From the Department of Biochemistry, School of Life
Sciences, Chungbuk National University, Cheongju 361-763, the
¶ Department of Biology, College of Science, Chonnam National
University, Kwangju 500-757, and the
Department of Life
Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
Received for publication, October 20, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Macrophage migration inhibitory factor (MIF) is
an important mediator that plays a central role in the control of the
host immune and inflammatory response. To investigate the molecular mechanism of MIF action, we have used the yeast two-hybrid system and
identified PAG, a thiol-specific antioxidant protein, as an interacting
partner of MIF. Association of MIF with PAG was found in 293T cells
transiently expressing MIF and PAG. The use of PAG mutants (C52S, C71S,
and C173S) revealed that this association was significantly affected by
C173S, but not C52S and C71S, indicating that a disulfide involving
Cys173 of PAG is responsible for the formation of
MIF·PAG complex. In addition, the interaction was highly
dependent on the reducing conditions such as dithiothreitol or
Macrophage migration inhibitory factor
(MIF)1 is a cytokine that
plays an important role in the regulation of host immune and inflammatory response (1-6). MIF is different from other cytokines in
that it is found preformed in MIF-expressing cells (7, 8). In addition,
MIF has been proposed to catalyze chemical reactions. Structural
studies of MIF have led to the suggestion that MIF bears a close
architectural similarity to microbial enzymes such as
5-carboxymethyl-2-hydroxymuconate, 4-oxalocrotonate tautomerase, and chorismate mutase, even though these proteins share little homology
in the amino acid sequence (9-12). On the other hand, based on the
amino acid sequence homology and structural similarity of MIF with
D-dopachrome tautomerase, which converts
D-dopachrome methyl ester to
5,6-dihydroxyindole-2-carboxymethylester, a D-dopachrome tautomerase activity also has been proposed for MIF (13, 14). However,
the physiological significance is currently unclear, because natural
substrates of MIF have not yet been found. MIF was demonstrated to
catalyze the keto-enol isomerization of both p-hydroxyphenylpyruvate and phenylpyruvate, a
hydroxyphenylpyruvate tautomerase activity (15). More recently, MIF has
been reported to possess a thiol-protein oxidoreductase activity (16),
which involves the reduction of insulin and 2- hydroxyethyldisulfide.
Several lines of evidence suggest that the intracellular redox
regulation might be involved in a variety of cellular functions, including cell proliferation, differentiation, tumor promotion, and
apoptosis (17, 18). Antioxidants govern the intracellular redox status.
PAG, a known thiol-specific antioxidant, is a member of the
peroxiredoxin (Prx) protein family, which was previously referred to as the alkyl hydroperoxide reductase/thiol-specific antioxidant family and is constitutively expressed in most human tissues, but its expression is higher in organs having a higher level
of proliferation (19, 20). Many Prx proteins are highly conserved in a
wide variety of mammalian species such as human, mouse, and bovine,
suggesting a biological importance of this type of enzyme (20-22).
Most Prx family members contain two conserved cysteines that correspond
to Cys47 and Cys170 of yeast thioredoxin
peroxidase. In yeast, both Cys47 and Cys170
were shown to be necessary for the formation of intermolecular disulfide bonds, and Cys47, but not Cys170, was
the primary site of oxidation by H2O2 (23).
Here we show that PAG binds specifically to MIF in vivo, and
we found that this interaction is dependent on the redox status in that
the interaction was significantly affected under reducing conditions.
Binding of PAG to MIF can repress the D-dopachrome tautomerase activity of MIF. Moreover, this binding resulted in the
suppression of the antioxidant activity of PAG.
Cell Lines and Reagents--
293T cells, a derivative of human
kidney embryonal fibroblast-containing SV40 T antigen, were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin-streptomycin and 1 mM
glutamine in an atmosphere of 5% CO2 at 37 °C (24). The
anti-FLAG (M2) antibody, dithiothreitol (DTT), aprotinin, and
phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co.
The mouse hybridoma cell line producing anti-GST antibody was
generously provided by Dr. D. S. Im (Korea Research Institute
of Science and Technology, Taejon). Rabbit anti-PAG antibody was raised
against a recombinant PAG produced in Escherichia coli (24).
Polyvinylidene difluoride membrane was purchased from Millipore Corp.
