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• W2134987889 abstract "Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr274 (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix. Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr274 (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix. Transglutaminases (EC 2.3.2.13) are a group of Ca2+-dependent enzymes that catalyze the post-translational modification of proteins through the incorporation of primary amines into the γ-carboxamide group of glutamine residues or by the cross-linking of proteins via ε-(γ-glutamyl)lysine bridges (1.Folk J.E. Finlayson J.S. Adv. Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (783) Google Scholar). Proteins cross-linked as a result of transglutaminase-catalyzed reactions are generally more resistant to mechanical, chemical, and proteolytic breakdown. Tissue transglutaminase (tTGase 1The abbreviations used are: tTGasetissue transglutaminaseECMextracellular matrixPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayFITCfluorescein isothiocyanate1The abbreviations used are: tTGasetissue transglutaminaseECMextracellular matrixPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayFITCfluorescein isothiocyanate ; type II) is the most widely distributed form of transglutaminase in mammalian tissues (1.Folk J.E. Finlayson J.S. Adv. Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (783) Google Scholar). In addition to its ability to cross-link proteins, the enzyme can also bind and hydrolyze GTP and ATP (2.Griffin M. Wilson J. Mol. Cell. Biochem. 1984; 58: 37-49Crossref PubMed Scopus (41) Google Scholar, 3.Lee K.N. Arnold S.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). Binding of GTP/GDP to the enzyme is thought to increase the tTGase tertiary structure stability and in a viable cell keeps the enzyme inactive as a transglutaminase (4.Smethurst P.A. Griffin M. Biochem. J. 1996; 313: 803-808Crossref PubMed Scopus (134) Google Scholar). It has been reported that Ca2+ and GTP induce opposite conformational changes in the protein tertiary structure, therefore suggesting that the mechanism by which tTGase activity is inhibited by GTP/GDP is essentially due to a protein conformational change that obscures access to the transglutaminase active site (5.Di Venere A. Rossi A. De Matteis F. Rosato N. Agro A.F. Mei G. J. Biol. Chem. 2000; 275: 3915-3921Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). More recent work has also suggested that a non-proline cis peptide bond close to the active-site cysteine (Cys277) may play a role in the conformational changes linked to the binding of Ca2+ and/or substrate during activation of the enzyme (6.Weiss M.S. Metzner H.J. Hilgenfeld R. FEBS Lett. 1998; 423: 291-296Crossref PubMed Scopus (136) Google Scholar). tissue transglutaminase extracellular matrix phosphate-buffered saline enzyme-linked immunosorbent assay fluorescein isothiocyanate tissue transglutaminase extracellular matrix phosphate-buffered saline enzyme-linked immunosorbent assay fluorescein isothiocyanate The ability of tTGase to create covalent protein cross-links suggests its involvement in maintaining tissue integrity; and as a consequence, the enzyme is thought to play an important role in various physiological as well as pathological situations such as wound healing, fibrosis, inflammation, and tumor metastasis (7.Bowness J.M. Henteleff H. Dolynchuk K.N. Connect. Tissue Res. 1987; 16: 57-70Crossref PubMed Scopus (16) Google Scholar, 8.Bowness J.M. Tarr A.H. Wong T. Biochim. Biophys. Acta. 1988; 967: 234-240Crossref PubMed Scopus (54) Google Scholar, 9.Haroon Z.A. Hettasch J.M. Lai T.S. Dewhirst M.W. Greenberg C.S. FASEB J. 1999; 13: 1787-1795Crossref PubMed Scopus (231) Google Scholar, 10.Johnson T.S. Griffin M. Thomas G.L. Skill J. Cox A. Yang B. Nicholas B. Birckbichler P.J. Muchaneta-Kubara C. Meguid El Nahas A. J. Clin. Invest. 1997; 99: 2950-2960Crossref PubMed Scopus (121) Google Scholar, 11.Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.D. J. Cell. Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar). Although tTGase was originally thought to be an intracellular enzyme, accumulating evidence indicates that the enzyme is externalized and capable of cross-linking a wide range of extracellular matrix (ECM) proteins, which is thought to be important in ECM deposition/stabilization and the cell attachment and spreading of a number of different cell types (12.Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (492) Google Scholar, 13.Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar, 14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar, 15.Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (417) Google Scholar). However, the link between ECM cross-linking and the role of the enzyme in cell attachment and spreading is still not fully understood. Also unknown is the mechanism of secretion of the enzyme from cells because tTGase does not possess a leader sequence, and there is no evidence of its glycosylation (1.Folk J.E. Finlayson J.S. Adv. Protein Chem. 1977; 31: 1-133Crossref PubMed Scopus (783) Google Scholar). It is therefore unlikely that the enzyme follows the classical endoplasmic reticulum-Golgi secretion route. Despite this observation, evidence for the presence of tTGase in the ECM and on the surface of different cell types is now increasing (12.Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (492) Google Scholar, 13.Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar, 14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar, 15.Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (417) Google Scholar, 16.Aeschlimann D. Kaupp O. Paulsson M. J. Cell Biol. 1995; 129: 881-892Crossref PubMed Scopus (182) Google Scholar, 17.Barsigian C. Stern A.M. Martinez J. J. Biol. Chem. 1991; 266: 22501-22509Abstract Full Text PDF PubMed Google Scholar, 18.Martinez J. Chalupowicz D.G. Roush R.K. Sheth A. Barsigian C. Biochemistry. 1994; 33: 2538-2545Crossref PubMed Scopus (112) Google Scholar, 19.Verderio E. Gaudry C. Gross S. Smith C. Downes S. Griffin M. J. Histochem. Cytochem. 1999; 47: 1-16Crossref PubMed Scopus (90) Google Scholar). It has been recently described that tTGase mediates cell adhesion and spreading by a mechanism that is independent of its catalytic activity (20.Isobe T. Takahashi H. Ueki S. Takagi J. Saito Y. Eur. J. Cell Biol. 1999; 78: 876-883Crossref PubMed Scopus (39) Google Scholar). The mechanism proposed suggests that tTGase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated co-receptor (15.Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (417) Google Scholar). The results from the latter study suggested that complexes of tTGase with integrins are formed inside the cell during biosynthesis, leading to its accumulation on the surface at sites of focal adhesion points (15.Akimov S.S. Krylov D. Fleischman L.F. Belkin A.M. J. Cell Biol. 2000; 148: 825-838Crossref PubMed Scopus (417) Google Scholar). It has also been demonstrated that in cells undergoing attachment and spreading, the enzyme is co-distributed with adhesion site markers, suggesting that these processes may coincide with the externalization of the enzyme (21.Gaudry C.A. Verderio E. Jones R.A. Smith C. Griffin M. Exp. Cell Res. 1999; 252: 104-113Crossref PubMed Scopus (89) Google Scholar). tTGase has a high affinity binding site for fibronectin that is localized to the first seven N-terminal amino acids (22.Jeong J.M. Murthy S.N. Radek J.T. Lorand L. J. Biol. Chem. 1995; 270: 5654-5658Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar); and this binding is independent of its cross-linking activity. The deletion of this N-terminal sequence from the tTGase gene abolishes binding of the enzyme to fibronectin and prevents its cell-surface localization, suggesting that secretion of tTGase from the cells could be associated with the assembly of fibronectin fibrils (23.Gaudry C.A. Verderio E. Aeschlimann D. Cox A. Smith C. Griffin M. J. Biol. Chem. 1999; 274: 30707-30714Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Other proteins lacking the classical signal sequence but efficiently secreted from the cells have been described to cross the membrane by a novel secretion pathway (24.Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 25.Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26.Piotrowicz R.S. Martin J.L. Dillman W.H. Levin E.G. J. Biol. Chem. 1997; 272: 7042-7047Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27.Miyakawa K. Hatsuzawa K. Kurokawa T. Asada M. Kuroiwa T. Imamura T. J. Biol. Chem. 1999; 274: 29352-29357Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar); but in most cases, the exact mechanism is still not fully understood. Because tTGase is involved in both cell attachment and spreading and, through its cross-linking activity, in wound healing and tissue fibrosis (7.Bowness J.M. Henteleff H. Dolynchuk K.N. Connect. Tissue Res. 1987; 16: 57-70Crossref PubMed Scopus (16) Google Scholar, 8.Bowness J.M. Tarr A.H. Wong T. Biochim. Biophys. Acta. 1988; 967: 234-240Crossref PubMed Scopus (54) Google Scholar, 9.Haroon Z.A. Hettasch J.M. Lai T.S. Dewhirst M.W. Greenberg C.S. FASEB J. 1999; 13: 1787-1795Crossref PubMed Scopus (231) Google Scholar, 10.Johnson T.S. Griffin M. Thomas G.L. Skill J. Cox A. Yang B. Nicholas B. Birckbichler P.J. Muchaneta-Kubara C. Meguid El Nahas A. J. Clin. Invest. 1997; 99: 2950-2960Crossref PubMed Scopus (121) Google Scholar, 11.Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.D. J. Cell. Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar, 28.Johnson T.S. Skill N.J. El Nahas A.M. Oldroyd S.D. Thomas G.L. Douthwaite J.A. Haylor J.L. Griffin M. J. Am. Soc. Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar), it is not unreasonable to assume that it might also be involved in cell migration, a process that is important to a number of cellular events, including embryogenesis, tissue repair, and tumor invasion. To explore this, we have used 3T3 fibroblasts transfected with a number of different tTGase constructs expressing the catalytically active or inactive forms of tTGase. We show that tTGase can regulate migration and that this novel function is independent of its cross-linking activity. We also demonstrate that mutation of the active-site cysteine prevents the enzyme from being deposited into the ECM and that mutation of Tyr274 to Ala, thought to providecis rather than the preferred trans peptide bond conformation, also leads to loss of tTGase activity and of enzyme secretion. We therefore conclude that tTGase controls cell motility by a process that does not require its deposition into and cross-linking of the ECM, but by acting as a novel cell-surface binding protein. All general chemicals and tissue culture reagents were obtained from Sigma (Dorset, UK) unless otherwise stated. The C277S mutation was introduced into tTGase cDNA (29.Gentile V. Saydak M. Chiocca E.A. Akande O. Birckbichler P.J. Lee K.N. Stein J.P. Davies P.J. J. Biol. Chem. 1991; 266: 478-483Abstract Full Text PDF PubMed Google Scholar) as previously described (30.Mian S. El-Alaoui S. Lawry J. Gentile V. Davies P.J. Griffin M. FEBS Lett. 1995; 370: 27-31Crossref PubMed Scopus (87) Google Scholar). The resulting mutant tTGase cDNA was inserted into the vector pUHD10.3 (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar) to generate the expression plasmid pUHD10.3-TG277. The Y274A mutation was introduced into tTGase cDNA using the GeneEditor in vitro site-directed mutagenesis kit (Promega, Southampton, UK) according to the manufacturer's protocol. Starting with the expression vector pSG5-TG containing the full-length tTGase cDNA (donated by P. J. A. Davies, University of Texas Health Center, Houston, TX), the TAT codon of Tyr274 was mutated to GCT for Ala utilizing the oligonucleotide primer 5′-AAGACCCAGCACTGGCCAGCCTTGACGCGCTGGCA-3′ (antisense orientation), which is complementary to nucleotides 940–974 of tTGase cDNA (29.Gentile V. Saydak M. Chiocca E.A. Akande O. Birckbichler P.J. Lee K.N. Stein J.P. Davies P.J. J. Biol. Chem. 1991; 266: 478-483Abstract Full Text PDF PubMed Google Scholar) and is mutated at positions 955 and 956 (underlined). The resulting recombinant plasmid encoding mutant tTGase was named pSG5-TG274. The presence of the base changes was confirmed by DNA sequencing. The establishment, by cell transfection, of Swiss 3T3 cell lines expressing catalytically active tTGase under the control of the tetracycline-regulated system (31.Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4232) Google Scholar) has been previously described (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar). Swiss 3T3 cell lines expressing inactive C277S mutant tTGase were generated following the same protocol. Briefly, clone tTA2, stably expressing the tetracycline-controlled transactivator (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar), was cotransfected with pUHD10.3-TG277and the xanthine-guanine phosphoribosyltransferase expression plasmid pUS1000 (donated by P. Sanders, University of Surrey) in the presence of tetracycline in the medium. Clones resistant to selection medium for the salvage enzyme xanthine-guanine phosphoribosyltransferase were analyzed for their capacity to overexpress tTGase antigen by standard Western blotting of cell homogenates using anti-tTGase monoclonal antibody Cub7402 (Neomarkers). Transfection of Swiss 3T3 fibroblasts with wild-type and inactive Y274A mutant tTGases was achieved by cotransfecting 0.5 × 106 cells with 4.5 μg of plasmid vector pSG5-TG274 and 0.5 μg of selection vector pSV2neo using the liposome-based transfection reagent ESCORT™ (Sigma) following the manufacturer's protocol. Clones resistant to 800 μg/ml active G418 (Geneticin, Calbiochem) were screened for overexpression of tTGase by Western blotting as described below. Cell lines of Swiss 3T3 fibroblasts expressing catalytically active tTGase (clone TG3) (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar) or inactive C277S mutant tTGase (clone TGI19) in a tetracycline-regulated manner were cultured as described (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar). Cell lines were continuously cultured in the presence of tetracycline (2 μg/ml) in the medium. Under this condition, they expressed only low levels of endogenous tTGase. To induce maximum expression of transfected tTGase cDNA, cells were cultured in the absence of tetracycline for 72 h. Cell lines of Swiss 3T3 fibroblasts (clones TG1, TG16, TGY274A1, TGY274A2, neo1, and neo3) expressing active or inactive Y274A mutant tTGase or the selection marker for G418 resistance, under the control of a non-inducible promoter, were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml G418. The cell migration assay used was a modification of the technique described by Akiyama et al.(32.Akiyama S.K. Yamada S.S. Chen W.T. Yamada K.M. J. Cell Biol. 1989; 109: 863-875Crossref PubMed Scopus (510) Google Scholar). Low-melting-point agarose (0.2% (w/v) final concentration), maintained just above 38 °C, was added to a suspension of cells (3.3 × 107 cells/ml) in bicarbonate-free Dulbecco's modified Eagle's medium (buffered with 25 mm Hepes, pH 7.4). Droplets (0.5 μl) of the cell/agarose mixture were seeded in the center of the fibronectin (15 μg/ml)-coated wells of a 96-well plate. After the agarose was allowed to set for 7 min at +4 °C, 100 μl of the growth medium was added to each well. In some cases, the growth medium was supplemented with anti-tTGase antibody or tTGase inhibitors. Cells were left to migrate for 48 h and then fixed and stained with 0.5% (w/v) crystal violet in 70% (w/v) ethanol as described below. The area of outwardly migrating cells was measured using an Optimas 5.2 image analysis system (DataCell Ltd., Yately, UK). Cell attachment was evaluated as previously described (13.Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar). Briefly, 100 μl of a cell suspension (5 × 105 cells/ml) was seeded in a 96-well plate coated with 5 μg/ml fibronectin, incubated in serum-free medium, and allowed to attach for 30 min. After this, the cells were gently washed with phosphate-buffered saline (PBS), and attached cells were fixed and stained by addition of 0.5% (w/v) crystal violet in 70% (v/v) ethanol at 100 μl/well. Following three washes with PBS to remove nonspecific staining, cells were solubilized by adding 30% (v/v) acetic acid at 100 μl/well. The absorbance of the solubilized cell mixture was read at 540 nm in a SpectraFluor plate reader (Tecan). The activity of tTGase in cell homogenates was measured by the incorporation of14C-labeled putrescine (Amersham Biosciences, Buckinghamshire, UK) into N,N′-dimethylcasein as previously described by Lorand et al. (33.Lorand L. Campbell-Wilkes L.K. Cooperstein L. Anal. Biochem. 1972; 50: 623-631Crossref PubMed Scopus (266) Google Scholar). One unit of tTGase activity equals 1 nmol of putrescine incorporated per h. The activity of tTGase associated with the extracellular surface of live cells in culture was measured by the incorporation of biotinylated cadaverine into deoxycholate-insoluble fibronectin using an assay described in detail by Jones et al. (13.Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar). For detection of tTGase in cell homogenates, after cell lysis in ice-cold buffer (0.25 m sucrose, 2 mm EDTA, and 5 mm Tris-HCl, pH 7.4) containing protease inhibitors (1 μg/ml pepstatin, 1 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride) and sonication, cell homogenates were mixed with 2× Laemmli loading buffer (34.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar) and boiled for 5 min at 100 °C, and then proteins were resolved as described below. For separation of membrane and cytosolic fractions, cell homogenates were first fractionated by ultracentrifugation at 100,000 ×g for 1 h at +4 °C. The precipitated pellets were washed once with the cell lysis buffer and centrifuged as described above. Proteins were resolved by SDS-PAGE under reducing conditions according to Laemmli (34.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar), and tTGase was detected by Western blotting using anti-tTGase monoclonal antibody Cub7402 and revealed by enhanced chemiluminescence (Amersham Biosciences) after incubation with an anti-mouse horseradish peroxidase conjugate. For direct comparison of cell homogenates or subcellular fractions, equal amounts of protein were loaded onto the gels prior to fractionation. To detect tTGase secreted into the growth medium, confluent cells were grown in serum-free AIMV culture medium (Gibco) for 8 h. The medium was then collected and centrifuged to remove any floating cells. Proteins from the cell growth medium were precipitated by addition of trichloroacetic acid to a final concentration of 10% (w/v), followed by centrifugation. The protein pellet was washed once with 10% (w/v) trichloroacetic acid followed by ethanol/acetone (1:1) and acetone, dried, and resuspended in Laemmli buffer (34.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). The presence of tTGase in the protein pellet was detected by Western blotting as outlined above. Alternatively, the cell growth medium was lyophilized, reconstituted in one-tenth of the initial volume, and analyzed for tTGase antigen by a modified enzyme-linked immunosorbent assay (ELISA) method according to Achyuthan et al. (35.Achyuthan K.E. Goodell R.J. Kennedye J.R. Lee K.N. Henley A. Stiefer J.R. Birckbichler P.J. J. Immunol. Methods. 1995; 180: 69-79Crossref PubMed Scopus (20) Google Scholar) as described below. The protein concentration was determined according to the method of Lowry et al. (36.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Detection of extracellular tTGase was performed using the modified ELISA technique described previously (19.Verderio E. Gaudry C. Gross S. Smith C. Downes S. Griffin M. J. Histochem. Cytochem. 1999; 47: 1-16Crossref PubMed Scopus (90) Google Scholar). Briefly, 1.5 × 104cells/well were seeded in a 96-well plate 1 day prior to the assay. Anti-tTGase antibody Cub7402 was diluted 1:1000 in the cell growth medium and added directly to cells in live culture. After a 3-h incubation, cells were washed with PBS and fixed in methanol. The antigen-antibody complex was revealed by incubation with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody. Bound horseradish peroxidase activity was detected by addition of 3,3′,5,5′-tetramethylbenzidine substrate. Color development was stopped with 2.5 n H2SO4, and the absorbance was read at 450 nm in a plate reader. For protein quantification, identical cell numbers were grown in parallel and solubilized in 0.1% (w/v) deoxycholate. Proteins were precipitated in 10% (w/v) trichloroacetic acid and assayed by the bicinchoninic acid method (37.Brown R.E. Jarvis K.L. Hyland K.J. Anal. Biochem. 1989; 180: 136-139Crossref PubMed Scopus (578) Google Scholar). The measured tTGase protein was then expressed asA 450 nm/1.0 mg of deoxycholate-soluble protein. For detection of total tTGase, a modification of the method of Achyuthan et al. (35.Achyuthan K.E. Goodell R.J. Kennedye J.R. Lee K.N. Henley A. Stiefer J.R. Birckbichler P.J. J. Immunol. Methods. 