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J Biol Chem, Vol. 275, Issue 18, 13228-13234, May 5, 2000
From the Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, Freiburg D-79104, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Large clostridial cytotoxins catalyze the
glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By
site-directed mutagenesis of Clostridium sordellii lethal
toxin and Clostridium difficile toxin B fragments, we
identified tryptophan 102, which is located in a conserved region
within the catalytic domain of all clostridial cytotoxins, to be
crucial for UDP-glucose binding. Exchange of Trp-102 with alanine
decreased the glucosyltransferase activity by about 1,000-fold and
blocked cytotoxic activity after microinjection. Replacement of Trp-102
by tyrosine caused a 100-fold reduction in enzyme activity, indicating
a partial compensation of the tryptophan function by tyrosine. Decrease
in glucosyltransferase and glycohydrolase activity was caused
predominantly by an increase in the Km for
UDP-glucose of these mutants. The data indicate that the conserved
tryptophan residue is implicated in the binding of the cosubstrate
UDP-glucose by large clostridial cytotoxins. Data bank searches
revealed different groups of proteins sharing the recently identified
DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J.,
Jeckel, D., and Aktories, K. (1998) J. Biol. Chem.
273, 19566-19572) and a conserved region defined by a tryptophan
residue equivalent to Trp-102 of C. sordellii lethal toxin.
From our findings, we propose a novel family of glycosyltransferases
which includes both prokaryotic and eukaryotic proteins.
The family of large clostridial cytotoxins consists of the toxins
A and B of Clostridium difficile, the lethal and hemorrhagic toxins of Clostridium sordellii, and the The toxins are glucosyltransferases that specifically modify and
thereby inactivate small GTP-binding proteins of the Rho and Ras
subfamily (5-8). Whereas the The large clostridial cytotoxins belong to the superfamily of
glycosyltransferases that have been classified in different families
depending on their sequence homologies (14). Some of these
glycosyltransferases share a DXD motif also conserved among all large clostridial cytotoxins. This motif is located within the
NH2-terminal part of the toxins, which has been shown to be the catalytic domain of the toxins (15, 16). Several groups showed that
the DXD motif is essential for the enzyme activity of the
glycosyltransferases (17-20) and is probably involved in the binding
of manganese ions (17).
Recently, crystal structures of two glycosyltransferases of families 2 and 7, exhibiting the DXD motif, have been solved (21, 22),
showing that the proteins share a similar topology of the catalytic
center and that most of the amino acid residues involved in nucleotide
recognition are conserved among all members of the respective families.
Based on these findings, we compared thoroughly the sequences of
glycosyltransferases showing sequence homology to the large clostridial
cytotoxins in the region around the DXD motif and identified
another homology region conserved in all of these proteins. Here we
report on a tryptophan residue located in this second conserved region
of many putative glycosyltransferases, including clostridial
cytotoxins, which is likely to be implicated in the binding of the
cosubstrate UDP-glucose.
Materials--
14C-Labeled UDP-hexoses were obtained
from NEN Life Science Products (Dreieich, Germany). Polymerase chain
reaction primers were from MWG Biotech (Ebersberg, Germany). All other
reagents were of analytical grade and purchased from commercial sources.
Polymerase Chain Reaction Amplification--
Amplification of
NH2-terminal C. difficile toxin B and C. sordellii lethal toxin fragments and construction of COOH-terminal truncated fragments B5461 and
LT546 was done as described previously (15, 16).
Site-directed Mutagenesis of Toxin Fragment LT546 and
B546--
The QuikChange KitTM (Stratagene) was used for
mutating one or two nucleotides in the pGEX 2T-LT546 construct or in
the pGEX 2T-B546 construct, respectively. Procedures were carried out
according to the manufacturer's instructions. Primers were constructed
as follows:
K96A.LT546: Primer pair S1K96A sense/antisense
(5'-CTGCTGCTTCTGCTATATTACGAATAT-3'/5'-GATATTCGTAATATAGCAGAAGCAGCA-3').
H99A.LT546: Primer pair S1H99A sense/antisense
(5'-CGAATATCTATGTTAGCAGAAGATGGTGG-3'/5'-CCACCATCTTCTGCTAACATAGATATTCG-3').
W102A.LT546: Primer pair S1W102A sense/antisense
(5'-AAAAATTTACATTTTATAGCTATTGGAGGACAA-3'/5'-TTGTCCTCCAATAGCTATAAAATGTAAATTTTT-3').
