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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 632-641
Structural and Functional Implications of the Intron/Exon Organization
of the Human Endothelial Cell Protein C/Activated Protein C Receptor
(EPCR) Gene: Comparison With the Structure of CD1/Major
Histocompatibility Complex  1 and 2 Domains
By
Rachel E. Simmonds and
David A. Lane
From the Department of Haematology, Imperial College School of
Medicine, London, UK.
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ABSTRACT |
The endothelial cell protein C/activated protein C receptor (EPCR)
is located primarily on the surface of the large vessels of the
vasculature. In vitro studies suggest that it is involved in the
protein C anticoagulant pathway. We report the organization and
nucleotide sequence of the human EPCR gene. It spans approximately 6 kbp of genomic DNA, with a transcription initiation point 79 bp
upstream of the translation initiation (Met) codon in close proximity
to a TATA box and other promoter element consensus sequences. The human
EPCR gene has been localized to 20q11.2 and consists of four exons
interrupted by three introns, all of which obey the GT-AG rule. Exon I
encodes the 5' untranslated region and the signal peptide, and
exon IV encodes the transmembrane domain, the cytoplasmic tail, and the
3' untranslated region. Exons II and III encode most of the
extracellular region of the EPCR. These exons have been found to
correspond to those encoding the  1 and 2 domains of the CD1/major
histocompatibility complex (MHC) class I superfamily. Flanking and
intervening introns are of the same phase (phase I) and the position of
the intervening intron is identically located. Secondary structure
prediction for the amino acid sequence of exons II and III corresponds
well with the actual secondary structure elements determined for the
1 and 2 domains of HLA-A2 and murine CD1.1 from crystal
structures. These findings suggest that the EPCR folds with a  -sheet
platform supporting two -helical regions collectively forming a
potential binding pocket for protein C/activated protein C.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PROTEIN C anticoagulant system is a
well-established pathway regulating thrombin generation and therefore
clot formation (reviewed in Esmon1 and Simmonds and
Lane2). Regulation is achieved via the degradation of
procoagulant activated factors V and VIII by a serine proteinase,
activated protein C (APC), in conjunction with its nonenzymatic
cofactor, protein S. When thrombin binds to the endothelial cell
transmembrane protein, thrombomodulin, its potent procoagulant
functions are reversed and its substrate specificity is redirected
towards protein C.1,3 Protein C (the zymogen of APC) is
then activated on the surface of endothelial cells by the
thrombin/thrombomodulin complex. Mutations in the genes for
thrombomodulin,4,5 protein C,6 and protein S7 have been identified in patients with venous and/or
arterial thrombosis, highlighting the importance of this pathway.
Recently, this model of protein C activation has been shown to be a
simplification; an additional endothelial cell-specific transmembrane
protein has been identified that binds protein C and APC on the cell
surface.8 This novel protein was named the endothelial cell
protein C/APC receptor (EPCR). Direct binding between protein C and the
EPCR (kd, ~30 nmol/L) has also been demonstrated,9 and this is dependent on the Gla domain of
protein C.8 The EPCR itself does not have any direct
anticoagulant effect in the absence of APC, because its addition to
plasma does not increase the clotting time,10 and APC bound
to the EPCR looses its anticoagulant ability to inactivate activated
factor V.10 However, the EPCR influences the rate of
protein C activation by the thrombin/thrombomodulin
complex.11 If protein C binding to the EPCR is blocked,
then the rate to protein C activation is reduced by approximately 80%.
The important function of the EPCR is thought to be anticoagulant, as
an accessory protein in the activation of protein C.
Thrombomodulin is known to have a uniform distribution on all
endothelial cells, which results in an effective 100-fold decrease in
thrombomodulin concentration in large vessels compared with capillaries
due to geometric considerations.12 On the surface of the
large vessels, this would have been expected to result in inefficient
activation of the protein C anticoagulant pathway, because the affinity
between thrombomodulin and protein C is weak. However, the EPCR has
since been found to be restricted primarily to the surface of the
endothelium of arteries and veins, with very little found on capillary
endothelial cells.13 Together with the high affinity of the
protein C/EPCR interaction, this should result in the effective
localization of protein C on the surface of the endothelium, leading to
an increase in local concentration. It has been suggested that this
ensures efficient protein C activation on the surface of the large vessels.
