From the Henry Ford Health Sciences Center, Division of Biochemical Research, Detroit, Michigan 48202-3450 and the § Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, B-3000, Belgium
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ABSTRACT |
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The serpin plasminogen activator inhibitor-1
(PAI-1) slowly converts to an inactive latent form by inserting a major
part of its reactive center loop (RCL) into its Plasminogen activator inhibitor-1
(PAI-1)1 is a serine
proteinase inhibitor of the serpin family, a class of inhibitory and noninhibitory proteins sharing a common tertiary structure. Its most
prominent features are the major, 5-stranded PAI-1 is the only serpin known to fold spontaneously to an inactive
latent form (7), and the t1/2 for this conversion is
9.5 h at 25 °C and pH 7.4 (6, 8). In this process, the P15-P3
portion of the RCL, which is normally exposed at the apex of the
molecule, is inserted as strand 4 in Two types of murine monoclonal antibodies against the human tPA·PAI-1
complex have been documented with respect to their effect on the
structure and function of PAI-1: one type inactivates PAI-1 so it
neither forms a covalent complex with its target enzymes nor is
hydrolyzed at the scissile bond; the second type inactivates PAI-1 by
making it a substrate for its target proteinases (12). The present work
focuses on one particular antibody of the first type, denoted MA-33B8,
which has been observed to inactivate PAI-1 rapidly (13).
Studies using a phage-displayed library of PAI-1 peptides or
site-directed mutagenesis have failed to identify the epitope for
MA-33B82 and the mechanism by
which it inactivates PAI-1 remains obscure. Binding of the antibody can
shield the reactive center bond in PAI-1 from proteinase by one of two
mechanisms. We must assume either that antibody binding interferes
sterically with binding of the proteinase or that antibody binding
induces a conformational change that translocates the reactive center
into a shielded position, which would be the case if the
active-to-latent conversion was triggered by the antibody.
We have previously shown that the S338C mutant PAI-1, with the cysteine
at P9 in the RCL, and labeled with NBD at the mercapto group (P9·NBD
PAI-1), responds to the spontaneous conversion to the latent form by a
marked increase and a distinct blue shift of the NBD fluorescence and
that these changes are caused by loop insertion (8). The fluorescence
enhancement is consistent with a translocation of the NBD probe from a
solvent exposed environment of the extracted RCL of active PAI-1 to the
more shielded position in the latent form between apolar groups in the
tip of the helix F loop and Using either the fluorescence increase of P9·NBD PAI-1 or the
inactivation of wild type PAI-1 as a signal, we measured second order
rate constants for MA-33B8 binding of the order of 104
M General Conditions, Reagents, and Procedures--
If not
otherwise indicated, spectral and kinetic measurements were performed
at pH 7.4 and 25 °C in a reaction buffer containing 0.1 M HEPES, 0.1 M NaCl, 1 mM EDTA,
0.1% polyethylene glycol 8000. Fluorescence measurements were
performed using an SLM 8000 spectrofluorometer. The excitation
wavelength used for studying NBD fluorescence was 480 nm. Sample
cuvettes were coated with polyethylene glycol 20,000 to reduce protein
adsorption. Protein analysis by SDS-PAGE was performed under
nonreducing conditions according to Laemmli (17). Pancreatic elastase
and analytical grade buffer chemicals were from Sigma. Commercial USP
heparin (Mr 16,000) was from Diosynth BV (Oss, The
Netherlands), and native vitronectin was from Dr. D. Mosher (University
of Wisconsin). SpectrozymetPA
(methyl-sulfonyl-D-cyclohexyltyrosyl-glycyl-L-arginine-p-nitroanilide) was from American Diagnostica Inc. (Greenwich, CT). Chromatography materials were from Amersham Pharmacia Biotech. The octapeptide NAc-TVASSSTA was provided by the University of Michigan
Protein Facility (Ann Arbor, MI).
PAI-1 Variants and tPA--
Human wild type PAI-1 was expressed
in Escherichia coli using the expression vector pET24d (9).
The T333R (P14 Arg) and S338C (P9 Cys) PAI-1 single mutants, as well as
a PAI-1 carrying both mutations, were obtained by site-directed
mutagenesis as described elsewhere (9). PAI-1 was purified and
separated into the active and latent forms according to a previous
report from this laboratory (18). PAI-1 protein concentrations were
measured at 280 nm, using an extinction coefficient of 0.93 ml
mg
Unlabeled and P9·NBD PAI-1 were complexed with the octapeptide
NAc-TVASSSTA as described elsewhere (6), and excess peptide was removed by gel filtration on a PD-10 column. In the preparation with native PAI-1, latent PAI-1, formed in the process, was removed by
chromatography on phenyl-Sepharose (18). Analysis of the product by
SDS-PAGE, before and after addition of tPA, indicated that it was free
of active PAI-1 and contained less than 1% (latent) PAI-1 that was not
hydrolyzed by tPA. The PAI-1·octapeptide complex is very stable and
does not release the octapeptide even during prolonged dialysis (6) or
after cleavage of the RCL by plasminogen activators, as demonstrated
below. PAI-1 hydrolyzed at the P3-P4 peptide bond in the RCL was
prepared with elastase, using 50 µM PAI-1 and 50 nM enzyme. Elastase was removed by affinity chromatography on immobilized heparin (9), which binds PAI-1 but not elastase. The
tPA·PAI-1 complex was generated by incubation of PAI-1 (5 µM) with two-chain tPA (8 µM) for 30 min in
the reaction buffer at room temperature. Excess tPA was inactivated by
treatment with p-amidino-phenylmethylsulfonyl fluoride (0.1 mM).
