| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, August 2, 1995; and in revised form, January 16, 1996) From the
Lethal toxin (LT) from Clostridium sordellii is one of
the high molecular mass clostridial cytotoxins. On cultured cells, it
causes a rounding of cell bodies and a disruption of actin stress
fibers. We demonstrate that LT is a glucosyltransferase that uses
UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in
vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In
Ras, threonine at position 35 was identified as the target amino acid
glucosylated by LT. Other related members of the Ras GTPase
superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT.
Incubation of serum-starved Swiss 3T3 cells with LT prevents the
epidermal growth factor-induced phosphorylation of mitogen-activated
protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras
function in vivo. We also demonstrate that LT acts inside the
cell and that the glucosylation reaction is required to observe its
dramatic effect on cell morphology. LT is thus a powerful tool to
inhibit Ras function in vivo.
Several different species of the genus Clostridium produce large molecular mass ( C. sordellii produces two toxins, LT
and hemorrhagic toxin, two major virulence factors inducing gas
gangrene and hemorrhagic diarrhea in humans and animals(6) .
These C. sordellii toxins have some similarities to toxins A
and B from C. difficile in terms of amino acid sequences and
immunological epitopes(7) . Despite these similarities, it
seems that LT and toxins A and B affect different intracellular target
proteins. LT causes morphological and cytoskeletal effects different
from those elicited by the C. difficile toxins. The effects
consist of the rounding of cell bodies with the reorganization of
F-actin structures into numerous cell-surface filopodia and a loss of
actin stress fibers(8, 9) . In addition, we have
recently shown that overexpression of RhoA, RhoB, or RhoC cDNA in HeLa
cells protects these cells from the effects of toxins A and B, but not
from those of LT(9) . These observations clearly pointed out
that Rho small GTP-binding proteins were the main substrate for the C. difficile toxins and that the targets of LT were distinct. A mutant hamster fibroblast cell line has been described that is
resistant to toxins A and B from C. difficile(10) .
This resistance was attributed to a low intracellular UDP-Glc content,
and the fact that this mutant cell line was not intoxicated by LT
indicated that LT is a glucosyltransferase(11) . In this
paper, we report that LT modifies and inactivates p21
Guanine nucleotide exchange and GTP hydrolysis of glucosylated versus unmodified Y64W Ras (0.5 µM) were measured
at 37 °C in 50 mM Hepes (pH 7.5), 1 mM MgCl
Figure 1:
Effects of LT on
actin and fimbrin/plastin cytoskeleton of HeLa cells. HeLa cells were
incubated for 3 h with 2.5 µg/ml LT and then processed for
immunofluorescence. Green, F-actin fluorescence (fluorescein
isothiocyanate-labeled phalloidin); red, fimbrin/plastin
fluorescence (Texas Red-labeled second antibodies); green and red, overlapping picture of the two fluorescences. Yellow spots indicate where the two fluorescences overlapped. A,
control cells; B, LT treated-cells. Bar = 5
nm.
Figure 2:
LT-induced glucosylation of 21-kDa
proteins in HeLa cell lysates. HeLa cell lysates were incubated with
UDP-[
Figure 3:
In vivo glucosylation of cellular
21-kDa proteins in rat fibroblasts by LT. Rat-1-EJ-Rap2.31.A8
fibroblasts were incubated with the concentrations of LT indicated
below for 120 min, detached from the culture dishes, lysed, and
glucosylated by LT with UDP-[
Figure 4:
Glucosylation of recombinant Ras-related
GTPases by LT. Ha-Ras, Rap2, Rac1, Cdc42, RhoA, Rab6, Ral-GST, and
Rac-GST (
Figure 5:
Localization of LT-catalyzed
Figure 6:
EGF-induced mobility shift of MAP kinases
in cells pretreated with LT. Serum-starved Swiss 3T3 cells were treated
with EGF and LT as shown. Cells were lysed, and
Figure 7:
Intracellular modulation of LT action. A and B, phase-contrast micrographs of Don-wt cells. A, typical cytopathogenic effect observed after treatment with
LT (1.25 µg/ml; 3 h) in the presence of preimmune serum (1:200
dilution). B, neutralization of the cytopathogenic effect by
adding rabbit anti-LT antibodies (1:200 dilution) to the medium
containing LT (1.25 µg/ml; 3 h). C-H, microinjection
experiments with Don-wt (C, D, G, and H) or Don-Q (E and F) cells. C, E, and G, fluorescence micrographs of D, F, and H (phase-contrast micrographs), respectively. Large arrowheads point to microinjected cells; small
arrowheads point to cells solely treated with substances added to
the medium. Fluorescein staining was due to fluorescein
isothiocyanate-labeled dextran added to the injected medium. C and D, typical rounding of Don-wt cells microinjected
with LT (concentration in the micropipette of 200 µg/ml) in medium
containing LT antibodies (1:200 dilution) to protect against any LT
molecules possibly leaking out from injected cells. E and F, typical rounding of UDP-Glc-deficient Don-Q cells exposed
to LT (1.25 µg/ml; 3 h) in the medium and then microinjected with
UDP-Glc (concentration in the micropipette of 100 mM). G and H, protection from rounding of Don-wt cells
microinjected with neutralizing anti-LT antibodies (serum diluted 1:10)
and exposed to LT (1.25 µg/ml; 3 h) in the medium. The toxin is
accessible to neutralizing anti-LT antibodies once it reaches the
cytosol.
