From the Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands and the Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
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Human C4b-binding protein (C4BP) is a regulator
of the complement system and plays an important role in the regulation
of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for
activated protein C. C4BP is a multimeric protein containing several
identical C4b-binding protein
(C4BP)1 is an important
regulator of the complement system (1-5). It accelerates C2a decay
from the classical pathway C3-convertase (C4b2a) complex (1, 6) and
promotes factor I-mediated degradation of C4b (1-3, 7). C4BP also has a high affinity for anticoagulant vitamin K-dependent
protein S, and together they form a noncovalent 1:1 stoichiometric
complex (8-11). Binding of protein S to C4BP results in a decreased
cofactor function of protein S for anticoagulant activated protein C
(APC) in the degradation of coagulation factors Va and VIIIa (12-14). Complex formation between protein S and C4BP has no effect on the
inhibition of complement activation. C4BP is a multimeric glycoprotein
(Mr 530,000-570,000), composed of six or seven
identical Proteins--
C4BP was immunopurified from human plasma as
described by Hessing et al. (32). Protein C was purified and
activated as described previously (33). Protein S was purified from
prothrombin concentrates as described by Hackeng et al.
(34).
Chimeric SCR-tPA Constructs--
Each individual Cell Culture, Transfection, and Purification of Recombinant
Constructs--
Transfection of baby hamster kidney cells was
performed as described previously (36). Expression of all constructs
was performed in conditioned medium (CHO-II-SFM; Life Technologies,
Inc., Paisley, U.K.), and harvested medium was stored at Binding of Protein S to Immobilized Chimeric SCR-tPA
Constructs--
The binding of protein S to immobilized chimeric
SCR-tPA constructs was performed as follows. Microtiter plates (96-well
vinyl assay plates; catalog no. 2595, Costar, Cambridge, MA) were
coated overnight at 4 °C with a polyclonal antibody against tissue
plasminogen activator (5 µg/ml; ImulyseTM-tPA, Biopool
AB) in coat buffer (15 mM Na2CO3.10
H2O, 35 mM NaHCO3, pH 9.6), 50 µl/well. Plates were washed three times with Tris-buffered saline
(TBS; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl)
containing 0.1% (v/v) Tween 20. Plates were blocked for 2 h at
37 °C with TBS containing 3% (w/v) bovine serum albumin (blocking
buffer), 100 µl/well. CHO-II-SFM supernatant derived from baby
hamster kidney cells transfected with the constructs described above
was then added to the wells and incubated for 2 h at 37 °C in
blocking buffer containing 0.1% Tween 20, 50 µl/well. After washing
three times with TBS containing 0.1% Tween 20, increasing
concentrations of protein S were added to the wells and incubated for
2 h at 37 °C in blocking buffer containing 0.1% Tween 20 and 5 mM CaCl2, 50 µl/well. Plates were washed
three times with TBS containing 0.1% Tween 20, and bound protein S was
detected using a polyclonal peroxidase-conjugated antibody against
protein S (1.3 g/liter IgG; Dako), 1:2000 diluted in blocking buffer
containing 0.1% Tween 20 and 5 mM CaCl2, 50 µl/well. After washing three times with TBS containing 0.1% Tween
20, staining solution consisting of 0.4 mg/ml
o-phenylenediamine and 0.002% H2O2
in 100 mM phosphate, 50 mM citric acid buffer
(pH 5.0) was added to the wells (100 µl/well). The reaction was
stopped by adding 50 µl/well 1 M
H2SO4, and absorbance was measured at 490 nm in
a Vmax microtiter plate reader (Molecular
Devices, Menlo Park, CA). Values were corrected for background absorbance.
