From the Department of Biological Sciences,
University of Wollongong, Wollongong 2522, Australia,
§ Biotech Australia Pty Ltd, P.O. Box 20, Roseville 2069, Australia and ¶ John Curtin School of Medical Research, Division
of Cell Biology, Australian National University,
Canberra 2601, Australia
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ABSTRACT |
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The physiological roles of plasminogen activator
inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies
suggest a role in the regulation of plasminogen activator-driven
proteolysis in many cell types. This study describes a monoclonal
antibody (2H5), which uniquely recognizes neoepitope determinants on
PAI-2 appearing after thermodynamic relaxation of the molecule.
Enzyme-linked immunosorbent assays and native polyacrylamide gel
electrophoresis immunoblotting confirmed the specificity of 2H5 for
urokinase type plasminogen activator·PAI-2 complexes. Examination of
the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue
plasminogen activator, or thrombin suggests that the 2H5 epitope is
determined exclusively by sequences found only on PAI-2 following
proteolytic cleavage of the
Arg380-Thr381 bond and insertion of the
reactive site loop into -sheet A. Peptides lacking both the P13
(Glu368) and P14 (Thr367) residues did not
induce a conformational change or affect the inhibitory activity of
PAI-2, indicating that one or both of these residues are critical for
PAI-2 function. To our knowledge, this is the first description of a
monoclonal antibody that can distinguish conformational changes in
PAI-2 related specifically to its potential biological function(s).
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INTRODUCTION |
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Plasminogen activator inhibitor type-2 (PAI-2)1 is a member of the serpin (serine protease inhibitor) family (1) and is an efficient inhibitor of either active two-chain urokinase type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA). Although definitive in vivo evidence of the function of PAI-2 remains to be established, kinetic data, molecular regulation, and expression of PAI-2 by a wide range of cell types strongly suggests that PAI-2 plays an important role in many physiological and pathological processes such as pregnancy, skin differentiation, apoptosis, inflammation, and cancer (2).
In general, serpins interact with their target proteases through an
exposed peptide loop, known as the reactive site loop (RSL), which acts
as a form of "suicide" substrate leading to the formation of a
serpin-protease complex. As the crystal structure of a serpin-protease
complex has yet to be determined, the precise nature of the interaction
between serpin and protease remains somewhat controversial. However, an
extensive review of the literature (3) has led to a proposed mechanism
involving formation of a tetrahedral intermediate in which the RSL is
partially inserted into -sheet A, followed sequentially by cleavage
of the P1-P1' bond, formation of an acyl intermediate, release of the
C-terminal part of the inhibitor, and complete insertion of the RSL as
the central strand (s4A) of
-sheet A. Complete RSL insertion is
considered essentially irreversible and, along with associated
conformational changes, may distort the active site geometry of the
enzyme, preventing deacylation and trapping both proteins in a stable
complex. Slow hydrolysis can then occur to regenerate active protease
and release a cleaved, inactive form of the serpin. Examination of the
molecular interactions involved in RSL insertion are essential for a
complete understanding of the serpin inhibitory mechanism and for the
development of biochemical probes to determine biologically relevant
expression of serpin activity.
Despite uncertainty over the precise nature of the serpin inhibitory
mechanism, it is clear that inhibition involves insertion of the RSL
into -sheet A of the serpin and transition to a more stable
thermodynamic state, known as the relaxed (R) form of the inhibitor
(4-8). Transition to the R state involves dramatic conformational
changes in the serpin molecule, with the P1 and P1' residues separated
by approximately 70 Å following complete loop insertion. Other
conformational shifts may involve
-sheet C,
-helix F, and other
strands of
-sheet A (5).
