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• W2003132014 abstract "Bordetella dermonecrotic toxin (DNT) causes the deamidation of glutamine 63 of Rho. Here we identified the region of DNT harboring the enzyme activity and compared the toxin with the cytotoxic necrotizing factor 1, which also deamidates Rho. The DNT fragment (ΔDNT) covering amino acid residues 1136–1451 caused deamidation of RhoA at glutamine 63 as determined by mass spectrometric analysis and by the release of ammonia. In the presence of dansylcadaverine or ethylenediamine, ΔDNT caused transglutamination of Rho. Deamidase and transglutaminase activities were blocked in the mutant proteins Cys1292 → Ala, His1307 → Ala, and Lys1310 → Ala of ΔDNT. Deamidation and transglutamination induced by ΔDNT blocked intrinsic and Rho- GTPase-activating protein-stimulated GTPase activity of RhoA. ΔDNT deamidated and transglutaminated Rac and Cdc42 in the absence and presence of ethylenediamine, respectively. Modification of Rho proteins by ΔDNT was nucleotide-dependent and did not occur with GTPγS-loaded GTPases. In contrast to cytotoxic necrotizing factor, which caused the same kinetics of ammonia release in the absence and presence of ethylenediamine, ammonia release by ΔDNT was largely increased in the presence of ethylenediamine, indicating that ΔDNT acts primarily as a transglutaminase. Bordetella dermonecrotic toxin (DNT) causes the deamidation of glutamine 63 of Rho. Here we identified the region of DNT harboring the enzyme activity and compared the toxin with the cytotoxic necrotizing factor 1, which also deamidates Rho. The DNT fragment (ΔDNT) covering amino acid residues 1136–1451 caused deamidation of RhoA at glutamine 63 as determined by mass spectrometric analysis and by the release of ammonia. In the presence of dansylcadaverine or ethylenediamine, ΔDNT caused transglutamination of Rho. Deamidase and transglutaminase activities were blocked in the mutant proteins Cys1292 → Ala, His1307 → Ala, and Lys1310 → Ala of ΔDNT. Deamidation and transglutamination induced by ΔDNT blocked intrinsic and Rho- GTPase-activating protein-stimulated GTPase activity of RhoA. ΔDNT deamidated and transglutaminated Rac and Cdc42 in the absence and presence of ethylenediamine, respectively. Modification of Rho proteins by ΔDNT was nucleotide-dependent and did not occur with GTPγS-loaded GTPases. In contrast to cytotoxic necrotizing factor, which caused the same kinetics of ammonia release in the absence and presence of ethylenediamine, ammonia release by ΔDNT was largely increased in the presence of ethylenediamine, indicating that ΔDNT acts primarily as a transglutaminase. Clostridium botulinum exoenzyme C3 E. coli cytotoxic necrotizing factor 1 the active fragment of CNF1 consisting of amino acid residues 709–1014 Bordetella dermonecrotic toxin the active fragment of DNT consisting of amino acid residues 1136–1451 GTPase-activating protein glutathioneS-transferase polyacrylamide gel electrophoresis Rho GTPases including Rho, Rac, and Cdc42 isoforms are regulators of the actin cytoskeleton and act as molecular switches in a large array of signaling processes (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5219) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2096) Google Scholar). The GTPases are the eukaryotic substrates for various bacterial protein toxins (3Aktories K. Trends Microbiol. 1997; 5: 282-288Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 4Schmidt G. Aktories K. Naturwissenschaften. 1998; 85: 253-261Crossref PubMed Scopus (19) Google Scholar). C31-like exoenzymes (e.g. Clostridium botulinum exoenzyme C3) ADP ribosylate RhoA, B, and C at asparagine 41 thereby inhibiting the biological functions of the GTPases (5Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar, 6Chardin P. Boquet P. Madaule P. Popoff M.R. Rubin E.J. Gill D.M. EMBO J. 1989; 8: 1087-1092Crossref PubMed Scopus (422) Google Scholar, 7Aktories K. Braun U. Rösener S. Just I. Hall A. Biochem. Biophys. Res. Commun. 1989; 158: 209-213Crossref PubMed Scopus (215) Google Scholar). Large clostridial cytotoxins (e.g. Clostridium difficile toxins A and B) inhibit Rho, Rac, and Cdc42 GTPases by monoglucosylation at threonine 37 and threonine 35, respectively (8Just I. Selzer J. Wilm M. Von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (883) Google Scholar, 9Just I. Wilm M. Selzer J. Rex G. Von Eichel-Streiber C. Mann M. Aktories K. J. Biol. Chem. 1995; 270: 13932-13936Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Rho family GTPases are also the targets for the Bordetella dermonecrotic toxin (DNT), which is produced by Bordetella strains (10Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar, 11Pullinger G.D. Adams T.E. Mullan P.B. Garrod T.I. Lax A.J. Infect. Immun. 