From the Cardiovascular Biology Research Program,
Oklahoma Medical Research Foundation and the Departments of
** Pathology and
Biochemistry and Molecular Biology, University
of Oklahoma Health Sciences Center and the ¶ Howard Hughes Medical
Institute, Oklahoma City, Oklahoma 73104
Received for publication, November 22, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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The endothelial cell protein C receptor (EPCR) is
an endothelial cell-specific transmembrane protein that binds both
protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C
activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two The protein C anticoagulant pathway serves as the major
physiologic control of clot formation (reviewed in Refs. 1-3). The pathway is initiated upon binding of thrombin to the endothelial cell
surface protein thrombomodulin
(TM)1 (4). The thrombin-TM
complex activates protein C to activated protein C (APC). APC, in
conjunction with its cofactor protein S, degrades factors Va and VIIIa
on the phospholipid surface, thereby attenuating the coagulation
cascade. Defects in the protein C anticoagulant pathway have been
implicated as the underlying risk factors for the development of venous
and arterial thrombosis (5-11).
Recent studies demonstrate that human endothelial cells express a
transmembrane protein that binds both protein C and APC with high
affinity (Kd EPCR has sequence homology to members of the MHC class I/CD1 family of
molecules. MHC class I/CD1 molecules are organized into the In this study, we set out to identify the residues within EPCR that are
involved in protein C/APC binding. Our approach was to combine data
from loss-of-function alanine substitution studies with that obtained
from gain-of-function epitope-mapping studies. Given that EPCR differs
from other members of the MHC class 1/CD1 family in that (a)
it lacks the Materials--
Human thrombin (25), human protein C and APC
(26), and recombinant Gla-domainless protein C (GDPC) (27) were
prepared as described previously. GDPC is a truncated form of protein C lacking residues 1-46 of the N terminus. Oligonucleotides were synthesized by Operon Technologies Inc. Dulbecco's modified Eagle's medium, fetal bovine serum, and G418 were from Life Technologies, Inc.
Sulfo-NHS-LC-biotin UltraLink and Immobilized NeutrAvidin Plus resin
were from Pierce. Effectene transfection reagent was from Qiagen, Inc.
(Valencia, CA).
H-D-( DNA Construction and Mutagenesis--
Human (12) and murine EPCR
(28) cDNAs were cloned as XhoI/NotI fragments
into the multiple cloning site of the eukaryotic expression vector
pcDNA3.1( Transient Expression of Wild-type and Variant Forms of EPCR in
293T Cells--
In the pcDNA3.1( Stable Expression of Wild-type and Variant Forms of EPCR in 293 Cells--
293 cells were transfected as described above. 48 h
post-transfection, the medium was changed to Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum and 400 µg/ml G418
(Life Technologies, Inc.). After 2 weeks of drug selection in which the
medium was changed every 3 days, drug-resistant colonies were isolated,
and the levels of cell surface EPCR expression were determined by flow
cytometric analysis as described below.
Fluorescent Labeling of APC--
APC labeled at the active site
with fluorescein (Fl-APC) was prepared as described by Bock (30).
Briefly, 6.5 ml of 0.2 mg/ml APC was incubated with 6-fold molar excess
of N Fluorescent Labeling of Protein C, JRK 1494, JRK 1535, JRK 1500, and JRK 1513 Monoclonal Antibodies--
Human protein C and four
monoclonal antibodies against human EPCR (JRK 1535, JRK 1494, JRK 1500, and JRK 1513) were labeled with fluorescein 5-isothiocyanate as
described by Goding (31).
Flow Cytometric Analysis of Fluorescein-labeled APC, Protein C,
JRK 1535, JRK 1494, JRK 1500, and JRK 1513 Binding to Transfected
Cells--
Adherent transfected cells were harvested by incubation at
room temperature for 5 min in phosphate-buffered saline (137 mM NaCl, 8 mM
Na2HPO4·7H20, 2.7 mM
KCl, 1.5 mM KH2PO4) containing 0.02% EDTA. Cells were resuspended in Hanks' balanced salt solution (HBSS) containing 1% bovine serum albumin, 25 mM Hepes, pH
7.5, 3 mM CaCl2, 0.6 mM
MgCl2, and 0.02% sodium azide (binding buffer). Cells
(1 × 105) were incubated at room temperature with 30 nM Fl-APC or Fl-protein C or 5 µg/ml fluorescein-labeled
monoclonal anti-EPCR antibodies for 15 min in the dark. After washing,
the cells were resuspended in binding buffer. Bound Fl-APC, Fl-protein
C, or fluorescein-labeled antibody was detected on the
fluorescence-1 channel on a FACSCalibur (Becton Dickinson). The
fluorescence intensity of each sample was analyzed twice.
