(Received for publication, July 6, 1995; and in revised form, September 6, 1995 )
From the
Eighteen mutants of recombinant staphylokinase (SakSTAR) in
which clusters of two or three charged residues were converted to
alanine (``clustered charge-to-alanine scan'') were
characterized. Fifteen of these mutants had specific
plasminogen-activating activities of >20% of that of wild-type
SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A (SakSTAR13),
SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakSTAR67) had
specific activities of 3%. SakSTAR13 had an intact affinity for
plasminogen and a normal rate of active site exposure in equimolar
mixtures with plasminogen. The plasmin-SakSTAR13 complex had a 14-fold
reduced catalytic efficiency for plasminogen activation but was 5-fold
more efficient for conversion of plasminogen-SakSTAR13 to
plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold
reduced affinity for plasminogen and a markedly reduced active site
exposure; their complexes with plasmin had a more than 20-fold reduced
catalytic efficiency toward plasminogen. Thus, plasminogen activation
by catalytic amounts of SakSTAR is dependent on complex formation
between plasmin(ogen) and SakSTAR, which is deficient with SakSTAR48
and SakSTAR67, but also on the induction of a functional active site
configuration in the plasmin-SakSTAR complex, which is deficient with
all three mutants. These findings support a mechanism for the
activation of plasminogen by SakSTAR involving formation of an
equimolar complex of SakSTAR with traces of plasmin, which converts
plasminogen to plasmin and, more rapidly, inactive plasminogen-SakSTAR
to plasmin-SakSTAR.
Staphylokinase, produced by certain strains of Staphylococcus aureus, activates the human plasma fibrinolytic
system indirectly(1, 2) . It forms a 1:1
stoichiometric complex with plasmin, which activates
plasminogen(3, 4) . Recombinant staphylokinase
(SakSTAR) ()was shown to induce fibrin-specific clot lysis
in a human plasma milieu in vitro(5, 6) , in
animal models of thrombosis(7) , and in patients with acute
myocardial infarction(8, 9) . This fibrin specificity
has been explained by specific molecular interactions between SakSTAR,
plasmin(ogen),
-antiplasmin, and fibrin. The
plasmin-SakSTAR complex is rapidly inhibited by
-antiplasmin(6, 10) , resulting in
dissociation of active SakSTAR from the complex and recycling to other
plasminogen molecules(11) . The inhibition rate of the complex
by
-antiplasmin (second-order rate constant
10
M
s
)
is, however, reduced more than 100-fold in the presence of
fibrin(12) . Thus, in the absence of fibrin,
-antiplasmin inhibits the activation of plasminogen by
SakSTAR by preventing generation of active plasmin-SakSTAR complex.
Fibrin stimulates plasminogen activation by SakSTAR via a mechanism
involving the lysine-binding sites of plasminogen, probably by
facilitating the generation of plasmin-SakSTAR complex and by delaying
its inhibition at the surface of a clot(13) .
SakSTAR consists of 136 amino acids, of which 45 are charged, in a single polypeptide chain without disulfide bridges; it consists of two widely separated domains of similar size linked by a flexible helix (14) . To investigate the structure-function relationships of SakSTAR, which determine its interaction with plasminogen in more detail, charged amino acids were mutagenized to alanine in clusters of 2 or 3 residues (clustered charge to alanine scan)(15) . Replacement of clusters of charged amino acids in the SakSTAR regions comprising amino acids 11-14, 46-50, and 65-69 resulted in a markedly reduced plasminogen activating capacity. Investigation of the mechanism of plasminogen activation by wild-type and mutant SakSTAR moieties supported a model involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts plasminogen to plasmin and, more rapidly, inactive plasminogen-SakSTAR to active plasmin-SakSTAR.
