From the Laboratory of Cellular Protein Science,
Department of Molecular and Structural Biology, Aarhus University,
8000 Aarhus C, Denmark, and ¶ AstraZeneca, R&D
Mölndal, 431 83 Mölndal, Sweden
Received for publication, October 3, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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We have characterized the neutralization of
the inhibitory activity of the serpin plasminogen activator inhibitor-1
(PAI-1) by a number of structurally distinct organochemicals, including compounds with environment-sensitive spectroscopic properties. In
contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in
complex with urokinase-type plasminogen activator (uPA), active PAI-1
strongly increased the fluorescence of the PAI-1-neutralizing compounds
1-anilinonaphthalene-8-sulfonic acid and
4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid. The fluorescence
increase could be competed by all tested nonfluorescent neutralizers,
indicating that all neutralizers bind to a common hydrophobic area
preferentially accessible in active PAI-1. Activity neutralization
proceeded through two consecutive steps as follows: first step is
conversion to forms displaying substrate behavior toward uPA, and
second step is to forms inert to uPA. With some neutralizers, the
second step was associated with PAI-1 polymerization. Vitronectin
reduced the susceptibility to the neutralizers. Changes in sensitivity
to activity neutralization by point mutations were compatible with the
various neutralizers having overlapping, but not identical, binding
sites in the region around Plasminogen activator inhibitor-1
(PAI-1)1 is a fast and
specific inhibitor of the serine proteinases urokinase-type (uPA) and
tissue-type plasminogen activator (tPA) and, as such, an important regulator of extracellular proteolysis in turn over of extracellular matrix and in fibrinolysis (for reviews see Refs. 1 and 2). PAI-1 binds
with high affinity to vitronectin (for reviews see Refs. 3 and 4) and
may regulate cell migration and adhesion by inhibition of vitronectin
binding of integrins and the uPA receptor (5-10). The PAI-1 level in
malignant tumors is one of the most informative biochemical markers of
a poor prognosis (for reviews see Refs. 11 and 12), and PAI-1 seems to
be causally involved in tumor invasion and angiogenesis (13). A high
PAI-1 level in blood plasma is a risk factor for ischemic
cardiovascular disease and venous thromboembolism (for review see Ref.
14). PAI-1 is therefore a potential target for both anti-cancer and anti-thrombotic therapy.
PAI-1 belongs to the serpin superfamily. Serpins are composed of 3 During RCL insertion, the so-called small serpin fragment, consisting
of s1A, s2A, s3A, -helices D and E and
-strand 1A, known
to act as a flexible joint when
-sheet A opens and the reactive
center loop inserts as
-strand 4A during reaction with target
proteinases. The defined binding area may be a target for development
of compounds for neutralizing PAI-1 in cancer and cardiovascular diseases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets and 9
-helices. Serpins and their target proteinases form
stable complexes by interaction of the active site of the proteinases
with the reactive center peptide bond (P1-P1')
in the solvent-exposed, ~20-amino acid long peptide loop, the
reactive center loop (RCL) (for reviews see Refs. 2 and 15-17). There is both structural and biochemical evidence that complex formation is
associated with the P1-P1' bond being cleaved,
the active site Ser of the proteinase linked to the carboxyl group of
the P1 residue by an ester bond, the N-terminal part of the
RCL inserted as strand 4 of the large central
-sheet A (s4A), and
the proteinase translocated across the plane of
-sheet A, toward the
other pole of the molecule (18-27). Under specific in vitro
conditions, however, the ester bond is hydrolyzed, and the serpin
exhibits substrate behavior. This also leads to insertion of the
N-terminal part of the RCL as s4A (for a review see Ref. 2). RCL
insertion also takes place during transition to the inactive, latent
state, occurring spontaneously only in PAI-1 (28). Latent PAI-1 can be
reconverted to the active form by denaturation and refolding (29).
-helix F (hF) and the loop connecting hF and s3A
(the hF/s3A-loop), moves relative to the rest of the molecule, the
large serpin fragment, to make space for the RCL between s3A and s5A.
The regions around hD and hE were proposed to form a flexible joint
during RCL insertion (30) (Fig. 1). The
idea of flexibility of this region was supported by the observation that it contains a cluster of peptide bonds with differential susceptibility to nontarget proteinases in active, latent, and reactive
center-cleaved PAI-1 (31). Comparison of the x-ray crystal structures
of latent, active, and reactive center-cleaved PAI-1 (28, 32-34)
demonstrated directly the conformational changes. Vitronectin binds
near the flexible joint region (35-37) (see Fig. 1) and delays the
conversion of PAI-1 to the latent state (for reviews see Refs.
1-3).
View larger version (76K):
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Fig. 1.
The three-dimensional structure of
s5A, the small serpin fragment, and the flexible joint region of active
PAI-1. Amino acids of relevance are shown in wire frame
presentations. The residues substituted in the triple mutant of
Björquist et al. (50) (Arg-97, Arg-136, and Arg-139)
are shown in red. The additional residues substituted in
this study (Pro-94, Arg-97, and His-98) are shown in blue.
