(Received for publication, December 1, 1994; and in revised form, January 9, 1995)
From the
Recently, we identified and cloned a human endothelial cell protein C/activated protein C receptor (EPCR). EPCR was predicted to be a type 1 transmembrane glycoprotein and a novel member of the CD1/major histocompatibility complex superfamily with 28% identity with CD1d. Even greater homology (62% identity) was detected with the murine protein, CCD41, which was previously characterized as a centrosome-associated, cell cycle-dependent protein. This raised the possibility that CCD41 was the murine homologue of EPCR. To address this possibility, to better understand structure-function relationships, and to facilitate physiological experiments on EPCR function, we cloned and sequenced murine and bovine EPCR from endothelial cell cDNA libraries. The nucleotide sequence of murine EPCR and CCD41 exhibited five differences corresponding to one base change, three single-base insertions, and one base deletion in the protein coding region. As a result, the predicted structures of EPCR and CCD41 differed in their amino and carboxyl termini but were identical in the central portion of the coding sequence. Based on comparison of the murine, bovine, and human EPCR sequences and the regions where discrepancies between murine EPCR and CCD41 were detected, we believe that CCD41 is probably identical to murine EPCR and that the reported sequence differences are likely the result of compression on the sequencing gel. Compared with human EPCR, the murine and bovine sequences were 69 and 73% identical, respectively, and 57% of the residues were identical between all three species. Both bovine and murine EPCR could bind human activated protein C when the cDNA clones were transfected into 293T cells. Like human EPCR, of the cell lines tested, the murine EPCR message was restricted to endothelium. Cloning of the murine and bovine homologue of EPCR will facilitate in vivo and in vitro studies of the role of EPCR in the protein C pathway.
Protein C is the zymogen of the key anticoagulant enzyme,
activated protein C (APC). ()Protein C is activated by the
thrombin-thrombomodulin (TM) complex on endothelial cell surfaces
(reviewed in (7) ). APC forms complexes with protein S and
inactivates the coagulation factors Va and VIIIa. Protein C, APC, and
protein S bind specifically to cell surfaces such as platelets (8, 9, 10, 11) and endothelial
cells(12, 13) . In a previous study, we identified a
specific, saturable, and calcium-dependent binding site for protein
C/APC on cultured HUVEC (K
= 30
nM, 7,000 sites/cell). Labeled APC was displaced by both
unlabeled APC and protein C equally but not by factor X or
protein S, two structurally similar, Gla domain-containing proteins.
Therefore, the binding sites were specific for protein C and APC. Since
APC blocked at the active center with covalent inhibitors can bind to
the receptor, it is likely that the APC is proteolytically active.
Expression cloning revealed a 1.3-kb cDNA clone, which was capable of
protein C and APC binding when transfected into mammalian
cells(14) . Of the cell types tested, only endothelium
expressed significant APC binding capacity and message levels.
Therefore, we designated the molecule as an endothelial cell protein
C/APC receptor (EPCR). Like TM, the APC binding function and message
expression of EPCR were down-regulated when endothelium was exposed to
tumor necrosis factor (TNF)
.
The predicted protein structure
of EPCR was that of a type 1 transmembrane glycoprotein with 238 amino
acids containing a signal sequence at the amino-terminal, a
transmembrane domain near the carboxyl-terminal, a short cytosolic
domain, and four potential N-glycosylation sites. Protein data
base searches indicated significant homology with CD1/MHC molecules.
The extracellular domain of the CD1/MHC family is composed of three
domains termed 1,
2, and
3(15) . In EPCR, the
extracellular region was predicted to contain only two domains
corresponding to the
1 and
2 domains but apparently lacks the
third domain, also known as the immunoglobulin domain. Therefore, EPCR
appeared to be a novel member of this family.
The physiological
function of EPCR is unknown, but based on homology with the CD1/MHC
family, it is likely to be involved in regulating inflammation. This
prediction is consistent with in vivo studies that suggest
that protein C/APC may be a negative regulator of inflammatory injury.
