Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin Characterization of different PAI-1 mutants Nuria Arroyo De Prada 1, *, Florian Schroeck 1, *, Eva-Kathrin Sinner 2 , Bernd Muehlenweg 1,3 , Jens Twellmeyer 1 , Stefan Sperl 3 , Olaf G. Wilhelm 3 , Manfred Schmitt 1 and Viktor Magdolen 1 1 Klinische Forschergruppe der Frauenklinik der Technischen Universita È tMu È nchen, Klinikum rechts der Isar, Germany; 2 Max-Planck-Institut fu È r Biochemie, Martinsried, Germany; 3 Wilex AG, Mu È nchen, Germany The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and ®brinolysis, a s w ell as pathophysiological processes such as thrombosis, tumor invasion and metas- tasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with d ierent components of the extracellular matrix, i.e. ®brin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important deter- minants of the Vn-binding site of PAI-1 appear to reside between amino acid s 110±147, which includes a helix E (hE, amino acids 1 09±118). Ten dierent PAI- 1 variants (mostly h arboring m odi®cations in hE) a s w ell as w ild-type PAI-1, the previously described PAI-1 mutant Q123K, and another s erpin, PAI-2, were recombinantly p roduced in Escherichia coli containing a His 6 tag and puri®ed by anity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all o f the newly generated mutants which retained inhibitory activity against u PA still bound to Vn. Mutant A114±118, in which all amino-acids at positions 114±118 of PAI-1 were exchanged for alanine, displayed a reduced anity to Vn as compared to wild- type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, s erved as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114±118 towards thrombin a s well as uPA were signi®- cantly reduced in the presence of Hep. Our results dem- onstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn. Keywords: plasminogen activator inhibito r t ype-1; vitronectin; heparin; mutational analysis; surface plasmon resonance. The urokinase-type plasminogen activation system plays an important role in tumor growth, invasion, and metastasis. The serine protease urokinase-type plasmino- gen activator (uPA) activates plasminogen, the zymogen of plasmin, thus generating a p rotease with broad substrate speci®city a nd leading t o d egradation of extra- cellular matrix (ECM) proteins [1±3]. The activity of uPA is focussed to the cell surface by interaction with its speci®c receptor uPAR (CD87). T issue-type plasminogen activator (tPA), the second type of human plasminogen activator, in contrast to uPA does not bind to a h igh af®nity receptor on tumor cell surfaces and therefore does not promote tumor cell-associated pericellular pro- teolysis. There are two main inhibitors of uPA and tPA, the serine protease inhibitors (serpin) plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) [4]. For inhibition, the surface-exposed reactive center loop (RCL) of PA I-1 or P AI-2 interacts with the r eactive site of the target protease. Initially, the P1±P1¢ bond of the inhibitor is cleaved and an intermediate enzyme±inhibitor complex is formed. This is followed by the insertion of part of the RCL as additional b strand 4A, which leads t o the translocation of the p rotease a cross t he p lane o f bsheet A of PAI-1 and formation of an SDS-stable enzyme± inhibitor complex [5]. This complex dissociates very slowly and is cleared from circulation before d isassembly can occur. In vitro, the inhibitor can be released from the protease in a substrate-like manner, generating the so-called RCL-cleaved form of the inhibitor [6]. Correspondence to V. Magdolen, Klinische Forschergruppe der Frauenklinik der Technischen Universita È tMu È nchen, Klinikum rechts der Isar, Ismaninger Str. 22, D-81675 Mu È nchen, Germany. Fax: + 49 89 4140 7410, Tel.: + 49 89 4140 2493, E-mail: viktor@magdolen.de Abbreviations: ECM, extracellular matrix; Hep, heparin; hE, helix E; HMW-uPA, high molecular weight urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor type-1; PAI-2, plasminogen activator inhibitor type-2; RCL, reactive center loop; RU, resonance units; SPR, surface plasmon resonance; tPA, tissue- type plasminogen activator; uPA, urokinase-type plasminogen acti- vator; uPAR, uP A r eceptor; Vn, vitronectin; IPTG, isop ropyl t hio-b- D -galactoside. *Note: these au thors contributed equally to the work. Note: web pages are available at h ttp://www.frauenklinik-tu-muen- chen.de, http://www.biochem.mpg.de/oesterhelt/ a nd http://www.wilex.com (Received 04 July 2001, re vised 22 O ctober 2001, accepted 29 Oc tober 2001) Eur. J. Biochem. 