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J Biol Chem, Vol. 274, Issue 41, 28999-29004, October 8, 1999
From the Institut für Pharmakologie und Toxikologie der
Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Straße 5, D-79104 Freiburg, Germany
The Escherichia coli cytotoxic
necrotizing factor 1 (CNF1) and the Bordetella
dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of
Gln63 of RhoA (Gln61 of Cdc42 and Rac). In
addition, both toxins possess in vitro transglutaminase
activity in the presence of primary amines. Here we characterized the
region of Rho essential for substrate recognition by the toxins using
Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho
was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12
cells microinjected with Ras55Rho developed formation of neurite-like
structures after treatment with the CNF1 holotoxin indicating
activation of the Ha-Ras chimera and Ras-like effects in intact cells.
The Ras59Rho78Ras chimera protein contained the minimal Rho sequence
allowing deamidation or transglutamination by CNF1. A peptide covering
mainly the switch II region and consisting of amino acid residues
Asp59 through Asp78 of RhoA was substrate for
CNF1. Changes of amino acid residues Arg68 or
Leu72 of RhoA into the corresponding residues of Ras
(R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of
the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp59 through
Asp78). Glucosylation of RhoA at Thr37 blocked
deamidation by DNT but not by CNF. The data indicate that CNF1
recognizes Rho GTPases exclusively in the switch II region, whereas the
substrate recognition by DNT is characterized by additional structural requirements.
Rho GTPases (e.g. Rho, Rac, and Cdc42) participate in
the regulation of the actin cytoskeleton (1, 2). Whereas Rho subtype proteins induce formation of stress fibers and adhesion complexes, Rac
is involved in formation of lamellipodia and Cdc42 induces microspikes
(3-5). Beside their roles in the organization of the actin
cytoskeleton, Rho proteins act as molecular switches in various
signal transduction processes (6, 7).
Rho proteins are the preferred substrates for several bacterial protein
toxins. Exoenzyme C3 from Clostridium botulinum and related
C3-like transferases ADP-ribosylate RhoA, B, and C at Asn41
thereby inhibiting the biological activity of the GTPases (8-11). Rho
proteins are monoglucosylated by members of the family of large
clostridial cytotoxins (e.g. Clostridium
difficile toxins A and B) (12-14). The toxins modify RhoA at
Thr37 (Thr35 of Rac and Cdc42), a modification
which blocks the interaction of the GTPases with their effectors (15,
16).
Rho family GTPases are also the targets for cytotoxic necrotizing
factors (CNF)1 1 and 2 from
Escherichia coli and the dermonecrotizing toxin (DNT)
produced by various Bordetella species. CNF and DNT are ~115 and ~165 kDa proteins which share a region of homology at their C termini harboring the enzyme domain of the toxins (17). In
culture cells, the toxins induce actin polymerization and inhibit cytokinesis resulting in formation of multinucleated cells (18-20). Recently it has been reported that CNF and DNT act on Rho GTPases by
deamidation of glutamine 63 of RhoA, thereby inhibiting the GTPase
activity of Rho. Because Gln63 is essential for GTP
hydrolysis, deamidation causes persistent activation of the GTPase
resulting in strong formation of stress fibers of CNF- or DNT-treated
cells. In addition to their deamidase activity, both toxins possess
in vitro transglutaminase activity to attach primary amines
onto Rho GTPases. Substrates of CNF and DNT are Rho subfamily members
including Rac and Cdc42.
In the present communication the substrate recognition of Rho GTPases
by CNF1 and DNT was studied. Using GTPase chimeras of RhoA and Ha-Ras
which is not a substrate of the toxins, we identified the switch II
region of Rho as being sufficient for recognition by CNF1. Accordingly,
a peptide consisting of amino acid residues Asp59 through
Asp78 of RhoA was deamidated and/or transglutaminated by
CNF1. By contrast, the structural requirements for substrate
recognition by DNT are more stringent.
