Interaction of plasminogen activator inhibitor type-1PAI-1 with vitronectin Characterization of different PAI-1 mutants Nuria Arroyo De Prada1,*, Florian Schroeck1,*, Eva-Kathrin Sinner2
Trang 1Interaction of plasminogen activator inhibitor type-1
(PAI-1) with vitronectin
Characterization of different PAI-1 mutants
Nuria Arroyo De Prada1,*, Florian Schroeck1,*, Eva-Kathrin Sinner2, Bernd Muehlenweg1,3,
Jens Twellmeyer1, Stefan Sperl3, Olaf G Wilhelm3, Manfred Schmitt1and Viktor Magdolen1
1 Klinische Forschergruppe der Frauenklinik der Technischen UniversitaÈt MuÈnchen, Klinikum rechts der Isar, Germany;
2 Max-Planck-Institut fuÈr Biochemie, Martinsried, Germany; 3 Wilex AG, MuÈnchen, Germany
The serpin plasminogen activator inhibitor type 1 (PAI-1)
plays an important role in physiological processes such as
thrombolysis and ®brinolysis, as well 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 dierent 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 acids 110±147, which includes a helix E
(hE, amino acids 109±118) Ten dierent PAI-1 variants
(mostly harboring modi®cations in hE) as well as wild-type
PAI-1, the previously described PAI-1 mutant Q123K, and
another serpin, PAI-2, were recombinantly produced in
Escherichia coli containing a His6 tag and puri®ed by
anity chromatography As shown in microtiter
plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA 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 anity 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, served as a control In contrast to other active PAI-1 mutants, the inhibitory properties of A114±118 towards thrombin as 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 inhibitor type-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 plasminoplasmino-gen, the zymoplasmino-gen
of plasmin, thus generating a protease with broad substrate speci®city and leading to degradation 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) Tissue-type plasminogen activator (tPA), the second type of human plasminogen activator, in contrast to uPA does not bind to a high 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 PAI-1 or PAI-2 interacts with the reactive 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 to the translocation of the protease across the plane of b sheet
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 disassembly 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 UniversitaÈt MuÈnchen, Klinikum rechts
der Isar, Ismaninger Str 22, D-81675 MuÈnchen, 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-tissue-type plasminogen
acti-vator; uPAR, uPA receptor; Vn, vitronectin; IPTG, isopropyl
thio-b-D -galactoside.
*Note: these authors contributed equally to the work.
Note: web pages are available at
http://www.frauenklinik-tu-muen-chen.de, http://www.biochem.mpg.de/oesterhelt/ and
http://www.wilex.com
(Received 04 July 2001, revised 22 October 2001, accepted 29 October
2001)
Trang 2Active PAI-1 is metastable and 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, heparin
(Hep), and vitronectin (Vn) [1,8,9] Binding to Vn doubles
the physiological half-life of active PAI-1 in solution
Moreover, by binding to Vn or Hep, the substrate speci®city
of PAI-1 is altered, because interaction with Vn or Hep
enables PAI-1 to inhibit another serine protease, thrombin
[10]
High levels of PAI-1 in tumor tissue indicate short
recurrence-free and overall survival of tumor patients
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 tumor cell adhesion and/or tumor
angiogenesis and, as a result of this, may support 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 detachment of these cells [11,14] This effect can
be reversed by uPA, as Vn-bound PAI-1 dissociates from
Vn upon interaction with uPA [9] Interestingly, as
demon-strated by Bajou et al [13], the in¯uence of PAI-1 on tumor
vascularization is due to the inhibition 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 research 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 110±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
M A T E R I A L S A N D M E T H O D S
Generation of PAI-1 variants
The coding regions of wild-type PAI-1 (amino acids 1±379
according to the numbering proposed 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-terminalHis6tagintotheE coliexpressionvectorpQE-30
(Qiagen, Hilden, Germany) as described previously [21]
Modi®cations in the wild-type cDNA sequence of PAI-1
were generated by reverse long-range PCR (ÔExpand High
Fidelity PCR System KitÕ; Roche, Mannheim, Germany)