269, 184±192 (2002) Ó FEBS 2002 Active PAI-1 is metastable a nd spontaneously converts to a latent form by inserting a major part of its RCL into the central b sheet A [7]. Latent PAI-1, as well as PAI-1 in complex with uPA or tPA, do not bind to ECM compo- nents. Active PAI-1, however, interacts with ®brin, h eparin (Hep), and vitronectin (Vn) [1,8,9]. Binding to Vn doubles the physiological half-life of active PAI-1 in solution. Moreover, b y b inding to Vn or Hep, the substrate speci®city of PAI-1 i s a ltered, because i nteraction with Vn or Hep enables PAI-1 to inhibit another serine protease, thrombin [10]. High levels of PAI-1 in tumor tissu e indicate short recurrence-free and overall survival of tumor p atients af¯icted with a broad variety of cancers, e.g. mammary, ovarian, cervical, colorectal, bladder, renal and lung carcinomas [2,3]. In line with this, it was demonstrated that PAI-1 affects t umor cell adhesion and/or tumor angiogenesis and, as a result of this, may s upport tumor invasion [11±13]. As the PAI-1-binding site on Vn partially overlaps with the binding sites of cellular adhesion proteins, e.g. uPAR and some integrins, addition of PAI-1 to Vn-bound tumor cells leads to d etachment of t hese cells [11,14]. This effect can be reversed by uPA, as Vn-bound PAI-1 dissociates from Vn upon interacti on w ith uPA [9]. Interestingly, as demon- strated b y Bajou et al. [13], the in¯uence of PAI-1 on tumor vascularization is due to the i nhibition of proteases and not due to its interaction with Vn. Thus, the balance of several tumor-associated factors seems to control the modulatory effects of PAI-1 on cell adhesion, migration, and angiogen- esis and may play a crucial role in tumor invasion and metastasis [13,15]. Various r esearch groups have attributed the Vn-binding site on PAI-1 to different epitopes. However, essential amino acids appear to be located in a region within amino acids 1 10±147 [16±19], most of them being localized in the a helix E (hE). The major aim of the present study was to further analyze the structure-function relationship of PAI-1 mainly regarding its interactions with Vn, but also with Hep. Therefore, a number of PAI-1 variants preferentially containing modi®cations in hE of PAI-1 were generated and characterized biochemically. MATERIALS AND METHODS Generation of PAI-1 variants The coding regions of wild-type PAI-1 (amino acids 1±379 according to the numbering pr oposed by Ny et al. [20]) and wild-type PAI-2 (amino acids 2±415; PIR protein sequence database: A32853) have been cloned in frame with an N-terminal His 6 tag into the E. coli expression vector pQE-30 (Qiagen, Hilden, Germany) as described previously [21]. Modi®cations in the w ild-type cDNA s equence of P AI-1 were generated by reverse long-range PCR ( ÔExpand High Fidelity PCR System KitÕ; Roche, M annheim, Germany) applying mutated primers (Metabion, Martinsried, Germa- ny). PCR p roducts were ph osphorylated (T4 polynucleotide kinase; R oche, M annheim, Germany), re-ligated (T4 DNA ligase; Roche, Mannheim, Germany) and transformed into the E. coli strain XL1 b lue (Stratagene, H eidelberg, Ger- many). The mutated sequences were veri®ed b y DNA sequencing performed by TopLab, Martinsried, Germany. Expression and puri®cation of wild-type PAI-1, wild-type PAI-2, and PAI-1 variants Expression of recombinant proteins w as induced by adding isopropyl thio-b- D -galactoside (IPTG; ®nal concentration: 2m M ) to an X L1 blue (variant) PAI-1 or PAI-2 culture, pregrown in Luria±Bertani medium supplemented with 100 lgámL )1 ampicillin ( D 600 0.6±0.7). P rotein ex pression occurred at 37 °C o vernight on an orbital shaker at 200 r.p.m. The bacterial culture was harvested by centrif- ugation at 5000 g at 4 °C for 10 min. Then, the bacterial pellet was frozen for 20 min at )80 °C and, subsequently suspe nded in 20 m M Na±acetate, 1 M NaCl, 0 .1% Tween-80 (v/v), pH 7.4 supplemented with the protease inhibitor mix ÔComplete EDTA-freeÕ (Roche, Mannheim, Germany). In the case of wild-type PAI-2, a slightly different buffer [20 m M Na 2 HPO 4 ,1 M NaCl,0.1%Tween-80(v/v),pH 7.4 supplemented w ith C omplete E DTA-free] was used. Bacte- ria were disrupted mechanically by addition of glass beads (Sigma, Taufkirchen, G ermany) t o the bacterial cell sus- pension and 10 subsequent cycles of vortexing and incuba- tion on ice for 1 min each. The lysate was centrifuged for 15 min (12 000 g,4°C), the supernatant recovered and subjected to Ni 2+ -nitrilotriacetic acid agarose af®nity column puri®cation. Ni 2+ -nitrilotriacetic acid af®nity chromatography The Ni 2+ -nitrilotriacetic acid af®nity column was prepared as described by the manufacturer (Qiagen, Hilden, Germany). Initially, the af®nity column was equilibrated with 20 m M Na-acetate, 1 M NaCl, 0.1% Tween-80 (v/v), pH 7.4. Then, t he cleared bacterial lysate was applied to the column and, subsequently, the column washed with equili- bration buffer followed by 20 m M Na-acetate, 1 M NaCl, 0.1% Tween- 80 (v/v), 20 m M imidazole, pH 5.6 until the absorption of the ef¯uent had r eturned to b aseline (D 280 < 0.001). F inally, t he a dsorbed recombinant proteins were eluted with 20 m M Na-acetate, 1 M NaCl, 0.1% Tween-80 (v/v), 200 m M imidazole, pH 5.6. The e luate containing the recombinant proteins was dialyzed in equilibration buffer and puri®ed by a second Ni 2+ -nitrilo- triacetic acid af®nity chromatography as described above. The supernatant of lysates of wild-type PAI-2 expr essing bacterial cells (in a buffer containing 20 m M Na 2 HPO 4 ,1 M NaCl, 0.1% Tween-80, pH 7.4) was also applied to a Ni 2+ - nitrilotriacetic a