Expression Cloning of a Novel Scavenger Receptor from Human Endothelial Cells*

Scavenger receptors mediate the endocytosis of chemically modified lipoproteins, such as acetylated low density lipoprotein (Ac-LDL) and oxidized LDL (Ox-LDL), and have been implicated in the pathogenesis of atherosclerosis. The evidence that endothelial cells possess scavenger receptor activity is substantial, and this property is widely used in the isolation of endothelial cells from vascular tissues. In the current study, we have isolated, by expression cloning, the cDNA encoding a novel type of scavenger receptor expressed by endothelial cells (SREC), which mediates the binding and degradation of Ac-LDL. The primary structure of the molecule has no significant homology to other types of scavenger receptors, including the recently cloned endothelial cell Ox-LDL receptor, a member of the C-type lectin family. The cDNA encodes a protein of 830 amino acids with a calculated molecular mass of 85,735 Da (mature peptide). Chinese hamster ovary cells stably expressing SREC bound 125I-labeled Ac-LDL with high affinity (K d = 3.0 μg/ml, approximately 1.7 nm) and degraded them via an endocytic pathway. Association of DiII-Ac-LDL were effectively inhibited by Ox-LDL, malondialdehyde-modified LDL, dextran sulfate, and polyinosinic acid, but not by natural LDL and heparin. The cloned receptor has several characteristic domain structures, including an N-terminal extracellular domain with five epidermal growth factor-like cysteine pattern signatures and an unusually long C-terminal cytoplasmic domain (391 amino acids) composed of a Ser/Pro-rich region followed by a Gly-rich region.

Scavenger receptors mediate the endocytosis of chemically modified lipoproteins, such as acetylated low density lipoprotein (Ac-LDL) and oxidized LDL (Ox-LDL), and have been implicated in the pathogenesis of atherosclerosis. The evidence that endothelial cells possess scavenger receptor activity is substantial, and this property is widely used in the isolation of endothelial cells from vascular tissues. In the current study, we have isolated, by expression cloning, the cDNA encoding a novel type of scavenger receptor expressed by endothelial cells (SREC), which mediates the binding and degradation of Ac-LDL. The primary structure of the molecule has no significant homology to other types of scavenger receptors, including the recently cloned endothelial cell Ox-LDL receptor, a member of the C-type lectin family. The cDNA encodes a protein of 830 amino acids with a calculated molecular mass of 85,735 Da (mature peptide). Chinese hamster ovary cells stably expressing SREC bound 125 I-labeled Ac-LDL with high affinity (K d ‫؍‬ 3.0 g/ml, approximately 1.7 nM) and degraded them via an endocytic pathway. Association of DiII-Ac-LDL were effectively inhibited by Ox-LDL, malondialdehyde-modified LDL, dextran sulfate, and polyinosinic acid, but not by natural LDL and heparin. The cloned receptor has several characteristic domain structures, including an N-terminal extracellular domain with five epidermal growth factor-like cysteine pattern signatures and an unusually long C-terminal cytoplasmic domain (391 amino acids) composed of a Ser/Pro-rich region followed by a Gly-rich region.
Chemically modified low density lipoprotein (LDL) 1 , such as acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL), can be rapidly taken up by cultured macrophages and some other cells via receptor-mediated endocytosis. The receptor involved in this pathway is called a scavenger receptor. The first scavenger receptors to be purified and cloned were the macrophage type I and type II receptors (1,2). Receptors that recognize chemically modified LDL, but which are distinct from the type I and II macrophage scavenger receptors, have been identified in macrophages and some other cell types (3)(4)(5)(6). It is well recognized that endothelial cells possess scavenger receptor activity, and the uptake by endothelial cells of 1,1Ј-dioctadecyl-3,3,3Ј,3Јtetramethylindocarbocyanine perchlorate (DiI)-labeled Ac-LDL (DiI Ac-LDL) is widely used to isolate endothelial cells from vascular tissues. However, several lines of evidence suggest that the receptor present on their surface is distinct from the scavenger receptors described to date (3)(4)(5)7).
Recently, an endothelial receptor for Ox-LDL was cloned, using an expression cloning strategy, from cultured bovine aortic endothelial cells (8). The cDNA for the receptor encodes a protein of 270 amino acid residues, with a calculated molecular mass of 30,872 Da. It has a type II membrane protein structure with a short N-terminal cytosolic domain and a long C-terminal extracellular domain and belongs to the C-type lectin family. Although the ligand specificity or the competitor sensitivity of the receptor is not yet known, the cloned receptor recognizes Ox-LDL with a high affinity, but not Ac-LDL. Similarly, it was demonstrated that rabbit venous endothelial cells in primary culture and a bovine aortic endothelial cell line had no detectable mRNA of type I and type II scavenger receptors (9). Based on these observations, we speculated that a scavenger receptor(s) other than those that have been cloned to date is expressed by endothelial cells.
In the present study, we have isolated, by expression cloning, the cDNA encoding a new type of scavenger receptor expressed by endothelial cells (SREC) which mediates the binding and degradation of Ac-LDL.

