From the Department of Vascular Biology, The Holland
Laboratory, American Red Cross, Rockville, Maryland 20855, ¶ Wyeth
Research, N2265A, Collegeville, Pennsylvania 19426, and
§ Wyeth Research, CN8000, Princeton, New Jersey 08543
Received for publication, August 17, 2002, and in revised form, February 20, 2003
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ABSTRACT |
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The mechanism for the conversion of plasminogen
activator inhibitor-1 (PAI-1) from the active to the latent
conformation is not well understood. Recently, a monoclonal antibody,
33B8, was described that rapidly converts PAI-1 to the latent
conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S.,
Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an
attempt to understand this interaction, and more broadly to understand
the mechanism of the natural transition of PAI-1 to the latent
conformation, we have used random mutagenesis to identify the 33B8
epitope in PAI-1. This site involves at least 8 amino acids scattered
over more than two-thirds of the linear sequence that form a compact
epitope on the PAI-1 three-dimensional structure. Surface plasmon
resonance studies indicate a high affinity interaction between
latent PAI-1 and 33B8 that is ~100-fold higher than comparable
binding to active PAI-1. Structural modeling results together with
surface plasmon resonance analysis of parental and site-directed PAI-1
mutants with disrupted 33B8 binding suggest the existence of a specific
PAI-1 intermediate structure that is stabilized by 33B8 binding. These
analyses strongly suggest that this intermediate form of PAI-1 has a
partial insertion of the reactive center loop into Plasminogen activator inhibitor-1
(PAI-1)1 is a member of the
serine protease inhibitor (serpin) gene family and is the principal inhibitor of the plasminogen activators tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in
vivo (1). In healthy individuals, PAI-1 is found at low levels in
the plasma (5-10 ng/ml), but it is elevated significantly in a number
of diseases, including atherosclerosis (2), deep vein thrombosis (3),
and non-insulin dependent diabetes mellitus (4). PAI-1 stabilizes both
arterial and venous thrombi, contributing, respectively, to coronary
arterial occlusion in post-myocardial infarction (5) and venous
thrombosis following post-operative recovery from orthopedic surgery
(6). Plasma PAI-1 is also elevated in postmenopausal women, and has
been proposed to contribute to the relative increased incidence of
cardiovascular disease in this population (7). The role of PAI-1 in
atherosclerosis is less well defined, but PAI-1 is found in high
concentrations in vascular plaque (8), and is involved in the in
vitro regulation of tissue proteolysis and smooth muscle cell
migration (9).
Whereas PAI-1 elevation is associated with disease progression, PAI-1
inhibition is associated with improvement in a number of
pathophysiologic processes. PAI-1 null mice are viable and have normal
coagulation and normal bleeding time (10). However, when subjected to
vascular injury, PAI-1 null mice develop unstable thrombi that lyse
spontaneously, indicating that revascularization of occluded arteries
is improved significantly in the absence of PAI-1 (11). With respect to
pathological fibrosis, PAI-1 null mice are protected from the
development of vascular plaque (12) and pulmonary fibrosis (13, 14).
These studies, together with the aforementioned clinical observations,
suggest that PAI-1 inhibition may restore endogenous stimulation of
fibrinolysis by plasminogen activation, re-establishing a critical
defense mechanism for the prevention of intravascular thrombosis and
tissue fibrosis.
Structurally, PAI-1 is a metastable protein exhibiting conformational
plasticity, and exists in active, latent, and cleaved forms (15). The
conversion of PAI-1 from the active to the latent conformation appears
to be unique among serpins in that it occurs spontaneously at a
relatively rapid rate. And while this transition has been known for
more than 15 years (16), and the latent structure has been known for 10 years (17), the mechanism of the conversion from the active to the
latent conformation is not well understood.
To gain a better understanding of the structural basis for the latent
transition we have analyzed the interaction of a murine monoclonal
antibody, 33B8, with PAI-1 (18). This antibody was raised against the
tPA·PAI-1 complex, and has previously been shown to rapidly
inactivate PAI-1 by accelerating the conversion of PAI-1 to the latent
conformation (19). The reported rate of inactivation by this antibody
is ~4000-fold faster than the spontaneous conversion of PAI-1 to the
latent structure. Based on these studies it was suggested that exposure
of the antibody epitope depended on an unfavorable equilibrium in PAI-1
that might involve an intermediate PAI-1 structure with partial
insertion of the RCL into Materials--
Murine monoclonal antibodies MA-33B8 (33B8) and
MA-31C9 (31C9) as well as rabbit polyclonal antibodies directed against
human PAI-1 were purchased from Molecular Innovations (Southfield, MI), horseradish peroxidase-conjugated goat anti-mouse and
anti-rabbit antiserum were from Bio-Rad. tPA was from Genentech (South
San Francisco, CA), and uPA was from Molecular Innovations. Polystyrene 96-well microtiter plates were purchased from Costar (Cambridge, MA).
