(Received for publication, September 6, 1995)
From the
The interaction of streptokinase (SK) with human plasminogen
(HPlg) was investigated using truncated SK peptides prepared by gene
cloning techniques. SK(16-414) and SK(16-378) could
activate HPlg as efficiently as the authentic SK. SK(60-414),
which had been preincubatd with SK(1-59), could also activate
HPlg. SK(91-414), SK(127-414), and SK(158-414), at a
concentration of one-tenth of HPlg, all failed to activate HPlg.
However, the truncated SK peptides in complexes with equimolar HPlg
could form amidolytically active virgin enzymes that slowly converted
to human plasmin (HPlm) after a lag period of 15 min. SK(16-316)
could not activate HPlg. No virgin enzyme was detected when
SK(16-316) was incubated with equimolar HPlg, but the HPlg in the
complex was modified to HPlm after reaction for 20 min.
SK(220-414) and SK(16-251) had no ability to transform HPlg
to virgin enzyme or to HPlm in equimolar complex with HPlg, although
they could bind to HPlg. The functions of five regions in the SK
molecule (a, Ile-Lys
; b,
Ser
-Asn
; c,
Val
-Arg
; d,
Tyr
-Ala
; e,
Ser
-Ala
) in interaction with HPlg are
deduced. Region a is important in stabilizing the conformation of the
SK molecule, and region b is essential for HPlg activation. Region c is
required for induction of the conformational changes of HPlg to virgin
enzyme. Regions c and d are required for the conversion of HPlg to HPlm
in the HPlg
SK equimolar complex. Coordination of regions c, d,
and e of SK is essential for a virgin enzyme formation, and
coordination of regions b, c, d and e is required for an effective
SK-type HPlg activator.
Streptokinase (SK) ()is a single-peptide secretory
protein of 414 (1) or 415 (2) amino acid residues
produced by various strains of
-hemolytic Streptococcus(1, 2, 3) . The SK and
human plasminogen (HPlg) can form an equimolar activator complex that
catalyzes the conversion of plasminogens (Plgs) to plasmins (Plms) of
different mammalian
species(4, 5, 6, 7, 8, 9, 10, 11) .
Plm is a potent protease that in turn catalyzes the hydrolysis of
fibrin, which causes the dissolution of blood clots. SK, therefore, is
used as a thrombolytic agent clinically to relieve thromboembolic
blockages in blood vessels, such as acute myocardial infarction.
Studies of the activation of HPlg by SK suggest that more than one
intermediate step is involved in the entire process. In the first step,
a 1:1 stoichiometric complex of HPlg and SK is formed, and the
conformation of the catalytic domain of HPlg is altered to expose its
enzyme-active center(6, 7, 9) . The HPlg
moiety in the complex has been named virgin enzyme since it has similar
catalytic activity to human plasmin (HPlm), but the activating peptide
bond of Arg-Val
is not
cleaved(12) . In the second reaction step, the HPlg
SK
complex is converted to HPlm
SK, and then in the final reaction
step, the HPlm
SK catalyzes the hydrolysis of the specific
activating peptide bond of Arg
-Val
on
substrate HPlg, resulting in the formation of
HPlm(5, 8, 13, 14, 15) .
The exact interaction sites of SK with HPlg and their functions,
however, have not been determined. Proteolytic SK fragments obtained in
the reactions of SK with human, rabbit, and dog Plg(m)s have been used
to study the functions of the SK
molecule(15, 16, 17, 18) . A 36 and
a 25.7-kDa fragment obtained in the reaction with HPlm and dog Plg,
respectively, can activate HPlg(15, 16, 17) .
A 17-kDa SK fragment consisting of Val (Glu
) to Arg
(Lys
)
obtained in the reaction with Sepharose-immobilized HPlg (HPlm) is the
smallest SK fragment that can bind to HPlg(18) . A SK fragment,
SK-o, Ser
-Lys
(or
Ser
-Lys
according to the numbering used in
this paper), is essential for minimal SK activator
activity(19) . Obviously, the COOH-terminal peptide of SK-p,
Ala
-Lys
(or
Ala
-Lys
), is required for strong binding
with HPlm. The NH
-terminal 59-amino acid peptide is
important in maintaining the proper conformation of SK for full
activator activity(19) . These studies imply that domain-like
structures may exist in the SK molecule that exert various functions in
HPlg activation. This study was undertaken to elucidate the functions
of various domains of SK and to determine more precisely the
interaction sites between SK and HPlg using a series of truncated SK
peptides lacking NH
and/or COOH-terminal amino acid
residues.
