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Analysis of Tissue Transglutaminase Function in the Migration of Swiss 3T3 Fibroblasts

THE ACTIVE-STATE CONFORMATION OF THE ENZYME DOES NOT AFFECT CELL MOTILITY BUT IS IMPORTANT FOR ITS SECRETION*
Open AccessPublished:February 26, 2002DOI:https://doi.org/10.1074/jbc.M109836200
      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 (
      • Folk J.E.
      • Finlayson J.S.
      ). Proteins cross-linked as a result of transglutaminase-catalyzed reactions are generally more resistant to mechanical, chemical, and proteolytic breakdown. Tissue transglutaminase (tTGase
      The abbreviations used are: tTGase
      tissue transglutaminase
      ECM
      extracellular matrix
      PBS
      phosphate-buffered saline
      ELISA
      enzyme-linked immunosorbent assay
      FITC
      fluorescein isothiocyanate
      1The abbreviations used are: tTGase
      tissue transglutaminase
      ECM
      extracellular matrix
      PBS
      phosphate-buffered saline
      ELISA
      enzyme-linked immunosorbent assay
      FITC
      fluorescein isothiocyanate
      ; type II) is the most widely distributed form of transglutaminase in mammalian tissues (
      • Folk J.E.
      • Finlayson J.S.
      ). In addition to its ability to cross-link proteins, the enzyme can also bind and hydrolyze GTP and ATP (
      • Griffin M.
      • Wilson J.
      ,
      • Lee K.N.
      • Arnold S.A.
      • Birckbichler P.J.
      • Patterson Jr., M.K.
      • Fraij B.M.
      • Takeuchi Y.
      • Carter H.A.
      ). 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 (
      • Smethurst P.A.
      • Griffin M.
      ). 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 (
      • Di Venere A.
      • Rossi A.
      • De Matteis F.
      • Rosato N.
      • Agro A.F.
      • Mei G.
      ). 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 (
      • Weiss M.S.
      • Metzner H.J.
      • Hilgenfeld R.
      ).
      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 (
      • Bowness J.M.
      • Henteleff H.
      • Dolynchuk K.N.
      ,
      • Bowness J.M.
      • Tarr A.H.
      • Wong T.
      ,
      • Haroon Z.A.
      • Hettasch J.M.
      • Lai T.S.
      • Dewhirst M.W.
      • Greenberg C.S.
      ,
      • 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.
      ,
      • Upchurch H.F.
      • Conway E.
      • Patterson Jr., M.K.
      • Maxwell M.D.
      ). 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 (
      • Aeschlimann D.
      • Paulsson M.
      ,
      • Jones R.A.
      • Nicholas B.
      • Mian S.
      • Davies P.J.
      • Griffin M.
      ,
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ,
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ). 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 (
      • Folk J.E.
      • Finlayson J.S.
      ). 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 (
      • Aeschlimann D.
      • Paulsson M.
      ,
      • Jones R.A.
      • Nicholas B.
      • Mian S.
      • Davies P.J.
      • Griffin M.
      ,
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ,
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ,
      • Aeschlimann D.
      • Kaupp O.
      • Paulsson M.
      ,
      • Barsigian C.
      • Stern A.M.
      • Martinez J.
      ,
      • Martinez J.
      • Chalupowicz D.G.
      • Roush R.K.
      • Sheth A.
      • Barsigian C.
      ,
      • Verderio E.
      • Gaudry C.
      • Gross S.
      • Smith C.
      • Downes S.
      • Griffin M.
      ).
      It has been recently described that tTGase mediates cell adhesion and spreading by a mechanism that is independent of its catalytic activity (
      • Isobe T.
      • Takahashi H.
      • Ueki S.
      • Takagi J.
      • Saito Y.
      ). The mechanism proposed suggests that tTGase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated co-receptor (
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ). 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 (
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ). 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 (
      • Gaudry C.A.
      • Verderio E.
      • Jones R.A.
      • Smith C.
      • Griffin M.
      ).
      tTGase has a high affinity binding site for fibronectin that is localized to the first seven N-terminal amino acids (
      • Jeong J.M.
      • Murthy S.N.
      • Radek J.T.
      • Lorand L.
      ); 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 (
      • Gaudry C.A.
      • Verderio E.
      • Aeschlimann D.
      • Cox A.
      • Smith C.
      • Griffin M.
      ). 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 (
      • Jackson A.
      • Tarantini F.
      • Gamble S.
      • Friedman S.
      • Maciag T.
      ,
      • Tarantini F.
      • Gamble S.
      • Jackson A.