Residues implicated in the binding of vitronectin (Phe-130, Met-131,
Leu-137, and Gln-144) (35) are indicated in green. The
figure is a RasMol display based on the coordinates of the x-ray
crystal structure of a quadruple mutant of PAI-1 in the active form
(33). Please notice that the RasMol program uses the same signature for
-helices and the short 310-helix found in the hF/s3A
loop.
A number of compounds have been found to inhibit the reaction of PAI-1 with uPA and tPA (38-50). Among these are some organochemicals, including 1-dodecyl sulfuric acid (42, 43), the diketopiperazine derivative XR5118 (49), and the anthranilic acid derivative AR-H029953XX (50). The flexible joint region was implicated in the PAI-1 neutralizing activity of the latter, by the demonstration that Glu substitutions of three basic residues in hD and hE (see Fig. 1) protected PAI-1 against activity neutralization (50).
We have now studied the mechanism of action of a few PAI-1-neutralizing
organochemicals, including the negatively charged AR-H029953XX and
1-dodecyl sulfuric acid and the positively charged XR5118. Basic
biochemical knowledge about the mechanism of PAI-1 neutralization is
necessary for development of clinically applicable inhibitors of PAI-1
function in cancer and cardiovascular diseases. We have characterized
neutralizer binding sites and neutralizer-induced molecular changes of
PAI-1.
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EXPERIMENTAL PROCEDURES |
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PAI-1--
PAI-1 residues were numbered by the
1-proteinase inhibitor (
1PI) template
numbering system, based on the alignment of PAI-1 and
1PI by Huber and Carrell (15).
Natural human PAI-1 was purified in the latent form from serum-free conditioned medium of dexamethasone-treated HT-1080 cells by immunoaffinity chromatography (43). Wild type (wt) human PAI-1 was expressed in the Escherichia coli strain TG1 by the use of the expression vector pAlter®-Ex1 and purified from bacterial lysates by ion exchange chromatography on a CM-50 Sephadex column (51), followed by immunoaffinity chromatography (43). Amino acid sequencing of the purified protein gave the expected, slightly modified N-terminal sequence MHVHPPSYVAHL. Expression of recombinant wt and mutant human PAI-1 in the yeast Pichia pastoris and its purification from the conditioned medium in the latent form were performed as described (52). Human wt and PAI-1 R97E/R136E/R139E were expressed in CHO cells as before (50).
Latent PAI-1 was converted to the active form by denaturation in 4 M guanidinium chloride and refolding by extensive dialysis against phosphate-buffered saline (PBS; 0.01 M sodium
phosphate, pH 7.4, 0.15 M NaCl) at 0 °C. Active PAI-1
was stored at 80 °C. Reactive center-cleaved PAI-1 (form C) and
uPA·PAI-1 complex were prepared as described (31).
uPA-- Human uPA was purchased from Wakamoto Pharmaceutical Co., Tokyo, Japan. Low molecular weight uPA (LMW-uPA), with N terminus at Lys-136, was prepared as described (53). The molar concentrations of active native and LMW-uPA were determined by active site titration with p-nitrophenyl-guanidinobenzoate.
Miscellaneous Proteins and Materials--
Human
vitronectin, in its multimeric form, was from Becton Dickinson (Le
Pont-de-Claix, France). Bovine serum albumin (BSA), -galactosidase,
and porcine gelatin were from Sigma. Restriction enzymes and other
materials for DNA technology were as described (52). Oligonucleotides
were purchased from DNA Technology (Aarhus, Denmark). AR-H029953XX was
described before (50). XR5118
(3Z,6Z)-6-benzylidene-3-(2-dimethylaminoethyl-thio)-2-(thienyl)methylene-2,5-piperazinedione hydrochloride (49) was a kind gift from Dr. Thomas Frandsen, Finsen
Laboratory, Copenhagen, Denmark. The following chemicals were purchased
from the indicated sources: anthranilic acid (2-aminobenzoic acid),
1-decanesulfonic acid, decanoic acid, dodecanoic acid, deoxycholic
acid, flufenamic acid
(N-(3-(trifluoromethyl)phenyl)anthranilic acid),
1-nonanesulfonic acid, 2-propylpentanoic acid, and suramin (Sigma); ANS
(1-anilinonaphthalene-8-sulfonic acid), bis-ANS
(4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid, dipotassium
salt), 2-anilinonaphthalene-6-sulfonic acid, and DapoxylTM
sulfonic acid (Molecular Probes, Eugene, OR); the sodium salt of
1-dodecyl sulfuric acid (Merck); 1,3,6-naphthalenesulfonic acid (Acros
Organics, Geel, Belgium); pyro-Glu-Gly-Arg-p-nitroanilide (S-2444) (Chromogenix, Mölndal, Sweden); blue dextran (Amersham Pharmacia Biotech). All other materials were of the best grade available.