W102Y.LT546: Primer pair S1W102Y sense/antisense
(5'-AAAAATTTACATTTTATATATATTGGAGGACAA-3'/5'-TTGTCCTCCAATATATATAAAATGTAAATTTTT-3').
W102R.LT546 Primer pair S1W102R sense/antisense
(5'-AAAAATTTACATTTTATAAGAATTGGAGGACAA-3'/5'-TTGTCCTCCAATTCTTATAAAATGTAAATTTTT-3').
G105A.LT546: Primer pair S1G105A sense/antisense
(5'-TTTATATGGATTGGAGCACAAATAAATGATAC-3'/5'-GTATCATTTATTTGTGCTCCAATCCATATAAA-3').
D109A.LT546: Primer pair S1D109A sense/antisense
(5'-GGAGGACAAATAAATGCTACCGC
TATCAAC-3'/5'-GTTGATAGCGGTAGCATTATTTATTTGTCCTCC-3').
W102A.B546: Primer pair B1W102A sense/antisense
(5'-AAAAATTTACATTTTGTTGCTATTGGAGGTCAA-3'/5'-TTGACCTCCAATAGCAACAAAATGTAAATTTTT-3').
Sequencing--
Sequencing of the mutated clones of LT546 and
B546 was done with the Applied Biosystems, Inc. PrismTM Dye
Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany) to check both for correct cloning and mutations caused by polymerase chain reaction amplification.
Expression of Recombinant Proteins--
The recombinant
GTP-binding proteins RhoA, Rac, Cdc42 and Ha-Ras were prepared from
their fusion proteins as described (7, 8). The recombinant toxin
fragments were expressed and purified as GST fusion proteins in
accordance with the manufacturer's instructions (see Fig. 2). GST
fusion proteins from the Escherichia coli expression vector
pGEX 2T were isolated by affinity chromatography with
glutathione-Sepharose (Amersham Pharmacia Biotech, Freiburg, Germany)
followed by removing GST fusion proteins with glutathione elution
buffer (10 mM glutathione in 50 mM Tris-HCl, pH
8.0). Recombinant Rap1a was a gift from Dr. A. Wittinghofer (Dortmund, Germany).
Glucosylation Assay--
Recombinant GTP-binding proteins
(50-250 µg/ml) were incubated with recombinant toxin fragment LT546,
B546, or mutated fragments at the indicated concentrations in a buffer
containing 50 mM Hepes, pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM MnCl2,
100 µg/ml bovine serum albumin, and the indicated concentrations of
UDP-[14C]glucose and unlabeled UDP-glucose at 37 °C
for the indicated periods. The total volume was 20 µl. Labeled
proteins were analyzed by SDS-PAGE and subsequently by PhosphorImaging
(Molecular Dynamics, Germany).
Glycohydrolase Assay--
LT546 and the mutated fragments were
incubated with 14C-labeled UDP-glucose and unlabeled
UDP-glucose at the indicated concentrations in a buffer containing 50 mM Hepes, pH 7.5, 100 mM KCl, 2 mM
MgCl2, 100 µM bovine serum albumin, 1 mM MnCl2. The total volume was 5-10 µl.
Samples of 1.5 µl were taken out at each time point and subjected to
thin layer chromatography with polyethylenimine cellulose plates
(Merck, Germany) and 0.2 M LiCl as mobile phase in order to
separate hydrolyzed glucose from UDP-glucose. The plates were dried and
analyzed by PhosphorImaging.
Tryptophan Quenching Experiments--
The decrease in intrinsic
protein fluorescence of the wild-type fragment LT546 or the mutant
W102Y.LT546 as a function of the UDP-glucose concentration was measured
at 25 °C (25). The proteins were diluted in a buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 2.5 mM CaCl2. The solutions were excited at 280 nm
(3.5-nm band pass), and the fluorescence intensity was measured at 340 nm (3.5-nm band pass) in a Perkin Elmer LS50B luminescence
spectrometer. The final concentrations of the fragments used in the
experiments were 0.8 µM for LT546 and 1 µM
for W102Y.LT546. The fluorescence decrease of L-tryptophan
was used to correct for the inner filter effect. All data points were
corrected additionally for dilution.
Kinetic Experiments--
Initial rate data for the
glucosyltransferase and glycohydrolase reaction were determined with
regard to UDP-glucose binding by varying the UDP-glucose concentration
from about 0.2 to 2 × Km. For the
glucosyltransferase reaction, experiments were performed at fixed
GST-Rac concentrations of 12.5 µM. The kinetic values
were obtained by analysis of Lineweaver-Burk plots of initial
velocities from three independent experiments.