We contend that PAI-2, as a member of the serpin family of proteins, undergoes the stressed to relaxed (S-R) conformational transition in association with inhibition of potential target proteases (e.g. uPA and tPA), as observed for several other inhibitory serpins. Detection of neoepitopes on complexes between antithrombin III and a synthetic RSL peptide (9) antithrombin-heparin complexes (10) and C1 inhibitor-protease complexes (11, 12) suggests that immunological approaches can be utilized to discriminate between the conformational states associated with expression of serpin activity. This study describes for the first time the induction of relaxed PAI-2 by insertion of a synthetic RSL peptide and characterization of a monoclonal antibody (mAb) directed specifically against an epitope expressed on this conformation of PAI-2. This immunological probe will assist in determining the mechanisms involved in the inhibition of potential target proteases and hence the physiological and/or pathological roles of the unique serpin PAI-2.
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant (Escherichia coli) human
PAI-2 and goat anti-human PAI-2 polyclonal antibody was a kind gift
from Biotech Australia (Sydney, Australia). Human two-chain uPA was
from Serono (Sydney, Australia). SDS-PAGE analysis showed that uPA in
this preparation existed mainly in the 55-kDa form. Human two-chain tPA
was from American Diagnostica Inc. (Sydney, Australia) and was
reconstituted in PBS and stored at 20 °C before use. Thrombin
(bovine, plasminogen-free) was from Calbiochem (Sydney, Australia) and
before use was reconstituted in PBS and stored at
20 °C.
Peptide Synthesis-- Synthetic PAI-2 RSL peptides were synthesized by the solid phase method using an ABI 430A peptide synthesizer and optimized tert-butyloxycarbonyl chemistry protocols (13) Crude product was purified to homogeneity by preparative reversed-phase high pressure liquid chromatography on a wide pore C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. Peptides were subsequently characterized by mass spectrometry and amino acid composition analysis. RSL peptides were acetylated at the N terminus and were synthesized corresponding to different length sections of the reactive site loop of PAI-2. Each peptide was successively shortened by two residues from the N terminus with the 14-mer having the sequence H-Ac-TEAAAGTGGVMTGR-OH.
Preparation of Protein Complexes--
uPA·PAI-2 complexes were
produced by incubating equimolar amounts of uPA and PAI-2 for 2 h
at 37 °C. uPA (55 kDa)·PAI-2 complexes were separated from
uncomplexed PAI-2, uPA, polymerized PAI-2, and uPA (33 kDa)·PAI-2
complexes by FPLC on a Mono-S HR5/5 column (Amersham Pharmacia Biotech)
using a 0-1 M NaCl gradient in 50 mM phosphate
buffer (pH 6). Fractions were analyzed by SDS-PAGE, pooled where
appropriate, concentrated, and desalted with Centricon-10 microconcentrators (Amicon, Sydney). PAI-2·RSL complexes were prepared by incubating active "stressed" PAI-2 at 1 mg·ml1 with a 100-fold molar excess of the RSL peptide
in 50 mM Tris, 50 mM NaCl, pH 8.0 at 37 °C
for 48 h. The tPA/PAI-2 reaction mixture was prepared by
incubating equimolar amounts of tPA and PAI-2 for 1 h at 37 °C.
Thrombin-treated PAI-2 was prepared by incubating equimolar amounts of
thrombin and PAI-2 for 1 h at 37 °C. Since PAI-2 does not form
complexes with thrombin (14), purification of PAI-2 from thrombin was
not deemed necessary.