cid af®nity column, and washed with the same buffer (at pH 6.5) supplemented with 20 m M imidaz- ole. For elution, the same buffer (at pH 6.0) containing 200 m M imidazole was used. Denaturation and refolding of the recombinant proteins The puri®ed recombinant proteins, with exception of wild- type PAI -2, were denatured f or 4 h in 4 M guanidinium/HCl at room temperature under light protection. Refolding of the proteins was achieved by dialysis (2 h, 4 °C) in 20 m M Na-acetate, 1 M NaCl, 0.01% Tween-80 (v/v), pH 5.6 followed by a second dialysis step (overnight, 4 °C). The proteins were subsequently concentrated in Centricon centrifugal ®lter devices (Millipo re, Eschborn, G ermany). Wild-type PAI-2 was dialyzed against NaCl/P i (pH 7.4) in order to remove residual i midazole, concentrated in Ó FEBS 2002 Interaction of PAI-1 variants with vitronectin (Eur. J. Biochem. 269) 185 Centricon centrifugal ®lter d evices, and then stored at )80 °C until use. Characterization of the recombinant proteins The protein c oncentration was determined ac cording to Bradford using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Krefeld, Germany). PAI-1 antigen was determined u sing the Imubind Tissue P AI-1 ELISA Kit (American Diagnostica, Pfungstadt, Germany). The iden- tity of the puri®ed proteins was demonstrated by Western blotting employing a polyclonal antibody (pAb) from chicken directed against human PAI-1 (a k ind gift of N . Grebenschikov, Institute of Ch emical Endocrinology, Uni- versity of Nijmegen, the Netherlands), a monoclonal mouse antibody (mAb) to human PAI-2 (#110 from American Diagnostica, Pfungstadt, Germany), and the ECL Western Blotting Detection R eagent (Amersham P h armacia, Frei- burg, Germany) for detection. N-terminal amino-acid sequence analysis p erformed by TopLab (Martinsried, Germany) was used t o con®rm the identity of the puri®ed recombinant wild-type PAI-1 and wild-type PAI-2. Amidolytic assay for determination of the inhibitory activity of the recombinant proteins against HMW-uPA The assay was performed in 96-well microtiter plates (Greiner, Frickenhausen, Germany). Puri®ed recombinant proteins were diluted in a buffer containing 100 m M Tris/ HCl, 0.05% T ween-20 (v/v), p H 7 .5, and 100 lgámL )1 BSA (ICN, Aurora, Ohio, US A), incubated with 1 0 U high molecular weight (HMW-)uPA (RheotrombÒ 500 000, Curasan Pharma GmbH, Kleinostheim, Germany) for 15 min at r oom temperature and then 10 lL o f chromo- genic substrate Bz-b-Ala-Gly-Arg-pNA.AcOH ( Pefa- chromeÒ uPA, Pentapharm Ltd, Basel, Switzerland, concentration 2 m M ) w ere a dded (30 min, 37 °C). The absorption was measured a t 405 nm. O ne unit PAI activity was d e®ned as the amount, w hich completely inhibited one unit of HMW-uPA activity. Complex formation of the recombinant proteins with HMW-uPA For complex formation, 100 U ( 0.7 lg) of HMW-uPA were incubated with t he recombinant proteins at room temperature for 10 min. The complexes were visualized by SDS/PAGE under nonreducing conditions and subsequent silver staining or Western blotting with the chicken pAb against PAI-1, mouse mAb #110 against PAI-2 (as mentioned above), and polyclonal chicken anti-uPA Ig (kindly provided by N. Grebenschikov, Institute of Chem- ical Endocrinology, Nijmegen, the Netherlands). POX- labeled chicken anti-(mouse IgG) Ig and POX-labeled goat anti-(chicken IgY) Ig w ere purchased from Dianova, Hamburg, Germany. Amidolytic assay for determination of the inhibitory activity of the recombinant proteins against thrombin The assay was performed i n 96-well microtiter plates. F ifty units of active recombinant PAI-1 (  35 n M ) i n the presence or absence o f 1 40 n M Vn (Promega GmbH, Mannheim, Germany) or 1 UámL )1 Hep (LiqueminÒ N 25 000, Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany) were incubated with 0.1 U of thrombin (from human plasma; Sigma, T aufkirchen, Germany) i n a total volume of 130 lL of Tris/NaCl/Tween buffer [20 m M Tris/HCl, 100 m M NaCl, 0.1% Tween-80 (v/v), pH 8.0] a t 37 °Cfor 1 h [10]. Then, 10 lL of chromogenic substrate (Chromo- zymÒ TH, Roche, Mannheim, Germany, concentration: 2m M ) were a dded and the thrombin a ctivity measured monitoring the change of D at 405 nm. Complex formation of the recombinant proteins PAI-1 with thrombin Fifty units of PAI-1 ( variant) in the p resence or absence of 600 n M Vn or 1 UámL )1 Hepwereincubatedwith0.5Uof thrombin in a total volume of 30 lLTris/NaCl/Tween buffer (1 h, 37 °C) and then subjected to nonreducing SDS/ PAGE followed by Western blotting employing chicken pAb against PAI-1 and a POX-labeled goat anti-(chicken IgY) Ig. Binding of recombinant PAI-1 to Vn-coated microtiter plates Vn or collagen type IV (Sigma, Taufkirchen, Germany) weredilutedtoaconcentrationof10lgámL )1 in a buffer containing 100 m M Na 2 CO 3 ,pH9.6.Forcoating,50lLof the V n or collagen type IV dilutions were poured into wells of a NuncMaxiSorp microtiter plate ( Nunc GmbH & Co. KG, Wiesbaden, Germany) and incubated overnight at 4 °C. After three washes with NaCl/P i /Tween [NaCl/P i containing 0.05% Tween-20 (v/v)], the wells were