To
demonstrate that the activity of LT is mediated through glucosylation
(of G-proteins), we took advantage of a mutant Don cell (Don-Q). This
cell has a low content of UDP-Glc, which renders it resistant to the
glucosylating toxins A and B from C. difficile and also to
LT(11) . Don-Q cells were incubated with LT, followed by
microinjection of UDP-Glc into some of them (those lighting up under
fluorescence microscopy). As shown in Fig. 7(E and F), only cells that were microinjected with UDP-Glc exhibited
the characteristic cytopathogenic effect of the toxin, suggesting that
the toxin and the cofactor act at the same side of the cell membrane.
The specificity of the effect was confirmed by microinjecting, instead
of UDP-Glc, UDP-Gal or UDP-GlcUA (100 mM) into cells similarly
treated with LT. Neither of the additionally used activated sugars
promoted any cytopathogenic effect. Finally, none of the three
UDP-sugars used in this study had any effect if the cells were not
pretreated with toxin (data not shown). Knowing that our rabbit anti-LT
serum neutralized the toxin, we microinjected Don-wt cells with this
serum and then incubated them with LT added to the medium. As shown in Fig. 7(G and H), microinjection of anti-LT
antibodies protected against LT, clearly indicating that the
neutralizing antibody and the toxin meet each other in the cytosol.
Accordingly, cells not injected exhibited the cytopathogenic effect
typical of LT (Fig. 7, G and H), as did cells
microinjected with nonimmune rabbit serum (data not shown). The
experiments shown in Fig. 7, together with those presented in Fig. 3, strongly suggest that LT acts from the cytosol by
glucosylating small GTP-binding proteins using UDP-Glc as a cofactor.
Figure 8:
Effect of LT-catalyzed glucosylation on
nucleotide dissociation, GTPase activity, and intrinsic fluorescence of
Y64W Ras. A, shown is GDP and GTP dissociation at low
magnesium concentration. Glucosylated or unmodified Y64W Ras-GDP (0.5
µM) was activated, in the presence of 0.8 µM free magnesium (1 mM MgCl
The effect of glucosylation on GTP hydrolysis by Ras is
shown in Fig. 8B. Y64W Ras-GDP was incubated with GTP
at 1 mM magnesium. Activation was triggered by the addition of
2 mM EDTA, which reduced the free magnesium concentration to
<1 µM. The first instantaneous fluorescence decrease
reflected the dissociation of magnesium from Y64W Ras-GDP, whereas the
slower fluorescence decrease reflected (as in Fig. 8A)
the exchange of GTP for GDP. After completion of GDP/GTP exchange,
magnesium was added back to the reaction (1 mM free
magnesium). Due to the intrinsic GTPase activity of the protein, the
fluorescence of the unmodified form of Ras slowly increased toward the
level of fluorescence initially observed for Ras-GDP (Fig. 8B). In the case of the glucosylated form of Ras,
much slower kinetics of GTPase activity was observed. Indeed, upon
glucosylation of threonine 35, Ras had a four times slower intrinsic
GTPase activity (Fig. 8B). Glucosylation of Y64W Ras
by LT slightly modified the fluorescence of the protein. As compared
with unmodified Y64W Ras, LT-glucosylated Y64W Ras exhibited, on one
hand, a larger absolute fluorescence level and, on the other hand, a
smaller fluorescence change upon GDP/GTP exchange or GTP hydrolysis (Fig. 8, A and B). Therefore, we looked for a
fluorescence signal that could correlate with the glucosylation of the
protein. When Y64W Ras-GDP was incubated with LT and UDP-Glc,
fluorescence was enhanced by 2% within 2 h (Fig. 8C).
Since this signal required both LT and UDP-Glc, it certainly reflects
the time course of UDP-Glc incorporation. Toxins A and B from C. difficile have been shown to
covalently modify and thereby inactivate the small GTP-binding protein
Rho, resulting in the disruption of F-actin
structures(20, 21) . In vitro and in vivo evidence indicates that toxins A and B modify RhoA by
UDP-Glc-dependent glucosylation of threonine
37(3, 4) . In addition to RhoA, toxins A and B from C. difficile also modify in vitro Rac1 and
Cdc42(3, 4) , two other proteins of the Rho subfamily
involved in the control of membrane ruffling and filopodia formation,
respectively (22, 23, 24) . Also, it has
recently been reported that the The effects induced by LT on the HeLa cell
actin cytoskeleton are obviously different from those elicited by
toxins A and B from C. difficile. The LT effects consist of
the disruption of actin stress fibers and the formation of filopodia
containing F-actin and fimbrin/plastin(9) . Glycosyltransferase
activity of both C. difficile and C. novyi toxins is
directed toward GTP-binding proteins of the Rho subfamily. With C.