      • Maciag T.
      ,
      • Piotrowicz R.S.
      • Martin J.L.
      • Dillman W.H.
      • Levin E.G.
      ,
      • Miyakawa K.
      • Hatsuzawa K.
      • Kurokawa T.
      • Asada M.
      • Kuroiwa T.
      • Imamura T.
      ); 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 (
      • Bowness J.M.
      • Henteleff H.
      • Dolynchuk K.N.
      ,
      • Bowness J.M.
      • Tarr A.H.
      • Wong T.
      ,
      • Haroon Z.A.
      • Hettasch J.M.
      • Lai T.S.
      • Dewhirst M.W.
      • Greenberg C.S.
      ,
      • 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.
      ,
      • Upchurch H.F.
      • Conway E.
      • Patterson Jr., M.K.
      • Maxwell M.D.
      ,
      • Johnson T.S.
      • Skill N.J.
      • El Nahas A.M.
      • Oldroyd S.D.
      • Thomas G.L.
      • Douthwaite J.A.
      • Haylor J.L.
      • Griffin M.
      ), 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.

      DISCUSSION

      The functional role played by tTGase in cell migration has never been clearly established despite observations indicating that tTGase is involved in cell adhesion (
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ,
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ,
      • Gentile V.
      • Saydak M.
      • Chiocca E.A.
      • Akande O.
      • Birckbichler P.J.
      • Lee K.N.
      • Stein J.P.
      • Davies P.J.
      ,
      • Johnson T.S.
      • Knight C.R.
      • El-Alaoui S.
      • Mian S.
      • Rees R.C.
      • Gentile V.
      • Davies P.J.
      • Griffin M.
      ). In this report, our objective was to ascertain whether the enzyme's effect on cell migration was due to its cross-linking activity, its action as a cell-surface binding protein, and/or its ability to act as a GTP-binding protein. The model used involved cell lines of Swiss 3T3 fibroblasts expressing catalytically active or inactive C277S mutant tTGase in a tetracycline-regulated manner. We have previously shown that this model allows maximum expression of tTGase following a 72-h period of induction (
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ,
      • Verderio E.
      • Gaudry C.
      • Gross S.
      • Smith C.
      • Downes S.
      • Griffin M.
      ,
      • Gaudry C.A.
      • Verderio E.
      • Aeschlimann D.
      • Cox A.
      • Smith C.
      • Griffin M.
      ). It does not suffer from clonal variation because each clone acts as its own control when induced; moreover, it gives homogeneous enzyme expression unlike the heterogeneous expression and low transfection efficiency often associated with transient transfection models.
      In transfected cells induced to express catalytically active tTGase (clone TG3), the activity of tTGase was increased in cell homogenates and at the cell surface of the cells, confirming that increased expression of tTGase leads to increased externalization of the enzyme as previously found (
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ). Following induction of catalytically inactive C277S mutant tTGase in clone TGI19, the tTGase activity of cell homogenates remained unchanged, as expected. The relative measure of the ability of the C277S mutant and wild-type proteins in the transfected cell lines to associate with the substrate fibronectin was found to be comparable. This is a significant result in support of our additional investigations, as we have recently shown that binding of tTGase to fibronectin is crucial for enzyme cell-surface localization (
      • Gaudry C.A.
      • Verderio E.
      • Aeschlimann D.
      • Cox A.
      • Smith C.
      • Griffin M.
      ). We demonstrated that fibroblasts induced to overexpress active and inactive forms of tTGase showed a decreased rate of migration on fibronectin, which was accompanied by enhanced cell attachment, suggesting that the cross-linking activity of tTGase is not responsible for the enzyme's effects on cell motility. This observation was confirmed by the inability of inhibitors of tTGase activity to affect cell migration. One of these inhibitors, the irreversible inhibitor Rob283, led to ∼90% inhibition of cell surface-related tTGase activity when used at 100 μm (data not shown). However, the ability of anti-tTGase monoclonal antibody Cub7402 to reduce cell migration in a dose-dependent manner indicates that the cell-surface enzyme is an essential component in the migration of cells. Complete loss of cell motility at 100 μg/ml Cub7402 could be explained by earlier findings indicating that the binding of the antibody to cell-surface tTGase completely inhibits cell attachment to fibronectin (
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ). As a consequence, cell movement is not possible without cell attachment. This ability of anti-tTGase antibodies to block both cell attachment and cell migration is comparable to the outcome observed when cells are incubated with antibodies directed against the cell-surface region of the β1- and α5-integrins (
      • Fogerty F.J.
      • Akiyama S.K.