blocked by addition of 200 lL p er well of NaCl/P i supplemented with 2% BSA ( w/v) and incubation at room temperature for 2 h. T he wells were washed three times with NaCl/P i / Tween. Afterwards 100 lL p er well of (mutant) PAI-1 in the desired concentration were added at room temperature for 1 h. Following three additional washing steps, 200 lLper well horseradish peroxidase labeled Ni 2+ -nitrilotriacetic acid (Qiagen, Hilden, G ermany) at a dilution of 1 : 1000 in NaCl/Pi/Tween containing 0.2% BSA (w/v) were added (1 h, room temperature). After another four times of washing, binding of PAI-1 to t he solid phase was visualized by addition of 100 lL per well of a TMB substrate mix (KPL, Gaithersburg, Maryland, USA). T he reaction was stopped after color development with 50 lLperwellof 0.5 M H 2 SO 4 . Optical density was measured at 450 nm. Surface plasmon resonance analysis of (mutant) PAI-1 binding to Vn Surface plasmon resonance (SPR) studies were conducted with a BIACORE 2000 (Biacore AB, Uppsala, Sweden). Approximately 2000 resonance units (RU) of collagen type IV (10 lgámL )1 in 10 m M Na-acetate, pH 4 .0) (lane 1) and Vn (10 lgámL )1 in 10 m M Na-formiate, pH 4.0 [22]) (lanes 2±4) were immobilized to a CM5 sensor chip (research grade, Biacore AB, Uppsala, Sweden) using the amino coupling kit accordin g to t he manufacturer's recommenda- tion. All experiments were performed in HBS-EP [10 m M Hepes, 150 m M NaCl, 3 m M EDTA, 0.005% Tween-20 (v/v), pH 7.4] at a ¯ow rate of 20 lLámin )1 . HMW-uPA was 186 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 used in a c oncentration o f 400 UámL )1 . Regeneration of the surface was achieved by injection of 1 0 m M HCl f or 8 min. In order to check for reproducibility during the measure- ment, at ®rst 80 lLofa200UámL )1 dilution of wild-type PAI-1 were injected fo r t wo subsequent experiments, followed by two subsequent measurements of 80 lLofa 200 UámL )1 dilution of a PAI-1 mutant. Then, wild-type PAI-1 was measured a third time in duplicate, followed b y a measurement in duplicate of the next mutant and so on. Thus, for each PAI-1 variant at least t wo binding pro®les were recorded. The kinetics obtained in the collagen type IV-coated ¯ow cell were subtracted from the kinetics derived from the Vn-coated ¯ow cell in order t o obtain binding pro®les without bulk effects. RESULTS Expression and puri®cation of recombinant PAI-1, PAI-2, and PAI-1 variants The coding sequences for w ild-type PAI -1 a nd wild-type PAI-2, respectively, have previously been cloned by us in expression vector pQE-30 [21]. By reverse PCR, a series of PAI-1 variants w as generated using pQE-30-wild-type P AI-1 as the template. Due to the fact that serpins have a very compact tertiary s tructure (Fig. 1A), large modi®cations of the molecule m ay r esult in misfolded, and t herefore inactive, proteins. B ecause of this, we designed various strategies such as introduction of point mutations, s ubstitution of few amino a cids by alanine and glycine, substitution of selected epitopes by the homologue PAI-2 sequence, and short deletions ( Fig. 1B). The g enerated PAI-1 v ariants are summarized in Table 1. IPTG-induced recombinant protein expression in the bacterial strain XL1 blue y ielded 5±10% of the total E. coli protein. The recombinant proteins contained a His 6 tag at their N-terminus, allowing puri®cation by Ni 2+ -nitrilotriacetic acid af®nity chromatography. Although one cycle o f af®nity chromatography substantially enriched the recombinant proteins from other bacterial proteins, a puri®cation grade of > 95% was only achieved after a second chromato- graphic cycle as demonstrated by SDS/PAGE (Fig. 2 ). Inhibitory activity of the recombinant proteins against HMW-uPA PAI-1 i s unique among serpins b y i ts metastability th at l eads to a s hort half-life of  2 h under physiological conditions. Therefore, it was not surprising that after puri®cation (performed at room temperature) most of the recombinant PAI-1 wild-type protein and variants were p resent in the inhibitory inactive latent conformation. However, denatur- ation a nd subsequent refolding by dialysis leads to the reactivation of latent PAI-1 [23]. An up to 87% inhibitory activity of wild-type PAI-1 and PAI-1 variants against HMW-uPA (100 000 Uámg )1 de®ned as t he maximum [ 24]) was reached after denaturation with 4 M guanidinium/HCl followed b y dialysis in a buffer of h igh ionic strength at pH 5.6. Moreover, inhibitory activities of the protein preparations remained stable for more than one year in this buffer when stored a t )80 °C. The inhibitory activity of the g enerated PAI-1 mutants is summarized in Table 1. All inhibitory active mutants were metastable (half-life £ 2h at 37 °C) and the half-life was roughly doubled in the presence of Vn. Furthermore, inhibitory active variants formed SDS-stable complexes with HMW-uPA; t he inactive Fig. 1 . Three-dimensional struc ture of ac tive PAI-1. (A) Important structural elements of active PAI-1 (PDB 1B3K). The central b sheet A consisting of strands 1A, 2A, 3A, 5A, and 6A is indicated in yellow, helix D in green, and helix E (hE) in cyan blue. The P1-residue of PAI-1 (R346) is also indicated. (B) Location o f amino acid alterations i n some of t he generated PAI-1 mutants. P73A, single amino acid exch an ge of P73 to alanine ; A114±118, exchange of the sequence 114FRLFR118 to ®ve a lanines; D109±112, deletion of the four amino acids 109MPHF112; Q123K, single amino acid exchange of Q123 to lysine. Ó FEBS 2002 Interaction of PAI-1 variants with vitronectin (Eur. J. Biochem. 