Microinjection Studies--
For microinjection studies, HeLa
cells were grown for 24 h in Dulbecco's modified Eagle's medium
supplemented with 10% calf serum, 4 mM
glutamine/penicillin/streptomycin and plated on Cellocate (Eppendorf,
Germany) coverslips at about 105 cells/dish at 37 °C and
5% CO2. Microinjection was performed with the
microinjector 5242 and micromanipulator 5171 from Eppendorf.
Electropermeabilization Experiments and Measurement of
Transepithelial Resistance--
Caco-2 cells (Cell Line Service,
Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, 1% non-essential
amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Cells were subcultured every week and seeded on filter inserts for cell
culture (12-mm diameter, Falcon) at a density of approximate 4 × 104 cells × cm Identification of Proteins with Sequence Homology to Members of the
Glucosyltransferase Family of Large Clostridial Cytotoxins--
We
have recently shown the importance of a highly conserved region
(DXD motif) in active fragments of large clostridial
cytotoxins for their glucosyltransferase activities (17). Besides the
DXD motif, which we found to be essential for enzyme
activity, this entire region contains 16 additional residues conserved
among all large clostridial cytotoxins (Fig.
1A). At least two of these additional residues appear to be important for enzyme activity (17).
When this whole region, which we refer to as the extended
DXD motif, is used to search protein data bases, several
further sequences can be identified, all of which share at least six
residues of this motif with the clostridial toxins. Fig. 1A
shows an alignment of some of these proteins in the mentioned section.
Furthermore, another short amino acid stretch located
NH2-terminal of the DXD motif is conserved in
all sequences (Fig. 1B). From these findings, we set out to
elucidate the significance of this second homology region for the
enzyme activity of large clostridial cytotoxins.
Effects of Site-directed Mutagenesis on Enzyme Activity of Toxin
Fragments--
Alanine mutants of all conserved amino acid residues in
the region of amino acids 96-109 of the enzymatically active
NH2-terminal fragment of C. sordellii lethal
toxin (LT546) were constructed. The mutants were expressed as GST
fusion proteins and purified as described. Fig.
2A shows a SDS-PAGE analysis
of the purified fusion proteins. Next, we tested the
glucosyltransferase activity of these mutants in a glucosylation
experiment with the substrate GST-Rac. We found that all mutations
(K96A, H99A, G105A, and D109A) had little effect on glucosyltransferase
activity (not shown) except for that of Trp-102, which resulted in a
reduced activity compared with the wild-type fragment (Fig.
3). This decrease in activity was most
likely not caused by major changes in the overall structure of the
protein because the susceptibility of the fragment toward trypsin
treatment was not changed (Fig. 2B). Replacement of Trp-102
with arginine or tyrosine also led to a decrease in enzyme activity.
Although the conservative mutation W102Y.LT546 resulted in an enzyme
activity higher than that of the alanine mutant, the arginine mutant
only showed a residual activity (Fig. 4A).
To exclude that the reduced activity was caused by an impaired binding
only to the substrate GST-Rac, we determined the substrate specificity
of the mutant W102A.LT546. As shown in Fig.
5, we found that W102A.LT546 was capable
of modifying all substrates of the wild-type fragment, even though to a
considerably lesser extent. When we compared LT546 and the mutant in
time courses of the modification of the GST-Rac or Ras-protein,
respectively, we observed an approximately 1,000-fold reduction of
enzyme activity of the mutant W102A.LT546 for both protein substrates
(Fig. 3).
To confirm the significance of Trp-102 for the glucosyltransferase
activity of the whole family of large clostridial cytotoxins, we
constructed the respective alanine mutant of Trp-102 of the enzymatically active C. difficile toxin B fragment.
Corroborating the results with the lethal toxin fragment, the mutant
W102A.B546 was found to be much less active than the wild-type fragment
in a glucosylation assay (Fig. 4B).
Kinetic Studies of Trp-102 Mutants--
The effects of replacing
Trp-102 with tyrosine or alanine on enzyme activity of LT546 were
evaluated by comparing the relative specific activities for the
glucosyltransferase and the glycohydrolase reaction
(kcat and
kcat/Km) of the wild-type and
mutant proteins. Furthermore, we also compared the Michaelis constant (Km) of the fragments toward UDP-glucose.