T4 DNA ligase, Klenow Fragment of E. coli DNA polymerase I and alkaline phosphatase were obtained from Boehringer Mannheim. The oligonucleotide-directed mutagenesis system and the pMa/c plasmids were kindly provided by Corvas (Ghent)(21) . The expression vector pMEX602SAKB was constructed as described previously(9) . M13KO7 helper phage was purchased from Promega (Leiden). Luria Broth growth medium was purchased from Life Technology (Merelbeke).
Protein concentrations were determined
according to Bradford(23) . SDS-PAGE was performed with the
Phast System(TM) (Pharmacia) using 10-15% gradient gels and
Coomassie Brilliant Blue staining. Reduction of the samples was
performed by heating at 100 °C for 3 min in the presence of 1% SDS
and 1% dithioerythritol. SakSTAR-related antigen was determined with a
specific enzyme-linked immunosorbent assay(24) , calibrated
with the respective purified SakSTAR moieties. NH-terminal
amino acid sequence analysis was performed on an Applied Biosystems
477A protein sequencer with identification of amino acids by high
performance liquid chromatography.
The specific plasminogen-activating activities of SakSTAR moieties were determined with a plasminogen-coupled chromogenic substrate assay, and were expressed in home units by comparison with an in-house standard of natural staphylokinase, which was assigned an activity of 100,000 home units/mg of protein as determined by amino acid analysis(25) .
Association rate constants (k), dissociation
rate constants (k
) and affinity constants (K
= k
/k
) for the interactions
between different staphylokinase moieties and rPlg-Ala
or
VFK-plasmin were determined by real time biospecific interaction
analysis using the BIAcore instrument (Pharmacia) as described
elsewhere(26) . The kinetic parameters for the hydrolysis of
S-2403 (final concentration, 0.05-1 mM) by
plasmin-SakSTAR complexes (final concentration, 5 and 10 nM)
were determined by Lineweaver-Burk analysis. Inhibition of
plasmin-SakSTAR complexes (final concentration, 5 nM) by
-antiplasmin (final concentration, 25 nM) was
monitored continuously in the presence of S-2403 (final concentration,
0.3 mM), and the apparent second-order inhibition rate
constants were calculated as described previously(6) .
Figure 1: Schematic representation of the primary structure of SakSTAR mutants. The clusters of charged amino acids that were mutagenized to Ala are identified by their mean residue number.
In addition, 9 mutants (Table 1) were constructed at the Institute for Molecular
Biotechnology in Jena, Germany. Mutants SakSTAR34, SakSTAR58,
SakSTAR94, SakSTAR97, SakSTAR100a, and SakSTAR109 were generated by
cloning of couples of tailor-made sakSTAR encoding fragments generated
by polymerase chain reaction in pMEX6. These fragments were
produced using primers derived from the ``left'' and
``right'' flanking regions of the pMEX6 vector outside the sak gene (l-primer, CGTATAATGTGTGGAATTGTGAGCGG and r-primer,
GGCTGAAAATCTTCTCTCATCCGCC) and ``upward'' or
``downward'' polymerase chain reaction primers in the sak gene (u-primer and d-primer) (Table 1). The l- and u-primers
were used to generate the NH-terminal SakSTAR-encoding
fragment (N-fragment). The d-primer carrying the coding sequence for
the charge to Ala mutation and the r-primer were used to generate the
modified COOH-terminal SakSTAR-encoding fragment. The fragments were
trimmed by appropriate restriction enzymes and cloned into the EcoRI-HindIII-digested expression vector pMEX6.
To construct mutants SakSTAR118 and SakSTAR120, the
NH-terminal SakSTAR encoding fragments were generated by
polymerase chain reaction in pMEX6 using the l- and u-primers listed in Table 1. The COOH-terminal SakSTAR encoding fragments were
generated by annealing the complementary oligonucleotides (l-1 and
l-2), encoding the charge to Ala modification and extending upstream to
a protruding end compatible with the XmaI site induced in the
codon for Pro
with the u-primer and downstream to the StyI site of SakSTAR. The EcoRI-XmaI digested
NH
-terminal SakSTAR-encoding fragment and the COOH-terminal
SakSTAR-encoding oligonucleotides were ligated in EcoRI-StyI-digested pMEX602SAKB. Mutant
SakSTAR135 was generated by digestion of pMEX602SAKB with StyI and PstI, and ligation of the annealed
complementary oligonucleotides l-1 and l-2 (Table 1), with
protruding StyI and PstI compatible ends, into the
linearized vector.