Monoclonal Antibody Production--
Mice were immunized with
uPA·PAI-2 complexes. Boosting and polyethylene glycol-induced fusion
of splenic lymphocytes with mouse plasmacytoma (NS-1) cells was
performed as described previously (15). Hybridomas were screened for
reactivity against purified uPA·PAI-2 complexes (and negative
reactivity against uPA or PAI-2 alone) by enzyme-linked immunosorbent
assay (ELISA), and the antibodies produced by the selected hybridoma
clone (2H5) were determined to be of the IgG1
subclass.2 The 2H5-producing
hybridoma clones were cultured in Dulbecco's modified Eagle's medium
(CSL, Melbourne, Australia) supplemented with 10% fetal calf serum
(CSL). Hybridoma-conditioned medium was centrifuged at 1000 × g for 10 min, precipitated with 50% ammonium sulfate with
continual stirring for 6 h at 4 °C, and then dialyzed against
three changes of PBS overnight. Further purification was achieved using
Protein G Superose FPLC affinity chromatography equilibrated in 20 mM sodium phosphate buffer (pH 7). Elution was achieved
with 0.1 M glycine-HCl (pH 2.7). Fractions (1 ml) were
neutralized by the addition of 200 µl of 1 M Tris-HCl (pH
9) and dialyzed against three changes of PBS at 4 °C overnight.
SDS-PAGE-- Unless otherwise stated, 4-20% Tris/glycine precast polyacrylamide gels (Bio-Rad, Sydney, Australia) under nonreducing conditions were used for analysis of protein-containing fractions collected from FPLC, checking the integrity of uPA and PAI-2 standards, or confirming the purity of all mAb preparations. Gels were routinely run at 150 V for 1 h in Tris/glycine running buffer (pH 6.8).
Nondenaturing Polyacrylamide Gel Electrophoresis (Native PAGE)-- In order to conserve the native conformation of proteins being investigated by Western blotting, nondenaturing (native) gel electrophoresis was employed. Samples were routinely run on 4-10% gradient gels for 1 h at 150 V in 90 mM Tris, 80 mM borate, 3 mM EDTA buffer (pH 8.2) (16).
Tranverse Urea Gradient Polyacrylamide Gel Electrophoresis (TUG-PAGE)-- Sample (100 µg of protein) was diluted 1:1 with native PAGE sample buffer and loaded onto 7% acrylamide gels containing a linear 0-8 M urea gradient. Gels were run at 30 mA for 70 min using Tris/glycine running buffer (17).
Fluorescence Spectroscopy--
Fluorescence spectroscopy was
performed on a Hitachi F4500 spectrofluorimeter at an excitation
wavelength of 295 nm. Measurements were performed at a PAI-2
concentration of 0.4 µM in 50 mM Tris, 50 mM NaCl. Data are presented as the fraction of protein
denatured, calculated as (native
)/(
native
denatured), where
native is the emission wavelength maximum of native
PAI-2,
is the emission wavelength maximum of the individual sample,
and
denatured is the emission wavelength maximum of
completely denatured PAI-2 (18).
PAI-2 Activity Assay--
Specific activity of PAI-2 was
determined by inhibition of uPA-mediated hydrolysis of
carbobenzoxy-L--glutamyl-(
-t-butoxy)-glycyl-arginine-p-nitroanilide-diacetate (2.0 mM) in the Spectrolyse® UK assay
(American Diagnostica). Statistical comparison of mean activity was
performed using Student's t test.
ELISA--
Titretek PVC microtiter plates (Flow Laboratories)
were routinely coated with 50 µl of antigen (3 µg·ml1) in PBS for 1 h at 37 °. Plates were
washed with PBS/Tween, and nonspecific binding sites were blocked with
50 µl of 1% casein in PBS/Tween at 37 °C for 1 h or at
4 °C overnight. 50 µl of purified mAb 2H5 in PBS was then applied,
and the plates were incubated at 37 °C for 1 h. Plates were
then washed and incubated with 50 µl of 1:3000 alkaline
phosphatase-conjugated goat anti-mouse (
-chain-specific) second
antibody (Sigma) at 37 °C for 1 h. Plates were finally washed
three times with 150 µl of carbonate buffer (pH 9.1). Color development was measured at 405 nm following the addition of 50 µl of
-nitrophenyl phosphate (Boehringer Mannheim, Sydney, Australia) at 1 mg·ml
1 in carbonate buffer (pH 9.1) at 37 °C. Color
development was stopped by the addition of 50 µl of 3 M
NaOH.