Chromogenic uPA substrate pGlu-Gly-Arg p-nitroanilide was purchased from Sigma. DNA restriction endonucleases (ScaI
and MluI) were purchased from Invitrogen and New
England Biolabs, Inc. (Beverly, MA), respectively. Synthetic
oligonucleotides (for mutagenesis and DNA sequencing) were synthesized
and PAGE-purified by Integrated DNA Technologies, Inc. (Coralville,
IA). All Escherichia coli strains used for subcloning,
plasmid replication, or expression of human PAI-1 were purchased from
Stratagene (La Jolla, CA), Clontech Laboratories,
Inc., and Novagen (Madison, WI) as competent cells. Nitrocellulose
filters and membranes for protein transfer were from Schleicher & Schuell, Inc. All supplies and reagents for SDS-PAGE including precast
gels and protein standards were from Novex (San Diego, CA). Bovine
serum albumin (BSA), sodium azide (NaN3),
isopropyl-1-thio- General DNA Techniques--
DNA manipulation techniques were
carried out according to standard procedures and following the
manufacturer's instructions. Plasmid DNA was isolated using QIAprep
Spin Miniprep kit from Qiagen Inc. (Valencia, CA). DNA sequencing was
done with the BigDyeTM Terminator Cycle sequencing ready
reaction kit on a ABI PRISMTM 310 genetic analyzer from
Applied Biosystems (Foster City, CA). PCR was performed using the
MastercyclerR Personal 5332 from Eppendorf Scientific, Inc. (Westburry,
NY). DNA sequences were analyzed with the Vector NTITM
Suite 6.0 molecular biology software for WindowsTM.
Site-directed Mutagenesis--
Nine individual site-directed
human PAI-1 mutants were constructed using either the
TransformerTM or QuikChangeTM site-directed
mutagenesis kits purchased from Clontech
Laboratories, Inc. or Stratagene (La Jolla, CA), respectively. The
pEX-human-PAI-1 plasmid DNA (20) was used as the template to generate
the required mutations. The following "mutagenic" and
"selective" primers were designed to make the desired replacements
(Table 1). All mutations were confirmed
by sequencing throughout the entire PAI-1 coding region of mutant
plasmids isolated from at least four separate clones of each individual
mutant.
Immunoscreening for 33B8 Binding Negative Variants--
The
expression Functional Analysis of Mutant PAI-1 Molecules--
PAI-1 mutants
from purified phage were automatically subcloned in pEX plasmids using
the Cre/lox P system (Novagen), and expressed in E. coli
BL21(DE3) or E. coli BL21(DE3) Tuner host strains along with
wild-type PAI-1. The cell-free extracts were prepared from the cells
pregrown to an A600 nm of 0.5-1.5 and then
induced by isopropyl-1-thio-
Aliquots of CE were serially diluted in 0.05 M Tris-HCl (pH
7.5) buffer, containing 0.15 M NaCl, 0.01% BSA, 0.0001%
Tween 20, 0.02% sodium azide at room temperature at concentrations of 0.016-40 µg/ml of protein in a volume of 100 µl and analyzed for functional activity as measured by inhibitory activity against plasminogen activators (uPA or tPA) using the single step chromogenic substrate assay as described (20, 22). 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 1.5 nM uPA in the reaction with the
chromogenic substrate. The total soluble protein contents in CE were
determined by BCA (Pierce). The level of expression of PAI-1 in the CE
was calculated as the percent ratio of active PAI-1 detected in the
activity assay to the total soluble protein contents of each individual mutant.
Immunoblot Assays--
To evaluate the 33B8 binding negative
phenotype of selected PAI-1 mutants, aliquots of CE of E. coli BL21(DE3) Tuner containing at least 5 ng of PAI-1, were
separated by SDS-PAGE in 4-20% Tris glycine gradient gels under
reducing conditions; electrotransferred to nitrocellulose membranes,
blocked with 1% BSA, 5% milk, Tris-buffered saline, and then probed
with either 33B8 or rabbit anti-PAI-1 polyclonal antibodies as the
primary antibodies. The formed complexes were detected with horseradish
peroxidase-conjugated goat anti-mouse or goat anti-rabbit
immunoglobulins by the ECL method according to the same methods used
for library screening (described above).
Surface Plasmon Resonance (SPR)--
The binding of purified
PAI-1 proteins and PAI-1 proteins in E. coli BL21(DE3) Tuner
CE to 33B8 was also analyzed by SPR using a BIAcoreTM 3000 optical biosensor from BIAcore AB. This method detects binding interactions in real time by measuring changes in refractive index at a
biospecific surface, allowing the calculation of association and
dissociation constants. Murine monoclonal antibody 31C9 served as the
control. This antibody is also directed toward PAI-1, but does not
cause PAI-1 to convert to the latent conformation and binds to the
active, latent, and cleaved forms of PAI-1 with similar affinity.
Control reactions were also analyzed with
Purified wild-type PAI-1 in its active, or latent conformations, and
E. coli CE containing functionally active PAI-1
diluted to a concentration of 62 nM were analyzed in HBS-P
buffer (pH 7.4) (BIAcore AB). Samples were injected for 120-600 s at a
flow rate of 10-20 µl/min followed by 10 min dissociation.
Regeneration of the sensor chip surface was done with 20 µl of 0.01 M glycine buffer (pH 1.5). All measurements were performed
at 25 °C, and BIAcore 3000 Bioevaluation software program package
(BIAcore AB) was used for analysis of data.
