From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050
Received for publication, January 31, 2001, and in revised form, April 25, 2001
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ABSTRACT |
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The fate of the catalytic subunit of the
Escherichia coli heat labile toxin (LTA1) was
studied after expression in mammalian cells to assess the requirement
for ADP-ribosylation factor (ARF) binding to localization and toxicity
and ability to compete with endogenous ARF effectors. A progression in
LTA1 localization from cytosol to binding Golgi stacks to
condensation of Golgi membranes was found to correlate with the time
and level of LTA1 expression. At the highest levels of
LTA1 expression the staining of LTA and both extrinsic and
lumenal Golgi markers all became diffuse, in a fashion reminiscent of
the actions of brefeldin A. Thus, LTA1 binds to the Golgi
and can alter its morphology in two distinct ways. However, point
mutants of LTA1 that are defective in the ability to bind
activated ARF were also unable to bind Golgi membranes or modify Golgi
morphology. Co-expression of mutants of ARF3 that regained binding
to these same mutant LTA1 proteins restored the localization and activities of the toxin. Thus, binding to ARF is
required both for the localization of the toxin to the Golgi and for
effects on Golgi membranes. A correlation was also seen between the
ability of LTA mutants to bind ARF and the increase in cellular cAMP
levels. These results demonstrate the importance of ARF binding to the
toxicity and cellular effects of the ADP-ribosylating bacterial toxin
and reveal that mutants defective in binding ARF retain basal
ADP-ribosylation activity but are the least toxic LTA1
mutants yet described, making them the best candidates for development
as mucosal adjuvants.
Escherichia coli heat labile
(LT)1 and cholera (CT) toxins
are heptameric protein complexes that share enzymatic functions, ADP-ribosyltransferase activity, and >80% primary sequence identity (1). During intoxication of mammalian cells, these enzymes catalyze the
covalent modification of the regulatory, stimulatory subunit of
adenylyl cyclase, Gs, resulting in persistent activation of
the cyclase (2-4). The high levels of cAMP generated in gut epithelial
cells promote the secretion of chloride and water into the gut lumen
and the resulting diarrhea that can be fatal if not treated. Neither
toxin is very specific in the choice of substrate and can transfer the
ADP-ribose moiety from NAD onto almost any arginine acceptor or even
water (NADase activity). Both the enzymatic activity and specificity
for Gs of each toxin can be shown in vitro to be
increased markedly (>50-fold) by the addition to the in
vitro reaction of the protein co-factor, ADP-ribosylation factor (ARF) (5, 6). ARFs were later found to be ubiquitous and essential
guanine nucleotide-binding proteins with a diverse array of functions
and activities in eucaryote, including the regulation of membrane
traffic (7, 8), phospholipase D (9, 10), and phosphatidylinositol
4-phosphate, 5-kinase activities (11, 12). The use of specific host
proteins by pathogenic organisms is unlikely to be arbitrary and has
led us to propose additional functional interactions between ARFs and
these bacterial toxins. For example, in a recent study we identified a
short sequence in the A2 subunit of LT with sequence
homology to the effector binding switch 2 domain of ARF that has weak
ARF mimetic activity (13). However, it has not yet been shown whether
ARFs play any role in the actions of these bacterial toxins in live cells.
LT and CT are AB5 type toxins in which the five identical B
subunits bind to receptors on target cells (1). The structure of the
holotoxin has been solved (14, 15) and reveals that the A subunit lies
in the center of a hole created by a ring of the five B subunits. The A
subunit is made up of the catalytic A1 and linker
A2 chains. Although it was originally thought that the B
subunit doughnut promoted penetration of the A subunits across the
plasma membrane and access to the Gs and adenylyl cyclase at the cell surface, it was later found that the toxins must transit the entire secretory pathway in reverse, probably making use of the
endoplasmic reticulum-retrieval (KDEL) sequence at the C
terminus of the A2 subunit (16-19). Access to the cytosol
is achieved only after traffic to the Golgi and then endoplasmic
reticulum (17, 20, 21). It was assumed that the toxin then finds its
way to the plasma membrane where it activates the Gs and
cyclase. The levels of toxin achieved in cytosol are normally quite low because toxicity prevents continued entry. This has limited the ability
to localize the A subunit in intact cells.
In this report we made use of a collection of recently described (13)
mutations in LTA and human ARF3 that are altered in their ability to
bind each other, to test the importance of ARF binding to toxin action
in intact cells. Our results provide the first experimental evidence
for a required role for ARFs in the actions of these toxins.
