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• W2051208708 abstract "Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme β-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal β-sandwich domain and that this interaction is crucial for cell surface association of tTG. Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme β-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal β-sandwich domain and that this interaction is crucial for cell surface association of tTG. The 80-kDa tissue transglutaminase (tTG) 1The abbreviations used are:tTGtissue transglutaminaseDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayECMextracellular matrixPCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisFNfibronectin is a member of a family of Ca2+-dependent enzymes which catalyze the formation of cross-links between the γ-carboxamide group of peptide-bound glutamine residues and either the amino groups of primary amines such as putrescine and cadaverine or the ε-amino group of peptide bound lysine residues (1Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Crossref PubMed Scopus (659) Google Scholar, 2Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (935) Google Scholar, 3Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar). Although initially believed to be an intracellular enzyme, there is growing evidence for the involvement of tissue transglutaminase in the assembly and stabilization of the pericellular matrix of cells (4Barsigian C. Stern A.M. Martinez J. J. Biol. Chem. 1991; 266: 22501-22509Abstract Full Text PDF PubMed Google Scholar, 5Aeschlimann D. Kaupp O. Paulsson M. J. Cell Biol. 1995; 129: 881-892Crossref PubMed Scopus (182) Google Scholar, 6Kleman J.P. Aeschlimann D. Paulsson M. van der Rest M. Biochemistry. 1995; 34: 13768-13775Crossref PubMed Scopus (103) Google Scholar, 7Martinez J. Chapulowicz D.G. Roush R.K. Sheth A. Barsigian C. Biochemistry. 1994; 33: 2538-2545Crossref PubMed Scopus (112) Google Scholar) and of various extracellular matrices including basement membranes (8Aeschlimann D. Paulsson M. J. Biol. Chem. 1991; 266: 15308-15317Abstract Full Text PDF PubMed Google Scholar, 9Raghunath M. Hopfner B. Aeschlimann D. Luthi U. Meuli M. Altermatt S. Gobet R. Brucker-Tuderman L. Steinmann B. J. Clin. Invest. 1996; 98: 1174-1184Crossref PubMed Scopus (88) Google Scholar). It has also been shown that the tissue-type enzyme binds to the extracellular matrix with high affinity (10Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.D. J. Cell Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar) independently of its cross-linking activity (11LeMosy E.K. Erickson H.P. Beyer W.F. Radek Jr., J.T. Jeong J.M. Murthy S.N. Lorand L. J. Biol. Chem. 1992; 267: 7880-7885Abstract Full Text PDF PubMed Google Scholar, 12Hohenadl C. Mann K. Mayer U. Timpl R. Paulsson M. Aeschlimann D. J. Biol. Chem. 1995; 270: 23415-23420Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Unlike the other members of the transglutaminase protein family, tTG is a GTP/GDP-binding protein which shows minimal transglutaminase activity in the GTP/GDP-bound form (13Mian S. El Alaoui S. Lawry J. Gentile V. Davies P.J.A. Griffin M. FEBS letters. 1995; 370: 27-31Crossref PubMed Scopus (87) Google Scholar, 14Achyuthan K.E. Greenberg C.S. J. Biol. Chem. 1987; 262: 1901-1906Abstract Full Text PDF PubMed Google Scholar). In the intracellular environment its binding to these nucleotides consequently prevents Ca2+activation of the enzyme (15Smethurst P.A. Griffin M. Biochem. J. 1996; 313: 803-808Crossref PubMed Scopus (135) Google Scholar), consistent with an extracellular function (16Johnson T.S. Griffin M. Thomas G.L. Skill N.J. Cox A. Yang B. Nicholas B. Birckbichler P.J. Muchaneto-Kubara C. El Nahas A.M. J. Clin. Invest. 1997; 99: 2950-2960Crossref PubMed Scopus (122) Google Scholar). Despite the growing evidence for an extracellular role for tTG, the enzyme presents the features of a cytosolic protein such as N-terminal acetylation, lack of disulfide bridges, and lack of glycosylation (17Ikura K. Nasu T. Yokota H. Tsuchiya Y. Sasaki R. Chiba H. Biochemistry. 1988; 27: 2898-2905Crossref PubMed Scopus (149) Google Scholar, 18Ikura K. Yokota H. Sasaki R. Chiba H. Biochemistry. 