Triton X-100 Lysis of Transfected 293T Cells--
Confluent
transfected 293T cells in a 6-well dish were harvested as described
above. The cells were lysed in 50 µl of phosphate-buffered saline
containing 1% Triton X-100 at room temperature for 10 min. The levels
of EPCR in the cell lysates were analyzed by electrophoresis and
immunoblotting as described below.
Biotinylation of Cell Surface Proteins and Precipitation of
Biotinylated EPCR--
Confluent transfected 293T cells in a 6-well
dish were harvested as described above and resuspended in 90 µl of
HBSS. The cells were surface-biotinylated with 10 µl of 5 mg/ml
sulfo-NHS-LC-biotin (Pierce) at room temperature for 10 min. After
pelleting, the cells were lysed in 200 µl of phosphate-buffered
saline containing 1% Triton X-100 for 10 min at room temperature. To
precipitate the biotinylated EPCR, 20 µl of UltraLink Immobilized
NeutrAvidin Plus resin (Pierce) was added to the lysate and mixed for
1 h at room temperature. The resin was washed 3 times with 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5, 0.5% Triton
X-100, then washed once with Tris-buffered saline. Cell surface
levels of EPCR were analyzed by electrophoresis of the resin and
immunoblotting as described below.
Electrophoresis and Immunoblotting--
Electrophoresis was
performed according to the method of Laemmli (32) using 4-20%
SDS-polyacrylamide gels. Immunoblotting was performed using JRK 1513 anti-EPCR monoclonal antibody.
Flow Cytometric Analysis of Fl-APC Binding to EPCR
Mutants--
The affinities of Fl-APC for EPCR mutants expressed on
the surface of 293 cells were determined as follows. Briefly, cells were grown to confluency in T-75 flasks, detached with 0.53 mM EDTA, and suspended in 3 ml of complete HBSS buffer
(HBSS containing 3 mM CaCl2, 0.6 mM
MgCl2, 1% bovine serum albumin, and 0.02%
NaN3). The cells were diluted 1:20 in complete HBSS buffer
and incubated with increasing concentrations of Fl-APC at 4 °C for
15 min in the dark. Binding was analyzed on a FACSCalibur flow
cytometer (Becton Dickinson). Values of Kd were
determined by fitting binding isotherms with a hyperbolic equation
using the TableCurveTM program (Jandel Scientific, San Rafael, CA).
Previous studies demonstrate that this approach yielded values similar
to those obtained with direct radioligand measurement (12).
Molecular Modeling of Human EPCR (hEPCR)--
The hEPCR model
was constructed by homology modeling following the method of Greer (33)
using the graphics program MAIN (34). The template structure was murine
MHC class I H-2Kb (35) (Protein Data Bank accession number 1VAB), with
which there was 51% amino acid similarity and 22% identity. The
insertion loops present in EPCR between residues Ale-31 and Glu-42,
Cys-101 and Glu-106, and Arg-156 and Leu-161 in EPCR were not
modeled, and a turn was constructed between Arg-10 and Gln-15, where
MHC-I has a 10-residue insert.
Protein C Activation on Transfected 293 Cells--
Stably
transfected 293 cells in 24-well plates were washed 3 times with
phosphate-buffered saline. The cells were preincubated for 5 min at
room temperature with 0.5 ml of HBSS containing 25 mM
Hepes, pH 7.5, 0.1% bovine serum albumin, 3 mM
CaCl2, and 0.6 mM MgCl2 before the
addition of 0.2 µM human protein C or 0.2 µM GDPC. Protein C or GDPC activation was initiated by
the addition of 10 nM thrombin. In some cases, 0.5 µM anti-EPCR mAb JRK 1494 was added before protein C and
preincubated with the cells for 10 min at room temperature. After 30 min at 37 °C, 100 µl of the reactions were stopped by the addition
of 20 µl of 1.66 mg/ml antithrombin containing 20 mM
EDTA. 50 µl of the supernatant was transferred into a 96-well
microplate, and amidolytic activities of APC were determined toward 0.2 mM Spectrozyme PCa substrate in 20 mm Tris-HCl, pH 7.5, 150 mM NaCl. The rates of substrate cleavage were measured in a
Vmax microplate reader (Molecular Devices). All
determinations were performed in duplicate. Under the conditions
employed in this assay, less than 10% of the protein C was activated
during the assay, as determined by reference to a standard curve of
fresh fully activated protein C versus absorbance units/min.