Yeast Two-hybrid Assay--
A genetic screen using the yeast
interaction trap was performed as previously described (25). The
full-length human MIF was cloned in-frame into an LexA coding sequence
to generate a bait plasmid, pEG202-MIF. A human HeLa cDNA library
in the pJG4-5 plasmid was screened for proteins that interact
with MIF using EGY48 yeast strain (Mat Cotransfection and in Vivo Interaction Assay--
293T cells
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum were plated in 6-well flat-bottomed microplates
(Nunc) at a concentration of 2 × 105 cells per well
the day before transfection. 1 µg of each plasmid DNA was transfected
into 293T cells with a calcium phosphate precipitation method. 48 h after transfection, cells were washed three times with ice-cold
phosphate-buffered saline and solubilized with 100 µl of lysis buffer
(20 mM Hepes (pH 7.9), 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl) containing 0.1% Nonidet
P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM
sodium fluoride, 2 µg/ml Expression Constructs--
The eukaryotic glutathione
S-transferase (GST) expression vector (pEBG) and pFLAG-CMV-2
vector with a FLAG epitope were obtained as described previously (25).
For expressing proteins in mammalian cells, full-length MIF and PAG and
their mutants were cloned into pEBG and pFLAG-CMV-2. The
BamHI/NotI and HindIII/XhoI
fragments of MIF cloned in pYESTrp (Invitrogen) were used to generate
pEBG-WT-MIF and pFLAG-WT-MIF, respectively. pEBG-WT-PAG containing a
full-length PAG cDNA was constructed in several steps. We first
cloned the EcoRI/XhoI fragment of PAG cloned in
pJG4-5 into pBacPAK9 (CLONTECH), and digested with
EcoRI plus NotI and subcloned into pBluescript KS
(Stratagene). Finally, the ClaI/NotI fragment of
the resulting plasmid was cloned into pEBG cut with ClaI and
NotI, yielding pEBG-WT-PAG. To generate the pFLAG-WT-PAG, a
PAG cDNA (pJG4-5-PAG) was subcloned as an
EcoRI/XhoI fragment into the
EcoRI/SalI site of pFLAG-CMV-2. Each of the
NdeI/BamHI fragments from four mutant PAG
proteins (C52S, C71S, and C173S), in which cysteines were individually
replaced by serines, cloned in pCR3.1 basic vector (Invitrogen) was
transferred to the pGEM-T vector (Promega), and each of the
SacI/BamHI fragments of the resulting plasmids
was cloned into pBacPAK9 cut with SacI and BglII
to yield pBacPAK9-C52S, pBacPAK9-C71S, and pBacPAK9-C173S. The
EcoRI/XhoI fragments from pBacPAK9-PAG mutants
were then transferred to the pFLAG-CMV-2 to produce pFLAG-PAG mutants
(pFLAG-C52S, pFLAG-C71S, and pFLAG-C173S). To generate pEBG-PAG
mutants, the EcoRI/NotI fragment of pBacPAK9-PAG mutants was first subcloned into pBluescript KS (Stratagene), and
digested with ClaI plus NotI and subcloned into pEBG.
Expression and Purification of Recombinant Proteins--
The
recombinant glutathione S-transferase (GST) fusion vector
containing human MIF or hexahistidine (His)-tagged vectors carrying human wild-type and mutant PAGs were constructed by subcloning the
cDNA fragments of MIF and PAGs into pGEX4T-1 (Amersham Pharmacia Biotech) or pQE30 (Qiagen), respectively. The resulting plasmids were
transformed into E. coli strains BL21(DE3) and BL21,
respectively. Transformed BL21(DE3) bacteria were grown in LB broth
containing 50 µg/ml ampicillin at 37 °C to
A600 of 0.5 and induced with 0.1 mM
isopropyl-1-thio- D-Dopachrome Tautomerase Assay--
To prepare
D-dopachrome substrate, 9.6 µl of 10 mM
D-3,4-dihydroxy phenylalanine and 6.4 µl of 20 mM sodium m-periodate were added to 800 µl of
an assay medium containing 10 mM sodium phosphate (pH 6.2)
and 1 mM EDTA, and, after 30 s, 5 µl of ethanol was
added to prevent a significant inhibition by the solvent. The reactions were analyzed at room temperature in a spectrophotometer with the
D-dopachrome substrate, 200 µg of human recombinant MIF,
and various concentrations of human recombinant wild-type and mutant PAG proteins. The tautomerase activity was measured by monitoring the
decrease in absorbance at 474 nm (26).