sordellii LT, we have the first toxin that mainly acts on the Ras
subfamily of GTPases. The specific effect of LT on the HeLa cell actin
cytoskeleton is fundamentally different from that observed with toxin A
or B from C. difficile(9) . Since both toxin B (or A)
and LT are able to glucosylate Rac(3, 4) , the
specific activity of LT on the cytoskeleton cannot be attributed to Rac
modification alone. Instead, we believe that the combination of the
modified GTPases causes LT to induce its cytopathogenic effect. We
would like to stress that since the physiological function of Rap is
still unknown, Rap modification by LT could be a key event in LT-
induced cytoskeletal disruption. In Swiss 3T3 cells, stimulation of
Ras activates membrane ruffling and actin stress fiber organization by
Rac- and Rho-dependent mechanisms(22) . Since LT inactivates
both Ras and Rac, this may in turn inhibit Rho, resulting in the
collapse of actin stress fibers. It is interesting to note that
filopodia induced by LT markedly resemble those generated by
microinjection of the activated form of Cdc42 into Swiss 3T3
cells(23, 24) . It is tempting to speculate that LT is
responsible for the formation of filopodia by indirectly activating
Cdc42, which in turn is a consequence of a toxin-induced Ras
inactivation. An alternative hypothesis to explain the formation of
filopodia due to LT could be that Cdc42 is already in an active state,
but formation of filopodia is masked by activation of Ras, leading to a
dominant phenotype of membrane ruffling and actin stress fibers.
Inactivation of Ras and Rac would therefore allow observation of the
Cdc42 phenotype. LT inactivates Ras by glucosylation of threonine
35, which corresponds to threonine 37 of Rho(25) , the residue
modified by toxins A and B(3, 4) . In addition, our
data strongly suggest that LT acts in the cytosol and glucosylates
small 21-kDa molecules in vivo, resulting in the inactivation
of Ras, since serum-starved Swiss 3T3 cells intoxicated with LT have no
Ras-dependent induced MAP kinase phosphorylation (see Fig. 6). At the present time, we do not understand the nature of the
substrate specificity of LT for Ras, Rap, and Rac. It seems reasonable
that amino acid sequences apart from the threonine 35 acceptor site of
glucosylation enable LT to specifically recognize the various small
G-proteins. LT glucosylation of Ras at threonine 35 induced a small
but significant decrease in the K It is remarkable that four out of
five members of the group of these large clostridial cytotoxins (toxins
A and B, LT, and Taking into account that LT is the first toxin that
inactivates the Ras small GTP-binding protein, it should soon become a
powerful laboratory reagent to explore cellular signaling pathways
stimulated by this molecule.
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10217-10224
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
250-300 kDa) cytotoxins
that cause effects on the actin cytoskeleton, including disruption of
actin stress fibers and rounding of cell bodies. This subgroup of
clostridial cytotoxins includes toxins A and B from Clostridium
difficile, lethal toxin (LT) (
)and hemorrhagic toxin
from Clostridium sordellii, and Clostridium novyi 
-toxin(1) . Recently, toxins A and B from C.
difficile, the causative agent of antibiotic-associated
diarrhea(2) , were shown to covalently modify the mammalian
protein Rho by UDP-Glc-dependent glucosylation of threonine
37(3, 4) . Rho is a small Ras-related GTP-binding
protein involved in the control of actin polymerization(5) .
Glucosylation of threonine 37 of Rho by C. difficile toxin A
or B apparently inactivates this protein and results in a loss of actin
stress fiber assembly.
by glucosylation of threonine 35. In addition, LT was also
found to glucosylate Rap and Rac proteins. No activity was found on
other Ras-related proteins including Ral, Rho, Cdc42, Arf1, and Rab.
Materials
C. sordellii LT was obtained from culture
supernatants of the pathogenic C. sordellii IP82 strain and
purified to homogeneity as described previously(8) .
Recombinant Ha-Ras, RhoA, Rac1, Rap2, Ral, Rab6, and Cdc42 proteins
were made either in baculovirus (Rac1, Rab6, and Arf1) or in Escherichia coli through either GST fusions (Ha-Ras, Rap1,
Ral, RhoA, and Rac1) or a histidine-tagged fusion (His-Cdc42). The
Ral-GST fusion could not be processed by thrombin to yield a 21-kDa
protein due to the fact that Ral itself was proteolyzed. Ral was
therefore tested for LT glucosylation as a 47-kDa fusion protein. The
Rho protein, used in this study, could be fully ADP-ribosylated by
exoenzyme C3 or glucosylated by C. difficile toxin B. Rac1 and
Cdc42, used in this work, could be fully glucosylated by C.
