(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 (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.
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 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.
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, 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.
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-[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.
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-[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.
Figure 4:
Glucosylation of recombinant Ras-related
GTPases by LT. Ha-Ras, Rap2, Rac1, Cdc42, RhoA, Rab6, Ral-GST, and
Rac-GST (2 µg/assay) were incubated with LT and
UDP-[
C]Glc. A, PhosphorImager picture; B, Coomassie Blue staining of the
gel.
Figure 5:
Localization of LT-catalyzed 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).
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 30 µg of total
protein/experiment was electrophoresed, blotted, and stained with the
monoclonal antibody MK12 (ERK1, 44 kDa (p44); ERK2, 42 kDa (p42)).
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 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.
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 -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.
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 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) .
It is remarkable that four out of
five members of the group of these large clostridial cytotoxins (toxins
A and B, LT, and -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. (
)
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.