Human recombinant tPA (Activase®) was a gift of Dr. B. Keyt (Genentech, South San Francisco, CA). The predominantly
single-chain enzyme was converted to the two-chain form by treatment
with immobilized plasmin (18).
MA-33B8--
The monoclonal antibody was raised against the
tPA·PAI-1 complex as described previously (12). The IgG fraction was
purified from ascitic fluid by affinity chromatography on protein
A-Sepharose (20). The antibody protein concentration was determined at
280 nm, using an extinction coefficient of 1.3 ml mg Effect of MA-33B8 on the Rate of PAI-1
Inactivation--
Unlabeled wild type and P9·NBD PAI-1 (0.3-0.5
µM) were incubated separately with MA-33B8 (2.8-4.2
µN) in reaction buffer at room temperature. At timed
intervals, a sample from each incubation was withdrawn and added to
excess tPA. Residual active PAI-1 was determined based on the
depression of the activity of tPA toward the chromogenic substrate
SpectrozymetPA and plotted against time.
P9·NBD PAI-1 Fluorescence Spectra--
The latent form of
P9·NBD PAI-1 was obtained by incubation of active P9·NBD PAI-1 (2.5 µM) in a sealed and foil-wrapped Eppendorf tube at
25 °C for 5 days at pH 8.2 in 0.1 M Bicine, 0.1 M NaCl, 0.1% sodium azide (6, 8). The content of the tube
was assayed for residual inhibitory activity and analyzed by SDS-PAGE
after incubation with tPA at pH 7.4. Antibody-induced inactivation of the labeled mutant was achieved by incubating P9·NBD PAI-1 (0.1 µM) with excess MA-33B8 (0.4 µN) and was
complete after 20 min. Active P9·NBD PAI-1 and the spontaneously
inactivated and MA-33B8-inactivated P9·NBD PAI-1 were adjusted to 0.1 µM in the reaction buffer, and the 500-600 nm
fluorescence emission spectrum of each species was recorded. The effect
of MA-33B8 (0.4 µN) on the NBD fluorescence of
spontaneously latent P9·NBD PAI-1 (0.1 µM), as well as
on that of the octapeptide-complexed and P14 Arg variants of P9·NBD
PAI-1 (0.1 µM), was studied by recording the respective
emission spectrum before and after adding antibody. The P9·NBD PAI-1
used in this study was obtained from the same stock.
Kinetics of MA-33B8 Binding Measured by P9·NBD PAI-1
Fluorescence--
The rate of the fluorescence increase induced by
treating P9·NBD PAI-1 with MA-33B8 was measured at 529 nm as a
function of antibody concentration up to 3 µN, keeping
P9·NBD PAI-1 at 0.03-0.3 µM to maintain first order
conditions. The corresponding rate constants were evaluated by
nonlinear least squares fitting of a single exponential to the
fluorescence progress curves. The apparent second order rate constant
for antibody binding was determined by plotting the rate constants
against the concentration of MA-33B8.
Competitive Binding of P9·NBD PAI-1 and Unlabeled PAI-1 Forms
to MA-33B8--
P9·NBD PAI-1 and MA-33B8 were reacted at essentially
equimolar concentrations (0.18 and 0.17 µM,
respectively), and the time course of the NBD fluorescence increase was
monitored at 529 nm in the absence and presence of unlabeled latent
PAI-1 at concentrations ranging from 0.06 to 1.2 µM. In
separate experiments, P9·NBD PAI-1 (0.1 µM) and MA-33B8
(0.08 µN) were mixed, and the time course of the NBD
fluorescence increase was monitored in the absence and presence of each
of the following unlabeled PAI-1 forms: active PAI-1 (0.1 µM), PAI-1 in complex with NAc-TVASSSTA (1 µM), elastase-cleaved PAI-1 (0.1 µM), and
the tPA·PAI-1 complex (0.1 µM). Similarly, the
fluorescence increase of P9·NBD PAI-1 (0.2 µM) reacting
with MA-33B8 (0.19 µM) was monitored in the absence and
presence of intact P14 Arg PAI-1 (2 µM).
Direct Binding of Unlabeled PAI-1 Forms to MA-33B8--
Direct
binding studies were performed based on the strong affinity of the
MA-33B8 FC region for protein A. Latent and
peptide-complexed PAI-1 were added to vials containing a slight excess
of MA-33B8 (3 µN) in 20 mM sodium phosphate,
pH 7.4, adjusted to 0.15 M ionic strength with NaCl. The
respective mixture (600 µl) was passed over 1 ml of protein
A-Sepharose (HiTrap, Amersham Pharmacia Biotech) equilibrated with the
pH 7.4 phosphate buffer. The column was rinsed with the equilibration
buffer, and material that eluted was collected. The antibody was
subsequently eluted with a pulse of 0.1 M citric acid
adjusted to pH 3 with NaOH. Void and antibody fractions were analyzed
by SDS-PAGE before and after treatment with excess tPA at pH 7.4.
Effects of MA-33B8 on the Susceptibility of PAI-1 to Hydrolysis
by Elastase--
The susceptibility of various PAI-1 forms to
hydrolysis by elastase was tested by incubating mixtures of inhibitor
(1 µM) and enzyme (0.1 µM) for 20 min at
room temperature. The reactions were quenched with SDS (1%) and
analyzed by SDS-PAGE. The species tested were active wild type and
P9·NBD PAI-1, latent PAI-1, and the antibody-inactivated wild type
and P9·NBD PAI-1 forms. The latter were generated by preincubating
the respective active PAI-1 form with MA-33B8 (3 µN) for
20 min.