difficile toxin B. Rabbit polyclonal antibodies against fimbrin
were a gift of Monique Arpin (Unité de Biologie
des Membranes, Institut Pasteur, Paris). The monoclonal antibody MK12
(Zymed Laboratories, Inc., S. San Francisco, CA) was used for
immunoblotting MAP kinases. The recombinant Y64W Ras mutant used for
tryptophan fluorescence experiments was produced in E. coli and purified as described previously(12) .Methods
Glucosylation Reactions
Incorporation of
LT-catalyzed [
C]Glc in the GTPases or cell
lysates was performed as described by Just et al.(3) in the case of C. difficile toxin B. Briefly,
10 µl of UDP-[C]Glc in ethanol (0.2 µCi,
300 mCi/mmol; DuPont NEN, Les Ulis, France) was dried down under
vacuum. Recombinant proteins (2 µg) dissolved in 15 µl of 50
mM triethanolamine HCl buffer (pH 7.5) containing 2 mM MgCl
, 150 mM KCl, 100 µM dithiothreitol, and 2 µM GDP were added to the dried
UDP-[C]Glc. LT (2 µg/ml) was then added to
start the reaction, which was carried out for 1 h at 37 °C. The
reaction was stopped by adding 5 µl of 2 
SDS sample buffer,
boiled, and electrophoresed on a 15% SDS-polyacrylamide gel. Upon
staining with Coomassie Blue followed by destaining, the gel was dried,
and radioactivity was recorded and counted using a PhosphorImager
system (Molecular Dynamics, Inc., Sunnyvale, CA). Glucosylation of HeLa
cell lysates by LT was performed as follows. HeLa cells (5
10
) were homogenized by three cycles of freeze-thawing in
200 µl of 50 mM triethanolamine HCl buffer (pH 7.5)
containing 100 µM dithiothreitol, 1 µg/ml leupeptin,
and 1 µg/ml pepstatin (glucosylation buffer). Cell lysates (20
µl) were added to 10 µl of dried
UDP-[C]Glc, and 2 µg/ml LT was added to
start the enzymatic reaction. After 1 h at 37 °C, further
processing and imaging were done as described above.In Vivo Glucosylation by LT of Small GTP-binding Proteins
in Rat Fibroblasts
Rat-1 fibroblasts (Rat-1-EJ-Rap2.31.A8)
stably transfected with G12V Ras and Rap2 (13) were grown in
60-mm Petri dishes to a subconfluent density. LT was added to the cells
at the indicated concentration in 5 ml of fresh medium containing 10%
fetal calf serum. After 2 h, the cells were removed from the dishes
with a rubber policeman and washed in 10 ml of PBS followed by
centrifugation at low speed. Washing was repeated five times to remove
residual LT, and finally, the cell pellets were resuspended in 50
µl of glucosylation buffer. Cells were then lysed by four cycles of
freeze-thawing. After homogenization, the amount of protein in each
cell lysate was estimated. For in vitro glucosylation of small
GTP-binding proteins with LT, 40 µl of cell lysate was added to 15
µl of dried UDP-[C]Glc with 5 µg/ml LT.
This mixture was incubated for 1 h at 37 °C. Then, 5 µl of each
reaction was added to 10 µl of sample buffer, boiled, and
electrophoresed on a 15% SDS-polyacrylamide gel. The gel was stained,
destained, dried, and analyzed for radioactivity by autoradiography.Localization of the Glucosylated Amino Acid in
Ha-Ras
This experiment was performed by microsequencing the
radioactively labeled protein. The Ha-Ras protein (10 µg) was first
radioactively glucosylated by LT with 40 µl of dried
UDP-[C]Glc (0.8 µCi) for 1 h (under reaction
conditions as described above). Then, 10 mM unlabeled UDP-Glc
was added; the reaction was further incubated for an additional 1 h at
37 °C; and the proteins were separated on a 12.5%
SDS-polyacrylamide gel. After migration and staining with Amido Black,
the band observed at 21 kDa was cut out from the gel and digested with
1 µg of trypsin in 200 µl of 100 mM Tris-HCl buffer
(pH 8.8) containing 0.01% Tween 20. The reaction was incubated for 18 h
at 35 °C. The resulting peptides were separated by HPLC using a
hydrophobic C
column with an acetonitrile/trifluoroacetic
gradient. Fractions eluted from the column were each analyzed for
radioactivity. Radioactive peptides were repurified by HPLC using the
C column with a sodium acetate buffer (pH 6.00) gradient.
The eluted peptides were analyzed for radioactivity. The radioactive
peaks were sequenced with an Applied Biosystems microsequencer, and the
product of each Edman degradation cycle was collected and counted for
radioactivity.Immunofluorescence Experiments
Cells grown on
coverslips were treated with LT and then fixed with 3% paraformaldehyde
for 20 min. After fixation, monolayers were washed three times with
PBS, and free aldehyde groups were quenched by incubation with 50
mM NH
Cl in PBS for 10 min. Cells were
permeabilized for 5 min at room temperature in PBS containing 0.2%
Triton X-100 and then incubated for 30 min at room temperature with the
first antibody. Coverslips were then washed extensively with PBS and
incubated with the secondary antibodies (Texas Red-conjugated sheep
anti-mouse, Amersham International, Buckinghamshire, United Kingdom)
together with fluorescein isothiocyanate-labeled phalloidin (Sigma,
L'Isle-d'Abeau, France) for 30 min. After three washes in
PBS, coverslips were mounted in Moviol (Calbiochem-Novabiochem GmbH,
Bad Soden, Germany), and fluorescence was observed with a confocal
microscope.MAP Kinase Activation
Experiments examining the
effects of LT on epidermal growth factor (EGF)-stimulated MAP kinase
phosphorylation were performed as follows. Swiss 3T3 cells were
cultured according to routine procedures in H21 medium supplemented
with 10% fetal calf serum. When the cells reached confluency, they were
serum-starved overnight in 0.1% fetal calf serum. After 3 h of
incubation with LT (1.7 µg/ml) in serum-free medium (the activity
of LT was monitored by the cytopathogenic effect on cells), EGF was
added (or not) at a 50 ng/ml final concentration for 5 min. Cells were
then scraped into polyacrylamide gel electrophoresis sample buffer, and
30 µg of total protein, for each experiment, was electrophoresed on
a 12.5% SDS-polyacrylamide gel. The gel was blotted onto nitrocellulose
and incubated with a monoclonal antibody directed against MAP kinases
(anti-ERK1 and anti-ERK2). Immune complexes were detected by
horseradish peroxidase-conjugated secondary antibody followed by the
ECL kit (Amersham International).Fluorescence Measurements
LT-catalyzed
glucosylation of Y64W Ras-GDP was performed in 50 mM triethanolamine HCl buffer (pH 7.5) containing 140 mM KCl, 1 mM MgCl, and 0.1 µM dithiothreitol. Y64W Ras-GDP (50 µM) was incubated
with 100 µM UDP-Glc and 2.5 µg/ml LT at 37 °C for
2 h. Control experiments were performed in the absence of LT. , and 1 mM dithiothreitol by monitoring
tryptophan fluorescence at 340 nm upon excitation at 292 or 300
nm(12) . When needed, 2 mM EDTA was added to reduce
free magnesium to 0.8 µM.Cell Microinjections
Diploid Chinese hamster lung
fibroblasts (Don cells; ATCC CCL16, Don-wt (where ``wt''
indicates wild-type)) and the C. difficile toxin A- and
B-resistant mutant of this cell line,
Cdt
-Q(10, 11) , referred to here as Don-Q
(a UDP-Glc-deficient mutant of these cells), were grown on 13-mm slides
for 48 h. Semiconfluent wild-type and mutant cells were microinjected
(Eppendorf microinjector) with the indicated concentrations of LT,
UDP-Glc, or anti-LT antibodies with fluorescein isothiocyanate-labeled
dextran (Sigma) in calcium-free PBS. Approximately 100 cells were
microinjected in each experiment. The cultures were further incubated
for 30 min at 37 °C and fixed with 3.7% paraformaldehyde for 10
min. Cells were visualized by phase-contrast and fluorescence
microscopy.