Screening of the PAI-1 Random Mutant Library for 33B8
Binding-deficient Clones--
A screen of the PAI-1 random mutant
library for clones with a specific 33B8 binding deficit was performed
as described above. Fig. 1A
shows 33B8 positive clones and Fig. 1B shows the same filter
reacted with polyclonal anti-PAI-1 antibodies. Plaques positive for
functional PAI-1 but negative for 33B8 binding were identified by
overlaying of these screens, and the 33B8-binding negative clones (Fig.
1C, note the circles) were isolated as described above. From three-hundred thousand DNA Sequence Analysis and Molecular Modeling--
DNA sequence
analysis of the entire PAI-1 coding sequence of each of the parental
mutants identified 34 independent mutations in 30 different positions
spread across the entire PAI-1 coding sequence (Fig.
2). This represents an average of 3.4 amino acid substitutions per variant with all clones having at least
two mutations and one clone having six. Two of the mutations, K88E and
S331R, were present in more than 1 identified clone with S331R being
present in 4 of the 10 clones (Table II).
This suggested that at least these 2 residues were part of the 33B8
epitope. By highlighting the positions of each of the 34 mutations on
the three-dimensional structure of latent and active PAI-1, we
identified a cluster of 8 mutations that suggested a potential surface
of contact with 33B8 (Fig. 3). The
residues of this putative epitope, Asn87,
Lys88, Asp89, Gln174,
Gly230, Thr232, Asn329, and
Ser331, were spread over two-thirds of the PAI-1 linear
sequence (244 amino acids), but encompassed only a small area on the
surface of latent PAI-1 (~18 Å × 8 Å). Furthermore, as would be
expected for the 33B8 epitope, this area contained at least 1 mutation from each of the 10 identified clones including the K88E and S331R mutations that were present on multiple parental clones. Together these
data suggested that these residues likely constituted a significant
portion of the 33B8 binding epitope.
Construction and Characterization of Site-directed Mutants--
To
see if the molecular modeling was indicating the correct localization
of the epitope, all of the 8 putative epitope mutations (N87D, K88E,
D89G, Q174R, G230V, T232S, N329I, and S331R) were constructed
individually by site-directed mutagenesis and each was analyzed for
33B8 binding by Western blot and for inhibitory activity against uPA
(Fig. 4). Data from these analyses
indicated that 4 of the 8 point mutations significantly impaired 33B8
binding. These mutants, K88E, D89G, G230V, and S331R, appear to account for most if not all of the binding defect in 8 of the 10 parental clones (Table II). Of the remaining 4 point mutations, 2 of these, Q174R and T232S, were the only 2 mutations present in the parental clone p7 that has a severe binding deficit as judged by immunoblot analysis (Fig. 1D). Therefore, these 2 mutations must act
together to disrupt 33B8 binding. The two remaining point mutations,
N87D and N329I, were also present in the same parental clone, p9, which also contained 2 additional mutations. Therefore, to see if these 2 mutations also act together they were constructed in tandem, and this
double mutant now showed a severe 33B8 binding deficit (Fig.
4A). Functional analysis of the inhibitory activity of each of these site-directed mutants toward uPA was also performed and these
data indicated that all of the mutants were active uPA inhibitors (Fig.
4C).
Together, these data demonstrate that these 8 residues must comprise at
least part of the 33B8 binding epitope, their location in active and
latent PAI-1 relative to one another is shown in Fig.
5. This analysis indicates the
conformation of the identified epitope is quite different in the two
conformations. It is also readily apparent from these structures that
in latent PAI-1 the putative epitope is much more linear and compact
than it is in active PAI-1. This result is consistent with earlier
studies suggesting that latent PAI-1 bound the antibody with much
higher affinity that did active PAI-1 (19).
SPR Analysis of PAI-1 Binding to 33B8--
To better characterize
the binding of PAI-1 in different conformations to 33B8 and to develop
methods to quantify and characterize the binding defect in our point
mutations, surface plasmon resonance experiments were performed. Fig.
6 shows the binding of purified active
and latent wild-type PAI-1 to 33B8 (panel A) and to a
control monoclonal antibody 31C9 (panel B). These data
indicate that the latent form of PAI-1 binds to 33B8 with much higher
affinity than active PAI-1 and that this difference in affinity is
because of a lower rate of active PAI-1 association with the antibody
and not to differences in the rate of dissociation. This is consistent with the observation that 33B8 converts active PAI-1 to the latent conformation and also with the relatively less accessible appearance of
the putative epitope in the active structure.
Similar experiments with the parental clones and with the site-directed
mutants were then performed and these data are shown in Fig.