A soluble form of the EPCR (43 kD) has been identified in human plasma
collected from healthy individuals.14 It circulates at a
concentration of approximately 2.5 nmol/L, well below the kd for the
protein C/EPCR interaction, suggesting that, in healthy individuals,
this form is physiologically unimportant. However, soluble EPCR
purified from plasma can bind both protein C and APC and blocks the
anticoagulant function of APC.14
The human EPCR cDNA is 1.3 kbp in size and encodes a protein of 238 amino acids.8 It has a molecular weight of approximately 46 kD, which is larger than that predicted based on amino acid sequence
(25 kD), probably due to the presence of carbohydrate located at 4 potential N-glycosylation sites in the coding region. After the removal
of 17 residues during processing, the mature protein is predicted to
consist of 221 amino acids.9,14 To date, additional cDNAs
for the murine and bovine EPCR have been described,15
although, until now, the gene encoding the EPCR has not been described
for any species.
The contribution of the EPCR to the regulation of coagulation and the
prevention of thrombus formation in vivo is, as yet, unknown. The
investigations outlined above strongly suggest that it is important.
Dysfunction of the human EPCR gene may lead to failure of endogenous
anticoagulation and thrombosis. Also, at present, there is little
information regarding the three-dimensional structure of the EPCR.
Amino acid sequence homology has been noted,8 which
suggested similarities to the CD1/major histocompatibility complex
(MHC) class I superfamily. The greatest homology is between the EPCR
and CD1d (17% at the amino acid level in humans). To facilitate
additional investigations of the role of the EPCR in physiology and
pathology and to further investigate the domain structure of the
protein, we have studied the organization and established the
chromosomal location of the human EPCR gene.
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MATERIALS AND METHODS |
Amplification of regions of the EPCR gene by polymerase chain reaction
(PCR).
Amplification of DNA samples by PCR was performed using Taq DNA
polymerase and standard methods.16 The template for
amplification was either genomic DNA isolated from peripheral blood
leukocytes of a healthy individual, P1 artificial chromosome (PAC), or
plasmid DNA isolated from transformed bacteria. All amplifications were primed by pairs of chemically synthesized gene-specific 17- or 18-mer
oligonucleotides (Fig 1 and
Table 1). These were designed based on
either the published human EPCR cDNA sequence8 or from
genomic sequence derived as described below. Reaction conditions have
been described elsewhere.17 Exon-specific primer pairs EPCR-3 and EPCR-4 as well as EPCR-5 and EPCR-8 (Fig 1 and Table 1) had
previously been identified. These were capable of amplifying regions of
genomic DNA that corresponded in size and DNA sequence to that expected
from the EPCR cDNA sequence (data not shown).
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| Fig 1.
Schematic representation of the human EPCR gene. The
upper diagram shows the overall intron/exon structure of the gene,
which consists of four exons (I to IV) and three introns (a to c).
Black regions represent the coding regions; white regions represent the
5' and 3' untranslated regions to the left and right,
respectively. The scale of this diagram is given below the gene. The
double headed arrows in the centre represent fragments of the EPCR gene
that were amplified from various templates by PCR. Fragment 3/4
resulted from amplification using primers EPCR-3 and EPCR-4; 5/8
resulted from amplification of EPCR-5 and EPCR-8 (Table 1). These
primer pairs were exon-specific. Fragments 1A/1B, 2C/2B, 3A/3B, and
4A/4B were amplified using primer pairs EP1A and EP1B, EP2C and EP2B,
EP3A and EP3B, and EP4A and EP4B (Table 1). Use of these primers
resulted in the amplification of each exon and its associated
intron/exon boundaries. The lower diagram represents the EPCR
polypeptide. The position of the introns within the amino acid sequence
are represented by dotted lines. The residue number is indicated above
the position of each intron. The signal peptide, extracellular regions,
transmembrane domain, and cytoplasmic tail are also indicated.
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DNA sequence analysis.
All DNA sequence analysis was performed using the dideoxynucleotide
chain termination method18 with the ABI PRISM Big Dye terminator cycle-sequencing ready reaction kit (Perkin Elmer, Applied
Biosystems Division, Foster City, CA) and an automated detection system
(ABI PRISM 373 stretch XL; Perkin Elmer). Each reaction was primed
using a gene-specific 17- or 18-mer oligonucleotide in an appropriate
position/orientation to obtain the DNA sequence of the newly
synthesized strand. To obtain the EPCR gene sequence from cloned
fragments, a primer walking approach was used in which new
oligonucleotides were designed based on the 3' sequence obtained from previous reactions. Initial reactions used the exon-specific primer pairs mentioned above. M13 forward and reverse primers were used
to sequence across vector/insert boundaries. To obtain the DNA sequence
of amplification products, the reactions were primed with the same
oligonucleotides used in the amplification reaction. Sequences were
assembled using Assemblylign software (Eastman Kodak Inc, Rochester,
NY). All DNA sequences reported were determined at least
once in each direction.
