From the
Shiga toxin has a single A subunit non-covalently associated
with a pentamer of B subunits. The toxin has a trypsin-sensitive region
near the COOH-terminal end of the A-chain, and upon cleavage, two
disulfide bonded fragments, A
Shiga toxin produced by Shigelladysenteriae type 1, is composed of an enzymatically active A-subunit in
non-covalent association with a pentamer of B-subunits responsible for
binding to cell surface receptors (for review, see Ref. 1). The
A-subunit is a specific N-glycosidase that cleaves off a
single adenine residue from 28 S rRNA of the 60 S ribosomal subunit
(2, 3) , resulting in inhibition of the protein
synthesis. After binding to cell surface receptors, the toxin is
endocytosed from clathrin-coated pits
(4, 5) . The
functional receptor is a
Gal
Shiga toxin A-chain
(ST-A)
Both diphtheria toxin
and anthrax toxin seem to be activated by furin
(11, 12) , which is a Ca
In the present article, we demonstrate that furin cleaves ST-A into
A
We have
shown earlier that a cell-permeable inhibitor of the cytosolic enzyme
calpain protected Vero cells against a trypsin-resistant mutant
(R248H/R251H) of Shiga toxin and inhibited the cell-mediated cleavage
of this mutant toxin, while the inhibitor had no effect on the
intoxication and processing of wild type toxin.
The main finding in this paper is that the enzyme furin is
responsible for cellular cleavage of Shiga toxin and that cleavage is
essential for efficient intoxication of cells. Furin has been reported
to nick proteins with the sequence Arg- X- X-Arg
(19) . Indeed, Shiga toxin A-chain contains this motif
(Arg
LoVo/fur cells were less sensitive to nicked Shiga toxin than
LoVo/neo cells. In the case of A431 cells, we have previously shown
that these cells can be sensitized to Shiga toxin by butyric acid
treatment, and following this treatment more Shiga toxin is transported
to the Golgi apparatus, and the toxin can be visualized not only in the
TGN but also in the endoplasmic reticulum
(20, 40) ,
where translocation to the cytosol may take place
(20, 40, 41) . Similarly, butyric acid treatment
sensitized both LoVo/neo cells and LoVo/fur cells to Shiga toxin, and
the toxicity of nicked toxin was then similar in LoVo/neo and LoVo/fur
cells.
Furin is expressed in a wide variety of cells
(14) ,
whereas other members of the family of subtilisin-like proteases,
including PC 1, PC 2, and PC 4, are expressed in specialized cells,
like neuroendocrine cells, and recognize dibasic sequences
(14) . Shiga toxin does not have any such sequence in the region
where cleavage occurs. Therefore, furin or a furin-like enzyme may
cleave and activate Shiga toxin in many different cell types.
Pseudomonas toxin, diphtheria toxin, and anthrax toxin clearly
require cleavage for successful translocation to the cytosol and for
intoxication of cells, and furin seems to be the enzyme responsible for
cleavage
(11, 12, 27, 42) . In contrast,
it has been questioned whether cleavage of Shiga toxin is important for
intoxication of cells. Olsnes et al.(9) showed that
Shiga toxin nicked between A
Furin is
reported to be localized primarily in the TGN
(15, 16) but may also be present in small amounts in endosomes and
at the cell surface
(11, 12, 16) . In the
presence of ATP-depleting agents, which inhibited the internalization
of Shiga toxin, there was essentially no cleavage at the cell surface
of LoVo/fur cells, whereas in the presence of BFA, which disrupts the
Golgi cisterns, processing occurred at a normal rate, indicating that
nicking of Shiga toxin may take place in TGN and/or in endosomes.
Diphtheria toxin has recently been reported to be nicked at the cell
surface by furin in transfected LoVo cells. This difference from our
results can be explained by the different pH optima for furin cleavage
of diphtheria toxin and Shiga toxin in vitro. Diphtheria toxin
is in contrast to Shiga toxin cleaved efficiently at neutral pH
(12, 27) . In LoVo/neo cells, cleavage of Shiga toxin
was much less extensive than in LoVo/fur cells. Also in these cells, we
could not detect any cleavage at the cell surface, and BFA in this case
partly inhibited the cleavage. To further characterize the inefficient
cleavage taking place in LoVo/neo cells, we tested the effect of a
cell-permeable inhibitor of the cytosolic enzyme calpain and the effect
of leupeptin, a protease inhibitor taken up by endocytosis and reported
to inhibit the lysosomal proteases cathepsin B and L
(39) .
