Background
Plasminogen activator inhibitor-1 (PAI-1) is a serine
proteinase inhibitor in the serpin superfamily [ 1 2 3 ] .
This 50 kDa glycoprotein is apparently the most important
physiological inhibitor of tissue-type plasminogen
activator (tPA) and of urokinase plasminogen activator
(uPA). [ 4 ] It was shown to play a crucial role in the
regulation of vascular thrombosis, tumor invasion,
neovascularization, inflammation and wound healing. [ 5 6 ]
Increased plasma levels of PAI-1 were found to be
correlated with an increased risk for cardiovascular
diseases. [ 6 7 8 ] The essential function of PAI-1 in the
fibrinolytic system has been confirmed by studies with
transgenic animals. [ 7 9 ]
PAI-1 shares a 35% homology with 40 other serpins. [ 10
] The X-ray structure of active PAI-1 [ 11 12 ] consists of
three sheets (A-C), nine helices (a-i) and a reactive
center loop (RCL). The RCL contains the residues Ser331 to
Arg356 (P16-P10'), and within it there is a peptide loop
(Glu 351 to Pro 357, P5'-P11'), which is defined as the
distal hinge (Table 1). It should be noted that the
placement and conformation of the RCL in active PAI-1 is
quite different from that of latent PAI-1. [ 13 ] The
movement of the distal hinge and the insertion of the RCL
into sheet A as strand 4A (s4A) should take place during
the transition from the active into the latent form of
PAI-1.
The catalytic center of tPA interacts with the reactive
site of PAI-1 with concominant formation of 1:1 molar,
SDS-stable complex. The P1 (Arg346) residue in the RCL of
PAI-1 is an essential determinant for its target
specificity and inhibitory activity. [ 14 15 ] Since many
serpins with identical amino acid residue at P1 position
display different specificities, it is unlikely that the P1
residue is the sole determinant for target protease
specificity, other regions of PAI-1 may also affect its
specificity toward target proteases. The variable region in
serine proteases domain determines their specificity for
PAI-1. Alignment of variable region 1 of the serine
proteases shows a remarkable correlation between the
composition of this area and their susceptibility to
inhibition by PAI-1 as shown in Table 2. [ 16 ] Both tPA
and uPA, the target proteases of PAI-1, contain a
relatively extensive variable region 1, consisting of four
to five positively charged amino acid residues present at
identical position. Plasmin and thrombin with moderate
reactivity toward PAI-1 have relatively short variable
regions which contain only two to three positively charged
residues. For example, positively charged amino acids in
the variable region of tPA, uPA and thrombin play dominant
roles in the specific interaction with PAI-1. [ 17 18 19 ]
Furthermore, Glu350 (P4') of PAI-1 has been shown to
mediate the interactions with tPA. [ 18 ]
In this study, By producing wtPAI-1 and several single
site mutations in the distal hinge of PAI-1 in a
baculovirus expression system, we were able to find out the
role of the distal hinge in conformational transition of
PAI-1 and to elucidate the role of Glu351 in PAI-1 for
serine protease specificities.
Results
Characterization of wtPAI-1
To confirm the validity of recombinant PAI-1 as a
model to study the native inhibitor secreted from human
fibrosarcoma cells, we compared the recombinant PAI-1 and
the fibrosarcoma PAI-1 first by SDS-PAGE and immunoblot
with anti-PAI-1 monoclonal antibodies. The molecular
weight of wtPAI-1 was found to be lower than native PAI-1
(~50 kDa, Fig. 1B). We assumed that this is due to a
lower (probably different) extent of glycosylation in the
baculovirus expression system. Indeed, when both proteins
were deglycosylated with N-glycanase they were found to
have an essentially identical M.W. (~48 kDa) and to
cross-react immunochemically (Fig. 1B). Furthermore, when
this wtPAI-1 was chromatographed on heparin-Sepharose it
was found to be > 98% pure by silver staining of the
gel (Fig. 1Alane 2).
Interestingly, the specific inhibitory activity of
wtPAI was 63 ± 15 U/ug just after purification, and was
increased to 84 ± 21 U/ug after denatured by 6 M Gdn-HCl
and refolded in 50 mM sodium acetate (pH 5.6) at 4°C. In
contrast, the specific activity of the fibrosarcoma PAI-1
was only 5 ± 3 U/ug before activation, and was only
increased to 45 ± 16 U/μg after denaturing and refolding.