applying mutated primers (Metabion, Martinsried,
Germa-ny) PCR products were phosphorylated (T4 polynucleotide
kinase; Roche, Mannheim, Germany), re-ligated (T4 DNA
ligase; Roche, Mannheim, Germany) and transformed into
the E coli strain XL1 blue (Stratagene, Heidelberg,
Ger-many) The mutated sequences were veri®ed by 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 was induced by adding isopropyl thio-b-D-galactoside (IPTG; ®nal concentration:
2 mM) to an XL1 blue (variant) PAI-1 or PAI-2 culture, pregrown in Luria±Bertani medium supplemented with
100 lgámL)1ampicillin (D600 0.6±0.7) Protein expression occurred at 37 °C overnight 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 suspended in 20 mMNa±acetate, 1MNaCl, 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 mMNa2HPO4, 1MNaCl, 0.1% Tween-80 (v/v), pH 7.4 supplemented with Complete EDTA-free] was used Bacte-ria were disrupted mechanically by addition of glass beads (Sigma, Taufkirchen, Germany) to 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 Ni2+-nitrilotriacetic acid agarose af®nity column puri®cation
Ni2+-nitrilotriacetic acid af®nity chromatography The Ni2+-nitrilotriacetic acid af®nity column was prepared
as described by the manufacturer (Qiagen, Hilden, Germany) Initially, the af®nity column was equilibrated with 20 mM Na-acetate, 1MNaCl, 0.1% Tween-80 (v/v),
pH 7.4 Then, the cleared bacterial lysate was applied to the column and, subsequently, the column washed with equili-bration buffer followed by 20 mM Na-acetate, 1M NaCl, 0.1% Tween-80 (v/v), 20 mM imidazole, pH 5.6 until the absorption of the ef¯uent had returned to baseline (D280< 0.001) Finally, the adsorbed recombinant proteins were eluted with 20 mM Na-acetate, 1M NaCl, 0.1% Tween-80 (v/v), 200 mM imidazole, pH 5.6 The eluate containing the recombinant proteins was dialyzed in equilibration buffer and puri®ed by a second Ni2+ -nitrilo-triacetic acid af®nity chromatography as described above The supernatant of lysates of wild-type PAI-2 expressing bacterial cells (in a buffer containing 20 mMNa2HPO4, 1M NaCl, 0.1% Tween-80, pH 7.4) was also applied to a Ni2+ -nitrilotriacetic acid af®nity column, and washed with the same buffer (at pH 6.5) supplemented with 20 mM imidaz-ole For elution, the same buffer (at pH 6.0) containing
200 mMimidazole was used
Denaturation and refolding of the recombinant proteins The puri®ed recombinant proteins, with exception of wild-type PAI-2, were denatured for 4 h in 4Mguanidinium/HCl
at room temperature under light protection Refolding of the proteins was achieved by dialysis (2 h, 4 °C) in 20 mM Na-acetate, 1M 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 (Millipore, Eschborn, Germany) Wild-type PAI-2 was dialyzed against NaCl/Pi(pH 7.4) in order to remove residual imidazole, concentrated in
Trang 3Centricon centrifugal ®lter devices, and then stored at
)80 °C until use
Characterization of the recombinant proteins
The protein concentration was determined according to
Bradford using the Bio-Rad Protein Assay Dye Reagent
Concentrate (Bio-Rad, Krefeld, Germany) PAI-1 antigen
was determined using the Imubind Tissue PAI-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 kind gift of N
Grebenschikov, Institute of Chemical 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 Reagent (Amersham Pharmacia,
Frei-burg, Germany) for detection N-terminal amino-acid
sequence analysis performed by TopLab (Martinsried,
Germany) was used to 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 mMTris/
HCl, 0.05% Tween-20 (v/v), pH 7.5, and 100 lgámL)1BSA
(ICN, Aurora, Ohio, USA), incubated with 10 U high
molecular weight (HMW-)uPA (RheotrombÒ 500 000,
Curasan Pharma GmbH, Kleinostheim, Germany) for
15 min at room temperature and then 10 lL of
chromo-genic substrate Bz-b-Ala-Gly-Arg-pNA.AcOH
(Pefa-chromeÒ uPA, Pentapharm Ltd, Basel, Switzerland,
concentration 2 mM) were added (30 min, 37 °C) The
absorption was measured at 405 nm One unit PAI activity
was de®ned as the amount, which 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 the 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-POX-labeled goat
anti-(chicken IgY) Ig were purchased from Dianova,
Hamburg, Germany
Amidolytic assay for determination of the inhibitory
activity of the recombinant proteins against thrombin
The assay was performed in 96-well microtiter plates Fifty
units of active recombinant PAI-1 ( 35 nM) in the presence
or absence of 140 nM 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, Taufkirchen, Germany) in a total volume of