Leupeptin inhibited the cleavage extensively but did not protect
against Shiga toxin, suggesting that some cleavage occurs in a
compartment from where the toxin cannot translocate to the cytosol,
most likely in the lysosomes. In contrast, the calpain inhibitor
inhibited both cleavage and toxicity, indicating that a fraction of the
toxin molecules can be cleaved and activated by another cellular
protease than furin, possibly by the cytosolic enzyme calpain. However,
from the experiments shown, it seems clear that furin, in contrast to
other enzymes in LoVo cells, efficiently cleaves and activates Shiga
toxin.
We are grateful to Anne-Grethe Myrann, Tove Lie Berle,
Marianne Lund, Anne Mette Ohlsen, Keld Ottesen, and Kirsten Pedersen
for excellent technical assistance. We thank Dr. Eisuke Mekada for
providing LoVo/neo and LoVo/fur cells, Dr. Gary Thomas for providing
purified furin, and Dr. Knut Sletten (Oslo University) for performing
protein sequencing. We are also grateful to Dr. Markus Lanzrein and Dr.
Sjur Olsnes for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and A
, are
generated. These fragments are also formed upon incubation with cells.
The disulfide loop contains the sequence (Arg- X-X-Arg), which
is a consensus motif for cleavage by the membrane-anchored protease
furin. We found that a soluble form of furin cleaves intact A-chain
producing A
and A
fragments, and furin also
seems to be responsible for rapid cellular cleavage of Shiga toxin.
LoVo cells, which normally do not produce functional furin, cleave
intact A-chain very efficiently when transfected with furin (LoVo/fur),
whereas a control cell (LoVo/neo) cleaves the toxin very slowly. To
investigate the role of this cleavage for intoxication of cells, we
studied the ability of unnicked and furin-nicked toxin to inhibit
protein synthesis in LoVo/fur and LoVo/neo cells. LoVo/fur cells were
intoxicated equally well with unnicked and nicked toxin, whereas in
LoVo/neo cells nicked toxin was about 20 times more active than
unnicked toxin. The results suggest that cleavage of Shiga toxin is
important for intoxication of cells, and they indicate that furin can
cleave and thereby activate Shiga toxin in cells.
(1, 2, 3, 4) Gal-containing
glycolipid, primarily globotriasyl ceramide
(4, 6, 7, 8) .
(
)
contains 2 cysteines that are linked by
a disulfide bond. The loop between the 2 cysteines contains the
sequence Arg-Val-Ala-Arg, which is recognized and nicked by trypsin,
separating the A-chain into A
(
27.5 kDa) and A
(
4.5 kDa) fragments
(9) . It has previously been
shown that Shiga toxin and Shiga-like toxin type IIv can be nicked by a
cellular protease
(10) ,
(
)
and we have in
this study tried to identify this protease.
-dependent
serine protease with a subtilisin-like catalytic domain
(13, 14) . Furin is expressed in most cells and tissues
(14) , and the enzyme seems to be localized primarily in the
trans-Golgi network (TGN)
(15, 16) but can
also be found in endosomes and at the cell surface in small amounts
(11, 12, 16) . Furin has been reported to cleave
precursors of various secretory and membrane proteins with the
consensus motif Arg- X-Arg/Lys-Arg in the secretory pathway
(17, 18) , the cleavage site being at the COOH terminus
of this sequence
(14) . Substrates with the sequence
Arg- X- X-Arg, which is the minimal sequence for
recognition by furin, have also been reported to be cleaved by furin,
although less efficiently
(19) . Shiga toxin contains this motif
in the disulfide loop between the A
and A
fragments. Furthermore, after endocytosis, the toxin can be
transported to the TGN in a number of different cell types
(4, 20, 21) . Thus, furin or a furin-like enzyme
could recognize and cleave Shiga toxin after endocytosis of the toxin.
and A
fragments both in vitro and
in vivo.We used LoVo cells, a cell line that does
not express functional furin
(22) , and LoVo cells transfected
with furin to provide evidence for the involvement of furin in cellular
cleavage and activation of Shiga toxin.
Materials
Brefeldin A (BFA) was
purchased from Epicentre Technologies (Madison, WI). Calpain inhibitor
I was purchased from Boehringer Mannheim GmbH (Mannheim, Germany).
Butyric acid, Hepes, diaminobenzidine, and 3-(2-pyridyldithio)propionic
acid N-hydroxysuccinimide ester were purchased from Sigma.