Antioxidant Assay--
The glutamine synthetase (GS) protection
by antioxidants was measured as described previously with a slight
modification (27). In brief, the 30-µl reaction mixture containing
1.5 unit/ml GS, 30 mM FeCl3, 100 mM
DTT, 500 mM Hepes (pH 7.0), and various concentrations of
PAG and MIF, was incubated at 37 °C for 15 min, and 500 µl of
MIF and PAG Physically Interact with Each Other in Mammalian
Cells--
In an effort to identify proteins that interacts with MIF,
the yeast two-hybrid system was employed using the LexA DNA-binding domain MIF as a bait. From 3 × 106 individual
transformants in a human HeLa cDNA library, ~100 clones specifically interacted with MIF. DNA sequencing and a BLAST algorithm of the positive clones revealed that 20 of 54 clones were found to
encode partial or full-length sequences of PAG (accession number X67951). To verify the interaction specificity of PAG, the PAG
library plasmid was rescued from the galactose-dependent
Leu+/lacZ+ yeast and reintroduced into the
original selection MIF bait strain as well as the other strains
containing nonspecific baits available in our laboratory. From
transformants, PAG cDNA was found to interact with both MIF and PAG
but not with the other baits tested, indicating that MIF interacts with
PAG specifically in the yeast two-hybrid system (data not shown).
To determine whether MIF and PAG interact in vivo, we
performed cotransfection experiments using GST- and FLAG-tagged
eukaryotic expression vectors. In these experiments, the wild-type PAG
and MIF were coexpressed as a GST fusion protein and a FLAG-tagged protein in 293T cells, respectively. The interactions of FLAG-tagged MIF proteins to the GST-PAG fusion proteins were analyzed by
immunoblotting with an anti-FLAG antibody. As shown in Fig.
1A, the MIF was detected in
the coprecipitate only when coexpressed with the GST-PAG but not with
the control GST alone. These observations support the results of the
two-hybrid screen and demonstrate that MIF physically interacts with
PAG in vivo. To further verify the interaction of MIF with
PAG in vivo, we performed coimmunoprecipitation experiments using 293T cells transiently transfected with the vector alone or
FLAG-tagged MIF. Endogenous PAG was immunoprecipitated from cell
lysates, and Western blot analysis shows that PAG was precipitated (Fig. 1B, lower). The binding of MIF was
subsequently analyzed using Western blotting with an anti-FLAG antibody
and, as shown in Fig. 1B, MIF is present in the PAG
immunoprecipitate. In addition, it has been shown that MIF crystallizes
as a trimer of three identical monomers (11, 28, 29). This observation
and the yeast two-hybrid specificity data (data not shown) led us to
examine the self-association of MIF in cells. To confirm the MIF
self-association, coprecipitations with glutathione-Sepharose beads
followed by immunoblotting with an anti-FLAG antibody were performed.
Cleared cell lysates were first purified with glutathione-Sepharose
beads, and then the coprecipitates were resolved by SDS-PAGE, and the
filters transferred were probed with an anti-FLAG antibody.
Accordingly, as shown in Fig. 1C, we found that GST-MIF
coprecipitated with FLAG-tagged MIF. These findings were consistent
with MIF existing as a homodimer in addition to its ability to
heterodimerize with PAG. Expression of amounts of GST- and FLAG-tagged
proteins in cells was also confirmed by protein immunoblot analysis
with the antibodies to GST and FLAG (Fig. 1, A and
C, lower and upper right,
respectively).
The MIF·PAG Complex Formation Is Affected by Cysteine Residues of
PAG--
Alignment of the peroxiredoxin (Prx) family members revealed
two highly conserved cysteine residues, which correspond to
Cys47 and Cys170 in yeast thiol-specific
antioxidant (TSA), and, especially, the N-terminal cysteine
(Cys47 in yeast TSA) was well conserved in all family
members (20, 21-23). To investigate the effect of conserved cysteine
residues of PAG on the MIF·PAG complex, 293T cells were transiently
cotransfected with wild-type GST-PAG and mutants C52S (corresponding to
Cys47 of yeast TSA), C71S, C83S, and C173S (corresponding
to Cys170 of yeast TSA), together with FLAG-tagged MIF, and
the complex formation between MIF and PAG was tested by coprecipitation
with glutathione-Sepharose beads followed by immunoblotting with an anti-FLAG antibody. As shown in Fig. 2,
expression of C173S mutant resulted in a dramatic decrease in the
complex formation, whereas wild-type PAG, C52S, C71S, and C83S did not
influence the significant change in the complex formation. These
results clearly demonstrate that the conserved Cys173 of
PAG plays a critical role in the association of MIF with PAG. Furthermore, these strongly indicate that the in vivo
association of MIF and PAG is not affected by PAG activity, because no
significant difference in the interaction was observed in the presence
of C52S mutant that lacks Cys52, which corresponds to
Cys47, essential for TSA activity, of yeast TSA (23). As a
control, expression levels of total PAG and MIF proteins were
determined by immunoblotting with antibodies to GST and FLAG using the
same blot and cell lysates, respectively, and the amount of PAG and MIF
in all lanes was similar (Fig. 2, middle and
bottom). These results indicate that the observed difference
in the complex formation was not due to differences in PAG and MIF
expression levels.