Western Blotting-- Following native PAGE, proteins were transferred onto Immobilon-P membrane (Millipore, Sydney, Australia) in prechilled Tris/glycine transfer buffer at 100 V for 1 h or 30 V overnight. Antibody-antigen interactions were detected using the Immuno-Blot assay kit (Bio-Rad), as per the manufacturer's instructions. Nonspecific binding sites on the membrane were blocked with 3% gelatin/Tris-buffered saline, and the membrane was incubated in primary antibody (mAb 2H5) diluted 1:1500 in 1% gelatin, 0.05% Tween-20, Tris-buffered saline for 1 h at room temperature, washed, and incubated for 1 h in alkaline phosphatase-conjugated goat anti-mouse second antibody diluted 1:3000.
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RESULTS |
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Insertion of a Synthetic RSL Peptide Induces the "Relaxed" Conformation of PAI-2-- To investigate the stability of PAI-2·RSL complexes, resistance to denaturation by urea was analyzed using TUG gels. Fig. 1A, a characteristic denaturation profile of native recombinant PAI-2, shows greatly reduced electrophoretic mobility of the protein at higher urea concentrations. This profile is indicative of increasing denaturation over the corresponding urea gradient. The denaturation curve of PAI-2 incubated with a 100-fold molar excess of 14-mer RSL peptide shows a major curve comprising a series of three bands. In contrast to native PAI-2, the electrophoretic mobility of the PAI-2·14-mer RSL complex is not significantly reduced at higher concentrations of urea, indicating increased resistance to denaturation and hence a more stable conformation. PAI-2 incubated with RSL peptides lacking both the P13 (Glu) and P14 (Thr) residues displayed denaturation curves similar to native PAI-2 (data not shown).
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CD Spectral Analysis of the PAI-2·RSL Complex-- CD spectra from 200 nm in the far UV region to 300 nm in the near UV region of stressed PAI-2 (i.e. no peptide) and PAI-2 incubated with a 100-fold molar excess of RSL peptides were measured. Fig. 3 clearly indicates a dramatic shift in the spectrum of PAI-2 following incubation with the 14-mer RSL peptide, indicating a significant conformational transition. No significant shift was observed in the CD spectra of PAI-2 incubated with shorter peptides lacking the P13 and P14 residues (data not shown).
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Relaxed PAI-2 Formed by RSL Peptide Insertion Does Not Inhibit uPA-- To ascertain if incubation of PAI-2 with the synthetic RSL peptides altered the ability of the serpin to act as an inhibitor, the uPA-specific inhibitory activities of native PAI-2 and PAI-2·RSL complexes were measured. The uPA-specific inhibitory activity of native PAI-2 was determined to be approximately 1.8 × 105 IU/mg (Fig. 4). The inhibitory activity of PAI-2 incubated with a 100-fold molar excess of RSL peptides ranging in size from 12 to 6 residues (i.e.. lacking both the P13 (Glu) and P14 (Thr) residues) was not significantly different from that of native PAI-2. However, incubation of PAI-2 with a 100-fold molar excess of 14-mer RSL peptide significantly decreased inhibitory activity to almost undetectable levels (p < 0.05) (Fig. 4). In addition, SDS-PAGE analysis and N-terminal amino acid sequencing indicated that the PAI-2·14-mer RSL complex behaved as a substrate of uPA. Two N-terminal sequences, corresponding to the N terminus of PAI-2 and the sequence from Thr381 (P1') onwards, were detected within 30 s of uPA addition, indicating cleavage of the P1-P1' bond of PAI-2. No evidence of formation of complexes between uPA and the PAI-2·14mer RSL complex was apparent by SDS-PAGE analysis (data not shown).