NaI was purchased from Du Pont (Belgium), and
[
H]leucine was purchased from Amersham
International (Amersham, United Kingdom). Shiga toxin was
I-labeled according to Fraker and Speck
(23) to a
specific activity of 30,000-40,000 cpm/ng. A conjugate of Shiga
toxin and horseradish peroxidase was prepared by the
3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester
method as previously described
(24) . Shiga toxin was a gift
from Dr. J. Kozlov (W. A. Engelhardt Institute of Molecular Biology,
Moscow) and Dr. J. E. Brown (U. S. Army Medical Research Institute of
Infectious Diseases, Frederick, MD). Furin was a gift from Dr. G.
Thomas (Vollum Institute, Oregon Health Sciences University, Portland,
OR).
Cell Culture
LoVo cells (obtained from
ATCC) were grown in HAM's F-12 media supplemented with 10% fetal
calf serum. LoVo cells transfected with mouse furin (LoVo/LF36) and
with control vector (LoVo/neo) were a gift from Dr. E Mekada (Kurume
University, Kurume, Fukuoka, Japan). LoVo/LF36 cells are called
LoVo/fur cells in this paper. Establishment of these cell lines are
described elsewhere
(25) .
Assay of Shiga Toxin Cytotoxicity
Cells
were transferred to 24-well plates at a density of 3 10
cells/well 2 or 3 days prior to the experiments. The cells were
incubated with increasing amounts of toxin for the indicated time.
Then, the cells were incubated 10 min in Hepes medium with 1 µCi/ml
[
H]leucine and no unlabeled leucine. The medium
was removed, and the cells were washed two times in 5% trichloroacetic
acid. Then, the cells were dissolved in 0.1 M KOH, and the
radioactivity was measured.
Cleavage of Shiga Toxin by Cultured
Cells
Cells were seeded out with a density of 6
10
cells/well in 12-well plates 3 days prior to the
experiments. On the second day, 1 mM butyric acid was added to
some of the wells, and the cells were incubated further for 48 h before
the experiments. The cells were washed in Hepes medium and preincubated
with BFA (2 µg/ml) or protease inhibitors for 20 min at 37 °C;
then,
I-labeled Shiga toxin was added, and the incubation
was continued for the indicated period of time. Control cells (no
inhibitors) were incubated in parallel. The cells were then washed
three times with phosphate-buffered saline and lysed in 1% Triton (1%
Triton X-100, 20 mM Hepes, 140 mM NaCl, 1 mM
phenylmethanesulfonyl fluoride, pH 7.4) on ice for 20 min. The cell
lysates were transferred to Eppendorf tubes, nuclei were removed by
centrifugation, and proteins were precipitated for 30 min on ice in the
presence of 5% trichloroacetic acid. After centrifugation, the pellet
was washed in ether, dissolved in sample buffer containing
2-mercaptoethanol, and then subjected to SDS-PAGE. After
autoradiography, the bands corresponding to A and A
fragments of Shiga toxin were quantified by densitometry (model
300 A, Molecular Dynamics).
In Vitro Cleavage of Shiga Toxin by Purified
Furin
The cleavage was performed in a reaction volume of 25
µl containing 5 mM CaCl, 1 mM
2-mercaptoethanol, 100 mM buffer (sodium acetate, pH 5.0; MES,
pH 5.5-7.5; Hepes, pH 8.0), 10 ng of
I-Shiga toxin,
and 6 ng of purified furin. In a parallel experiment at pH 6.0, 5
mM EDTA was added instead of CaCl
. The reaction
mixture was incubated for 3 h at 30 °C, and the reaction was
stopped by adding SDS sample buffer with 2-mercaptoethanol. The samples
were boiled and subjected to SDS-PAGE. For use in toxicity experiments,
unlabeled Shiga toxin was cleaved by furin at a toxin concentration of
100 µg/ml at pH 6.0. The reaction mixture was the same as above,
but without the addition of 1 mM 2-mercaptoethanol. After the
incubation, the sample was frozen at -20 °C before use.
Acrylamide Gel
Electrophoresis
Electrophoresis was carried out as
described by Laemmli
(26) . After electrophoresis, the gels were
fixed for 30 min in 4% acetic acid and 27% methanol. For
autoradiography, Kodak XAR films were exposed to dried gels at
-80 °C.
NH
Shiga toxin cleaved by furin, as described above,
was applied to a protein sequenator (Applied Biosystems 477A). As
expected, the reaction mixture contained three peptides due to nicking
of the A-chain and the presence of the B-chain.