The SK gene of 1.3 kilobases was amplified by PCR with
standard procedures from S. equisimilis H46A and was
constructed into pGEM-3Z and pET3 plasmid. Unidirectional deletion of
SK gene from either end was carried out with exonuclease III. Nine
truncated SKs were prepared and designated SK(16-414),
SK(60-414), SK(91-414), SK(127-414),
SK(158-414), SK(220-414), SK(16-378),
SK(16-316), and SK(16-251). The numbers in the parenthesis
indicate positions of the initial and terminal amino acids of the
corresponding truncated SK peptide according to the published amino
acid sequence deduced from the nucleotide sequence of SK
gene(1, 33) . In sequencing the PCR-amplified
full-length SK DNA, one silent mutation at Lys (AAA
AAG) and one mutation at Arg
to Pro (CGT
CCT) were
detected as compared with the SK gene sequences(1) . The
slightly modified SK DNA was used for preparation of truncated SK
peptides without correction of these mutations. Apparently the
alteration had no effect on the HPlg activator activity of recombinant
SK since purified SK(16-414) had a specific activity of 118,755
IU/mg (Table 1), which is comparable with purified commercially
available native SK. The expressed recombinant SK and its truncated
peptides were also sequenced and found to be identical to the published
sequence (1, 2) except that an additional fusion
peptide of 14-18 amino acid residues derived from the pET-3
plasmid was attached at their NH
termini. The
SK(16-414) DNA was used to prepare COOH-truncated SK, since
SK(16-414) protein had the same HPlg activator activity as native
SK, and the SK peptides without NH
-terminal 15 amino acids
were more easily overexpressed in the E. coli cells than the
full-length SK. In general the NH
- and COOH-truncated SK
peptides overexpressed in the E. coli system consisted of more
than 70% of the total amount of proteins in the crude extracts of the E. coli cells. Homogeneous SK peptides were obtained after
ammonium sulfate precipitation and DEAE column chromatography as shown
in SDS-PAGE (Fig. 1). All of the SK peptides could be detected
by a polyclonal antiserum raised against the native SK. The recovery
and specific activities of SK peptides during each purification step
are summarized in Table 1. The specific HPlg activator activities
of purified SK(16-414) and SK(16-378) were higher than
100,000 IU/mg and were comparable with that of the purified commercial
native SK. SK(60-414), SK(91-414), SK(127-414),
SK(158-414), and SK(16-316) had low specific HPlg activator
activities (Table 1). The HPlg activator activities of the two
short peptides, SK(16-251) and SK(220-414), were too low to
detect.
Figure 1: SDS-PAGE of purified truncated SK peptides. Lane M, molecular mass marker; lane a, purified native SK; lane b, SK(16-414); lane c, SK(60-414); lane d, SK(91-414); lane e, SK(127-414); lane f, SK(158-414); lane g, SK(220-414); lane h, SK(16-378); lane i, SK(16-316); lane j, SK(16-251).
SK(16-414) and SK(16-378) at a catalytic
concentration (0.02 µM) could effectively catalyze the
conversion of HPlg (2 µM) to HPlm, and an increasing rate
of substrate hydrolysis was observed as more HPlm was produced (Fig. 2A). The conversion of HPlg to HPlm was completed in
10 min as confirmed by SDS-PAGE. The bands of HPlg (94 kDa)
disappeared, and both the heavy (65 kDa) and light (26 kDa) chains were
detected (Fig. 2B). The second-order rate constants of HPlg
activation, k/K
, of
native SK, SK(16-414), and SK(16-378) were similar (Table 2). SK(60-414) had low HPlg activator activity (Table 1). However, its activator activity could be
dose-dependently enhanced by incubating with a catalytic amount of
SK(1-59), which was prepared by limited digestion of native SK
with immobilized HPlm and purified by HPLC (Fig. 2A).
After 10 min of incubation, about half of HPlg was activated by
SK(1-59) modified SK(60-414)*, while complete activation
occurred after 30 min of incubation (Fig. 2B). This
result suggests that SK(1-59) may induce a conformational change
in SK(60-414) to form an efficient activator. SK(91-414),
SK(127-414), or SK(158-414) alone at a catalytic amount
could not activate HPlg nor in the presence of SK(1-59).