130 lL of Tris/NaCl/Tween buffer [20 mM Tris/HCl,
100 mMNaCl, 0.1% Tween-80 (v/v), pH 8.0] at 37 °C for
1 h [10] Then, 10 lL of chromogenic substrate (Chromo-zymÒ TH, Roche, Mannheim, Germany, concentration:
2 mM) were added and the thrombin activity 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 presence or absence of
600 nMVn or 1 UámL)1Hep were incubated with 0.5 U of thrombin in a total volume of 30 lL Tris/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) were diluted to a concentration of 10 lgámL)1in a buffer containing 100 mMNa2CO3, pH 9.6 For coating, 50 lL of the Vn 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/Pi/Tween [NaCl/Pi containing 0.05% Tween-20 (v/v)], the wells were blocked
by addition of 200 lL per well of NaCl/Pisupplemented with 2% BSA (w/v) and incubation at room temperature for
2 h The wells were washed three times with NaCl/Pi/ Tween Afterwards 100 lL per well of (mutant) PAI-1 in the desired concentration were added at room temperature for
1 h Following three additional washing steps, 200 lL per well horseradish peroxidase labeled Ni2+-nitrilotriacetic acid (Qiagen, Hilden, Germany) 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 the solid phase was visualized
by addition of 100 lL per well of a TMB substrate mix (KPL, Gaithersburg, Maryland, USA) The reaction was stopped after color development with 50 lL per well of 0.5MH2SO4 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)1in 10 mMNa-acetate, pH 4.0) (lane 1) and
Vn (10 lgámL)1in 10 mMNa-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 according to the manufacturer's recommenda-tion All experiments were performed in HBS-EP [10 mM Hepes,150 mMNaCl,3 mMEDTA,0.005%Tween-20(v/v),
pH 7.4] at a ¯ow rate of 20 lLámin)1 HMW-uPA was
Trang 4used in a concentration of 400 UámL)1 Regeneration of the
surface was achieved by injection of 10 mMHCl for 8 min
In order to check for reproducibility during the
measure-ment, at ®rst 80 lL of a 200 UámL)1dilution of wild-type
PAI-1 were injected for two subsequent experiments,
followed by two subsequent measurements of 80 lL of a
200 UámL)1dilution of a PAI-1 mutant Then, wild-type
PAI-1 was measured a third time in duplicate, followed by a
measurement in duplicate of the next mutant and so on
Thus, for each PAI-1 variant at least two 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 to obtain
binding pro®les without bulk effects
R E S U L T S
Expression and puri®cation of recombinant PAI-1,
PAI-2, and PAI-1 variants
The coding sequences for wild-type PAI-1 and 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 was generated using pQE-30-wild-type PAI-1
as the template Due to the fact that serpins have a very
compact tertiary structure (Fig 1A), large modi®cations of
the molecule may result in misfolded, and therefore inactive,
proteins Because of this, we designed various strategies such
as introduction of point mutations, substitution of few
amino acids by alanine and glycine, substitution of selected
epitopes by the homologue PAI-2 sequence, and short
deletions (Fig 1B) The generated PAI-1 variants are
summarized in Table 1 IPTG-induced recombinant protein
expression in the bacterial strain XL1 blue yielded 5±10% of the total E coli protein
The recombinant proteins contained a His6tag at their N-terminus, allowing puri®cation by Ni2+-nitrilotriacetic acid af®nity chromatography Although one cycle of 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 is unique among serpins by its metastability that leads
to a short 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 present in the inhibitory inactive latent conformation However, denatur-ation and 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)1de®ned as the maximum [24]) was reached after denaturation with 4Mguanidinium/HCl followed by dialysis in a buffer of high 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 at )80 °C The inhibitory activity of the generated PAI-1 mutants is summarized in Table 1 All inhibitory active mutants were metastable (half-life £ 2 h
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; the inactive
Fig 1 Three-dimensional structure of active 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 of amino acid alterations in some of the generated PAI-1 mutants P73A, single amino acid exchange of P73 to alanine; A114±118, exchange of the sequence 114FRLFR118 to ®ve alanines; D109±112, deletion of the four amino acids 109MPHF112; Q123K, single amino acid exchange of Q123 to lysine.