7. These results demonstrate that all of
the mutants show a significant and specific deficit in binding to 33B8
compared with wild-type PAI-1 (Fig. 7), whereas no difference in
binding of the mutants and wild-type PAI-1 to the 31C9 control antibody was observed (not shown). However, the extent of the deficit and the
type of defect vary for each mutant. For example, four of the single
point mutants show either no binding (K88E, and G230V), or very large
increases in the rates of dissociation (D89G and S331R). These are the
same four point mutations that showed no reactivity in the immunoblots
(Fig. 4). In contrast, 3 of the point mutations (N87D, T232S, and
N329I) show clear reductions in their rates of association with 33B8
but only very modest changes in their dissociation rates, whereas the
final point mutation (Q174R) shows modest reductions on both the
association rates and the dissociation rates. In each of these latter
cases, where the dissociation rate is not severely reduced, all 4 point
mutations retained significant reactivity in the immunoblots. Together, these data support the conclusion that this site is the 33B8 binding epitope.
Together, our data demonstrate that the identified 8 residues must
comprise at least a significant part of the 33B8 binding epitope on the
surface of PAI-1. From our modeling of this epitope on the active and
latent conformations (Fig. 5) it is readily apparent that in latent
PAI-1 the putative epitope is much more linear and consolidated than it
is in active PAI-1. The surface plasmon resonance experiments also show
that the latent form of PAI-1 binds 33B8 with much higher apparent
affinity than does active PAI-1, and that the difference in apparent
affinity is because of a large reduction in the rate of active PAI-1
association with the antibody and not to differences in the rates of
dissociation. This observation is consistent with earlier suggestions
that active PAI-1 may be in equilibrium between a structure with the
RCL fully exposed and an intermediate form with partial RCL insertion
into Nonetheless our data do provide significant support for active PAI-1
being in equilibrium with an intermediate form. This comes from
modeling of the 33B8 epitope on the structures of two different serpins
with partial RCL insertion. The first is native antithrombin III (24,
25) and the second is a mutant of -sheet A, and
together, these data have significant implications for the general
serpin mechanism of proteinase inhibition.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-sheet A. Therefore, to see if this
epitope requires partial RCL insertion into
-sheet A we have used a
random PAI-1 mutant library (20) to identify this binding epitope, by
screening the library for functional PAI-1 mutants that no longer
react with 33B8.
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-D-galactoside, carbenicillin, tetracycline, and kanamycin were from Sigma. Reagents for protein concentration detection and enhanced chemiluminescence (ECL) assay were
from Pierce. All supplies and buffers for binding assays with BIAcore
3000TM Biosensor were purchased from BIAcore AB (Uppsala,
Sweden). All other chemical reagents used in the study were purchased
from Fisher.
Mutagenic primers for construction of site-directed mutants of PAI-1
EX(lox) library of random PAI-1 mutants with
>2 × 106 independent clones, constructed and
described by Berkenpas (20), was screened for mutant PAI-1 molecules
that were negative for 33B8 binding. Briefly, 200 µl of exponentially
growing host cells of E. coli BL21(DE3) resuspended in 10 mM MgSO4 at an A600 nm of 2.0 were infected with ~1000 plaque forming units of
EX(lox) PAI-1 library and plated. After 5.5 h of
growth at 37 °C, the plates were overlaid with nitrocellulose
filters previously coated with tPA at 10 µg/ml, blocked with 1%
BSA, 5% milk in Tris-buffered saline and saturated with 10 mM isopropyl-1-thio-
-D-galactoside. The
plates were then incubated for an additional hour at 37 °C. During
this time lytically infected cells expressing PAI-1 mutants release the
protein into the plaques, where functionally active inhibitor binds tPA
on the filter. The filters were then removed from the plates and washed
in Tris-buffered saline containing 0.5% SDS to remove any PAI-1 that
was not covalently bound to tPA (21). The filters were then incubated
with 33B8 as the primary antibody, and filter-bound tPA·PAI-1·33B8
complexes were detected immunologically with goat anti-mouse
horseradish peroxidase-conjugated immunoglobulins and ECL. The dark
spots revealed on ECL Hyperfilms indicated plaques positive for 33B8
binding. The filters were then washed with water and Tris-buffered
saline several times, and then incubated with rabbit anti-PAI-1
polyclonal antibodies as primary and goat anti-rabbit alkaline
phosphatase-conjugated antibodies as secondary. The filters were then
developed in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate with dark purple spots corresponding to plaques expressing
functionally active PAI-1 molecules. To select 33B8 binding negative
plaques, ECL hyperfilms were overlaid with the nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate-developed
nitrocellulose filters, and phage negative for 33B8 binding but
positive for binding to the polyclonal antibody were picked from
relevant bacterial plates. These isolated phage were then subjected to
two sequential rounds of double screening to obtain a pure phage
culture for each parental mutant.
-D-galactoside (1 mM) for 2 h. Pellets from 1.5 ml of culture were
resuspended in 0.4 ml of 0.05 M KPi buffer (pH 5.1),
containing 0.001 M EDTA, 0.15 M NaCl, and
0.02% sodium azide, then sonicated using a microtip on an
Ultrasonic processor XL from Misonix Inc. (Farmingdale, NY) for 1 min
with a 50% pulse in an ice slurry. Homogenates were then centrifuged
for 15 min at 14,000 rpm, and supernatant fractions were denoted as
cell-free extracts (CE) and tested in inhibitory activity and antibody
binding assays.
mouse Fc
fragments.