Fluorescence Measurements--
Fluorescence emission spectra for
ANS and bis-ANS in the absence and presence of PAI-1 were recorded with
a SFM 25 spectrofluorimeter (Kontron Instruments), using excitation
wavelengths of 386 and 395 nm, respectively. The emission was recorded
over the range of 400-600 nm. The change in fluorescence was measured
about 10 min after mixing PAI-1 with the fluorescent ligands in a
buffer of 0.1 M Tris-HCl, pH 8.1, at 14 °C, unless
otherwise indicated. The observed fluorescence intensities,
Fobs, were corrected for the dilution effect of
the added ANS and bis-ANS solutions, for the low background
fluorescence of ANS and bis-ANS in buffer alone, and for the inner
filter effect of the varying concentrations of ANS and bis-ANS. The
correction for the inner filter effect was performed using the
equation F = Fobs·((2.303·Ex·[ANS or
bis-ANS]T)/(1-10
Ex·[ANS
or bis-ANS]T)), where
Ex is
the molar extinction coefficient of ANS and bis-ANS at the excitation
wavelength; [ANS or bis-ANS]T is the total concentration of
ANS or bis-ANS; and F and Fobs are the corrected and the observed fluorescence intensities, respectively (54).
Estimation of the parameters for bis-ANS-PAI-1 binding was initiated by
determination of the fluorescence intensity per 1 M
PAI-1-bound bis-ANS, FM, as the slope of the line
relating the fluorescence intensity at 480 nm to the bis-ANS
concentration at 6 µM PAI-1 and bis-ANS concentrations
below 6 µM. The concentrations of PAI-1-bound bis-ANS at
any PAI-1 and bis-ANS concentrations were then calculated from
the fluorescence intensity measured at these conditions by
Equation 1,
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(Eq. 1) |
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(Eq. 2) |
The estimation of Kd for ANS-PAI-1 binding
was obtained by measuring the fluorescence intensity at 470 nm at
several ANS concentrations ([ANS]T) in the presence of 1 µM PAI-1 and expressing them as RFI1,
i.e. a fraction of the fluorescence intensity at saturating
ANS concentrations. Assuming a simple binding equilibrium, one arrives
at Equation 3,
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(Eq. 3) |
Estimation of the dissociation constants for nonfluorescent
neutralizers was done by competition studies, in which 0.5 or 1 µM PAI-1 was preincubated with ANS (100 µM), and the fluorescence intensity at 470 nm was then
recorded 10 min after addition of nonfluorescent competitors in various
concentrations. The fluorescence intensity at max was
expressed relative to that obtained in the absence of nonfluorescent
competitor (RFI2). Assuming competition of fluorescent and
nonfluorescent ligands for binding to one site, an excess of
nonfluorescent ligand I over PAI-1, and simple binding equilibria for
both ANS and I, the following Equation 4 is true.
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(Eq. 4) |
Measurements of the Effects of Neutralizers on Specific Inhibitory Activity of PAI-1-- The specific inhibitory activity of PAI-1, i.e. the fraction of the total amount of PAI-1 forming a stable complex with uPA at the conditions used, was measured by titration of PAI-1 against uPA in a peptidyl anilide assay, in the presence of several concentrations of each neutralizing compound. Shortly, PAI-1 was serially diluted with a buffer of 0.1 M Tris-HCl, pH 8.1, 0.25% gelatin at 37 °C, corresponding to concentrations between 0.02 and 40 µg/ml and a volume of 100 µl. To each dilution series, a particular neutralizing compound was added in a particular concentration and incubated with PAI-1 for 10 min at 37 °C. Portions of uPA solutions of the same temperature and in the same buffer were then added, corresponding to a final uPA concentration of 0.25 µg/ml, a final volume of 200 µl, and final PAI-1 concentrations between 0.01 and 20 µg/ml. Incubation was then continued until the process of inhibition of uPA had come to an end. Control experiments showed that this was in all cases achieved in less than 2 min. The remaining uPA enzyme activity was determined by incubation with the peptidyl anilide substrate S-2444 at 37 °C and measurement of the increase in absorbance at 405 nm. The specific inhibitory activity of PAI-1 was calculated from the amount of PAI-1 that had to be added to inhibit 50% of the 0.25 µg/ml uPA. The IC50 values for the compounds being tested, i.e. the concentrations causing a 50% reduction in the PAI-1 specific inhibitory activity, were determined from plots of the specific inhibitory activity against the concentration of the compounds in the final assay mixture.
Measurement of Time Course of Changes in the Specific Inhibitory Activity of PAI-1-- PAI-1 was incubated at various concentrations between 0.33 and 330 µg/ml in 0.1 M Tris-HCl, pH 8.1, 0.25% gelatin, at 0 or 37 °C, without or with neutralizers. After various times, samples were removed for assay of specific inhibitory activity, which in this case was performed by diluting all samples to a final PAI-1 concentration of 0.33 µg/ml, using the same buffer and the same temperature as in the original incubation. uPA was added to a final concentration equivalent to 90% of the activity of PAI-1 in the absence of neutralizers. The uPA solutions were without or with BSA in a concentration corresponding to a final concentration, in the assay, of 1%. After incubation for a time sufficient for the process of inhibition of uPA to come to an end (2 min), the remaining uPA activity was estimated with S-2444 and used to calculate the specific inhibitory activity of PAI-1 in the samples.