The stability of equimolar plasmin-SakSTAR complexes was analyzed by incubation of 0.5 ml of a 3 µM solution in 0.1 M phosphate buffer, pH 7.4, for 1 h at 4 °C with 75 mg of suction-dried lysine-Sepharose gel. After excessive washing, the gel was eluted with 50 mM 6-aminohexanoic acid in 0.1 M phosphate buffer, pH 7.4, and the distribution of SakSTAR between the unbound fraction and the eluate was monitored by enzyme-linked immunosorbent assay and SDS-PAGE.
For
kinetic analysis of plasminogen activation, equimolar plasmin-SakSTAR
complexes (final concentration, 2 µM) were prepared by
incubation of plasminogen with the SakSTAR mutants at 37 °C for
5-30 min in 0.1 M phosphate buffer, pH 7.4, containing
25% glycerol; the mixtures were then stored on ice. Plasmin-SakSTAR
complex (final concentration, 10-20 nM) was incubated
with plasminogen (1-33 µM) at 37 °C in 0.1 M phosphate buffer, pH 7.4, and generated plasmin was measured at
different time intervals (0-4 min) with S-2403. Initial
activation rates were obtained from linear plots of the concentration
of generated plasmin versus time. Kinetic parameters (K and k
) were determined
from Lineweaver-Burk plots by linear regression analysis. With
wild-type SakSTAR, SakSTAR13, SakSTAR48, and SakSTAR67, a similar
kinetic analysis was performed with low M
plasminogen.
In separate experiments, the rate of the
conversion of single chain rPlg-Ala to two chain
rPli-Ala
either free or in an equimolar complex with
SakSTAR13 (final concentration, 3 µM) by addition of
preformed plasmin-SakSTAR13 complex (final concentration, 20
nM) was monitored as a function of time. Therefore, samples
were removed from the incubation mixtures at different time points
(0-35 min) and subjected to SDS-PAGE under reducing conditions
followed by quantitation of generated rPli-Ala
by
densitometric scanning.
All purified proteins were homogeneous as shown by SDS-PAGE using 10-15% gradient gels with or without reduction, and migrated with an apparent molecular mass of 18 kDa (not shown).
Binding of SakSTAR13
or SakSTAR67 to VFK-plasmin occurred with a 3.5- or a 2.3-fold lower
affinity than binding of wild-type SakSTAR. Binding of SakSTAR13 was
characterized by (mean ± S.D.; n = 3) k = 4.4 ± 0.12
10
M
s
and k
= 19 ± 0.9
10
s
, yielding K
= 2.4 ± 0.2
10
M
, as compared with k
= 21 ± 4.3
10
M
s
and k
= 58 ± 1.2
10
s
, resulting in K
= 3.6 ± 0.67
10
M
for SakSTAR67. For
wild-type SakSTAR, k
= 3.4 ± 0.42
10
M
s
, k
= 4.3 ± 1.2
10
s
, and K
= 8.4 ± 1.6
10
M
. Binding of SakSTAR48 to VFK-plasmin was
characterized by a 9-fold lower K
(0.93 ±
0.37
10
M
) as compared
with binding of wild-type SakSTAR, mainly as a result of a higher k
value (130 ± 4.0
10
s
) with a comparable k
value (12 ± 4.4
10
M
s
).