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Purification of uPA·PAI-2 Complexes and Generation of mAb 2H5-- To screen hybridomas for antibodies that specifically detected relaxed PAI-2 but not native PAI-2 or uPA, it was necessary to produce purified uPA·PAI-2 complexes free of any traces of uPA or native stressed PAI-2 remaining in the reaction mixture. Purification of uPA (55 kDa)·PAI-2 complexes from residual uncomplexed uPA and PAI-2, dimerized PAI-2, and uPA (33 kDa)·PAI-2 complexes remaining in the reaction mixture was achieved by ion exchange FPLC. A significant peak eluted from the column prior to increasing the salt concentration and was shown by SDS-PAGE to represent residual uncomplexed PAI-2 (data not shown). A peak eluting at 0.2 M NaCl represented uPA (33 kDa)·PAI-2 complexes, while another at 0.3-0.35 M NaCl contained only uPA (55 kDa)·PAI-2 complexes (data not shown). At higher salt concentrations (>0.5 M), two smaller peaks were visible, representing uncomplexed uPA (33 kDa) and uPA (55 kDa), respectively (data not shown). This method provided sufficient quantities of highly pure uPA (55 kDa)·PAI-2 antigen for subsequent hybridoma screening2 and immunological studies.
Detection of Neoepitopes Expressed on Relaxed PAI-2 Using mAb 2H5-- SDS-PAGE analysis of uPA incubated with PAI-2 in increasing molar ratios showed a corresponding increase in the relative amount of uPA·PAI-2 complex formed, reaching a maximum when the molar ratio of uPA to PAI-2 was 1:1 (Fig. 5A). A strong relationship was observed between mAb 2H5 binding to uPA·PAI-2 complexes by ELISA and the amount of uPA·PAI-2 complex observed by SDS-PAGE analysis. Maximum binding of mAb 2H5 was observed when the molar ratio of uPA to PAI-2 was approximately 1:1 (Fig. 5B).
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DISCUSSION |
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Understanding the mechanism of action of serpins is critical not only for understanding how this family of proteins functions but also to assist in the identification of cognate target proteases where the physiological activity of particular serpins is unknown. Additionally, the production of immunological probes capable of distinguishing the different serpin conformations may prove invaluable for identifying the temporal and spatial factors affecting serpin physiological function.
Despite uncertainty over the precise nature of the serpin inhibitory
mechanism, recent studies support the following model: (a)
cleavage of the P1-P1' bond and formation of a covalent acyl intermediate with the protease active site serine; (b) rapid
insertion of the RSL into -sheet A of the inhibitor, preventing
deacylation by distorting the active site of the protease, hence
trapping the molecules in a stable complex; and (c)
associated conformational changes in the inhibitor resulting in
transition to a more thermodynamically stable or relaxed state (3-9,
20).
Previous studies have shown that serpin action requires RSL insertion
(6, 7). These studies used CD spectroscopy, activity assays, and
thermal and chemical denaturation to show that insertion of synthetic
RSL peptides induced the relaxed conformations of 1-antitrypsin and
antithrombin III, respectively. The present study confirms and advances
these works by focusing on the biomolecular interactions and
conformational changes associated with the serpin activity of the
putative plasminogen activator inhibitor, PAI-2. In summary, our data
show induction of the relaxed conformation of PAI-2 by insertion of a
synthetic RSL peptide by several methods. TUG-PAGE and fluorimetry
showed that PAI-2·RSL complexes had greatly increased resistance to
urea denaturation, indicating a significant increase in conformational
stability of PAI-2 following peptide insertion. Insertion of synthetic
RSL peptides or formation of serpin-protease complexes has previously
been shown by several authors (21, 22) to increase resistance to
denaturation in urea. Additionally, a significant shift in the CD
spectra of PAI-2 was observed following incubation with 14-mer RSL
peptide. This shift was similar to that observed for formation of
relaxed
1-antitrypsin by cleavage of the P1-P1' bond (6) and is
further evidence of relaxed PAI-2 formation by insertion of RSL
peptide.