Preparation of Recombinant Proteins--
For protein
purification, E. coli strains carrying pGEX plasmids with
the coding sequence for the respective GTPases, GTPase chimera, CNF1
(either as full-length or as the catalytic C-terminal part ( Construction of GTPase Chimeras--
The GTPase chimeras were
constructed using the splicing by overlap extension method described
previously (21). In brief, two polymerase chain reactions (PCR) were
performed, each amplifying the sequences to be fused. The products of
the two first PCRs were pooled and cycled in a third PCR, in which the
overhanging sequences act as primers. We used pGEX2T-RhoA (wild-type,
WT) and pGEX2TGL-Ha-Ras (wild-type) as templates with the different primers listed in Table I and pGEX
primers either annealing 5' or 3' of the multiple cloning site of pGEX
(Table I). The product of the second PCR was subjected to
BamHI and EcoRI cleavage and subcloned into a
pGEX2TGL vector. The sites of splicing are numbered by Rho
nomenclature. The "sandwich" chimeras Ras55Rho70Ras and Ras59Rho70Ras were constructed with splicing by overlay extension by using the primers for Ras55Rho and Ras59Rho, respectively, with
Rho70Ras serving as template. Ras59Rho115Ras was constructed by using
the primers for Ras59Rho with Rho115Ras as a template. All constructs
were checked for proper sequences.
Mutagenesis--
Mutants were constructed from pGEX2T-RhoA
(wild-type) or pGEX2T-Ha-Ras (wild-type) by PCR in the presence of two
primers (sense and corresponding antisense) carrying a base mismatch
encoding the proper mutant. The parental DNA was eliminated using the
restriction enzyme DpnI, which digests methylated DNA. The
PCR products were transformed into Epicurean Blue XL-1 ultracompetent
cells (Stratagene). After verifying the mutation by sequencing, the
plasmids were transformed into E. coli BL21 for protein
expression. The sense primers for the various mutants are listed in
Table II.
Cell Culture and Microinjection--
Rat pheochromocytoma cells
(PC12) were cultivated on plastic dishes in Dulbecco's modified
Eagle's medium supplemented with 10% horse serum, 5% fetal calf
serum, penicillin (4 mM), and streptomycin (4 mM) and kept at 37 °C in a humidified atmosphere with
10% CO2. The cells were microinjected with 0.5 µg/µl
GST-Ras55Rho or buffer by means of a microinjector 5242 (Eppendorf) and
subsequently treated for 48 h with full-length GST-CNF1 (300 ng/ml).
Transglutamination Assay--
GTPase or chimeric protein (3-6
µg) was incubated with or without CNF1 or DNT in a buffer containing
50 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM
EDTA, and 50 mM monodansylcadaverine for 15 min at
37 °C. The proteins were separated by SDS-PAGE and the
transglutaminated proteins were detected under UV light (206 nm).
Subsequently, the gel was Coomassie-stained to check the amount of
proteins in each lane.
Nucleotide Binding Assay--
RhoA, Ha-Ras, RhoA mutants, or
Rho/Ras chimeras (each 0.5 µM) were incubated at 37 °C
in a buffer containing 150 mM NaCl, 2.5 mM
MgCl2, 10 mM triethanolamine (pH 7.5). After
addition of 2 µM
2'(3')-O-(N-methylanthraniloyl)-GDP which was
synthesized as described (22), the increase in light emission at 444 nm, due to the higher intensity of bound
2'(3')-O-(N-methylanthraniloyl)-nucleotide excited at 357 nm, was monitored in a Perkin-Elmer LS 50B luminescence spectrometer.
Release of Ammonia--
To determine the release of ammonia from
the GTPases during incubation with CNF1, we utilized a coupled
enzymatic reaction. In the presence of glutamate dehydrogenase and
NADH, ammonia reacts with Glucosylation and ADP-ribosylation of RhoA--
RhoA was
incubated in a glucosylation buffer (50 mM Hepes (pH 7.5),
100 mM KCl, 0.1 mg/ml bovine serum albumin, 2 mM MgCl2, and 1 mM
MnCl2) in the presence or absence of C. difficile toxin B (6 ng/µl) or in an ADP-ribosylation buffer (40 mM Hepes (pH 7.5), 200 µM NAD, 0.1 mg/ml
bovine serum albumin, 100 µM GDP, and 2 mM
MgCl2) in the presence or absence of C. botulinum C3 toxin (1 ng/µl) at 37 °C for 30 min.
