Immunological Detection of Conformational Neoepitopes Associated with the Serpin Activity of Plasminogen Activator Inhibitor Type-2*

Darren N. SaundersDagger , Kathy M. L. ButtigiegDagger , Alison Gould§, Virginia McPhun, and Mark S. BakerDagger parallel

From the Dagger  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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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).

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 beta -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 beta -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 beta -sheet C, alpha -helix F, and other strands of beta -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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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·ml-1 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 IgG1kappa 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 (lambda native - lambda )/(lambda native - lambda denatured), where lambda native is the emission wavelength maximum of native PAI-2, lambda  is the emission wavelength maximum of the individual sample, and lambda 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-gamma -glutamyl-(alpha -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·ml-1) 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 (gamma -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 rho -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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   Urea denaturation analysis of PAI-2·RSL complexes. A, TUG-PAGE analysis of PAI-2 and PAI-2 incubated with 14-mer RSL peptide over a linear urea gradient (0-8 M). B, intrinsic tryptophan fluorescence emission (excitation, 295 nm) of PAI-2 (open squares), PAI-2 incubated with a 100-fold molar excess of 14-mer RSL peptide (open circles), and PAI-2 incubated with a 100-fold molar excess of 12-mer RSL peptide (open diamonds) over a range of urea concentrations. Data are presented as the fraction of protein denatured, calculated as (lambda native - lambda )/(lambda native - lambda denatured), where lambda native is the emission wavelength maximum of native PAI-2, lambda  is the emission wavelength maximum of the individual sample, and lambda denatured is the emission wavelength maximum of completely denatured PAI-2.

Measurement of the intrinsic tryptophan fluorescence spectrum of PAI-2 over a urea gradient (Fig. 1B) shows an unfolding pattern comparable with that observed by TUG-PAGE. The urea concentration at half-maximal unfolding of native PAI-2 ([urea]1/2) was calculated to be 6.4 M, and a predicted Delta G(H2O) of 4.693 kcal/mol was obtained. In contrast, PAI-2 incubated with a 100-fold molar excess of 14-mer RSL peptide was extremely resistant to denaturation even at high concentrations of urea, confirming results obtained by TUG-PAGE. Because an unfolding transition was not observed for this complex, the Delta G(H2O) and [urea]1/2 could not be confidently predicted. PAI-2 incubated with 12-mer RSL peptide, lacking both the P13 (Glu) and P14 (Thr) residues, displayed a denaturation curve and predicted Delta G(H2O) matching native PAI-2. These data show that the interaction of PAI-2 with the 14-mer RSL peptide induced resistance to urea denaturation, indicating a significant increase in conformational stability of native PAI-2.

Incubation of PAI-2 with varying molar ratios of 14-mer RSL peptide for 48 h at 37 °C and subsequent analysis by fluorescence spectroscopy showed that resistance to urea denaturation increased as the molar ratio of peptide increased, approaching a maximum at a 100-fold excess (Fig. 2). Similarly, inhibition of PAI-2 activity increased as the molar ratio of peptide increased, reaching a maximum at a 100-fold excess (data not shown).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Dose dependence of urea denaturation of PAI-2·RSL complexes on RSL:PAI-2 ratio. Resistance to denaturation by 8 M urea of PAI-2 incubated with 14-mer RSL peptide (open circles) or 12-mer RSL peptide (open squares) over a range of RSL:PAI-2 molar ratios. Data is presented as the fraction of protein denatured, calculated as (lambda native - lambda )/(lambda native - lambda denatured), where lambda native is the emission wavelength maximum of native PAI-2, lambda  is the emission wavelength maximum of the individual sample, and lambda denatured is the emission wavelength maximum of completely denatured PAI-2.

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   CD spectral analysis of PAI-2·RSL complexes. CD spectra of native PAI-2 and PAI-2 incubated with a 100-fold molar excess of 14-mer RSL peptide.

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).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Relaxed PAI-2 formed by RSL peptide insertion does not inhibit uPA. uPA-specific inhibitory activity (IU/mg × 105) of untreated recombinant PAI-2 and PAI-2 incubated with a 100-fold molar excess of RSL peptide. Data are expressed as mean ± S.E. (n = 6).

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).


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 5.   Formation of uPA·PAI-2 complex in vitro by titration of uPA against PAI-2. A, nonreducing SDS-PAGE analysis of the production of uPA (55 kDa)·PAI-2 complex over a range of uPA:PAI-2 molar ratios as follows: uPA:PAI-2 = 1:16 (lane 1); uPA:PAI-2 = 1:8 (lane 2); uPA:PAI-2 = 1:4 (lane 3); uPA:PAI-2 = 1:2 (lane 4); uPA:PAI-2 = 1:1 (lane 5); and uPA:PAI-2 = 2:1 (lane 6). B, graph showing relative density of the ~92-kDa band corresponding to uPA (55 kDa)·PAI-2 complex (open circles) and binding of mAb 2H5 (open squares) over a range of uPA:PAI-2 molar ratios. Binding was measured by ELISA at a primary antibody dilution of 1:50 (from a stock of 478 µg·ml-1). Data are presented as mean ± S.E. (n = 3).