      • Yamada K.M.
      • Mosher D.F.
      ). Integrin ligand-binding properties are thought to govern cell migration speed through the degree of cell-substratum adhesiveness (
      • Palecek S.P.
      • Loftus J.C.
      • Ginsberg M.H.
      • Lauffenburger D.A.
      • Horwitz A.F.
      ). The data reported here indicate that the effects of tTGase on the migration of fibroblasts are brought about by a similar mechanism. Our studies on cell attachment to fibronectin show that overexpression of tTGase (active and inactive forms), which was accompanied by a decreased rate of cell migration, led to a small but significant increase in cell adhesiveness. Previous work has also demonstrated that changes in expression of tTGase in NIH 3T3 fibroblasts (
      • Cai D.
      • Ben T.
      • De Luca L.M.
      ), fibrosarcoma cells (
      • Ball D.J.
      • Mayhew S.
      • Vernon D.I.
      • Griffin M.
      • Brown S.B.
      ), and endothelial like cells (
      • Jones R.A.
      • Nicholas B.
      • Mian S.
      • Davies P.J.
      • Griffin M.
      ) can lead to similar changes in cell adhesion.
      Using immunogold electron microscopy, we have recently provided evidence for a preferential extracellular location of tTGase in dense clusters close to the cell surface/pericellular matrix and in association with fibronectin (
      • Gaudry C.A.
      • Verderio E.
      • Aeschlimann D.
      • Cox A.
      • Smith C.
      • Griffin M.
      ). It is therefore possible that tTGase might be involved in cell attachment and migration as a cell-surface binding protein. In agreement with our data, Akimov et al.(
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ) recently reported that the adhesive function of tTGase does not require its cross-linking activity, but is thought to be dependent on its stable noncovalent association with integrins. A close association of tTGase with β1-integrin was also demonstrated by Gaudry et al. (
      • Gaudry C.A.
      • Verderio E.
      • Jones R.A.
      • Smith C.
      • Griffin M.
      ) in the early stages of cell attachment by immunofluorescent staining. However, in this case, the localization became less prominent as cells spread more. Therefore, tTGase could function as a cell-surface molecule independent of its catalytic activity, directly through its close association with fibronectin (
      • Gaudry C.A.
      • Verderio E.
      • Aeschlimann D.
      • Cox A.
      • Smith C.
      • Griffin M.
      ), or by interacting with the integrin cell-surface receptors (
      • Akimov S.S.
      • Krylov D.
      • Fleischman L.F.
      • Belkin A.M.
      ), promoting cell interaction with the matrix and therefore slowing down cell migration. Alternatively, tTGase could still act as a GTP-binding protein in controlling cell migration (the C277S mutant retains GTP-binding activity), although the ability of cell surface-directed anti-tTGase antibodies to block migration strongly suggests it to be a cell-surface event.
      In previous work using 96-h-old cultures of Swiss 3T3 cells induced to overexpress tTGase, we reported a clear increase in ε-(γ-glutamyl)lysine cross-links, the majority of which are likely to be present in the ECM (
      • Verderio E.
      • Nicholas B.
      • Gross S.
      • Griffin M.
      ). It may therefore be plausible that tTGase contributes early to cell adhesion, acting from the cell surface independent of its cross-linking activity; but once released in the extracellular space, possibly by a “piggyback” mechanism via its binding to fibronectin, it starts to accumulate and in doing so contributes to the cross-linking of ECM proteins when and if appropriate substrates become available. This suggests that stabilization of ECM proteins by tTGase is more of a “long-term” process as a result of the progressive accumulation of secreted tTGase and availability of substrate proteins rather than an immediate event capable of mediating cell adhesion and migration. However, in a pathological situation such as in a wounded area, an increased amount of tTGase might also be deposited into the matrix as a result of increased expression of the enzyme (
      • 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.
      ) or as a result of leakage following cell stress (
      • Johnson T.S.
      • Skill N.J.
      • El Nahas A.M.
      • Oldroyd S.D.
      • Thomas G.L.
      • Douthwaite J.A.
      • Haylor J.L.
      • Griffin M.
      ) or cell death and exert its cross-linking-mediated role of matrix stabilizer directly, thus contributing to the maintenance of tissue integrity.