All antibodies were coupled to a N-hydroxy
succinimide/N-ethyl-N'-(dimethylaminopropyl)carbodiimide-activated CM-5 research grade sensor chip surface, to yield ~1000
resonance units response.
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EX(lox) phage
subjected to primary screening, 22 phage exhibited a negative
33B8-binding phenotype in the first round of selection, and of these 10 isolates remained 33B8-binding negative following two more rounds of
selection. These 10 independent clones were then subjected to
Cre-mediated recombination and the pEX-PAI-1 mutant plasmids
coding for the parental PAI-1 mutants were isolated. PAI-1 protein from
each of these mutants was then expressed in E. coli and
their 33B8 deficit confirmed by immunoblotting (Fig. 1D).
Nine of the 10 parental PAI-1 mutants showed a complete loss of 33B8
binding in this assay and only one of the 10 parental mutants (p5)
showed any residual binding to 33B8 by immunoblot. The functional
activity of each of these mutants was also examined and as expected,
because the screening assay selected only clones that produced active, soluble PAI-1, all of the clones effectively inhibited uPA (Fig. 1F). However, the amount of active PAI-1 produced by the
different clones varied by ~20-fold.
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Fig. 1.
Screen of 33B8 binding deficient PAI-1
mutants. Panels A-C, filter series
showing EX(lox) plaques. Panel A, plaques immunoreactive
with murine monoclonal 33B8; panel B, plaques reactive with
rabbit anti-PAI-1 polyclonal antibodies; panel C, a merged
image of A and B. The same filter is shown in
each case. The dark spots indicate PAI-1·antibody
complexes, and the circles indicate examples of PAI-1
mutants that are specifically disrupted in 33B8 binding. Panels
D and E show immunoblots of cell-free extracts of
wild-type and parental PAI-1 mutants selected from the screen.
Panel D was probed with 33B8 and panel E with
anti-PAI-1 polyclonal antibodies. Panel F shows the
inhibitory activity toward high molecular weight uPA of wild-type and
each of the PAI-1 parental mutants in cell-free extracts. The
inhibitory activity is expressed as the percentage of active PAI-1
protein relative to the total soluble protein of the cell
extracts.
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Fig. 2.
Frequency and distribution of mutations
identified by DNA sequence analysis of the parental clones.
PAI-1 mutants with reduced binding to monoclonal antibody 33B8
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Fig. 3.
Mapping of mutations identified in the
parental clones on the three-dimensional structures of active and
latent PAI-1. Mutations from each parental clone are shown in a
different color. Panels A and B show different
orientations of the PAI-1 structures, and the arrows
indicate the location of the mutation cluster on each view.
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Fig. 4.
Analysis of site-directed PAI-1 mutants.
Panels A and B show immunoblots of cell-free
extracts of wild-type and site-directed PAI-1 mutants. Panel
A was probed with 33B8 and panel B with anti-PAI-1
polyclonal antibodies. Panel C shows the inhibitory activity
toward high molecular weight uPA of wild-type and each of the PAI-1
site-directed mutants in cell-free extracts. The inhibitory activity is
expressed as the percentage of functionally active PAI-1 relative to
the total soluble protein of the cell extracts.
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Fig. 5.
Structure of the identified 33B8 binding
epitope in latent and active PAI-1. The relative positions of each
of the identified residues in either the latent conformation
(red) or the active conformation (blue) are
shown.
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Fig. 6.
Real time affinity binding of active and
latent PAI-1 to 33B8 (panel A) or to control
monoclonal antibody 31C9 (panel B). Binding was
measured by SPR with a biosensor BIAcore 3000. In each case 62 nM of purified active or latent PAI-1 in HBS-P buffer (pH
7.4) was analyzed on a CM 5 chip with amine-coupled murine 33B8 and
31C9 at a flow rate of 20 µl/min at 25 °C. The binding association
and dissociation phases were monitored for 3 min, then the chip surface
was regenerated by a 20-µl pulse of 10 mM glycine (pH
1.5) after each injection. The SPR response of control activated
surfaces without antibodies or with mouse Fc fragments was subtracted
from each curve.
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Fig. 7.
Real time affinity binding of parental
(panel A) and PAI-1 site-directed mutants (panel
B) to 33B8 was measured by SPR as described in the legend to
Fig. 6 except that the samples were cell-free extracts of wild-type,
parental, or site-directed PAI-1 mutants, diluted to 62 nM
PAI-1 in HBS-P buffer (pH 7.4). The individual site-directed
mutants are indicated as follows: N87D, N329I, K88E, D89G, Q174R,
T232S, G230V, S331R; double mutant N87D-N329I; wt indicates
wild type PAI-1.
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-sheet A (19, 23). If this equilibrium exists then there are
two possible ways in which the PAI-1 mutants identified in this study
could show reduced binding to 33B8. The first, which has already been
discussed, is by directly disrupting the epitope, and the second is by
stabilizing PAI-1 in a structure with low affinity for 33B8. In the
latter case we would expect that these mutants would also show a
reduced rate of conversion to the latent form because the putative
intermediate should be on the pathway from active PAI-1 to the latent
structure. To test this possibility we determined the rate at which
each of the point mutants converted to the latent conformation. These
data indicated that there was no correlation between the rate of PAI-1
conversion to the latent form and the 33B8 binding defect.