Expression of EPCR Variants in 293 Cells--
hEPCR, bovine EPCR,
and murine EPCR (mEPCR) are single-chain transmembrane glycoproteins
containing 221, 222, and 225 amino acids, respectively. The
amino acid comparisons of human, bovine, and murine EPCR are shown in
Fig. 1. cDNAs encoding human and mouse EPCR were cloned into the eukaryotic expression vector
pcDNA3.1( Selection of Amino Acid Residues on EPCR for
Mutagenesis--
Human, bovine, and murine EPCR all bind saturated and
in a Ca2+-dependent manner to Fl-APC (28). Fig.
1 shows the locations of the residues in the extracellular domain of
EPCR that we selected for individual or multiple mutations to alanine.
Most of the alanine substitutions were directed at conserved residues,
but some were also directed at nonconserved regions of EPCR.
Interaction of EPCR Alanine Mutants with Fl-APC and
Fluorescein-labeled Anti-hEPCR Monoclonal Antibodies--
The
affinities of EPCR variants for Fl-APC were first qualitatively
assessed by flow cytometric analysis. The cDNAs of the variants
were transiently transfected into 293 cells, and Fl-APC binding was
monitored on a FACSCalibur flow cytometer. The analyses were performed
in the presence of 30 nM Fl-APC, which is the dissociation constant for the interaction of hEPCR with APC (36). The ligand binding
properties of the cell surface EPCR variants were compared with 293 cells transfected with hEPCR (positive control) and pcDNA3.1(
The transiently transfected cells were also screened for the ability to
bind fluorescein isothiocyanate-labeled anti-human EPCR monoclonal
antibodies. Our laboratory has raised a panel of anti-human EPCR
monoclonal antibodies, four of which are used in this study. These
antibodies recognize human EPCR but not its mouse counterpart. As shown
in Fig. 3, JRK 1494 and JRK 1535 mAbs block hEPCR/Fl-APC interactions, whereas JRK 1500 and JRK 1513 mAbs do
not. As expected, protein C also blocks hEPCR/Fl-APC interactions, consistent with previous studies demonstrating that EPCR binds to the
Gla domain of both protein C and APC (13). The effect of these four
mAbs on the interaction between hEPCR and Fl-protein C is identical to
that observed on the interaction between hEPCR and Fl-APC (data not
shown).
The results of the flow cytometric analyses and Western blotting
analyses are summarized in Fig. 1. Residues enclosed in green circles are those that, when mutated to alanine, do not affect the
binding of Fl-APC to the transfected cells. These residues include the
four N-linked carbohydrate attachment sites at Asn-30, Asn-47, Asn-119, and Asn-115, suggesting that the carbohydrate moieties
of EPCR are not critical for protein C/APC interactions. The 10 residues marked with red asterisks (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160) are
those that, when mutated to alanine, result in cell surface expression
but no detectable Fl-APC binding, suggesting that these residues are
involved in EPCR-APC interactions. As expected, these 10 mutants do not
bind to Fl-protein C. Residues enclosed in blue circles are
those that, when mutated to alanine, result in the loss of detectable
intracellular and cell surface expression, suggesting that these
residues are critical for secondary structure integrity of EPCR. Fig. 1
also shows residues that, when mutated to alanine, result in the loss
of binding to fluorescein isothiocyanate-labeled anti-EPCR monoclonal
antibodies (denoted by arrows).
Determination of the Affinities of EPCR Variants for
Fl-APC--
As mentioned above, the initial flow cytometric
characterization of the variants was performed in the presence of 30 nM Fl-APC. Thus, to determine qualitatively the affinities
of EPCR variants for Fl-APC, stably transfected 293 cells were
generated. The affinities of 11 EPCR variants for Fl-APC were
determined by monitoring the changes in cell fluorescence during Fl-APC
titration. These variants were chosen to represent a wide range of
ligand binding properties. Binding was analyzed by flow cytometry, and
Kd values were determined by fitting binding
isotherms to a hyperbolic equation. To correct for nonspecific binding,
293 cells transfected with pcDNA3.1(
As shown in Table I, hEPCR, mEPCR, and
clone 64-3 (a hEPCR-mEPCR chimera) all have similar binding affinities
to Fl-APC (Kd = 31 ± 28, 46 ± 15, and
51 ± 22 nM, respectively). Alanine substitutions that
result in the removal of the epitope for JRK 1500 do not influence the
binding of hEPCR to Fl-APC (Kd = 55 ± 11 nM). Alanine substitution of 5 residues in the groove of
hEPCR (clone L1) or removal of two N-linked carbohydrate
attachment sites (clone A/D sugar) result in a modest decrease in
binding affinity to Fl-APC (Kd = 161 ± 52 and
87 ± 4 nM, respectively). In contrast, the mutations
E86A, R87A, F146A, Y154A, and R158A, all of which result in variants
that do not bind Fl-APC as demonstrated in the initial qualitative
screens (Fig. 1), decreased the affinity of hEPCR for Fl-APC greater
than 30-fold (Kd values of Mutagenesis to Map Monoclonal Antibody Epitopes on hEPCR--
A
potential weakness of alanine substitution mutagenesis is that lack of
ligand recognition may be a consequence of destabilizations in tertiary
structure or loop conformations of the native molecule rather than in
the removal of ligand binding residues. We thus aimed to merge the
above loss-of-function data with data obtained from gain-of-function
studies. Since our anti-hEPCR mAbs do not recognize mEPCR, we generated
human/mouse chimeric proteins to delineate the mAb epitopes. The
overall goal is to see if the epitopes for the blocking mAbs colocalize
with the 10 residues that, when mutated to alanine, lose Fl-APC binding
ability. If so, this would increase our confidence in the definitive
assignment of a role for Arg-81, Leu-82, Val-83, Glu-86, Arg-87,
Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160 in protein C/APC
binding. The cDNAs of five human-mouse chimeras were transfected
into 293 cells, and ligand binding was monitored by flow cytometry.