Trang 5mutants did 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) prior to 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 KD value for wild-type PAI-1 (KD 0.18 nM) as well as for P73A (KD 0.33 nM) is similar to that previously reported for the interaction of human PAI-1 recombinantly expressed in Chinese hamster ovary cells with vitronectin (KD 0.1 nM), also applying
Table 1 PAI-1 variants and binding to Vn PAI-1 mutants with corresponding speci®c inhibitory activity towards uPA and their ability to interact with Vn measured by use of Vn-coated microtiter plates (MP) and surface plasmon resonance (SPR) + Indicates binding to Vn; ± indicates no binding to Vn NT, not tested.
PAI-1 variant Modi®cation a Inhibitory activity
towards uPA (Uámg )1 )
Binding to Vn
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 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
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 of recombinant wild-type PAI-1 Human
recombi-nant wild-type PAI-1 equipped with an N-terminal His 6 tag (wild-type
PAI-1) was puri®ed from an E coli cell lysate by Ni 2+ -nitrilotriacetic
acid anity chromatography and analyzed by SDS/PAGE M,
marker; lane 1, E coli lysate; lane 2, euent of the ®rst Ni 2+
-nitrilo-triacetic acid anity chromatography cycle; lane 3, eluate of the ®rst
Ni 2+ -nitrilotriacetic acid anity chromatography cycle; lane 4, eluate
of the second Ni 2+ -nitrilotriacetic acid anity chromatography cycle.
Fig 3 Formation of SDS stable complexes Wild-type PAI-1 or variants thereof in the presence (+ uPA) or absence (± uPA) of 100 U ( 0.7 lg) HMW-uPA were incubated for 10 min at room temperature and then subjected to nonreducing SDS/PAGE Subsequently, the gels were silver-stained 200 U of PAI-1, 110 U of Q123K, 50 U of P73A and of A114±118 were applied; alternatively 1.4 lg of (inactive) M4 and 10 U of PAI-1 were used.