MIF·PAG Interaction Is Dependent on the Redox Status--
PAG, a
thiol-specific antioxidant, is known to exist as a homo- or heterodimer
using disulfide linkages of cysteine residues (24). Additionally, the
conserved Cys57-X-X-Cys60
motif of MIF was shown to form an intramolecular disulfide bond and the
oxidoreductase and macrophage activating activities of MIF were found
to be dependent on the presence of the cysteines in this motif (16, 30,
31). These observations led us to investigate the importance of the
redox status in MIF·PAG complex formation. The cell lysates from 293T
cells transiently cotransfected with GST-PAG and FLAG-tagged MIF were
treated with DTT, PAG Is a Negative Regulator of MIF Activity--
To establish
whether MIF·PAG complex formation is involved in the regulation of
functional specificities of both proteins, we first analyzed the effect
of PAG on MIF activity using a D-dopachrome tautomerase
assay. Human PAG contains two cysteines, at amino acid positions 71 and
83, in addition to the conserved Cys52 and
Cys173 (19). To investigate whether PAG indeed influences
the MIF activity and, if so, whether the conserved Cys52
and Cys173 are necessary for the regulation of MIF
activity, we used PAG mutants in which each of the cysteine residues
was individually replaced by serine. The corresponding recombinant
mutant (C52S, C71S, and C173S) and wild-type PAG proteins were
expressed in E. coli and purified from the soluble extract
of the bacterial cells. Although wild-type PAG, C52S, and C71S
decreased the D-dopachrome tautomerase activity of MIF in a
dose-dependent manner (Fig.
4A and data not shown), we did
not detect a significant decrease in MIF activity in the presence of
various concentrations of C173S (Fig. 4B). In addition,
similar results were obtained when MIF activity was measured by a
D-dopachrome tautomerase assay using equivalent amounts of
the PAG mutants (Fig. 4C). This observation is in agreement
with the in vivo binding data obtained with PAG mutants (see
Fig. 2), showing that MIF is present in the C52S and C71S but not in
the C173S. Taken together, these results indicate that the direct
binding of PAG with MIF is very important for regulation of the
D-dopachrome tautomerase activity of MIF and suggest that
MIF activity is negatively regulated by PAG.
MIF Is Also Required for Negative Regulation of PAG
Activity--
To determine whether the MIF, an interacting partner of
PAG, also regulates PAG activity using the same approach, His-tagged proteins, including MIF, wild-type PAG, and PAG mutants (C52S, C71S,
C173S) were expressed and purified from E. coli BL21 cells. Purified His-tagged proteins were assessed by reducing SDS-PAGE and
were all detected at the corresponding molecular sizes (data not
shown). PAG belongs to the Prx protein family of thiol-specific antioxidants, and the standard assay for thiol-specific antioxidants is
the protection of glutamine synthetase from inactivation by a
thiol/Fe3+/O2 mixed-function oxidase system
(27). In this assay, the recombinant wild-type PAG and C71S mutant
showed similar antioxidant activities, whereas mutants C52S and C173S
had no effect (data not shown). We next asked whether MIF could
influence the antioxidant activity of PAG. Increasing amounts of MIF
were added to 30 µg of wild-type PAG, which resulted in a 100%
protection of inactivation of glutamine synthetase. As shown in Fig.
5, this shifts the protection value from
100% (PAG alone) to 0% (5 µg of MIF), and the inhibitory effect of
MIF on the antioxidant activity of PAG is
concentration-dependent. These results clearly indicate
that MIF and PAG do not only interact physically, but that binding of
MIF also inhibits the antioxidant activity of PAG.