Fig. 4A shows the results of
this epitope-mapping strategy. The epitope for JRK 1494, a blocking
mAb, is localized to hEPCR (Trp-26-Val-116). Consistent with
this map, mutation of Arg-81 to alanine abolishes Fl-JRK 1494 and
Fl-APC binding but does not affect the binding of the other three mAbs
(Fig. 1). Residues Val-25 to Leu-52 contain the epitope for JRK 1513, a
nonblocking mAb. Mutations of Leu-37, Thr-38, and His-39 to alanine
abolishes Fl-JRK 1513 binding but does not affect the binding of Fl-APC
nor the other three mAbs (Fig. 1). The C-terminal half of hEPCR
(Phe-113 to Cys-222) contains the epitopes for both JRK 1500 (nonblocking) and JRK 1535 (blocking). The epitope for JRK 1500 likely
included residues Arg-127, Glu-129, and Arg-130 since clone 64-3 (R127A, E129A, R130A) does not bind Fl-JRK 1500 but does bind to Fl-APC
as well as to the other three mAbs (Fig. 1).
Fig. 4B summarizes the results of the loss-of-function and
gain-of-function studies. Five of the 10 candidate residues for protein
C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with
the epitope for the blocking mAb JRK 1494. In contrast, the epitopes
for JRK 1513 and JRK 1500, both of which are nonblocking mAbs, do not
colocalize with any of the 10 candidate residues for APC/protein C
binding. Western blot analysis revealed that hEPCR is immunoreactive
only with JRK 1513 mAb, suggesting that JRK 1494, JRK 1500, and JRK
1535 mAbs recognize conformation-dependent epitopes (data
not shown).
Fig. 5 shows a three-dimensional ribbon
model of the extracellular domain of human EPCR built using mouse CD1
as a structural template. The domain consists of an eight-stranded
antiparallel Influence of EPCR Variants on Protein C Activation
Rates--
Previous studies demonstrated that binding of protein C to
EPCR enhances the rate of protein C activation by the thrombin-TM complex (14-16). In this study, we coexpressed EPCR variants and TM in
293 cells to confirm the requirement for protein C binding in the
EPCR-dependent enhancement in protein C activation. The following cell lines were used in these studies: (a) 293 cells, (b) 293 cells stably transfected with TM cDNA,
(c) 293 cells stably transfected with both TM and hEPCR,
(d) 293 cells stably transfected with both TM and E86A
hEPCR, and (e) 293 cells transfected with both TM and A/D
sugar hEPCR. Protein C activation was performed on the cell surface in
the absence or presence of JRK 1494, a mAb that blocks protein C
binding to EPCR. We also determined the activation rates of
Gla-domainless protein C, which is a derivative of protein C that
cannot bind to EPCR but in solution is activated by the thrombin-TM
complex at the same rate as full-length protein C (37). As shown in
Fig. 6, the levels of protein C and GDPC activation on 293 cells is low, and protein C activation is not influenced by pre-incubation with JRK 1494. In 293 cells expressing either TM alone or TM and EPCR variants, the activation rate of GDPC is
~7-fold higher than that observed in 293 cells. This rate is
relatively constant between the four TM-expressing cell lines, as is to
be expected since the cell lines express similar levels of TM as
measured by 125I-radiolabeled CTM 1009 anti-human TM Fab
fragments (Fig. 6, bottom of graph). Interestingly, the addition
of JRK 1494 to 293 cells expressing TM alone inhibited protein C
activation rates approximately 2-fold. A likely explanation for this
observation is that transfection of 293 cells with TM cDNA also
increases the levels of cell surface EPCR. Indeed, cell surface EPCR
antigen is higher in TM-expressing cells compared with 293 cells as
measured by 125I-radiolabeled JRK 1535 anti-human EPCR Fab
fragments (Fig. 6, bottom of graph) and by flow cytometry using
fluorescently-labeled JRK 1500 and JRK 1535 mAbs (data not shown).