Mass Spectrometry--
RhoA switch II peptide (1 mM)
was incubated for 3 h at 37 °C with or without CNF1 (1 µM) in the presence of 20 mM ethylenediamine in a buffer containing 25 mM triethanolamine (pH 7.5), 75 mM NaCl, 1 mM CaCl2, 2 mM dithiothreitol, 10 mM MgCl2, and
1 mM EDTA. To obtain a concentration of the RhoA switch II
peptide of 10 µM, the reaction mixture was diluted with
TA (acetonitrile, 0.1% trifluoroacetic acid, 1:1). A saturated
matrix solution of recrystallized 4-hydroxy- Rho But Not Ras Is Deamidated by CNF1--
It has been shown that
Rho, Rac, and Cdc42 are deamidated by CNF1 (23). To test whether Ha-Ras
is a substrate for CNF1, the release of ammonia occurring concomitantly
with deamidation was determined in a coupled enzyme assay (see
"Experimental Procedures"). Fig. 1
shows that addition of the active fragment of CNF1 ( Identification of the Substrate Recognition Site of Rho--
By
exploiting the finding that Ha-Ras is not a substrate for
Hall and co-workers reported that amino acid residues 23 through 46 of
Ras are sufficient to elicit Ras-dependent transformation in NIH3T3 cells (24). Constitutively active Ras is known to induce
neurite formation in PC12 cells (25). Therefore, we tested whether CNF1
was able to induce neurite formation in PC12 cells microinjected with
Ras55Rho prior to toxin treatment. PC12 cells were grown on plastic
dishes, microinjected with 0.5 µg/µl GST-Ras55Rho, and treated with
GST fusion protein of the CNF1 holotoxin (300 ng/ml) for 48 h.
Whereas Ras55Rho-injected cells without CNF1 showed only small
processes (Fig. 4A), injected
cells developed long neurite extensions after CNF1 treatment (Fig.
4B) indicating an activation of the Ras chimera by CNF1.
Because experiments with the Ras55Rho and Rho115Ras chimeras suggested
that neither the N terminus nor the C terminus of Rho are essential for
substrate recognition by Identification of Amino Acid Residues in RhoA Which Are Essential
for CNF1 Substrate Recognition--
Deduced from recent crystal
structure analysis of Rho (26) and by means of the "Rasmol" program
we identified four amino acids (Arg68, Leu72,
Lys98, and His105) between residues 59 through
115 of Rho which are surface exposed and are identical in Rho, Rac, and
Cdc42. These amino acids were changed to the corresponding Ras
residues. All mutant proteins were capable of binding
2',3'-O-N-methylanthraniloyl-GDP in the nucleotide binding assay (not shown) indicating a correct protein folding. We tested the deamidation of the mutant RhoA proteins by
A Peptide Corresponding to the RhoA Switch II Region Is Deamidated
by CNF1--
A peptide of the sequence
59DTAGQEDYDRLRPLSYPDTD78 which
covers the switch II region of RhoA was tested for its ability to serve as substrate for CNF1 in the ammonia release assay. The switch II
peptide was compared with RhoA (10 µM) at two
different concentrations (10 and 100 µM) in the presence
of 1 µM Comparison of CNF1 with the Related Toxin DNT--
Next we
compared
To investigate whether DNT requires the same amino acid residues of Rho
for substrate recognition as CNF1, the transglutaminase activities of
both toxins were studied in the presence of monodansylcadaverine with
the RhoA mutants (R68A, L72Q, K98Q, and H105R) mentioned above (Fig.
10). Like
We tested the Ras55Rho and Rho115Ras chimeras with GST- In contrast to various bacterial protein toxins inactivating small
GTPases including large clostridial cytotoxins and exoenzyme C3 (28),
the E. coli cytotoxic necrotizing factors CNF1 and CNF2 activate Rho GTPases. The latter toxins deamidate Rho
GTPases at Gln63 of Rho (Gln61 of Rac and
Cdc42) to inhibit the endogenous and GAP-stimulated GTP hydrolysis
thereby blocking the switch off reaction of the GTPase (29, 30).
Recently, we reported that CNF1 possesses in vitro
transglutaminase activity in addition to deamidase activity (31, 32).
Here we attempted to identify the recognition site of Rho GTPases
for CNF1.