Preparation of an EPCR cDNA probe.
The probe used to screen the genomic DNA library, in Southern blots and
colony lifts (see below) was an expressed sequence tag (EST)
representing the human EPCR cDNA, obtained as an I.M.A.G.E. clone.19 It was identified by searching the National Centre for Biotechnology Information EST database with the first 40 nucleotides of the published human EPCR cDNA sequence.8
I.M.A.G.E. clone 327665 was found to have a DNA sequence identical to
the published EPCR cDNA sequence, with the exception of three
nucleotides at positions 679, 1043, and 1044. An additional 44 bp of
5' untranslated sequence was present in the clone and the site of
poly-A addition to the cDNA was different from that previously reported
at nucleotide 1118. This was 16 bp downstream of an alternative
polyadenylation signal sequence (AATAAA) at position 1097 to 1102 in
the cDNA (Fig
2). In view of these small differences, it was felt that the clone
327665 represented the human EPCR cDNA and it is referred to as such
throughout this communication. It should be noted that all cDNA and
amino acid numbering used here is in accordance with Fukudome and
Esmon.8 The probe for subsequent hybridization reactions
was prepared by restriction digestion of I.M.A.G.E. clone 327665 with
Not I and Xho I, releasing a 1.2-kbp fragment that
included the entire coding region of the EPCR. This fragment was
gel-purified and radioactively labeled with 32P-dCTP using
the random priming method [Ready-To-Go DNA kit (-dCTP); Pharmacia,
Uppsala, Sweden]. After removal of unincorporated nucleotides and
primers, the probe was used directly in hybridization reactions.
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| Fig 2.
Complete nucleotide sequence of the human endothelial
cell protein C/APC receptor gene. Numbering is relative to the first
nucleotide of the translation start (Met) codon (+1). Numbers on the
left-hand side denote the number of the first nucleotide on that line.
Uppercase letters denote a sequence that has been isolated as mRNA or
cDNA, lowercase letter denote 5' and 3' flanking sequence
and introns. The bullet point under the sequence represents a
transcription initiation site (C-79). Putative regulatory elements in
the 5' region of the gene are indicated by broken lines
and the name of the element. Two alternative polyadenylation sites in
the 3' region of the gene are also indicated by broken lines. The
exon sequences correspond with the EPCR cDNA sequence previously
published, except for 3 nucleotides (double underlined). Intronic
sequences that are potentially polymorphic are indicated with italics.
Three Alu repetitive elements within introns and in the 3'
flanking sequence are underlined. An EcoRI site (GAATTC) in
exon III is also indicated. CEBP, CAAT enhancer binding protein.
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Determination of the complete nucleotide sequence of the human EPCR
gene.
To obtain the complete sequence of the gene encoding the human EPCR, a
human genomic PAC library was screened for clones that hybridized with
the EPCR cDNA. The library, RPCI1,20 was obtained from the
Human Genome Mapping Project (HGMP) and was constructed in the vector
pCYPAC2. The library was screened by hybridization to the EPCR cDNA
probe using standard techniques.21 Three clones hybridized
strongly: 198-F17, 212-C5, and 212-F6 (data not shown). The presence of
exonic regions of the EPCR gene in these clones was confirmed by
amplification with primers EPCR-3 and EPCR-4 (data not shown).
Southern blot analysis of the genomic PAC clones that hybridized with
the EPCR cDNA probe was performed using standard
techniques.21 PAC DNA was digested with EcoRI and
Not I. Not I was included in the reaction, because
restriction sites for this endonuclease were present in pCYPAC2,
flanking the cloning site. Cleavage at these positions therefore
resulted in the release of the entire genomic DNA insert. Filters were
screened with the EPCR cDNA probe, as described above. PAC 212-C5
contained the largest portion of genomic DNA with the EPCR gene and was
chosen for further analysis. Two fragments of DNA (9.5 and 1.5 kbp)
that hybridized with the EPCR cDNA were identified in this clone (data
not shown). The overall size of the EPCR gene was therefore predicted
to be less than 11 kbp.