Analysis of Functional Behavior of PAI-1 by Reaction with LMW-uPA and SDS-PAGE-- After incubation at various conditions, samples of 5 µg PAI-1 were mixed with 10 µg LMW-uPA. After 2 min, the samples were precipitated with trichloroacetic acid and subjected to SDS-PAGE in gels with 6-16% polyacrylamide. The gels were stained with Coomassie Blue. In this analysis, inhibitory active PAI-1 will be recovered as a complex with LMW-uPA. PAI-1 exhibiting substrate behavior will be recovered as the large N-terminal fragment produced by reactive center cleavage, whereas the only 33 amino acid long C-terminal fragment will not be recovered by the gel system used here. Inert PAI-1, for instance the latent form, will be recovered in the position of native full-length PAI-1 (45). Control experiments (not shown) ensured that no further changes in the reaction between PAI-1 and LMW-uPA occurred by prolonging the incubation time beyond the routinely used 2 min.
Gel Filtration--
PAI-1 was analyzed by fast protein liquid
chromatography gel filtration on a Superdex 200 HR10/30 column
(Amersham Pharmacia Biotech) in 0.1 M Tris-HCl, pH 8.1, 0.5 M NaCl at 4 °C, using a flow rate of 0.4 ml per min. The
following marker proteins were used: BSA (Mr
67,000), murine IgG (Mr 150,000), and
-galactosidase (Mr 540,000). The void volume
was determined with blue dextran. Before the chromatography, the
samples were passed through a layer of 200 µl of BSA-coupled
Sepharose 4B and a filter with a pore size of 0.22 µm, resulting in
removal of more than 98% of the organochemicals present in the
samples. A quantitative estimation of the distribution of PAI-1 between
polymer and monomer peaks was obtained by cutting out the areas
representing the peaks on the recordings of A280
and weighing them.
Native Gel Electrophoresis-- The electrophoresis was performed at 4 °C in gradient gels with 5-15% polyacrylamide, in the absence of SDS (55). Coomassie Blue was added to the cathode buffer to a final concentration of 0.02% (56). The following marker proteins were used: BSA (Mr 67,000), beef heart lactate dehydrogenase (Mr 140,000), beef liver catalase (Mr 232,000), horse spleen ferritin (Mr 440,000), and hog thyroid thyroglobulin (Mr 669,000).
Molecular Graphics-- RasMol version 2.6 (Roger Sayle, Glaxo Research and Development, Greenford, Middlesex, UK) was used to display three-dimensional protein structures. The coordinate (Protein Data Bank) files corresponding to the x-ray crystal structure of active PAI-1 (33) were kindly provided by Dr. R. J. Read, Cambridge, UK.
Statistical Analysis--
Data were evaluated by Student's
t test, and differences in results with a p value
below 0.005 were considered statistically significant. Nonlinear
regression analysis, by the method of least squares, were performed by
the SigmaPlot program (Jandel Scientific Software, San Rafael, CA).
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RESULTS |
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Neutralization of the Inhibitory Activity of PAI-1 by Various
Compounds--
We measured the IC50 values for the effect
on the specific inhibitory activity of PAI-1 of a variety of
amphipathic compounds, including the negatively charged PAI-1
neutralizers 1-dodecyl sulfuric acid and AR-H029953XX (42, 43, 50) and
the positively charged PAI-1 neutralizer XR5118 (49). About half of the
compounds tested were found to neutralize PAI-1 in the concentration
ranges used, with IC50 values between 0.6 and 300 µM (Fig. 2).
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Routinely, the assays were performed with guanidinium chloride-activated HT-1080 PAI-1, but the compounds also neutralized guanidinium chloride-activated P. pastoris PAI-1, guanidinium chloride-activated CHO cell PAI-1, and spontaneously active E. coli PAI-1 (see also below). Routinely, we performed the experiments at pH 8.1, but the same results were obtained at pH 7.4 (data not shown).
ANS, AR-H029953XX, bis-ANS, 1-dodecyl sulfuric acid, and XR5118 were selected for further characterization, ANS and bis-ANS because the fluorescence of these compounds generally increases at transfer from a hydrophilic to a hydrophobic environment (57) and they therefore can be used for fluorimetric binding assays, AR-H029953XX, bis-ANS, 1-dodecyl sulfuric acid, and XR5118 because of their relatively low IC50 values.
Fluorimetric Analysis of Neutralizer-PAI-1 Binding--
In the
presence of 1 µM active PAI-1, the fluorescence emission
intensities of ANS in concentrations above 10 µM and of
bis-ANS in concentrations above 0.1 µM were strongly
increased over those attained in the absence of protein. The
wavelengths with maximal fluorescence intensity (max)
shifted from 490 to 470 nm for ANS and from 490 to 480 nm for bis-ANS.
In contrast, only a small fluorescence increase was observed with 1 µM latent, 1 µM reactive center-cleaved,
and 1 µM uPA-complexed PAI-1 (Fig.
3). These observations are consistent
with ANS and bis-ANS binding to a hydrophobic area preferentially
accessible in active PAI-1.