269) 187 mutants d id not (Fig. 3). PAI-2 was stable (no loss of inhibitory activity after incubation for 24 h at 37 °C) and exerted an inhibitory activity against HMW-uPA of 90% (equivalent to 90 000 Uámg )1 ) after the two-step af®nity chromatography. Interaction of (mutant) PAI-1 with Vn All inhibitory active mutants but mutant Q123K [16] did interact with Vn as veri®ed by measuring (mutant) PAI-1 binding to Vn-coated microtiter plates and by surface plasmon resonance analysis (Table 1). This binding was demonstrated to be highly speci®c, as binding of (mutant) PAI-1 was completely abolished by preincubation of recombinant PAI-1 with soluble Vn (10 lgámL )1 )priorto adding it to the wells or before injection in a reproducible manner ( data not shown). Furthermore, latent PAI-1 (data not shown) as well as heat-denatured PAI-1 (Fig. 4) did not bind to the immobilized Vn. The observed K D value for wild-type PAI-1 (K D  0.18 n M )aswellasforP73A(K D  0.33 n M )is similar to t hat previously reported for the interaction of human PAI-1 recombinantly expressed in Chinese hamster ovary cells with vitronectin (K D  0.1 n M ), also applying Table 1. PAI-1 variants and binding t o V n. PAI-1 mutants with corresponding sp eci®c inhibitory activity towards uPA and their ability to interact with Vn measured b y use of Vn-coated microtiter plates ( M P) and surface plasmon r esonanc e (SPR). + Indicates bind ing to Vn; ± indicates n o binding to Vn. NT, not tested. PAI-1 variant Modi®cation a Inhibitory activity towards uPA (Uámg )1 ) Binding to Vn MP SPR Wild-type PAI-1 87 000 + + Wild-type PAI-2 90 000 ± NT Mutant 1 (D109-123) D F109-Q123 Inactive ± NT Mutant 2 (M2) F109-Q123 vs. AAGAGAA Inactive ± NT Mutant 3 (M3) F109-Q123 vs. homologue Inactive ± ± PAI-2 sequence b and E128G Mutant 4 (M4) F109-Q123 vs. AAAA Inactive ± ± Mutant 5 (D109-112) D F109-H112 10 000 + NT Mutant 6 (A114-115) F114A and R115A 57 000 + NT Mutant 7 (A114±118) F114-R118 vs. AAAAA 20 800 + + Mutant 8 (M8) F114-R118 vs. AAAAA and D68G 7 700 + + Mutant 9 (M9) V284-G294 vs. homologue PAI-2 sequence c Inactive ± ± Mutant 10 (P73A) P73A 56 250 + + Mutant 11 (Q123K) Q123K 47 800 ± ± a Numbering of PAI-1 according to Ny et al. [20]; b 141YIRLCQKYYSSEPQA155, and c 318YELRSILRSMG328, PAI-2 according to PIR protein sequence database: A32853. Fig. 2. Puri®cation o f recombinant wild-type PAI-1. Human recombi- nant wild-type PA I-1 e quipped w ith a n N-terminal His 6 tag ( wild-type PAI-1) was puri®ed from an E. coli cell lysate by Ni 2+ -nitrilotriacetic acid anity chromatography and analyzed by SDS/PAGE. M, marker; lane 1, E. coli ly sate; lane 2, euent o f the ®rst Ni 2+ -nitrilo- triacetic a cid anity chro matography cycle; lane 3, elu ate of the ®rst Ni 2+ -nitrilotriacetic acid anity chromatography cycle; lane 4, eluate of the second Ni 2+ -nitrilotriacetic acid anity c hromatography c ycle. Fig. 3. Formation of S DS stable complexes. Wild-type PAI-1 o r variants thereof i n the presen ce (+ uP A) or absenc e (± uPA) of 100 U ( 0.7 lg) HMW-uPA were incubated for 10 min at room tem per ature and then subjected to n onred ucing SDS/PAGE. Subsequently, the gels were silver- stained. 200 U o f PAI-1, 1 10 U o f Q123K, 50 U of P73A and of A114±118 were applied; alternatively 1.4 lg of ( inactive ) M 4 a nd 1 0 U of PAI-1 were used. 188 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 SPR analysis [22]. Mutant A114±118 displayed a slower association to (on-rate: 5.35 ´ 10 5 M )1 ás )1 )andafaster dissociation from Vn (off-rate: 1.0 ´ 10 )3 s )1 ) c ompared t o wild-type PAI-1 (on-rate: 1.0 ´ 10 6 M )1 ás )1 ; off-rate: 1.9 ´ 10 )4 s )1 ). Only a low amount of mutant Q123K associated to V n and d issociated immediately a fter washing with buffer , indicating an unspeci®c binding to Vn (Fig. 4). Moreover, mutant Q123K did not show any change in binding to solid phase Vn in the presence or a bsence of soluble V n (data not shown). All Vn-binding mutants dissociated immediately from Vn after uPA injection (Fig. 4). Thus, these results clearly demonstrate that (mutant) PAI-1, but not Q123K, s peci®cally binds t o Vn immobilized to the dextran matrix of the CM5 chip, and then still was able to form complexes with uPA. Inhibition of thrombin by PAI-1 and binding to Hep Binding to Vn provides wild-type P AI-1 with thrombin inhibitory properties [10]. Therefore, we tested whether the PAI-1 mutants generated were also able to inhibit thrombin in the p resence of Vn. I n line w ith our data obtained by SPR, the Vn-interacting mutants A114±118 and P73A, but not Q123K, inhibited thrombin in t he presence of Vn to about 40%. Vn alone