The PAC clone 212-C5 was digested with the restriction endonuclease
EcoRI, and all resulting fragments were subcloned into the
cloning vector pGEM-3Z (Promega, Madison, WI) using highly competent
JM109 Escherichia coli (Promega) and standard methods. Clones
with the EPCR gene containing fragments were identified by colony
hybridization.22 The probe in the hybridization was the
EPCR cDNA, which is described above. One clone selected by hybridization, termed EPCRg6, contained solely the 9.5-kbp fragment mentioned above. To isolate the 1.5-kbp fragment, a further subcloning step was required, producing in the clone EPCRg11. The sequence of the
entire EPCR gene was obtained by sequencing clones EPCRg6 and EPCRg11
in both directions, as described above. Intron/exon boundaries were
located by comparison of the cDNA and genomic DNA sequences. The
integrity of exon sequence was investigated by amplification of each
identified exon and its associated intron/exon boundaries. The
templates were genomic DNA samples from healthy individuals, which were
amplified using primer pairs EP1A and EP1B, EP2C and EP2B, EP3A and
EP3B, and EP4A and EP4B (Fig 1 and Table 1). Amplification products
were sequenced as described above.
Determination of transcription initiation sites by 5' rapid
amplification of cDNA ends (RACE).
The transcription initiation site(s) were identified by 5' RACE
using a commercially available kit (5' RACE system Version 2;
GIBCO BRL, Gaithersburg, MD). Poly-A+ RNA was isolated from
human umbilical vein endothelial cells using the Oligotex direct mRNA
kit (QIAgen, Valencia, CA). This was used as a template for
first-strand cDNA synthesis in a reaction primed by the oligonucleotide
RACE-2 (Table 1). After removal of RNA from the reaction and poly-C
anchor addition, cDNA was then used as the template in heminested PCR,
primed by the Abridged Anchor Primer supplied with the kit and the
oligonucleotide RACE-9 (Table 1). Amplification products were diluted 1 in 500 and used as the template in a further nested amplification
reaction. Here, the primers used were Abridged Universal Amplification
Primer and the oligonucleotide RACE-11 (Table 1). The products of
5' RACE were analyzed by electrophoresis on a 2% agarose gel and cloned directly into the TA cloning vector pCR 2.1-TOPO (Invitrogen, Carlsbad, CA) using the topoisomerase method. The complete DNA sequence
of the 5' RACE product(s) was then obtained, as described above.
Determination of the chromosomal location of the EPCR gene.
The chromosomal location of the EPCR gene was determined by use of a
human monochromosomal somatic cell hybrid DNA panel obtained from
HGMP.23 This consisted of a panel of DNA samples from
mouse/human or hamster/human somatic cell hybrids, with each being
almost monochromosomal. DNA in the panel was amplified by PCR, as
described above, using primers EPCR-3 and EPCR-4 (Fig 1 and Table 1).
Amplified DNA was analyzed by electrophoresis on a 1% agarose gel. The
regional assignment of the EPCR gene was determined by fluorescent in
situ hybridization (FISH), which was performed according to standard procedures.24 Slides were prepared from
phytohemagglutinin-stimulated peripheral blood cultures. PAC DNA was
labeled with biotin by nick-translation and was detected with
fluoroscein isothiocyanate after hybridization. The chromosomes were
background-stained with 4,6-diamidino-2-phenylindole (DAPI). Images
were captured using an Olympus Vanox fluorescence microscope equipped
with a CCD camera and SmartCapture software (Vysis, Downers Grove,
IL). The DAPI-banding pattern was enhanced and converted
to greyscale with SmartCapture software to enable chromosome and band assignation.
Computer analysis.
Potential transcription factor binding sites were identified in the
5' flanking sequence of the EPCR gene using a transcription factor database TRANSFAC25 (Molecular Bioinformatics of
Gene Regulation, Braunschweig, Germany) in conjunction with
MatInspector software (GSF-National Center for Environment and Health,
Neuherberg, Germany).26 The secondary
structure of the EPCR, based on amino acid sequence, was predicted by
the use of six different computational algorithms:
PHD,27,28 ssp,29 Gibrat,30
Levin,31 DPM,32 and SOPMA.33 The
consensus for assigning a secondary structure element ( -helix [H]
or -sheet [E]) to a particular residue was if three or more
algorithms predicted that element. Amino acid sequences were optimally
aligned using clustalW software.34 To calculate the degree
of residue conservation between exons II and III of the EPCR and the
1 and 2 domains of CD1 proteins and HLA-A2, each pair of
sequences was first optimally aligned. The number of conserved
(identical) residues was then counted and expressed as a percentage of
the total compared regions (including gaps introduced for optimal alignment).