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At PAI-1 concentrations above 5 µM and bis-ANS
concentrations below 5 µM, the fluorescence intensity of
bis-ANS at 480 nm was independent of the PAI-1 concentration, showing
that practically all added bis-ANS was bound to PAI-1 at these
conditions. Therefore, the fluorescence intensity per 1 M
bound bis-ANS, FM, could be determined from the
bis-ANS concentration dependence of the fluorescence intensity at these
conditions (Fig. 4A). It was
then possible to use fluorescence intensity measurements to estimate
the concentrations of bound and free bis-ANS in the presence of lower
PAI-1 concentrations (0.125-1 µM), at which there is an
equilibrium between bound and free ligand. By analysis of the relationship between bound and free bis-ANS, we found a
Kd of 0.57 ± 0.29 µM and a
number of binding sites per PAI-1 molecule of 1.66 ± 0.62 (n = 12) (Fig. 4B and Table
I).
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With ANS, only a small fraction of the ligand added to µM concentrations of PAI-1 was bound to the protein, rendering calculations of the stoichiometry impossible. However, an estimate of the Kd was obtained by studying the ANS concentration dependence of the fluorescence intensity in the presence of PAI-1 (Table I).
To measure the dissociation constants for the binding to PAI-1 of the
nonfluorescent neutralizers, PAI-1 was preincubated with 100 µM ANS, and the fluorescence intensity was recorded after addition of increasing concentrations of the nonfluorescent compounds. AR-H029953XX, 1-dodecyl sulfuric acid, or XR5118 caused a decrease in
the ANS fluorescence intensity down to background levels. In contrast,
two non-neutralizing, nonfluorescent compounds, 1-nonanesulfonic acid
and 2-propylpentanoic acid, caused very little inhibition of the
fluorescence in concentrations up to 1 mM (Fig.
5). Ki values for the
nonfluorescent compounds were determined by analysis of the
displacement curves (Table I).
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Similar competition experiments were done with bis-ANS. The results obtained (not shown) were in full agreement with those obtained with ANS.
The fluorimetrically determined dissociation constants for ANS, bis-ANS, 1-dodecyl sulfuric acid, and XR5118 (Table I) were not significantly different from the corresponding IC50 values (Fig. 2). Only in the case of AR-H029953XX, the dissociation constant was somewhat lower than the IC50 value. This disagreement may be related to the irreversible reaction of PAI-1 with uPA leading to dissociation of AR-H029953XX·PAI-1 complex during the determination of PAI-1-specific inhibitory activity.
The results of the fluorimetric analyses are most readily explained by the hypothesis that all the tested neutralizers have overlapping binding sites.
Analysis of Activity Neutralization by Site-directed Mutagenesis-- The five neutralizers were tested on a series of PAI-1 variants, expressed in P. pastoris or CHO cells (Table II). The IC50 values for neutralization of P. pastoris and CHO PAI-1 wt were not significantly different from those for neutralization of HT-1080 PAI-1. The susceptibility to the neutralizers was changed by substitution of specific amino acids in the flexible joint region. The P94K substitution, in hD (see Fig. 1), caused a 10-fold increase in the IC50 for XR5118 but had no effects on the IC50 for the negatively charged neutralizers. The double substitution R97K/H98K was without effect. The triple substitution R97E/R136E/R139E (see Fig. 1) increased the IC50- value for AR-H029953XX from about 5 µM to more than 20 µM and increased the IC50 values for ANS-, bis-ANS-, and 1-dodecyl sulfuric acid neutralization about 3-fold, but did not affect the IC50 value for XR5118 neutralization. Conclusively, the IC50 values for all neutralizers were sensitive to mutations in the flexible joint region, consistent with the conclusion from the fluorimetric measurements of these compounds having overlapping binding sites. Still, the substitutions in the flexible joint region affecting the susceptibility to the negatively charged neutralizers had no effect on the susceptibility to the positively charged neutralizer and vice versa.
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Competition between Vitronectin and PAI-1 Neutralizers-- Preincubation of PAI-1 with vitronectin decreased its susceptibility to all five neutralizers but to a variable extent. Thus, the IC50 value for bis-ANS increased about 50-fold, to 27 ± 9 µM (n = 3), but the IC50 value for 1-dodecyl sulfuric acid increased only about 7-fold, to 110 ± 4 µM (n = 4). The IC50 value for XR5118 increased to more than 250 µM and that for ANS to more than 1000 µM.
Time Course of Changes of PAI-1-specific Inhibitory Activity-- The PAI-1-specific inhibitory activity was determined after incubations for various times at 0 or 37 °C, at various PAI-1 concentrations, without or with neutralizers in concentrations of severalfold the IC50 values and severalfold the PAI-1 concentrations. To distinguish between reversible and irreversible neutralization, we took advantage of the fact that none of the five neutralizers affected PAI-1 in buffers with 1% BSA (data not shown), in agreement with the ability of BSA to bind several molecules of a variety of hydrophobic compounds (58, 59). Accordingly, the assays of the specific inhibitory activity were performed as follows: 1) in the presence of a neutralizer concentration equal to that used in the incubation (reversible plus irreversible neutralization); and 2) in the presence of 1% BSA, the BSA being added at the same time as uPA, to remove free and reversibly bound neutralizers (irreversible neutralization).