-terminal Sequence
Analysis
Processing for Electron
Microscopy
LoVo/fur cells grown as monolayers in T-24
flasks were incubated 2 h with Shiga toxin horseradish peroxidase
conjugate. The cells were fixed and processed for electron microscopy,
as earlier described
(24) .
Furin Cleaves Intact Shiga Toxin A-chain to
A
Shiga toxin has a trypsin-sensitive region near the
COOH terminus of the A-chain, containing the motif
Arg and A
Fragments in
Vitro
-Val-Ala-Arg
, the minimal recognition
sequence for furin
(19) . Upon cleavage, two disulfide-bonded
fragments A
(
27.5 kDa) and A
(
4.5
kDa) are generated. The pH optimum for proteolytic activity by furin is
variable depending on the substrate
(27) , and we therefore
measured the ability of furin to cleave Shiga toxin at different pH
values. As shown in Fig. 1, a soluble form of furin cleaves
intact Shiga toxin with a pH optimum of 5.5-6.0, generating
A
(
27.5 kDa) and A
(
4.5 kDa)
fragments (the A
fragment is in the front of the gel).
Furin requires calcium for enzymatic activity
(13) , and in
accordance with this, EDTA completely inhibited the cleavage at pH 6.0
(data not shown). To locate the cleavage site in the toxin, we
performed amino-terminal sequencing of furin-nicked A-chain. The
resulting sequence was Met-Ala- X-Asp-Glu-Phe-Pro, identical
with residues 252-258. This indicates that furin cleaves Shiga
toxin at the COOH-terminal side of Arg
, as expected.
Figure 1:
pH-dependent cleavage of
Shiga toxin by furin. I-Labeled Shiga toxin was incubated
with a soluble form of furin for 3 h at 30 °C at different pH
values (see ``Experimental Procedures''). The reaction
products were analyzed by SDS-PAGE (13.5%) and autoradiography.
Lane1, pH 7.0 without furin; lane2, pH 5.0; lane3, pH 5.5; lane4, pH 6.0; lane5, pH 6.5; lane6, pH 7.0; lane7, pH 7.5; lane8, pH 8.0.
Proteolytic Cleavage of Shiga Toxin by
Cells
To examine whether furin is involved in cleavage of
Shiga toxin in vivo, we used a stable, transfected cell line
of LoVo cells expressing furin (LoVo/fur) and a control cell
transfected with the vector alone (LoVo/neo). Cells were incubated with
I-labeled Shiga toxin and then subjected to SDS-PAGE and
autoradiography; the A
and A
fragments were
then quantified by densitometry. In LoVo/fur cells, ST-A was cleaved
rapidly into A
and A
fragments. After 1 h of
incubation, 40% of the cell-associated toxin was cleaved, while after 5
h, 80% was cleaved (Fig. 2, lanes8 and
9). In LoVo/neo cells, ST-A was cleaved much more slowly and
not as extensively. After a 1-h incubation cleavage was not observed,
while after 5 h of incubation, 30% of the cell-associated toxin
molecules were cleaved (Fig. 2, lanes2 and
3). The data suggest that in vivo, furin is the
cellular protease mainly responsible for cleavage of Shiga toxin.
However, other cellular proteases can also cleave Shiga toxin in cells
without furin, although much less efficiently.
Figure 2:
Processing of Shiga toxin in LoVo/neo and
LoVo/fur cells. The cells were preincubated with or without calpain
inhibitor ( Calp. Inh.) (100 µg/ml) or brefeldin A (2
µg/ml) in Hepes medium for 20 min at 37 °C. Then
I-toxin (100 ng/ml) was added, and the incubation was
continued for 1 or 5 h more. After the indicated period of time, the
cells were washed and lysed in buffer containing 20 mM Hepes,
150 mM NaCl, 1% Triton X-100, and 1 mM
phenylmethanesulfonyl fluoride, pH 7.2. Nuclei were removed by
centrifugation, and proteins in the supernatant were precipitated with
5% (w/v) trichloroacetic acid, washed in ether, and dissolved in sample
buffer containing 6% (v/v) 2-mercaptoethanol and analyzed by SDS-PAGE
(13.5%) and autoradiography. Lanes2-7,
LoVo/neo cells; lanes8-13, LoVo/fur cells. In
lane1,
I-labeled Shiga toxin that had
not been incubated with cells.