Binding of Chimeric SCR-tPA Constructs to Immobilized Protein
S--
Microtiter plates were coated overnight at 4 °C with a
polyclonal antibody against protein S (3 g/liter IgG; Dako), 1:1000 diluted in coat buffer, 50 µl/well. Plates were washed three times with TBS containing 0.1% Tween 20. Plates were blocked for 2 h at
37 °C with blocking buffer, 100 µl/well. Purified human protein S
(1 µg/ml) was added to the wells and incubated in blocking buffer containing 0.1% Tween 20 and 5 mM CaCl2 for
2 h at 37 °C, 50 µl/well. Plates were washed three times with
TBS containing 0.1% Tween 20, and increasing concentrations of
purified chimeric SCR-tPA constructs were added and incubated for
2 h at 37 °C in blocking buffer containing 0.1% Tween 20 and 5 mM CaCl2, 50 µl/well. After washing the
plates three times with TBS containing 0.1% Tween 20, sheep anti-tPA
antibodies (1 µg/ml; Enzyme Research Laboratories Inc.) were added in
blocking buffer containing 0.1% Tween 20 and 5 mM
CaCl2, 50 µl/well. After 1 h of incubation at
37 °C, plates were washed three times with TBS containing 0.1%
Tween 20. Bound sheep antibodies against tPA were detected by adding a
polyclonal peroxidase-conjugated rabbit antibody against sheep
antibodies (Dako), 1:1000 diluted in blocking buffer containing 0.1%
Tween 20 and 5 mM CaCl2, 50 µl/well. After
washing three times with TBS containing 0.1% Tween 20, the wells were
developed and measured as described above. Values were corrected for
background absorbance.
Stoichiometry of the Interaction between Protein S and SCR-tPA
Constructs--
A fluid phase binding assay was used to investigate
the stoichiometry of the interaction between protein S and the SCR-tPA constructs that bound to protein S. For this assay, 5 mg of purified rabbit antibodies directed against human protein S (Dako) was coupled
to 2.5 ml of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech,
Uppsala, Sweden) according to the manufacturer's instructions. Fluid
phase binding was performed by allowing increasing concentrations of
SCR-tPA constructs to bind to 10 nM human protein S in a
volume of 100 µl of TBS containing 5% bovine serum albumin and 10 mM CaCl2. Binding was performed overnight at
4 °C with constant rotation. Then 100 µl of rabbit anti-human
protein S-Sepharose beads in TBS was added (final CaCl2
concentration 5 mM). After 90 min of incubation with
constant rotation at room temperature, total protein S was removed from
the incubation mixture by spinning down the Sepharose beads in an
Eppendorf centrifuge for 3 min at 14,000 rpm. The supernatant was
analyzed with a polyclonal tPA ELISA (ImulyseTM-tPA,
Biopool AB) for nonbound (free) SCR-tPA constructs. As a control, the
supernatant was also analyzed with a polyclonal protein S ELISA to
confirm that all of the protein S was precipitated by the Sepharose
beads. In this polyclonal ELISA, rabbit anti-protein S antibodies were
used as catching antibodies, and peroxidase-conjugated rabbit
anti-protein S antibodies were used as detecting antibodies (antibodies
from Dako).
Competition Experiments Using Chimeric SCR-tPA Constructs
for Binding of Protein S to Immobilized C4BP--
Microtiter plates
were coated overnight at 4 °C with a monoclonal antibody against
C4BP Protein S Cofactor Activity in Plasma--
Protein S cofactor
activity was determined with an activated partial thromboplastin
time-based assay using a KC-10A microcoagulometer (Amelung, Lemgo,
Germany). For this assay, plasma deficient in protein S and C4BP was
prepared by immunoadsorption as described previously (37). Purified
human protein S (160 nM) was preincubated for 30 min at
37 °C with serial dilutions of chimeric SCR-tPA constructs in TBS
containing 0.3% (w/v) bovine serum albumin plus 3 mM
CaCl2. Aliquots of 12.5 µl from the preincubation
mixtures were added to a mixture of 25 µl of plasma deficient in
protein S and C4BP and 12.5 µl of 240 nM activated
protein C solution (final APC concentration 30 nM). After
adding 25 µl of PTT reagent (Roche Molecular Biochemicals),
coagulation was initiated by adding 25 µl of 25 mM
CaCl2 (final volume 100 µl). In the range of protein S
used in this assay (0-20 nM protein S), there was a linear
relationship between clotting time and protein S concentration (data
not shown). The protein S cofactor activity was expressed as a
percentage of the maximum cofactor activity in the absence of chimeric
SCR-tPA constructs.
Expression and Purification of Chimeric SCR-tPA Constructs--
In
order to study the role of each individual C4BP Binding of Protein S to Immobilized Chimeric SCR-tPA
Constructs--
The interaction of chimeric SCR-tPA constructs with
protein S was investigated using a direct binding assay in which
protein S was allowed to bind to immobilized chimeric SCR-tPA
constructs (Fig. 3). The results are
expressed as a percentage of maximum binding
(Bmax) for each construct. Apparent dissociation
constants of the binding of protein S to chimeric SCR-tPA constructs
were 10.3 nM for SCR-1, 1.9 nM for SCR-1+2, and
13.7 nM for SCR-1+3, respectively. Protein S did not bind
to SCR-2, SCR-3, SCR-2+3, or tPA. The apparent dissociation constant
for the binding of protein S to plasma C4BP was 1.7 nM,
which is identical to published values (10). This indicates that
protein S has an affinity for SCR-1+ 2 that is comparable with the
affinity for plasma C4BP, whereas the affinity of protein S for SCR-1
and SCR-1+3 was approximately 5 times lower.