The high specific inhibitory activity of wt-PAI-1 could
be due to the fact that it was secreted from insect
High-5 cells cultured at 27°C, while the fibrosarcoma
preparation of PAI-1 was obtained from the medium of
fibrosarcoma cells cultured at 37°C [ 20 ] . Exposure to
the higher temperature could accelerate the conversion of
the inhibitor into its latent form.
The functional stability of wtPAI-1 and the
fibrosarcoma PAI-1 in the absence or in presence of
vitronectin are illustrated in Figure 1C. wtPAI-1 lost
its inhibitory activity at the same pace as the
fibrosarcoma PAI-1, indicating that its functional half
life as an inhibitor and its ability to be stabilized by
vitronectin are similar to those of the fibrosarcoma
PAI-1.
The second-order rate constants (in M -1s -1units) of
the fibrosarcoma PAI-1 and the wtPAI-1 towards t-PA were
1.4 ± 0.5 × 10 7and 1.7 ± 0.6 × 10 7, and towards uPA
were 4.1 ± 1.4 × 10 7and 4.4 ± 1.6 × 10 7, respectively.
These results show that the wtPAI-1 could inhibit tPA or
uPA at the same rate as the fibrosarcoma PAI-1. In
addition, it is known that PAI-1 inhibits either tPA or
uPA by forming a 1:1 SDS stable complex [ 4 ] , wtPAI-1
indeed could form SDS stable complex with tPA or uPA
(Fig. 3A) just as fibrosarcoma PAI-1.
Single site mutations within the distal hinge of
PAI-1
Since a proline occurs not only in position 357 of
PAI-1, but also in the corresponding position of several
other serpins (Table 1) we attempted to find out how does
a mutation of P357 affect the inhibitory activity of
PAI-1. Mutant of Pro357Gly blocked the inhibitory
activity of PAI-1 (Fig. 2A). Similarly, a mutation into
proline of either Asp355 or Arg356 also inactivated the
inhibitor. It should be noted that all these mutants
could be activated to a similar extent by 6 M Gnd-HCl
(~20% inhibitory activity of the wtPAI-1, activated under
the same conditions). These results suggest that a single
site mutation at either one of the positions 355, 356 or
357 significantly inactivates PAI-1.
To elucidate the mechanism through which such
mutations affect the activity of PAI-1, we determined
their effect on the functional stability of PAI-1 and on
its ability to form an SDS stable complex with either tPA
or uPA. As seen in Fig. 2B, the rate of the spontaneous
inactivation of the inhibitor was significantly increased
upon mutation: the functional half life of the mutants
Asp355Pro, Arg356Pro and Pro357Gly was 23.8 ± 4.5 min,
26.2 ± 3.7 min and 24.7 ± 4.8 min, respectively, while
that of wtPAI-1 was 89.8 ± 8.7 min. Interestingly, there
was no significant difference between the mutants tested
and the wtPAI-1 in their ability to inactivate either tPA
or uPA (Table 3). They could also form SDS-stable
complexes with tPA or uPA just as well as wtPAI-1 (Fig.
3A).
An interesting structural similarity among several
serpins is known to occur at the position corresponding
to 355 in PAI-1. While in PAI-1 this position
accommodates a negatively charged aspartic acid residue,
in other serpins it sometimes accommodates its non
charged analog asparagine (Table 1). Asp355His,
Asp355Lys, Asp355Glu, Asp355Gln and Asp355Asn were
prepared and used to show that the negatively charged
aspartic acid in PAI-1 is essential (though not
sufficient) for its inhibitory activity. Mutation of
Asp355 by asparagine (the non charged analog of aspartic
acid), or by histidine or lysine (that have a positively
charged side chain) or by alanine (neutral), inactivated
PAI-1. Replacement of this aspartic acid residue even by
the negatively charged glutamic acid (a very close analog
of aspartic acid) did not significantly suffice to
restore a full inhibitory activity of PAI-1 (Fig. 3B).