Effects of Heparin and Vitronectin on the P9·NBD PAI-1-MA-33B8
Interaction--
P9·NBD PAI-1 (0.17 µM) was reacted
with MA-33B8 (0.17 µN), in the absence and presence of
heparin (5 µM), and the resulting fluorescence
enhancement monitored as a function of time using the SLM 8000 fluorescence spectrophotometer. PAI-1 was preincubated with heparin,
and the reactions were initiated by adding the antibody. As a control,
heparin and P9·NBD PAI-1 were reacted in the absence of antibody.
Similarly, the fluorescence enhancement caused by reacting MA-33B8
(0.08 µM) with P9·NBD PAI-1 (0.1 µM) was
monitored both in the absence and presence of 1 µM
vitronectin. To probe the effect of vitronectin on PAI-1 stability,
active native and P9·NBD PAI-1 (1.5 µM) were incubated
with vitronectin (5 µM), and the activity of the two
PAI-1 forms toward tPA was determined at timed intervals.
Effect of MA-33B8 on the Rate of Inactivation of Wild Type and
P9·NBD PAI-1--
The effects of MA-33B8 on wild type and P9·NBD
PAI-1 were compared by incubating the respective PAI-1 form with excess
MA-33B8 and measuring residual inhibitory activity as a function of
time. Fig. 1 demonstrates that with 4.2 µN MA-33B8, the antibody-induced inactivation of wild
type and P9·NBD PAI-1 occurred at comparable rates, corresponding to
inactivation rate enhancement factors of 4000 and 8000, respectively.
The larger factor for P9·NBD PAI-1 derives from its 2-fold lower rate
of spontaneous inactivation. Based on the rates of inactivation of wild
type and P9·NBD PAI-1 induced by excess MA-33B8 at three different
concentrations, the apparent second order rate constant for antibody
binding to the respective PAI-1 form was 1.8 ± 0.2 and 1.4 ± 0.2 × 104 M Comparative Characterization of Latent and Antibody-inactivated
PAI-1--
To compare the inactive forms of P9·NBD PAI-1 generated
in the absence and presence of antibody, respectively, the emission spectra of P9·NBD PAI-1 before and after complete spontaneous or
MA-33B8-induced inactivation were recorded. The data in Fig. 2 show that antibody-induced
inactivation of P9·NBD PAI-1 causes changes to the NBD emission
spectrum within experimental precision identical to the changes
observed following spontaneous inactivation of P9·NBD PAI-1
(i.e. a 13-nm blue shift of the emission maximum and a
6.7-fold fluorescence enhancement). The NBD fluorescence of latent
P9·NBD PAI-1 was not affected by addition of MA-33B8.
The spontaneously and antibody-induced inactive forms of P9·NBD PAI-1
were compared also with respect to hydrolysis by elastase. Data in Fig.
3B show that when subjected to
catalytic amounts of elastase and treated with SDS, active P9·NBD
PAI-1 releases a ~3-kDa peptide, consistent with hydrolysis of the
P3-P4 RCL peptide bond, as previously reported for hydrolysis of wild
type PAI-1 by elastase (11). Following inactivation by antibody, neither native PAI-1 nor P9·NBD PAI-1 was hydrolyzed by elastase (Fig. 3C).
Kinetics of MA-33B8-induced P9·NBD PAI-1 Fluorescence
Increase--
The time course of the NBD fluorescence increase
triggered by reacting 0.1 µM P9 NBD PAI-1 with 0.08 µN MA-33B8 is shown in Fig.
4. The first order rate constant for a
set of such curves increased linearly with antibody concentration up to
3 µN, the highest concentration tested (Fig. 4,
inset). The concentration dependence gave an apparent second
order rate constant for antibody-induced fluorescence enhancement of
1.3 × 104 M Relative Rates of Binding of P9·NBD PAI-1 and Wild Type PAI-1
Variants to MA-33B8--
Data presented above indicate that MA-33B8
binding to PAI-1 promotes insertion of the RCL into Rates of Loop Insertion in Peptide-complexed and P14 Arg P9·NBD
PAI-1--
To assess directly the constraints against loop insertion
in PAI-1 caused by mutating the threonine at P14 in the RCL to an arginine or by binding the NAc-TVASSSTA octapeptide to
position 4 in sheet A, these modifications were introduced into
P9·NBD PAI-1. This enabled loop insertion to be monitored by means of the NBD fluorescence. As anticipated based on the effects of the same
modifications on wild type PAI-1, the modified P9·NBD PAI-1 forms
were substrates for tPA and hydrolyzed completely at the scissile bond.
Following hydrolysis, insertion of the RCL was extremely slow with P14
Arg P9·NBD PAI-1 (kobs = 2.3 h Relative Rates of Binding P9·NBD PAI-1 and Loop Constrained PAI-1
Variants to MA-33B8--
The comparatively slow binding of MA-33B8 to
active PAI-1 could be caused by an unfavorable equilibrium in PAI-1
that exposes the epitope in a conformation of the inhibitor associated
with insertion of the RCL into Direct Assessment of the Affinities of MA-33B8 for Latent and
Peptide-complexed PAI-1--
The contrasting high and low affinities
of MA-33B8 for latent and peptide blocked PAI-1, respectively, were
demonstrated directly by attempts to separate each PAI-1 form from a
slight excess of antibody binding sites on immobilized protein A. Protein A binds the immunoglobulin via the FC region.