For the glucosyltransferase reaction, the kinetic parameters were
determined by varying the UDP-glucose concentrations from 0.2 to 2 × Km at a fixed GST-Rac concentration of 5 µM. The values obtained by Lineweaver-Burk plots are
summarized in Table I.
Conservative substitution of Trp-102 with tyrosine resulted in a
20-fold increase in Km, whereas
kcat was reduced 4-fold. The mutant was about
100-fold less efficient (9 × 103-fold lower
kcat/Km) compared with the
wild-type fragment. Glycohydrolase activity was less influenced by the
replacement with tyrosine. As can be seen in Table I, the activity
(kcat) was unchanged, whereas the binding
affinity was reduced about 10-fold, resulting in a 10-fold less
efficient enzyme.
On the other hand, the replacement of Trp-102 with alanine resulted in
an enzyme that was altered much more dramatically in its kinetic
properties. W102A.LT546 showed no detectable glycohydrolase activity
(Fig. 6). Moreover, the
Km value for the glucosyltransferase reaction was
reduced more than 200-fold, whereas the specificity constant
kcat was reduced 16-fold. Reflecting these
alterations, the mutant W102A.LT546 was more than 3,500-fold less
efficient (2.7 × 10 Fluorescence Studies--
To determine the dissociation constants
for UDP-glucose binding independently of enzyme activity, we measured
the decrease in intrinsic protein fluorescence caused by quenching of
the tryptophan fluorescence (excitation at 280 nm, emission at 340 nm)
as a function of UDP-glucose concentration. The values recorded at the
fluorescence maximum at 340 nm were corrected for dilution and the
inner filter effect.
We tested the wild-type toxin fragment and observed a quenching of
about 20% at a concentration of 23 µM UDP-glucose (Fig. 7). A dissociation constant
(Kd) of 6 µM was determined for this
interaction. Similar results were obtained for UDP-mannose, a
nucleotide sugar that inhibits the glucosylation reaction with UDP-glucose (not shown). GDP-mannose is neither a cosubstrate for the
glucosylation reaction of the lethal toxin, nor does it inhibit the
glucosylation reaction. When we tested this nucleotide sugar with the
wild-type fragment, we observed a much weaker quenching effect (less
than 10%). The quenching curve of the mutant W102Y.LT546 was
superimposable on that of the wild-type fragment with GDP-mannose (Fig.
7). This was also found for higher concentrations of the respective
nucleotide sugars (not shown). Because of the low quenching effect, no
dissociation constant could be determined.
Effects of Lethal Toxin Mutants on Cultured Cells--
To
characterize further the mutants W102Y.LT546 and W102A.LT546, we tested
their biological activity by microinjection into HeLa cells. As shown
in Fig. 8, we found the tyrosine mutant
to elicit the same morphological effects as the wild-type fragment after 30 min when applied in a high concentration of 800 nM, whereas the alanine mutant showed no effect even after
more than 4 h of treatment. To obtain more quantitative data, we
measured the effect of LT546 and the mutants W102A.LT546 and
W102Y.LT546 on transepithelial resistance of Caco-2 cells. For this
purpose, the fragments were introduced in the cells by
electropermeabilization. As can be seen in Fig.
9, treatment with the wild-type toxin
fragment LT546 led to an 80% reduction of transepithelial resistance
after 450 min, whereas only a 20% decrease was observed with the
tyrosine mutant of Trp-102. The mutant W102A.LT546 was without effect
on the integrity of the monolayer.
The large clostridial cytotoxins belong to the superfamily of
glycosyltransferases that use nucleotide sugars as cosubstrates. However, their homology to glycosyltransferase families as established by Campbell et al. (14) is restricted to the small peptide
motif DXD, which can be found in several otherwise unrelated
families of glycosyltransferases. This motif has recently been shown to be crucial for enzyme activity of different types of
glycosyltransferases (17-20). Elucidation of crystal structures of two
glycosyltransferases has revealed the involvement of the aspartates of
the DXD motif in the binding of the nucleotide sugar (21,
22).
Based on the DXD motif and its conserved surrounding, the
large clostridial cytotoxins appear to be related to some recently described putative glycosyltransferases. Among these proteins are a
number of hypothetical enzymes that are part of capsular polysaccharide
synthesis clusters in bacteria, such as Streptococcus pneumoniae cap8J and a related sequence in the C. difficile genome (23, 24). Furthermore, a group of yeast proteins
related to the mannosyltransferase Och1p and a family of closely
related proteins of E. coli EPEC and EHEC strains and a
number of hypothetical proteins of Chlamydia trachomatis
serotype D are homologous to large clostridial cytotoxins. All of these
proteins share at least an extended form of the DXD motif
and another short peptide motif containing a conserved tryptophan
residue with the toxins (Fig. 1).