Trang 6SPR analysis [22] Mutant A114±118 displayed a slower
association to (on-rate: 5.35 ´ 105 M)1ás)1) and a faster
dissociation from Vn (off-rate: 1.0 ´ 10)3s)1) compared to
wild-type PAI-1 (on-rate: 1.0 ´ 106 M)1ás)1; off-rate:
1.9 ´ 10)4s)1) Only a low amount of mutant Q123K
associated to Vn and dissociated immediately after 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 absence of
soluble Vn (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, speci®cally binds to 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 PAI-1 with thrombin
inhibitory properties [10] Therefore, we tested whether the
PAI-1 mutants generated were also able to inhibit thrombin
in the presence of Vn In line with our data obtained by
SPR, the Vn-interacting mutants A114±118 and P73A, but
not Q123K, inhibited thrombin in the presence of Vn to
about 40% Vn alone did not reduce thrombin activity
signi®cantly (Fig 5)
We also tested the effects of Hep on the inhibitory activity
of PAI-1 towards thrombin All mutants tested but A114±
118 displayed similar 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 results determined by amidolytic assays measuring
(residual) thrombin activity were supported by the results
seen in Western blots visualizing the formation of SDS stable complexes between thrombin and PAI-1 with or without Vn or Hep Detection of a higher residual thrombin activity correlated with a lower amount 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 units 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 buer at 37 °C for 1 h Then, 10 lL of 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 set to 100%, the other activities were calculated accordingly Data shown are from three independent experiments, each measured in duplicate ( SD) As a control, the eect of 140 n M Vn or 1 UámL )1 Hep without inhibitor was measured Black bars, buer, 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 RU of collagen type IV or Vn were immobilized to a CM5 sensor chip inserted in the BIAcore 2000 system Then, wild-type PAI-1 (200 UámL )1 ) was injected and allowed to bind to Vn, which was followed by a washing step with buer Finally, HMW-uPA (400 UámL )1 ) was injected Two subsequent measurements of the binding 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 least two independent binding pro®les were obtained All experiments were performed at a ¯ow rate of 20 lLámin )1 ; regeneration of the surface was achieved by treatment with
10 m M HCl for 8 min 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 to obtain binding pro®les without bulk eects Heat denatured controls were measured to compare the binding signal with the unspeci®c binding to the identical surface conditions.
Trang 7Hep, as in SPR we observed that A114±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 ef®ciently than the other tested
mutants, it also reduced the inhibitory property of A114±
118 towards uPA in a dose dependent manner Hep had
only marginal effects on the inhibition of uPA by other
inhibitory active PAI-1 proteins (wild-type PAI-1, P73A,
Q123K, data not shown)
D I S C U S S I O N
Puri®cation and inhibitory activity of recombinant
PAI-1, PAI-2, and PAI-1 variants
Recombinant expression in a bacterial system is an easy and
quick method for the production of large amounts of
human PAI-1 and PAI-2 It has been shown previously,
that, although PAI-1 and extracellular PAI-2 are
glycosy-lated proteins, expression of both proteins in 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
histidine6-sequence at the N-terminus of the recombinant
proteins [21], thus allowing puri®cation by Ni2+
-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 modi®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 buffer that had
been described previously for the isolation of PAI-1 from
conditioned medium of human endothelial cells [26] This
buffer is characterized by a low pH (5.6) as well as a
relatively high ionic strength (1MNaCl) These conditions
resemble the inner milieu of the a-granula contained in
thrombocytes, which are the main reservoir of PAI-1 in
blood [27] Sancho et al [28] reported the isolation of
signi®cant amounts of active PAI-1 under native conditions
by using such a buffer However, we did not obtain such
high amounts of active PAI-1 under similar conditions, but
the inhibitory activity of PAI-1 was dramatically enhanced
by denaturation, refolding, and storage in a buffer at pH 5.6
containing 1MNaCl
The generated PAI-1 variants 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, and Q123K, in which only one or
two amino-acid were exchanged, showed the highest
inhibitory capacity against HMW-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) about 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 contained more than seven modi®ed amino-acid positions did not display any inhibitory activity towards uPA, indicating that larger modi®cations at these positions are not tolerated and lead to
a loss of activity most 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 their metastability and the stabiliza-tion of the active form by Vn In contrast to wild-type PAI-1 and its active variants, recombinant PAI-2 did not show any signi®cant loss of activity after puri®cation under native conditions, underlining the uniqueness of the metastable PAI-1 among serpins
Interaction of (mutant) PAI-1 with Vn Using Vn-coated microtiter plates and SPR, we analyzed speci®c binding of wild-type PAI-1 and PAI-1 variants to