Latent PAI-1 alone was not retained on the column. Latent PAI-1 and
antibody, however, were retained completely on the column and were
eluted together at pH 3. Under the same conditions, most of the
peptide-complexed PAI-1 was separated from the antibody in the
equilibration buffer and eluted in the column void. The elution peak,
however, was extended at its trailing end, in keeping with the
concentration of antibody used and the Kd reported
above. These results, as well as electrophoresis gels demonstrating the
contents of each elution peak, are documented in Fig.
7.
Effects of Heparin and Vitronectin on Binding MA-33B8 to
PAI-1--
PAI-1 has high affinity for heparin and vitronectin (21,
22). Experiments were performed to determine whether binding of these
macromolecular ligands interferes with binding of MA-33B8. Heparin and
vitronectin had no effect per se on the NBD fluorescence of
P9·NBD PAI-1, consistent with the suggested binding sites remote from
the exposed loop. Neither ligand reduced the rate or amplitude of the
fluorescence enhancement observed after addition of MA-33B8, indicating
that the binding sites for heparin and vitronectin on PAI-1 (22, 23) do
not overlap with that for MA-33B8 and that the two ligands do not
noticeably interfere with the associated step of loop insertion.
Effect of Native Monomeric Vitronectin on PAI-1
Stability--
Vitronectin binding has been reported to reduce the
rate by which PAI-1 becomes latent (24). The absence of an effect of vitronectin on the rate of change of the P9·NBD fluorescence induced by MA-33B8 therefore had potential implications for the mechanisms by
which vitronectin stabilizes and MA-33B8 destabilizes PAI-1. However,
the monomeric vitronectin (Mr 70,000) used in this
study was shown to have a 1.3-fold stabilizing effect on native PAI-1,
less than the factor of 2 reported for multimeric vitronectin (~ 400 kDa) (24). The effect of monomeric vitronectin on P9·NBD PAI-1 was
even further reduced.
We have presented results that strongly suggest that the murine
monoclonal antibody MA-33B8, directed against the human tPA·PAI-1 complex, rapidly converts inhibitory PAI-1 to its latent form. Antibody-induced inactivation of the P9 Ser Hypothetically, MA-33B8 could bind to an epitope on PAI-1 in the
vicinity of the reactive center and thereby change the P9·NBD fluorescence and prevent proteinases from attacking the RCL. The fact
that antibody binding to active PAI-1 required displacement of the RCL
does not prove in itself that this results in latency. However, it is
highly unlikely that displacement of the RCL of P9·NBD PAI-1, without
insertion into sheet A, would result in changes of the fluorescence of
the environmentally sensitive NBD-probe that are within experimental
error identical to those resulting from formation of the latent labeled
PAI-1.
The experiments that determined the relative rates of binding to
MA-33B8 of PAI-1 forms other than active P9·NBD PAI-1 provided additional insights. Wild type PAI-1 at a concentration equal to that
of the P9·NBD mutant decreased the amplitude of the antibody-induced fluorescence enhancement by 55%. This indicates that P9 NBD and wild
type PAI-1 bind to MA-33B8 at similar rates, consistent with the
observation that the antibody inactivated the two PAI-1 forms at
comparable rates. The loop-inserted forms of PAI-1, however, including
latent PAI-1, elastase-cleaved PAI-1, and the tPA·PAI-1 complex,
bound much more rapidly to MA-33B8 than active PAI-1, indicating that
loop insertion leads to a greater exposure of the epitope. The fact
that the octapeptide-complexed PAI-1 and the P14 Thr A significant aspect of the antibody effect is the speed with which it
causes conversion to latency. At 25 °C, the spontaneous active-to-latent conversion of wild type and P9·NBD-labeled PAI-1 has
a t1/2 of 9.5 and 23.5 h respectively, whereas
in the presence of 4.2 µN MA-33B8 the
t1/2 was reduced to 9 s for both forms. No
evidence for saturation was seen in the antibody concentration
dependence of the rate of conversion to the latent form, indicating
that inactivation of PAI-1 could be substantially faster at saturating conditions.
To accomplish this rate enhancement, antibody binding must either
devise a new and faster path for latency in PAI-1, or make the existing
path faster by reducing the energy gap between the ground state of the
active PAI-1 molecule and a major transition state for its conversion
to the latent form. In the first alternative (Scheme
1), the epitope is fully exposed in
active PAI-1 (I) and no binding energy is reserved for changing the
conformation of the inhibitor or supporting a transition state.
Antibody binding, however, generates a species of the inhibitor (I'Ab),
the molecular dynamics of which are different from that of the parent
molecule and which is less stable at the input of the same amount of
thermal energy.
-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA)·PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1
labeled with the fluorescent probe
N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9·NBD PAI-1 to the latent form. Like latent PAI-1,
antibody-inactivated PAI-1 was protected from cleavage by elastase. The
rate constants for MA-33B8 binding, measured by NBD fluorescence or
inactivation, were similar (1.3-1.8 × 104
M
1 s
1), resulting in a
4000-fold faster inactivation at 4.2 µM antibody binding
sites. The apparent antibody binding rate constant, at least 1000 times
slower than one limited by diffusion, indicates that exposure of its
epitope depends on an unfavorable equilibrium of PAI-1. Our
observations are consistent with this idea and suggest that the
equilibrium involves partial insertion of the RCL into sheet A: latent,
RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted
forms, bound much faster than active PAI-1 to MA-33B8, whereas two
loop-extracted forms of PAI-1, modified to prevent loop insertion, did
not bind or bound much more weakly to the antibody.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet A, which can
accommodate an additional strand in position 4, and a flexible reactive
center loop (RCL) of about 20 residues denoted P15-P5' (1, 2).