Investigating the latter homology region by site-directed
mutagenesis of an enzymatically active NH2-terminal
fragment of C. sordellii lethal toxin, we observed only a
small reduction (up to 50%) of enzyme activity with the alanine
mutants of Lys-96, His-99, Gly-105, and Asp-109 (not shown). This
decrease in activity might be caused by minor alterations of the
tertiary structure, whereas a direct involvement of these residues in
the catalytic process or substrate binding seems unlikely.
Substitution of Trp-102, however, was found to have a major impact on
enzyme activity of both the lethal toxin and the toxin B fragment. We
observed a decrease in enzyme activity for all constructed mutants of
this residue (Figs. 4 and 5). Because both glucosyltransferase and
glycohydrolase activity were affected, an involvement of Trp-102 in the
binding of the cosubstrate or the catalytic reaction was assumed. In
line with this hypothesis, a similar decrease in glucosyltransferase
activity was observed with two different protein substrates (Fig.
3).
Kinetic studies of different mutants of Trp-102 provided further
insight into the role of this residue (Table I). As for the
glycohydrolase reaction, we found about a 10-fold increase in the
Km for the tyrosine mutant compared with the
wild-type fragment, whereas kcat was slightly
increased. From these results, a direct involvement of Trp-102 in the
catalytic process could be excluded when kcat is
taken as a measure for enzyme activity. The decrease in activity is
rather caused by a reduction in binding affinity as the
Km value is increased. The arginine and the alanine
mutants showed no detectable glycohydrolase activity in our system,
presumably reflecting the lower sensitivity of the glycohydrolase
compared with the glucosyltransferase assay.
With respect to the glucosyltransferase reaction of the tyrosine
mutant, both the catalytic activity and the binding affinity to
UDP-glucose were reduced. The alanine mutant showed an even more
impaired efficiency, mainly because the Km for
UDP-glucose was increased about 200-fold. This low in vitro
activity of the alanine mutant is reflected by its lack of cytotoxic
activity when introduced into cells by microinjection or
electroporation. The tyrosine mutant, however, showed a clearly
detectable, albeit reduced, cytotoxic activity (Figs. 8 and 9). The
decrease in kcat of the latter mutant observed
with the glucosyltransferase reaction is contradictory to the findings
with the glycohydrolase reaction. However, it is conceivable that the
complex interactions in the three-component glucosyltransferase
reaction are more sensitive to alterations in the enzyme's catalytic
center than the glycohydrolase reaction. In contrast to recent
findings, we found the glucohydrolase activity to be similar to the
glucosyltransferase activity as the respective
kcat values were similar. This can be explained, however, by our using the relatively bad substrate GST-Rac for the
determination of enzyme activity. From the experimental data shown in
Fig. 3, turnover rates of the modification of GST-Rac or Ras were
determined to be approximately 260 h In conclusion, the results of the kinetic studies give evidence that
the reduction in enzyme activity of Trp-102 mutants is mainly the
result of a decrease in binding affinity for the cosubstrate UDP-glucose. These findings were corroborated further by tryptophan quenching experiments. We observed a quenching of about 20% with the
cosubstrate UDP-glucose or the competitive inhibitor UDP-mannose, whereas the quenching effects of all other nucleotide diphosphate sugar
used such as GDP-mannose (Fig. 7), UDP-GlcNAc, or GDP-glucose (not
shown) were considerably lower. Because the quenching effect of
UDP-glucose on the mutant protein W102Y.LT546 was reduced
significantly, we conclude that the specific binding affinity of the
mutant for the cosubstrate was decreased. Because of the low quenching
effect we were unable to determine the Kd for the
mutant accurately. The overall low quenching of tryptophan fluorescence
observed in our experiments implies that not all of the five tryptophan residues in the toxin fragment are involved in the interaction with the cosubstrate.
Concerning the mode of the interaction of Trp-102 with the cosubstrate,
it should be noted that the replacing Trp-102 with tyrosine resulted in
the most active mutant, whereas the arginine mutant only exhibited a
weak, hardly detectable activity (Fig. 4). Interestingly, in the
Och1p-related yeast protein Sur1p, which shares the equivalents of
Lys-96 and His-99 with the clostridial cytotoxins, the tryptophan
residue is replaced by a tyrosine (Fig. 1B). This leads us
to hypothesize that Trp-102 is involved in a hydrophobic interaction
with the cosubstrate UDP-glucose.