Rozengurt and co-workers (33) reported that CNF1 does not activate the
mitogen-activated protein kinases p42mapk or p44mapk in
Swiss 3T3 cells, suggesting that Ras is not activated by the toxin. In
line with this notion, we confirmed that recombinant Ras is not
modified by CNF1. To get more information about the structural
requirements for substrate recognition by CNF1, we constructed several
chimeras consisting of Rho and Ras. The enzymatically active toxin
fragment These results indicate that the switch II region of Rho is sufficient
for substrate recognition by CNF1 and neither the switch I region nor
the insert region (Asp124 through Gln136) are
required for interaction with CNF1. This notion was corroborated by the
findings that the peptide Asp59 through Asp78
covering the switch II region of RhoA was capable to serve as a
substrate for the active fragment of CNF1. Similar as found for the
chimera Ras59Rho78Ras, the peptide was a poorer substrate for CNF1 than
wild-type RhoA, suggesting that additional residues, although not
essential for modification by the toxin, increase the substrate
properties of RhoA. With one exception (Asp76 of Rho, which
is glutamine in Rac and Cdc42), all Rho proteins including Rac and
Cdc42 possess highly conserved amino acid sequences in the switch II
region. Thus, the substrate requirements observed in this study are in
agreement with recent findings that Rac and Cdc42 are modified by CNF1
(23) and it seems likely that all Rho GTPases, including those not
studied so far, are also substrates of the toxin.
To identify amino acids which are possibly involved in interaction with
CNF1, we selected surface-exposed amino acids (Arg68,
Leu72, Lys98, and His105) for
site-directed mutagenesis by using the crystal structure data of RhoA
(26). Replacement of Arg68 or Leu72 of RhoA
with the corresponding amino acid in Ha-Ras (R68A and L72Q) prevented
modification of the mutant proteins. By contrast, replacement of
His105 and Lys98, which are located outside the
switch II region, with the corresponding residues of Ha-Ras did not
alter the modification of RhoA, and the rate of ammonia release from
H105R RhoA was greater than that from wild-type Rho. Although both
Arg68 and Leu72 of RhoA are essential for
recognition by CNF1, they are not sufficient for modification. This is
concluded from the finding that replacement of the corresponding amino
acids in Ras by arginine and leucine did not make it a substrate for
CNF1.
The observation that the switch I region of Rho is not important for
substrate recognition by CNF1 in vitro was confirmed in
intact cells. Microinjection of the Ras55Rho chimera into PC12 cells
and subsequent treatment with the CNF1 holotoxin, which in contrast to
The Bordetella dermonecrotizing toxin DNT is another toxin
which activates Rho GTPases. This toxin shares significant similarity at its C terminus (amino acids 1136-1451) with the active region of
CNF1 (17, 34). Like CNF1, DNT catalyzes deamidation of Gln63 of Rho (35). Moreover, DNT possesses high
transglutaminase activity (36). We compared the structural requirements
for substrate recognition by In summary, the switch II region of Rho was identified as the substrate
recognition site for CNF1. Minimal structural requirements for
substrate recognition by CNF1 are provided by a peptide covering amino
acid residues Asp59 through Asp78 of Rho. By
contrast, the substrate recognition by DNT is much more complex
requiring, in addition to the switch II region, further sites including
the switch I region of Rho.
We thank Fred Wittinghofer for the E63D
Ha-Ras mutant and Iris Misicka for excellent technical assistance.
*
This work was supported by Grant SFB 388 from the Deutsche
Forschungsgemeinschaft.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
CNF1, E.
coli cytotoxic necrotizing factor 1;
Identification of the Region of Rho Involved in Substrate
Recognition by Escherichia coli Cytotoxic Necrotizing
Factor 1 (CNF1)*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CNF1,
amino acid residues 709-1014), or DNT (the catalytic C-terminal part
(DNT, amino acid residues 1136-1451) were grown in LB medium and
induced with 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside at OD 0.5. The
cells were harvested after 3-6 h and the glutathione S-transferase (GST) fusion proteins were purified by means
of glutathione-Sepharose (Amersham Pharmacia Biotech). Unstable
proteins were kept as GST fusion proteins (Rho115Ras, Ras59Rho115Ras,
DNT, and full-length CNF1); otherwise, the proteins were subjected to thrombin cleavage to remove GST.
