(Received for publication, January 16, 1997, and in revised form, February 25, 1997)
From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Plasminogen activator inhibitor-1
(PAI-1) is a unique member of the serpin superfamily. The alternative
behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a
non-reactive latent form has been shown to be dependent on the initial
conformation. In this study, we have evaluated the effect of a
substitution outside the reactive site loop (P18) or in the reactive
site loop (P6 and P10) on proteinase specificity and conformational
transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same
conformational distribution pattern toward tissue-type plasminogen
activator (t-PA) as toward urokinase-type plasminogen activator (u-PA)
(i.e. 53 ± 6.9% active, 36 ± 6.8% latent, and
12 ± 1.9% substrate). Inactivation of wtPAI-1 resulted in the
conversion of the labile active form into the latent form while the
stable substrate form remained unchanged. PAI-1-P6 (Val Pro at
P6) revealed a target specificity for t-PA (39 ± 7%
versus 3 ± 2% of the theoretical maximal value
toward t-PA and u-PA, respectively), PAI-1-P10 (Ser
Pro at P10)
was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18
(Asn
Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 ± 10%). Surprisingly, inactivation of these
mutants revealed functional and conformational transitions distinct
from those observed for wtPAI-1. Inactivation of
PAI-1-P6(Val
Pro) resulted in a total conversion of the active
form into the latent form and in a partial conversion of the substrate
form into the latent form. The active forms of both
PAI-1-P10(Ser
Pro) and PAI-1-P18(Asn
Pro) are also labile
but, in contrast to the active form of wtPAI-1, convert into substrate
forms. Based on the existence of various conformations of PAI-1, we
propose an alternative reaction scheme describing the putative
interactions between serpins and their target proteinases. The unusual
conformational and functional flexibility of PAI-1 that, according to
the current study, appears not to be restricted to the reactive site
loop further underlines the importance of potential structural
rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in
general) for its functional behavior at particular biological
sites.
Plasminogen activator inhibitor 1 (PAI-1),1 a glycoprotein with an apparent
molecular weight of 50,000 (1), is the most important physiological
inhibitor of tissue-type plasminogen activator (t-PA) and
urokinase-type plasminogen activator (u-PA) and inhibits both
proteinases very rapidly with second-order rate constants of more than
2 × 107 M1 s
1
(2, 3). Increased plasma levels of PAI-1 have been shown to correlate
with an increased risk for cardiovascular disease (4; for review, see
Ref. 5).
PAI-1 is synthetized as an active molecule that converts spontaneously
to a latent form that can be partially reactivated by denaturants such
as guanidinium chloride, sodium dodecyl sulfate or urea (6). In
addition, a stable non-inhibitory form with substrate properties has
been identified (7-9). PAI-1 is a member of the serine proteinase
inhibitor (serpin) superfamily (10-13). The serpins comprise more than
40 single-chain proteins, each containing 370-390 residues with an
overall amino acid homology of approximately 35% (14, 15). All serpins
have the same highly ordered tertiary structure consisting of three
beta sheets, A, B, and C, nine -helices, and a reactive site loop
containing residues P16 to P10
, which are highly variable (16). The
reactive site loop is situated 30-40 amino acids from the
carboxyl-terminal end and provides a "bait" peptide bond (P1-P1
)
that mimics the normal substrate of the target proteinase (17). In
PAI-1, the Arg346-Met347 bond has been
identified as the P1-P1
bond (18). In latent PAI-1, the amino-terminal part of the reactive site loop (P16-P1) is
inserted into beta sheet A forming s4A and rendering P1-P1
inaccessible to the target proteinase. The substrate form of PAI-1 reacts with its target proteinases, e.g. t-PA or u-PA, resulting in a
cleavage of the P1-P1
bond but, in contrast to the active form,
without formation of a covalent complex and without inhibition of the
proteinase. The target specificity of serpins is mainly determined by
the nature of the residues at the P1 and P1
position (19-27).
However, residues at positions P3 and P2 (26, 28-30) or the presence
of cofactors (31) have also been reported to influence the target
specificity of serpins.