Vn In general, SPR measurements are considered to be very quantitative and association/dissociation constants are normally easily analyzed by Langmuir binding isotherms
to obtain the respective binding constants However, in the case of PAI-1 and variants thereof, this kind of measure-ments may not be accurate for the following reasons: PAI-1 spontaneously converts from its active to a latent form To determine the proportion of active PAI-1, we measured the speci®c inhibitory activity against uPA with a theoretical maximum de®ned as 100 000 Uámg)1[24] This method 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 the amount of active PAI-1 in a given preparation to the time-point when PAI-1 is used in the SPR analysis, another as yet unknown part of active PAI-1 converts to the latent 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 be semiquantitative It has to be emphasized, however, that in the present study the main aim was to analyze whether mutants with variations in hE are still able to bind to Vn and not 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 variations covering the whole area of hE (D109-123, M2, M3, M4) yielded inactive PAI-1 variants However, D109-112, in which the N-terminal amino acids of
hE were deleted, as well as A114±118, with changes in the C-terminal region of hE, still bound to Vn and inhibited uPA Thus, mutations within hE of PAI-1 are tolerated to 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 concluded that the mutants still interact functionally with Vn Moreover, concerning Q123K and its dramatically reduced af®nity to Vn, we reproduced the results of Lawrence et al [16], which again underlines the functionality of our test systems Lawrence et al [16] also reported about another mutation (L116P) in 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,
Trang 8still 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 the Vn-binding site
on PAI-1 to amino acids 115±130 employing PAI-1/PAI-2
chimera and protease-digested PAI-1 This seems to be
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 monoclonal antibodies which inhibited Vn/
PAI)1-interaction The region encompassing amino acids
110±145 not only comprises hE but also the region of the
strand 1 edge of b sheet A, where Q123 is located [29]
Cross-linking studies reported by Deng 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 with 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 af®nity binding of PAI-1 to Vn 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 around strand 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 that wild-type PAI-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], 1 UámL)1 was determined as the ideal Hep
concentration for inhibition of thrombin by wild-type
PAI-1 In addition to wild-type PAI-1, we also tested the 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 expected, Hep-bound Q123K inhibited thrombin
exactly like wild-type PAI-1 A114±118 did not display a
signi®cantly altered af®nity to Hep in SPR, although it was
not able to inhibit thrombin as ef®ciently as wild-type PAI-1
together with Hep This contrasts with the results of Sui and
Wiman [30] who detected a change in af®nity to Hep in their
mutant R118D and proposed that mainly ionic interactions
occur between PAI-1 and Hep However, a change of 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, the inhibitory 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 conformational changes occur, affecting the inhibition
characteristics of this PAI-1 mutant towards both thrombin
and uPA
C O N C L U S I O N S
Comparison of the main protease 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 Rather, it seems that further interactions of one of the proteases, uPA, with other molecules support tumor invasion and metastasis Whereas 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 machinery most likely due to effects excerted upon interaction with its speci®c receptor, uPAR [3] Similarly, the additional binding 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 adhesion and, thus, may facilitate tumor cell invasion via a balanced interference/induction of tumor cell attachment/migration [11,12,33,34]
Detailedknowledgeofthestructuralregion(s)ofthePAI-1 molecule implicated in the PAI-1/Vn-interaction is the basis for the rational development of site-speci®c PAI-1 modu-lators [35] Surface-exposed loop structures, such as hE in PAI-1, represent attractive targets for the development of such modulators because of their high accessibility hE has been implicated in the binding of PAI-1 to Vn [16±19] An hE-blocking compound may not block further PAI-1 activities (inhibition of serine proteases or binding to ®brin) and, thus, would not alter functions of PAI-1 important for physiological processes such as ®brinolysis However, in the present paper, we demonstrate that the region around hE in PAI-1 is not a prerequisite for binding to Vn and, thus, may not be a target for the development of a therapeutically applicable PAI-1 modulator
A C K N O W L E D G E M E N T S
The excellent technical assistance of S Creutzburg is gratefully acknowledged We thank J StuÈrzebecher, J Krol, and S Sato for stimulating discussions Part of this work was supported by grants of the Graduiertenkolleg 333, the Sonderforschungsbereich 469 (A4), and the Sonderforschungsbereich 456 (B9) of the Deutsche Forschungs-gemeinschaft.
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