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RESULTS |
Organization and complete nucleotide sequence of the human EPCR gene.
Approximately 6 kbp of cloned genomic DNA (EPCRg6 and EPCRg11) has been
sequenced, including 5' and 3' flanking sequence, exons,
and introns (Fig 2). The nucleotides of the gene have been numbered
relative to the first nucleotide of the translation initiation (Met)
codon. The EPCR gene consists of four exons of 138, 252, 279, and 659 bp (determined from both cloned and amplified genomic DNA; Figs 1 and
2). Exon I (amino acids 1 to 24) encodes the 5' untranslated
region, the signal peptide, and 7 additional residues. Exons II (amino
acids 24 to 108) and III (amino acids 108 to 201) encode the
extracellular region of the EPCR. Exon IV (amino acids 201 to 238)
encodes an additional 10 residues of the extracellular portion of the
EPCR, the transmembrane domain, the cytoplasmic tail, and the 3'
untranslated region. The exons are interrupted by three introns of
2477, 1217, and 251 bp (Fig 2). All of the splice donor and acceptor
sites obey the GT-AG rule and largely agree with consensus
sequences35 (Table 2). All
splice junctions of the EPCR gene are in phase I, ie, after the first
nucleotide of the triplet codon for amino acids 24, 108, and 201. Introns a and b both contain an Alu repetitive element present on the complementary strand. These span from 1230 to 1590 in intron a and from
3417 to 3747 in intron b (Fig 2).
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Table 2.
Comparison of the Intron/Exon Boundaries of the Human
EPCR Gene With the Consensus Sequences for These Regions
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Two potentially polymorphic sites have been identified within the
intronic regions of the human EPCR gene (Fig 2). The sequence of the
amplified genomic DNA fragment containing exon II was heterozygous at
position 2532, 16 bp upstream of the start of exon II, where both C and
T nucleotides were observed. This polymorphism is close enough to the
intron/exon boundary to suggest potential differences in splicing
efficiency, although this has not been tested. The amplified genomic
DNA fragment containing this sequence variation was also heterozygous
at position 2894, 85 bp downstream of the end of exon II, where both A
and G were observed. This latter change is unlikely to have an effect
on splicing.
The clone EPCRg6 contained sequence located upstream of an
EcoRI site (GAATTC) located in exon III of the EPCR gene,
whereas EPCRg11 contained sequence downstream of this position (Fig 2). The absence of additional gene sequence between EPCRg6 and EPCRg11 was
confirmed by amplification and sequencing of a genomic DNA sample with
EP3A (located in EPCRg6) and EP3B (located in EPCRg11).
To identify a transcription start site for the human EPCR gene,
5' RACE analysis was used. After two rounds of heminested PCR
amplification, an amplification product of approximately 240 bp was
identified by agarose gel electrophoresis (data not shown). Using DNA
sequence analysis, the transcription initiation site was identified as
nucleotide C-79 for this fragment (Fig 2). Under lower stringency
amplification conditions, products corresponding to transcripts
terminated at A-83 were also found. The entire sequence of the cloned
fragment corresponded to EPCR gene sequence obtained from EPCRg6. The
transcription start site is 26 bp downstream of an SP1 binding site (on
the reverse strand, CCGCCC) and 84 bp downstream of a TATA box element
(TATAA). Additional potential transcription factor binding sites were
identified in the 5' flanking sequence of the EPCR gene using
computer analysis. Complete agreement with consensus sequences of the
core binding sites and surrounding nucleotides was found in several
locations (Fig 2). In the 3' region of the human EPCR gene, there
are two alternative polyadenylation sites (AATAAA) at positions 5017 and 5186 (Fig 2). There is an additional Alu repeat, also on the
complementary strand, in this region of the gene (position 5280 to
5594; Fig 2).
The EPCR gene was initially assigned to a chromosome by amplification
of a monochromosomal cell hybrid DNA panel with exon-specific primers
(EPCR-3 and EPCR-4; Fig 1 and Table 1). Amplification of mouse and
hamster control DNAs was not possible under the conditions used
(Fig 3, lanes 26 and 27). An amplification
product corresponding to the EPCR gene was observed in the human
genomic DNA controls (Fig 3, lanes 25 and 28) and also the hybrid that
contained chromosome 20 (Fig 3, lane 20). This hybrid may also contain
fragments of other chromosomes. However, the absence of an
amplification product in the hybrids containing all other whole
chromosomes confirmed that the EPCR gene is located on chromosome 20. This finding was confirmed by FISH and further localized the gene to
position 20q11.2, near the centromere, in all of the cells examined
(Fig 4). Results for PAC 212-C5
hybridization to a single typical cell have been shown here. These
findings were reproduced in all cells examined and when another PAC
clone (198-F17) was used. The single hybridization position confirmed
that these clones were not chimeric.