The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate or a non-reactive latent form, has been shown to be mainly dependent on the initial conformation of this serpin (7). However, in general, the functional behavior of serpins (i.e. inhibition versus substrate) is often explained by the occurrence of one reactive conformation resulting in the generation of common intermediates until the branching point between substrate and inhibitory pathways (32). According to this hypothesis, the product distribution (i.e. complex formation or cleaved serpin) depends on the reaction conditions influencing this branching (32-35).
In the current study, the characterization of PAI-1 variants carrying
proline mutations at positions P6, P10, or P18 reveals (a) a
t-PA target specifity for PAI-1-P6(Val Pro) and a u-PA target
specificity for PAI-1-P10(Ser
Pro) and PAI-1-P18(Asn
Pro), (b) an unexpected transition of the active form of
PAI-1-P10(Ser
Pro) and PAI-1-P18(Asn
Pro) into a substrate
form, and (c) the existence of two distinct substrate
populations in PAI-1-P6(Val
Pro). These observations have allowed
us to draw an alternative reaction scheme for the interactions between
serpins and their target proteinases.
The chromogenic substrate S-2403 was obtained from Chromogenix (Mölndal, Sweden). t-PA (predominantly single chain) was a kind gift from Boehringer Ingelheim (Brussels, Belgium); low molecular weight two-chain u-PA (tcu-PA) was prepared from single-chain u-PA (scu-PA) kindly provided by Dr. Lijnen (University of Leuven, Belgium). Alternatively, u-PA (consisting of high and low molecar weight tcu-PA in a 75:25 ratio) kindly provided by Bournonville Pharma (Braine l'Alleud, Belgium) was used.
Construction, Expression, and Purification of PAI-1 and PAI-1 VariantsPAI-1-P6 (Val Pro at P6) and PAI-1-P10
(Ser
Pro at P10) were constructed as described before (36).
PAI-1-P18 (Asn
Pro at P18) was constructed in a similar way using
the synthetic oligonucleotide 5
-CACCGTGCCACTCTCCGGGACCTCGATCTT-3
to obtain the desired
mutation. Expression and purification of wtPAI-1 and variants was
performed in Escherichia coli as described earlier (37).
PAI-1 activity was determined using the method described by Verheijen (38) by adding a fixed amount of t-PA or u-PA to the PAI-1 containing samples. t-PA was calibrated versus the international reference preparation for t-PA (NIBSC 86/670). All PAI-1 activity data are expressed as percentage of the theoretical maximal activity, i.e. ~745,000 units/mg (t-PA inhibitory units) and 120,000 units/mg (u-PA inhibitory units) calculated on the basis of a specific activity for t-PA of 500,000 units/mg and for u-PA of 100,000 units/mg and molecular masses of 67, 54, and 45 kDa for t-PA, u-PA, and PAI-1, respectively.
Determination of the Conformational Distribution of PAI-1 and PAI-1 VariantsSamples of wtPAI-1 and PAI-1 mutants were incubated with a 2-fold molar excess of t-PA or u-PA. PAI-1 samples were diluted with phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4) to a concentration of 0.25 mg/ml. T-PA and u-PA were diluted with PBS to a concentration of 0.7 mg/ml and 0.5 mg/ml, respectively. Equal volumes of t-PA (or u-PA) and PAI-1 were mixed and incubated at 37 °C for 2 h. The reaction was terminated by adding SDS (final concentration of 1%) and heating during 30 s at 100 °C. Reaction products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent densitometric scanning with the ImagemasterTM (Pharmacia, Uppsala, Sweden).
Kinetic Analysis of the Inhibition of t-PA and u-PA by PAI-1 and PAI-1 VariantsThe rate of inhibition of t-PA (predominantly single chain) and u-PA (consisting of high and low molecar weight tcu-PA in a 75:25 ratio) by PAI-1 and PAI-1 variants was determined as follows. u-PA and t-PA were used at concentrations of 0.2 nM, and PAI-1 and PAI-1 variants were used at concentrations of 0.8, 1.2, or 1.6 nM (pseudo first-order conditions). Residual u-PA and t-PA activity was quantified (38) after blocking the reaction by addition of a 200-fold molar excess of the monoclonal antibodies MA-8H9D4 (for u-PA reaction) and MA-56A7C10 (for t-PA reaction). Both antibodies inhibit PAI-1 activity very rapidly and completely, without interference on the u-PA or the t-PA activity assay.