did not reduce thrombin activity signi®cantly (Fig. 5). We also tested the e ffects of Hep on the inhibitory activity of PAI-1 towards thrombin. All mutants tested but A114± 118 d isplayed s imilar properties as wild-type PAI-1 (> 90% inhibition; Fig. 5). A114±118 inhibited thrombin in the presence of Hep clearly less effectively (about 40%; Fig. 5). These r esults determ ined by amidolytic assays measuring (residual) thrombin activity were supported by the results seen in Western blots visualizing the formation of SDS stable c omplexes between thrombin and PAI-1 with or without Vn o r H ep. Detection o f a higher residual thrombin activity correlated with a lower a mount of SDS stable complexes (data not shown). The reduced capacity of A114±118 to inhibit thrombin with Hep as a cofactor is not related to an altered af®nity to Fig. 5. Inhibition of thrombin. Fifty un its of recombinant PAI-1 ( 35 n M ) in the presence or absence of 140 n M Vn or one UámL )1 Hep were incubated with 0.1 U of thrombin in a total volume of 130 lL of Tris/NaCl/Tween buer a t 3 7 °C for 1 h . T hen, 10 lLof chromogenic substrate were added and the thrombin activity measured monitoring the change of optical density at 405 nm. The activity of thrombin in the absence of inhibitor was se t to 100%, the othe r activities were calculated accord ingly. Data shown are from three independent experiments, each measured in duplicate (  SD). As a control, the eect of 140 n M Vn or 1 UámL )1 Hep witho ut inhib itor was measured. Black bars, buer, only; hatched bars, plus Vn (140 n M ); grey bars, plus Hep (1 UámL )1 ). Fig. 4. Surface plasmon resonance: binding of (mutant) PAI-1 to VN. Approximately 2000 R U of collagen type I V or V n were immobilized to a CM5 sensor chip i nserted in the BIAcore 2 000 system. Then, wild-type PAI-1 (200 UámL )1 ) was injected and a llowed to bind to Vn, w hich was followed by a washing step w ith buer. Finally, H MW-uPA ( 400 UámL )1 ) was i njected. Two subsequent measurements of the b inding kinetics of wild-type PAI-1 were followed by two independent measurements of a PAI-1 variant (200 UámL )1 ). After that, wild-type PAI-1 was measured a third time, followed by a measurement in duplicate of the next mutant and so on. Thus, for each PAI-1 variant at l east two independent binding pro®les we re o btained. All experiments were pe rf ormed a t a ¯ow rate of 2 0 lLámin )1 ; regeneration of the surface was achieved by treatment with 10 m M HCl f or 8 min. The k inetics obtained in the collagen t ype IV-coated ¯ow c ell w ere subtracted f rom t he k inetics de rived f rom t he Vn -coated ¯ow cell in o rd er to obtain binding pro®les without bulk eects. Heat d enature d controls were measured to compare the binding signal w ith the unspeci®c binding to the identical surface conditions. Ó FEBS 2002 Interaction of PAI-1 variants with vitronectin (Eur. J. Biochem. 269) 189 Hep, as in SPR we observed that A 114±118 displayed similar binding to biotinylated Hep immobilized on a SA-5 sensor chip as wild-type PAI-1 (data not shown). Interest- ingly, Hep did not only provide A114±118 with thrombin inhibitory properties less e f®ciently than t he other t ested mutants, it also reduced the inhibitory property of A114± 118 towards uPA in a dose dependent manner. Hep had only marginal e ffects on t he inhibition of uPA by other inhibitory active PAI-1 proteins (wild-type P AI-1, P 73A, Q123K, data not shown). DISCUSSION Puri®cation and inhibitory activity of recombinant PAI-1, PAI-2, and PAI-1 variants Recombinant expre ssion i n a bac terial s ystem i s a n easy a nd quick method for the productio n of large a mounts of human PAI-1 and PAI-2. It has been s hown p reviously, that, although PAI-1 and extracellular PAI-2 are glycosy- lated proteins, expression of both proteins i n a nonglycosy- lated form in prokaryotes does not affect production and inhibitory activity of these proteins [25]. We cloned the cDNA sequence of both wild-type PAI-1 and wild-type PAI-2 in an expression vector that provides an additional histidine 6 -sequence at t he N-terminus of the r ecombinant proteins [21], thus allowing puri®cation by Ni 2+ -nitrilotri- acetic acid af®nity chromatography. The obtained speci®c activities of wild-type PAI-1 and wild-type PAI-2, respec- tively, strongly indicate that this mo di®cation does not have any signi®cant effect on the inhibitory activity of the recombinant in comparison to the natural proteins. This is most likely due to the fact that the RCL of both serpins is located close to the C-terminus. The recombinant proteins were puri®ed under native conditions using a modi®ed version of a b uffer t hat had been described previously for the isolation of PAI-1 from conditioned medium of human endothelial cells [26]. This buffer i s characterized by a l ow pH (5.6) as well as a relatively high ionic strength (1 M NaCl). These conditions resemble the inner milieu of the a-granula contained in thrombocytes, which are the main reservoir o f PAI-1 in blood [27]. Sancho et al. [28] reported the isolation of signi®cant amounts of a ctive PAI-1 under native