The present study demonstrates that MIF interacts with PAG
in vivo, and that the MIF·PAG interaction is dependent on
the redox status and the cysteine residues of PAG. In addition, we
found that both D-dopachrome tautomerase activity of
MIF and antioxidant activity of PAG were negatively regulated
by direct association of MIF with PAG.
MIF has been proposed as an unusual cytokine containing a combined
function both as a conventional cytokine and as an enzyme (1, 13, 16).
Recently, several studies have reported a variety of enzymatic
functions for MIF, including D-dopachrome tautomerase
(13, 14), phenylpyruvate tautomerase (15), and a thiol protein
oxidoreductase (16). D-dopachrome tautomerase was
discovered during the study of melanin biosynthesis (13). This enzyme
was found to have an amino acid sequence that is highly homologous with
that of MIF. It has previously been shown that the N-terminal proline
of MIF is required for the D-dopachrome tautomerase and
phenylpyruvate tautomerase activities (32-34). However, based on the
observed sequence homology with known thiol-protein oxidoreductases
(35), it is conceivable that MIF may exhibit a
cysteine-dependent enzymatic oxidoreductase activity and
that this activity is dependent on the redox-active conserved sequence motif (Cys-X-X-Cys). Analysis of the amino acid
sequence of MIF revealed that there was a conserved sequence motif
consisting of Cys57-Ala-Leu-Cys60 that was
found to be present in the catalytic center of thiol-protein oxidoreductases such as thioredoxin, protein disulfide isomerase, and
glutaredoxin. Consistent with this fact, recently a critical role of
the conserved cysteine sequence motif
(Cys-X-X-Cys) in the oxidoreductase and
macrophage-activating activities of MIF was reported (16). The
existence of an intramolecular disulfide bridge was also demonstrated
from studies of the conserved cysteine sequence motif, even though
previous studies had shown that neither recombinant E. coli-derived MIF nor native MIF from natural cell sources
contained an intermolecular disulfide structure (7, 16, 36). In
addition, MIF was crystallized as a trimer of three identical subunits
(11, 28, 29). This raises the possibility that MIF may heterodimerize
with other cellular proteins in addition to its ability to homodimerize
through disulfide linkages between cysteine residues, and that redox
regulation may be also involved in the control of this association. To
address this question, we sought to identify cellular target proteins
that directly associate with MIF. In this study, we report isolation of
PAG as an MIF-interacting protein. We performed the coprecipitation
experiments of transiently expressed MIF and wild-type PAG, as well as
PAG mutants, and demonstrated that MIF associates with wild-type PAG
and PAG mutants tested, except for C173S, in mammalian cells (Fig. 2),
suggesting that the conserved Cys173 of PAG plays a pivotal
role in the formation of intermolecular disulfide linkages between MIF
and PAG. However, we cannot rule out the other possibility that the
other conserved Cys52 is also involved in the
intermolecular disulfide linkages, because we are able to detect a
significant decrease in the association of MIF with C52S, although to a
somewhat lesser extent, by coprecipitation studies (Fig. 2). To gain
more insight into the roles of cysteine residues in the MIF·PAG
association, we examined the in vivo binding of MIF and PAG
under various redox conditions. As shown in Fig. 3, upon reducing
conditions the association was remarkably decreased in a
dose-dependent manner, suggesting that the in
vivo association of MIF and PAG may be mediated through disulfide
linkages of cysteine residues. Recent studies have suggested that MIF
and thiol antioxidants could be bridged through inter-chain disulfides
(24, 37, 38). It is necessary to determine whether the conserved
cysteine residues of PAG could influence homo- and heterodimerization
and whether this complex formation contributes toward the regulation of
functional activities of interacting partners.