Inhibitory serpins function as suicide substrate inhibitors and acylate
their target proteinases (3, 4). The acyl-enzyme complex is stabilized
by insertion of the cleaved RCL as strand 4 of
-sheet A, accompanied
by a translocation of the acylated enzyme from its initial binding site
to its "locking" site. If RCL insertion is hindered by mutating the
P14 threonine to an arginine (5), or by blocking position 4 of
-sheet A with a peptide analog of the P14-P7 part of the RCL (6),
PAI-1 becomes a substrate that is completely hydrolyzed at the scissile bond by its target proteinases.
-sheet A, forcing the remainder
of the loop, including the reactive center, to adopt an extended
conformation alongside the protein scaffold. The term "latent" was
introduced because PAI-1 traditionally has been isolated in the
inactive form, and its activity was partly restored by transient
treatment with chaotropic agents (9). In the absence of such treatment,
however, formation of latent PAI-1 is a virtually irreversible process
(6). Latent PAI-1 has greater thermal stability than native PAI-1 (10),
and its reactive center is unreactive with proteinase. Another marker for the conversion is that in the latent form, the RCL is protected from cleavage by elastase (11).
-sheet A, respectively. Similar
fluorescence changes were observed after reacting P9·NBD PAI-1 with
tPA, urokinase-type plasminogen activator, and trypsin, as well as with
elastase, all of which cleave the RCL and trigger insertion of the RCL
into
-sheet A, whereas no changes resulted from complexing P9·NBD PAI-1 with trypsin and tPA after these had been inactivated by converting their catalytic triad serine residues to dehydroalanine and
alanine, respectively (8,
14).3 In the present study,
we demonstrate that the antibody-accelerated inactivation of P9·NBD
PAI-1 occurs concomitant with changes in the NBD fluorescence that were
within experimental error identical to those characterizing the
spontaneous inactivation of the labeled mutant. We also show that the
differences between active and latent PAI-1 in susceptibility to
hydrolysis by elastase are reproduced by the antibody-induced
inactivation of PAI-1.
1 s
1. The second order rate
constants determined for the antibody antigen interaction usually fall
within the range 106-107
M
1 s
1 (15, 16), approaching the
limit set by molecular diffusion. The much lower rate constant for
binding MA-33B8 to PAI-1 suggests an extra step in the binding of
MA-33B8 to PAI-1, involving an unfavorable conformational change in
PAI-1. To determine whether this conformational change involves loop
insertion, various loop-inserted forms of PAI-1, as well as two
loop-exposed variants with a documented reduced rate of loop insertion,
were tested for their ability to compete with P9·NBD PAI-1 for
binding to MA-33B8. These measurements revealed a striking difference
in reactivity, consistent with antibody binding to a site on PAI-1 that
is exposed only when the RCL is inserted into
-sheet A. Studies
involving the complexes of P9·NBD PAI-1 with heparin and vitronectin
indicate that binding of these ligands does not interfere with binding
of MA-33B8.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 cm
1 and a Mr of
43,000 (19). The mercapto group of the P9 Cys PAI-1 mutant was labeled
with NBD (Molecular Probes) as described previously (8). To quantitate
latent PAI-1, formed in storage or handling, unlabeled and
P9·NBD-labeled PAI-1 preparations were analyzed by SDS-PAGE after
reaction with excess two-chain tPA.
1
cm
1 and a Mr of 150,000. The antibody
binding site concentrations is given as normality (N),
assuming two independent and noninteracting binding sites on each molecule.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
s
1.
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Fig. 1.
The time courses of MA-33B8-induced
inactivation of wild type and P9·NBD PAI-1. The mouse monoclonal
anti-human tPA·PAI-1 antibody MA-33B8 (4.2 µN) was
incubated with wild type and P9·NBD recombinant human PAI-1 (0.5 µM) at room temperature, and residual PAI-1 activity was
measured at the intervals indicated. Data for wild type ( ) and
P9·NBD PAI-1 (
) were analyzed by nonlinear regression. The
solid and dashed lines are exponential decay
functions with the best fit rate constants 0.083 ± 0.005 s
1 (PAI-1) and 0.075 ± 0.003 s
1
(P9·NBD PAI-1), respectively.
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Fig. 2.
Changes in the fluorescence of P9·NBD PAI-1
following spontaneous or antibody-induced inactivation. Shown are
the Fluorescence emission spectra of P9·NBD PAI-1 (0.1 µM) before (trace 1) and after
complete spontaneous (trace 2) or antibody-induced
(trace 3) inactivation. Excitation was at 480 nm.
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Fig. 3.
The reactivity of wild type and P9·NBD
PAI-1 with tPA and elastase. A demonstrates the
reactivity of active wild type and P9·NBD PAI-1 toward excess tPA.
Lanes 1 and 3 were obtained with wild type and
P9·NBD PAI-1, respectively, and lanes 2 and 4 were obtained with the same species after treatment with tPA. The bands
at a and b are latent and reactive center bond
hydrolyzed PAI-1, respectively. The tPA·PAI complex is at
c, and the surplus tPA is at d. B
demonstrates the susceptibility of active PAI-1, active P9·NBD PAI-1,
and latent PAI-1 (all at 1 µM) to hydrolysis by elastase
(0.1 µM). Lanes 5-7 contain the
elastase-treated PAI-1, P9·NBD PAI-1, and latent PAI-1, respectively.