Specifically, APC has been shown to block the lethal effects of Escherichia coli infusion in baboons(1) , and blocking
the protein C pathway increases the circulating levels of tumor
necrosis factor that occur in response to low level E. coli infusion(2) . Preliminary clinical studies suggest that
protein C infusion can improve the clinical outcome of patients with at
least certain forms of septic shock (3, 4). In addition, protein C and
APC have been shown to inhibit selectin-mediated adhesion of
neutrophils to endothelium(5) , and APC has been reported to
diminish TNF secretion from monocytes exposed to endotoxin(6) .
EPCR is a candidate to be involved in these functions since it is a
receptor for APC, has homology with the CD1/MHC molecules, and is
down-regulated by TNF.
In addition to the homology to the CD1/MHC family, the EPCR cDNA exhibited 75% identity in nucleotide sequence and 62% in protein sequence with a murine protein, CCD41(16) , raising the possibility that EPCR is the human homologue of CCD41. However, the characteristics of CCD41 were quite different from those of EPCR. CCD41 was characterized as a centrosome-associated protein that was prevalent at the G2/M phase of the cell cycle. Comparison of the predicted amino acid sequences of the mature CCD41 and EPCR proteins revealed that the amino-terminal region and the cytosolic tail were quite different, while the middle portions of the molecules were nearly identical. CCD41 was predicted to be involved in cell cycle regulation because it was originally cloned from libraries enriched in cell cycle-specific messages, and it contained both a PEST motif (Pro-, Glu-, Ser-, and Thr-rich region) and potential phosphorylation sites. The PEST motif and the phosphorylation sites were not conserved in human EPCR. The high degree of homology between human EPCR and CCD41 raised the question of whether EPCR structure and function were conserved among different mammalian species. To resolve these issues, we cloned and expressed the murine and bovine EPCR proteins and demonstrate that the sequence of murine EPCR, which is shown to bind human APC, is distinct from that reported for CCD41.
The transformed bacteria (2
10
) were cultured on eight dishes (150 mm) of LB
plates containing ampicillin. Screening by colony hybridization was
carried out on a Hybond-N nylon membrane filter (Amersham Corp.) by
standard methods(17) . An EcoRI fragment (560 bp) from
the 5`-end of human EPCR cDNA was labeled with
-[
P]dCTP (Amersham) using a multi-prime
labeling kit (Amersham). High stringency conditions were used for
hybridization. Specifically, hybridization was performed at 42 °C
in 50% formamide containing buffer. Washing was carried out at 45
°C in 5
SSC containing 0.1% SDS.
Figure 1: Nucleotide sequence and predicted protein sequence of murine EPCR. A, the nucleotide sequence of murine clone MEC4 is shown above the predicted amino acid sequence. The putative signal sequence is underlined. The transmembrane region is underlinedtwice. Potential N-glycosylation sites are boxed. Extracellular cysteine residues are circled. B, hydropathy plots of murine EPCR were generated by the method of Engelman et al.(27) (solidline) and Kyte and Doolittle (28) (dottedline). The signal sequence and transmembrane region are indicated with the solidbars. The potential N-glycosylation sites are indicated by a circledN.
The nucleotide sequence was almost identical with that of CCD41 (Fig. 2A). Only five single-base differences were detected. When differences in the sequences were observed, we used at least two and usually all three of the sequencing methods described under ``Experimental Procedures'' to confirm the differences. The first difference was one base change from T to C at position 75 in the murine EPCR nucleotide sequence. The substitution was in the second base in the codon for Pro in EPCR and Leu in CCD41 corresponding to amino acid residue seven in the signal sequences of both proteins (Fig. 2B). In the next three differences, the C base was inserted in the murine EPCR sequence at positions 105, 133, and 195. These insertions resulted in different usage of the coding frame, leading to major differences between the protein sequences of EPCR from residues 18 to 47 and of CCD41 from residues 18 to 46 (Fig. 2B). The third insertion resulted in the realignment of the reading frame. As a result, the middle portion of the two molecules have identical sequences. Relative to the nucleotide sequence of CCD41, the murine EPCR cDNA also exhibited a deletion of 1 C base between base 775 and 776. The three separate sequencing methods described under ``Experimental Procedures'' all failed to detect the C base found in the CCD41 sequence. This 1-base deletion resulted in the carboxyl-terminal sequence of murine EPCR (Arg-Arg-Cys) being different from that of CCD41 (Arg-Leu-Leu-Ile-Ile). The sequences of the cytoplasmic tails of murine and human EPCR were identical.