Disruption of Actin Stress Fibers and Formation of
Filopodia Induced in HeLa Cells by LT
The cytopathic effect of C. sordellii LT consists of the rounding of cell bodies and
profound alteration of F-actin-containing
structures(8, 9) . After a 3-h incubation with 2
µg/ml LT, HeLa cells became round, displayed F-actin structures
rearranged into cell-surface filopodia, and exhibited a loss of actin
stress fibers (Fig. 1). Using a polyclonal rabbit antibody that
reacts against all known isoforms of the actin-bundling protein
fimbrin/plastin(14) , we observed that fimbrin/plastin was
present in LT-induced filopodia (Fig. 1).
LT Catalyzes the UDP-Glc-dependent Glucosylation of
21-23-kDa Proteins in HeLa Cell Lysates
Incubation of HeLa
cell lysates with LT in the presence of
UDP-[C]Glc followed by gel electrophoresis of
the reaction products showed that the toxin induced labeling of
proteins in the range of 21-23 kDa (Fig. 2). This reaction
could be displaced by adding an excess of nonradioactive UDP-Glc, but
not UDP-glucuronic acid (Fig. 2). No modification of proteins by
LT was found with [C]Glc alone (data not shown).
C]Glc in the absence (lane A) or
presence (lanes B-D) of LT. Specificity of UDP-Glc
labeling by LT was tested by incubating HeLa lysates with a 10-fold
excess of unlabeled UDP-Glc (lane C) or UDP-glucuronic acid (lane D) together with
UDP-[C]Glc.
LT Glucosylates 21-kDa Proteins in Vivo
To
demonstrate that small GTP-binding proteins were glucosylated by LT in vivo, Rat-1-EJ-Rap2.31.A8 fibroblasts were incubated with
increasing amounts of LT (from 0.005 to 5 µg/ml). The highest
concentration of toxin caused the characteristic cytopathogenic effect
of LT in 100% of the cells within 1 h. All cells were then lysed, and
the lysates were glucosylated with LT a second time, now in vitro in the presence of radioactive UDP-Glc. If LT acts from inside the
cell, there should be an inverse correlation between the LT dose used
for in vivo pretreatment of cells and the amount of
[C]Glc incorporated into small G-proteins in
vitro. As shown in Fig. 3, the highest rate of
glucosylation by LT of a 23-kDa protein was observed in control cells.
Two minor bands of 21 and 25 kDa glucosylated by LT were also noticed
in control lysates (Fig. 3). Fig. 3also demonstrates
that a clear decrease to a total absence of labeling of these bands was
observed when the cells had been preincubated in vivo with
increasing concentrations of LT prior to the in vitro LT
glucosylation. Assuming that LT reacts with small G-proteins, in
accordance with its homology to C. difficile toxin
B(15) , this dose-dependent activity of LT suggests that the
toxin exerts its action from within the cell.
C]Glc as described
under ``Experimental Procedures.'' Lane A, labeling
of 21-kDa proteins in cells not incubated with LT in vivo prior to the in vitro radioactive LT glucosylation of the
cell lysate; lanes B-E, labeling of 21-kDa proteins in
cells incubated first in vivo with 5, 0.5, 0.05, and 0.005
µg/ml LT, respectively, prior to the in vitro radioactive
LT glucosylation of the cell lysates.
LT Glucosylates Ras, Rap, and Rac Small GTP-binding
Proteins in Vitro
Specificity of LT was studied by incubating
UDP-[C]Glc and LT with different members of the
p21
superfamily of small GTP-binding proteins. As shown
in Fig. 4, Ha-Ras, Rap2, and Rac1 were substrates for
LT-catalyzed glucosylation. In contrast, RhoA, Cdc42, and Rab6 were not
modified in vitro by LT. Since Ral was only available as a GST
fusion protein, we tested a possible influence of the fusion with GST
by adding a Rac-GST construct to the series. As evidenced by Fig. 4, Rac1-GST was a substrate for LT glucosylation, whereas
Ral-GST was not modified by the toxin. This suggests that Ral is not
modified by LT. Finally, no incorporation of glucose catalyzed by LT
could be found on Arf1 (data not shown).