We hypothesized that the affinity of mAb 2H5 for the uPA·PAI-2 complex resulted from expression of neoepitopes on PAI-2 following conformational changes induced by interaction with target proteases. This implies that the epitope recognized by mAb 2H5 is not determined by sequences found on uPA. Fig. 6A shows that mAb 2H5 bound at low titer to purified uPA·PAI-2 complex but not to either native recombinant (i.e. active, stressed) PAI-2 nor uPA. Although considered to be the principal in vivo inhibitor of uPA, PAI-2 is also a very efficient inhibitor of tPA (20). Fig. 6C shows that mAb 2H5 also interacts at low titers with a mixture of tPA and PAI-2 but not with either tPA or PAI-2 protein alone.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   ELISA detection of neoepitopes expressed on relaxed PAI-2 using mAb 2H5. Shown is binding of mAb 2H5 to uPA (square ), stressed PAI-2 (open circle ), and FPLC-purified uPA:PAI-2 complexes (bullet ) (A); stressed PAI-2 (open circle ), RSL peptide (triangle ), and PAI-2·RSL complexes (black-triangle) (B); and PAI-2 (open circle ), tPA (diamond ), bovine thrombin (down-triangle), and tPA·PAI-2 complexes (black-diamond ) and stressed PAI-2 treated with thrombin (black-triangle) (C) measured by ELISA (as described under "Experimental Procedures"). The stock mAb 2H5 concentration was 478 µg·ml-1 soluble protein. Data are presented as mean ± S.E. (n = 3).

It was expected that the formation of a complex between PAI-2 and the 14-mer RSL peptide would mimic the interaction of PAI-2 with uPA by insertion of the RSL analogue into the beta -sheet A of PAI-2, as previously observed with other serpins (6-9), hence inducing the relaxed conformation of the molecule. mAb 2H5 binding to PAI-2·14-mer RSL complexes was relatively high. In contrast, mAb 2H5 showed no interaction with either active recombinant PAI-2 or intact RSL peptide alone (Fig. 6B). All synthetic RSL peptides lacking both the P13 (Glu) and P14 (Thr) residues failed to induce any detectable changes in the conformation of PAI-2 by ELISA (data not shown). Analysis of the peptide sequence of PAI-2 revealed the presence of a predicted thrombin cleavage site at the P1-P1' bond (Arg380-Thr381). Fig. 6C shows that treatment of PAI-2 with bovine thrombin for 1 h at 37 °C resulted in a significant increase in mAb 2H5 binding. Bovine thrombin alone did not interact with mAb 2H5.

To further investigate the specificity of mAb 2H5 while conserving protein conformation, nondenaturing (native) PAGE immunoblotting analysis was used. The absence of reaction against native PAI-2 (Fig. 7B) confirms that mAb 2H5 does not interact with active, stressed PAI-2. In contrast, mAb 2H5 reacts strongly with uPA·PAI-2 complexes (Fig. 7B). Analysis of PAI-2·RSL complexes by native PAGE and silver staining (Fig. 7A) showed the presence of several distinct protein bands, all of which were detectable by mAb 2H5 on Western blots (Fig. 6B). Native PAGE and Western blotting analysis of uPA alone was not possible because the protein has a net positive charge at pH 8.2, making it unsuitable in the buffer system used; however, uPA applied directly to the membrane did not react with mAb 2H5 (data not shown). These data confirm that RSL insertion induces an immunologically distinct conformation of PAI-2, which is detectable by mAb 2H5.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 7.   Native immunoblot detection of neoepitopes expressed on relaxed PAI-2 using mAb 2H5. Native PAGE immunoblot to examine the specificity of mAb 2H5 (A). Silver-stained native PAGE gel (pH 8.2) showing PAI-2 (lane 1); FPLC-purified uPA (55 kDa)·PAI-2 complexes (lane 2); and PAI-2·RSL complexes (lane 3). B, corresponding duplicate Western blot using a 1:1500 dilution of mAb 2H5 as primary antibody (from 478 µg·ml-1 stock) with identical lane sequence to A.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 alpha 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 alpha 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 beta -sheet A, preventing deacylation. It is likely that the synthetic RSL peptide occupies a similar, if not identical, position in beta -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 alpha 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 beta -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 beta -sheet A and drive insertion of the remainder of the strand through electrostatic interactions. The amino acid residues on strands 3 and 5 of beta -sheet A, predicted to be in close proximity to P13, are positively charged and may facilitate the insertion of the RSL into beta -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-alpha -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 alpha 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 beta -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 beta -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