conditions by using such a buffer. However, we did not obtain such high amounts of a ctive PAI-1 under similar conditions, but the inhibitory activity of PAI-1 was dramatically enhanced by denaturation, refolding, and storage in a buffer a t p H 5.6 containing 1 M NaCl. The generated PAI-1 var iants did also not display any inhibitory activity after puri®cation under native conditions. Again, the inhibitory activity of at least some of the PAI-1 mutants could be restored by denaturation and refolding. The achieved activities among the variants were highly related to the number of modi®cations introduced. The variants A114±115, P73A , a nd Q 123K, i n w hich only one or two amino-acid were exchanged, showed the highest inhibitory capacity against H MW-uPA among the PAI-1 variants ( 50 000 Uámg )1 ). Mutant A114±118 containing ®ve amino-acid substitutions displayed 24%, D109±112 (four deleted amino acids) and M8 (six amino-acid substi- tutions) a bout 10% of the inhibitory activity of recombinant wild-type PAI-1, only. All of the other PAI-1 variants (D109-123, M2, M3, M4, M9), which c ontained more than seven modi®ed amino-acid positions did not display a ny inhibitory activity towards uPA, indicating that larger modi®cations at these positions are not tolerated and lead to a loss o f activity m ost likely due to misfolding of the compact PAI-1 structure. The active PAI-1 variants behaved similarly to wild-type PAI-1 with respect to the ir metastability and the stabiliza- tion of the active f orm b y V n. In contrast to wild-type PAI-1 and its active variants, r ecombinant P AI-2 did not show any signi®cant loss of activity after puri®cation under native conditions, underlining the uniqueness of the metastable PAI-1 a mo ng se rp ins. Interaction of (mutant) PAI-1 with Vn Using Vn-coated microtiter plates and SPR, we a nalyzed speci®c binding of wild-type PAI-1 and PAI-1 variants to Vn. I n general, SPR measurements are c onsidered to be very quantitative and association/dissociation constants are normally easily analyzed by Langmuir binding isotherms to obtain t he respective bin ding constants. H owever, in the case of PAI-1 and variants thereof, this kind of measure- ments may not be accurate for t he following reasons: PAI-1 spontaneously converts from its active to a latent form. To determine t he p roportion o f active PAI-1, w e m easured t he speci®c inhibitory activity against uPA with a theoretical maximum de®ned a s 100 000 Uámg )1 [24]. This m ethod can be used for wild-type PAI-1, but for the PAI-1 mutants a different maximum for the speci®c activity may exist. Furthermore, within the time frame of determination of t he amount of active PAI-1 in a given preparation to the time- point when PAI-1 is u sed in the SPR analysis, anoth er a s y et unknown part of active PAI-1 converts to the l atent form and thus cannot bind to Vn anymore. Therefore, the analysis of the interaction of PAI-1 and, especially, PAI-1 variants with Vn can only b e semiquantitative. It has t o be emphasized, however, that in the present s tudy the main a im was to analyze whether mutants with variations in hE are still able to bind to Vn and n ot to compare the binding af®nities of the various mutants in a quantitative manner. As only inhibitory active PAI-1 binds to Vn [8], conclusions about the Vn-binding site on PAI-1 can only be drawn from the mutants with inhibitory activity. All mutants with var iations covering the w hole area of hE (D109-123, M2, M3, M4) yielded inactive PAI-1 variants. However, D109-112, in which t he N-t erminal amino a cids of hE were deleted, as well as A114±118, with changes in the C-terminal region of hE, still bo und to Vn and inhibited uPA. Thus, m utations within hE of PAI-1 are tolerated t o some extent. Our results from measuring binding of wild- type PAI-1 and its variants to Vn-coated microtiter plates were reproduced in SPR. Furthermore, as these results are in line with those obtained in thrombin inhibition experi- ments, it can be c oncluded that the mutants still interact functionally with Vn. Moreover, concerning Q123K and its dramatically reduced af®nity to Vn, we r eproduced the results of Lawrence et al. [16], which again underlines the functionality of our test systems. Lawrence et al.[16]also reported a bout another mutation (L116P) i n hE that led to Vn-binding de®ciency. In two of our mutants (A114±118, M8), L116, among other amino acid changes, was substi- tuted by alanine. The resulting mutant proteins, however, 190 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 still displayed Vn-binding activity. This may indicate that the rather conservative alteration of L116 to alanine ( as compared to proline) may not be dramatic enough to eliminate the Vn-binding capacity of these PAI-1 variants. Padmanabhan and Sane [19] located t he Vn-binding site on PAI-1 to amino acids 115±130 employing PAI-1/PAI-2 chimera a nd protease-digested P AI-1. This seems to b e contradictory to our results, but one has to keep in mind that proteolytic treatment of PAI-1 most likely leads to an altered overall structure in the