2 µg/assay) were incubated with LT and
UDP-[C]Glc. A, PhosphorImager picture; B, Coomassie Blue staining of the
gel.
LT Glucosylates Threonine 35 of Ha-Ras
To identify
the acceptor amino acid glucosylated by LT, Ha-Ras protein was modified
by LT in the presence of UDP-[C]Glc,
electrophoresed on SDS-polyacrylamide gel, and digested with trypsin,
and the resulting peptides were separated as described under
``Experimental Procedures.'' As shown in Fig. 5A, 47 fractions were obtained. The radioactivity
was exclusively associated with fractions 39 and 40 (Fig. 5A). As shown in Fig. 5(B and C), repurification of fraction 39 or 40 gave rise to a major
peptide (peptide D for fraction 39 and peptide E for fraction 40)
containing the radioactivity and several other small peptides. Peptides
D and E were microsequenced and gave exactly the same amino acid
sequence. Each cycle of Edman degradation was collected and counted for
radioactivity. We found the following unambiguous sequence for these
peptides: SALTIQLIQNHFVDEYDPTIEDSYR. Cycle 19 corresponding to a
threonine gave a very small signal. The small amount of threonine
detected in position 19 may be the consequence of the LT-catalyzed
glucosylation of most of the Ras molecules present in the reaction. A
decrease in or absence of threonine 37 of RhoA in automated amino acid
sequencing, after glucosylation by toxin A or B, has been already
reported(3, 4) . The amino acid sequence found for
both peptides D and E corresponds exactly to a sequence found in the
Ha-Ras protein between amino acids 17 and 41(16) .
Radioactivity was associated first with cycle 19 and decreased
thereafter (Fig. 5E). The rise in radioactivity at
cycle 19 establishes threonine 35 (of the Ha-Ras molecule) as the
unique amino acid glucosylated by LT.
C-glucosylated Ha-Ras by microsequencing. A,
separation by HPLC of the peptides generated by trypsin and
radioactivity of each fraction (on a 15-µl aliquot). B and C, purification by HPLC of fractions 39 and 40. Radioactivity
associated with each peptide was counted on 50-µl aliquots. D, radioactivity associated with each Edman degradation cycle
(each Edman cycle of peptides D and E was combined and
counted).
Inhibition of EGF-induced Phosphorylation of MAP Kinases
in Swiss 3T3 Cells by LT
In serum-starved Swiss 3T3 cells, the
mitogenic signaling pathway involving tyrosine phosphorylation of
growth factor receptors such as the EGF receptor and the subsequent
Ras-dependent activation of MAP kinase phosphorylation is reduced to a
basal level(17) . After incubation with EGF, Ras-dependent
activation of MAP kinases ERK1 and ERK2 can be followed by a shift in
electrophoretic mobility resulting from phosphorylation(18) .
If the toxin blocks Ras activity, serum-starved Swiss 3T3 cells
incubated with LT before the addition of EGF should not activate MAP
kinases. As shown in Fig. 6, serum-starved Swiss 3T3 cells
incubated with EGF had MAP kinases shifted toward higher molecular mass
compared with MAP kinases of cells not incubated with EGF. In contrast,
when serum-starved Swiss 3T3 cells were incubated with LT, prior to
incubation with EGF, the growth factor was not able to induce a shift
in electrophoretic mobility of the MAP kinases (Fig. 6).
30 µg of total
protein/experiment was electrophoresed, blotted, and stained with the
monoclonal antibody MK12 (ERK1, 44 kDa (p44); ERK2, 42 kDa (p42)).
LT Acts in the Cytosol by Glucosylation
To further
substantiate the notion that LT reaches the cytosol and acts by
glucosylation of small GTP-binding proteins, a series of microinjection
experiments was performed. Don-wt cells were incubated with LT in
medium containing nonimmune rabbit serum. The expected characteristic
cytopathogenic effect was observed in the whole cell population (Fig. 7A). When rabbit anti-LT antibodies were added to
the medium, the same amount of LT as used in Fig. 7A did not affect the cells (Fig. 7B). Drugs blocking
the endocytic pathway acidification (bafilomycin A,
chloroquine, or monensin), known to prevent many bacterial toxins from
penetration into the cytosol(19) , blocked the activity of LT
on cells (data not shown). When Don-wt cells in medium containing
anti-LT antibodies were microinjected with LT, they rapidly exhibited
the cytopathogenic effect characteristic of LT (Fig. 7, C and D). Successful microinjection was monitored by a
yellow-green fluorescence of fluorescein-labeled dextran added to the
solutions microinjected (see ``Experimental Procedures'').
This showed that LT can exert its activity from the cytosol.
LT Glucosylation of Ras Enhances the GTP Dissociation
Rate and Reduces GTP Hydrolysis of the GTP-binding Protein
The
effects of LT glucosylation on the intrinsic properties of Ras was
studied using the Y64W Ras mutant. This mutant has the same intrinsic
biochemical properties as wild-type Ras, but its
activation-deactivation cycle can be followed in real time by
monitoring changes in the fluorescence of tryptophan 64(12) .