Figure 2:
Time
course of active site generation in equimolar mixtures of plasminogen
with wild-type SakSTAR (), SakSTAR13 (
), SakSTAR48
(
), and SakSTAR67 (
). The generation of plasmin-SakSTAR
complex in equimolar mixtures of plasminogen and SakSTAR (final
concentration, 3.6 µM), as quantitated by titration with
NPGB at different time points after mixing, is shown as a function of
time. The data represent one representative experiment. The inset shows SDS-PAGE (10-15% gradient gels) under reducing
conditions of samples taken at time 0 (lane 1) and at 15 min (lane 2) for wild-type SakSTAR, at 8 min for SakSTAR13 (lane 3), at 30 min for SakSTAR48 (lane 4), and at 50
min for SakSTAR67 (lane 5). Lane 6 represents a
protein calibration mixture consisting of phosphorylase b (97 kDa),
albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa),
trypsin inhibitor (20.1 kDa), and
-lactalbumin (14.4 kDa). Plg, plasminogen; PliA, plasmin A-chain; PliB, plasmin B-chain.
Figure 3:
Activation rate of plasminogen (final
concentration, 1 µM) by wild-type SakSTAR (),
SakSTAR13 (
), SakSTAR48 (
), and SakSTAR67 (
) (final
concentration, 5 nM each). The concentration of generated
plasmin was quantitated as a function of time with S-2403. The data
represent mean ± S.D. of three experiments. The inset shows SDS-PAGE (10-15% gradient gels) under reducing
conditions of samples taken at time 0 (lane 1) and 20 min (lane 2) for wild-type SakSTAR and at 50 min for SakSTAR13 (lane 3), SakSTAR48 (lane 4) and SakSTAR67 (lane
5). Lane 6 represents a protein calibration mixture, as
in Fig. 2. Plg, plasminogen; PliA, plasmin
A-chain; PliB, plasmin B-chain.
Activation of plasminogen by preformed plasmin-SakSTAR complexes
obeyed Michaelis-Menten kinetics, as revealed by linear
double-reciprocal plots of the initial activation rate versus the plasminogen concentration (data not shown). The K ranged between 5 and 17 µM for the
mutants, including SakSTAR13 (K
= 11
µM), SakSTAR48 (K
= 7
µM), and SakSTAR67 (K
= 5
µM), as compared with 12 µM for wild-type
SakSTAR. In contrast the k
values of SakSTAR13 (k
= 0.11 s
),
SakSTAR48 (k
= 0.03
s
), and SakSTAR67 (k
=
0.03 s
) were significantly lower than that of
wild-type SakSTAR (k
= 1.7
s
) or of the other mutants (k
ranging between 0.9 and 2.3 s
). As a result,
the catalytic efficiencies (k
/K
) for plasminogen
activation of complexes of plasmin with SakSTAR13 (0.01
µM
s
),
SakSTAR48 (0.005
µM
s
) and
SakSTAR67 (0.006
µM
s
) were
14-28-fold lower than that of wild-type SakSTAR (0.14
µM
s
), whereas k
/K
of the other mutants
was comparable with wild-type SakSTAR (0.11-0.24
µM
s
). The
catalytic efficiencies of the mutant plasmin-SakSTAR complexes for
plasminogen, relative to wild-type SakSTAR, are represented in Fig. 4.
Figure 4: Catalytic efficiency of preformed plasmin-SakSTAR complexes for the activation of plasminogen. The values obtained with the mutants (identified by the mean residue number) are expressed as a ratio with wild-type SakSTAR.
Conversion of rPlg-Ala (final
concentration, 3 µM) to rPli-Ala
by
catalytic amounts (20 nM) of preformed plasmin-SakSTAR13
complex occurred with an initial rate of 0.37
nM
s
and reached a maximum of 270
nM after 12 min. In contrast, conversion to rPli-Ala
in the rPlg-Ala
SakSTAR13 complex was much
more efficient, occurring with an initial rate of 1.4
nM
s
to reach a maximum of 1,700
nM after 20 min. Plasmin did not significiantly convert
rPlg-Ala
to rPli-Ala
(Fig. 5).