C demonstrates the susceptibility of wild type and P9·NBD
PAI-1 (both at 1 µM), preinactivated by MA-33B8 (3 µN, band e), to hydrolysis by elastase.
Lanes 9 and 10 contain antibody-inactivated and
elastase-treated PAI-1 and P9·NBD PAI-1, respectively. Lane
8 is antibody and wild type PAI-1 before treatment with
elastase.
1
s
1, similar to the value of 1.4 × 104
M
1 s
1 presented above and
calculated based on the rate of inactivation of P9·NBD PAI-1 induced
by the antibody.
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Fig. 4.
The time course of the antibody-induced
fluorescence enhancement of P9·NBD PAI-1. P9·NBD PAI-1 (0.2 µM) was reacted with MA-33B8 (0.8 µN), and
the fluorescence was monitored at 529 nm (excitation at 480). The
regression curve is a first order growth function with
kobs = 0.011 s 1. The
inset shows the MA-33B8 concentration dependence of
kobs. The slope of the regression line
corresponds to a second order rate constant of 1.3 ± 0.3 × 104 M
1s
1, and its
intercept with the y axis to an apparent dissociation rate
constant indistinguishable from zero.
-sheet A. To
determine whether there is a reciprocal enhancement of antibody binding
when the RCL is preinserted into sheet A, unlabeled active PAI-1 and
RCL-inserted variants of the inhibitor were compared for their ability
to compete kinetically with P9·NBD PAI-1 for binding to MA-33B8. When
PAI-1, P9·NBD PAI-1, and MA-33B8 were mixed in equimolar proportions, the amplitude of the NBD fluorescence progress curve was decreased to
about half of that registered in the absence of the wild type inhibitor
(Fig. 5). This demonstrates that P9·NBD
and wild type PAI-1 compete equally for binding to MA-33B8. The
loop-inserted wild type PAI-1 forms (i.e. latent,
elastase-cleaved, and tPA-bound), on the other hand, decreased the
amplitude of the NBD fluorescence progress curve in a stoichiometric
fashion. This is most convincingly shown by the set of curves displayed
in Fig. 6, which exhibit a linear
decrease in the MA-33B8-induced fluorescence enhancement of P9·NBD
PAI-1 with the concentration of latent PAI-1. It is also demonstrated
by the proportionate depression of the reaction of MA-33B8 with
P9·NBD PAI-1 by the other loop-inserted PAI-1 forms shown in Fig. 5.
The stoichiometric effect indicates that binding of the loop-inserted
PAI-1 forms to MA-33B8 is faster than binding of active PAI-1 by a
factor considerably larger than 10, that is, that the corresponding
second order rate constants are close to or exceed 106
M
1 s
1.
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Fig. 5.
Kinetic competition for MA-33B8 between
P9·NBD PAI-1 and unlabeled PAI-1 forms. All fluorescence
progress curves were obtained at essentially equimolar concentrations
of P9·NBD PAI-1 and MA-33B8 binding sites (0.2 and 0.19 µM in curves 1 and 2; 0.1 and 0.08 µM in all other curves). The two superimposed
curves 1 and 2 were obtained in the absence and
presence of P14-Arg PAI-1 (2 µM), and the two
superimposed curves 3 and 4 were obtained in the
absence and presence of monomeric vitronectin (1 µM).
Curve 5 was recorded in the presence of
NAc-TVASSSTA·PAI-1 (1 µM), and curves
6-8 were recorded in the presence of active wild type PAI-1, the
tPA·PAI-1 complex, and elastase-cleaved PAI-1, respectively (all at
0.1 µM).
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Fig. 6.
Stoichiometric suppression of MA-33B8-induced
inactivation of P9·NBD PAI-1 by latent PAI-1. A, the
fluorescence enhancements recorded after reacting 0.18 µM
P9·NBD PAI-1 with 0.17 µN MA-33B8 in the absence
(topmost trace) and presence of 0.06, 0.12, 0.15, and 1.2 µM latent PAI-1 (traces in descending order).
B, the amplitudes of the curves in A plotted
against the concentration of latent PAI-1. The regression line
indicates complete suppression of the fluorescence enhancement at a
concentration of latent PAI-1 equal to that of antibody binding
sites.
1)
and too slow to be measured with the peptide-complexed P9·NBD form.
In contrast, the RCL of inhibitory P9·NBD PAI-1 inserts with a rate
constant of 3.4 s
1 in the reaction with saturating tPA
(8).
-sheet A. To investigate this
possibility, the competitive binding measurements were extended to
include P14 Arg PAI-1 and PAI-1 in complex with
NAc-TVASSSTA, which, by inference from the data presented
above on the corresponding variants of P9·NBD PAI-1, are prevented
from inserting the RCL into
-sheet A at a significant rate. With the
P14 Arg PAI-1, no effect on the rate of binding MA-33B8 to P9·NBD
PAI-1 could be observed, even at 2 µM P14 Arg PAI-1,
representing a 10-fold molar excess over P9·NBD PAI-1 and MA-33B8
binding sites (Fig. 5, curves 1 and
2). A small effect on the rate of the fluorescence change, but not on its amplitude, was observed when peptide-complexed PAI-1 (1 µM) was added to a mixture of P9·NBD (0.1 µM) and MA-33B8 (0.1 µN) (Fig. 5,
curve 5). Based on this rate depression and the assumption
that binding of peptide-complexed PAI-1 to MA-33B8 equilibrates fast
compared with the duration of the experiment, the Kd
for binding the peptide-complexed PAI-1 to MA-33B8 was estimated to be
1-2 µM. The absence of an effect of P14 Arg PAI-1 on
MA-33B8 binding to P9·NBD PAI-1 indicates that the antibody binds
this species with a Kd that exceeds 10 µM.