Binding of Chimeric SCR-tPA Constructs to Immobilized Protein
S--
A binding assay was performed in which chimeric SCR-tPA
constructs were allowed to bind to immobilized protein S. The results of the binding experiments of chimeric SCR-tPA constructs to
immobilized protein S are presented in Fig.
4. Binding of each chimeric SCR-tPA construct is expressed as a percentage of Bmax
to protein S. The apparent dissociation constants for the binding of
chimeric SCR-tPA constructs to protein S are 207.4 nM for
SCR-1, 24.9 nM for SCR-1+2, and 124.0 nM for
SCR-1+3, respectively. SCR-2, SCR-3, SCR-2+3, and tPA did not bind to
protein S. The apparent dissociation constant for the binding of
plasma-purified C4BP to protein S was 2.9 nM, which is in
the range of previously published values between 2 and 5 nM
(28). This implies that in this system, SCR-1+2 has an affinity for
protein S approximately 5-10 times lower compared with plasma-purified
C4BP.
Stoichiometry of the Interaction between Protein S and SCR-tPA
Constructs--
The stoichiometry of the interaction between protein S
and the SCR-tPA constructs that bound to protein S was analyzed using a
fluid phase binding assay. Increasing concentrations of SCR-tPA constructs were allowed to bind to protein S, after which total protein
S was immunoprecipitated using rabbit anti-protein S antibodies coupled
to Sepharose beads. After immunoprecipitation, no protein S could be
detected in the supernatant using a polyclonal protein S ELISA. Free
SCR-tPA in the supernatant was determined using a polyclonal tPA ELISA.
Bound SCR-tPA was calculated by subtracting values for free SCR-tPA
from the concentrations of SCR-tPA added. Values for bound SCR-tPA were
plotted against free SCR-tPA (data not shown), and the
Bmax of each SCR-tPA construct was calculated. The stoichiometry of the interaction of protein S with each protein S-binding SCR-tPA construct is presented in Table
II as the ratio of
Bmax and the protein S concentration used. The
values for Bmax/protein S displayed in Table II
are presented as means of two separate experiments. One protein S
molecule was found to bind to 1.0 molecule of SCR-1, 1.0 molecule of
SCR-1+2, and 1.1 molecules of SCR-1+3, respectively. These findings
show that each SCR-tPA construct that is able to bind to protein S
(SCR-1, SCR-1+2, and SCR-1+3) contains one binding site for protein S,
confirming the existence of a single binding site for protein S on the
Competition Experiments Using Chimeric SCR-tPA Constructs for
Binding of Protein S to Immobilized C4BP--
Chimeric SCR-tPA
constructs were allowed to compete with immobilized plasma purified
C4BP to bind protein S. In these experiments, purified human protein S
(0.5 nM) was preincubated with increasing concentrations of
chimeric SCR-tPA constructs and then allowed to bind to immobilized
purified human C4BP. Bound protein S was expressed as a percentage of
maximum binding in the absence of chimeric SCR-tPA constructs in Fig.
5. Preincubation of protein S with
SCR-1+2 resulted in inhibition of protein S binding, and a 50%
inhibition of protein S binding was observed at 22 nM
SCR-1+2. SCR-1 and SCR-1+3 had only a minor effect on the binding of
protein S to C4BP, and a 50% inhibition of protein S binding was
observed at 212 and 179 nM, respectively. In agreement with
the results of the direct binding assays (Fig. 3 and 4), SCR-2, SCR-3,
SCR-2+3, and tPA did not compete for protein S binding.
Protein S Cofactor Activity in Plasma--
The effect of the SCR
constructs on the cofactor activity of protein S was tested by
preincubation of 20 nM protein S (final concentration) with
increasing concentrations of chimeric SCR-tPA constructs. Subsequently,
C4BP- and protein S-depleted plasma was added, and the clotting time
was determined in the presence of 30 nM APC. The residual
cofactor activity of protein S after preincubation with chimeric
SCR-tPA constructs is shown in Fig. 6.