Primers used for construction of chimeras
Sense primers used for site-directed mutagenesis
-ketoglutarate to form
L-glutamate, thereby oxidating the NADH. The amount of NADH
oxidized in the reaction is stoichiometric to the amount of ammonia
produced. RhoA, Ha-Ras, or Rho/Ras chimeras (each 10 µM)
were incubated in a buffer containing 5 mM Tris-HCl (pH 7.4), 10 mM MgCl2,, 1 mM
dithiothreitol, 1 mM EDTA and the components of the coupled
enzyme reaction system (Roche Molecular Biochemicals, Ammonia Test
Combination for Food Analysis): 140 µM NADH, 7 mM -ketoglutarate, and 10 units of glutamate
dehydrogenase. The samples were equilibrated at 37 °C prior to
addition of CNF1 as indicated in the figure legends. The decrease in
NADH concentration was measured fluorimetrically (excitation at
340 nm, emission at 460 nm) in a Perkin-Elmer LS 50B luminescence spectrometer.
-cyanocinnamic acid
(Aldrich) in TA was freshly prepared and marker peptides (ACTH 18-39
clip, human, and angiotensin II, human) were added for internal
calibration. Matrix and peptide solution were mixed in equal amounts.
Using the dried-drop method of matrix crystallization, 1 µl of the
sample matrix solution was placed on the MALDI stainless steel target
and allowed to air-dry several minutes at room temperature. MALDI/TOF-MS was performed on a Bruker Biflex mass spectrometer equipped with a nitrogen laser (
= 337). Mass spectra were
recorded in the reflector positive mode in combination with delayed extraction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CNF1) caused
release of ammonia in the presence of RhoA but not in the presence of
Ras indicating that the latter GTPase is not modified by CNF1. Because
Rho and Ha-Ras differ in the amino acid residues flanking the site of
modification in Rho (Fig. 2), we tested a Ha-Ras mutant with an aspartic acid residue instead of glutamic acid at
position 63 of Ha-Ras. Like wild-type Ha-Ras, the mutant E63D Ras was
not deamidated by CNF1 (not shown).
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Fig. 1.
Release of ammonia from Rho induced by
CNF1. RhoA or Ha-Ras (each 10 µM) were incubated in
a buffer containing the components of the coupled enzymatic reaction:
NADH, -ketoglutarate, and glutamate dehydrogenase. After the samples
had equilibrated at 37 °C, CNF1 (2 µM, final
concentration) was added. Release of ammonia is measured by the
decrease in NADH due to the coupled reaction. In one control, CNF1 was
added to buffer without GTPase. In another control, 10 µM
NH4+ was added to buffer without GTPase.
The experiment was repeated 3 times with similar results.
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Fig. 2.
Alignment of amino acids of RhoA, Cdc42,
Rac1, and Ha-Ras that cover the switch II region of the GTPases.
Gln63 (bold letter, Rho nomenclature) is the
target for deamidation or transglutamination by CNF and DNT. Mutant
RhoA proteins were constructed with amino acids Arg68 or
Leu72 changed to the respective residues of Ha-Ras. The
peptide Asp59 through Asp78 was studied as a
substrate for CNF1.
CNF1,
chimeras of Rho and Ha-Ras were constructed to identify the minimal
amino acid sequence of Rho allowing the modification by CNF1. First,
we constructed Rho115Ras and Ras55Rho chimeras (note that the Rho
nomenclature is used), which both have the switch II region of RhoA.
The chimeras were tested in a transglutaminase assay with
monodansylcadaverine as a co-substrate. This primary amine contains a
naphthalene group, which is excitable by UV light at 206 nm. Fig.
3 shows the proteins analyzed by SDS-PAGE
and photographed under UV light. As observed for wild-type Rho, the Ras55Rho chimera was transglutaminated by CNF. Also the Rho115Ras chimera was substrate for CNF. This chimera was less stable than Ras55Rho. Therefore, we studied Rho115Ras as a GST fusion protein and
compared it with GST fusion proteins of wild-type RhoA and Ha-Ras.
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Fig. 3.
Transglutamination of Rho/Ras chimeras.
GST-RhoA, GST-Rho115Ras, or GST-Ha-Ras (each 3 µg, upper
panel) and RhoA or Ras55Rho (each 3 µg, lower panel)
were incubated with or without CNF1 (1 µg) at 37 °C for 15 min
in a buffer containing monodansylcadaverine. The samples were subjected
to SDS-PAGE and the gels were photographed under UV light
(A). Subsequently, the gels were stained with Coomassie Blue
(B). The lower band of Rho115Ras, which is also
modified by CNF1, may be a degradation product of the protein. The
experiment was repeated more than 3 times with similar results.