Figure 2: Comparisons of the nucleotide and predicted amino acid sequences of murine EPCR and CCD41. A, comparison of the nucleotide sequences of murine EPCR (mEPCR) and CCD41. Sites of differences in the nucleotide sequences are indicated by the numbers marked with the asterisk above the mEPCR sequence. B, comparison of the amino acid sequences. The predicted amino acid sequence of murine EPCR (upperline) is compared with that of CCD41 (lowerline).
Protein data base searches revealed that the extracellular domain of
murine EPCR, like its human counterpart, had significant homology with
1 and
2 domains of the CD1/MHC class I family. For instance,
28% identity was observed between murine EPCR and human CD1d.
Figure 3: Nucleotide sequence and predicted protein structure of bovine EPCR. A, nucleotide and predicted amino acid sequence of bovine EPCR clone, BEC3. The putative signal sequence, transmembrane domain, N-glycosylation sites, and extracellular cysteine residues are indicated in the same manner as Fig. 1. B, hydropathy plots of bovine EPCR were constructed as in Fig. 1. The signal sequence and transmembrane region are indicated by solidbars, and the potential N-glycosylation sites are indicated by a circledN.
Figure 4: Amino acid sequence comparisons of human, bovine, and murine EPCR. Amino acid sequences of human (firstline), bovine (secondline), and murine (thirdline) are compared. The sequences conserved in these three species are boxed.
The inserts of the other two clones, BEC5 and BEC9, were 1.8 kb instead of the 1.1-1.4 kb characteristic of the majority of the inserts. To identify the basis for these differences, the nucleotide sequence of BEC5 was determined. The sequence at the 5`- and 3`-end was identical with EPCR, except that a 259-bp insertion was found between position 745 (G) and 746 (G) (Fig. 5). The sequence at the 5`-end of the insertion was GT, and the sequence at the 3`-end was AG. Therefore, this clone probably arose due to an alternative splicing event in which the intron was not removed. As a result, a stop codon was detected prior to the membrane-spanning domain. Thus, the predicted protein structure appears to be that of a soluble form of the receptor. We are yet to identify a comparable alternatively spliced message in human or murine EPCR.
Figure 5: Nucleotide and predicted protein sequence of the larger bovine EPCR clone. The sequence of the 1.8-kb bovine clone (BEC5) was determined. The nucleotide and predicted protein sequence, which differed from the smaller clones, is shown in the figure. The nucleotide differences are indicated by Intron above the sequence and by lowercaseletters, and the protein coding differences are indicated by underlining. The probable GT and AG sites that were not removed by message splicing are indicated by outlining the gt and ag. The stop codon for protein translation is indicated by a star.
Figure 6: Flow cytometric analysis of Fl-APC binding. cDNAs of bovine (upper) and murine (middle) EPCR were transfected into 293T cells. After 48 h, Fl-APC binding in the absence (brokenlines) and presence of 1.3 mM calcium (solidlines) was determined by flow cytometric analysis. The dottedlines indicate the background (293T cells incubated without Fl-APC). As a control, transfected cells with pEF-BOS without the insert were used. For details, see ``Experimental Procedures.''
Figure 7:
Northern blot analysis of murine EPCR.
Northern blot analysis was performed with RNAs from various murine cell
lines (each 15 µg). As the probe, a 969-bp fragment of murine EPCR
was used (upper). The membrane was rehybridized with a control
probe of -actin (lower).
Sequence comparisons among proteins of different species can make a number of significant contributions, especially when the structure-function relationships of the protein in question remain to be elucidated. In the case of EPCR, the analysis of the murine EPCR was particularly germane since the data base search had revealed that the sequence of the human EPCR was highly homologous to a murine protein, CCD41, previously described as a cell cycle-specific centrosome-associated protein. In human endothelial cell cultures, EPCR is expressed on the cell surface and is capable of binding protein C/APC. Therefore, identification of the murine EPCR gained added importance for determining whether EPCR was conserved structurally and functionally across species. This was especially relevant since, if CCD41 were the murine homologue of human EPCR, it would suggest major differences in apparent function of the proteins between species. By hybridization screening, four independent murine clones were isolated and were found to contain identical nucleotide sequences. These clones appear to code for murine EPCR because of the significant structural homology with human and bovine EPCR (Fig. 4), the ability of cells transfected with these cDNAs to bind APC (Fig. 6), and the endothelium-specific message expression (Fig. 7). We identified only five nucleotide differences between murine EPCR and CCD41. This extreme similarity in nucleotide sequence suggests that murine EPCR and CCD41 are identical proteins and that the differences were likely to have arisen from sequencing or cloning errors arising at these limited number of sites. Since murine, bovine, and human EPCR are so homologous and since we used multiple approaches to sequence murine EPCR, we presume our sequence to be correct. We cannot, of course, exclude the possibility that CCD41 is a distinct gene product that we simply failed to identify.