In Fig. 8A, Y64W Ras-GDP, glucosylated or not, was
first activated by the addition of GTP. After several minutes, the
protein was converted again to the GDP-bound form by addition of a
large excess of GDP. This experiment was performed at a low magnesium
concentration in order to favor the dissociation of the bound
nucleotide (the rate-limiting step of nucleotide exchange) and to
prevent GTP hydrolysis. Similar fluorescence changes were observed for
the nonglucosylated and glucosylated forms of Ras (Fig. 8A). Indeed, binding of GTP in place of GDP
induced a decrease in fluorescence, and conversely, binding of GDP in
place of GTP induced an increase in fluorescence. Upon GTP addition,
the time course of the fluorescence decrease was similar for the two
forms of Ras, indicating that glucosylation did not greatly modify the
GDP dissociation rate. In contrast, the increase in fluorescence by GDP
addition was four times faster for glucosylated Ras than for unmodified
Ras (Fig. 8A). This result demonstrates that
glucosylation weakened GTP binding in the nucleotide site of Ras by
accelerating its dissociation rate. Similar effects of glucosylation
were observed for the dissociation rate of GTP
S either at low (1
µM) or high (1 mM) magnesium concentration (data
not shown).
and 2 mM EDTA), by the addition of 10 µM GTP (first
arrow). Deactivation was achieved by the addition of 500
µM GDP while its intrinsic fluorescence at 340 nm was
continuously monitored. GDP dissociation rate constants were as
follows: glucosylated, 0.0125 s
; and control, 0.017
s. GTP dissociation rate constants were as follows:
glucosylated, 0.0125 s; and control, 0.0033
s. B, shown is GTP hydrolysis at 1 mM magnesium. Glucosylated or unmodified Y64W Ras-GDP (0.5
µM) was incubated with 10 µM GTP in the
presence of 1 mM magnesium. GDP/GTP exchange was initiated by
the addition of 2 mM EDTA. After 6 min, GTP hydrolysis was
initiated by the addition of 2 mM MgCl (1 mM free magnesium). Note the change in time scale after magnesium
addition. C, glucosylation of Y64W Ras-GDP induces a small
increase in the intrinsic fluorescence of the protein. The fluorescence
of Y64W Ras-GDP was continuously monitored while 0.8 µg/ml LT and
100 µM UDP-Glc were sequentially added to the fluorescence
cuvette. In A and C, the samples were excited at 300
nm to minimize light absorption due to the large amount of nucleotides
used in these experiments. In B, the excitation was set at 292
nm.
-toxin from C. novyi is a
glycosyltransferase that acts on the cytoskeleton through modification
of Rho. However, in this case, UDP-Glc was not the cofactor required
for modification. ()We report here that LT, like toxins A
and B from C. difficile, is also a glucosyltransferase that
uses UDP-Glc to modify small GTP-binding proteins. However, the
substrate specificity of LT is different from that of toxins A and B.
LT glucosylates Ras, Rap2, and Rac1 in vitro. LT had no effect
on Rho or on Cdc42, two of the main substrates for C. difficile toxins A and B.
of GDP, most
likely due to a higher affinity of glucosylated Ras for magnesium. Such
a difference in magnesium affinity has not been observed for the T35A
mutant of Ras (26) . Apart from this small difference, the
Thr-35 glucosylated form of Ras in the GTP-bound form has properties
very similar to those of the T35A mutant: a 4-fold increase in the GTP K and a four to five times slower rate of GTP
hydrolysis(26) . It is thus extremely likely that the Thr-35
glucosylation of Ras, as the T35A mutant of Ras, has a much decreased
affinity for the Raf Ras-binding domain(27) . The T35A mutant
of Ras has a 200-fold reduced affinity for the Raf Ras-binding domain (27) and represents the mutation that has the most drastic
effect on the Ras/Ras-binding domain interaction(27) .
Threonine 35 contacts both magnesium and -phosphate in the
GTP-bound form and a water molecule that also makes a hydrogen bound
with aspartic acid 38 in the Rap
Raf Ras-binding domain
complex(28) . Threonine 35 is conserved in all of the small
G-proteins and is an essential residue of the switch I
region(29) . Thus, the modification of threonine 35 either by
mutation (T35A) or by glucosylation would result in the inability of
Ras to interact with its effector(27) . Even the conservative
T35S mutation greatly decreases (20-fold) the transforming
potential of an oncogenic Ras, pointing to the importance of this
residue in switching to the active conformation and/or interacting with
the Raf effector(30) .-toxin) have glycosyltransferase activities on
small GTP-binding proteins. Recently, we have found that C.
sordellii hemorrhagic toxin, the fifth member of this large
clostridial cytotoxin group, is also a glucosyltransferase. (
)
)S, guanosine
5`-3-O-(thio)triphosphate.))
We thank Jacques d'Alayer (Institut Pasteur),
who performed microsequencing of the LT-modified form of Ha-Ras; Keith
Ireton (Institut Pasteur) for stimulating discussions; Bruno Goud
(Institut Curie, Paris) for the gift of Rab6; Pierre Vignais and
Alexandra Fuchs (Centre Biologie Moleculaire et Structurale, Grenoble,
France) for the gift of Rac1; and Martina Schmitd (Institut
für Pharmakologie, Universität
GH Essen, Essen, Germany) for the gift of Arf1. We specially thank M.