Materials--
The FuGene6 transfection reagent was obtained
from Roche Molecular Biochemicals. Ham's F-12 medium was
purchased from Life Technologies, Inc. Other reagents and chemicals
were obtained from Sigma.
ARF and LTA Mutants--
Each of the mutants used in this study
were constructed as described in Zhu et al. (13). All DNA
sequences were confirmed by sequencing.
Transient Expression of Wild Type and Mutant LTA in CHO
Cells--
Chinese hamster ovary cells were cultured in Ham's F-12
medium with 10% fetal bovine serum. The cells were grown in six-well culture dishes to 30-40% confluence for transient transfection with 1 µg of pcDNA3-myc-His vector carrying either LTA1 or
LTA1 mutants and FuGene 6 transfection reagents. Mutants of
LTA1 and human ARF3 were generated and subcloned into the
indicated vectors as described in Zhu et al. (13). The
expression of LTA1 proteins was confirmed by immunoblotting
with either monoclonal anti-Myc or polyclonal anti-His6
antibodies. The cells were fixed with 2% formaldehyde, permeabilized
with 0.2% saponin, and labeled with antibodies as described
previously (22, 23). The monoclonal ARF antibody, 1D9, was used for
detecting endogenous ARF (22). Mannosidase II (Mann II),
Data Collection--
The images were obtained using an Olympus
BX-60 with 100× objective and B-max filter cubes. Images were
processed using Image Pro software. Fluorescent cell staining studies
were each performed at least three times with similar results. The
levels of protein expression were compared by using the same
integration time within each experiment.
Intracellular cAMP Determinations--
Intracellular cAMP was
determined using the nonacetylation enzyme immunoassay procedure, as
provided by Amersham Pharmacia Biotech, according to the
manufacturer's directions. CHO cells were grown and transfected as
described above. 18 h post-transfection the cells were trypsinized
and washed twice with Ham's F-12 medium, and cell density was
determined. Cells (2.5 × 105) were added to wells of
a standard 96-well microtiter plate and spun at 1500 × g for 3 min, and the supernatant was discarded. The cell
pellets were resuspended in 200 µl of lysis reagent and agitated for
10 min. Samples (100 µl) and standards (100 µl; 12.5-3200 fmol/well) were incubated at 4° C for 2 h with 100 µl of
rabbit anti-cAMP antiserum before the addition of 50 µl of
cAMP-peroxidase conjugate, and the reaction was allowed to proceed for
an additional hour. The peroxidase substrate
(3,3',5,5'-tetramethylbenzidine) was added at room temperature, and the
colored product was allowed to accumulate for 1 h before stopping
with 1 M sulfuric acid. The optical density was determined
in a plate reader at 450 nm within 30 min. The standard curve was
plotted and filtered by the exponential decay equation
y = y0 + Aee LTA1 Expressed in Mammalian Cells Binds to Golgi
Membranes--
To study the fate of the catalytic subunit of LT,
LTA1, once in the cytosol, and assess the importance of ARF
binding to cell toxicity and the ability of LTA to compete with
endogenous ARF effectors, we transiently transfected a plasmid
(pCDNA3-LTA1-Myc-His6), carrying
C-terminally Myc- and His6-tagged LTA1 under
control of the strong, constitutive cytomegalovirus promoter, into CHO cells. The location of LTA1 within transfected cells was
determined by indirect immunofluorescence using a monoclonal antibody
directed against the Myc tag or polyclonal antibody to the
His6 epitope. Fluorescence intensity was used as a measure
of the level of protein expression and was classified as low,
intermediate, or high. As expected, the level of LTA1
expression in CHO cells was time-dependent (Fig.
1). 12 h after transient
transfection, we found 57% of expressing cells exhibiting low level
expression, 31% had intermediate expression, and 11% high expression
of LTA1 (Fig. 1). At later times (e.g. 24 h), the number of cells with low level of expression of
LTA1 decreased and almost disappeared, whereas the number
of cells with medium or strong expression of LTA1 increased
from 31 and 11% to 49 and 48%, respectively (Fig. 1). This temporal
progression is important to document both as an indication that the
expressed protein is stable in these cells and because later analyses
of phenotypes depend on similar temporal progressions and
correlations.
When LTA1 can first be detected (low level) we observed a
diffuse signal in cytosol (Fig.