1996; 64: 4163-4171Crossref PubMed Google Scholar). DNT induces stress fiber formation, focal adhesion assembly, and tyrosine phosphorylation of focal adhesion kinase and paxillin (10Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar, 12Horiguchi Y. Senda T. Sugimoto N. Katahira J. Matsuda M. J. Cell Sci. 1995; 108: 3243-3251PubMed Google Scholar, 13Lacerda H.M. Pullinger G.D. Lax A.J. Rozengurt E. J. Biol. Chem. 1997; 272: 9587-9596Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Recent studies indicate that DNT causes deamidation of glutamine 63 of RhoA (10Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar). Glutamine 63 is essential for GTP hydrolysis by Rho. Deamidation of glutamine by DNT inhibits the GTPase activity of Rho and renders the Rho protein constitutively active. The same mechanism of Rho activation by deamidation was reported for the cytotoxic necrotizing factor CNF1 from Escherichia coli(14Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 15Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar). Also CNF deamidates Rho at glutamine 63 and causes similar cytotoxic effects such as multinucleation of cells and stress fiber formation. CNF1 and DNT share a region of homology (amino acid residues 1250–1351 of DNT) located at the C termini of the toxins (16Walker K.E. Weiss A.A. Infect. Immun. 1994; 62: 3817-3828Crossref PubMed Google Scholar). Other parts of the protein sequences are not significantly similar. Recently, it was shown that a C-terminal fragment of CNF1 (ΔCNF), covering the region of homology, causes the typical cytotoxic effects after microinjection and possesses full Rho-deamidating activity in vitro. In addition to deamidase activity, ΔCNF possesses transglutaminase activity. However, this activity is observed only in the presence of high concentrations of primary amines and is apparently lower than the deamidase activity (17Schmidt G. Selzer J. Lerm M. Aktories K. J. Biol. Chem. 1998; 273: 13669-13674Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Here we attempted to identify the region of DNT that harbors the enzyme activity of the toxin and characterized its biological and biochemical activities. We report that ΔDNT covering amino acid residues 1136–1451 possesses full deamidating activity. Cysteine 1292, histidine 1307, and lysine 1310 are essential for enzyme activity. As found for ΔCNF, the active fragment of DNT possesses transglutaminase activity. In contrast to CNF1, ΔDNT exhibits a higher transglutaminase than deamidase activity, indicating that DNT acts preferentially as a transglutaminase. Another difference between ΔCNF and ΔDNT is the nucleotide dependence of the deamidation/transglutamination reaction. Whereas ΔCNF modifies GDP- and GTP-loaded Rho proteins, ΔDNT exclusively accepts GDP-bound RhoA. RhoA and p50RhoGAP (obtained from A. Hall, London) were prepared from their fusion proteins as described. Dansylcadaverine and ethylenediamine were purchased from Sigma. Methanol and chloroform were of analytical grade, and trifluoroacetic acid and acetonitrile were of high pressure liquid chromatography grade. For production of ΔDNT consisting of amino acid residues 1136–1451, the DNA was amplified from the plasmid DNT 103 (16Walker K.E. Weiss A.A. Infect. Immun. 1994; 62: 3817-3828Crossref PubMed Google Scholar) by polymerase chain reaction with the following primers: ΔDNT sense, 5′-GGATCCGCTTCCGGCGGGGGGCCG-3′; ΔDNT antisense, 5′-GAATTCTCAGACCGGCGCCGGAAACAA-3′. The PCR product was purified from agarose gel (Jet sorb, Genomed) and amplified in the pCRTMII vector (Invitrogen) by means of TA cloning. From this vector the DNT fragment was cut withBamHI and EcoRI, purified, and ligated into the digested pGEX vector. The proper construct was checked by DNA sequencing. The vector was transformed into BL21 cells by heat shock at 42 °C. Expression of the GST fusion protein in E. coliBL21 cells growing at 37 °C was induced by adding 0.2 mmisopropyl-1-thio-β-d-galactopyranoside (final concentration) at OD 0.5. 6 h after induction, cells were collected and lysed by sonication in lysis buffer (20 mmTris-HCl, pH 7.4, 10 mm NaCl, 5 mmMgCl2, 1% Triton X-100) and purified by affinity chromatography with glutathione-Sepharose (Amersham Pharmacia Biotech). Loaded beads were washed two times in washing buffer A (20 mm Tris-HCl, pH 7.4, 10 mm NaCl, 5 mm MgCl2) and washing buffer B (150 mm NaCl, 50 mm Tris-HCl, pH 7.5) at 4 °C. ΔDNT was eluted from the beads as a GST fusion protein with glutathione (10 mm glutathione, 50 mm Tris-HCl, pH 7.5) for 10 min at room temperature. Mutagenesis of ΔDNT was performed by round circle polymerase chain reaction-based site-directed mutagenesis (Quick changeTM, Stratagene) with the following sense primers and corresponding antisense primers (MWG): C1292S