After activation with 6 M Gnd-HCl, each of the six
mutants exhibited a relatively high inhibitory activity,
but their specific activities were still much lower than
that of wtPAI-1 (Fig. 3B). Among them, Asp355Lys (5.1 ±
4.1 U/ug) had the lowest specific activity, while the
Asp355Asn (20 ± 4.2 U/μg) had the highest specific
activity (Fig. 3B). Although they had lower specific
activities, all of the mutants mentioned above could
still form SDS-stable complexes with tPA or uPA just as
the wtPAI-1 (data not shown).
Two additional mutations, Arg356Ala and Arg356Glu,
were prepared to further elucidate the role of Arg356.
The specific inhibitory activity of Arg356Ala was 7.2 ±
2.5 U/μg, while Arg356Glu exhibited essentially no
inhibitory activity. This specific activity was increased
to 35.4 ± 8.7 U/ug for Arg356Ala and 6.4 ± 2.3 U/μg for
Arg356Glu after denature and refolding. Just like
ARG356Pro, Arg356Ala and Arg356Glu were much more labile
than wtPAI-1. These results suggest that the role of the
Asp355-Pro357 segment is to stabilize the inhibitory
conformation of PAI-1.
Specific activity, functional stability and complex
formation of Glu351Ala and Glu351Arg
The specific inhibitory activities of wtPAI-1,
Glu351Ala and Glu351Arg were assessed with uPA and
S-2444. As shown in Fg. 4A, the specificity of wtPAI-1,
Glu351Ala and Glu351Arg after denatured by 6 M Gdn-HCl
and dialyzed against 50 mM sodium acetate, pH 5.6, was 84
± 15, 112 ± 18 and 68 ± 9 U/ug, respectively. Glu351Ala
had a higher, and Glu351Arg lower specific activity than
wtPAI-1. The specific activities of PAI-1s determined
immediately after purification displayed similar pattern
as that of inhibitors reactivated. There was about 15-20%
increase after reactivation compared to each untreated
protein itself. As shown in Fig. 4B, the two mutants also
spontaneously lost their inhibitory activity(convert to
latency) as wtPAI-1, but at different rates. The
functional stability of wtPAI-1, Glu351Ala, Glu351Arg was
about 18 ± 5, 90 ± 8 and 14 ± 3 minutes, respectively. It
suggested that the specific activities measured above, to
some extent, proportionally represent the functional
stability. More importantly, amount complexes formed by
tPA and PAI-1s remained after incubation at 37°C (Fig.
4C) were proportionally concordant with the results
obtained from enzymatic stability assay (Fig. 4B). This
proves that results from enzymatic assay are valid. The
result of functional stability suggests that Glu351 is
indeed involved in the conformational transition of
PAI-1.
Inhibition of thrombin by PAI-1s and Neutralization
of PAI-1s by Thrombin
Both mutants inhibited thrombin more efficiently than
wtPAI-1, more importantly, Glu351Ala was better than
Glu351Arg in inhibiting thrombin (Fig. 5A); Inhibition of
thrombin by Glu351Arg was almost as same as by Glu351Ala
in presence of vitronectin (Fig. 5B), which facilitated
the thrombin inhibition by both mutants. When excess
equal amount of thrombin was incubated with equal amount
of PAI-1s in term of inhibitory activity against uPA,
mutant inhibitors were neutralized faster by thrombin
than wtPAI-1, and vitronectin also facilitated the
neutralization of Glu351Ala or Glu351Arg by thrombin,
just similar to the wtPAI-1 (Fig. 5C).
Inhibition of plasmin by PAI-1s
When plasmin was incubated with increased amount of
equal active PAI-1s in absence or in presence of
vitronectin, it was inhibited faster by mutants than by
wtPAI-1 (Fig. 6upper panel); and both of mutant also
displayed improved inhibition toward plasmin in presence
of vitronectin (Fig. 6lower panel).