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Fig. 7.
Comparison of the binding of latent and
peptide-complexed PAI-1 to MA-33B8 by chromatography on protein
A-Sepharose. Antibody (3 µN) was mixed with
NAc-TVASSSTA·PAI-1 (2.8 µM) or latent PAI-1
(2.8 µM) and subjected to chromatography on immobilized
protein A. The top graph of panel A is the
chromatogram for antibody and peptide-complexed PAI-1. The
arrow marks the change of buffer to elute the antibody. The
gel in panel B shows SDS-PAGE analysis of the
peptide-complexed PAI-1, before (lane 1) and after
(lane 2) treatment with equimolar tPA. The gel in
panel C demonstrates the composition of material eluted in
the void and antibody peaks, before (lanes 3 and
5, respectively) and after (lane 4 and
6, respectively) treatment with excess tPA. The bottom
graph of panel A shows the chromatogram for antibody
mixed with latent PAI-1 (black) and for latent PAI-1 alone
(gray). The gel in panel D demonstrates the
composition of the mixture of antibody and latent PAI-1 applied to the
column (lane 7) and of the antibody peak (lane
9). Lane 8 demonstrates the absence of protein in the
void fraction. The close bands at migration positions a and
b are intact and RCL-hydrolyzed PAI-1, respectively;
d and e are intact and hydrolyzed
peptide-complexed PAI-1, respectively; and g is latent
PAI-1. The diffuse band at migration position c is tPA, and
the bands at f represent the antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Cys mutant PAI-1, labeled with the fluorescent probe NBD at the mercapto group, resulted
in a 6.7-fold enhancement and 13-nm blue shift of the NBD fluorescence,
within experimental precision identical to the changes documented for
the spontaneous conversion of the labeled mutant to the latent form.
Like latent PAI-1, antibody-inactivated PAI-1 was unreactive with
target proteinases, as well as with elastase (11). The rate of the
fluorescence enhancement of P9·NBD PAI-1 was proportional to antibody
concentration up to 3 µN, the highest concentration
tested, with an apparent bimolecular rate constant of 1.3 × 104 M
1 s
1. Similar
rate constants (1.4 and 1.8 × 104
M
1 s
1) were calculated based on
the rate of inactivation of P9·NBD PAI-1 and wild type PAI-1 by
MA-33B8, which at 4.2 µN antibody resulted in rates of
inactivation 4000 and 8000 times faster than the spontaneous
inactivation of the respective PAI-1 form.
Arg mutant,
which cannot rapidly insert the loop, reacted poorly or not at all with
MA-33B8 is consistent with this assumption.
If the epitope were fully exposed in active PAI-1, as implicated
by the mechanism in Scheme 1, MA-33B8 would bind free active PAI-1 as
fast as it binds the loop inserted PAI-1 forms. The data in Fig. 5 and
6, however, show that whereas binding of MA-33B8 to loop-inserted forms
of PAI-1 occurred at a rate comparable to that typically reported for
antibody antigen reactions (i.e. ~106
M
1 s
1), the second order rate
constant for MA-33B8 binding to active PAI-1 was reduced approximately
1000-fold. To account for an apparent second order rate constant for
the overall process in Scheme 1 as low as 104
M
1 s
1, we would have to assume
that the second step is much slower than the reversal of the
bimolecular binding step. If this were the case, however, obvious signs
of rate saturation would be observed at concentrations of MA-33B8
corresponding to the typical antibody-antigen dissociation constant,
which is lower by far than 4.2 µM (15, 16). For these
reasons, the mechanism in Scheme 1 can be considered unlikely.
In the alternative mechanism, presented in Scheme
2, the dominant form of active PAI-1 (I)
obscures the epitope for MA-33B8. The epitope becomes completely
exposed in a conformation (I*) that is also a transition state for
formation of the latent species. Antibody binding traps this transition
state and reduces its energy relative to the active inhibitor,
resulting in an enhanced rate of PAI-1 inactivation. Scheme 2 is
analogous to that used to explain the rate enhancement caused by
binding a substrate (I) to the surface of an enzyme (Ab) (25).
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The spontaneous formation of latent PAI-1 was negligible for the duration of our antibody binding experiments. The documented strong binding of MA-33B8 to latent PAI-1 is logical in view of the mechanism in Scheme 2 but does not explain the rate enhancement. Although one of the two paths leading to I*Ab may be preferred kinetically, they are thermodynamically equivalent.
According to Scheme 2, the maximal rate enhancement factor (fmax) is linked thermodynamically to the relative affinities of I* and I for the antibody and is given by fmax = KIAb/KI*Ab, where KIAb and KI*Ab are the equilibrium constants for dissociation of IAb and I*Ab, respectively. Our results indicate that fmax exceeds 4000, which means that MA-33B8 binds I* more than 4000 times stronger than it binds I. The rate enhancement was not saturated at 4.2 µM antibody binding sites, which precludes the possibility that at this or lower concentrations, MA-33B8 will exhibit significant binding to PAI-1 without changes, which would bring the conformation of I closer to that of I*. Not indicated in Scheme 2 but possibly suggested by the weak binding of MA-33B8 to the loop exposed octapeptide blocked PAI-1 is binding of the antibody in several discrete steps to an increasingly exposed epitope. Other modifications to Scheme 2 may include slightly different rates for the collapse of I* and I*Ab into the latent species. None of these modifications, however, will significantly alter the conclusions based on the mechanism depicted in Scheme 2.