Without neutralizers, active PAI-1 lost its activity, due to conversion to the latent form, with a half-life of 44 ± 9 min (n = 6) at 37 °C and of more than 5 days at 0 °C, without or with BSA in the assay and independently of the PAI-1 concentration (data not shown).
Fig. 6 shows representative experiments
on the time course of neutralizer-induced activity loss, with 1-dodecyl
sulfuric acid. By using assays without BSA, more than 85% of the PAI-1
activity was lost in less than 5 min at 0 as well as 37 °C. By using
assays with BSA, the activity loss was slower and less complete. The PAI-1 activity measured in the assays with BSA did not change by
increasing the BSA concentration from the routinely used 1% to 5% or
by prolonging the incubation time with BSA, before the addition of uPA,
from 0 to 10 min, indicating that the large excess of BSA rapidly
removed both free and PAI-1-bound neutralizer. A relatively fast and a
relatively slow activity loss could therefore be distinguished, the
fast one requiring the continued presence of neutralizer and the slow
one being irreversible upon removal of neutralizer. The rate of the
irreversible activity loss increased with increasing temperature and
increasing PAI-1 concentration. Thus, the rate-limiting step of the
irreversible activity loss seemed to be bi- or multimolecular.
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The time courses of the activity losses induced by the other negatively charged neutralizers had the same characteristics. Only, the rate of the ANS-induced irreversible activity loss did not depend on the PAI-1 concentration, indicating that the rate-limiting step was monomolecular in this case (data not shown).
The time course of the activity loss induced by the positively charged XR5118 was not significantly different in assays without and with BSA, showing that the XR5118-induced activity loss was totally irreversible. The rate of XR5118-induced activity loss was strongly temperature-dependent. We found no evidence that the rate of XR5118-induced neutralization increased with increasing PAI-1 concentration, suggesting that the neutralization process proceeded monomolecularly (data not shown).
Analysis of the Functional Behavior of Neutralizer-induced Forms of
PAI-1 by Reaction with LMW-uPA and SDS-PAGE--
The effect of the
neutralizers on the functional behavior of PAI-1 was analyzed by
reacting neutralizer-treated PAI-1 with a molar excess of LMW-uPA and
separating the reaction products by SDS-PAGE. A representative
experiment, with bis-ANS, is shown in Fig.
7. Without incubation with neutralizers,
most of the PAI-1 formed a stable complex with LMW-uPA, whereas minor
fractions were latent or exhibited substrate behavior, respectively
(Fig. 7, lane to the left). Complex formation was totally
abolished within 2 min of incubation of PAI-1 with bis-ANS at 37 °C.
An inactive form with substrate behavior predominated for the first 2 min, after which time PAI-1 was gradually converted to an inert form.
At 0 °C, complex formation was abolished more slowly, and substrate
behavior predominated for at least 2 h (data not shown). Identical
observations were done with ANS, AR-H029953XX, and 1-dodecyl sulfuric
acid. Neutralization by the positively charged XR5118 was at 0 °C
associated with a fast conversion to a form with a substrate behavior,
followed by conversion to an inert form, but at 37 °C, there was no
detectable increase in substrate behavior, but an immediate increase in
the amount of the inert form (data not shown). HT-1080 PAI-1 and
E. coli PAI-1 reacted in the same manner to all neutralizers
(data not shown).
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Characterization of Neutralizer-induced PAI-1 Forms by Gel
Filtration--
PAI-1 was preincubated at 0 or 37 °C in the absence
or the presence of neutralizers, for practical reasons with a
relatively high PAI-1 concentration (330 µg/ml), before being
analyzed by gel filtration. Without neutralizers, both latent and
active PAI-1 migrated as one major symmetrical peak, in a position
which by comparison to the migration of the marker proteins was that
expected for monomeric PAI-1 (Fig.
8).
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Preincubation of active but not latent PAI-1 with ANS, AR-H029953XX, bis-ANS, or 1-dodecyl sulfuric acid resulted in conversion of PAI-1 to larger species, the sizes of which increased with increasing incubation time, with a strongly temperature-dependent rate. Eventually, about 85% of the material migrated in the void volume (Fig. 8 and data not shown). Five hundred µM of the negatively charged, but non-neutralizing 1-nonanesulfonic acid and 2-propylpentanoic acid did not induce polymerization (data not shown). We concluded that the negatively charged neutralizers were able to induce PAI-1 polymerization.
In contrast, the positively charged XR5118 did not cause polymerization
(Fig. 8). Moreover, preincubation with XR5118 inhibited the
polymerization induced by bis-ANS (Fig.
9) or any of the other negatively charged
neutralizers (data not shown). Analyzing the XR5118 concentration
dependence of the inhibition under the assumption of competition
between XR5118 and the negatively charged neutralizers for a single
binding site, a Ki value could be calculated that
agreed well with the fluorimetrically determined dissociation constant
for XR5118 binding (Fig. 9 and Table I).
|
A representative analysis of the functional behavior of PAI-1 in
individual peaks of the gel filtration profiles is shown in Fig.