1989; 28: 2344-2348Crossref PubMed Scopus (18) Google Scholar). A further feature of this protein and other members of this protein family with an extracellular function including factor XIIIa is the lack of a classical leader sequence necessary for the translocation of proteins into the endoplasmic reticulum for their secretion (19Ichinose A. Bottenus R.E. Davie E.W. J. Biol. Chem. 1990; 265: 13411-13414Abstract Full Text PDF PubMed Google Scholar). It is conceivable that tTG is released passively from cells through stress-induced transient ruptures in the plasma membrane or it may be actively secreted by one of the more recently proposed alternative mechanisms (for review, see Ref. 3Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar). tissue transglutaminase Dulbecco's modified Eagle's medium phosphate-buffered saline enzyme-linked immunosorbent assay extracellular matrix polymerase chain reaction polyacrylamide gel electrophoresis fibronectin A number of studies (10Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.D. J. Cell Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar, 11LeMosy E.K. Erickson H.P. Beyer W.F. Radek Jr., J.T. Jeong J.M. Murthy S.N. Lorand L. J. Biol. Chem. 1992; 267: 7880-7885Abstract Full Text PDF PubMed Google Scholar, 20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar) have shown a high affinity of tTG for fibronectin and a putative fibronectin-binding site has been localized on tTG (21Jeong 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). Cell culture experiments have indicated that polymerization of fibronectin by cells is promoted by cell surface-associated tTG (4Barsigian C. Stern A.M. Martinez J. J. Biol. Chem. 1991; 266: 22501-22509Abstract Full Text PDF PubMed Google Scholar, 7Martinez J. Chapulowicz D.G. Roush R.K. Sheth A. Barsigian C. Biochemistry. 1994; 33: 2538-2545Crossref PubMed Scopus (112) Google Scholar, 20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar, 22Zhang Q. Mosher D.F. J. Biol. Chem. 1996; 271: 33284-33292Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 23Jones R.A. Nicholas B. Mian S. Davies P.J.A. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar). Antisense experiments (23Jones R.A. Nicholas B. Mian S. Davies P.J.A. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar) have also suggested that the cross-linking of cell surface-associated fibronectin by tTG may be related to the proposed role for the enzyme in cell adhesion and spreading (24Gentile V. Thomazy V. Piacentini M. Fesus L. Davies P.J.A. J. Cell Biol. 1992; 119: 463-474Crossref PubMed Scopus (230) Google Scholar), in agreement with the observation that adhesion and spreading of cells on a fibrin-fibronectin matrix formed with a mutant fibronectin lacking its transglutaminase cross-linking site is greatly reduced as compared with a matrix formed with wild type fibronectin (25Corbett S.A. Lee L. Wilson C.L. Schwarzbauer J.E. J. Biol. Chem. 1997; 272: 24999-25005Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These findings together with the reported close association of tTG and fibronectin in confluent cell monolayers as detected by immunocytochemistry (Ref. 20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar, and references therein) raise the possibility that the externalization of tTG from cells could be associated with the assembly of fibronectin fibrils. To study the relationship between fibronectin and cell surface association of tTG, two lines of investigation were undertaken. In the first, a Swiss 3T3 cell line stably transfected with the cDNA of tTG under the control of a tetracycline inducible promoter (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar) was used to establish colocalization of tTG and fibronectin at the cell surface on the ultrastructural level. In the second, fusion proteins between tTG and the bacterial reporter enzyme β-galactosidase were engineered, one of which carried a truncated tTG which lacked the first seven N-terminal amino acids. These amino acids have been proposed byin vitro analysis to be essential for fibronectin binding (21Jeong 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). COS-7 cells transiently expressing the fusion constructs provided a tool to analyze the dependence of the cell surface-associated tTG pool on the integrity of its binding site for fibronectin. Our results demonstrate that the introduced deletion abolishes binding of the enzyme to fibronectin and prevents its cell surface localization. We conclude that an intact N-terminal domain of tTG is required for its association with the extracellular matrix. Swiss 3T3 fibroblasts, COS-7, and