed Scopus (27) Google Scholar). The C-terminal eight amino acid residues of tTG were recently reported to associate with the recognition and stimulation of phospholipase C (20Hwang K.-C. Gray C.D. Sivasubramanian N. Im M.-J. J. Biol. Chem. 1995; 270: 27058-27062Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).The purpose of this study was to perform C-terminal deletion analysis of the recombinant human tTG and determine the effect on GTP/ATP hydrolysis (nucleotide triphosphatase, NTPase) activity of human tTG. We report that C-terminal deletion causes a loss of TGase activity and a major increase in NTPase activity. All mutants retained the fibronectin binding property. The importance of the C terminus in regulating the NTPase function will be discussed.
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