SCR-1+2 yielded a 50% inhibition of protein S cofactor activity at a
concentration of approximately 70 nM (SCR-tPA:protein S
ratio of 3.5). A 50% inhibition of protein S cofactor activity by
SCR-1 and SCR-1+3 was obtained at 320 nM SCR-1
(SCR-tPA:protein S ratio of 16) and 210 nM SCR-1+3
(SCR-tPA:protein S ratio of 10.5), respectively. Preincubation of
protein S with SCR-2, SCR-3, SCR-2+3, or tPA had no effect on the
cofactor activity of protein S. The inhibition of protein S cofactor
activity by SCR-1+2 is in concert with the competition experiments
shown in Fig. 5. The maximum inhibitory effect in both experiments is
accomplished at approximately 300 nM SCR-1+2. At these
concentrations, maximum binding of SCR-1+2 occurred in the direct
binding assays of chimeric SCR-tPA constructs to immobilized protein S
shown in Fig. 4. This suggests that the decrease in protein S cofactor
activity can be attributed to the complex formation of protein S with
SCR-1, SCR-1+2, and SCR-1+3, respectively.
The binding site of protein S on C4BP has been localized before on
the Binding assays of chimeric SCR-tPA constructs and protein S showed that
constructs containing SCR-1 bind to protein S with a stoichiometry of
1:1 (Table II), whereas constructs lacking SCR-1 did not bind to
protein S (Figs. 3 and 4). These studies confirm previous results that
have localized the protein S-binding site to the
NH2-terminal SCR (SCR-1) of C4BP Interestingly, constructs containing SCR-1 but lacking SCR-2 (SCR-1 and
SCR-1+3) had an affinity that was approximately 5 times lower for
protein S than SCR-1+2. This was found in the binding of protein S to
immobilized SCR-tPA constructs (Fig. 3) as well in the binding of
SCR-tPA constructs to immobilized protein S (Fig. 4). These results
were confirmed by competition experiments in which soluble chimeric
SCR-tPA constructs were allowed to compete with immobilized C4BP for
binding to protein S (Fig. 5). A possible explanation for the
difference found between SCR-1 and SCR-1+2 is that SCR-2 in the latter
construct could function as a spacer, localizing SCR-1 distant from the
tPA module and thereby yielding better binding properties of this SCR
module. However, such an additional SCR unit is also present in the
construct SCR-1+3, while this construct had a binding affinity for
protein S as low as SCR-1 alone. This implies that the contribution of
SCR-2 in the interaction between protein S and SCR-1+2 was specific and cannot be explained by a spacer function alone. Instead, SCR-2 could
cause a conformational change in SCR-1 that yields a higher affinity
binding of SCR-1 to protein S. Alternatively, when SCR-2 is adjacent to
SCR-1 and SCR-1 is bound to protein S, SCR-2 may bind to protein S as
well, thereby yielding a higher affinity binding of SCR-1+2 to protein S.
In previous studies using synthetic peptides, a peptide comprising
It is known that the The finding that A role of the In our activated partial thromboplastin time-based coagulation assay,
SCR-1+2 had the strongest effect in the inhibition of cofactor activity
of protein S (Fig. 6). Maximum inhibition of protein S cofactor
activity was accomplished at concentrations at which also maximum
binding was accomplished in the direct binding assays (Fig. 4). Hence,
inhibition of protein S cofactor activity can be attributed to the
complex formation between chimeric SCR-tPA constructs and protein S. Nishioka and Suzuki (46) have shown that protein S in complex with C4BP
is still able to bind to APC, excluding the possibility that C4BP
prevents binding of protein S to APC by steric hindrance. Studies using
fluorescence resonance energy transfer have shown that protein S
alters the active site location of APC closer to the membrane surface,
and as a mechanism, a change in topography and/or conformation of the
active site of APC has been postulated (47). A possible mechanism for
the decrease in protein S cofactor activity due to the complex
formation of protein S with C4BP could be that C4BP prevents protein S
to induce this topographical and/or conformational change in the active
site of APC. The mechanism by which C4BP prevents this change in
topography and/or conformation could be by altering the conformation of
protein S or vice versa, preventing protein S from changing
into a conformation that is necessary to express cofactor activity. In
the mechanism of inhibition of protein S cofactor activity, it is most
likely that the SCR-tPA chimeras SCR-1, SCR-1+2, and SCR-1+3 act in a
way comparable with C4BP. The exact contribution of the SCR-tPA
constructs in the decrease in cofactor activity of protein S for APC in
the inhibition of factors Va and VIIIa remains to be elucidated.