endothelial ECV304 cells were obtained from the European Collection of Animal Cell Cultures and cultured in Dulbecco's modified Eagle's medium (DMEM) complemented with 10%(v/v) fetal calf serum, 2 mmglutamine, 20 units/ml penicillin, and 20 μg/ml streptomycin (Sigma, United Kingdom). The Swiss 3T3 cell line which was inducible for tTG under a tetracycline regulatable promoter was grown in DMEM containing 10% (v/v) fetal calf serum, 2 mm glutamine, 20 units/ml penicillin, 20 μg/ml streptomycin, 400 μg/ml geneticin, 250 μg/ml xanthine, 15 μg/ml hypoxanthine, 10 μg/ml thymidine, 2 μg/ml aminopterin, and 10 μg/ml mycophenolic acid (Sigma). The cells were usually cultured in the presence of 2 μg/ml tetracycline when no induction of tTG was required (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar). The presence of tTG antigen in conditioned culture medium was investigated using a modification of the quantitative ELISA method of Achyuthan et al. (26Achyuthan K.E. Goodell R.J. Kennedye J.R. Lee K.N. Henley A. Stiefer J.R. Birckbichler P.J. J. Immun. Met. 1995; 180: 69-79Crossref PubMed Scopus (20) Google Scholar). The plates were precoated with 3%(w/v) bovine serum albumin in PBS for 1 h at 37 °C prior to the assay as an additional step. Culture supernatants were concentrated approximately 10-fold on 30-kDa cut-off columns (Falcon Ltd., Oxford, United Kingdom) prior to assay. Stably transfected Swiss 3T3 cells (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar) which had been induced to express tTG by withdrawal of tetracycline from the culture medium for 72 h were seeded on glass slides and incubated overnight to obtain a subconfluent monolayer. Cells were fixed in 1% (w/v) paraformaldehyde in PBS for 15 min, blocked for 1 h in 3% (w/v) bovine serum albumin in PBS, and incubated overnight at 4 °C with a mixture of primary antibodies in blocking solution. The primary antibodies used were Cub7402 (Neomarkers, Union City, CA), a mouse monoclonal antibody targeting the active site of tTG (27Birckbichler P. Upchurch H.F. Patterson M.K. Conway E. Hybridoma. 1985; 4: 179-186Crossref PubMed Scopus (59) Google Scholar) and rabbit polyclonal antibodies against fibronectin (Sigma). A similar method was used for immunostaining of transiently transfected COS-7 cells using a mouse monoclonal antibody to β-galactosidase (Promega) with the exception that cells were permeabilized in 0.1% Triton X-100 in PBS for 15 min prior to blocking. After thorough rinsing in PBS, secondary antibodies, fluorescein isothiocyanate-conjugated anti-mouse, and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit (DAKO) were applied for 2 h at room temperature. Slides were rinsed in PBS, mounted in Vectashield mountant (Sigma), and examined on a LEICA laser confocal microscope. Cells were cultured to confluency on 0.5-cm squares of Melinex, previously conditioned by overnight incubation in serum containing DMEM. For immunolabeling of tTG in the ECM, cultures were labeled prior to fixation by addition of monoclonal antibody Cub7402 into the culture medium at a final dilution of 1/300 for 2 h (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar). Unbound antibodies were removed by extensive rinsing in PBS and cells were then fixed in 1% (w/v) paraformaldehyde and 0.05% (w/v) glutaraldehyde in PBS, dehydrated through increasing concentrations of ethanol and placed in hydrophilic resin (LRGold resin and glycolmethacrylate (low acid) (6:4), plus 0.1% bezoinethylether (Taab, Berks, UK)). Following several changes of resin, the samples were placed in plastic molds and embedded by polymerization of the resin using ultraviolet light (360 nm) (under nitrogen gas) for 24 h at room temperature. The Melinex support was removed to allow for vertical sectioning of the cells. Ultrathin sections (60–90 nm) were collected on collodion (2% w/v in amyl acetate)-coated nickel grids. Sections were blocked for nonspecific binding with 0.5% (w/v) bovine serum albumin in TBS (20 mmTris/HCl, pH 7.6, 225 mm NaCl) prior to being exposed to the primary antibody as indicated. Fibronectin was detected with rabbit polyclonal anti-fibronectin antibodies (Sigma) diluted 1/200 in blocking solution. For labeling of intracellular tTG, sections were also incubated with mouse monoclonal anti-tTG antibody (Cub7402) diluted 1/500 in blocking buffer containing 0.1% (v/v) Tween 20. Grids were then incubated with the respective colloidal