Specifically, the three point mutations that show major reductions in
their rates of association with 33B8 but only small changes in their
dissociation rates (N87D, T232S, and N329I) were either as stable, or
less stable than wild-type PAI-1 (data not shown). These results
support the conclusion that the identified mutations map the 33B8
binding epitope, and that the loss of binding by the mutants is not
because of effects of the mutations outside the epitope that shift the
putative equilibrium of active PAI-1 away from a form with high
affinity for 33B8.
1-antichymotrypsin in
the
conformation (26). These structures are shown in Fig. 8 along with the active and latent forms
of PAI-1. The 8 residues identified in our studies are purple in each
PAI-1 structure, as are residues at the homologous positions in
antithrombin III and
1-antichymotrypsin. Examination of
the four structures demonstrates that in active PAI-1 the putative
epitope is less compact than in the other structures, and that one of
the residues (Ser331) is essentially entirely
blocked by residues of the RCL in active PAI-1 (red arrow,
Fig. 8, A and E). In contrast, both latent PAI-1 (Fig. 8, D and H) and the mutant form of
1-antichymotrypsin (Fig. 8, C and
G) show a compact coherent epitope with all the residues visible on the surface of the molecule. In the case of antithrombin III
the epitope structure is intermediate between active PAI-1 (Fig. 8,
A and E) and the
conformation (Fig. 8,
C and G) with only the equivalent of the
Asn329 position being partially obscured by the RCL
(blue arrow, Fig. 8, B and F). These
structures demonstrate that serpins can exist in conformations with
partial RCL insertion and that the extent of this insertion can vary.
Furthermore, our modeling studies suggest that because the 33B8 epitope
contains residues that are located on both sides of the RCL insertion
site in the fully RCL expelled form of active PAI-1
(Asn329-
-strand 5A, Gln174-
-strand 3A,
and Asp89-
-strand 2A), then the RCL must be partially
inserted in a structure similar to those shown in Fig. 8, panels
B, F, C, and G, before the
antibody binds, otherwise the most likely effect of antibody binding
would be to block RCL insertion, not to promote it.
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Fig. 8.
Structures of active PAI-1 (panels
A and E; Protein Data Bank
ID 1b3k), native antithrombin III (panels B and
F; Protein Data Bank ID 1e05), a
mutant form of 1-antichymotrypsin
in the
conformation (panels C and
G; Protein Data Bank ID 1qmn)
and latent PAI-1 (panels D and
H; Protein Data Bank ID
1c5g). The identified 33B8 binding epitope in PAI-1 and modeled on
antithrombin III and
1-antichymotrypsin is shown in
purple and the RCL from P7-P15 is
highlighted in yellow in each structure. The red
arrows indicate where the RCL covers residue Ser331 of
the epitope in the active form of PAI-1, and the blue arrows
indicate where the RCL partially covers the residue equivalent to PAI-1
Asn329 in antithrombin III. The amino acids shown in
purple in antithrombin III and
1-antichymotrypsin were identified by sequence alignment
with PAI-1 and residues 353-357 (P7-P15) were
not visible in
1-antichymotrypsin and are presented as a
thin line.
Based on all of these observations, we hypothesize that PAI-1 is in a
state of dynamic equilibrium between a "closed" conformation, similar to the active structure seen in Fig. 8, panels A and
E, and an "open" conformation, with -sheet A at least
partially open between
-strands 3A and 5A. The open conformation
likely also includes partial insertion of the RCL similar to Fig. 8, panels B, F, C, and G. A
schematic presentation of the equilibrium is shown in Scheme 1.
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This hypothesis is consistent with current models of the serpin
mechanism, which suggest that active serpins have flexible RCLs, and
that this flexibility is essential for inhibitor function (21, 27-33).
Serpin activity can also be blocked by synthetic peptides that are
homologous to the serpin RCL. These peptides incorporate into the
serpin, as strand 4 of -sheet A and convert the inhibitor to a form
analogous to the latent structure but with the RCL exposed where it
becomes a non-inhibitory substrate for its target proteinase (34-37).
Presumably the first step in binding of the synthetic peptide would be
to the open conformation that we propose. During the normal inhibition
reaction, proteinase cleavage of the RCL to the point of an acylenzyme
intermediate is coupled with a rapid conformational rearrangement of
the serpin involving complete RCL insertion, which in turn causes a
distortion of the catalytic center of the enzyme and consequent
trapping of the proteinase (21, 29, 31, 32, 38-41). This mechanism, which was predicted in 1990 (38), was largely confirmed when the
crystal structure of a serpin-proteinase complex was recently solved
(42). This structure demonstrates that in the stable inhibited complex
the serpin RCL is fully inserted into
-sheet A, and that large
regions of the proteinase are distorted including the active site. This
distortion prevents the efficient deacylation of the complex and
kinetically traps the proteinase. Thus, full insertion of the RCL is
essential for stable inhibition. However, the initial steps of RCL
insertion are not well defined. For example, it has been suggested that
antithrombin III can undergo an allosteric switch between two
conformations of its RCL, one that is partially inserted and one that
is fully expelled from
-sheet A (43). Furthermore, this transition
regulates the activity of antithrombin toward different proteinases
(44, 45). These studies, together with the results described here,
suggest that a dynamic equilibrium between partial RCL insertion and an
uninserted form may be a common feature of serpins, and that this
equilibrium may play an important role in regulating serpin inhibitory activity.