We conclude that the second NH2-terminal SCR unit of
C4BP-chains and a single
-chain (C4BP
), each chain being
composed of short consensus repeats (SCRs). Previous studies have
localized the protein S binding site to the
NH2-terminal SCR (SCR-1) of C4BP
. To further
localize the protein S binding site, we constructed chimeras containing
C4BP
SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to
tissue-type plasminogen activator. Binding assays of protein S with
these chimeras indicated that SCR-2 contributes to the interaction of
protein S with SCR-1, since the affinity of protein S for SCR-1+2 was
up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay
that measures protein S cofactor activity, we showed that cofactor
activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no
additional effect on SCR-1, and therefore the effect of SCR-2 was
specific. In conclusion,
-chain SCR-2 contributes to the interaction
of C4BP with protein S.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chains, and approximately 80-85% of C4BP contains an
additional single
-chain that binds protein S (8, 15, 16). In their COOH-terminal regions, the
- and
-chains contain cysteine
residues that form the interchain disulfide bridges in the so-called
core region (17). In electron microscopy studies, C4BP has an
octopus-like appearance (18-20). Under normal conditions,
approximately 60% of total protein S is bound to C4BP, and 40% is
free (21). During an acute phase response, C4BP levels can increase up
to 4-fold. Due to a mechanism of differential regulation of
- and
-chain expression, an increase of
-chains predominates during
such an acute phase response, and hence free protein S is held at
stable levels (21). The
-chains (Mr 70,000)
are composed of eight homologous domains called short consensus repeats
(SCRs) (17, 22). SCRs are commonly found structures in complement
regulatory proteins such as factor H and decay acceleration factor, in
which the SCR units have complement C3b/C4b binding properties (23). However, noncomplement regulatory proteins have also been found containing SCR units such as
2 glycoprotein I and the
-subunit of
coagulation factor XIII, in which the function of the SCR units are
unknown (for reviews, see Refs. 23 and 24). The
-chain (Mr 45,000) is composed of three SCR units, and
previous studies have shown the protein S binding site to be localized
within the NH2-terminal SCR unit (SCR-1) of the
-chain
(25-28). In this study, recombinant chimeras were constructed composed
of each individual
-chain SCR unit and combinations of SCR units
(SCR-1+2, SCR-1+3, and SCR-2+3) fused to the NH2 terminus
of a modified tissue plasminogen activator (tPA) in which the serine
residue was replaced by an alanine residue (29). This inactive tPA
module is well characterized and has been proven in previous studies to
be a useful tool to investigate the function of protein domains
(29-31). The aim of this study was to investigate the role of each
individual
-chain SCR unit in the interaction between protein S and
C4BP. Studies using chimeric SCR-tPA constructs show that SCR-2 of C4BP
-chain is involved in the interaction of protein S with SCR-1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain
SCR unit and adjacent SCR units (SCR-1+2 and SCR-2+3) were amplified
using wild type recombinant C4BP
-chain. This construct was made by
PCR amplification from a human liver cDNA library using
oligonucleotides C4BP
F
(5'-TTTGAATTCTGGGGAGAGGACTTTGATCAC-3') and C4BP
R
(5'-TTTGAATTCTATTACATCTGCTCAGCTGTA-3'). After
amplification, the PCR product was cleaved with EcoRI
(underlined) and cloned in EcoRI-cleaved expression vector
pcDNA3 (Invitrogen, Leek, The Netherlands). The sequence and
orientation of the amplified region of this construct was confirmed by
dideoxy sequencing. This construct was designated pcDNA3-C4BP
.
PCR strategies for amplification of SCR units from pcDNA3-C4BP
were based on the intron/exon organization of the C4BP
gene as
described (35) with the primers depicted in Table I. For the
amplification of SCR-1+3, a modified C4BP
was used that lacked SCR-2
(pcDNA3-C4BP
SCR-2 in Table I)
that will be described
elsewhere.2 After
amplification, PCR products were cleaved with
BglII/XhoI and cloned in
BglII/XhoI-cleaved expression vector ZpL7
containing a modified tPA (30). These chimeric SCR-tPA constructs were designated SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3 and SCR-2+3, respectively. The sequence of the amplified regions of all constructs was confirmed by dideoxy sequencing.