In the presence of hEPCR and A/D sugar hEPCR, the protein C activation
rate by the thombin-TM complex is increased by 12.1- and 10.5-fold,
respectively (Fig. 6). Preincubation with JRK 1494 blocked protein C
activation rates to near that observed with 293 cells transfected with
TM cDNA alone. In contrast, even at an EPCR:TM ratio of 8:1, E86A
hEPCR does not augment protein C activation by the thrombin-TM complex
on the cell surface. Taken together, these studies confirm that binding
of protein C to EPCR is necessary for the EPCR-dependent
enhancement in protein C activation by the thrombin-TM complex.
Although EPCR has been implicated as an important regulatory
protein of coagulation and inflammation, until now no information was
available related to the critical contact sites between EPCR and
protein C/APC. In this study, we have identified 10 amino acids
(Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157,
Arg-158, and Glu-160) that, when individually mutated to alanine
residues, result in EPCR variants that lose protein C/APC binding
ability. We believe that these alanine substitutions reflect the
removal of ligand binding residues rather than the disruption of
structural integrity of EPCR for the following reasons. First, using
human-mouse chimeric EPCR constructs, half of the point mutations
responsible for loss of protein C/APC binding were mapped to the
epitope responsible for binding one of the inhibitory antibodies (Fig.
4). Second, each of the 10 EPCR variants was screened for the ability
to bind four anti-EPCR mAbs, three of which recognize
conformation-dependent epitopes (Fig. 1). All of these EPCR
variants retain the ability to bind the anti-EPCR mAbs, suggesting that
the mutations have not perturbed the three-dimensional conformation of
EPCR. The only exception is hEPCR R81A, which does not bind to JRK
1494, suggesting that alanine substitution of Arg-81 removes both
protein C/APC and JRK 1494 binding. Third, molecular modeling of EPCR
indicates that the 10 candidate residues are clustered in the distal
end of the two This study has also identified several residues in EPCR that, when
mutated, result in the loss of intracellular and cell surface expression (Fig. 1). Given the importance of the protein C pathway and
the roles that EPCR plays in this pathway, it follows that mutations in
EPCR that impair protein C/APC binding or impair EPCR expression would
likely increase thrombotic risk. This assumption appears to be
supported by preliminary clinical studies by Merati et al.
(38), in which an EPCR loss-of-function mutation has been identified
that is more prevalent in patients with deep vein thrombosis compared
with controls.
We have also provided evidence of glycosylation at all four
N-glycosylation consensus sites of hEPCR (Asn-30, Asn-47,
Asn-119, and Asn-155). The glycosylation contributes to nearly half of the apparent molecular mass of the molecule. Human EPCR variants containing mutations in the carbohydrate attachment sites exhibited decreased molecular masses compared with wild-type EPCR (Fig. 2). These
findings are consistent with previous studies in our laboratory showing
that treatment of hEPCR with endoglycosidase F/peptide-N-glycosidase reduces the apparent molecular mass
from 46 to 28.5 kDa on SDS-polyacrylamide gel electrophoresis (36). The
fact that glutamine substitutions at the glycosylation sites do not
affect Fl-APC nor Fl-protein C binding significantly (Fig. 1) suggests
that the carbohydrate moieties of EPCR are not critical for APC/protein
C recognition. Instead, the N-glycosylation of hEPCR may
contribute to the protection of EPCR from proteolytic degradation or
may serve as specific recognition domains for other, as yet
unidentified, ligands.
Our three-dimensional molecular model of hEPCR was constructed by
homology modeling using the murine MHC class 1 H-2Kb as the structural
template (35). The model suggests that hEPCR is folded into a ligand
binding groove composed of two anti-parallel MHC class 1 molecules bind to octamer and nonamer peptides (35, 40),
whereas CD1 molecules recognize lipids and glycolipids (reviewed in
Ref. 41). In the case of MHC class 1 receptors, peptide ligands are
tethered by hydrogen bonds between backbone atoms of the peptide and
side chain residues in the Unlike conventional MHC class 1/CD1 proteins, EPCR lacks the The present study is the first to identify the protein C/APC binding
region of EPCR. This information may provide a framework to help guide
interpretation of future genetic screening studies. This work also
suggests that EPCR has exploited the MHC class 1 fold for an
alternative and possibly novel mode of ligand recognition.