In Scheme 2, exposure of the epitope in I* occurs by an unfavorable equilibrium. This predicts that the second order rate constant measured for formation of latent PAI-1 should be greatly reduced compared with that measured for antibody-antigen interactions in general, and for binding of MA-33B8 to the fully exposed epitope in PAI-1 in particular, a prediction in agreement with our results. This reduction is independent of whether I*Ab is formed via binding of Ab to I* or I. Scheme 2 also explains why at an antibody binding site concentration as high as 4.2 µM, the rate of inactivation is not saturated. The overall equilibrium dissociation constant of I*Ab is the product of the two equilibria of each path and involves either tight binding to an epitope exposed in an unfavorable equilibrium or unfavorable binding to a partly exposed epitope, coupled to a compensatory more favorable equilibrium for its complete exposure.
In keeping with Scheme 2, our results show that MA-33B8 binds at a fast
rate to latent, tPA-complexed, and elastase-cleaved PAI-1; at a
significantly reduced rate to active PAI-1; and not at all or only
weakly to the P14 Arg and peptide-complexed PAI-1 variants. The first
group is characterized by complete or partial insertion of strand 4 into sheet A; active PAI-1 has its RCL exposed but the potential for
loop insertion; and the RCL of the substrate PAI-1 forms is exposed and
cannot insert at a significant rate. Taken together, this suggests that
the conformational change that exposes the epitope for MA-33B8 is
associated with some insertion of the RCL into sheet A. Because the
spontaneous formation of latent PAI-1 is virtually irreversible and
because antibody binding increases the rate of this process by a factor
larger than 4000, the epitope must be exposed in an intermediate (I*)
that, in the absence of antibody, has greater tendency to revert to the
active loop extracted conformation (I) than to proceed to form latent PAI-1, as indicated in Scheme 2. Because insertion of strand 4A should
be increasingly prone to proceed to completion for each additional
residue inserted, it is probable that insertion of only a few of the 14 residues of strand 4A is sufficient to expose the epitope. A tentative
prediction is that the epitope for MA-33B8 is exposed in an
intermediate that has inserted the proximal part of the RCL into sheet
A to a point such that further insertion would require complete
displacement of -strand 1C at its distal end.
Our results do not enable us to identify specifically the epitope for MA-33B8. Loop insertion can unmask an epitope that is directly shielded by the extracted RCL, or it could be associated with a conformational change that exposes an epitope elsewhere on the molecule. To start with, however, the epitope cannot involve residues that are not exposed in the antigen, the tPA·PAI-1 complex. This should exclude residues in the P15-P1 portion of the RCL, which are either inserted into sheet A, covered by the helix F loop, or in close contact with the proteinase. Also excluded are residues covered by the proteinase at its final locking site. The fact that latent P9·NBD PAI-1 binds MA-33B8 without changes in NBD fluorescence argues against the epitope being within 10 Å of the label on P9. This would exclude residues in the loop that carries helix F, which has been shown to harbor the epitopes for the murine antibodies that convert PAI-1 to a substrate for tPA (26, 27). Our data also indicate that the vitronectin and heparin binding sites on PAI-1 do not overlap the epitope for MA-33B8.
Binding of a multimeric form of vitronectin to PAI-1 has been reported to reduce the rate of latency by a factor 2 (24). This effect, however, has not been documented with monomeric vitronectin, which was used in this study. Direct measurements of the inactivation of native and P9·NBD PAI-1 in the presence of monomeric vitronectin, at a concentration that was saturating based both on our own observations and on data presented in the literature (28), revealed a stabilizing effect on native PAI-1 of only 1.3, whereas no significant effect on P9·NBD PAI-1 was observed. The absence of an effect of monomeric vitronectin on the rate of change of the P9·NBD PAI-1 fluorescence induced by antibody binding is consistent with these results.
In summary, we have found that binding of MA-33B8 induces at least a
4000-fold decrease of the half-life of active PAI-1 and have provided
evidence that the inactive species generated is latent PAI-1. We have
presented data that suggest that binding of antibody depends on partial
insertion of the RCL into -sheet A of the inhibitor and that
antibody binding stabilizes an intermediate that represents or is close
to a transition state on the pathway to latent PAI-1. Our results
should provide the basis for studies aiming at identifying the epitope
for MA-33B8 and determining the mechanism for the antibody-induced, as
well as the spontaneous, formation of latent PAI-1.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL45930A07 (to J. D. S.), American Heart Association Grant 9650683N (to J. D. S.), and Fund of Scientific Research-Flanders (Belgium) Grant G.0266.97 (to P. J. D.).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 work.
¶ To whom correspondence should be addressed: Henry Ford Health Sciences Center, Division of Biochemical Research, One Ford Place, 5D, Detroit, MI 48202-3450. Tel.: 313-876-3196; Fax: 313-876-2380; E-mail: jshore1{at}hfhs.org.
2 P. Declerck., S. Debrock, and N. Vleugels, unpublished observations.
3 I. Verhamme, J.-O. Kvassman, D. Day, and J. D. Shore, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PAI, plasminogen activator inhibitor; NBD, N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine; Bicine, N,N-bis(2-hydroxyethyl)glycine; PAGE, polyacrylamide gel electrophoresis; RCL, reactive center loop; tPA, human tissue-type plasminogen activator; Ab, antibody.
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