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Fig. 4.
Neurite outgrowth of PC12 cells after
microinjection of the GST-Ras55Rho chimera and activation by CNF1.
Subconfluent PC12 cells growing on plastic dishes were microinjected
with 0.5 µg/µl GST-Ras55Rho (A and B) and
subsequently incubated for 48 h without (A) or with
(B) 300 ng/ml full-length GST-CNF1. The experiment was
repeated 3 times with similar results.
CNF1, we studied whether sandwich
(Ras/Rho/Ras) chimeras are modified by the toxin. The sandwich chimera,
Ras59Rho115Ras, was substrate for deamidation by CNF1 (not shown).
Further reduction of the length of the RhoA insert in Ha-Ras resulted
in the chimera Ras59Rho78Ras. We tested this chimera in the
transglutamination assay at increasing concentrations of CNF1. The
chimera was a substrate for transglutamination, however, whereas
wild-type Rho was significantly transglutaminated at 10 nM
CNF1, higher concentrations of CNF1 (about 10-fold) were required
for transglutamination of Ras59Rho78Ras (Fig.
5). Chimeras containing very short Rho
sequences of only 8 or 5 amino acid residues (Ras55Rho70Ras and
Ras59Rho70Ras) did not serve as substrates of CNF1 (data not shown). To
exclude that these results were due to incorrect protein folding,
we tested the ability of the chimeras to bind nucleotide. Both chimeras
(Ras55Rho70Ras and Ras59Rho70Ras) were able to bind
2'(3')-O-(N-methylanthraniloyl)-GDP indicating a
proper folding of the proteins (not shown). The results obtained with
the different chimeras are summarized in Fig.
6.
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Fig. 5.
Transglutamination of the GTPase chimera
Ras59Rho78Ras. Wild-type RhoA (RhoWT) or the chimera Ras59Rho78Ras
(each 20 µM) were incubated for 15 min in the presence of
monodansylcadaverine at the indicated concentrations of CNF1. The
samples were separated on SDS-PAGE and the gel was photographed under
UV light (A), thereafter, the gels were stained with
Coomassie Blue (B). Repetition of the experiment gave
similar results.
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Fig. 6.
Properties of Rho/Ras chimeras to serve as
substrates for CNF1. Substrate properties
were tested at least 3 times by the ammonia release and/or the
transglutamination assay. ++, substrate properties similar as wild-type
RhoA; +, lower rate of transglutamination than with wild-type RhoA; 
,
no substrate for CNF1.
CNF1 in the ammonia release assay. As shown in Fig.
7, A and B, whereas
the K98Q and the H105R mutants were deamidated by CNF1, L72Q and
R68A mutants did not serve as substrates or were only marginally
modified by the toxin. The rate of the modification of the mutant K98Q
varied between different experiments and was not consistently slower
than the wild-type. To investigate whether arginine 68 and leucine 72 are sufficient for CNF1 substrate recognition, a Ha-Ras double mutant
carrying an arginine residue at position 66 and a leucine residue at
position 70 was constructed and tested for activity in the
transglutaminase and ammonia release assay. However, this mutant was
not modified by CNF1 (not shown).
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Fig. 7.
Release of ammonia from RhoA mutants induced
by CNF1. Wild-type RhoA (RhoWT,
A and B), R68A RhoA (A), K98Q RhoA
(A), L72Q RhoA (B), or H105R RhoA (B)
were incubated in a buffer containing the components of the coupled
enzymatic reaction: NADH, -ketoglutarate, and glutamate
dehydrogenase. After equilibration of the samples at 37 °C, CNF1
(0.5 µM) was added. Ammonia release was determined by the
decrease in NADH fluorescence (arbitrary units) caused by the coupled
reaction. In control experiments (A and B),
CNF1 was added to buffer without Rho protein. The experiment was
repeated with similar results.
CNF1. As shown in Fig. 8, after addition of CNF1, ammonia was
released from the peptide, however, at a slower rate than with RhoA.