2A) and markers of the Golgi
apparatus reveal the normal tight, perinuclear staining, as visualized
by immunostaining with Mann II (Fig. 2B), ARF Was Recruited to the Golgi by LTA1--
We have
previously reported (24) that overexpression of the ARF-binding protein
GGA1 caused increased accumulation of ARF at Golgi membranes through a
novel mechanism of feed forward activation. To begin to examine the
relationship between LTA1 and ARF binding to Golgi
membranes we first asked whether the presence of LTA1 at
Golgi membranes altered the staining of ARF on those same membranes. As
seen in Fig. 3, low to intermediate
levels of LTA1 consistently produced increases in the
intensity of staining of ARF at the Golgi, as visualized with the use
of the monoclonal ARF antibody, 1D9 (Fig. 3B). Note in this
panel that the surrounding cells not expressing LTA1 have
lower intensity staining for ARF at the Golgi. When the expression of
LTA1 was higher (Fig. 3C), the ARF staining behaved just like the other markers of the Golgi, and only the diffuse
staining was observed throughout the cytosol (Fig. 3D). Thus, like increased expression of GGA1, the presence of
LTA1 in cells can influence the activation status of the
ARF in the cell.
The Presence of LTA1 at Golgi Membranes Delays the
Response to Brefeldin A--
A central tenet in models of ARF action
is its ability to recruit soluble proteins onto membranes in concert
with its own translocation that occurs upon activation (GTP binding).
To test the requirement for ARF binding to LTA1
localization, we examined the sensitivity to brefeldin A. CHO cells
expressing LTA1 were treated with brefeldin A for different
times, then fixed, and stained with anti-Myc or anti-His6
antibodies against epitopes on the expressed LTA1 proteins
(Fig. 4, A and C)
or with Mann II (Fig. 4B), LTA1 Mutants Failed to Localize to the Golgi--
We
have recently described (13) a series of LTA1 mutants, both
point and deletion mutations, that have lost the ability to bind
activated ARF3. We made use of these mutants to ask whether LTA1 proteins with decreased affinity for ARF could still
bind to Golgi membranes. Each of the deletion and point mutants of LTA1 that are used below were previously shown (13) to be
expressed as soluble, folded proteins capable of forming specific
protein-protein interactions, at least in yeast cells. One deletion,
(1)LTA1, and three point mutants of
LTA1, D43G, N93I, and W179R, were cloned into the
pCDNA3-Myc-His6-based plasmid for expression in mammalian cells. CHO cells were transiently transfected with plasmids directing expression of LTA1 mutants, and the cells were examined
after 16 h. We observed levels of fluorescent intensity that were
comparable with those of low, medium, or high expressors, as described
above (data not shown). In each case only cytosolic staining was
observed when cells were probed with antibodies to the Myc tag on the
LTA1 proteins. In addition, markers of the Golgi
compartment were indistinguishable in transfected and untransfected
cells, revealing the typical, punctate, perinuclear Golgi staining
(data not shown). Thus, the loss of ARF binding correlated precisely
with the loss of Golgi staining in intact cells in four of four mutants tested.
A further test of the importance of ARF to LTA1
localization at the Golgi was provided by three point mutants of
LTA1. A flexible loop in LTA1 contains two
residues, Phe31 and Arg33 that make extensive
contacts with LTA2 in the holotoxin and are involved in
binding ARF (13, 15). In contrast, the intervening residue,
Asp32, faces away from the protein binding surface, and
thus mutations at this residue did not alter ARF binding (13). We
tested the ability of three mutants in the flexible loop, F31L, D32H,
and R33A, to influence LTA1 localization in CHO cells and
found that the two mutants that cannot bind ARF (F31L and R33A) show
only cytosolic staining (Fig. 5,
top left and bottom left panels) and did not
produce any changes in Golgi morphology, as indicated by staining for
Mann II (Fig. 5, right panels). In contrast, the mutation
with unaltered ARF binding ((D32H)LTA1) behaved like the
wild type or (E112D)LTA1 proteins with regard to Golgi
localization and disruption of Golgi structures when expressed at
higher levels (Fig. 5, middle panels). These data further
support the conclusion that the interaction between LTA1
and ARF is required for LTA1 to bind to Golgi membranes and
subsequently alter the morphology of the Golgi.
Gain of Interaction LTA1 Mutants Relocalized to Golgi
in the Presence of the Corresponding ARF Mutants--
In the study of
the binding sites for the ARF-LTA1 interaction (13), we
identified mutations in ARF3 that resulted in a loss of binding and
also used two of these mutants to screen for mutations in
LTA1 that had regained the ability to bind to the mutated
ARF3. It turned out that these mutants of LTA1 bound the mutated ARF3 but not wild type ARF3. Such pairs of mutated binding partners allowed a more rigorous test of the importance of ARF binding
to LTA actions in cells.