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| Fig 3.
Chromosomal assignment of the human EPCR gene. A human
monochromosomal somatic cell hybrid DNA panel and controls were
amplified by PCR to determine the chromosomal location of the EPCR
gene. The exon-specific primers EPCR-3 and EPCR-4 (Table 1) were used
and the products were separated on a 1% agarose gel. The templates in
lanes 1 to 24 were mouse/human or hamster/human somatic cell hybrids
containing chromosomes 1 to 22, X and Y chromosomes, respectively.
Lanes 25 and 28 were human genomic DNA samples; one was provided with
the panel (lane 25) and one was our internal control (lane 28). Lanes
26 and 27 were mouse and hamster genomic DNA, respectively. The marker
lane (M) is pHC624/Taq I-pMJ/Nci I (Advanced
Biotechnologies, Leatherhead, UK). This contains fragments of 1444, 696, 475, 411, 358, 352, 212, and 96 bp (the 358- and 352-bp fragments
comigrate).
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| Fig 4.
Determination of the regional assignment of the EPCR gene
by FISH. Slides were prepared from phytohemagglutinin-stimulated
peripheral-blood cultures and hybridized with biotin labeled PAC DNA.
The chromosomes were background-stained with DAPI. The DAPI-banding
pattern was enhanced and converted to greyscale with SmartCapture
software to enable chromosome and band assignation (left panel).
Hybridized PAC DNA was detected using fluorescein isothiocyanate (right
panel). Specific fluorescent signals and the locus 20q11.2 are
indicated by arrows.
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Similarity between the human EPCR, CD1, and MHC class I genes.
An optimal alignment of the amino acid sequences encoded by exons II
and III of the EPCR gene and those exons that encode the 1 and 2
domains of murine CD1.1 and HLA-A2 is displayed in
Fig 5. Interestingly, the location of the
intervening intron is identical and those of the flanking exons are
similar. The phase of these introns in murine CD1.136 and
HLA-A237 are identical to human EPCR (see above and Fig 2).
The secondary structure elements of these exons of the EPCR gene (II
and III), once expressed, were predicted using six different
computational algorithms (see Materials and Methods). The consensus
predicted secondary structure from this analysis is also displayed in
Fig 5, together with the secondary structure elements of murine CD1.1
and HLA-A2 taken from the previously resolved crystal
structures.38,39 It can clearly be seen that the secondary
structure motifs found in murine CD1.1 and HLA-A2 are predicted to be
conserved in the EPCR. Furthermore, two cysteine residues in the 2
domains of both murine CD1.1 and HLA-A2 (known to form a disulphide
bridge from both crystal structures) are conserved in the precisely the
same positions in the EPCR.
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| Fig 5.
Similarities between the EPCR and the 1 and 2
domains of CD1 and MHC class I antigen-presenting molecules. The amino
acids encoded by exons II and III of the human EPCR gene (hEPCR) and
the 1 and 2 domains of murine CD1.1 (mCD1.1, homologous to human
CD1d40) and HLA-A2 (hHLA-A2) have been optimally
aligned using clustalW.34 The position of flanking and
intervening introns are indicated with a solid line. In all cases, the
introns are in phase I (ie, after the first nucleotide of the triplet
code). Secondary structure elements are indicated by light (-helix)
and dark (-sheet) shaded areas. For CD1.1 and HLA-A2, these were
taken from crystal structures.38,39 For hEPCR, these
represent the consensus from six secondary structure prediction
algorithms (see Materials and Methods). The location of a disulphide
bond identified in the crystal structures of murine CD1.1 and HLA-A2 is
indicated by a solid line. This is likely to link the two highly
conserved cysteine residues in the EPCR.