Figure 2:
Activation of HPlg by SK(16-414),
SK(16-378), and SK(60-414)*. A, rate of
activation; B, SDS-PAGE analysis. HPlg (2 µM) was
activated by incubation with a catalytic amount of activator, 0.02
µM SK(16-414) (), 0.02 µM SK(16-378) (
), or 0.2 µM SK(60-414)*
(
,
), at 37 °C in 0.05 M Tris, 0.1 M NaCl, pH 7.4, containing 0.5 mM S-2251. SK(60-414)*
was prepared by premixing SK(60-414) (0.2 µM) and
SK(1-59) 0.01 µM (
) or SK(1-59) 0.005
µM (
) at 25 °C for 1 min. An incubation with
SK(1-59) 0.01 µM was used as control (
). The
change in absorbance at 405 nm was monitored. Parallel samples of HPlg
activation for 10 min and 30 min at 37 °C were also taken for
SDS-PAGE analysis (B). Lane M, molecular mass marker; lane a, HPlg control; lane b, SK(16-414), 10
min; lane c, SK(16-414), 30 min; lane d,
SK(16-378), 10 min; lane e, SK(16-378), 30 min; lane f, SK(60-414)*, 10 min; lane g,
SK(60-414)*, 30 min; lane h, HPlm
control.
SK(91-414) could form a catalytically active virgin enzyme
with HPlg in a one-to-one equimolar complex. The equimolar HPlg and
SK(91-414) had a maximal amidolytic activity after reaction for 7
min (Fig. 3A). However, no cleavage of the activating
peptide bond, Arg-Val
, of HPlg was
observed at up to 13 min determined at intervals as shown by SDS-PAGE
analysis (Fig. 3B), suggesting that a virgin enzyme,
which consisted of intact HPlg and SK(91-414) equimolar complex,
was induced. However, after reaction for more than 15 min, some HPlg
was hydrolyzed, heavy and light chains of HPlm were detected, and
SK(91-414) was degraded (Fig. 3B, lanes e and f). The cleavage of the activating peptide bond was
also confirmed by NH
-terminal amino acid determination of
the HPlm light chain. Virgin enzymes could also be induced in
stoichiometric complexes of HPlg and other truncated SK peptides,
although different durations of incubation were required to reach
maximal amidolytic activities. For example, to achieve the maximal
amidolytic activities, it took 4 min for HPlg and SK(16-414) or
SK(16-378), but 7 min for HPlg and SK(91-414),
SK(127-414), or SK(158-414). The rates for hydrolysis of
S-2251 by the virgin enzyme complexes of HPlg and truncated SK peptides
are shown in Fig. 4A. The HPlg
SK(16-414) and
HPlg
SK(16-378) had similar second-order reaction constant
(k
/K
) as HPlm in hydrolysis of
substrate S-2251 (Table 3). The virgin enzymes of
HPlg
SK(91-414), HPlg
SK(127-414), and
HPlg
SK(158-414) had lower reaction constants (Table 3). Initially, no HPlm conversion was observed in each
HPlg
SK-peptide virgin enzyme complex, since only HPlg and the
corresponding SK peptide were detected by SDS-PAGE (Fig. 4B). However, after incubation for 15 min, HPlm
formation was also observed in the equimolar complexes of
HPlg
SK(127-414) and HPlg
SK(158-414) as that of
HPlg
SK(91-414), suggesting that the activation peptide bond
in these complexes was also cleaved.
Figure 3: Discontinuous assay for measuring the rates of active-site generation in HPlg and SK(91-414) complex. A, rate of amidolytic activity; B, SDS-PAGE analysis. HPlg (final concentration, 2 µM) was incubated with equimolar of SK(91-414) at 25 °C in 10 mM HEPES/NaOH, pH 7.4. Aliquots (final concentration, 0.2 µM) were removed at intervals for assay of amidolytic activity using S-2251 at a final concentration of 0.5 mM. Amidolytic activity was expressed as µM S-2251 hydrolyzed per min using an extinction coefficient (1 M, 1 cm, 405 nm) of 9559 as shown in A, parallel samples were also taken for SDS-PAGE as shown in B. Lane M, molecular mass marker; lane a, 1 min; lane b, 5 min; lane c, 11 min; lane d, 13 min; lane e, 15 min; lane f, 30 min.