Inactivation of PAI-1 SamplesPAI-1 samples (except PAI-1-P18) were inactivated by diluting the samples to a final concentration of 150-190 µg/ml using the appropiate diluent (containing Tween 80 and Na2HPO4) to obtain a buffered solution with 45 mM phosphate, 70 mM NaCl, and 0.01% Tween 80, pH 7.4, followed by incubation at 37 °C for 24 h. Under these conditions, PAI-1-P18 exhibited extensive precipitation. Inactivation of PAI-1-P18 was, therefore, carried out at a concentration of 125 µg/ml in PBS containing 0.01% Tween 80 and 2 mM glutathione and incubation at 4 °C for 60 h.
The half-lives for inactivation were calculated with the program
GraphPad PrismTM using "one-phase exponential decay"
according to the equation Y = Span*eK*X + Plateau. The half-life of the decay is then equal to 0.693/K.
The statistical significance of differences was evaluated using Student's t-test; p values >0.05 were considered non-significant.
wtPAI-1 revealed similar activities toward t-PA (68 ± 10% (mean ± S.D., n = 6)) as toward u-PA
(68 ± 6.0%) (Fig. 1). PAI-1-P6(Val Pro) was
significantly less active (p < 0.0001) toward u-PA
(3.9 ± 2.2%) than toward t-PA (45 ± 10%)
(n = 6). In contrast, PAI-1-P10(Ser
Pro) and
PAI-1-P18(Asn
Pro) were significantly less active
(p < 0.01) toward t-PA (5.9 ± 0.33 and 0.20 ± 0.04%, respectively) than toward u-PA (19 ± 4.9 and 41 ± 10%, respectively, n = 3).
Reaction Products Formed after Incubation of wtPAI-1 and PAI-1 Mutants With t-PA and u-PA at 37 °C
Incubation of wtPAI-1 at
37 °C with a 2-fold molar excess of t-PA revealed the formation of
t-PA·PAI-1 complexes (49 ± 6%, mean ± S.D.,
n = 4), small amounts of cleaved derivative (13 ± 1%), and residual non-reactive material (38 ± 5%) (Fig.
2A and Table I). Under these
conditions, the amount of complexes formed with
PAI-1-P6(Val Pro), PAI-1-P10(Ser
Pro), and
PAI-1-P18(Asn
Pro) were compatible with their respective
activity data. In addition, for all these mutants, a decreased
inhibitory activity was associated with an increased (3-10-fold,
p < 0.0001 versus wtPAI-1) substrate behavior. Surprisingly, in contrast to wtPAI-1,
PAI-1-P6(Val
Pro), and PAI-1-P10(Ser
Pro), virtually no
latency was observed for PAI-1-P18(Asn
Pro), demonstrating that
the absence of any inhibitory activity of this mutant toward t-PA was
totally attributed to substrate behavior. (Fig. 2A, Table
I)
|
The amount of complexes formed after incubation of PAI-1-P10 with a
2-fold molar excess of u-PA (Fig. 2B, Table I) was
compatible with the u-PA inhibitory activity data. Comparison of the
amount of complexes formed in the presence of either t-PA or u-PA
further confirmed the proteinase specificity (i.e. 4-fold higher
activity toward u-PA than toward t-PA). In agreement with the activity data, no detectable complexes were formed between
PAI-1-P18(Asn Pro) and t-PA, whereas up to 40% complex formation
could be observed in the presence of u-PA. Complex formation of
PAI-1-P6(Val
Pro) with t-PA was 10-15-fold higher compared with
complex formation with u-PA. Importantly, from the data in Table I and
Fig. 2, it can be deduced that, for each mutant, the differences in
proteinase specificity (with respect to inhibitory activity) were
mainly attributed to a concomitant inversed difference in substrate
behavior. In all cases, no proteinase-dependent differences
in non-reactive material could be observed.