Oligonucleotide sequences used for PCR amplification
is depicted as
pcDNA3-C4BP
. Recombinant C4BP
lacking the second
NH2-terminal SCR unit is depicted as
pcDNA3-C4BP
SCR-2. In the left column, forward primers are
denoted by the letter F, and backward primers are denoted by the letter
R. In the second column, nucleotide sequences of the primers are shown.
Boldface, BglII restriction site. Underlined,
XhoI restriction site. On top of the six outmost right
columns, the different
-chain SCR unit chimeras are depicted by
numbers equal to the corresponding
-chain SCR units contained within
the chimeras. Combinations of template DNA and primers used for
amplification of the different chimeras are indicated by
dots.
20 °C
until needed for further use. Purification of chimeric SCR-tPA
constructs was performed as described previously (29) using a
monoclonal antibody against tPA. Concentrations of chimeric SCR-tPA
constructs were determined using an ELISA system that determines tPA
concentration (ImulyseTM tPA; Biopool, Umeå, Sweden).
Purified constructs were applied to 10% SDS-PAGE under reducing and
nonreducing conditions and stained by Coomassie Brilliant Blue or
transferred to polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA) for standard Western blotting procedures using sheep
anti-tPA antibodies (1 µg/ml, Enzyme Research Laboratories Inc.)
followed by a polyclonal peroxidase-conjugated antibody against sheep
antibodies (Dako, Glostrup, Denmark).
(8C11) in coat buffer, 2 µg/ml, 50 µl/well. Plates were
washed three times with TBS containing 0.1% Tween 20. Plates were
blocked for 2 h at 37 °C with blocking buffer, 100 µl/well.
Purified human C4BP (2 µg/ml) was incubated in blocking buffer for
1 h at 37 °C. Purified human protein S (0.5 nM) was preincubated with chimeric SCR-tPA constructs (0-400 nM)
for 1 h at 37 °C in blocking buffer containing 5 mM
CaCl2. After washing the plates three times with TBS
containing 0.1% Tween 20, aliquots of 50 µl from the preincubation
mixtures were applied to the plates and incubated for 2 h at
37 °C. After washing three times with TBS containing 0.1% Tween 20, bound protein S was detected using a polyclonal peroxidase-conjugated
antibody against protein S (1.3 g/liter IgG; Dako), 1:2000 diluted in
blocking buffer containing 0.1% Tween 20 and 5 mM
CaCl2, 50 µl/well. After washing three times with TBS
containing 0.1% Tween 20, the wells were developed and measured as
described above. Values were corrected for background absorbance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SCR unit in the
interaction of C4BP with protein S, chimeras were constructed of
-chain SCR units fused to the NH2 terminus of a modified
tPA. Baby hamster kidney cells were transfected with the expression vector containing the chimeric SCR-tPA constructs. Expression levels in
the medium were detected using a tPA ELISA system and were 1-5 µg/ml
after 3 days of culture. After purification of the chimeric constructs
with an immobilized monoclonal antibody against tPA, the constructs
were applied to 10% SDS-PAGE under reducing and nonreducing conditions
and stained by Coomassie Brilliant Blue (Fig.
1, A and B) or
transferred to polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA) for standard Western blotting procedures using a
polyclonal antibody against tPA (Fig. 2,
A and B). The chimeric SCR-tPA constructs
appeared as diffuse bands, which is probably caused by heterogeneous
glycosylation of the proteins, because the
-chain of C4BP and the
tPA module are both highly glycosylated (17, 29). Constructs containing
single
-chain SCR units had molecular weights of approximately
73,000 with the exception of SCR-3, which had an estimated molecular weight of 65,000. Constructs containing two
-chain SCR units had
molecular weights of approximately 80,000. The chimeric SCR-tPA constructs were also detected by Western blotting using a polyclonal antibody against tPA (Fig. 2, A and B).
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Fig. 1.
SDS-PAGE of chimeric SCR-tPA constructs.
Purified chimeric SCR-tPA constructs (1.5 µg) were applied to 10%
SDS-polyacrylamide gel under nonreducing (A) and reducing
conditions (B). Proteins were visualized by Coomassie
Brilliant Blue staining. Lane 1, SCR-1-tPA;
lane 2, SCR-2-tPA; lane 3,
SCR-3-tPA; lanes 4 and 8, molecular
weight markers; lane 5, SCR-1+2-tPA;
lane 6, SCR-1+3-tPA; lane
7, SCR-2+3-tPA; lane 9, tPA.
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Fig. 2.