Recently, two crystal structures of glycosyltransferases containing the
DXD motif have been solved. These are the family 2 glycosyltransferase spsA from B. subtilis and the bovine
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-toxin from
Clostridium novyi (1, 2). C. difficile toxins A
and B are responsible for symptoms in C. difficile-induced
antibiotic-associated diarrhea and pseudomembranous colitis, whereas
lethal toxin, hemorrhagic toxin, and -toxin are virulence factors in
gas gangrene (3, 4).
-toxin from C. novyi
catalyzes the transfer of an N-acetylglucosamine moiety from
UDP-GlcNAc to its substrates (9), all other members of the toxin family share UDP-glucose as a cosubstrate. The modification occurs at amino
acid Thr-37 in Rho or at Thr-35 in the other GTPases, resulting in
inhibition of the interaction of the GTPases with their effectors (10,
11). Because Rho proteins are regulators of the actin cytoskeleton,
their inactivation results in a breakdown of cytoskeletal structures
within target cells (12). Furthermore, various other signal
transduction processes controlled by the small GTPases are inhibited
(13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 for
electropermeabilization experiments and determination of transepithelial resistance. Caco-2 cells on filter inserts, cultured in
12-well plates, were washed once with Hanks' balanced salt solution
and equilibrated at 37 °C for 30 min in HBSS containing a 10 nM concentration of the toxin fragment to be inserted into cells in the apical and basolateral reservoir. Before electroporation, the transepithelial electrical resistance was measured using an epithelial volts/
meter (EVOM, World Precision Instruments,
Sarasota, FL) supplemented with a chamber for filter inserts (Endohm
24). Only cells with an initial transepithelial resistance of at least 200 cm2 were chosen for experiments. The
electroporation was performed in Hanks' balanced salt solution in a
purpose-built electroporation chamber where electrodes were positioned
apically and basally of the cell monolayer. For the capacitative
discharge a pulse generator (Gene Pulser II, Bio-Rad) with a
capacitance extender was used. According to the initial transepithelial
electrical resistance of the filter, the parallel shunt resistor of the
pulse controller was adjusted to 400 for filters with 200-400 cm2 or to 600 for filters with 400-600 cm2 to parallel the resistance of cell monolayer. After the
pulse (200 V/cm; 10 microfarads; 1.2 ms), the filter inserts were
transferred back into 12-well plates and incubated in Hanks' balanced
salt solution at 37 °C. The transepithelial electrical resistance
was measured after the indicated times.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignments of the large clostridial
cytotoxins and related proteins. Panel A, alignment of
the region around the DXD motif. Panel B,
alignment of the second homology region including the conserved
tryptophan. LT, C. sordellii strain 6018 lethal toxin
(GenBank X82638); Toxin B, C. difficile strain VPI 10463 cytotoxin B (GenBank X53138); Toxin BF, C. difficile strain
1470 variant cytotoxin B (GenBank Z23277); Toxin A, C. difficile strain VPI 10463 cytotoxin A (GenBank M30307);
Alpha-tox, C. novyi -toxin (GenBank Z48636); EHEC,
E. coli enterohemorrhagic strain O157:H7 toxin B (sptrembl
o82916); Chlam., C. trachomatis CT166 (sptrembl
o84168). Yeast proteins: Och1p, Saccharomyces
cerevisiae och1 mannosyltransferase (GenBank D11095); Hoc1p,
S. cerevisiae putative glycosyltransferase Hoc1 precursor
(pir s57094); YDB6, Schizosaccharomyces pombe hypothetical
protein C17G8.11C (EMBL z69795); Sur1p, S. cerevisiae
hypothetical protein sur1p (GenBank M96648). Cps proteins:
bacterial capsular polysaccharide synthesis proteins; C. diff., C. difficile contig 1343 (these sequence data
were produced by the C. difficile Sequencing Group at the
Sanger Center; S. pneum. cap8J, S. pneumoniae
type 8 gene cluster cap8J gene (EMBL 239004); capp33fI,
S. pneumoniae type 33F capsular gene cluster
capp33fI gene (EMBL spaj6986); cps 14K, S. pneumoniae cps14 locus gene cps14K (sptrembl o07341);
ORF4, Haemeophilus influenzae hypothetical protein 4 (capsulation locus; pir s60905).
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Fig. 2.