sense, 5′-GGCTCCTTGAGCGGGTCCACGACGATGGTTGGG-3′; C1292A sense, 5′-GGCTCCTTGAGCGGGGCCACGACGATGGTTGGG-3′; H1307A sense, 5′-GGCTACCTGGCCTTCTACGCCACTGGCAAGTCGACC-3′; and K1310A sense, 5′-GCCTTCTACCACACTGGCGCGTCGACCGAACTCGGG-3′. Mutations were verified by DNA sequencing using a dye terminator sequencing kit with AmpliTaq DNA polymerase (Applied Biosystems). Activation of FXIII occurs through thrombin cleavage of the a-chains in the presence of calcium ions. 10 μm human factor XIII a-chains (Centeon) were incubated with 2 μg/μl thrombin for 30 min at room temperature in reaction buffer containing 150 mm NaCl, 50 mmtriethanolamine, and 8.5 mm CaCl2. Thrombin was then removed by incubation with benzamidine-Sepharose for 10 min at room temperature. The activity of FXIII was tested with fibronectin as a substrate. For qualitative measurement of ammonia a coupled enzymatic reaction was used that was based on the ammonia test combination for food analysis (Roche Molecular Biochemicals). NADH was diluted to give a concentration of 50 μm with triethanolamine buffer containing 2-oxoglutarate, 20 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 1 mm dithiothreitol, and 1 mm EDTA. Ten units of GlDH and RhoA (final concentration 10 μm) were added. After the addition of ΔCNF1 or ΔDNT (each 1 μm), the decrease in NADH fluorescence was monitored in a Perkin-Elmer LS-50B luminescence spectrometer. The emission was measured at 460 nm with excitation at 340 nm. For quantitative analysis of the ammonia release, Rho proteins (200 μm) were incubated with ΔCNF1 (1 μm) or ΔDNT (1 μm) in a reaction buffer containing 20 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 8 mm CaCl2, 1 mm dithiothreitol, and 1 mm EDTA at 37 °C. The reaction was stopped at different time points by incubation for 1 min at 95 °C. Denatured proteins were removed by centrifugation, and ammonia produced was measured in the supernatant. Decrease in absorbance was measured following the instructions given for the ammonia test combination for food analysis (Roche Molecular Biochemicals). For microinjection, NIH3T3 cells were seeded subconfluently on glass coverslips (CELLocate, Eppendorf) and cultivated for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 5% CO2 at 37 °C. After serum starvation GST-ΔDNT (2 mg/ml) or buffer was microinjected into NIH3T3 cells with a Microinjector 5242 (Eppendorf). 6 h after microinjection, cells were fixed with 4% formaldehyde and 0.1% Tween 20 in phosphate-buffered saline at room temperature for 10 min. For actin staining, formaldehyde-fixed cells were intensively washed with phosphate-buffered saline. The cells were then incubated with rhodamine-conjugated phalloidine (1 unit/coverslip) at room temperature for 1 h, washed again, and applied for fluorescence microscopy (as bleaching preservative KAISER'S glycerol gelatin (Merck) was used). Small GTPases were incubated with GST-ΔCNF1 or GST-ΔDNT in the presence of monodansylcadaverine or ethylenediamine (50 mm) in transglutamination buffer (20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 8 mm CaCl2, 1 mm dithiothreitol, 1 mm EDTA) for the indicated times at 37 °C. As a control, RhoA was incubated without the toxins but in the presence of a cosubstrate. The molar ratio of toxin:RhoA was 1:20. Labeling of proteins with the fluorescent lysine analog dansylcadaverine was analyzed by fluorescence activity under UV light before staining and drying the gel. Recombinant Rho proteins were modified by ΔCNF1 or transglutaminase in the presence or absence of primary amines. The reaction was stopped by freezing in liquid nitrogen. After thawing the proteins were loaded with [γ-32P]GTP for 5 min at 37 °C in loading buffer (50 mm Tris-HCl, pH 7.5, 10 mm EDTA, 2 mm dithiothreitol). MgCl2 (12 mm, final concentration) and unlabeled GTP (2 mm, final concentration) were added. For stimulation of GTPase activity by Rho-GAP, 50 nmp50RhoGAP were added to 1 μm Rho, and incubation was for 4 min at 37 °C. GTPase activity was analyzed by filter binding assay as described (15Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar). GST-ΔDNT was incubated with different concentrations of N-ethylmaleimide in 50 mm Tris-HCl, pH 7.5, for 30 min at room temperature. N-ethylmaleimide was than inactivated by adding dithiothreitol in a molar ratio of 10:1 (dithiothreitol:N-ethylmaleimide) for 10 min. For modification of RhoA, the GTPase was incubated withN-ethylmaleimide-treated or -untreated toxin in the presence of 50 mm ethylenediamine in transglutamination buffer (20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 8 mm CaCl2, 1 mm dithiothreitol, 1 mm EDTA) for 30 min at 37 °C. The excised gel plugs of Rho A were destained for 1 h at 50 °C in 40% acetonitrile, 60% hydrogen carbonate (50 mm, pH 7.8) to remove Coomassie Blue, gel buffer, SDS, and salts. The plug was subsequently dried in a vacuum centrifuge for 15 min. Thereafter, 30 μl of digestion buffer with trypsin was added, and digestion was carried out for 12 h at 37 °C. 4-Hydroxy-α-cyanocinnamic acid (Aldrich) was recrystallized from hot methanol and stored in the dark. Saturated matrix solution of 4-hydroxy-α-cyanocinnamic acid in a 1:1 solution of acetonitrile/aqueous 0.1% trifluoroacetic acid was prepared. 