Figure 5:
Conversion of rPlg-Ala (final concentration, 3 µM) either free (
) or
in the rPlg-Ala
-SakSTAR13 complex (
) to
rPli-Ala
by plasmin-SakSTAR13 complex (final
concentration 20 nM) monitored as a function of time. A
control experiment with addition of plasmin to rPlg-Ala
is also shown (
). The data were obtained by densitometric
scanning of SDS-PAGE (10-15% gradient gels) under reducing
conditions, as illustrated in the inset for samples taken at
16 min from the mixture of rPlg-Ala
and plasmin-SakSTAR13 (lane 1), the mixture of rPlg-Ala
-SakSTAR13
complex, and plasmin-SakSTAR13 (lane 2), or the mixture of
rPlg-Ala
and plasmin (lane 3). Lane 4 represent a protein calibration mixture, as in Fig. 2. Plg, rPlg-Ala
; PliA, A-chain of
rPli-Ala
; PliB, B-chain of
rPli-Ala
.
Activation of low M plasminogen by preformed
plasmin-SakSTAR complexes also obeyed Michaelis-Menten kinetics. The
kinetic parameters of the plasmin-SakSTAR13 complex (K
= 4.0 µM and k
= 0.08 s
) and of the wild-type
plasmin-SakSTAR complex (K
= 5.3 µM and k
= 1.6 s
)
for activation of low M
plasminogen were
comparable with those for the activation of intact plasminogen, as
reported above. In contrast, activation of low M
-plasminogen by the plasmin-SakSTAR48 and
plasmin-SakSTAR67 complexes was characterized by higher K
values (25 µM and
60
µM, respectively) and comparable or higher k
values (0.2 s
and
0.2
s
) than those for activation of intact plasminogen.
Thus, the catalytic efficiencies for activation of low M
plasminogen were about 2-fold higher for wild-type
plasmin-SakSTAR, plasmin-SakSTAR13, and plasmin-SakSTAR48 and about
2-fold lower for plasmin-SakSTAR67, as compared with those for
activation of intact plasminogen.
The activity of preformed
plasmin-SakSTAR complexes toward S-2403, as determined by
Lineweaver-Burk analysis, was comparable for wild-type SakSTAR and all
the mutants. K values for the mutants ranged
between 120 and 390 µM, as compared with 240 µM for wild-type SakSTAR, and k
values ranged
between 50 and 100 s
, as compared with 63
s
for wild-type SakSTAR, yielding catalytic
efficiencies of 0.16-0.62
µM
s
for the
mutants, as compared with 0.26
µM
s
for
wild-type SakSTAR (data not shown).
In the present study, structure-function relationships in SakSTAR, which determine its interaction with plasminogen, were investigated by construction of mutants in which clusters of two or three charged amino acids were mutagenized to alanine. A clustered charge to alanine scan approach has previously been used to study structure-function relationships in other plasminogen activators, e.g. tissue-type plasminogen activator (30) and urokinase-type plasminogen activator(31) .
Twenty mutants
were designed, two of which could not be obtained because of a very low
expression level or of protein instability (SakSTAR100a with E99A and
E100A substitution, and SakSTAR100b with E99A, E100A, and K102A
substitution). Out of 18 mutants that were studied in detail, only
three (SakSTAR13, SakSTAR48, and SakSTAR67) were markedly different
from wild-type SakSTAR with respect to their interaction with
plasmin(ogen). This was revealed by a specific activity of 3% of
that of wild-type, the absence of measurable plasminogen activation by
catalytic amounts of the mutants, and a 10-20-fold lower
catalytic efficiency of preformed complexes with plasmin for the
activation of plasminogen. Furthermore, two of these mutants (SakSTAR48
and SakSTAR67) had a 10-20-fold reduced affinity for binding to
plasminogen as compared with wild-type SakSTAR, and one mutant
(SakSTAR67) did not induce active site exposure in equimolar mixtures
with plasminogen.