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The conservation between the amino acids encoded by exons II and III of
the EPCR gene and between the 1 and 2 domains of murine CD1.1 and
HLA-A2 is summarized in Table 3. Human CD1d has been included in this analysis because it has the closest similarity to the EPCR8 and it is the human homologue of
murine CD1.1,40 with 59% conservation in the 1 and 2
domains (Table 3). For all other pairwise comparisons, the 1 and
2 domains have between 11% and 29% conservation when these domains
are considered separately and between 16% and 27% if they are
considered together (Table 3). The proportion of conserved residues
between a CD1 and an MHC class I protein are therefore of the same
order as that found between either the EPCR and CD1 or EPCR and MHC
class I in the 1 and 2 domains. The greatest conservation in
these regions was still found with human CD1d (Table 3).
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DISCUSSION |
The human EPCR gene spans approximately 6 kbp genomic DNA and consists
of four exons (I to IV) interrupted by three introns (a to c). In the
5' region of the gene, we identified a transcription start site
by 5' RACE. This technique uses a gene-specific primer to
generate first-strand cDNA and further gene-specific primers to amplify
this. The template for first-strand cDNA synthesis was
poly-A+ RNA purified from human umbilical vein endothelial
cells, which express the EPCR on the cell surface.8 The
identification of sequence that corresponded to the human EPCR gene
(and previously unidentified as cDNA) indicated that the gene is active
in these cells. The presence of multiple potential transcription factor binding sites surrounding the transcription start site further emphasizes its functionality. The transcription initiation site was
localized to nucleotide C-79 (Fig 2). This is 84 bp downstream of a
TATA box element (TATAA), which may be important in initiation of EPCR
gene expression. Complete agreement with consensus sequences of the
core binding sites and surrounding nucleotides for other transcription
factors was found in several locations (referred to by the 5'
nucleotide of the core consensus). Of particular interest were SP1
binding sites at positions  236 and 105, two CAAT box
elements (positions 436 and 243), and a CAAT enhancer binding protein recognition sequences (positions 462). Also, three Ets-1 binding sites (positions 293, 194, and
3) were found that matched the consensus sequence
G/CA/CGGAA/TGC/T in 7, 6, and 8 nucleotides, respectively (Fig 2).
During computer analysis of the putative promoter region, a TATA box
element containing all 15 nucleotides of the consensus was identified
at position 474 (Fig 2). A possible translation initiation codon
(Met) is present at position 374, approximately 100 bp
downstream of this element, but its significance is uncertain. It is
unlikely that this TATA box element is important for EPCR expression
due to its distance from the transcription initiation site (389 bp). No
5' RACE products were identified corresponding to initiation near
this TATA box and including the Met codon of the EPCR gene. It
therefore seems that the TATA box element at 474, if active, is
unrelated to the EPCR.
Similarity between the EPCR and the CD1/MHC class I superfamily has
previously been noted,8 particularly in the 1 and 2
domains of these latter proteins. This lead to the suggestion that the
EPCR extracellular region also consisted of two discreet modules.
However, detailed consideration of the EPCR structure was not possible,
because the boundaries of structural units were undefined. The
relationship between the EPCR and antigen-presenting molecules has been
confirmed and extended in the present study.
Exons II and III of the EPCR gene code for amino acids 24 to 201 of the
EPCR protein (Fig 2) and account for almost the entire extracellular
domain. The amino acids encoded by these regions will be referred to as
the extracellular region of the EPCR in this discussion, although it
should be noted that an additional 7 and 10 residues of the EPCR
(encoded by exons I and IV, respectively) are likely to be exposed on
the cell surface. The flanking and intervening introns of exons II and
III are all in phase I. This is identical with the phase of the
corresponding introns (ie, adjacent to the 1 and 2 domains) of
all the CD1 and MHC class I genes studied to date.40 When
the amino acids in the 1 and 2 domains (defined by the exons
encoding them) of a typical CD1 (murine CD1.1, homologue of human CD1d)
and MHC class I protein (HLA-A2) were optimally aligned with the
extracellular region of the EPCR, the position of the intervening
intron was found to be in an identical position (Fig 5).
A previous search with the complete polypeptide chain of the human EPCR
identified the greatest sequence similarity with human CD1d,8 with an overall residue conservation of 17%.
Comparison of the 1 and 2 exons of CD1d36 with exons
II and III of the EPCR (this report) shows 27% conservation (Table 3).