gold-conjugated secondary antibodies (BioCell, Cardiff, UK), a goat anti-rabbit antibody (5-nm gold conjugate, diluted 1/200), and a goat anti-mouse antibody (15-nm gold conjugate, diluted 1/100). The grids were silver enhanced (Silver Enhancement Kit, BioCell), prior to counterstaining, with 2% aqueous uranyl acetate and alkaline lead citrate. Samples were viewed on a JEOL transmission electron microscope (100 CX-II). Fusion protein constructs were engineered by subcloning the human tTG cDNA into the KpnI restriction site of the pCHK vector which was designed to fuse proteins with the enzyme β-galactosidase (28Shreiber V. De Murcia G. Menissier de Murcia J. Gene ( Amst. ). 1994; 150: 411-412Crossref PubMed Scopus (7) Google Scholar). The following primers were used to amplify by PCR the complete tTG cDNA, with bold letters indicating the KpnI restriction site added to the cDNA to allow subcloning into pCHK: sense primer, 5′-CAGTGGTACCCATGGCCGAGGAGCTG-3′; antisense primer, 5′-TGAGGTACCGTGGCGGGGCCAATGATGAC-3′. To amplify a truncated tTG cDNA which lacked its first 21 bases, the following sense primer was used together with the above antisense primer: 5′-CGATGGTACCCAGGTGTGATCTGGAG-3′. The PCR reactions were carried out with 2.5 units of Taq DNA polymerase (Roche Molecular Biochemicals) and 0.3 μg of DNA template (pSG5/hTG-1 kindly provided by Dr. Peter J. A. Davies, Houston, TX) in a total volume of 100 μl containing 1.25 mm of each dNTPs and 60 pmol of each primer. The PCR cycles were 1 min at 95 °C (denaturation), 1 min at 60 °C (annealing), and 1 min at 72 °C (elongation) for a total of 30 cycles. The PCR products were cleaved with KpnI and ligated following standard protocols. The engineered constructs were sequenced to confirm proper integration of the cloned DNA fragment and the absence of mutations in the PCR amplified tTG coding sequence. Transient transfection of COS-7 cells with the generated plasmids (pCHKTG, pCHKTG/ΔN) and the control pCHK was carried out using 30 μl of the transfection reagent DOTAP (Roche Molecular Biochemicals) and 5 μg of plasmid DNA per 28.3 cm2 of cells following the manufacturer's instructions. Cells were grown on plastic to 80% confluency for transfection and transfected cells were cultured for another 48 h in 5% CO2 at 37 °C in phenol red-free culture medium before being processed for different assays as indicated. Transfection efficiencies were calculated by determining the number of transfected cells using the β-galactosidase in situ staining system (Promega) according to the manufacturer's instructions. Transfection efficiencies were established from parallel cultures (duplicates) to the experimental cultures in every experiment. Transiently transfected COS-7 cells were harvested by trypsinization in PBS containing 5 mm EDTA and extracted by sonication in 0.25 msucrose, 2 mm EDTA, 5 mm Tris-HCl, pH 7.4, and protease inhibitors: 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride. Cells were cleared from particulate material by centrifugation at 10,000 × gfor 5 min. Western blot analysis was performed following standard procedures (29Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing & Wiley-Interscience, New York1990Google Scholar). Proteins were separated in 8% SDS-polyacrylamide gels under reducing conditions and transferred to a nitrocellulose membrane. β-Galactosidase and fusion proteins were detected using a mouse monoclonal antibody against β-galactosidase (Promega). A horseradish peroxidase-conjugated anti-mouse antibody was used in combination with the ECL kit (Amersham International Plc.) to develop the blots. Cell extracts from 2 × 106 transiently transfected COS-7 cells were clarified by centrifugation at 20,000 × g for 20 min. The resultant supernatant (150 μl containing approximately 2000 μg of protein) was incubated with GTP-agarose previously washed and equilibrated in 50 mm Tris-HCl, pH 7.5 (volume of GTP-agarose used, equivalent to 0.5 ml of original suspension) (Sigma), and then incubated overnight at 4 °C with gentle shaking. The agarose beads were pelleted by centrifugation, the supernatant removed, and the beads washed twice in cold (4 °C) 50 mm Tris buffer, pH 7.5, and once more in PBS. The washed beads were boiled in 2 × strength Laemmli sample buffer for 5 min to solubilize GTP-binding proteins and the extracted proteins were then fractionated by SDS-polyacrylamide gel electrophoresis as described