Figure 4:
Virgin enzyme formation of HPlg with
various SK peptides in stoichiometric complexes. A, continuous
assay for determination of virgin enzyme activity; B, SDS-PAGE
analysis. HPlg (final concentration, 2 µM) was
preincubated with equimolar of SK(16-414) () or
SK(16-378) (
) for 4 min, or with SK(91-414) (
),
SK(127-414) (
), or SK(158-414) (
) for 7 min
in 10 mM HEPES/NaOH, pH 7.4, at 25 °C to form a virgin
enzyme complex. The complex (final concentration, 0.2 µM)
was then added into 150 µl of 0.05 M Tris-HCl/0.1 M NaCl, pH 7.4, containing 0.5 mM S-2251 at 37 °C. The
increment of absorbance at 405 nm of the mixture was recorded. The
amidolytic activity of human plasmin (0.2 µM) (
) was
also shown as control. Parallel samples of virgin enzyme complex were
also taken for SDS-PAGE as shown in B. Lane M,
molecular-mass marker; lane a, HPlg
SK(16-414), 30
s; lane b, HPlg
SK(16-414), 4 min; lane c,
HPlg
SK(16-378), 30 s; lane d,
HPlg
SK(16-378), 4 min; lane e,
HPlg
SK(91-414), 30 s; lane f,
HPlg
SK(91-414), 7 min; lane g,
HPlg
SK(127-414), 30 s; lane h,
HPlg
SK(127-414), 7 min; lane i,
HPlg
SK(158-414), 30 s; lane j,
HPlg
SK(158-414), 7 min.
The COOH-truncated peptide,
SK(16-316) at a concentration one-tenth of HPlg, had no HPlg
activator activity. It also failed to induce a virgin enzyme formation
in an equimolar stoichiometric complex with HPlg under the same
conditions described previously. However, enzyme activity of HPlm
converted from HPlg in HPlgSK(16-316) equimolar complex
could be detected after incubation for 20 min and reached its maximum
after 30 min (Fig. 5A). Cleavage of the activating peptide
of HPlg in the HPlg
SK(16-316) complex was observed after 20
min of incubation as analyzed by SDS-PAGE (Fig. 5B). The
SK(16-316) in the complex was degraded as the HPlm appeared (Fig. 5B). The truncated peptides, SK(220-414)
and SK(16-251), could neither induce catalytically active virgin
enzyme in the stoichiometric complexes with HPlg nor activate HPlg to
HPlm in the complexes. However, the isotope-labeled SK(220-414)
and SK(16-251) could bind to HPlg (Fig. 6).
Figure 5: Discontinuous assay for measuring the amidolytic activity of HPlg and SK(16-316) complex. A, rate of amidolytic activity; B, SDS-PAGE analysis. HPlg (final concentration, 2 µM) was incubated with equimolar of SK(16-316) at 25 °C in 10 mM HEPES/NaOH, pH 7.4. Aliquots (final concentration, 0.2 µM) were removed at intervals for assay of amidolytic activity using S-2251 at a final concentration of 0.5 mM. Amidolytic activity was expressed as µM S-2251 hydrolyzed per min using an extinction coefficient (1 M, 1 cm, 405 nm) of 9559 (A). Parallel samples were also taken for SDS-PAGE as shown in B. Lane M, molecular-mass marker; lane a, 1 min; lane b, 20 min; lane c, 30 min; lane d, 45 min; lane e, 60 min; lane f, HPlm control.
Figure 6:
Binding of [I]-SK,
[
I]-SK(220-414), and
[
I]-SK(16-251) to HPlg. HPlg (2 mg/ml)
was coated on a radioimmunoassay strip plate.
I-Labeled
native SK (
),
I-labeled SK(220-414) (
),
and
I-labeled SK(16-251) (
) at various
concentrations from 0.03 to 1 µM were added to the wells
and incubated at 4 °C for 1 h. After washing out extensively, the
radioactivity was determined. Each point represents the mean ±
S.D. of three independent determinations and the nonspecific binding
has been subtracted.
The secondary structures of truncated SK peptides used in
this study were determined by circular dichroism (CD) spectroscopy and
were all found to be similar to that of the corresponding regions of
the native SK moiety reported (34) except for SK(60-414).
This result suggests that the recombinant truncated SK peptides were
suitable for the study of structure-function relationship of SK.