The difference in the modification rates was calculated as a 110-fold
decrease for the peptide in comparison with the recombinant protein
(Sigma Plot). A shorter peptide with the sequence
59DTAGQEDYDRLR70 did not release ammonia after
addition of CNF1 (not shown). To study whether the switch II peptide
(Asp59-Asp78) was modified by
transglutamination, the peptide was treated with CNF1 in the presence
of ethylenediamine. Thereafter, the sample was analyzed by MALDI-TOF
mass spectrometry. As shown in Fig. 9,
after CNF1 treatment a new peptide characterized by an increase in
mass by 43 Da was detected indicating the attachment of
ethylenediamine onto the switch II peptide.
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Fig. 8.
Release of ammonia from the RhoA peptide
Asp59 through Asp78. RhoA (10 µM) or the RhoA peptide (10 and 100 µM)
were equilibrated at 37 °C in a buffer containing the components of
the coupled enzymatic reaction, then CNF1 (1 µM) was
added. Ammonia release was determined by the decrease in NADH
fluorescence (arbitrary units) caused by the coupled reaction. In
controls, CNF1 was added to buffer without Rho protein or peptide.
Repetition of the experiment gave similar results.
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Fig. 9.
MALDI-TOF-MS spectra of the transglutaminated
RhoA peptide Asp59-Asp78. The RhoA peptide
Asp59-Asp78 was treated in the presence of
ethylenediamine (20 mM) without (upper line) or
with CNF1 (1 µM, lower line) for 3 h.
Thereafter, the peptides were analyzed by MALDI-TOF-MS. Shown is the
Rho peptide Asp59-Asp78 (2326 Da) and a new
peptide with a mass of 2369.1 Da caused by CNF1-catalyzed attachment
of ethylenediamine (43 Da) onto the Rho peptide. Note the increase in
mass of 1 Da by deamidation was not detected, because the reaction was
not complete.
CNF with the related Bordetella deamidase DNT in
respect to substrate specificity and substrate recognition. The active
C-terminal part of Bordetella DNT (amino acids 1136-1451, DNT) was applied as a GST fusion protein because GST-DNT proved to be more stable than DNT. For comparison of the two enzymes, CNF1 was also used as a GST fusion protein. As observed for
GST-CNF1, GST-DNT deamidated RhoA, Rac 1, and Cdc42 (not shown).
CNF1, DNT did not
modify the mutants R68A RhoA and L72Q RhoA with alterations in
the switch II region. K98Q RhoA and H105R RhoA were
transglutaminated by both toxins.
View larger version (100K):
[in a new window]
Fig. 10.
Transglutamination of Rho mutants by DNT. The Rho mutants R68A, L72Q, K98Q, and
H105R (each 6 µg) were incubated for 15 min in the presence of
monodansylcadaverine and GST-DNT (2 µg). The samples were
separated on SDS-PAGE and the gel photographed under UV light
(A) before staining the gels with Coomassie Blue
(B). The experiment was repeated more than 3 times.
CNF1 and
GST-DNT in the transglutamination assay. In contrast to GST-CNF1,
both chimeras were not modified by GST-DNT (not shown). It has been
reported that unlike CNF1, DNT modifies RhoA only in the GDP-bound form
(27). To exclude the possibility that GTP binding caused inhibition of
modification by DNT, chimera Ras55R40 was loaded with GDP prior to
toxin treatment. However, the Rho chimeras were not modified by DNT
even after loading with GDP (not shown). The above findings suggested
that the substrate recognition by DNT is more stringent than for CNF1.
Because nucleotide binding causes major conformational changes in the
switch I region of Rho, we studied the effects of glucosylation of RhoA
at Thr37 by C. difficile toxin B on modification
by DNT. As shown in Fig. 11, prior
glucosylation of Rho by toxin B inhibited the transglutamination by DNT
but not by CNF1. By contrast, ADP-ribosylation of Rho at Asn41 by exoenzyme C3, a modification that occurs
downstream of the switch I region did not affect transglutamination by
CNF1 or DNT.
View larger version (74K):
[in a new window]
Fig. 11.