The Q71L mutation in ARF proteins produces a GTPase-deficient protein
that is more persistently active in cells. The second site
mutants, (V53M,Q71L)ARF3 and (I74S,Q71L)ARF3, are deficient in binding
or activation of LTA1 but retain interactions with at least
some other ARF effectors and also bind both GDP and GTP (13).
(Y145H)LTA1 and (Y149C)LTA1 were identified in
yeast two-hybrid screens as mutants that bind to (V53M,Q71L)ARF3 and
(I74S,Q71L)ARF3, respectively, but neither LTA1 double
mutant binds to the parental (Q71L)ARF3 (13). We expressed each of
these LTA1 mutants, either alone or in combination with the
corresponding ARF3 mutant to which it binds. As expected for toxins
that do not bind ARF, (Y145H)LTA1 and
(Y149C)LTA1 alone were found in cytosol as diffuse staining (Fig. 6, A and C,
respectively) and failed to co-localize with or alter the location of
Golgi markers (Fig. 6, B and D). However, the
co-expression of (Y145H)LTA1 with (I74S,Q71L)ARF3 (Fig.
6E) or (Y149C)LTA1 with (V53M,Q71L)ARF3 (Fig.
6G) resulted in the re-establishment of the Golgi
localization of each LTA1 protein and condensation of the
Golgi compartment (Fig. 6, F and H), comparable with that seen with wild type or (E112D)LTA1. Because
expression of (V53M,Q71L)ARF3, (I74S,Q71L)ARF3, or (Q71L)ARF3 caused
Golgi expansion, we used this change in Golgi morphology as an
indicator of cells expressing the mutant ARF. These observations
provide strong evidence that LTA1 binds to ARF directly and
in so doing is directed to and concentrated at the surface of Golgi
membranes and that subsequent to this interaction the morphology of the Golgi and its contents are first altered into a compressed area and
later the integrity is compromised in a fashion that resembles treatment with brefeldin A. Thus, each of the activities described for
LTA1 in cells was found to be dependent on its ability to bind ARF.
Changes in Cell Morphology upon Expression of
LTA1--
Another consequence of medium or high levels of
LTA1 but not low level expression was a change in cell
morphology from the flattened cuboidal shape, typical of CHO cells,
into elongated, spindle-shaped cells (Fig.
7, C and D). This
same response has been noted previously for externally applied LT or CT
toxins and has been shown to result from increased production of cAMP
(25). This explanation is likely and supported by the observation that at similar levels of expression the wild type LTA1 yielded
a higher percentage of cells that displayed the spindle shaped
morphology than did the(E112D)LTA1 mutant, which has
decreased ability to ADP-ribosylate Gs in vitro
and thereby activate adenylyl cyclase (26), although see below. These
results also indicate that the E112D mutation, although clearly
decreased in ADP-ribosyltransferase activity (26, 27), is still active
in live cells.
When co-transfected with both LTA1 and (Q71L)ARF3, cells
became much more asymmetric and dendritic in appearance as they sent out processes that extend to twice the diameter of the cell body or
more (Fig. 8, C, D,
and F), even in cells expressing low levels of
LTA1 and (Q71L)ARF3 (Fig. 8, A-C). Although
elongated, spindle shaped cells were prevalent with expression of
LTA1, and any mutant that bound ARF, the dendritic
processes were only observed with the expression of both
LTA1 and an activated ARF that bound the toxin. Note that
the intense staining of LTA1 seen in Fig. 8D is
the result of overexposure in capturing the image to visualize the
processes and is not the result of gross overexpression of the protein,
in contrast to other images shown.
The cells co-expressing both LTA1 gain of interaction
mutants and their corresponding ARF3 double mutants also showed
dendritic-like morphology, whereas the cells expressing these
LTA1 mutants alone did not (Fig.
9). Although the expression of (Q71L)ARF3
alone had dramatic effects on the morphology of the Golgi, it did not change the cell morphology (Fig. 9). The percentage of spindle-like cells was increased from 0 to 2% in cells expressing LTA1
mutants that lost ARF binding, to 91.7% of cells expressing
LTA1 alone (Fig. 9). Similarly, the percentage of
dendritic-like cells changed from 8.3% with expression of
LTA1 alone, to 87% of cells expressing both toxin and
(Q71L)ARF3 (Fig. 9). Again, we found that we could at least partially
restore this effect by pairing the LTA1 and ARF3 mutants that bound
each other. Thus, like the localization to the Golgi and consequences
to that organelle, the effects of the toxin to alter cell morphology
were also dependent on the direct binding of LTA1 and ARF
proteins.