The observed
processing could occur at the cell surface, in a membrane-bounded
organelle, or in the cytosol. To study whether cleavage could occur at
the cell surface, the cells were depleted for ATP by incubation with
NaN and 2-deoxyglucose. Endocytosis is then inhibited, but
the toxin molecules can still bind to cell-surface receptors (data not
shown). In the presence of ATP-depleting reagents, there was
essentially no cleavage of cell-surface bound Shiga toxin in LoVo/neo
or LoVo/fur cells (data not shown), indicating that Shiga toxin is
cleaved after endocytosis. To further localize the site of cleavage, we
used the drug BFA, which disrupts the Golgi stacks in a number of
different cell lines
(28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and which also protects these cells against Shiga toxin
(34) . In BFA-treated cells, toxin molecules can still be
endocytosed and exposed to proteases in endosomes and TGN, whereas
retrograde transport through the Golgi cisterns and translocation to
the cytosol cannot occur. BFA protected also LoVo/neo and LoVo/fur
cells against Shiga toxin (data not shown), and importantly BFA
treatment strongly inhibited the processing of Shiga toxin in LoVo/neo
cells (Fig. 2, lanes4 and 5), whereas
there was no effect on the processing in LoVo/fur cells (Fig. 2,
lanes10 and 11). Thus, processing by furin
seems to occur in endosomes and/or TGN, while furin-independent
cleavage seems to occur in another location in the cells.
We
therefore decided to investigate whether calpain inhibitor would
interfere with cleavage of wild type Shiga toxin in the LoVo cells. The
inhibitor in fact strongly reduced the processing of Shiga toxin in
LoVo/neo cells (Fig. 2, lanes6 and
7), whereas in LoVo/fur cells the processing was not inhibited
(Fig. 2, lanes12 and 13). The calpain
inhibitor has also been reported to inhibit cathepsin B and L
(38) , which are also inhibited by leupeptin
(39) .
Indeed, leupeptin partly inhibited the processing of Shiga toxin in
LoVo/neo cells (data not shown), suggesting that cathepsins are
responsible for some of the nicking activity seen in the absence of
furin.
Furin-induced Nicking of Shiga Toxin Is Important for
Intoxication of Cells
To test whether cleavage of ST-A with
furin is important for intoxication of cells, we measured the ability
of intact and furin-nicked Shiga toxin to intoxicate Vero cells and
LoVo cells. Vero cells are highly sensitive to Shiga toxin and cleave
intact toxin very efficiently
(10) , whereas LoVo
cells lack functional furin
(22) . In Vero cells
(Fig. 3 A), there was no detectable difference between
unnicked and nicked toxin, while in LoVo cells
(Fig. 3 B), nicked toxin was 30-50 times more
active than unnicked toxin, suggesting that cleavage is important for
intoxication of cells. We also studied the ability of unnicked and
furin-nicked Shiga toxin to inhibit protein synthesis in LoVo/neo and
LoVo/fur cells. In LoVo/neo cells, nicked toxin was 20-30 times
more active than unnicked toxin (Fig. 4 A), in accordance
with the results in Fig. 3 B. In LoVo/fur cells, where
the toxin is efficiently cleaved by furin, there was no difference
between unnicked and furin-nicked toxin (Fig. 4 B).
Unfortunately, LoVo/fur cells were less sensitive to Shiga toxin than
LoVo/neo cells. Such clonal difference in sensitivity to Shiga toxin
has been observed previously in other cell populations.
(
)
Since butyric acid is known to sensitize A431 cells to
Shiga toxin and induce increased transport of Shiga toxin to the TGN
and the endoplasmic reticulum
(20, 40) , from where
translocation to the cytosol may take place
(20, 40, 41) , we tested if butyric acid could
sensitize LoVo/neo and LoVo/fur cells. As shown in Fig. 5, that
was indeed the case, and both cell types became equally sensitive to
nicked Shiga toxin. In butyric acid-treated LoVo/neo cells,
furin-nicked toxin was still about 20 times more active than unnicked
toxin (Fig. 5 A), whereas in the sensitized LoVo/fur
cells, there was (as seen in the untreated cells) no difference between
unnicked and nicked toxin (Fig. 5 B). Electron
microscopical investigations confirmed that butyric acid induced Shiga
toxin transport to the Golgi apparatus in LoVo/fur cells (data not
shown). It should be mentioned that during culture, the LoVo/fur cells
occassionally became quite sensitive to Shiga toxin even without
treatment, and also then nicked and unnicked toxin were equally toxic
to the cells (data not shown). In parallel experiments, we also showed
that Shiga toxin was cleaved to about the same extent in butyric
acid-treated cells as in untreated cells and that protease inhibitors
inhibited cleavage in a similar way as in cells without butyric acid
treatment.