2 μl of the proteolytic peptide mixture were mixed with 2 μl of saturated matrix containing marker peptides (5 pmol of human ACTH (18–39) clip (MW 2466, Sigma) and 5 pmol of human angiotensin II (MW 1047, Sigma), respectively) for internal calibration. Using the dried-drop method of matrix crystallization, 1 μl of the sample matrix solution was placed on the matrix-assisted laser desorption ionization stainless-steel target and was allowed to air dry several minutes at room temperature resulting in a thin layer of fine granular matrix crystals. Matrix-assisted laser desorption ionization/time of flight-mass spectrometry was performed on a Bruker Biflex mass spectrometer equipped with a nitrogen laser (l = 337 nm) to desorb and ionize the samples. Mass spectra were recorded in the reflector positive mode in combination with delayed extraction. External calibration was routinely used, and internal calibration with two points that bracketed the mass range of interest was additionally performed to consolidate peptide masses further. The computer program mass spectrometry-digest (Peter Baker and Karl Clauser, UCSF Mass Spectrometry Facility) was used for computer-assisted comparison of the tryptic peptide mapping data with the expected set of peptides. Recently, it was shown that the CNF1 of E. coli activates Rho proteins by deamidating glutamine at position 63 of RhoA or 61 of Rac and Cdc42. Moreover, it has been reported that CNF1 possesses transglutaminase activity. CNF1 and DNT share a region of homology (amino acid residues 1250–1351 of DNT) located at their C termini (16Walker K.E. Weiss A.A. Infect. Immun. 1994; 62: 3817-3828Crossref PubMed Google Scholar). Therefore, we studied whether the C-terminal fragment ΔDNT (amino acid residues 1136–1451 of the holotoxin) is sufficient for the enzyme activity. To analyze the activity of GST-ΔDNT, we constructed the vector pGEX-ΔDNT, expressed the toxin fragment as a GST fusion protein, and purified it by affinity chromatography. Because the fusion toxin exhibited full activity and was not cleavable without degradation of ΔDNT, we used the fusion toxin (which is termed ΔDNT in the text) throughout the entire study. After incubation of RhoA with ΔDNT for 15 min, the GTPase shifted to an apparent higher molecular mass in SDS-PAGE indicating deamidation of Rho (15Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar) (Fig.1). In the presence of the transglutaminase cosubstrate ethylenediamine, however, RhoA shifted slightly to an apparent lower molecular mass. Recently, we reported that a downward shift of RhoA in SDS-PAGE corresponding to transglutamination was obtained when the GTPase was incubated with ΔCNF and ethylenediamine (17Schmidt G. Selzer J. Lerm M. Aktories K. J. Biol. Chem. 1998; 273: 13669-13674Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Therefore, we analyzed tryptic peptides of ΔDNT-treated RhoA by mass spectrometry. As shown in Fig.2 B, the mass analysis of the tryptic digest of the upper band of ΔDNT-treated RhoA revealed the RhoA peptide Gln52-Arg68 exhibiting a mass shift of one dalton in comparison to untreated RhoA (Fig.2 A). Tryptic digest of the downward shifted band of RhoA resulted in identification of the same peptide (Gln52-Arg68) but with a mass shift of 43 Da as compared with the control protein. This increase in mass indicated the transglutamination of Gln63 by ethylenediamine (Fig.2 C). Also dansylcadaverine, a fluorescent primary amine (18Takagi J. Aoyama T. Ueki S. Ohba H. Saito Y. Lorand L. Eur. J. Biochem. 1995; 232: 773-777Crossref PubMed Scopus (0) Google Scholar), served as a cosubstrate for the transglutamination of RhoA by ΔCNF1 (note that also ΔCNF1 was used as the GST fusion protein) and ΔDNT. To compare the transglutamination activity of the toxin fragments, RhoA was incubated with the enzymes in the presence of dansylcadaverine, and the amount of GTPase modified was analyzed in SDS-PAGE under UV light. As shown in Fig.3, RhoA was dansylated by ΔDNT to a larger extent than by ΔCNF. To compare the transglutaminase activities of both toxins in more detail, kinetic studies were performed.Figure 2Matrix-assisted laser desorption ionization/time of flight-mass spectrometry spectra of in gel digestion of modified RhoA. Gel plugs of unmodified Rho A (A) and Rho A modified by