10. PAI-1 was incubated with 1-dodecyl
sulfuric acid for 30 min at 0 or 37 °C before gel filtration, both
incubations leading to complete neutralization when assayed in the
continued presence of neutralizer. The polymeric forms in the gel
filtration profile had little or no inhibitory activity, and with
increasing size, they contained an increasing fraction of inert PAI-1.
The monomeric peak seen after preincubation at 0 oC had
regained an activity similar to that of PAI-1 not exposed to
neutralizers, whereas the small monomeric peak left after incubation at
37 °C was devoid of activity. It should be noticed that at least a
fraction of the inert material in the monomeric peak is latent PAI-1
contaminating the preparation from the start.
|
Characterization of Neutralizer-induced Forms of PAI-1 by Native
Gel Electrophoresis--
PAI-1 was subjected to native polyacrylamide
gel electrophoresis after incubation at a concentration of 330 µg/ml
for 30 min at 37 °C without or with neutralizers (Fig.
11). Without neutralizers, PAI-1
migrated mainly as a monomer in the expected position relative to the
Mr markers. With the negatively charged
neutralizers, but not with XR5118, most PAI-1 was converted to
distinct, slower migrating bands. By comparison to the migration of the
markers, the PAI-1 bands seem to represent dimers, trimers, tetramers, etc. The gel system did not allow resolution of polymer species with an
Mr above ~300,000. We concluded that PAI-1
neutralization by the negatively charged neutralizers, but not XR5118,
is associated with formation of distinct PAI-1 polymers.
|
A Two-step Neutralization Mechanism-- Comparing the time course of the change of PAI-1-specific inhibitory activity (Fig. 6), the time course of the changes in the functional behavior (Fig. 7), the time course of polymerization (Fig. 8), and the functional behavior of individual peaks of the gel filtration profiles (Fig. 10), we concluded that PAI-1 neutralization follows variations over a basic two-step mechanism, by which neutralizer-complexed PAI-1 (N~PAI-1) is rapidly converted to a form exhibiting substrate behavior (N~PAI-1S) and subsequently to an inert form (N~PAI-1I):
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DISCUSSION |
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On the basis of the fluorimetric binding assays and the site-directed mutagenesis studies reported here, we concluded that four negatively charged (ANS, AR-H029953XX, bis-ANS, and 1-dodecyl sulfuric acid) and one positively charged (XR5118) organochemical PAI-1 neutralizers have overlapping but not identical binding sites in a hydrophobic area in the flexible joint region of PAI-1. Neutralization proceeds through two steps as follows: first conversion to forms with substrate behavior and second to inert forms. With the negatively charged neutralizers, but not the positively charged one, the second step was associated with PAI-1 polymerization.
Besides the five neutralizers studied in detail, a number of other amphipathic organochemicals also neutralized PAI-1 (Fig. 2). Some of the compounds, including 1-dodecyl sulfuric acid and deoxycholic acid, are generally used as detergents. However, the neutralizing effect of the five compounds studied in detail did not seem to depend on their detergent properties. First, the critical micelle concentration for 1-dodecyl sulfuric acid is 8 mM (60, 61), much higher than its 15 µM IC50 value for PAI-1 neutralization. Second, although structurally very different compounds were able to neutralize PAI-1 with a relatively low IC50 value, a certain specificity was observed. For instance, among the two carboxylic acids with unbranched aliphatic side chains, the length of the side chain appeared to be decisive for activity (Fig. 2). Third, the observations that XR5118 inhibited the polymerization induced by the negatively charged neutralizers (Fig. 9) are difficult to reconcile with a detergent mechanism. Fourth, the well defined PAI-1 polymers observed by native gel electrophoresis (Fig. 11) are in contrast to the expectancies from a detergent-induced, nonspecific aggregation.
Rather, our findings point to the five neutralizers exerting their effect by binding to overlapping sites in a hydrophobic area with a relatively low binding specificity. This notion is supported by the observation that all nonfluorescent neutralizers competed the binding of ANS and bis-ANS (Fig. 5) and by the largely good agreement between the IC50 values (Fig. 2) and the fluorimetrically determined dissociation constants (Table I). Likewise, our bis-ANS binding studies were compatible with a single class of binding sites with respect to binding affinity. Although the binding studies led to determination of the number of bis-ANS-binding sites per PAI-1 molecule to a value between 1 and 2 and therefore did not exclude the existence of two independent bis-ANS-binding sites, the induction of about 80% polymerization of 6 µM PAI-1 by 5 µM bis-ANS (Fig. 8) is a strong argument for a single binding and effector site for bis-ANS. Furthermore, the observed XR5118 inhibition of the polymerization induced by the negatively charged neutralizers (Fig. 9) is most readily explained by XR5118 and the negatively charged neutralizers having overlapping binding sites. Thus, it seems most likely that both the reversible and irreversible neutralizer effects are caused by binding of one neutralizer molecule per PAI-1 molecule. The possibility of several effector sites in PAI-1, each causing neutralization by a specific mechanism, seems remote.