Immunoblot of chimeric SCR-tPA
constructs. Purified chimeric SCR-tPA constructs (200 ng) were
applied to 10% SDS-PAGE gel under nonreducing (A) and
reducing conditions (B). Proteins were transferred to
polyvinylidene difluoride membranes and visualized using polyclonal
antibodies against tPA. Lane 1, SCR-1-tPA;
lane 2, SCR-2-tPA; lane 3,
SCR-3-tPA; lanes 4 and 8, molecular
weight markers; lane 5, SCR-1+2-tPA;
lane 6, SCR-1+3-tPA; lane
7, SCR-2+3-tPA; lane 9, tPA.
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Fig. 3.
Binding of human protein S to immobilized
chimeric SCR-tPA constructs. Chimeric SCR-tPA constructs were
immobilized to microtiter wells using polyclonal antibodies against
tPA. After incubation with increasing concentrations of protein S,
bound protein S was detected using peroxidase-conjugated polyclonal
antibodies against protein S. Bound protein S was expressed as a
percentage of Bmax. Values are displayed as
means of three experiments ± S.D. , SCR-1;
, SCR-2;
,
SCR-3;
, SCR-1+2;
, SCR-1+3;
, SCR-2+3; *, tPA.
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Fig. 4.
Binding of chimeric SCR-tPA constructs to
immobilized human protein S. Protein S was immobilized to
microtiter wells using polyclonal antibodies. After incubation with
increasing concentrations of chimeric SCR-tPA constructs, bound
chimeric SCR-tPA constructs were detected using polyclonal antibodies
against tPA. Bound chimeric SCR-tPA constructs were expressed as
percentages of Bmax. Values are displayed as
means of three experiments ± S.D. , SCR-1;
, SCR-2;
,
SCR-3;
, SCR-1+2;
, SCR-1+3;
, SCR-2+3; *, tPA.
-chain of C4BP.
Overview of apparent dissociation constants and concentrations of 50%
inhibition
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Fig. 5.
Competition of chimeric SCR-tPA constructs
with immobilized human C4BP for protein S binding. C4BP was
immobilized to microtiter wells using monoclonal antibodies against
C4BP -chain. After preincubation of human protein S with chimeric
SCR-tPA constructs, protein S was allowed to bind to immobilized C4BP.
Bound protein S was detected using peroxidase-conjugated polyclonal
antibodies against protein S. Bound protein S was expressed as a
percentage of maximum binding in the absence of chimeric SCR-tPA
constructs. Values are displayed as means of three experiments ± S.D.
, SCR-1;
, SCR-2;
, SCR-3;
, SCR-1+2;
, SCR-1+3;
, SCR-2+3; *, tPA.
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Fig. 6.
Inhibition of protein S cofactor activity by
chimeric SCR-tPA constructs. Human protein S (final concentration
20 nM) was preincubated with chimeric SCR-tPA constructs
and added to a mixture of C4BP- and protein S-depleted plasma and 30 nM activated protein C. After the addition of 25 mM CaCl2, the clotting time was determined.
Protein S cofactor activity was expressed as percentage of maximum
activity in the absence of chimeric SCR-tPA constructs. Values are
displayed as means of two experiments. , SCR-1;
, SCR-2;
,
SCR-3;
, SCR-1+2;
, SCR-1+3;
, SCR-2+3; *,
tPA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain of C4BP (16, 32, 38), and residues within the first
NH2-terminal SCR unit were shown to be important for the
interaction between protein S and C4BP (25, 26, 28, 39). In this
report, the possible role of the second and third
-chain SCR unit in
this interaction was investigated using chimeric constructs containing
-chain SCR units fused to a modified tPA module. This tPA module is
well characterized and has been proven in previous studies to be a
useful tool to investigate the function of protein modules (29-31).
The SCR units in these SCR-tPA chimeras were recognized by several
monoclonal antibodies directed against the
-chain of
C4BP.3 The binding of protein
S to the SCR-tPA constructs and the effect on the cofactor function of
protein S for activated protein C due to the interaction with these
chimeric SCR constructs were investigated.
(25, 26, 28, 39). The
binding of protein S to immobilized SCR-1+2 occurred with the same
affinity as for binding to plasma C4BP, whereas the binding of SCR-1+2
to immobilized protein S occurred with an affinity that was
approximately 5-10 times lower compared with plasma C4BP. The reason
for this discrepancy is not known.