Kinetic analysis
The rate constants of tPA for wtPAI-1, Glu351Ala and
Glu351Arg were similar (Table 4), however, The rate
constants of inhibition toward uPA by Glu351Ala and
Glu351Arg were 4 and 7-fold higher than by wtPAI-1,
respectively. Moreover, The rate constants of Glu351Ala
and Glu351Arg for thrombin inhibition were 2 to 4 folds
higher than that of wtPAI-1 and rate constants of
Glu351Ala and Glu351Arg to plasmin were 2 fold higher
than that of wtPAI-1. This indicates that Glu351 is not
essential for the interaction with tPA, but it is an very
important determinant for the optimal interaction with
uPA, thrombin and plasmin.
Disscusion
Cognate protease specificity
Improvements in specificity of mutants for uPA
indicates that Glu351 involve in the interaction with
uPA. To the best of our knowledge, this is the first time
that the specificity of PAI-1 toward uPA is partially
mediated by the negatively charged residue located in the
C-terminal of cleavage site in PAI-1. The interaction of
Glu351 with uPA is much more important than that of
Glu351 with tPA, which is demonstrated by improved
reactivity of Glu351 mutants toward uPA, but not toward
tPA, showing that tPA is not directly interact with
Glu351 of PAI-1, whereas Glu351 is necessary for the
optimal interaction with uPA. Our results are concordant
with previous study, [ 25 ] which suggests that serpin
body-protease body interactions play significant roles in
determining serpin inhibitory activity against target
proteases.
Non-cognate protease
Substitution of Glu351 also altered the specificity
toward non-cognate proteases: plasmin and thrombin. Glu
at 351 may provide steric hindrance that slows the rate
of thrombin or plasmin inhibition. Mutations at Glu351
may decrease the original repulsive interactions with
variable region of thrombin, which may slow the rate of
thrombin inhibition, or results in favorable interactions
that may not exist when wtPAI-1 react with thrombin. So,
both Glu351Arg and Glu351Arg are better thrombin
inhibitors than wtPAI-1. Similar results were observed
for plasmin. With single mutation at position Glu351, we
improved reactivity of PAI-1 to thrombin and plasmin
without significantly affecting its specificity to tPA.
This substantiates previous mutagenesis studies, which
demonstrated that repulsive interactions and/or lack of
productive electrostatic interactions between Glu39 and
Glu192 of thrombin and PAI-1 are responsible for the slow
reaction of thrombin with this serpin. [ 26 ]
Furthermore, the rapid inhibition of plasmin requires a
non-covalent interaction between an amino-terminal site
of plasmin and a carboxyl-terminal on anti-plasmin, and a
second site outside the RCL of PAI-1 contributes to its
specific interaction with proteases. [ 15 27 ] The
specificity of the two mutants toward thrombin and
plasmin implicates that Glu351 of PAI-1 is involved in
its specificity toward thrombin and plasmin.
Conclusion
Single site mutations within the segment
Asp355-Arg356-Pro357 of PAI-1 yield Gdn-HCl activatable
inhibitors that can still form SDS stable complexes with
plasminogen activators, with second order inhibition
constants that are similar to the native inhibitor.
Interestingly, the conversion of these mutants to latent
forms was ~3-4 fold faster than wtPAI-1, suggesting that
the Asp355-Pro357 segment is involved in maintaining the
inhibitory conformation of PAI-1. Glu351 contributes to the
optimal functional stability of PAI-1 and participates its
conformational stability. More importantly, Glu351 of PAI-1
is a specificity determinant for uPA, thrombin and plasmin,
but not for tPA.
Methods
General DNA techniques
Plasmid DNA was purified using either Promega or
QIAGEN miniprep kits. The DNA was sequenced using the
di-deoxy chain termination reaction method using an ABI
373 DNA sequencer. Oligonucleotides were synthesized
using a 380 B DNA synthesizer (ABI model). DNA
manipulation techniques were carried out according to
standard procedures [ 29 ] .