parallel 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966[Medline] [Order article via Infotrieve]
  2. Sumiyoshi, K., Serizawa, K., Urano, T., Takada, Y., Takada, A., and Baba, S. (1992) Int. J. Cancer 50, 345-348[Medline] [Order article via Infotrieve]
  3. Gettins, P. G. W., Patston, P. A., and Olson, S. T. (1996) Serpins: Structure, Function, and Biology, Chapman & Hall, New York
  4. Gettins, P., Patston, P. A., and Schapira, M. (1992) Hematol. Oncol. Clin. North. Am. 6, 1393-1408[Medline] [Order article via Infotrieve]
  5. Stein, P. E., and Carrell, R. W. (1995) Nat. Struct. Biol 2, 96-113[Medline] [Order article via Infotrieve]
  6. Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R., and Laurell, C. B. (1990) Eur. J. Biochem. 194, 51-56[Abstract]
  7. Bjork, I., Ylinenjarvi, K., Olson, S. T., and Bock, P. E. (1992) J. Biol. Chem. 267, 1976-1982[Abstract/Free Full Text]
  8. Carrell, R. W., Evans, D. L., and Stein, P. E. (1991) Nature 353, 576-578[CrossRef][Medline] [Order article via Infotrieve]
  9. Bjork, I., Nordling, K., and Olson, S. T. (1993) Biochemistry 32, 6501-6505[Medline] [Order article via Infotrieve]
  10. Dawes, J., James, K., and Lane, D. A. (1994) Biochemistry 33, 4375-4383[Medline] [Order article via Infotrieve]
  11. Eldering, E., Verpy, E., Roem, D., Meo, T., and Tosi, M. (1995) J. Biol. Chem. 270, 2579-2587[Abstract/Free Full Text]
  12. de Agostini, A., Patston, P. A., Marottoli, V., Carrel, S., Harpel, P. C., and Schapira, M. (1988) J. Clin. Invest. 82, 700-705[Medline] [Order article via Infotrieve]
  13. Kent, S. B. (1988) Annu. Rev. Biochem. 57, 957-989[CrossRef][Medline] [Order article via Infotrieve]
  14. Kruithof, E. K., Vassalli, J. D., Schleuning, W. D., Mattaliano, R. J., and Bachmann, F. (1986) J. Biol. Chem. 261, 11207-11213[Abstract/Free Full Text]
  15. Kohler, G., and Milstein, C. (1975) Nature 256, 495-497[Medline] [Order article via Infotrieve]
  16. Blanche, P. J., Gong, E. L., Forte, T. M., and Nichols, A. V. (1981) Biochim. Biophys. Acta 665, 408-419[Medline] [Order article via Infotrieve]
  17. Goldenberg, D. P., and Creighton, T. E. (1984) Anal. Biochem. 138, 1-18[Medline] [Order article via Infotrieve]
  18. Pace, C. N., Shirley, B. A., and Thomson, J. A. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., ed), pp. 311-329, IRL Press, Oxford
  19. Hamilton, J. A., Whitty, G. A., Wojta, J., Gallichio, M., McGrath, K., and Ianches, G. (1993) Cell. Immunol. 152, 7-17[CrossRef][Medline] [Order article via Infotrieve]
  20. Gettins, P., Patston, P. A., and Schapira, M. (1993) BioEssays 15, 461-467[Medline] [Order article via Infotrieve]
  21. Mast, A. E., Enghild, J. J., and Salvesen, G. (1992) Biochemistry 31, 2720-2728[Medline] [Order article via Infotrieve]
  22. Enghild, J. J., Valnickova, Z., Thogersen, I. B., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 20159-20166[Abstract/Free Full Text]
  23. Stein, P. E., Leslie, A. G., Finch, J. T., Turnell, W. G., McLaughlin, P. J., and Carrell, R. W. (1990) Nature 347, 99-102[CrossRef][Medline] [Order article via Infotrieve]
  24. Kumar, S., and Baglioni, C. (1991) J. Biol. Chem. 266, 20960-20964[Abstract/Free Full Text]
  25. Jensen, P. H., Cressey, L. I., Gjertsen, B. T., Madsen, P., Mellgren, G., Hokland, P., Gliemann, J., Doskeland, S. O., Lanotte, M., and Vintermyr, O. K. (1994) Br. J. Cancer 70, 834-840[Medline] [Order article via Infotrieve]
  26. Dickinson, J. L., Bates, E. J., Ferrante, A., and Antalis, T. M. (1995) J. Biol. Chem. 270, 27894-27904[Abstract/Free Full Text]
  27. Tsatas, D., Baker, M. S., and Rice, G. E. (1997) J. Histochem. Cytochem. 45, 1593-1602[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.