      Important to the hypothesis that a cell surface-related tTGase (active or inactive) can mediate changes in cell migration is that both the active and inactive C277S mutant enzymes have similar cellular distributions and that mutation of the enzyme does not affect this distribution. Our data show that both forms of tTGase were detected at the cell surface. However, only cells overexpressing the catalytically active form of tTGase showed increased and detectable amounts of tTGase antigen deposited into the ECM and culture medium. This novel finding indicates that only cell surface-associated tTGase and not tTGase deposited into the matrix is required to affect the cell migratory process; moreover, this function of tTGase does not require cross-linking activity. Our preliminary studies have indicated so far that incubation of cells with the active site-directed inhibitor Rob283 or the competitive primary amine cystamine did not significantly reduce the amount of enzyme deposited into the matrix (data not shown). This initially suggests that the cross-linking activity of the enzyme is not required for the complete secretory process. However, these inhibitors may not access the active site until the enzyme is in its Ca2+-mediated active conformation, which is when the enzyme is already at the cell surface. We therefore cannot rule out that the active-site Cys277has two important roles in the secretory mechanism: one that is essential to the folding of the protein to achieve a conformation necessary for the secretion and the other in the cross-linking mechanism of the enzyme.
      Recently, on the basis of crystallographic studies of Factor XIIIa, a novel mechanism for transglutaminase activation has been proposed based on the identification of two non-prolinecis peptide bonds, which are thought to act as a conformational switch between catalytically active/inactive states of transglutaminase (
      • Weiss M.S.
      • Metzner H.J.
      • Hilgenfeld R.
      ). In Factor XIIIa, one of these bonds is thought to be necessary for close association of the two activea subunits, whereas the other is close to the active-site cysteine. According to this work, the conformational rearrangements necessary to expose the hidden active site would depend on the cis-to-trans isomerization of these peptide bonds, which may be linked to substrate, or calcium binding. The fact that these bonds are very rare in protein structures (
      • Stewart D.E.
      • Sarkar A.
      • Wampler J.E.
      ) and that one of them is found close to the active site, which is a highly conserved region among transglutaminases, strongly suggests a functional role for them. We therefore transfected Swiss 3T3 cells with tTGase in which the potential tTGase cis peptide close to the active-site region at position 274 was mutated from Tyr to Ala (Y274A). Analysis of these transfected cells indicated that the Y274A mutation abolished the activity of tTGase in both clones examined, as previously predicted (
      • Weiss M.S.
      • Metzner H.J.
      • Hilgenfeld R.
      ). Comparison of wild-type clone TG16 and mutant clone TGY274A2, which express similar amounts of total enzyme, indicated that both the active and inactive Y274A mutant forms of the enzyme could be found in the membrane-rich and cytosolic fractions of the cells; however, cells expressing the mutant form of tTGase showed lower levels of membrane-associated enzyme. Measurement of cell-surface tTGase by flow cytometry confirmed that the clones expressing the Y274A mutant form of the enzyme had relatively small amounts of cell-surface tTGase, although the levels were greater than those in the transfected negative controls (neo1 and neo3). The Y274A mutation was also found to prevent secretion of the enzyme into the cell culture medium.
      Our data therefore show that mutations in the Cys277active-site region of the enzyme and at Tyr274, which lies in a newly predicted non-proline cis peptide bond region thought to be critical for the exposure of the enzyme active site, both lead to loss of cross-linking activity. We also demonstrate that the conformation of this active-site region of the enzyme or possibly the cross-linking activity of the enzyme is a major factor in the mechanism that governs the secretion and deposition of the enzyme into the ECM. Hence, secretion of the enzyme may be connected to thecis-to-trans isomerization of the non-prolinecis peptide bonds. We hypothesize that the activetrans conformation may occur in the enzyme upon the binding of Ca2+ and substrate. This process would occur once the enzyme reaches the cell surface, where both Ca2+ and substrates such as fibronectin are available to the enzyme. Interestingly, we have recently reported that the fibronectin-binding site in the N-terminal β-sandwich domain of tTGase is also important in the secretion mechanism of this enzyme (
      • Gaudry C.A.
      • Verderio E.
      • Aeschlimann D.
      • Cox A.
      • Smith C.
      • Griffin M.
      ).
      In conclusion, this work shows the importance of cell-surface tTGase in the regulation of cell migration. This novel function of the enzyme does not require tTGase catalytic activity and can be correlated with a tTGase-mediated increase in cell adhesion strength. We have also demonstrated for the first time that the active-site cysteine and the tertiary structure of the active-site region, including a putative non-proline cis peptide bond, are key players in determining enzyme secretion.

      Acknowledgments

      We thank John Bladon (Rotherham General Hospital) for help with flow cytometry and Rob Saint and Ian Coutts (Nottingham Trent University) for synthesis of the transglutaminase inhibitor Rob283.

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