Increases in Cellular cAMP Levels Are Dependent on LT Binding to
ARF--
The ability of wild type and mutants of LTA1 to
increase intracellular cAMP was determined directly, 18 h after
transfection of CHO cells with different combinations of
LTA1 proteins with or without mutants of human ARF3. As
shown in Table I, expression of
LTA1 results in a >30-fold increase in cAMP. The magnitude of this increase would be even more dramatic if we factored in the fact
that only about 10% of transfected cells express the toxin. The
NAD-binding site mutant (E112D) repeatedly produced increases in cAMP
levels that were at least 90% those of the wild type toxin, despite
being only 5-10% as active in in vitro assays of
ADP-ribosyltransferase activity. In contrast, the ARF binding mutants
(R33A)LTA1 and (Y145H)LTA1 had lost the ability
to increase cellular cAMP in
cells.2 However,
co-expression of the mutant pair that have at least partially recovered
interaction in two-hybrid assays, (V53M,Q71L)ARF3 and
(Y145H)LTA1, resulted in partial recovery recovery of cAMP responsiveness.
In this study we have expressed wild type and mutants of LTA
within the cytoplasm and followed the events that then unfold. More
toxin is likely expressed than would enter a eucaryotic cell during
pathophysiological conditions, thus creating an artificial situation.
However, the events described can clearly be linked to specific
protein-protein interactions and offer several insights into the events
that occur during intoxication of eucaryotic cells. The first
experimental evidence for the requirement of ARF proteins in the
actions of LT in live cells is provided. Through its binding to ARF the
LTA becomes concentrated at Golgi membranes where it can first alter
the morphology of the Golgi into a more condensed structure and later
disperse the Golgi stacks in a fashion highly reminiscent of the
actions of brefeldin A (28, 29). Finally, we identified point mutants
of LTA1 with reduced binding to ARF that resulted in
proteins that retained basal catalytic (ADP-ribosyltransferase) activity but lacked all evidence of toxicity to mammalian cells. Such
mutants make attractive nontoxic alternatives to the native toxins and
NAD-binding site mutants for development, should they retain mucosal
adjuvant properties (30-33).
A required role for ARF proteins in the intoxication and toxicity of LT
in mammalian cells is evident first from the finding that LTA mutants
that are deficient in binding ARF no longer 1) localize to the Golgi,
2) cause the condensation or dissociation of Golgi elements, 3) promote
changes in cell morphology to either elongated spindles or
dendritic-like outgrowths, or 4) increase intracellular cAMP levels.
Compelling evidence that the defects in LTA activity were a direct
result of the loss of ARF binding was provided by the finding that each
of these effects could be restored by the co-expression of LTA mutants
with mutants of ARF3 that bind to the mutant LTA proteins. Thus, we
conclude that the binding of LTA to ARF is required for localization
and activation of adenylyl cyclase in mammalian cells.
The complex routing of LT and CT along the (reverse) secretory path
leaves open the site within cells where the toxins catalyze the
covalent modification of Gs, although the presence of the latter at the plasma membrane still makes this the most likely site of
action. Early studies of CT and LT action included the assumption that
the toxins acted at the plasma membrane, where the receptors for the
toxins on the outside of cells and the Gs and adenylyl
cyclase targets were found on the cytosolic side. However, the finding
that once inside cells LTA concentrates at Golgi membranes offers an
alternative site of action. Although the expressed LTA accumulates at
the Golgi, it is clear that it is a soluble protein that can also
migrate to other sites in the cell but the dependence on ARF for cell
toxicity (see below) suggests that co-localization with an ARF is
required for activity. No major differences have been described in the
specific activities of the six mammalian ARF isoforms as co-factors for
CT or LT in in vitro assays so they appear equally likely to
act with the toxins in cells. Even though the bulk of the expressed
toxin is seen at the Golgi, where ARF1-5 can be found, there is likely sufficient toxin in cytosol or at the plasma membrane, where ARF6 predominates, to fully activate the Gs and cyclase there.
From examination of cells at different times after transfection with
plasmids directing expression of the toxin we have concluded that there
is a progression from 1) synthesis in the cytosol to 2) association
with a morphologically normal appearing Golgi to 3) induction of a
highly condensed Golgi (that contains all the extrinsic and lumenal
markers of this organelle) to (4) dispersion of the Golgi throughout
the cytosol, similar in appearance to the effects of brefeldin A. As
described above, we found that the association to the Golgi was
ARF-dependent and likely limited only by the diffusion of
the toxin to the site where activated ARF is most abundant. But what is
the explanation for the effects on Golgi morphologies?