-helices that sit upon an 8-stranded
-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the
loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment
sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the
active site with fluorescein) interactions, whereas two do not. These
epitopes were localized by generating human-mouse EPCR chimeric
proteins, since the mAbs under investigation do not recognize mouse
EPCR. We found that 5 of the 10 candidate residues for protein C/APC
binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the
epitope for one of the blocking mAbs. Three-dimensional molecular
modeling of EPCR indicates that the 10 protein C/APC binding candidate
residues are clustered at the distal end of the two
-helical
segments. Protein C activation studies on 293 cells that coexpress EPCR
variants and thrombomodulin demonstrate that protein C binding to EPCR
is necessary for the EPCR-dependent enhancement in protein
activation by the thrombin-thrombomodulin complex. These studies
indicate that EPCR has exploited the MHC class 1 fold for an
alternative and possibly novel mode of ligand recognition. These
studies are also the first to identify the protein C/APC binding region
of EPCR and may provide useful information about molecular defects in
EPCR that could contribute to cardiovascular disease susceptibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 nM) (12). This
molecule, named endothelial cell protein C receptor (EPCR), is an
endothelial cell-specific, type 1 transmembrane protein that binds to
the Gla domain of protein C and APC (13). EPCR enhances the rate of
protein C activation by the thrombin-thrombomodulin complex on the
endothelial cell surface (14, 15) and when reconstituted into
phosphatidylcholine liposomes (16), primarily by decreasing the
Km for protein C. A soluble form of EPCR is found in
normal human plasma (17) and has been shown to bind to protein C and
APC with an affinity similar to that of intact membrane-bound EPCR (15,
17). In contrast to the membrane-bound form, soluble EPCR blocks APC
anticoagulant activity (18, 19) by blocking phospholipid interactions
(19). Interestingly, soluble EPCR alters the active site of APC,
suggesting that the macromolecular specificity of APC may be altered by
complex formation with soluble EPCR (19). EPCR also appears to aid in
the host response to sepsis since blocking EPCR-protein C interactions
in baboons exacerbates the coagulation and inflammatory responses to
Escherichia coli (20). Preliminary clinical studies suggest
that protein C and APC supplementation is beneficial in sepsis or
septic shock (21-23).
1,
2,
and
3 domains followed by a transmembrane region and a short
cytoplasmic tail. The
3 domain associates noncovalently with
-2
microglobulin, although in EPCR this domain is absent. The
1 and
2 domains form a ligand binding groove composed of two antiparallel
-helices that sit upon an 8-stranded
-sheet platform. Although
most members of the MHC/CD1 family utilize this groove to bind short
peptides, it should be noted that there are exceptions. For example,
the neonatal Fc receptor, which shares the MHC fold, has a closed
groove that is incapable of binding peptides (24). Instead, the ligand
binding interface is on the side of the neonatal Fc receptor.
3 domain and (b) its ligands are 62-kDa
proteins rather than short peptides, these studies will also provide
insight into how the MHC structural motif may have evolved to serve
different modes of ligand recognition.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Carbobenzoxy)-L-prolyl-L-arginine-p-nitroanilide diacetate (Spectrozyme PCa) was from American Diagnostica (Greenwich, CT). All other chemicals were of the highest grade commercially available.
) (Invitrogen, San Diego, CA). In vitro
mutagenesis to generate and select point mutations was performed using
the QuickChange site-directed mutagenesis system as described by the
supplier (Stratagene, La Jolla, CA). DNA manipulations to generate
deletion mutants or human-murine chimeric cDNA molecules were
carried out using standard DNA cloning techniques (29). Double-stranded
DNA sequencing was used to verify the authenticity of the mutations.
) vector, expression of EPCR
cDNA is under the control of the human cytomegalovirus
immediate-early promoter. Transient transfection of 293T cells was
performed in 6-well dishes in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum using Qiagen-purified
pcDNA3.1(
) constructs employing the Effectene transfection
reagent as described by the supplier (Qiagen).
-((acetylthio)acetyl)-FPR chloromethyl
ketone (ATA-PPACK) (Molecular Innovations Inc., Royal Oak MI) in 100 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM
EDTA. The labeling reaction was allowed to proceed until the APC was
99% inactive as monitored by the loss of enzymatic activity using
Spectrozyme PCa (typically 1 h at room temperature). Excess
ATA-PPACK was removed by centrifuging the sample in a molecular mass
10-kDa cut-off Centricon 10 filter (Amicon Inc.) for 15 min at
6,000 × g. All subsequent steps were performed in the
dark. A 10-fold molar excess of 5-iodoacetamidofluorescein and
one-tenth volume of 1 M hydroxylamine (in 1 M
Hepes, pH 7.4) was added to the ATA-PPACK-APC and incubated at room
temperature for 2 h. Free fluorescein was removed by gel
filtration on a PD-10 column (Amersham Pharmacia Biotech), and the
labeled APC was stored at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and expressed in 293 cells. The apparent molecular
mass of wild-type hEPCR, as determined by SDS-polyacrylamide gel
electrophoresis and immunoblot analysis (~46 kDa), is approximately
twice that of its predicted molecular mass (~24 kDa), consistent with
the presence of carbohydrate moieties on EPCR (Fig.