If murine EPCR and CCD41 are identical, the question
arises as to whether EPCR is a centrosome-associated protein as
characterized in the CCD41 report. The nuclear localization and
centrosome association of CCD41 were demonstrated by using antibodies
against bacterially expressed recombinant protein. The fusion protein
contained -galactosidase, part of the signal peptide of CCD41, and
the mature protein sequence. It is possible that the antibodies react
with sequences present in this fusion protein that are absent from the
mature molecule and that these cross-react with other proteins within
the cell. In support of the possibility, the antibodies prepared
against the CCD41 fusion protein recognized a 50-kDa protein in cell
lysate, but the molecular size of the in vitro translation
product was smaller than 45 kDa(16) . When appropriate
antibodies to murine and human EPCR become available, the localization
of EPCR should be reexamined.
Among the cell lines tested, message and expression of murine EPCR, like its human counterpart, seemed to be relatively specific for the endothelium. On the other hand, CCD41 was cloned from a cDNA library of Ehrlich ascites tumor cells. In the case of human EPCR, the message expression was readily demonstrable only with endothelial cells, but a weak signal could also be detected with an osteosarcoma cell line, HOS, and lymphoma line, U937. Assuming that CCD41 and murine EPCR are identical, abnormal expression of EPCR might be caused due to transformation of the cells. In fact, TM (21) and several proteases (22) that modulate blood coagulation have been reported to be expressed in tumor cell lines but to be essentially absent from normal cells from which the tumor lines originated.
CCD41 was originally isolated as a G2 phase prevalent
clone by differential screening with cell cycle phase-specific
probes(16) . Given that EPCR and CCD41 appear to be identical
proteins, it seems quite likely that EPCR message expression in
endothelium is also regulated by the cell cycle. TM and EPCR are both
down-regulated by TNF-. In addition to sharing this property, TM
expression has also been reported to be cell cycle
dependent(23) .
The relationship of EPCR to the anticoagulant functions of the endothelium remains unknown. With respect to the protein C pathway, the endothelium catalyzes protein C activation mediated by the thrombin-thrombomodulin complex and facilitates factor Va inactivation mediated by APC. In the case of bovine endothelium, protein S was found to be essential for both the binding of APC and rapid factor Va inactivation(13) . On the other hand, human APC binding to endothelial cells was protein S independent(14, 24) . Cultured human endothelium also exhibits less dependence on protein S to accelerate factor Va inactivation(25) . Whether EPCR expression levels are related to these specific differences can now be addressed.
Comparison of
the predicted sequences of human, bovine, and murine EPCR reveals
several conserved structural features of this molecule. Based on the
observation that all three species bind human APC, it is likely that
the APC binding site is conserved. Absolute conservation of the primary
sequence of surface regions suggests that these regions are involved in
critical interactions, possibly including protein C binding. The 2 Cys
residues in the second domain were well conserved in the EPCR and
CD1/MHC family of proteins. Both of these domains are predicted to be
globular and rich in -sheet structure. Between the
2 domain
and the membrane-spanning domain, there are 24 amino acids that may
form an extended structure. Because this region is serine and threonine
rich, it is likely to be the site of O-glycosylation, which
might help stabilize the extended structure as it appears to do in
thrombomodulin(26) . In addition, the transmembrane region and
the carboxyl-terminal Cys were conserved among the three species,
suggesting that they may play important biological functions. The
availability of the three primary structures provides a useful
framework for further investigation of structure-function relationships
in this receptor.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L-39017 and L-39065.