To understand how the interaction contributes to the activity of the
respective proteins, the examination of how MIF or PAG binding
modulates the interacting protein function seems to be important. As
shown in this report, it can be concluded that PAG is a negative
regulator of MIF activity. Moreover, we confirmed this observation with
the PAG mutants such as C52S, C71S, and C173S. From these mutation
experiments, all mutants, except for C173S, resulted in a decrease of
D-dopachrome tautomerase activity of MIF (Fig. 4 and data
not shown). These findings can be explained either by a physical
binding of PAG to MIF or by an enzymatic function of PAG. However, the
fact that C52S mutant, but not C173S, indeed decreases the MIF
activity, together with our observation that both Cys52 and
Cys173 are equally critical for the antioxidant activity of
PAG (data not shown), does not favor the second model describing the
importance of the enzymatic function of PAG in the regulation of MIF
activity. In addition, it has recently been shown that several MIF
residues near the N-terminal proline are perturbed upon addition of
S-hexylglutathione and affected by
p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF (33, 38). Because our studies reported here
demonstrated that PAG could inhibit the D-dopachrome tautomerase activity of MIF, it is tempting to speculate that this
effect is mediated through the perturbation of the residues surrounding
the N-terminal proline on the three-dimensional structure of MIF by
direct binding of PAG. Based on our observed results, we imagine that
rather than direct contact with the residues surrounding the N-terminal
proline, the conformational effect that is caused by intermolecular
disulfide linkages between cysteine residues of PAG and MIF likely
plays a role that is important in the regulation of MIF
D-dopachrome tautomerase activity, probably by structural changes within the region near the N-terminal proline.
A recent study (30) demonstrated that Aop1, a human thioredoxin
peroxidase, and cyclophilin18 do not only interact physically, but binding of cyclophilin18 also stimulates the antioxidant activity of Aop1, suggesting an important role for the direct interaction in the
regulation of the antioxidant activity of thioredoxin peroxidases. To
test a possible influence of binding of MIF on the enzymatic activity
of PAG, we used a similar approach. As shown in Fig. 5, a dramatic
decrease was observed in the antioxidant activity of PAG when
increasing amounts of MIF were added to the PAG. This also indicates
that MIF negatively regulates the PAG activity. In this regard, the
mechanism of interaction of MIF and PAG will be the interest of future
study. Further understanding of the mechanism of this interaction will
result from the identification of other interacting proteins associated
with MIF or PAG and detailed analyses of the binding region in the
MIF·PAG interaction.
-mercaptoethanol but not in the presence of
H2O2. Analysis of the activities of the
interacting proteins showed that the D-dopachrome
tautomerase activity of MIF was decreased in a
dose-dependent manner by coexpression of wild-type PAG,
C52S, and C71S, whereas C173S was almost ineffective, suggesting that
the direct interaction may be involved in the control of
D-dopachrome tautomerase activity of MIF. Moreover, MIF has
been shown to bind to PAG and it also inhibits the antioxidant activity
of PAG.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
trp1 ura3-52
leu2::pLeu2-lexAop6(
GUAS leu2)). Yeast transformation
was performed by the lithium acetate method. Positive clones were
selected, and the cDNA inserts were recovered and sequenced with
dideoxy sequencing according to the manufacturer's instructions
(Amersham Pharmacia Biotech). A fish plasmid, pJG4-5 harboring PAG, was
transformed back into yeast along with either the bait plasmid or other
nonspecific bait plasmids to verify the specificity of the two-hybrid
assay. [*]åtrfend 1043163641[*]åtrfend 1043163642The
-galactosidase activity was used for selection of proteins
interacting with the MIF.
-1-antitrypsin, 2 mM sodium
pyrophosphate, 25 mM sodium
-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM
phenylmethylsulfonyl fluoride. Detergent-insoluble materials were
removed by centrifugation at 13,000 rpm for 15 min at 4 °C.
Approximately 80 µl of the cleared lysates was mixed with 15 µl of
glutathione-Sepharose beads (Amersham Pharmacia Biotech) and rotated
for 2 h at 4 °C. Beads were washed three times with the lysis
buffer. The bound proteins were eluted by boiling in SDS sample buffer,
subjected to SDS-polyacrylamide gel electrophoresis, and then
transferred to polyvinylidene difluoride membranes. The membranes were
probed with an anti-FLAG (M2) antibody and then developed using an
enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech).
-D-galactopyranoside at 37 °C for
4 h. Cells were collected by centrifugation and lysed by
sonication in a buffer containing 20 mM potassium phosphate
(pH 7.0). The bacterial lysates were centrifuged at 13,000 rpm for 40 min at 4 °C, and the recombinant GST-MIF fusion protein was purified
from the soluble extracts of bacterial lysates by affinity
chromatography on glutathione-Sepharose 4B columns (Amersham Pharmacia
Biotech). His-tagged proteins were induced by a modification of the
procedures described above. In brief, BL21 cells expressing His-tagged
proteins were grown at 37 °C to A600 of 0.7, induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside at 37 °C for
4 h, and lysed by sonication in a binding buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris HCl (pH 7.9). The clear lysates were incubated with
a charge buffer (50 mM NiSO4) and loaded in an
His-Bind Resin column (Novagen), equilibrated with the binding buffer.