Construction of wtPAI-1 and its mutants
The pPAI-1-AI was digested with EcoRI and BglII to
release a 1426 basepair (bp) PAI-1 coding sequence, which
was isolated, purified, and ligated into EcoRI and BglII
digested pVL1393. The resultant pVL1393-PAI-1 was
analyzed by restriction mapping and sequencing. The
mutants at E351A, E351R, D355, R356, and P357 of PAI-1
were generated by PCR. The occurrence of the resulting
mutations D355A, D355E, D355H, D355K, D355N, D355P,
D355Q, R356P, P357G as well as E351A and E351R in pRSET
was confirmed by sequencing. The PAI-1 sequence
containing the desired mutations was digested with EcoR1
and BglII, and then subcloned into pVL1393. The
recombinant baculoviruses containing either wtPAI-1, or
one of the above mutants was obtained as deccribed by
Kjoller et al. [ 30 ]
Expression and purification of wtPAI-1 and its
mutants
High 5 cells, grown at 27°C in SF-900 serum-free
medium, were infected with high-titer virus stocks (1-3 ×
10 8/ml). The medium was collected 48 hrs after the
infection (found to be the optimal harvesting time for
wtPAI-1) and then centrifuged for 5 minutes (125 × g).
The supernatents (50 ml) were collected and sodium
chloride (final concentration 0.25 M) was added. They
were then applied on a heparin-Sepharose column (bed
volume 5 ml) that was equilibrated and run (flow rate 20
ml/h) with a buffer consisted of 50 mM sodium acetate, pH
5.6, containing 0.25 M NaCl and 0.01% Tween-20. The
columns were washed and the PAI-1s were eluted with a 40
ml gradient of 0.25 - 1 M NaCl in the same buffer.
SDS-PAGE, immunoblotting and deglycosylation of
PAI-1
The medium of the infected cells and the elutent from
heparin-Sepharose were separated on 10% SDS-PAGE, and
monitored by silver staining. Fibrosarcoma PAI-1 and
wtPAI-1 were transferred onto nitrocellulose paper and
the proteins were revealed with anti-human PAI-1
monoclonal antibodies, followed by horseradish peroxidase
(HRP) conjugated rabbit anti-mouse antibody, and enhanced
chemiluminescence (ECL). 5 ug of Fibrosarcoma PAI-1, or
wtPAI-1 was denatured by boiling for 5 min in a buffer
consisted of 0.5% SDS, 50 mM β-mercaptoethanol and 50 mM
Tris-HCl, pH 7.4. They were then digested (18 hr, at
37°C) with 0.5 units of recombinant N-glycanase in the
presence of 2.5% NP-40, and subjected to SDS-PAGE and
silver staining. Protein concentrations were determined
by the Bio-Rad protein assay reagent.
Functional stability of fibrosarcoma PAI-1, wtPAI-1
and its mutants
The inhibitory activities of fibrosarcoma PAI-1,
wtPAI-1, and its mutants were measured by a direct
chromogenic assay using uPA (100 U/ug) and its substrate
S-2444 in a microtiter plate as described earlier [ 20 ]
. Briefly, fibrosarcoma PAI-1, wtPAI-1, or its mutants
were diluted 1:10 in the activity measuring assay buffer
(50 uM Tris-HCl, pH 7.5, containing 0.15 M NaCl and 100
ug/ml BSA). The PAI-1 samples were incubated in the
absence or in the presence of a two molar excess of
vitronectin (the fibrosarcoma PAI-1 and the wtPAI-1 were
incubated with vitronectin) at 37°C. At the times
indicated, aliquots were removed and analyzed immediately
for their inhibitory activity. One unit of PAI-1 activity
is defined as the amount that completely neutralizes 1 U
of uPA. The specific activity obtained at time zero was
assigned a value of 100%.
Activation of PAI-1 and kinetic analysis
Fibrosarcoma PAI-1, wtPAI-1 or its mutants were
denatured with 6 M guanidine-hydrochloride (Gdn-HCl) (45
min at 37°C), followed by dialysis (4-16 hours at 4°C)
against 50 mM sodium acetate, pH 5.6, containing 0.5 M
NaCl and 0.01% Tween-20. This process which PAI-1
regained its activity is called activation by Gdn-HCl.