GST-ΔDNT in the absence (B) or presence of 20 mm ethylenediamine (C) were excised and destained for 1 h in 40% acetonitrile, 60% hydrogen carbonate (50 mm, pH 7.8). The plugs were subsequently dried in a vacuum centrifuge for 15 min. Thereafter, trypsin digestion was carried out for 12 h at 37 °C. A, the RhoA peptide Gln52-Arg68 (2009 Da) is shown. B, deamidation of Gln63 of Rho A by GST-ΔDNT results in a mass shift of the peptide of 1 Da. C, transglutamination of Gln63 of Rho A by GST-ΔDNT in the presence of ethylenediamine results in a mass shift of the peptide of 43 Da. aa, amino acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Dansylation of RhoA proteins with GST -Δ CNF1 or GST -Δ DNT . RhoA (20 μm) was incubated with GST-ΔCNF1 (1 μm) or GST-ΔDNT (1 μm) in the presence of dansylcadaverine for 15 min at 37 °C. Labeled proteins were analyzed by SDS-PAGE (5 μg RhoA/lane). Dansylated proteins were visualized by exposure to UV light (shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To compare kinetics of the deamidation and transglutamination reaction of ΔCNF1 and ΔDNT, the time course of ammonia release induced by the toxins was studied. The ammonia release assays were performed with a substrate concentration of 200 μm RhoA and an enzyme (GST-ΔDNT or GST-ΔCNF1) concentration of 1 μm. As shown in Fig.4 A, no difference in the production of ammonia was observed with or without ethylenediamine when RhoA was modified by ΔCNF1. On the contrary, ΔDNT released a higher amount of ammonia in the presence of ethylenediamine than in the absence of the primary amine (Fig. 4 B). A similar result was obtained in the presence of increasing concentrations of ethylenediamine. As shown in Fig.5 A, with ΔDNT the production of ammonia increased in an ethylenediamine concentration-dependent manner. In contrast, the addition of ethylenediamine at increasing concentration had no effect on ammonia production by ΔCNF1. Thus, all these data indicate that ΔDNT is preferentially a transglutaminase. Similarly, blood clotting factor FXIII, which is a mammalian transglutaminase (19Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar), released a higher amount of ammonia in the presence of the primary amine than in its absence (not shown).Figure 5Production of ammonia and GTPase activity of modified RhoA. Rho proteins (200 μm) were incubated with GST-ΔCNF1 (1 μm) (●) or GST-ΔDNT (1 μm) (▪) in transglutamination buffer in the presence of different concentrations of ethylenediamine at 37 °C. A,production of ammonia. The reaction was stopped after 10 min by heating for 1 min at 95 °C. Denatured proteins were removed by centrifugation, and the ammonia produced was measured in the supernatant. Shown is the ammonia produced at each ethylenediamine concentration as mean ± S.D. of three independent experiments. B, GTPase activity. The reaction was stopped by freezing an aliquot of the proteins in liquid nitrogen. Toxin-treated RhoA was loaded with [γ-32P]GTP. Thereafter, the GTPase activity was stimulated by adding p50GAP. The hydrolysis of GTP was determined after 4 min by filter binding assay. Shown is the remaining bound radioactivity as percent of loaded radioactivity as mean + S.D. of three independent experiments. ED, ethylenediamine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It is known that the activity of mammalian transglutaminases including FXIII is dependent on calcium ions (19Hettasch J.M. Greenberg C.S. J. Biol. Chem. 1994; 269: 28309-28313Abstract Full Text PDF PubMed Google Scholar). To test whether Ca2+ ions affect the activity of ΔDNT, we measured the ammonia release induced by the toxin fragment in the absence and presence of EGTA. Ammonia release caused by FXIII was dependent on the presence of Ca2+ ions, whereas the presence of EGTA had no (5 mm) or a very small (10 mm) effect on the activity of ΔDNT (not shown). In the presence of EGTA, both ΔDNT and ΔCNF1 modified RhoA by dansylation (not shown). Blood clotting factor FXIII, which modifies various protein substrates such as fibronectin, actin, and casein, transglutaminates three of the five glutamine residues of RhoA, whereas CNF1 is specific for Gln63 of RhoA and Gln61 of Cdc42 and Rac1 (17Schmidt G. Selzer J. Lerm M. Aktories K. J. Biol. Chem. 1998; 273: 13669-13674Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). To investigate the specificity of ΔDNT, we incubated RhoA, Rac1, Cdc42, the respective Q63E/Q61E mutants, actin, and casein in the presence of dansylcadaverine with ΔDNT for 30 min at 37 °C. Thereafter, transglutaminated proteins were analyzed by SDS-PAGE and UV light exposure. As shown in Fig.6, ΔDNT modified the wild-type GTPases RhoA, Cdc42, and Rac1 but not the Q63E/Q61E mutants, actin, or casein. In contrast, FXIII modified wild-type and mutant