The finding that mutations in the regions of amino acids 11-14, 46-50, and 65-69 affected the interaction with plasmin(ogen) in different aspects suggested that mutants SakSTAR13, SakSTAR48, and SakSTAR67 may be useful to study different steps in the mechanism of the interaction of SakSTAR with plasminogen. The following kinetic model has been proposed for the activation of plasminogen by SakSTAR (4) .
Plasminogen (P) and SakSTAR (S) produce an
inactive 1:1 stoichiometric complex (PS), which
does not activate plasminogen, as demonstrated by titration with NPGB.
The activation reaction appears to be initiated by trace amounts of
contaminating plasmin (p), which generates p
S, which converts P to p and P
S to p
S. According to
this model, SakSTAR48 and SakSTAR67, with a 10-20-fold reduced
affinity for binding to plasminogen, have an impaired formation of p
S and P
S complexes,
and thus of subsequent conversion of P to p and of P
S to p
S. This is
confirmed by our findings that no active site exposure occurred in
equimolar mixtures of plasminogen with SakSTAR67, whereas with
SakSTAR48, active site exposure was markedly delayed as compared with
wild-type SakSTAR (Fig. 2). With SakSTAR13, which had a normal
affinity for plasminogen, formation of p
S and
of P
S was apparently normal, as was conversion
of P
S to p
S, but
conversion of P to p was markedly impaired.
Preformed complexes of SakSTAR13, SakSTAR48, or SakSTAR67 with
plasmin (pS) had a comparable affinity for the
plasminogen substrate as wild-type SakSTAR (K
values for plasminogen activation of 5-12 µM)
but a much lower catalytic rate constant for plasminogen activation (k
of 0.03-0.11 s
, as
compared with 1.7 s
), resulting in 14-28-fold
lower catalytic efficiencies. These findings explain why catalytic
amounts of SakSTAR13 (normal formation of p
S complex, but low enzymatic activity) or of SakSTAR48 and SakSTAR67
(delayed p
S formation) do not induce measurable
plasminogen activation.
The finding of rapid and quantitative
formation of pS in equimolar mixtures of
plasminogen and SakSTAR13, despite the low catalytic efficiency of the p
S complex of this mutant for activation of P, can be explained by the observation that the conversion of P
S to p
S by catalytic
amounts of preformed p
S is much more efficient
than conversion of P to p. The finding that the rate
of plasminogen activation by catalytic amounts of SakSTAR48 and
SakSTAR67 is enhanced by the addition of traces of plasmin, from
undetectable to a rate similar to that observed with their preformed
complexes with plasmin, suggests that, under these conditions, active p
S is formed, not as a result of conversion of P
S to p
S but of direct
formation of the p
S complex between added
plasmin and SakSTAR. However, the rate of plasmin generation is still
much lower than that observed in control experiments with wild-type
SakSTAR because of the low catalytic efficiency of the mutant p
S complexes. The affinities of SakSTAR48 and
SakSTAR67 for VFK-plasmin are 10-20-fold higher than for
plasminogen, which is confirmed by the observation that preformed
complexes of plasmin with SakSTAR13, SakSTAR48, and SakSTAR67 are
stable following adsorption onto lysine-Sepharose.
Taken together,
these data further demonstrate that the running concentration of active pS determines the rate of conversion of P to p and of P
S to p
S. In the presence of excess p inhibitor (e.g. NPGB), P
S and P cannot be converted because of lack of active p
S. In the absence of inhibitor, the reaction
most likely is initiated by trace amounts of p that form p
S that converts both P to p and, more rapidly, P
S to p
S. Indeed, a contamination of P with
30 ppm of p is sufficient to explain the kinetics of
activation of P by SakSTAR (4) .
In summary, mutagenesis in the regions 11-14, 46-50, or 65-69 of SakSTAR resulted in impairment of its interaction with plasmin(ogen). These mutants have allowed to identify different steps of the interaction between plasminogen and SakSTAR in functional isolation.