Finally, our results demonstrate a novel approach for identifying the
binding epitope of a PAI-1 inactivator and for distinguishing its
mechanism of action. It is likely that this approach will be useful for
examining other PAI-1 ligands, and ultimately, this experimental design
may be applicable to the characterization of binding epitopes in other
metastable proteins, among which serpins are a predominant family that
presents many challenges, yet offer unique opportunities for drug discovery.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Ken Ingham for helpful discussions and for critically reading the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL55374 and HL55747 and Wyeth Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Vascular
Biology, The Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-517-0356; Fax: 301-738-0794; E-mail: Lawrenced@usa.redcross.org.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M208420200
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ABBREVIATIONS |
---|
The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; SPR, surface plasmon resonance; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; serpin, serine protease inhibitor; BSA, bovine serum albumin; CE, cell extracts.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Vaughan, D. E. (1998) J. Invest. Med. 46, 370-376[Medline] [Order article via Infotrieve] |
2. | Schneiderman, J., Sawdey, M. S., Keeton, M. R., Bordin, G. M., Bernstein, E. F., Dilley, R. B., and Loskutoff, D. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6998-7002[Abstract] |
3. | Juhan-Vague, I., Valadier, J., Alessi, M. C., Aillaud, M. F., Ansaldi, J., Philip-Joet, C., Holvoet, P., Serradimigni, A., and Collen, D. (1987) Thromb. Haemostasis 57, 67-72[Medline] [Order article via Infotrieve] |
4. | Juhan-Vague, I., and Alessi, M. C. (1997) Thromb. Haemostasis 78, 656-660[Medline] [Order article via Infotrieve] |
5. | Hamsten, A., de Faire, U., Walldius, G., Dahlen, G., Szamosi, A., Landou, C., Blombäck, M., and Wiman, B. (1987) Lancet 2, 3-9[Medline] [Order article via Infotrieve] |
6. | Siemens, H. J., Brueckner, S., Hagelberg, S., Wagner, T., and Schmucker, P. (1999) J. Clin. Anesth. 11, 622-629[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Koh, K. K.,
Mincemoyer, R.,
Bui, M. N.,
Csako, G.,
Pucino, F.,
Guetta, V.,
Waclawiw, M.,
and Cannon, R. O., III
(1997)
N. Engl. J. Med.
336,
683-690 |
8. | Taatjes, D. J., Wadsworth, M., Absher, P. M., Sobel, B. E., and Schneider, D. J. (1997) Ultrastruct. Pathol. 21, 527-536[Medline] [Order article via Infotrieve] |
9. | Stefansson, S., and Lawrence, D. A. (1996) Nature 383, 441-443[CrossRef][Medline] [Order article via Infotrieve] |
10. | Carmeliet, P., Stassen, J. M., Schoonjans, L., Ream, B., van den Oord, J. J., De Mol, M., Mulligan, R. C., and Collen, D. (1993) J. Clin. Invest. 92, 2756-2760[Medline] [Order article via Infotrieve] |
11. |
Farrehi, P. M.,
Ozaki, C. K.,
Carmeliet, P.,
and Fay, W. P.
(1998)
Circulation
97,
1002-1008 |
12. |
Eitzman, D. T.,
Westrick, R. J.,
Xu, Z.,
Tyson, J.,
and Ginsburg, D.
(2000)
Blood
96,
4212-4215 |
13. |
Hattori, N.,
Degen, J. L.,
Sisson, T. H.,
Liu, H.,
Moore, B. B.,
Pandrangi, R. G.,
Simon, R. H.,
and Drew, A. F.
(2000)
J. Clin. Invest.
106,
1341-1350 |
14. |
Eitzman, D. T.,
McCoy, R. D.,
Zheng, X.,
Fay, W. P.,
Shen, T.,
Ginsburg, D.,
and Simon, R. H.
(1996)
J. Clin. Invest.
97,
232-237 |
15. | Sharp, A. M., Stein, P. E., Pannu, N. S., Carrell, R. W., Berkenpas, M. B., Ginsburg, D., Lawrence, D. A., and Read, R. J. (1999) Structure Fold. Des. 7, 111-118[Medline] [Order article via Infotrieve] |
16. |
Hekman, C. M.,
and Loskutoff, D. J.
(1985)
J. Biol. Chem.
260,
11581-11587 |
17. | Mottonen, J., Strand, A., Symersky, J., Sweet, R. M., Danley, D. E., Geoghegan, K. F., Gerard, R. D., and Goldsmith, E. J. (1992) Nature 355, 270-273[CrossRef][Medline] [Order article via Infotrieve] |
18. | Debrock, S., and Declerck, P. J. (1997) Biochim. Biophys. Acta 1337, 257-266[Medline] [Order article via Infotrieve] |
19. |
Verhamme, I.,
Kvassman, J. O.,
Day, D.,
Debrock, S.,
Vleugels, N.,
Declerck, P. J.,
and Shore, J. D.