2). Glutamine substitutions at the four
N-glycosylation consensus sites (Asn-30, Asn-47, Asn-119,
Asn-155) result in EPCR variants with decreased molecular masses (Fig.
2). The electrophoretic mobility of the mutant proteins increases
proportionally with the number of N-glycosylation sites
ablated. In contrast, hEPCR variant L1, which contains alanine substitutions at five residues (Ser-71, Gln-75, Thr-145, Arg-156, glu-163), exhibits the same electrophoretic mobility as wild-type hEPCR
(Fig. 2).
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Fig. 1.
Amino acid sequence comparisons of human,
bovine, and murine EPCR. Residues enclosed in green
circles are those that, when mutated to alanine, retain APC
binding activity. Residues enclosed in blue circles are
those that, when mutated to alanine, result in the loss of all mAb
epitopes as well as APC binding and, hence, appear to have global
effects on EPCR conformation. The 10 residues marked with red
asterisks are those that, when mutated to alanine, resulted in
loss of APC binding but retain mAb epitopes. The only exception is
Arg-81, which resulted in the loss of APC as well as JRK 1494 binding.
The seven residues marked with arrows are alanine
substitutions that result in the loss of either the JRK 1513 epitope
(Leu-37, Thr-38, His-39) or the JRK 1494 epitope (Arg-81) or the JRK
1500 epitope (Arg-129, Glu-130, Arg-131).
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Fig. 2.
Immunoblot analysis of cell surface proteins
of 293 cells transfected with hEPCR variants. 293 cells were
transiently transfected with cDNAs encoding wild-type hEPCR
(WT), hEPCR mutant L1, hEPCR mutant N47Q, hEPCR mutant
N119Q, and hEPCR mutant N30Q/N155Q. 48 h post-transfection, the
cells were harvested, and cell surface proteins were isolated as
described under "Experimental Procedures." The proteins were
subjected to electrophoresis in a 4-20% SDS-polyacrylamide gel under
nonreducing conditions and transferred to nitrocellulose. The
immunoblot was probed with JRK 1513, a monoclonal antibody against
hEPCR. Molecular mass standards are on the left as indicated.
) vector (negative control). Western blotting analysis of whole cell
lysates and cell surface proteins was performed in parallel to
qualitatively monitor the EPCR antigen levels of the variants.
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Fig. 3.
Flow cytometric analysis of Fl-APC binding to
hEPCR in the presence of protein C and anti-hEPCR mAbs. 293 cells
stably expressing hEPCR were pre-incubated at room temperature with 500 nM protein C, JRK 1494, JRK 1535, JRK 1500, or JRK 1513 in
the presence of 3 mM CaCl2 and 0.6 mM MgCl2. After 15 min, 30 nM
Fl-APC was added to the cells and incubated at room temperature for an
additional 15 min. Binding of Fl-APC to the cells was analyzed by flow
cytometry.
) vector alone were utilized.
1000
nM).
Dissociation constants for the interaction of Fl-APC with EPCR variants
expressed in 293 cells
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Fig. 4.
Gain-of-function mutagenesis to map mAb
epitopes on hEPCR. A, schematic representation of
human-mouse EPCR chimeras. The amino acid residues at the junction
between the human and mouse cDNAs are shown. The cDNAs of
hEPCR, mEPCR, and the six human-mouse EPCR chimeras were transfected
into 293 cells, and binding to Fl-APC and fluorescein
isothiocyanate-labeled mAbs was monitored by flow cytometry as
described under "Experimental Procedures." The symbols + and designate binding and lack of binding to the ligands, respectively.
B, summary of the results of the loss-of-function alanine
mutagenesis studies and the gain-of-function epitope-mapping studies.
The 10 candidate residues for protein C/APC binding are shown in the
top schematic diagram. The epitopes for the four mAbs are shown
below.