1995; 180: 69-79Crossref PubMed Scopus (20) Google Scholar) was used. Cell homogenates were added to the fibronectin-coated wells of a 96-well plate, and the binding of tTGase to fibronectin was allowed to proceed for 1 h at 37 °C. Wells were then blocked with blocking buffer (5% (w/v) dried skimmed milk in PBS, pH 7.4) and incubated with Cub7402 (diluted 1:1000 in blocking buffer) for 2 h at room temperature. After three washes with PBS, incubation with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (diluted 1:1000 in blocking buffer) was carried out for 2 h at room temperature. Bound horseradish peroxidase activity was measured as described above. The amount of tTGase protein was expressed as A 450 nm/1.0 mg of total protein. Detection of extracellular tTGase was done as previously described (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar) by staining cells in culture. Immunolabeling of tTGase was carried out using anti-tTGase primary monoclonal antibody Cub7402 and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibody. Staining was visualized by confocal fluorescent microscopy using a Leica TCSNT confocal laser microscope system (Leica Laserechnik, Heidelberg, Germany). For flow cytometry, transfected Swiss 3T3 fibroblasts were detached from tissue culture dishes with 2 mm EDTA in PBS, pH 7.4. Live non-permeabilized cells in suspension (2 × 106 cells/ml) were stained for cell-surface tTGase with anti-tTGase antibody Cub7402 (3 μg/ml) in serum-free medium for 3 h at +4 °C. After washing cells with serum-free medium and incubation with FITC-conjugated anti-mouse IgG secondary antibody, cells were washed once more, fixed in 0.5% (v/v) formaldehyde, and analyzed with a Dako Galaxy flow cytometer. Values represent the mean fluorescence intensity. Student's t test was used to compare data. When p was <0.05, the difference between sets of data was considered to be statistically different. The amount of tTGase antigen expressed in cell homogenates of induced transfected cells was detected by SDS-PAGE and Western blot analysis. The Western blot shown in Fig. 1 A shows clear induction of both forms of the enzyme in TG3− and TGI19−. Densitometric analysis indicated that expression of clone TG3 increased between 7- and 10-fold, as previously documented (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar). For the inactive C277S mutant, quantitation of induction by densitometry was not possible because of the low endogenous background. Measurement of antigen by a modified ELISA that first involves attachment of the enzyme to a fibronectin-coated plate indicated a 2–3-fold induction for clone TG3 and a 4–5-fold increase for clone TG19. Overexpression of the active form of the enzyme in clone TG3 led to an increase in total tTGase activity in cell homogenates (∼12-fold) as measured by the [14C]putrescine incorporation assay (Fig. 1 C) and an increase in cell surface-related extracellular activity (∼3–4-fold) as measured by the cell-mediated incorporation of biotinylated cadaverine into fibronectin (Fig. 1 D) (14.Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (140) Google Scholar). As expected, no tTGase activity was observed in clone TGI19 when induced to overexpress the inactive form of the enzyme (Fig. 1,C and D). By the agarose droplet method (Fig. 2 A), the transfected clone TG3 overexpressing tTGase showed a decreased rate of cell migration on fibronectin compared with the non-induced control (Fig. 2 B), suggesting that increased expression of tTGase affects cell motility. A reduced rate of migration was also observed in clone TGI19 induced to overexpress inactive tTGase compared with the non-induced cells (Fig. 2 B), indicating that cell motility is not dependent on the cross-linking a" @default.
• W2134987889 created "2016-06-24" @default.
• W2134987889 creator A5001559765 @default.
• W2134987889 creator A5018380234 @default.
• W2134987889 creator A5028486853 @default.
• W2134987889 creator A5056473106 @default.
• W2134987889 creator A5063021163 @default.
• W2134987889 creator A5072962526 @default.
• W2134987889 date "2002-05-01" @default.
• W2134987889 modified "2023-10-17" @default.
• W2134987889 title "Analysis of Tissue Transglutaminase Function in the Migration of Swiss 3T3 Fibroblasts" @default.
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• W2134987889 doi "https://doi.org/10.1074/jbc.m109836200" @default.
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