The dispersion of Golgi is seen at later times and correlates with
higher levels of LTA expression. As indicated already the effect is
very similar to that described previously for the actions of brefeldin
A that result from the inhibition of ARF exchange factors and
consequent deficiency in activated ARF (34-36). We believe a similar
scheme can explain the dispersion of Golgi by LTA. But instead of
decreasing the available activated ARF by inhibiting the activity of
ARF exchange factor(s), the presence of high levels of LTA is predicted
to effectively compete with endogenous effectors that are required for
maintenance of Golgi integrity by simple mass action. The result would
thus be the same as that of brefeldin A, an insufficient amount of
activated ARF to bind and activate the key effectors that maintain the
normal Golgi morphology.
The condensation of the Golgi is a more difficult observation to
explain. At least three different things may influence or cause this
effect; increased expression of ARF effectors has been shown to
increase the amount of ARF at the Golgi (24), the presence of LTA and
ARF could activate Gs and adenylyl cyclase either at other
sites or potentially locally (see above), and the LTA may be competing
with endogenous effectors important to Golgi morphology (see above).
The increase in cAMP levels in these cells will activate protein kinase
A and may increase the binding of ARF to Golgi membranes, as described
by Martin et al. (37). Thus, two of these responses likely
produce more activated ARF on Golgi membranes. An excess of activated
ARF, by expression of the activating mutant (Q71L)ARF1, has been shown
to result in vesiculation of the Golgi (23), so it is possible that the
combination of increased ARF and PKA activities modulate the effects on
Golgi structure to this condensed structure. The presence of eight or
more ARF-binding partners on Golgi membranes (38) makes it risky to
ascribe this phenomenon to any one mechanism.
The effects of CT and LT on mammalian cells have been described for
over 25 years, and to date all of the toxic effects on cells or the
organism result from the increased production of cAMP. Although some
cultured cells are killed by increases in cAMP, others respond with
changes in cell shape or not at all. Changes in cell morphology,
e.g. in CHO cells, have been used throughout as sensitive
indicators of toxin action at the cellular level (25). The finding that
LTA mutants with reduced affinity for ARF have also lost this activity
is remarkable, given the higher levels of the LTA1 proteins
in cells induced to express the toxin, compared with those in which the
toxin must enter through the endocytic pathway. We conclude that the
loss of ARF binding has a more marked effect on the intracellular
activities of LTA than do mutations that cripple the catalytic site,
e.g. E112D (26, 27, 39) or cleavage of the A1-A2 subunits
(40).
CT and LT are the most potent mucosal adjuvants known, but toxicity to
mammals has slowed their clinical development (41, 42). Although
controversial, some studies have demonstrated the retention of potent
mucosal adjuvancy with mutants that are deficient in
ADP-ribosyltransferase activity (40, 43). However, toxicity of at least
some of these toxins is still problematic (31). The E112D mutant of LTA
has previously been shown to retain only 5-10% the in
vitro ADP-ribosyltransferase activity of the wild type protein
(26), so we were surprised to see only slight decreases in toxicities
on our cells and cAMP levels similar to those produced by the wild type
toxin. We concluded that the higher levels of toxin achieved in cytosol
in our system overcame the decrease in enzymatic activity and resulted
in full activation of adenylyl cyclase. Thus, the lack of effects of
our mutants, e.g. F31L, R33A, Y145H, or Y149C, which retain
ADP-ribosyltransferase activity but have lost the
ARF-dependent activity, is even more remarkable. We are
currently testing for retention of adjuvant properties of these mutants
and should one or more retain that activity they should immediately
become the most promising modified toxins for the development of
mucosal adjuvant therapies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP, and AP-1 antibodies were used as markers of the medial,
cis-, and trans-Golgi compartments, respectively.
Texas Red-conjugated anti-mouse IgG and fluorescein isothiocyanate-conjugated anti-rabbit IgG (Cappel) were the secondary antibodies used in immunofluorescence studies.
x/t,
where y0 stands for offset and A
stands for amplitude, using Origin 4.0 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The level of LTA1 expression in
CHO cells increased with time between 12 and 48 h. CHO cells
were transiently transfected with Myc-His6-LTA1
for different periods of time and labeled with antibodies to the Myc
epitope or Mann II, as described under "Experimental Procedures."
The levels of expression of LTA1 were determined by visual
inspection of the intensity of anti-Myc staining and were scored 12-48
h after transfection as weak, medium, or strong.