-chain SCR-1 residues 34-42 was able to completely inhibit the
binding of a monoclonal antibody directed against residues 420-434 of
protein S (26), and also C4BP was found to bind to a peptide comprising
protein S residues 408-434 (40), implying that
-chain residues
34-42 bind near residues 420-434 of protein S. In another study, a
synthetic peptide comprising protein S residues 605-614 was shown to
inhibit the binding of protein S to C4BP, and in addition C4BP was
shown to bind also to this immobilized peptide (41). Interestingly,
deletion variants of protein S lacking residues 607-635 (42) and
residues 583-635 (43) were shown to have markedly reduced affinities
for binding to C4BP. Peptides comprising a third region in protein S
that inhibit binding of protein S to C4BP have been found (44). It is
tempting to speculate that C4BP
SCR-1 and SCR-2 each have their own
binding region within protein S.
-chain is highly glycosylated (17) and that the
multiple carbohydrate side chains present in the
-chain of C4BP are
not involved in the protein S binding (27). As shown in Figs. 1 and 2,
the SCR-tPA chimeras are also highly glycosylated. Since the
carbohydrate side chains are not involved in the interaction between
protein S and C4BP, a possible different glycosylation of the chimeric
SCR-tPA chimeras is excluded as the cause of the different binding
affinities reported here for the interactions between the chimeric
SCR-tPA constructs and protein S.
-chain SCR-2 is involved in the interaction of
protein S with SCR-1 is not in agreement with the results of
Härdig and Dahlbäck, who found that SCR-1 of C4BP
bound to protein S with an affinity comparable with plasma-purified C4BP
(28). In their study, Härdig and Dahlbäck composed chimeras of C4BP
-chains with one, two, or three of the
NH2-terminal SCR modules replaced by the
-chain
counterpart. Hence, next to the
-chain SCR-1 unit in all of their
constructs, an additional second NH2-terminal SCR unit from
the
-chain or
-chain was present, and this may explain the
different results compared with our study. It is possible that the
second NH2-terminal SCR unit derived from the
-chain in
their constructs was able to exert a function comparable with SCR-2 of
C4BP
, yielding the same affinity of protein S for all of the
-chain chimeras. A possible explanation for this may be found in the
homology between the SCR units from the
-chain (22) and the
-chain (17). A specific sequence present in the second
NH2-terminal SCR unit of both the
-chain and the
-chain may result in an optimal folding of
-chain SCR-1 or,
alternatively, may contain sites that contribute to the binding of
protein S to
-chain SCR-1.
-chains in the interaction with protein S was
previously proposed in studies by Suzuki and Nishioka (45). In their
study, a COOH-terminal core fragment derived from C4BP (Mr 160,000) was found to contain a protein S
binding site, and after reduction and carboxymethylation of the
COOH-terminal fragment, a peptide of Mr 2500 corresponding to Ser447-Tyr467 was identified
as the protein S binding region. Possibly, this region (within the
eighth SCR unit of the
-chain) may also contain a sequence
homologous to the sequence discussed above that in combination with
-chain SCR-1 could contribute to the binding affinity of
-chain
SCR-1 with protein S. Hessing et al. (20) have identified
monoclonal antibodies directed against the
-chain that inhibited the
binding of protein S to C4BP. The inhibitory effect of these antibodies
on the interaction between protein S and C4BP is most likely to be due
to steric hindrance but does not exclude the possibility that the
-chains can also play a role in the interaction between C4BP and
protein S.
contributes to the interaction between protein S and the first NH2-terminal SCR unit of the C4BP
. This is the first
time that an SCR unit other than
-chain SCR-1 has been shown to be
involved in the interaction of C4BP with protein S.
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FOOTNOTES |
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* This work was supported in part by The Netherlands Heart Foundation Grant 94.044.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Thrombosis and
Haemostasis Laboratory, G03.647, Dept. of Haematology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The
Netherlands. Fax: 31-30-2511893; E-mail:
r.h.l.vandepoel{at}med.uu.nl.
§ Established Investigator of the Netherlands Heart Foundation.
2 R. H. L. van de Poel, J. C. M. Meijers, and B. N. Bouma, manuscript in preparation.
3 R. H. L. van de Poel, J. C. M. Meijers, and B. N. Bouma, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
C4BP, complement
C4b-binding protein;
C4BP, C4BP
-chain;
APC, activated protein C;
SCR, short consensus repeat;
tPA, tissue plasminogen activator;
ELISA, enzyme-linked immunosorbent assay;
PAGE, polyacrylamide gel
electrophoresis;
TBS, Tris-buffered saline.
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REFERENCES |
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