Investigation of the conditions required for PAI-2·RSL complex formation showed that resistance to urea denaturation increased as the molar ratio of RSL·PAI-2 present during incubation increased, approaching a maximum at a 100-fold molar excess of RSL. These data are in complete agreement with previous studies (6, 7), which found that similar excesses of RSL peptide were required to produce equimolar complexes. Together, these data indicate that the insertion of synthetic RSL peptide into native, stressed PAI-2 is kinetically unfavorable and may require partial unfolding of the molecule to overcome possible obstruction of the peptide binding site by the RSL of PAI-2.
Formation of PAI-2·RSL complexes was accompanied by an almost
complete loss of uPA inhibitory activity. However, uPA was still able
to cleave the P1-P1' bond of PAI-2 when complexed to RSL peptide,
indicating the conversion of PAI-2 from an inhibitor to a substrate of
uPA. The model outlined above predicts that inhibition of proteases by
serpins results from distortion of the protease active site through
conformational changes accompanying insertion of the RSL into -sheet
A, preventing deacylation. It is likely that the synthetic RSL peptide
occupies a similar, if not identical, position in
-sheet A to that
which the RSL of PAI-2 would occupy following stable complex formation
with uPA or other proteases. Binding of the synthetic RSL peptide to
this site may block insertion of the RSL of PAI-2 following cleavage at
the P1-P1' bond and prevent trapping of the protease in a stable complex, hence preventing inhibition. These data support analogous findings for antithrombin III (7) and provide strong evidence that RSL
insertion is a critical event in the inhibition of uPA, or other
potential target proteases, by PAI-2.
Examination of the molecular interactions involved in RSL insertion is
essential for a complete understanding of the serpin inhibitory
mechanism. Unlike the aforementioned studies on antithrombin III and
1-antitrypsin, this study employed a series of shortened RSL
peptides to investigate the interactions required for RSL insertion in
PAI-2. We did not observe the formation of relaxed PAI-2 by incubation
with any RSL peptides lacking the P14 (Thr367) and P13
(Glu368) residues, indicating that either/both of these
residues are critical for insertion of strand s4A into
-sheet A of
PAI-2 during protease inhibition. Previous studies have suggested that
the size and charge of the P14 residue (among others) may be critical in determining the favorability of RSL insertion and hence inhibitory activities of the different serpin molecules (23). For example, the
noninhibitory serpin ovalbumin has a large, positively charged amino
acid (arginine) at the P14 position, possibly preventing RSL insertion
by unfavorable spatial and electrostatic interactions and hence
preventing protease inhibition. Interestingly, our results indicate
that P13 (Glu368) may also play a very important role in
strand insertion. The negative charge of the glutamic acid may
stabilize interactions between the RSL and
-sheet A and drive
insertion of the remainder of the strand through electrostatic
interactions. The amino acid residues on strands 3 and 5 of
-sheet
A, predicted to be in close proximity to P13, are positively charged
and may facilitate the insertion of the RSL into
-sheet A. Recent
analysis of several inhibitory and noninhibitory serpins indicates
that, along with P13, the P8 residue may also be critical for RSL
insertion and protease
inhibition.3 Crystallization
of a serpin-protease complex should provide the necessary evidence to
elucidate the precise nature of molecular interactions involved in RSL
insertion during inhibition by serpins.
The precise physiological roles of PAI-2 are somewhat controversial.
Its expression by a wide variety of cells with invasive phenotypes
suggests that it is a regulator of cell-mediated extracellular matrix
degradation and tissue remodeling in processes as diverse as
inflammation, skin differentiation, metastasis, and pregnancy (2). More
recently, intracellular PAI-2 has been implicated in protection from
tumor necrosis factor--induced apoptosis (24-26), although the
mechanism responsible for this protection remains unknown. Previously,
polyclonal antibodies had been used to detect relaxed antithrombin III
and
1-antitrypsin (6, 9), suggesting the possibility of
using immunological methods to distinguish the different conformations
of serpins. In order to better understand the biochemistry and
physiology of PAI-2, we generated and characterized a monoclonal
antibody (2H5) specifically detecting only the relaxed form of PAI-2.