resulting fragments. Fur- thermore, all of the chimera that did not bind to Vn were not only modi®ed around hE but additionally in the area around Q123 of PAI-1. Van Meijer et al. [ 17] localized the Vn-binding region of PAI-1 to amino acids 110±145 using epitope-mapped m onoclonal antibodies which inhibited V n/ PAI)1-interaction. The region encompassing amino acids 110±145 not only comprises hE but also the region of the strand 1 edge o f bsheet A, where Q123 is located [29]. Cross- linking studies reported b y D eng et al. [18] localized the Vn-binding region of PAI-1 to the same region. However, Sui and Wiman [30] did not report any changes in the Vn-binding behavior of mutants w ith single amino-acid substitutions in the region of F113 to D138, which is in line with our results. Summarizing these data, it is much likely that the importance of hE for Vn-binding has been overestimated previously. Although hE still plays a role for high a f®nity binding of PAI-1 to V n as demonstrated in the altered association to and especially dissociation from Vn in the case of A114±118 in SPR, there seems to exist some cooperation of the region a round s trand 1 of b sheet A with hE in Vn-binding. Inhibition of thrombin by PAI-1 and binding to Hep Ehrlich et al. [10] demonstrated th at w ild-t ype P AI-1 inhibits thrombin in the presence of Vn. This is also true for all tested variants in the present study that did interact with Vn. Furthermore, in line with the results from Ehrlich et al .[31],1UámL )1 was determined as the ideal Hep concentration f or inhibition of thrombin by wild-type PAI- 1. In addition to wild-type PAI-1, we a lso t ested t he mutants A114±118, Q123K, and P73A at this concentration. P73A did not show any differences in interaction with Hep, although this amino acid is located in helix D, which was previously reported to contribute to the Hep binding site [32]. As e xpected, Hep-bound Q123K inhibited thrombin exactly like w ild-type PAI-1. A 114±118 did not display a signi®cantly altered af®nity to Hep i n SPR, although it was not able to inhibit t hrombin a s e f®ciently a s w ild-type P AI-1 together with Hep . This contras ts w ith the results o f Sui and Wiman [30] who detected a change in a f®nity to Hep in their mutant R118D and proposed that mainly ionic interactions occur between PAI-1 and Hep. However, a change o f R118 to alanine (as in the mutant A114±118) and not to aspartate (as in R118D) may not change the surface charge of this region signi®cantly enough to reduce af®nity to Hep. Surprisingly, t he i nhibitory activity of A114±118 towards uPA was reduced in a dose dependent manner by addition of Hep. This may suggest that upon Hep-binding to A114± 118 c onformational c hanges occur, affecting the inhibition characteristics of this PAI-1 mutant towards both thrombin and uPA. CONCLUSIONS Comparison of the m ain p rotease targets of PAI-1, uPA and tPA, has previously shown that the proteolytic activity of these enzymes is not exclusively the relevant feature for cancer spread. Rath er, it seems that further interactions of one of the proteases, uPA, w ith other molecules support tumor i nvasion a nd metastasis. W hereas tPA, at least in breast and ovarian cancer, does not play a major role in tumor cell invasion, uPA is an important, multifunctional component of the invasion m achinery most likely due to effects excerted upon interaction with its speci®c receptor, uPAR [3]. Similarly, the additional b inding partners of PAI- 1 (Vn, Hep, and ®brin) strongly differentiate it from PAI-2, which extracellularly targets serine proteases only. Especial- ly, interaction of PAI-1 with Vn is strongly related to the modulation of cancer cell adhe sion and, thus, m ay facilitate tumor cell invasion via a balanced interference/induction of tumor cell attachment/migration [11,12,33,34]. Detailed knowledge of the structural region(s) of the PAI-1 molecule implicated in the P AI-1/Vn-interaction is the basis for the rational development of site-speci®c PAI-1 modu- lators [35]. Surface-exposed loop structures, s uch as hE in PAI-1, represent attractive targets for the development of such modulators because of their high ac cessibility. hE has been implicated in the binding of PAI-1 to Vn [16±19]. An hE-blocking compound m ay not block further PAI-1 activities (inhibition of serine proteases o r binding to ®brin) and, thus, would not alter functions of PAI-1 important for physiological processes such a s ®brinolysis. 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[ epub ahead of print]. 1 192 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin Characterization of different PAI-1 mutants Nuria Arroyo De Prada 1, *, Florian Schroeck 1, *,. and exerted an inhibitory activity against HMW-uPA of 90% (equivalent to 90 000 Uámg )1 ) after the two-step af®nity chromatography. Interaction of (mutant) PAI-1 with Vn All inhibitory active mutants. 1 M NaCl. The generated PAI-1 var iants did also not display any inhibitory activity after puri®cation under native conditions. Again, the inhibitory activity of at least some of the PAI-1 mutants could