above. Gel loadings were adjusted to take differences in transfection efficiency into account. The reaction was performed by incubation of samples in β-galactosidase assay buffer (120 mm Na2HPO4, 80 mm NaH2PO4, 2 mmMgCl2, 100 mm β-mercaptoethanol, 1.33 mg/mlo-nitrophenyl-β-d-galactopyranoside (Promega) at 37 °C for 1 h. The hydrolysis of the β-galactosidase substrate o-nitrophenyl-β-d-galactopyranoside into o-nitrophenol was assessed by measuring the absorbance at 420 nm. Standards were made up in DMEM without phenol red following the kit's instructions (Promega). Knowing the corresponding transfection efficiency (see above), the specific β-galactosidase activity for each cell extract could be calculated and is given in β-galactosidase milliunits per 100,000 transfected cells. 150 μl extract of transfected COS-7 cells was diluted to 500 μl with PBS and incubated on a fibronectin-coated plastic surface (24-well tissue culture plates (Corning) coated overnight with 10 μg/ml fibronectin in 50 mm Tris-HCl, pH 7.4) for 2 h at 37 °C. The plates were thoroughly rinsed with PBS and bound β-galactosidase activity measured as described. COS-7 cells were cultured in 10-cm dishes and transfected as described. The conditioned culture media were harvested, centrifuged at 800 × g for 5 min to eliminate any cell debris, and analyzed for β-galactosidase activity (see below). The cells were recovered by brief trypsinization and immediately resuspended in serum containing medium to stop further protease action. Cells were collected by centrifugation, resuspended in 1 ml of serum-free DMEM, and counted. After incubation in suspension for a total of 30–35 min, the cells were fixed in 3.7% (w/v) paraformaldehyde in PBS for 15 min at room temperature. The fixed cells were washed 3 times in culture medium and finally resuspended in 150 μl of phenol red and serum-free DMEM. The cell suspensions, as well as the harvested media, were analyzed in the β-galactosidase enzyme assay described above with β-galactosidase standard solutions prepared in phenol red and serum-free DMEM. After a 1-h incubation at 37 °C, the cells were pelleted and the absorbance of the supernatant and the conditioned media were measured at 420 nm. To check the membrane integrity of the cell preparation, the fixed cells were incubated for 10 min at room temperature in a solution of trypan blue (Sigma) diluted 1/4 in PBS and analyzed for exclusion of the dye. Adherent transfected COS-7 cells (180,000 cells were originally plated out in 6-well plates for each assay) were rinsed with PBS before solubilization in 0.1% deoxycholate in PBS containing 5 mm EDTA. The remaining ECM on the plastic surface was washed thoroughly with PBS containing 5 mm EDTA and the associated β-galactosidase activity was determined as described above. Initial investigations were undertaken to assess any direct secretion of tTG into the growth medium of cells expressing high levels of the enzyme either constitutively or after transfection with an expression construct. Cells chosen for this study included stably transfected Swiss 3T3 fibroblasts in which tTG expression is under control of the tetracycline regulatable promoter (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar) and the human endothelial cell line ECV304 (23Jones R.A. Nicholas B. Mian S. Davies P.J.A. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar). Immunochemical analysis by quantitative ELISA was conducted on concentrated culture medium and did not reveal any detectable levels of tTG (data not shown). Since it has previously been shown that tTG is present at the surface of cells in suspension (4Barsigian C. Stern A.M. Martinez J. J. Biol. Chem. 1991; 266: 22501-22509Abstract Full Text PDF PubMed Google Scholar, 23Jones R.A. Nicholas B. Mian S. Davies P.J.A. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar) but is only released on the basal side of adherent cells (7Martinez J. Chapulowicz D.G. Roush R.K. Sheth A. Barsigian C. Biochemistry. 1994; 33: 2538-2545Crossref PubMed Scopus (112) Google Scholar), the experiment was repeated with cells that were trypsinized and kept in suspension in serum-free medium (to prevent scavenging of the enzyme by serum components including fibronectin) for 3 h at 37 °C. Although tTG has been shown by several authors to be implicated in the processing of the pericellular matrix in different cell types by providing evidence for its activity at the cell surface (4Barsigian C. Stern A.M. Martinez J. J. Biol. Chem. 1991; 266: 22501-22509Abstract Full Text PDF PubMed Google Scholar, 5Aeschlimann D. Kaupp O. Paulsson M. J. Cell Biol. 1995; 129: 881-892Crossref PubMed Scopus (182) Google Scholar, 6Kleman J.P. Aeschlimann D. Paulsson M. van der Rest M. Biochemistry. 