Future use of this probe may facilitate identification of potential
target proteases and establish patterns of temporal and spatial
expression of PAI-2 inhibitory activity in various physiological and
pathological processes.
Characterization of the epitope recognized by mAb 2H5 by ELISA and
native PAGE immunoblotting showed that the epitope existed on
uPA·PAI-2 complexes, tPA·PAI-2 complexes, PAI-2 treated with thrombin, and complexes of PAI-2 with 14-mer RSL peptide. The binding
of mAb 2H5 to a crude reaction mixture of uPA and PAI-2 was found to
increase as uPA was titrated with PAI-2. Increased binding corresponded
with levels of uPA·PAI-2 complex formation, approaching a maximum at
a 1:1 molar ratio of uPA to PAI-2, where maximal levels of uPA·PAI-2
complex were observed. mAb 2H5 did not recognize uPA, native stressed
PAI-2, an RSL peptide, or thrombin alone. Together, these data indicate
that mAb 2H5 recognizes neoepitopes on PAI-2 following the stressed to
relaxed conformational transition associated with cleavage at the
P1-P1' bond and insertion of the reactive site loop into -sheet A
of the inhibitor. Analysis of various mutant serpins (5) suggests the
presence of several functionally related antigenic mobile domains that
may be involved in immunological recognition of relaxed serpins. These
include the proximal and distal hinge regions, F helix, and
-sheets
A and C. However, proteolytic or cyanogen bromide fragmentation of
PAI-2 did not produce any mAb 2H5-reactive fragments (data not shown),
suggesting that the epitope recognized by mAb 2H5 may be a
conformational epitope rather than a single linear sequence. Further
investigation of the mAb 2H5 epitope may assist in detailing the
structural changes associated with protease inhibition by PAI-2 and
lead to a better understanding of the inhibitory mechanism of serpins
in general.
This study describes for the first time the generation and characterization of relaxed PAI-2 by insertion of a synthetic RSL peptide, mimicking the conformation of PAI-2 induced by inhibition of its target protease uPA. The generation of a monoclonal antibody to specifically detect this conformation of PAI-2 provides an immunological probe for precise localization of in vivo sites (i.e. skin, placenta, tumors) in which PAI-2 is actively inhibiting uPA or other target proteases and may lead to a better understanding of the physiological and/or pathological roles of this molecule. For example, mAb 2H5 has recently been used to show differential expression of relaxed PAI-2 in human gestational tissues (27), leading to a better understanding of the role of the plasminogen activation cascade in pregnancy.
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ACKNOWLEDGEMENTS |
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We thank Bernie McInerney and Michelle Gleeson (Biotech Australia) for assistance with peptide synthesis and PAI-2·RSL complex production, respectively, Sue Butler (Department of Chemistry, University of Wollongong) for assistance with CD spectroscopy, and Dr. Toni Antalis for critical review of the manuscript.
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FOOTNOTES |
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* 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
Biological Sciences, University of Wollongong, Wollongong 2522, Australia. Tel.: 61-42-214356; Fax: 61-42-214135; E-mail:
m.s.baker{at}uow.edu.au.
1 The abbreviations used are: PAI-2, plasminogen activator inhibitor type 2; uPA, urokinase type plasminogen activator; tPA, tissue type plasminogen activator; mAb, monoclonal antibody; RSL, reactive site loop; PAGE, polyacrylamide electrophoresis; FPLC, fast protein liquid chromatography; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TUG, tranverse urea gradient.
2 V. McPhun, L. Maxwell, and M. S. Baker, manuscript in preparation.
3 P. Curmi, personal communication.
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REFERENCES |
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