Panel A, purified recombinant lethal
toxin fragment mutants. The NH2-terminal toxin mutants were
constructed as GST fusion proteins, expressed in E. coli,
and purified by affinity chromatography. Lethal toxin fragment mutants
as fusion proteins were wild-type LT546, K96A.LT546, H99A.LT546,
W102Y.LT546, W102A.LT546, W102R.LT546, G105A.LT546, and D109.LT546
(approximately 1 µg of protein was loaded on each lane). Panel
B, NH2-terminal toxin B fragment mutants as GST fusion
proteins B546 and W102AB546 (approximately 0.5 µg was loaded on each
lane). Panel C, protease digest of toxin fragment
and mutants by trypsin. Lethal toxin fragment LT546 and mutant
W102A.LT546 (each 20 µg) were incubated with 180 ng of trypsin in a
buffer containing 10 mM glutathione and 50 mM
Tris-HCl, pH 8.0, in a total volume of 26 µl. At the indicated time
points, aliquots of 7 µg of protein were taken out and analyzed by
SDS-PAGE.
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Fig. 3.
Glucosyltransferase activities of LT546
mutant W102A.LT546. Panel A, time course of the
glucosylation of GST-Rac by LT546 and by the mutant W102A.LT546.
GST-Rac (3 µg) was incubated with LT546 (1 nM, 
) or
toxin fragment mutants W102A.LT546 (1 µM, 
) in the
presence of UDP-[14C]glucose (10 µM) for
the indicated times. Labeled proteins were then analyzed by SDS-PAGE
and PhosphorImaging. Data are given as means ± S.E.
(n = 3). Panel B, time course of the
glucosylation of Ras by LT546 and the mutant. Ras (3 µg) was
incubated with LT546 (1 nM, ) or toxin fragment mutants
W102A.LT546 (1 µM, ) in the presence of
UDP-[14C]glucose (10 µM) for the indicated
times. Then labeled proteins were analyzed by SDS-PAGE and
PhosphorImaging (shown). Data are given as means ± S.E.
(n = 3).
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Fig. 4.
Glucosyltransferase activities of toxin
mutants. Radioactively labeled proteins were analyzed by SDS-PAGE
and PhosphorImaging (shown). Panel A, glucosyltransferase
activities of W102Y.LT546 and W102R.LT546. GST-Rac1 (2 µg) was
glucosylated by LT546, W102Y.LT546, and W102R.LT546 at the indicated
concentrations for 20 min. Panel B, glucosyltransferase
activities of toxin B fragment mutant W102A.B546. GST-Rac1 (2 µg) was
glucosylated by LT546, W102Y.LT546, or W102R.LT546 at the indicated
concentrations for 10 min.
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Fig. 5.
Protein substrate specificity of
glucosylation by LT546 and W102A.LT546. Recombinant Ras, Rho,
Rac1, Cdc42, Ral, and Ras (each 1 µg) were glucosylated by LT546 and
W102A.LT546 at the indicated concentrations in the presence of
UDP-[14C]glucose for the indicated times. Labeled
proteins were then analyzed by SDS-PAGE and PhosphorImaging
(shown).
Kinetic studies of Trp-102 mutants
4-fold lower
kcat/Km) than the wild-type
toxin fragment.
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[in a new window]
Fig. 6.
Time course of glycohydrolase activity of by
LT546 and mutant W102A.LT546. LT546 (100 nM, 
) and
W102A.LT546 (1 µM, 
) were incubated with 20 µM UDP-[14C]glucose and 100 µM UDP-glucose in a total volume of 10 µl. At the
indicated time points, 1.5-µl samples were taken and analyzed by thin
layer chromatography and PhosphorImaging. Data are given as means ± S.E. (n = 3).
View larger version (18K):
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Fig. 7.
Quenching of intrinsic protein fluorescence
by nucleotide diphosphate sugars for LT546 and W102A.LT546.
Tryptophan quenching assays of LT546 by UDP-glucose ( ) or
GDP-mannose () and of W102Y.LT546 by UDP-glucose () were
performed as described under "Experimental Procedures." Data were
corrected for dilution and the inner filter effect.
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Fig. 8.
Microinjection into HeLa cells. HeLa
cells were injected with control buffer (panel A) or with
buffer containing 800 nM LT546 (panel B),
W102Y.LT546 (panel C), or W102A.LT546 (panel D).
Photographs were taken 30 min (panels B and C) or
4 h (panels A and D) after
microinjection.
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Fig. 9.