Weidmann for critically reading the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. S. Tait, M. Dalton, B. Geny, F. D'Agnillo, M. R. Popoff, and E. M. Sternberg The Large Clostridial Toxins from Clostridium sordellii and C. difficile Repress Glucocorticoid Receptor Activity Infect. Immun., August 1, 2007; 75(8): 3935 - 3940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Jank, T. Giesemann, and K. Aktories Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function Glycobiology, April 1, 2007; 17(4): 15R - 22R. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Geny, H. Khun, C. Fitting, L. Zarantonelli, C. Mazuet, N. Cayet, M. Szatanik, M.-C. Prevost, J.-M. Cavaillon, M. Huerre, et al. Clostridium sordellii Lethal Toxin Kills Mice by Inducing a Major Increase in Lung Vascular Permeability Am. J. Pathol., March 1, 2007; 170(3): 1003 - 1017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. I. Iliev, J. R. Djannatian, R. Nau, T. J. Mitchell, and F. S. Wouters Cholesterol-dependent actin remodeling via RhoA and Rac1 activation by the Streptococcus pneumoniae toxin pneumolysin PNAS, February 20, 2007; 104(8): 2897 - 2902. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Jank, U. Pack, T. Giesemann, G. Schmidt, and K. Aktories Exchange of a Single Amino Acid Switches the Substrate Properties of RhoA and RhoD toward Glucosylating and Transglutaminating Toxins J. Biol. Chem., July 14, 2006; 281(28): 19527 - 19535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Jank, D. J. Reinert, T. Giesemann, G. E. Schulz, and K. Aktories Change of the Donor Substrate Specificity of Clostridium difficile Toxin B by Site-directed Mutagenesis J. Biol. Chem., November 11, 2005; 280(45): 37833 - 37838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mazharian, S. Roger, P. Maurice, E. Berrou, M. R. Popoff, M. F. Hoylaerts, F. Fauvel-Lafeve, A. Bonnefoy, and M. Bryckaert Differential Involvement of ERK2 and p38 in Platelet Adhesion to Collagen J. Biol. Chem., July 15, 2005; 280(28): 26002 - 26010. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Citro, S. Ravasi, G. E. Rovati, and V. Capra Thromboxane Prostanoid Receptor Signals Through Gi Protein to Rapidly Activate Extracellular Signal-Regulated Kinase in Human Airways Am. J. Respir. Cell Mol. Biol., April 1, 2005; 32(4): 326 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Mesmin, K. Robbe, B. Geny, F. Luton, G. Brandolin, M. R. Popoff, and B. Antonny A Phosphatidylserine-binding Site in the Cytosolic Fragment of Clostridium sordellii Lethal Toxin Facilitates Glucosylation of Membrane-bound Rac and Is Required for Cytotoxicity J. Biol. Chem., November 26, 2004; 279(48): 49876 - 49882. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Fukui and Y. Horiguchi Bordetella Dermonecrotic Toxin Exerting Toxicity through Activation of the Small GTPase Rho J. Biochem., October 1, 2004; 136(4): 415 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Barbier, M. R. Popoff, and J. Molgo Degeneration and Regeneration of Murine Skeletal Neuromuscular Junctions after Intramuscular Injection with a Sublethal Dose of Clostridium sordellii Lethal Toxin Infect. Immun., June 1, 2004; 72(6): 3120 - 3128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Blake, F. Mitsikosta, and M. A. Metcalfe Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants J. Med. Microbiol., March 1, 2004; 53(3): 197 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Boyer, S. Travaglione, L. Falzano, N. C. Gauthier, M. R. Popoff, E. Lemichez, C. Fiorentini, and A. Fabbri Rac GTPase Instructs Nuclear Factor-{kappa}B Activation by Conveying the SCF Complex and IkB{alpha} to the Ruffling Membranes Mol. Biol. Cell, March 1, 2004; 15(3): 1124 - 1133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Masamura, K. Oida, H. Kanehara, J. Suzuki, S. Horie, H. Ishii, and I. Miyamori Pitavastatin-Induced Thrombomodulin Expression by Endothelial Cells Acts Via Inhibition of Small G Proteins of the Rho Family Arterioscler. Thromb. Vasc. Biol., March 1, 2003; 23(3): 512 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Chaves-Olarte, E. Freer, A. Parra, C. Guzman-Verri, E. Moreno, and M. Thelestam R-Ras Glucosylation and Transient RhoA Activation Determine the Cytopathic Effect Produced by Toxin B Variants from Toxin A-negative Strains of Clostridium difficile J. Biol. Chem., February 28, 2003; 278(10): 7956 - 7963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Belyi, M. R. Popoff, and N. P. Cianciotto Purification and Characterization of a UDP-Glucosyltransferase Produced by Legionella pneumophila Infect. Immun., January 1, 2003; 71(1): 181 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Vidal, B. Geny, J. Melle, M. Jandrot-Perrus, and M. Fontenay-Roupie Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin Blood, December 15, 2002; 100(13): 4462 - 4469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Marvaud, B. G. Stiles, A. Chenal, D. Gillet, M. Gibert, L. A. Smith, and M. R. Popoff Clostridium perfringens Iota Toxin. MAPPING OF THE Ia DOMAIN INVOLVED IN DOCKING WITH Ib AND CELLULAR INTERNALIZATION J. Biol. Chem., November 8, 2002; 277(46): 43659 - 43666. [Abstract] [Full Text] [PDF]
|
|