It was previously suggested that the flexible joint region of PAI-1
contains a regulatory, hydrophobic, ligand-binding area (50). The x-ray
crystal structures of active PAI-1 and 1PI are in
agreement with a hydrophobic cavity in this area (33, 34, 62, 63). Our
present findings are consistent with the hypothesis that the five
neutralizers bind in this cavity. First, amino acid substitutions in hD
and hE changed the IC50 values. Second, the stronger
binding of ANS and bis-ANS to active than to latent, reactive
center-cleaved and uPA-complexed PAI-1 is in agreement with the
expectancies from the x-ray crystal structures, showing that the
distance between s2A and hD and hE decreases upon insertion of RCL as
s4A (28, 32-34, 40). The volume of the hydrophobic cavity in active
PAI-1 was estimated to 676 Å3 (63). Each of the bulky and
and in most cases rigid neutralizer molecules would therefore be
expected to occupy a large fraction of the volume of the cavity. Thus,
the binding competition between the negatively and the positively
charged neutralizers is not in conflict with the observation that the
amino acid substitutions in hD and hE, respectively, affected the
IC50 values for the negatively and the positively charged
neutralizers differently. This observation is, in fact, in full
agreement with their different structures and the charge changes
associated with the substitutions. We therefore suggest that the
variation in neutralization kinetics, in the induced molecular changes,
and in the differential response to vitronectin is due to different
neutralizers occupying different subsites within the same hydrophobic
area. In this way, the different neutralizers may have different
effects on the conformation of
-sheet A and thereby different
effects on the movements of the RCL and the tendency to polymerization.
This situation is reminiscent of estrogen receptor binding of estrogen
agonists, antagonists, and partial agonists, which have overlapping
binding sites, but induce different conformational changes in the
receptor protein (64-66).
Induction of serpin polymerization by small organochemical ligands is a
novel finding. Previously described serpin polymerization all implicate
the RCL of one serpin molecule forming an additional strand in
-sheet A or
-sheet C of another molecule. There is evidence for
three different modes of loop-sheet polymerization. 1) Heating or
mutations in the so-called shutter region can lead to polymerization by
the RCL of one molecule inserting as s4A of another molecule (for
reviews see Refs. 2 and 17). 2) The RCL of one molecule can insert as
s1C in another molecule, in which the intrinsic s1C has been extracted
(67-69). 3) The RCL of one molecule can hydrogen-bond to s6A of
another molecule and thus form an s7A (33, 34). It is striking that the
fluorescence observed by addition of ANS and bis-ANS to active PAI-1
remained high after incubation under conditions leading to
polymerization, whereas the fluorescence induced by addition of these
compounds to latent, reactive center-cleaved and uPA-complexed PAI-1
was much lower. This observation argues against the polymerization involving insertion of the RCL as s4A and favors the other modes of
polymerization. The mode of the polymerization described here is
clearly different from the recently reported PAI-1 polymerization induced in both latent and active PAI-1 at the strongly acidic pH of 4 (70).
An important perspective is the possibility of utilizing the
hydrophobic area as a target for anti-cancer and anti-thrombotic drugs.
To do so, strategies must be developed to circumvent certain problems
that have become apparent from the studies described here. First, the
strong binding to serum albumin common to all the compounds studied
here must be avoided. Second, since PAI-1 is expected to be bound to
vitronectin in vivo, pharmacologically potentially
interesting molecules must have a high affinity not only to free PAI-1
but also to PAI-1 in its vitronectin-associated state. Third, other
serpins have similar hydrophobic areas, and the specificity of PAI-1
neutralizers of potential interest for in vivo use must
therefore be ensured.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. T. Frandsen for the gift of reagents; Dr. Kees W. Rodenburg for help in constructing PAI-1 mutants; Dr. R. J. Read for providing the coordinate file for active PAI-1; Dr. Michael Ploug for valuable suggestions; Ida Thøgersen and Dr. Jan Enghild for their help with native gel electrophoresis; and Janne D. Krogh, Lissy Nielsen, and Tina B. Nielsen for competent technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from the Danish Cancer Society, the Danish Natural Science Research Council, the Novo-Nordisk Foundation, and the Danish Heart Foundation.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.
§ These authors contributed equally to this study.
To whom correspondence should be addressed: Dept. of Molecular
and Structural Biology, Aarhus University, 10C Gustav Wied's Vej, 8000 Aarhus C, Denmark. Tel.: 4589425080; Fax: 4586123178; E-mail:
pa@mbio.aau.dk.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009024200
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ABBREVIATIONS |
---|
The abbreviations used are:
PAI-1, plasminogen
activator inhibitor-1;
ANS, 1-anilinonaphthalene-8-sulfonic acid;
1PI,
1-proteinase inhibitor;
bis-ANS, 4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid;
BSA, bovine serum
albumin;
h,
-helix;
IC50, concentration giving 50%
inhibition;
max, fluorescence emission maximum
wavelength;
LMW-uPA, low molecular weight-uPA;
PBS, phosphate-buffered
saline;
s,
-strand;
tPA, tissue-type plasminogen activator;
uPA, urokinase-type plasminogen activator;
wt, wild type;
CHO, Chinese hamster ovary;
RCL, reactive center loop;
PAGE, polyacrylamide
gel electrophoresis.
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