The column was washed once with the binding buffer and once with a wash
buffer (60 mM imidazole, 500 mM NaCl, 20 mM Tris HCl (pH 7.9)), and finally eluted with an elution
buffer (1 M imidazole, 500 mM NaCl, 20 mM Tris HCl (pH 7.9)). The fractions from the elute
containing His-tagged proteins were pooled and concentrated with a
vivaspin-6 concentrator (Vivascience) following the manufacturer's instructions.
-glutamyltransferase assay mixture containing 150 mM
glutamine, 0.4 mM ADP, 20 mM potassium
arsonate, 20 mM NH2OH, 0.4 mM
MnCl2, and 50 mM imidazole HCl (pH 7.0), was
added and incubated at 37 °C for 10 min to measure the remaining
activity of GS. The reaction was terminated by adding 500 µl of stop
solution (55 g of FeCl3·6H2O, 20 g of
trichloroacetic acid, 21 ml of concentrated HCl per liter), and the GS
protection was measured at 540 nm.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of MIF with PAG and
self-association of MIF in vivo. A and
B, in vivo association of MIF with PAG.
A, GST alone (pEBG), as a control, and pEBG-PAG
(GST-PAG) were cotransfected with pFLAG-MIF
(FLAG-MIF) into 293T cells. After 48 h, cells were
extracted in a lysis buffer containing 0.1% Nonidet P-40. GST fusion
proteins were purified on glutathione-Sepharose beads (GST
puri.) and analyzed on a SDS-polyacrylamide gel, and the complex
formation (upper left, GST puri.) and the
FLAG-tagged MIF of the amount used for the in vivo binding
assay (upper right, Lysate) were determined by
anti-FLAG antibody immunoblot. The same blot was stripped and reprobed
with an anti-GST antibody (lower panel) to confirm
expression of the GST fusion protein (GST-PAG) and the GST
control (GST). B, 293T cells were transiently
transfected with the vector alone (CMV), as a control, or
FLAG-MIF, and lysed and immunoprecipitated with an anti-PAG antibody.
The immunoprecipitate of PAG was analyzed for the presence of MIF by
Western blot using anti-FLAG antibody. Bands representing
coimmunoprecipitating MIF by anti-PAG antibody are indicated
(upper left, IP: -PAG). The amount
of immunoprecipitated PAG was analyzed using anti-PAG antibody
(lower left, IP:
-PAG). Total cell
lysates were analyzed for MIF (upper right,
Lysate) and PAG (lower right, Lysate).
C, in vivo self-association of MIF. 293T cells
were cotransfected with pEBG or pEBG-MIF (GST-MIF), together
with pFLAG-MIF (FLAG-MIF). MIF was coprecipitated from cell
lysates using the glutathione-Sepharose beads (GST puri.)
and subsequently separated by SDS-PAGE. The binding of MIF was detected
by immunoblotting using anti-FLAG antibody.
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Fig. 2.
Effect of wild-type and mutant PAG proteins
on MIF·PAG interaction. 293T cells were transiently transfected
with the appropriate expression plasmids, and GST fusion proteins were
purified on glutathione-Sepharose beads (GST purification)
and resolved by SDS-PAGE, and visualized by ECL. A complex formation
between MIF and PAG was determined by Western analysis using anti-FLAG
antibody (top panel). The same blot was reprobed with an
anti-GST antibody to demonstrate the coprecipitation of an equivalent
amounts of the GST fusion proteins (middle panel), and the
expression level of FLAG-tagged proteins in total cell lysates was
analyzed by Western analysis using anti-FLAG antibody (bottom
panel).
-mercaptoethanol, or H2O2,
and the MIF·PAG complexes were then coprecipitated with glutathione-Sepharose beads and analyzed by immunoblotting with an
anti-FLAG antibody. As shown in Fig.
3A, reductants such as DTT and
-mercaptoethanol markedly decreased the amount of coprecipitated MIF, whereas H2O2, an oxidant, did not,
suggesting that the redox-dependent interaction of MIF with
PAG can occur in vivo. As a control, the expression levels
of the transiently expressed proteins were analyzed in total cell
lysates (for FLAG-tagged MIF, data not shown) and coprecipitates (Fig.