As expected,
inactivation of wtPAI-1 occurred with a half-life of 60 ± 20 min
(mean ± S.D., n = 6) and was associated with a
concomitant increase in latent PAI-1. The amount of substrate remained
unchanged. (Fig. 3 and Table II)
|
Inactivation of PAI-1-P6(Val Pro) occurred with a similar
half-life (64 ± 18 min) and a conversion of the active form to the latent form. In contrast to what was observed for wtPAI-1, the
decrease in inhibitory activity was partly associated with a decrease
in substrate behavior. However, as observed for wtPAI-1, a significant
amount (~15%) remained as stable substrate. From the
time-dependent changes in inhibitory and substrate forms of PAI-1-P6(Val
Pro), it could be calculated that the conversion of
this unstable substrate form of PAI-1-P6(Val
Pro) to latent PAI-1-P6(Val
Pro) occurred with a different half-life than the conversion of inhibitory PAI-1-P6(Val
Pro) to latent
PAI-1-P6(Val
Pro) (t1/2 = 118 ± 34 min
versus 64 ± 18 min, p < 0.05). In contrast to wtPAI-1 and PAI-1-P6(Val
Pro), loss of inhibitory activity of
PAI-1-P10(Ser
Pro) or PAI-1-P18(Asn
Pro) was almost
exclusively associated with an increase in substrate behavior (Fig. 3
and Table II).
The second-order rate constants of inhibition of
t-PA and u-PA by PAI-1 and PAI-1 variants are shown in Table
III. The t-PA specific mutant PAI-1-P6(Val Pro) as
well as the u-PA specific mutant PAI-1-P10(Ser
Pro) inhibit t-PA
and u-PA with similar rate constants of ~107
M
1 s
1, showing that the
kinetics of inhibition are not related with the target specificity of
the PAI-1 variants.
|
Serpins form a large group of proteins with important regulatory
properties toward a wide variety of serine proteinases. Naturally occurring deficiencies and/or mutants with aberrant functional properties often give rise to severe pathologies. Even though from a
structural point of view serpins are very similar, functionally they
can be divided into two groups: (a) inhibitory serpins
forming stable covalent complexes with their target proteinases, and
(b) non-inhibitory serpins that are susceptible to cleavage
upon reaction with their putative target proteinase. Today, a wide
variety of mutants has been characterized (39) with the aim to
delineate domains that are important for the structure-function
relationship in serpins. The reactive site loop (P16 to P10) is
considered as one of the most important domains regarding the
interaction between the serpin and its target proteinase. The
importance of the P3 to P1
region for target specificity of serpins
has been reported extensively before (19-30, 40). While a basic
residue at the P1 position of PAI-1 is required for inhibition of u-PA (25), the presence of neutral or hydrophobic amino acids at this
position does not affect t-PA inhibition properties (27). Inhibition of
u-PA by PAI-1 still occurs when P1
is substituted with any amino acid
except proline (25). Alteration of the residues at postion P2 and P3 of
PAI-1 revealed the possibility for target specific inhibition, with
t-PA being more tolerant than u-PA for structural diversity at the P2
and P3 positions (30). The present study describes the functional
effects of PAI-1 proline mutations that are remote of the P3-P1
region. The current study reveals that substitution of the residue at
position P6 by a proline (PAI-1-P6(Val
Pro)) results in a t-PA
target specificity, whereas substitution by a proline at position P10
or P18 yields an increased u-PA target specificity
(PAI-1-P10(Ser
Pro)) or even a PAI-1 variant with exclusively
u-PA inhibitory properties (PAI-1-P18(Asn
Pro)). The
target-specific mutants inhibit t-PA and u-PA with similar rate
constants of ~107 M
1
s
1, illustrating that the kinetics of inhibition do not
contribute to this target specificity. To the best of our knowledge,
this is the first report of modulation of the proteinase specificity of
serpins by mutations remote of the P3-P1
region. Analysis of the
reaction products formed between the mutants studied and t-PA or u-PA
also revealed significant changes in the inhibitor versus
substrate ratio compared with wtPAI-1. The behavior of a serpin as an
inhibitor or as a substrate has been reported to be influenced (in
part) by external reaction conditions such as temperature, ionic
strength, presence of cofactors, etc. (32-34, 41). These effects are
generally explained through the existence of a branched pathway as
shown in Fig. 4. It should be realized that in those
studies, even though large conformational changes could be excluded,
minor conformational changes in the reactive site loop could have
influenced this reaction behavior. In addition, recent studies have
demonstrated that decreased kinetics of insertion (through mutations at
position P14) (42, 43) are associated with an increased substrate
behavior. This might also explain the substrate-type properties of
various serpin mutants in which bulky or charged residues have been
introduced in the reactive site loop. (36, 37, 43-47). However, for
the serpin PAI-1, it had also been shown that, in the wild-type
molecule, different initial conformations exist, which predetermine the
reaction pathway (7). The mutants described in the current study
further substantiate these findings and provide evidence for the
existence of two different substrate conformations. Indeed,
inactivation of PAI-1-P6(Val
Pro) results in a disappearance of
part of the substrate-reacting population with a different half-life
compared with the active form. Based on the branched pathway mechanism,
conversion of the one (and only) reactive form would have resulted in a
perfectly parallel decrease of substrate and inhibitor reaction.