The inhibitory activity of each of the PAI-1s was assayed
immediately. The second-order rate constants for the
interaction of the fibrosarcoma PAI-1, wtPAI-1 or the
indicated mutants with single-chain tPA or uPA were
determined in a single-step assay as previously
described. [ 20 ]
Specific activity, functional stability and complex
formation of PAI-1s
The inhibitory activities of wtPAI-1 and mutants were
measured by a direct chromogenic assay using uPA (100
U/μg) and substrate S-2444. [ 17 ] Briefly, serially
diluted wtPAI-1, or mutants were incubated with equal
volume of uPA (50 μl, 50 U/ml) for 30 min at 37°C,
followed by addition of 100 μl, 0.5 mM S-2444. Residual
uPA activity was quantitated by measuring the change in
absorbance at 405 nm with ELISA reader. The specific
activity of PAI-1 was calculated based on the amount of
uPA inhibited by PAI-1. The assay for PAI-1 inhibitory
activity toward two-chain tPA was the same for uPA except
that the samples were incubated with tPA and S-2288.
PAI-1 inhibitory activity is expressed as the number of
international units of PAs inhibited by PAI-1. Specific
activity of uPA and two-chain tPA are 100 U/μg and 764
U/μg, respectively. For the functional stability,
wtPAI-1, or mutants were incubated at 37°C. Aliquots were
taken out at times indicated and analyzed immediately as
described above. The specific activity obtained at time
zero was assigned a value of 100%. To provide the
evidence to support the conclusion obtained from enzyme
assay, certain amount of wtPAI-1, E351R and E351A were
incubated with equal amount of tPA at 37°C for 30 minutes
before put on ice, at same time these PAI-1s were
incubated in PBS at 37°C for 3 hours, respectively,
before the PAI-1s were incubated with same amount of tPA
at 37°C for 30 minutes, then the protein samples prepared
at two time points were subjected to SDS-PAGE and western
blot as described before.
Inhibition of thrombin and plasmin by PAI-1s
Thrombin (0.025 U), or plasmin (0.1 μg/ml) was
incubated at 37°C for 30 min with same amount of wtPAI-1,
E351A and E351R in term of inhibitory activity toward uPA
(20 U in the concentration range of 4-10 μg/ml) in
absence or presence of vitronectin (60 μg/ml). Linear
increase of absorbance at 405 nm were recorded after
adding S-2238 (final concentration 0.25 mM) for thrombin,
or N-p-Tosyl-Gly-Pro-Lys-p-Nitroanilide (final
concentration 0.5 mM) for plasmin. Thrombin, or plasmin
activity without PAI-1 was taken as 100%.
Neutralization of PAI-1s by Thrombin
wtPAI-1 and mutants were incubated with four molar
excess of thrombin (40 nM) in the absence or presence of
vitronectin (30 nM). Control experiments were carried out
in the absence of thrombin. After 20 min at 37°C, the
activity of thrombin was quenched by the addition of
hirudin (final concentration, 60 units /ml), resulting in
inhibition of more than 95% of the amidolytic activity of
thrombin. Subsequently, the residual PAI-1 inhibitory
activity was determined by titration of aliquots on uPA,
using S2444 as substrate. The increase of the optical
density at 405 nm in the samples was corrected for the
values measured with (hirudin-inactivated) thrombin
alone, since the amidolytic activity of thrombin on S2444
was not completely blocked by hirudin,
Complex formation of PAI-1s with tPA or uPA
Complex formation of the fibrosarcoma PAI-1, wtPAI-1
or its mutants with single-chain tPA or uPA were
performed as described earlier [ 20 ] . Samples of PAI-1s
(5 U) were incubated in the absence or in the presence of
a 2-fold excess of uPA or tPA in the activity assay
buffer. Following a 30 min incubation at room
temperature, the samples were subjected to a 10% SDS-PAGE
and immunoblotting as described above.
Abbreviations
tPA, tissue-type plasminogen activator; uPA,
urokinase-type plasminogen activator; PAI-1, plasminogen
activator inhibitor type 1; serpin, serine protease
inhibitor; RCL, reactive center loop; Vn, vitronectin, SDS,
sodium dodecylsulfate; PAGE, polyacrylamide gel
electrophoresis. Gdn-HCl, guanidine-hydrochloride; HRP,
horseradish peroxidase; ECL, enhanced chemiluminescence;
PBS, phosphate-buffered saline; BSA, bovine serum
albumin.
Authors' contribution
Qingcai Wang carried out the experiments and drafted the
manuscript;
Professor Shmuel Shaltiel sponsored and supervised the
project and revised the manuscript.