1995; 34: 13768-13775Crossref PubMed Scopus (103) Google Scholar, 7Martinez J. Chapulowicz D.G. Roush R.K. Sheth A. Barsigian C. Biochemistry. 1994; 33: 2538-2545Crossref PubMed Scopus (112) Google Scholar, 20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar, 23Jones R.A. Nicholas B. Mian S. Davies P.J.A. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar), several replicates of ELISA on the conditioned culture medium showed that there is no detectable secretion of tTG under these conditions from either cell type (data not shown). tTG binds with high affinity to ECM proteins, and in particular to cell surface-associated fibronectin (10Upchurch H.F. Conway E. Patterson Jr., M.K. Maxwell M.D. J. Cell Physiol. 1991; 149: 375-382Crossref PubMed Scopus (141) Google Scholar) which could result in sequestration of tTG at the cell surface and prevent it from being released into the culture medium. This is consistent with the fact that cells are known to contain a pericellular coat of fibronectin which is very rapidly re-established after trypsin treatment (less than 1 h) or which might only be partially removed by proteolytic cleavage (30Burridge K. Turner C.E. Romer L.N. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar). A logical working hypothesis is that tTG is released to the cell surface but effectively and tightly bound at the cell surface, as suggested by the presence of tTG activity at the cell surface but its absence in the culture medium. To test this hypothesis, immunocytochemical analysis of the cell surface was undertaken on stably transfected Swiss 3T3 cells which had been induced by withdrawal of tetracycline from the medium for 72 h to obtain a maximal level of tTG expression. These cells together with their non-induced controls were seeded at low density on culture slides 12 h prior to fixation and labeled with antibodies to tTG and fibronectin without permeabilization. We have previously shown that these cells secrete fibronectin and over time elaborate fibronectin fibrils on their surface as part of the organization of their ECM (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar). Detection of tTG and FN in the induced non-confluent cells showed a punctate pattern of staining which seemed to colocalize (Fig.1). In the non-induced cells, which showed negligible cell surface staining for tTG, FN staining did not appear altered (Fig. 1). This result showed first of all that tTG is detectable at the cell surface, and second, it showed a close association between tTG and fibronectin during the early stages of the organization of a pericellular fibronectin matrix. This close association of tTG with fibronectin at the cell surface as shown by confocal microscopy was further confirmed when stably transfected 3T3 cells induced to overexpress tTG were analyzed at the electron microscope level using immunogold labeling (Fig.2). Specific intracellular labeling for tTG and fibronectin was obtained as well as in the pericellular matrix of the cells. In the case of tTG, detection of the enzyme in the pericellular matrix was only made possible by addition of the primary antibody to live cells in culture prior to fixation and embedding, suggesting that the epitope of the enzyme recognized by the monoclonal antibody becomes occluded or destroyed during the processing techniques for immunogold labeling. We have previously used this technique to detect extracellular tTG by immunofluorescence in non-induced and induced transfected cells (20Verderio E. Nicholas B. Gross S. Griffin M. Exp. Cell Res. 1998; 239: 119-138Crossref PubMed Scopus (141) Google Scholar) which indicated very little labeling in the non-induced cells thus demonstrating" @default.
• W2051208708 created "2016-06-24" @default.
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• W2051208708 date "1999-10-01" @default.
• W2051208708 modified "2023-10-14" @default.
• W2051208708 title "Cell Surface Localization of Tissue Transglutaminase Is Dependent on a Fibronectin-binding Site in Its N-terminal β-Sandwich Domain" @default.
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