Importantly, even though the substrate behavior of
PAI-1-P6(Val
Pro) decreases during incubation at 37 °C, a
significant portion appears to be stable (as observed previously with
wtPAI-1), clearly demonstrating the existence of both a labile and a
stable substrate conformation of PAI-1-P6(Val
Pro). The existence
of an initial conformation pathway responsible for the formation of
cleaved serpin (PAI-1), independent of the possible phenomenon of
"branching," is further proven by the observation that inactivation
of PAI-1-P10(Ser
Pro) and PAI-1-P18(Asn
Pro) results in an
increased substrate behavior demonstrating that the active, inhibitory
form is converted to a distinct, substrate form. The proposed
conformational transitions are further substantiated by the observation
of changes in tryptophan fluorescence characteristics upon inactivation
of these mutants (data not shown).
Taken together, these observations demonstrate that, in contrast to
what is generally accepted, more conformations and conformational changes do occur. We propose an alternative reaction scheme of the
reaction between PAI-1 and its target proteinase (Fig.
5). It should be emphasized that this scheme might be
extrapolated to other serpin/proteinase interactions whereby cofactors,
changes in pH, etc. induce subtle conformational changes in the serpin, resulting in a conversion between an active (Ia)
and a substrate (Is) form.
PAI-1-P6(Val Pro) revealed the existence of both a labile and a
stable substrate form (Is), which can be cleaved (Ic) by target proteinases, and an active form
(Ia), which can form an inactive complex
(EIa) with the target proteinase or that
converts spontaneously into the latent form
(Il). In contrast, the active form
(Ia) of PAI-1-P10(Ser
Pro) and
PAI-1-P18(Asn
Pro) converts spontaneously to the substrate form
(Is). It has to be emphasized that, in our
current study, all reactions carried out before and after pretreatment
were performed under identical conditions and, consequently, exclude
the possibility that any observed "shift" in formed reaction
products (i.e. changes in active versus substrate ratio;
cf. Tables I and II) would have arisen from changes in the
k4/k3 ratio in the
branched reaction pathway. On the other hand, it should also be
realized that our data obviously do not allow us to exclude the
possible existence of a branched pathway under certain conditions with
certain serpins. Therefore, this possibility is still included in our
alternative reaction scheme. However, in this respect, it is important
to note that the hypothesis of a branched pathway responsible for the
formation of two possible reaction products (complexed or cleaved),
first proposed in 1991 (32) and applied in later studies, was based on
indirect evidence.
In conclusion, our study reveals (a) three target specific
PAI-1 variants in which in each variant only one residue, located outside the P3 to P1 region, is replaced by a proline; (b)
the existence of two different substrate populations in the
PAI-1-P6(Val
Pro) mutant; and (c) a conformational
transition from the active form into a substrate form for
PAI-1-P10(Ser
Pro) and PAI-1-P18(Asn
Pro). These
observations lead to the conclusion that the reaction pathway between
PAI-1 and its target proteinase is much more complex than originally
anticipated based on the previously proposed branched pathway for
serpins.