3A, c and d), and similar expression
levels were found for all lanes. Furthermore, a complex formation
between GST alone used as a control and MIF was not detectable (Fig.
3A, b). The redox-dependent
interaction of MIF and PAG in vivo was further confirmed by
transient expression of FLAG-tagged MIF in 293T cells (Fig.
3B). Endogenous PAG was immunoprecipitated using anti-PAG
antibody, and coimmunoprecipitation of MIF was analyzed by
immunoblotting with an anti-FLAG antibody. As expected, under the
reducing conditions the amount of coimmunoprecipitated MIF was also
decreased (Fig. 3B, a). As a control, the same
blot was incubated with an anti-PAG antibody showing that similar
amounts of endogenous PAG were immunoprecipitated (Fig. 3B,
b). Taken together, these data strongly suggest that the
in vivo association of MIF and PAG requires the
participation of cysteine residues.
View larger version (21K):
[in a new window]
Fig. 3.
Redox status-dependent
interaction of MIF and PAG. A, FLAG-tagged MIF
expression plasmid (FLAG-MIF) was transiently cotransfected
into 293T cells with pEBG (b, d), as a GST
control, or pEBG-PAG expression plasmid (GST-PAG)
(a, c). Purified GST fusion proteins immobilized
on the beads were treated with the indicated concentrations of DTT,
-mercaptoethanol (
-ME), or
H2O2, and the binding was determined as
described in Fig. 1. To verify the amount of GST fusion and FLAG-tagged
proteins in the coprecipitates, the purified GST-fusion proteins were
analyzed by immunoblotting with anti-FLAG (a, b)
and anti-GST antibodies (c, d). B, effects of reductants
(DTT and
-ME) and an oxidant
(H2O2) on the interaction of MIF
with endogenous PAG. 293T cells were transiently transfected with
FLAG-MIF, and MIF·PAG complexes were then immunoprecipitated with an
anti-PAG antibody and analyzed by immunoblotting with an anti-FLAG
antibody (a). The amount of immunoprecipitated PAG was
analyzed by anti-PAG antibody immunoblot (b).
View larger version (15K):
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Fig. 4.
PAG has an inhibitory effect on MIF
activity. D-Dopachrome tautomerase activity of
human recombinant MIF was spectrophotometrically measured by the method
as previously described (26) with slight modifications.
D-Dopachrome substrate was prepared as described under
"Experimental Procedures." Assays were carried out with
D-dopachrome substrate solutions containing 200 µg of MIF
and indicated concentrations of wild-type PAG (A) and mutant
C173S (B). PAG mutants were used at a concentration of 50 µg and compared with wild-type PAG at the same concentration
(C). 50 µg of wild-type PAG only without MIF (PAG
only) and 10 mM sodium phosphate buffer, pH 6.2, containing 1 mM EDTA (Buffer) were used as
negative controls. The data shown are the mean ± S.D. of
triplicate assays and are representative of at least three independent
experiments.
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Fig. 5.
PAG activity is negatively regulated by
MIF. Protection of glutamine synthetase (GS) against
thiol-specific oxidative inactivation was measured as described under
"Experimental Procedures." The extent of protection is expressed as
a percentage relative to the inactivation apparent in the absence of
PAG used in the assays. In this assay increasing amounts of MIF were
added to 30 µg of wild-type PAG. Control represents the GS protection
activity of wild-type PAG in the absence of MIF. The data shown are the
mean ± S.D. of triplicate assays and are representative of at
least three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. I. H. Kim, Department of Biochemistry, Paichai University, for helpful instructions on GS protection assay.
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FOOTNOTES |
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* This work was supported by the Korea Research Foundation (Grant KRF-99-041-D00405 D4013) and by the Regional Center for Health and Life Science, Chungbuk National University (Grants 98R-L-5 and 99R-L-11).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.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Biochemistry, School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea. Tel.: 82-43-261-3233; Fax: 82-43-267-2306; E-mail: hyunha@cbucc.chungbuk.ac.kr.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009620200
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ABBREVIATIONS |
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The abbreviations used are: MIF, macrophage migration inhibitory factor; TSA, thiol-specific antioxidant; Prx, peroxiredoxin; PAGE, polyacrylamide gel electrophoresis; PAG, proliferation-associated gene; GST, glutathione S-transferase; DTT, dithiothreitol; CMV, cytomegalovirus; GS, glutamine synthetase.
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