Effects of LT546 and mutants on
transepithelial resistance of Caco-2 cells. Cell monolayers were
electropermeabilized and treated with toxin fragment or mutants as
described under "Experimental Procedures." The transepithelial
electrical resistance of cell monolayers treated with 10 nM
LT546 ( ), W102A.LT546 (), and W102Y.LT546 () was measured
after the indicated times. The data are given as means of two
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 or 17,700 h1, respectively.
4-galactosyltransferase of family 7 (21, 22). Although the homology
of the amino acid sequences of these proteins is restricted to the
DXD motif, they exhibit a similar fold of the catalytic core
(26). Interestingly, both crystal structures comprise an aromatic
residue that is involved in the stacking of the uracil ring of the
cosubstrate in the catalytic fold. As found for the Trp-102 analog in
the cytotoxin-related proteins, this conserved aromatic residue is
located NH2-terminally of the DXD motif, and
there is no strictly defined distance between this residue and the
motif (Fig. 10). Therefore, it is
tempting to speculate that Trp-102 represents the analogous residue to the mentioned conserved aromatic residues.
View larger version (22K):
[in a new window]
Fig. 10.
Schematic representation of
glycosyltransferases of family 2, 7, and of the proteins related to
large clostridial cytotoxins. The length and location of the
conserved regions involved in nucleotide binding are indicated for each
glucosyltransferase family (light gray boxes). In each
sequence shown, the conserved DXD motif or the aromatic
amino acid presumably involved in the stacking of the uracil ring is
highlighted by a white or black box,
respectively. As for family 7 glycosyltransferases, bovine
4-galactosyltransferase (swissprot P08037) is given as an example.
Amino acids involved in the binding of the nucleotide moiety as
determined by crystal structure analysis are indicated (22). Family 2 glycosyltransferases are represented by the spore coat polysaccharide
synthesis protein spsA of Bacillus subtilis (swissprot
P39621), the N-acetylglucosaminyltransferase nodC of
Rhizobium meliloti (swissprot P04341), and the cellulose
synthase of Acetobacter pasteurianus (swissprot P19449).
Amino acid residues found to be involved in the binding of the
nucleotide in spsA are indicated. The putative catalytic amino acid of
spsA is marked as such (21). Domain nomenclature was adopted from
Griffiths et al. (20). Domain A represents the nucleotide
binding region (21), whereas domain B (dark box) is only
present in processive glycosyltransferases (20). The capsular
polysaccharide synthesis protein cap8J (EMBL AJ239004.1) and the
enzymatically active toxin fragment of C. sordellii lethal
toxin (GenBank X82638) are examples for the large clostridial
cytotoxins and proteins related to them. These proteins share two small
homology regions, a short stretch in the vicinity of the conserved
tryptophan, and an extended DXD motif (light gray
boxes). Two conserved amino acids of the extended DXD
motif are shown which were found to be important for enzyme activity of
the lethal toxin fragment LT546 (17). The homology region of bacterial
capsular polysaccharide synthesis proteins and Och1p-related yeast
proteins (not shown) in addition to the tryptophan and the
DXD motif is shown as a dark gray box.
Furthermore, all glucosyltransferase family 2 members share several other conserved amino acids, all of which appear to be located within the nucleotide binding domain as determined by the crystal structures. This binding domain is most often located within the NH2-terminal half of the transferases, with the DXD motif representing its COOH-terminal end (Fig. 10). As shown in Fig. 10, this structure can also be found in the proteins related to large clostridial cytotoxins.
In conclusion, the large clostridial cytotoxins and the glucosyltransferase family 2 might share a similar architecture of their glucosyltransferase domain, with the nucleotide binding region located NH2-terminally of the region that binds to the sugar molecule and the acceptor.
In this study, we have identified a conserved tryptophan residue that
is essential for enzyme activity of large clostridial cytotoxins. A
small group of proteins shares both the small motif in the vicinity of
the tryptophan residue and an extended DXD motif with the
toxins. Given the significance of those two motifs, it is conceivable
that these proteins are also structurally related to large clostridial
cytotoxins. Thus, we propose a family of large clostridial
cytotoxin-related proteins.
| |
FOOTNOTES |
|---|
* This work was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.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.
To whom correspondence should be addressed. Tel.: 49-761-203-5301;
Fax: 49-761-203-5311; E-mail: aktories@ruf.uni-freiburg.de.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
B546, NH2-terminal C. difficile toxin B fragment of
amino acid residues 1-546;
LT546, NH2-terminal C. sordellii lethal toxin fragment of amino acid residues 1-546;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
, ohm(s).
| |
REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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