Transglutamination of glucosylated and
ADP-ribosylated RhoA by CNF1 and DNT. RhoA was incubated in a
glucosylation buffer in the presence (1 and 2) or
absence (3 and 4) of C. difficile
toxin B (6 ng/µl) and subsequently transglutaminated by GST- CNF1
(1 µM, 1 and 3) or GST-DNT (2 µM, 2 and 4). Similarly, Rho A was
incubated in a ADP-ribosylation buffer in the presence
(5 and 6) or absence (7 and
8) of C3 toxin (1 ng/µl) prior to CNF1 (5 and
7) or DNT (6 and 8)
transglutamination. The samples were subjected to SDS-PAGE and the gel
was photographed under UV light (A). The Coomassie staining
is shown in B. Repetition of the experiment gave
similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CNF1 (amino acid residues 709-1014 of CNF1) deamidated and
transglutaminated all chimeras consisting of Ras harboring the switch
II region (Ala61 through Asp78) of Rho.
CNF1 (amino acids 709 through 1014) is able to enter cells, caused a
typical Ras-like response, e.g. neurite outgrowth. Addition
of CNF1 to buffer-injected cells did not lead to this phenotype (not
shown). These results are explained by interaction of the Ras55Rho
chimera with Ras effectors mediated by the intact switch I region of
Ras and the deamidation of the chimera at Gln63 (Rho
nomenclature) thereby preventing the inactivation of the chimeric
GTPase. These findings are in agreement with the recent report by Hall
and co-workers (24) that a constitutively active Ras/Rho chimera,
containing the switch I region of Ras caused the typical Ras effects
such as cell transformation (24). Moreover the observed neurite
outgrowth is clearly a Ras phenotype, because in contrast to
microinjection of dominant active Ras (G12V) the injection of dominant
active RhoA (G14V) does not induce neurite outgrowth in PC12 cells (not shown).
DNT and CNF1. As with CNF1,
changes of Arg68 and Leu72 to the equivalent
amino acids of Ras inhibited modification by DNT. However, in
contrast to CNF1 (comparison between GST-DNT and GST-CNF1),
DNT did not accept any of the Rho/Ras chimeras as substrates for
deamidation or transglutamination. Accordingly, the peptide
representing the switch II region of RhoA (Asp59 through Asp78) was not
modified by DNT. Because neither the Ras55Rho nor the Rho115Ras
chimera were modified by DNT, it can be concluded that structural
determinants located both N- and C-terminal to the switch II region are
essential for substrate recognition by DNT. Thus, it is obvious that
the structural requirements for substrate recognition by DNT are more
stringent than for CNF1. In line with this notion are recent reports
that modification of Rho by DNT, but not by CNF1, depends on the
nucleotide bound to Rho (27). Using the active fragment DNT, we
confirmed that GDP, but not the GTP
S bound form of Rho is a
substrate for toxin-catalyzed deamidation and transglutamination. These
findings suggest that in addition to the switch II region, the switch I
region, which undergoes major changes upon nucleotide binding, is
involved in substrate recognition by DNT. A role of the switch I region
in substrate recognition by DNT is also supported by the finding that
prior glucosylation of Rho by C. difficile toxin B impaired DNT-induced deamidation or transglutamination. Toxin B glucosylates Rho
at Thr37 which is located in the switch I region (13).
ADP-ribosylation of Rho by C3 at Asn41, which is located
outside the switch I region, did not alter DNT effects on Rho. By
contrast, as substrate recognition by CNF1 depends almost exclusively
on the sequence of the switch II region, it seems logical that
nucleotide binding of Rho and glucosylation or ADP-ribosylation by
toxins do not alter modification of the GTPase by CNF1.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Institut für
Pharmakologie und Toxikologie, der Albert-Ludwigs-Universität
Freiburg, Herman-Herder-Str. 5, D-79104 Freiburg, Germany. Tel.:
49-761-2035301; Fax: 49-761-2035311; E-mail:
aktories@uni-freiburg.de.
ABBREVIATIONS
CNF1, the active fragment
of CNF1 consisting of amino acid residues 709 through 1014;
DNT, Bordetella dermonecrotic toxin;
DNT, the active fragment
of DNT consisting of amino acid residues 1136 through 1451;
GST, glutathione S-transferase;
PC12, pheochromocytoma cells;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
MALDI-TOF-MS, matrix-assisted laser
desorption-ionization time of flight mass spectrometry;
GTPS, guanosine 5'-3-O-(thio)triphosphate.
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