GTPases, actin, and casein (not shown). In line with the above observations, no ammonia was released during incubation of the Q63E mutant with the toxins (not shown). To compare kinetics of the deamidation/transglutamination reactions of RhoA, Rac1, and Cdc42 induced by the toxin fragments, we measured ammonia release in a time course. The reactions were performed with a protein substrate concentration of 200 μm, an enzyme concentration of 1 μm and 20 mm ethylenediamine. In Fig.7, the time courses of ΔDNT-induced ammonia release of RhoA, Cdc42, and Rac1 are shown as the mean of three independent experiments. All Rho proteins exhibited similar kinetics of ammonia release. Similarly, ΔCNF1 did not show major differences in the kinetics of ammonia release between the three GTPases (not shown). Gln63 of RhoA is known to be important for the intrinsic and GAP-stimulated GTPase mechanism of the protein (20Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar). To analyze whether transglutaminated protein is still able to hydrolyze GTP, we measured its p50RhoGAP-stimulated GTPase activity. Fig.5 B illustrates the effects of ΔCNF1 and ΔDNT on the GTPase activity of RhoA in the presence of increasing concentrations of ethylenediamine. Similar as observed for the ammonia release (Fig.5 A), inhibition of the GTPase with ΔCNF1 was independent of the ethylenediamine concentration, whereas the blockade of the GTP hydrolysis with ΔDNT increased with increasing concentration of the primary amine. Thus, inhibition of GTPase activity and ammonia release induced by ΔDNT correlated very well, indicating that the transglutamination inhibits GTPase activity of RhoA. It has been shown by Horiguchi et al. (10Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar) that treatment of cells with DNT leads to actin polymerization and stress fiber formation because of activation of RhoA. To investigate whether ΔDNT possesses the same cytotoxic effect in intact cells, we microinjected the toxin fragment as a GST fusion protein into quiescent NIH3T3 cells. The toxin fragment caused formation of stress fibers after 6 h of incubation. However this effect was not as strong as observed with ΔCNF1 (not shown). This may be because of instability of the GST-ΔDNT fusion protein, which significantly decreased in activity after a few days of storage at 4 °C or after incubation for 30 min at 37 °C. Recently, cysteine was identified to be a functionally essential residue in ΔCNF1, which is most likely located in the active site of the enzyme. Like ΔCNF, ΔDNT contains a single cysteine residue in a protein region highly similar to ΔCNF (Fig. 9). According to the findings with CNF, treatment of the toxin fragment with iodoacetamide orN-ethylmaleimide blocked the enzyme activity of ΔDNT (not shown). Exchange of cysteine 1292 with serine or alanine largely decreased or completely inhibited the enzyme activity of ΔDNT, respectively. Moreover the exchange of histidine 1307 with alanine blocked the enzyme activity of ΔDNT in analogy to CNF1 (not shown). A nucleotide binding motif has been described for DNT (not present in CNF) covering residues 1304–1311 (AFYHTGKS) with the consensus (A/G)XXXXGK(S/T) (16Walker K.E. Weiss A.A. Infect. Immun. 1994; 62: 3817-3828Crossref PubMed Google Scholar). To study the relevance of this motif for ΔDNT activity, we changed lysine 1310 to alanine. This mutation blocked ΔDNT activity of the toxin fragment as already reported for the holotoxin (11Pullinger G.D. Adams T.E. Mullan P.B. Garrod T.I. Lax A.J. Infect. Immun. 1996; 64: 4163-4171Crossref PubMed Google Scholar). We observed differences between the toxins in respect to the nucleotide dependence of the deamidation/transglutamination reactions. Fig.8 shows the dansylation of V14RhoA previously loaded with GDP or GTP and of wild-type RhoA loaded with GDP or GTPγS. Free nucleotide was removed by gel filtration before modification by the toxins. Whereas ΔCNF catalyzed the deamidation reaction independently of the nucleotide bound, ΔDNT accepted GDP-loaded RhoA or GDP-loaded V14RhoA as a substrate but did not modify GTPγS-bound RhoA or GTP-loaded V14RhoA. To test whether the activity of ΔDNT was regulated by nucleotides via a direct interaction, the toxin was pretreated with nucleotides or nucleotides were added to the reaction mixture. In both cases, we were not able to obtain any evidence for an inhibition of ΔDNT activity by a direct interaction of the enzyme with GTPγS (not shown). Recently Horiguchi et al. (10Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar) showed that DNT fromBordetella modifies Rho GTPases by deamidation of Gln63. A similar deamidation of Rho at Gln63was reported for CNF1 from E. coli (14Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 15Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar). DNT and CNF share a significant sequence homology in a rather small part of the proteins, suggesting that the deamidase activity is located in this region of the toxins. Therefore, we constructed ΔDNT, which covered this homologous region (Fig. 9). This fragment consisting of amino acid residues 1136–1451 possessed full deamidase activity and typically caused an upward shift of RhoA in SDS-PAGE. This change in migration in SDS-PAGE was not observed with the Q63E mutant of RhoA, confirming that exclusively Gln63was deamidated. Thus, the active fragment ΔDNT exhibited the same biochemical properties as reported for the holotoxin DNT. Moreover, similarly as observed for the holotoxin but to a smaller extent microinjection of GST-DNT caused formation of stress fibers in fibroblasts. Recently, we reported that CNF1 possesses transglutaminase activity and modifies Rho GTPases in the presence of primary amines (17Schmidt G. Selzer J. Lerm M. Aktories K. J. Biol. Chem. 1998; 273: 13669-13674Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In the presence of ethylenediamine, transglutamination of RhoA by CNF caused a downward shift of the GTPase in SDS-PAGE. However, this activity of CNF was only observed at high concentrations of the primary amine and occurred slower than deamidation. Similarly as with CNF, we detected a downward shift of RhoA in SDS-PAGE after treatment with ΔDNT in the presence of ethylenediamine. The transglutamination of Rho by ΔDNT was verified by mass spectrometric analysis. To further characterize the enzyme activity of ΔDNT in more detail and to compare it with ΔCNF, we applied an ammonia release assay. Interestingly, we observed that the release of ammonia by ΔDNT was largely dependent on the presence of ethylenediamine. Almost no ammonia was released in the absence of the primary amine. Increasing concentration of ethylenediamine also increased ammonia production. In contrast, CNF-induced ammonia release was hardly changed in the presence and absence of the primary amine. These data suggest that (at least under the conditions used) DNT acts preferentially as a transglutaminase, whereas CNF behaves preferentially as a deamidase. In fact, differences in the activities of CNF and DNT are obvious from studies in intact cells. Treatment of intact cells with CNF causes an upward shift of RhoA in SDS-PAGE indicating a deamidase reaction (15Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar, 21Oswald E. Sugai M. Labigne A. Wu H.C. Fiorentini C. Boquet P. O'Brien A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3814-3818Crossref PubMed Scopus (169) Google Scholar). By contrast, Horiguchi et al. (10Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar) reported that DNT caused a downward shift of Rho after treatment of cells for 1–3 h. Longer incubation of cells with DNT (e.g. for up to 6 h) resulted in an occurrence of an additional upward shift. These data can be interpreted to indicate that DNT causes preferentially a transglutamination reaction also in intact cells. Because we did not succeed in the expression of a recombinant full-length DNT preparation, which was biologically active, we are at present not able to verify this hypothesis. Because deamidation- or transglutamination-induced changes in migration of GTPases in SDS-PAGE are less pronounced with Rac and Cdc42, we used the ammonia release assay to study the substrate specificity of ΔDNT. These data indicated that all Rho GTPases including Rac and Cdc42 are modified by ΔDNT. We did not detect major differences in the ability of the various Rho proteins to serve as substrate for ΔDNT. A similar substrate specificity was recently reported for CNF1 (17Schmidt G. Selzer J. Lerm M. Aktories K. J. Biol. Chem. 1998; 273: 13669-13674Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, we observed differences between ΔCNF1 and ΔDNT in respect to the nucleotide dependence of the deamidation/transglutamination reactions. Whereas ΔCNF1 catalyzed the deamidation reaction with a similar velocity in the presence of GDP or GTPγS-loaded RhoA, ΔDNT accepted GDP-loaded RhoA or GDP-loaded V14RhoA but did not modify GTPγS-bound RhoA or V14RhoA that is not able to hydrolyze GTP. The slight modification of GTPγS-loaded RhoA with ΔDNT and the low modification of V14RhoA GDP may be because of an incomplete exchange of the nucleotides. As binding of nucleotides largely changes the conformation of the switch II region of the GTPases, these findings suggest that the structural requirements for modification by DNT are more restricted. Another possibility would be that free nucleotides interact with the enzyme to alter its activity. In fact, a nucleotide binding motif has been described for DNT but not for CNF covering residues 1304–1311 (AFYHTGKS) with the consensus (A/G)XXXXGK(S/T) (11Pullinger G.D. Adams T.E. Mullan P.B. Garrod T.I. Lax A.J. Infect. Immun. 