Although these figures are quite low, previous studies have shown that these functional protein domains can tolerate a large variation in
amino acid content without affecting their overall structure. For
example, murine CD1.1 and HLA-A2 have 16% amino acid sequence conservation in the 1 and 2 domains (Table 3). The x-ray crystal structures of both these proteins have been
determined.38,39,41 Given the low amino acid conservation
between the two, an unexpected structural similarity is
observed.39
Crystal structures of several other MHC class I proteins have been
determined, although at present, this has not been achieved for the CD1
family. In all structures, the 1 and 2 domains have a
characteristic three-dimensional structure, located distal from the
transmembrane region and separated from it by the 3 domain and -2
microglobulin.42 In simplified terms, 1 and 2 form a
platform of 7 or 8 -sheets supporting 2 -helical regions (1 from
1 and 1 from 2). The exons encoding each of the 1 and 2
domains encode amino acids that adopt a secondary structure organization EEE(E)H (where E is -sheet and H is -helix; Fig 5).
There is a conserved disulphide bond between a cysteine residue adjacent to the first -sheet and -helical region of the 2
domain. The peptide binding pocket is provided by the groove between
the 2 -helical regions and residues of the -sheets that are
exposed to solvent at the bottom of the groove.42
The degrees of amino acid conservation between the human EPCR
extracellular region and the murine CD1.1 or HLA-A2 1 and 2 domains are of the same order as those between HLA-A2 and murine CD1.1
(Table 3). Like these latter two proteins, and despite the low amino
acid conservation, the secondary structure seems to have been conserved
for the EPCR: secondary structure prediction for the EPCR (Fig 5)
corresponds well with the determined secondary structure elements of
the related proteins shown by the crystal structures, ie, in the
pattern HEEEEH (1) then EEEHH (2). Both conserved cysteine
residues involved in the 2 disulphide bond of CD1/MHC class I
proteins are also present in the EPCR and are highly likely to be
bonded similarly (Fig 5). Taking all of this information together, it
can be predicted that the structure of the EPCR is similar to that of
the 1 and 2 domains of the CD1/MHC class I superfamily. The
protein C/APC binding site may therefore be provided by a groove
similar to that found in CD1/MHC class I proteins. It also seems likely
that the EPCR and CD1/MHC class I are evolutionarily related.
The similarity between the EPCR and antigen-presenting molecules may
well extend to the signal peptide (ie, exon I; data not shown),
although the more widely conserved nature of this structural unit makes
this difficult to evaluate. However, there is no region homologous to
the 3 domain in the EPCR gene, and in CD1/MHC class I genes, the
transmembrane, cytoplasmic, and 3' untranslated regions are
encoded by between 2 and 4 exons.40,43 The EPCR gene may therefore have evolved by selective insertion of the 1 and 2 domains and possibly the signal peptide. The greater similarity between
the EPCR and CD1d suggests that duplication/insertion occurred after
the divergence of the CD1 gene family. It might be expected that the
Alu repetitive elements identified here in the intronic sequences of
the EPCR gene played a role in the insertion event; however, no Alu
repetitive elements flanking the 1 and 2 domains of the human CD1
genes have been reported.36,44 This implies that the Alu
elements were inserted at a later time. This is supported by the
finding that the intron between 1 and 2 is much larger in the
EPCR gene (1.2 kbp compared with ~0.5 kbp in the CD1
family36,44). It will be interesting to see whether Alu
elements are present in analogous positions in the EPCR gene of other species.
We have localized the EPCR gene to chromosome 20 at position q11.2
(Figs 3 and 4). The CD1 and MHC gene clusters are found on chromosomes
1 and 6, respectively, and are therefore not expected to be linked to
the EPCR. However, the gene for thrombomodulin, the other endothelial
cell receptor essential for protein C activation, is also located on
chromosome 20 in position 20p12 to cen.45,46
The predicted structure of the extracellular region of the EPCR in this
study gives a basis for further experiments into its function.
Furthermore, the complete nucleotide sequence will facilitate clinical
studies into the role of the EPCR in pathological states.
| |
NOTE ADDED IN PROOF |
The complete nucleotide sequence of the murine EPCR gene has recently
been published: Liang Z, Rosen ED, and Castellino FJ: Thromb Haemost
81:585, 1999.
| |
ACKNOWLEDGMENT |
The authors thank Dr Andrew Chase and Antonella Adami for help with FISH.
| |
FOOTNOTES |
Submitted January 22, 1999; accepted March 17, 1999.
Supported by a grant from the British Heart Foundation (Grant No.
PG/97029).
The nucleotide sequence reported here has been submitted to GenBank
with accession no. AF106202.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rachel E. Simmonds, PhD,
Department of Haematology, Imperial College School of
Medicine, Charing Cross Campus, St Dunstan's Road, London W6
8RP, UK; e-mail: r.simmonds{at}ic.ac.uk.
| |
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ABSTRACT
INTRODUCTION