SK(60-414) had a majority of disordered secondary structure
according to the CD spectroscopy. It also had a lower HPlg activator
activity compared with the SK-p, Ser-Lys
(or
Ser
-Lys
according to the numbering used in
this study) obtained by limited digestion of native SK(19) .
The fusion peptide of 14 amino acid residues in the
NH
-terminal of SK(60-414) might interrupt the proper
conformation of SK(60-414). However, after incubation with
SK(1-59), SK(60-414) was shifted to SK(60-414)*, with
elevation of HPlg activator activity. This might be due to a
conformational change in SK(60-414) induced by SK(1-59).
Therefore, SK(1-59) was important in maintaining the proper
conformation of the core region of SK (19) .
Since
SK(16-414) and SK(16-378) were as active as native SK in
HPlg activation, the peptides Ile-Asn
and
Ser
-Lys
were of little functional
importance for SK. The study in which two SK gene products,
cSK
, Ile
-Ala
(or
Ile
-Ala
according to the numbering used in
this study), and cSK
, Ile
-Leu
(or
Ile
-Leu
according to the numbering used in
this study), were cloned suggested that Ala
(or
Ala
) was essential for activator activity of
SK(35) , since Ile
-Ala
(or
Ile
-Ala
) had full activator activity, while
Ile
-Leu
(or
Ile
-Leu
) retained only 25% activity. However,
SK(16-378) prepared in this study was as active as native SK in
HPlg activation and also had a properly folded secondary structure. The
low activator activity of cSK
might be due to reasons other
than Ala
(or Ala
) directly involved in
activation of HPlg.
In this report, we defined five important
regions in SK molecule as a HPlg activator. These regions were as
follows: a, Ile-Lys
; b,
Ser
-Asn
; c,
Val
-Arg
; d,
Tyr
-Ala
; e, Ser
-Ala
as shown in Fig. SI.
Figure SI: Scheme I
Region a functioned to stabilize
the conformation of SK in maintaining its full activator activity.
SK(60-414), which contained regions b, c, d, and e, was a
competent HPlg activator, although the activation rate was slower than
those of SK(16-414) and SK(16-378) (Fig. 2A). SK(91-414), at a molar ratio of
one-tenth of HPlg, could not activate free HPlg. However, an equimolar
complex of HPlg and SK(91-414) was amidolytically active. The
complex of HPlg and SK(127-414) or SK(158-414) had
properties similar to SK(91-414). Therefore, the SK peptide,
which contained regions c, d, and e, had the ability to form a
so-called virgin enzyme complex with HPlg, and region b was essential
for HPlg activation. The conversion of HPlg to HPlm in these one-to-one
complexes of SK peptides and HPlg was detected after a lag period of 15
min (Fig. 3B). The reason of this slow HPlg conversion
to HPlm in the complexes remains unclear. It is possible that the
conformation of HPlg is transformed in the complex and the activating
peptide bond is more vulnerable for hydrolysis by the less effective
activators. In regard to the formation of virgin enzyme complex with
HPlg, SK(220-414), which contained regions d and e, lost this
ability. Therefore, the region c, Val-Arg
,
was thought to possess one of the essential interaction cores for
virgin enzyme formation. The COOH-terminal truncated SK(16-378)
had all the essential regions to form a virgin enzyme complex with HPlg
and to catalyze HPlg activation. The SK(16-316), which consisted
of regions a, b, c, and d but not e, could not form virgin enzyme and
could not catalyze the activation of HPlg into HPlm as a typical
SK-type HPlg activator. However, the HPlg moiety was slowly converted
to HPlm in equimolar HPlg
SK(16-316) complex but not in
HPlg
SK(16-251), in which SK(16-251) contained only
regions a, b, and c. Therefore, region d apparently was involved in the
induction of the conformational changes in HPlg, so that it could be
activated in the complex.
In conclusion, by studying with the truncated SK peptides, five regions of defined functions in SK molecules were deduced. The functional studies of the truncated SK peptides provided the evidence that more than one region on SK could interact with HPlg. By comparing the functions of the truncated SK, we were able to define the function of each region of SK. These findings were consistent with the results of NMR and CD spectroscopy studies of SK in which a flexible structure of SK with existence of at least three or four domains was proposed(36, 37) . The elucidation of the function of each of these specific regions of SK molecule is very important for understanding the molecular mechanism of its interaction with HPlg. Further studies on the functions of specific point mutation of SK in different regions might provide more critical information needed for the delineation of the intriguing interaction between SK and HPlg.