-COP, ARF, or
AP-1 (data not shown). Even at this low level of expression there was
often evidence of more concentrated staining of LTA1 around
the nucleus. At intermediate levels of expression (Fig. 2,
C-F), LTA1 was found to concentrate adjacent to
the nucleus, in one of two configurations. In one of these the
LTA1 was found to co-localize with Mann II staining that
could not be distinguished from untransfected cells. In the other the
LTA1 and Golgi markers had condensed into a single area
(Fig. 2E) that remained adjacent to the nucleus. Staining of
LTA1 and Mann II were largely superimposable at this
intermediate level of expression (Fig. 2F), although
fainter, diffuse staining of LTA1 in the cytosol was also
apparent. At the highest levels of LTA1 expression the
staining again became diffuse (Fig. 2G). However, with high
LTA1 expression the markers of the Golgi also became
diffuse (Fig. 2H). This dramatic change appears to include all stacks of the Golgi as markers for the cis-, medial, and
trans-Golgi (
-COP, Mann II, and AP-1, respectively) all
became diffuse. Such a response is reminiscent of that to the drug,
brefeldin A. The effects of brefeldin A result from the inhibition of
guanine nucleotide exchange factors for ARFs that leads to insufficient
ARF activity for maintenance of Golgi integrity. We conclude that there
was a progression in LTA1 localization from cytosol to
Golgi stacks to condensation of Golgi membranes into one area to a
dispersion of both LTA1 and the Golgi compartments that
correlated with the time and level of LTA1 expression.
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Fig. 2.
Patterns of localization of LTA1
in CHO cells. CHO cells were prepared for indirect
immunofluorescence 16 h after transient transfection with
Myc-His6-LTA1, as described under
"Experimental Procedures." Mouse monoclonal Myc and rabbit
polyclonal Mann II antibodies were used as primary antibodies to
identify the LTA1 and Golgi compartments, respectively.
Weak, medium, and strong levels of expression of LTA1 are
shown in A, C, E, and G,
respectively. The staining of the same cells for Mann II are shown in
the panels on the right. Note the co-localization
of LTA1 with Man II in B-F and the diffuse
staining of Mann II in H, seen at high levels of
LTA1 expression.
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Fig. 3.
Localization of LTA1 at Golgi is
ARF-dependent. Myc-His-LTA1 was
transiently transfected into CHO cells and indirect immunofluorescence
with the primary antibodies anti-His6 (A and
C) and 1D9 (B and D) for detecting
LTA1 and endogenous ARF in cells, respectively. Note the
increased staining of ARF at the Golgi in the one cell in A
that is expressing LTA1 in comparison with control cells in
the field shown in B.
-COP (Fig. 4D) or
ARF antibodies (data not shown). Untransfected cells in the same field
served as controls for the action of brefeldin A. The dissociation of
COP-I, visualized with the antibody against the
subunit, and
ARF are rapid, typically within 1-3 min, whereas that of lumenal Golgi
markers, e.g. Mann II, occurs later (typically 5-30 min).
The tight perinuclear staining can be seen in control cells that were
not exposed to the drug (Fig. 4, A and B). After
2 min of brefeldin A (10 µM) treatment, the staining of
Mann II in untransfected or transfected cells were unaffected and
remained in the condensed, perinuclear pattern typical for these cells
(data not shown). The staining of
-COP or ARF in untransfected cells
became diffuse within 2-3 min. In contrast, in those cells that had
expressed LTA1 and had a condensed Golgi the staining of
-COP (Fig. 4D), ARF, and LTA1 (not shown) were still evident at the Golgi. After 5 min or more of brefeldin A
treatment, the staining of all three proteins, as well as
LTA1 was diffuse (data not shown). Thus, the presence of
LTA1 and induction of a condensed Golgi confers a degree of
resistance to brefeldin A at concentrations that were sufficient to
cause the dissociation of ARF and COP-I and (later) dissolution
of the compartment. The finding that this partial resistance extends to
ARF and
-COP suggests that the toxin may be acting through a
stabilization or promotion of the active conformation of ARF (24). The
changes in Golgi morphology and actions of the toxin to increase
activated ARF while the brefeldin A acts in the opposite direction
makes clear-cut conclusions from this approach difficult, so a more definitive test of the role of ARF binding in toxin action was sought.
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Fig. 4.
Localization of LTA1 at Golgi is
brefeldin A-resistant. Transfected and control cells were either
untreated (A and B) or exposed to 10 µM brefeldin A for 2 min (C and D)
and then immediately fixed and indirect immunofluorescence with primary
antibodies to Myc (A and C), Mann II
(B), or -COP (D). Note the persistence of
staining of LTA1 and
-COP at the perinuclear region in
contrast to untransfected cells in the same field.