(1999)
J. Biol. Chem.
274,
17511-17517 |
20. | Berkenpas, M. B., Lawrence, D. A., and Ginsburg, D. (1995) EMBO J. 14, 2969-2977[Abstract] |
21. |
Lawrence, D. A.,
Ginsburg, D.,
Day, D. E.,
Berkenpas, M. B.,
Verhamme, I. M.,
Kvassman, J.-O.,
and Shore, J. D.
(1995)
J. Biol. Chem.
270,
25309-25312 |
22. | Lawrence, D., Strandberg, L., Grundström, T., and Ny, T. (1989) Eur. J. Biochem. 186, 523-533[Abstract] |
23. | Olson, S. T., Swanson, R., Day, D., Verhamme, I., Kvassman, J., and Shore, J. D. (2001) Biochemistry 40, 11742-11756[CrossRef][Medline] [Order article via Infotrieve] |
24. | Schreuder, H. A., de Boer, B., Dijkema, R., Mulders, J., Theunissen, H. J. M., Grootenhuis, P. D. J., and Hol, W. G. J. (1994) Nat. Struct. Biol. 1, 48-54[Medline] [Order article via Infotrieve] |
25. | Skinner, R., Abrahams, J. P., Whisstock, J. C., Lesk, A. M., Carrell, R. W., and Wardell, M. R. (1997) J. Mol. Biol. 266, 601-609[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Gooptu, B.,
Hazes, B.,
Chang, W. S.,
Dafforn, T. R.,
Carrell, R. W.,
Read, R. J.,
and Lomas, D. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
67-72 |
27. | Carrell, R. W., and Evans, D. L. I. (1992) Curr. Opin. Struct. Biol. 2, 438-446[CrossRef] |
28. |
Lawrence, D. A.,
Olson, S. T.,
Palaniappan, S.,
and Ginsburg, D.
(1994)
J. Biol. Chem.
269,
27657-27662 |
29. | Wright, H. T., and Scarsdale, J. N. (1995) Proteins 22, 210-225[Medline] [Order article via Infotrieve] |
30. | Fa, M., Karolin, J., Aleshkov, S., Strandberg, L., Johansson, L. B. Å., and Ny, T. (1995) Biochemistry 34, 13833-13840[Medline] [Order article via Infotrieve] |
31. |
Stratikos, E.,
and Gettins, P. G. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
453-458 |
32. | Wilczynska, M., Fa, M., Karolin, J., Ohlsson, P.-I., Johansson, L. B. Å., and Ny, T. (1997) Nat. Struct. Biol. 4, 354-357[Medline] [Order article via Infotrieve] |
33. |
Lawrence, D. A.,
Olson, S. T.,
Muhammad, S.,
Day, D. E.,
Kvassman, J. O.,
Ginsburg, D.,
and Shore, J. D.
(2000)
J. Biol. Chem.
275,
5839-5844 |
34. | Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R., and Laurell, C.-B. (1990) Eur. J. Biochem. 194, 51-56[Abstract] |
35. |
Björk, I.,
Ylinenjärvi, K.,
Olson, S. T.,
and Bock, P. E.
(1992)
J. Biol. Chem.
267,
1976-1982 |
36. |
Björk, I.,
Nordling, K.,
Larsson, I.,
and Olson, S. T.
(1992)
J. Biol. Chem.
267,
19047-19050 |
37. |
Kvassman, J.-O.,
Lawrence, D. A.,
and Shore, J. D.
(1995)
J. Biol. Chem.
270,
27942-27947 |
38. |
Lawrence, D. A.,
Strandberg, L.,
Ericson, J.,
and Ny, T.
(1990)
J. Biol. Chem.
265,
20293-20301 |
39. |
Wilczynska, M.,
Fa, M.,
Ohlsson, P.-I.,
and Ny, T.
(1995)
J. Biol. Chem.
270,
29652-29655 |
40. | Plotnick, M. I., Mayne, L., Schechter, N. M., and Rubin, H. (1996) Biochemistry 35, 7586-7590[CrossRef][Medline] [Order article via Infotrieve] |
41. | Kvassman, J. O., Verhamme, I., and Shore, J. D. (1998) Biochemistry 37, 15491-15502[CrossRef][Medline] [Order article via Infotrieve] |
42. | Huntington, J. A., Read, R. J., and Carrell, R. W. (2000) Nature 407, 923-926[CrossRef][Medline] [Order article via Infotrieve] |
43. | van Boeckel, C. A., Grootenhuis, P. D., and Visser, A. (1994) Nat. Struct. Biol. 1, 423-425[Medline] [Order article via Infotrieve] |
44. | Huntington, J. A., Olson, S. T., Fan, B., and Gettins, P. G. (1996) Biochemistry 35, 8495-8503[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Belzar, K. J.,
Zhou, A.,
Carrell, R. W.,
Gettins, P. G.,
and Huntington, J. A.
(2002)
J. Biol. Chem.
277,
8551-8558 |