-pleated sheet with two antiparallel
-helices (helix
1= residues 58 to 83, helix 2= residues137 to 179). In the left
panel, areas shown in green are residues that can be
mutated to alanine without loss of Fl-APC binding. Regions in
blue, when mutated, result in loss of cell surface
expression and, hence, are likely mutations that influence protein
folding. Regions in red are the amino acid side chains of 7 of the 10 protein C-APC binding candidate residues. Thr-157, Arg-158,
and Glu-159 are not shown since they reside in a region of EPCR that
was not modeled due to lack of homology to mouse CD1. The right
panel shows the epitopes for JRK 1494 (red), JRK 1535 (red), JRK 1513 (white), and JRK 1500 (pink). Based on our three-dimensional molecular model, the
candidate residues for protein C/APC binding are located at the distal
end of the two
-helical segments that form the putative ligand
binding groove.
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Fig. 5.
Molecular model of hEPCR based on the murine
MHC class 1 H-2Kb structure. Left panel, regions in
red are the amino acid side chains of 7 of the 10 protein
C/APC binding candidate residues. Thr-157, Arg-158, and Glu-159 are not
shown, since they reside in a region of EPCR that was not modeled due
to lack of homology to mouse MHC class 1 H-2Kb. Areas shown in
green, many of which are in the groove region, could be
mutated to alanine without loss of APC binding. Regions in
blue, when mutated, result in loss of cell surface
expression and, hence, are likely mutations that influence protein
folding. Right panel, the epitopes for the nonblocking
antibodies are shown in white and pink (JRK 1513 and JRK 1500, respectively) and the epitopes for the blocking
antibodies are shown in red (JRK 1494 and JRK 1535).
Black line, with competitor; gray area, without
competitor.
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Fig. 6.
Protein C activation of 293 cells expressing
EPCR variants and TM. Protein C activation was performed on
confluent 293 cells and on confluent 293 cells expressing either TM or
TM and EPCR variants as described under "Experimental
Procedures." Gray bars, activation rate of protein C;
black bars, activation rate of protein C in the presence of
500 nM inhibitory anti-hEPCR mAb JRK 1494; white
bars, activation rate of GDPC. The bars represent the
mean, whereas the lines above the bars reflect the S.E. of at least two
determinations. The number of EPCR and TM molecules expressed per cell
surface are indicated on the figure as are the EPCR:TM ratios of each
cell line. WT, wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical segments of EPCR.
-helices that sit upon
an eight-stranded
-sheet platform (Fig. 5), a fold that is
characteristic of members of the MHC class1/CD1 family of receptors.
The overall structure of our hEPCR model is similar to that developed
by Villoutreix et al. (39) using the x-ray structure of
mouse CD1 as template.
-helices that line the groove (42). These
interactions are further supplemented by contacts of polymorphic MHC
groove side chains with a few "anchor" side chains in the peptide
(40). The anchor residues of the peptide occupy depressions within the
MHC groove. In contrast, the interaction between CD1 molecules and
lipids involves extensive hydrophobic interactions in deeply buried
depressions within the CD1 groove (43).
3
domain, and its ligands are large proteins rather than short peptides
or lipids. The results of this study indicate that the APC/protein C
binding region of EPCR is located in the distal end of the two
-helical segments that form the putative binding groove. To our
knowledge, this is the first report of such a binding motif for a
member of the MHC class 1/CD1 family of molecules. There are other
examples of ligand binding versatility of the MHC class1/CD1 fold. For
example, the neonatal Fc receptor has a proline residue in the
2
helix that produces a kink in the
2 helix, resulting in a closed
groove (24). Although the peptide binding groove is lost, the neonatal
Fc receptor has evolved to recognize the Fc portion of immunoglobulins
on the side of the Fc receptor. In the case of MIC-A, a
stress-inducible antigen restricted to gut epithelium, the peptide
binding groove is absent due to disordering in one of the
groove-defining helices (44). Potential receptor interaction surfaces
are on the "underside" of the
-sheet platform.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Naomi L. Esmon for critical reading of the manuscript and for many helpful discussions. We are grateful to Nici Barnard in the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by NHLBI, National Institutes of Health Grant P01 HL54804.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.
§ Recipient of a Research Fellowship from the Heart and Stroke Foundation of Canada.
An investigator with the Howard Hughes Medical Institute. To
whom correspondence should be addressed: Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104. Tel.: 405-271-7571; Fax: 405-271-3137; E-mail:
Charles-Esmon@omrf.ouhsc.edu.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M010572200
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ABBREVIATIONS |
---|
The abbreviations used are:
TM, thrombomodulin;
APC, activated protein C;
EPCR, endothelial cell protein C receptor;
hEPCR and mEPCR, human and murine EPCR, respectively;
HBSS, Hanks'
balanced salt solution;
mAb, monoclonal antibody;
GDPC, Gla-domainless
protein C;
Fl-APC, APC labeled at the active site with fluorescein;
ATA-PPACK, N-((acetylthio)acetyl)-FPR
chloromethyl ketone;
MHC, major histocompatibility complex.
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