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Fig. 5.
Mutants of LTA1 that are
defective in ARF binding fail to bind to the Golgi. CHO cells were
transiently transfected with plasmids directing the expression of
(F31L)LTA1 (top panels), (D32H)LTA1
(middle panels), or (R33A)LTA1 (bottom
panels). The cells were doubly labeled with Myc (left
panels) and Mann II (right panels) antibodies. Note
that the two mutants defective in binding active ARF (F31L and R33A)
are present throughout the cytosol, but the mutant that retained ARF
binding (D32H) behaves like the wild type protein and co-localized with
Mann II.
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Fig. 6.
Co-expression of both LTA1
mutants and ARF mutants that have regained binding resulted in the
re-localization of LTA1 to Golgi membranes. CHO cells
were transiently transfected with plasmids directing expression of
(Y145H)LTA1 (A, B, E, and
F) or (Y149C)LTA1 (C, D, G,
and H) either alone (A-D) or with doubly mutated
activated ARF3 to which they bind, (V53M,Q71L)ARF3 (E and
F) or (I74S,Q71L)ARF3 (G and H). The
left panels were stained with the Myc monoclonal antibody to
visualize the LTA1, and the right panels were
stained with anti-Mann II. Note the loss of Golgi staining in
A and C and its recovery in E and
G.
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Fig. 7.
CHO cells expressing LTA1 have a
spindly cell shape. CHO cells were fixed, permeabilized, and
stained with Myc and Mann II antibodies 16 h after transfection
with the Myc-His6-LTA1 plasmid. A
shows low level of expression of LTA1, and B
shows the typical shape of CHO cells. C shows the medium
level of expression of LTA1, and D reveals an
elongated, spindle-like cells expressed with LTA1 under the
phase contrast microscopy.
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Fig. 8.
CHO cells extend dendritic-like processes
after co-expression of LTA1 and (Q71L)ARF3. CHO cells
were transfected with both LTA1 and (Q71L)ARF3 and 16 h later were fixed for doubly labeled indirect immunofluorescence to
visualize LTA1 (A and D) and Mann II
(B and E). C and F are the
same fields viewed by phase contrast. The level of LTA1 in
the cells shown in D is comparable with that shown in
A, but the image was overexposed to reveal the processes
extending from the cell body.
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Fig. 9.
Cells expressing LTA1 become
elongated, but cells expressing both ARF and LTA1 extend
dendritic-like processes. CHO cells were transfected with
plasmid(s) directing expression of the indicated proteins and prepared
for indirect immunofluorescence after 16 h, as described under
"Experimental Procedures." The percentage of cells displaying
spindly or dendritic-like morphologies were then determined by counting
100-200 cells. Spindly cells were defined as those with one axis at
least twice the length of the other (and typically much more than twice
the length). Dendritic-like cells were those with a thin process that
extended to a length at least equal to the diameter of the cell body.
Cells expressing (Y145H,E112D)LTA1, (Y149C,E112D)LTA1, and (E112D)LTA1
with or without the corresponding ARF mutants to which they bind
((I74S,Q71L)ARF3, (V53M,Q71L)ARF3, and (Q71L)ARF3, respectively) were
assayed separately. Those conditions with no bars evident had no
spindly or dendritic-like cells. This experiment was repeated twice,
and the data from one set are shown.
LTA mutants with defects in ARF binding are also unable to stimulate
the production of cAMP in CHO cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Kelly Moremen and Marilyn Farquhar for providing the mannosidase II antibody and Witold Cieplak, Jr. for the original LTA plasmid. The secretarial assistance of Wendy Smith-Oglesby is gratefully acknowledged as are the many helpful discussions with members of the Kahn laboratory.
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FOOTNOTES |
---|
* 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: Dept. of Biochemistry,
Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA
30322-3050. Tel.: 404-727-3561; Fax: 404-727-3746; E-mail:
rkahn@emory.edu.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M100923200
2 The volumes of cell extracts were held constant within each experiment to prevent cell debris from interfering with the assay. When the volume of the extracts assayed was increased (data not shown), the values for the empty vector and these LTA1 mutants were within the standard curve, allowing accurate values to be determined, and still there were no differences.
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ABBREVIATIONS |
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The abbreviations used are: LT, heat labile toxin; CT, cholera toxin; ARF, ADP-ribosylation factor; CHO, Chinese hamster ovary; Mann II, mannosidase II.
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REFERENCES |
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