Figure 3:
Effect of unnicked and furin-nicked Shiga
toxin on Vero cells and LoVo cells. The cells were incubated in the
presence of increasing concentrations of Shiga toxin. The protein
synthesis was measured after 2 h in Vero cells and after 3 h in LoVo
cells, as described under ``Experimental Procedures.''
A, Vero cells; , unnicked toxin;
, nicked toxin.
B, LoVo cells;
, unnicked toxin;
, nicked toxin.
In A, bars indicate deviations between two
parallels.
Figure 4:
Effect of unnicked and furin-nicked Shiga
toxin on LoVo/neo and LoVo/fur cells. The cells were incubated in the
presence of increasing concentrations of Shiga toxin. After 3 h
incubation at 37 °C, the protein synthesis was measured as
described under ``Experimental Procedures.'' A,
LoVo/neo cells; , unnicked toxin;
, nicked toxin;
B, LoVo/fur cells;
, unnicked toxin;
, nicked
toxin.
Figure 5:
Effect
of unnicked and furin-nicked Shiga toxin on butyric acid-sensitized
LoVo/neo and LoVo/fur cells. Cells were incubated in the presence of 1
mM butyric acid for 48 h. Then, the cells were incubated with
or without calpain inhibitor (50 µg/ml) in Hepes medium for 20 min
at 37 °C. Increasing concentrations of Shiga toxin was added at 37
°C, and the protein synthesis was measured after 3 h as described
under ``Experimental Procedures.'' A, LoVo/neo
cells; , unnicked toxin;
, unnicked toxin and calpain
inhibitor;
, nicked toxin;
, nicked toxin and calpain
inhibitor. B, LoVo/fur cells;
, unnicked toxin;
, unnicked toxin and calpain inhibitor;
, nicked toxin;
, nicked toxin and calpain inhibitor.
As shown in Fig. 5 A, the calpain inhibitor
described above protected the butyric acid-treated LoVo/neo cells only
against unnicked toxin, and it had no effect on the intoxication of
LoVo/fur cells. On the other hand, the protease inhibitor leupeptin,
which similarly to the calpain inhibitor reduced nicking somewhat in
LoVo/neo cells, had no effect on the toxicity (data not shown).
-Val-Ala-Arg
) in the COOH-terminal
region in the loop bridged by a disulfide bond. Here, we provide
evidence that purified furin cleaves Shiga toxin A-chain between
Arg
and Met
in vitro, generating
A
(
27.5 kDa) and A
(
4.5 kDa)
fragments, and we show that the pH optimum for this reaction is
slightly acidic, pH 5.5-6.0. In LoVo cells that do not express
functional furin
(22) , Shiga toxin was cleaved very slowly and
to a small extent into A
and A
fragments, and
furin-nicked toxin was 20-30 times more toxic than unnicked
toxin, whereas in furin-transfected LoVo cells (LoVo/fur), Shiga toxin
was efficiently cleaved, and there was no difference in toxicity
between unnicked and nicked toxin. These results suggest that furin is
responsible for cleavage and activation of Shiga toxin in vivo but also implicate that other cellular proteases can cleave Shiga
toxin in cells without furin activity, although less efficiently.
and A
with trypsin
had increased enzymatic activity when inhibition of protein synthesis
was measured in a cell-free system, whereas there was no difference in
cytotoxic effect on HeLa S
cells. Kongmuang et al.(43) reported similar findings with Shiga-like toxin on
Vero cells and suggested that cleavage did not affect the biological
properties of the toxin. Similarly, we found no difference between
furin-nicked and unnicked Shiga toxin on Vero cells. This lack of
difference can be explained by the ability of these cells to cleave
Shiga toxin. It was recently shown that Shiga toxin
and
Shiga-like toxin type IIv are easily nicked in Vero cells
(10) .
Furthermore, we have produced mutants of Shiga toxin where the
furin-sensitive site has been eliminated. These mutants were less
efficient than wild type Shiga toxin in provoking rapid intoxication in
Vero cells, and the mutants where cleaved inefficiently compared to
wild type Shiga toxin.
Similar results were obtained by
Burgess and Roberts
(44) . All of these results indicate that
cleavage of Shiga toxin is important for intoxication.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.