1996; 64: 4163-4171Crossref PubMed Google Scholar, 22Wilk-Blaszczak M.A. Singer W.D. Quill T. Miller B. Frost J.A. Sternweis P.C. Belardetti F. J. Neurosci. 1997; 17: 4094-4100Crossref PubMed Google Scholar). To study the relevance of this motif for DNT activity, we changed lysine 1310 to alanine. This mutation blocked ΔDNT activity as reported earlier for the holotoxin (11Pullinger G.D. Adams T.E. Mullan P.B. Garrod T.I. Lax A.J. Infect. Immun. 1996; 64: 4163-4171Crossref PubMed Google Scholar). The role of lysine 1310 is not clear because this residue is not present with similar spacing in CNF1. It is conceivable that the loss in activity of the K1310A mutant is caused by structural changes of the toxin not directly involving catalysis, because K1310 is located in the vicinity of the catalytic important residue His1307. Although a putative nucleotide binding motif is present in DNT, we did not obtain evidence for a control of ΔDNT activity by direct interaction of the enzyme with nucleotides. All eukaryotic transglutaminases are characterized by a catalytic cysteine and histidine residue. Recently, we identified cysteine 866 in CNF1 as essential for deamidase and transglutaminase activity (17Schmidt G. Selzer J. Lerm M. Aktories K. J. Biol. Chem. 1998; 273: 13669-13674Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Suggesting that a similar catalytic mechanism is functional in DNT, we changed cysteine 1292 of DNT to serine or alanine. These mutations caused inhibition of the enzyme activity indicating an essential role in catalysis. Thus, as assumed from the amino acid sequence alignment of DNT and CNF1, cysteine 1292 of DNT is functionally equivalent to cysteine 866 of CNF1. In contrast to transglutaminases, like the blood clotting factor FXIII, the activity of ΔDNT or ΔCNF was not dependent on calcium ions. Another Ca2+-independent transglutaminase was recently cloned fromStreptoverticillum (23Kanaji T. Ozaki H. Takao T. Kawajiri H. Ide H. Motoki M. Shimonishi Y. J. Biol. Chem. 1993; 268: 11565-11572Abstract Full Text PDF PubMed Google Scholar). The preferential transglutamination of Rho allowed studies on the GTPase activity of the cross-linked Rho protein. Gln63 of RhoA is essential for the intrinsic and GAP-stimulated GTPase mechanism of the protein. Recent crystal structure analysis of Rho and Rho-GAP in a complex with a transition state analogue GDP-AlF4−explains the function of Gln63 in stabilizing the transition state of GTP hydrolysis. To this end, the nitrogen of the carboxamide group of Gln63 is bonded to the main chain carbonyl of Arg85 of Rho-GAP and to one of the fluorides of AlF4− (20Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar). If Gln63 is deamidated (e.g. by CNF), this interaction with GAP is not possible resulting in the blockade of GAP-stimulated GTP hydrolysis (14Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 15Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar). After transglutamination of Gln63, however, the pivotal nitrogen residue is still present. Therefore, we were surprised that after transglutamination both intrinsic and GAP-stimulated GTPase activity of Rho were blocked. The reason for this inhibition is not entirely clear but may be based on structural changes that are the prerequisite for catalysis of GTP hydrolysis. For example, binding of Rho-GAP and subsequent activation of Rho GTPase activity are accompanied by conformational changes to allow the introduction of the catalytic Arg85 of RhoGAP into Rho (20Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar). It is feasible that this interaction is hindered by transglutamination of Gln63. Further studies are underway to analyze the influence of smaller transglutaminase cosubstrates like methylamine on the GAP-stimulated and intrinsic GTPase activity of RhoA after modification with ΔDNT. In summary, we localized the enzyme domain of DNT to a C-terminal fragment covering amino acid residues 1136–1451 with cysteine 1292, histidine 1307, and lysine 1310 as essential residues. This active fragment acts as a deamidase and/or transglutaminase to modify Gln63 of Rho or Gln61 of Rac and Cdc42, respectively, and to activate the GTPases. Kinetic analysis indicates that ΔDNT acts preferentially as a transglutaminase. In contrast to ΔCNF, which effectively modifies Rho proteins in the GDP- and GTP-bound form, GDP-bound Rho proteins are the preferred substrates of ΔDNT. We gratefully acknowledge the excellent technical assistance of Iris Misicka. We thank Dr. H. Metzner (Centeon Pharma, Marburg, Germany) for providing FXIIIa." @default.
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