EXPERIMENTAL PROCEDURES
Expression Cloning-A cDNA library was constructed using the pcDNA3 vector (Invitrogen) with poly(A) ϩ mRNA isolated from human umbilical vein endothelial cells (HUVECs). To obtain long cDNA, the entire pooled cDNA library was digested with NotI and size-fractionated using 0.7% agarose gel electrophoresis. Large DNAs (Ͼ8 kb, corresponding to Ͼ3 kb of cDNA) were recircularized with a DNA ligation kit (Takara, Tokyo, Japan). The resultant transformants were divided into small pools (approximately 1,000 clones/pool), and we obtained the plasmid DNA using a plasmid mini-kit (QIAGEN). Plasmid DNA was transfected into Chinese hamster ovary (CHO) cells, and the transiently transfected cells were screened visually for endocytosis of fluorescent DiI Ac-LDL. On day 0, CHO cells were plated into six-well plates (1 ϫ 10 5 cells/well) in minimum essential medium ␣ (medium A) (Life Technologies, Inc.) with 10% fetal calf serum. On day 1, the cells in each plate were transfected with 1 g of expression library plasmid DNA from a single pool employing LipofectAMINE reagent (Life Technologies, Inc.). On day 3, the monolayers were refed with medium A containing 2 g/ml protein of DiI Ac-LDL (Biomedical Technologies Inc.) and 5% lipoprotein deficient serum. After a 3-h incubation at 37°C, the plates were washed twice with medium A containing 10% fetal calf serum and then twice with PBS, and the cells were fixed with 3.0% formaldehyde in PBS for 15 min at room temperature. The pres-* This work was supported in part by a grant for "Biodesign Research Program" from RIKEN. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ ence of fluorescent DiI in the fixed cells was determined by visual inspection using fluorescence microscopy. A positive pool was serially subdivided and retested to permit the purification of a single positive plasmid.
The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EBI, and NCBI nucleotide sequence data bases with the following accession number D86864.
Specific Binding of 125 I-Ac-LDL to SREC-expressing CHO Cells-CHO cells expressing the SREC (CHO-SREC) were established by a standard method. CHO-SREC and control cells grown to confluence in 12-well plates were incubated with 0.52-16.5 g/ml 125 I-Ac-LDL at 4°C for 2 h. A standard protocol for quantifying the binding of 125 I-Ac-LDL was employed as described previously (10). Nonspecific binding was measured in the presence of a 20-fold excess of Ac-LDL and subtracted from the data.
Uptake and Degradation of 125 I-Ac-LDL by CHO-SREC Cells-CHO-SREC cells and control cells grown to confluence in 24-well plates were incubated with 2 g/ml 125 I-Ac-LDL. After incubation at 37°C for the indicated time, degradation and association were determined in triplicate wells. Degradation and association in the presence of 100 M chloroquine were also determined.
Inhibition of DiI Ac-LDL Binding to CHO-SREC Cells by Various Compounds-The CHO-SREC cells were incubated with 2 g/ml DiI Ac-LDL at 4°C for 2 h in the presence of various inhibitors at 200 g/ml. LDL (d ϭ 1.019 -1.063 g/ml) was prepared as described previously (6). High density lipoprotein (HDL) was obtained from Biomedical Technologies Inc. Acetylation of LDL was achieved by the addition of acetic anhydride, whereas oxidation was carried out by incubating at 100 g/ml LDL in 5 mM CuSO 4 for 24 h at 37°C according to a previously described methods (10,11). Malondialdehyde-modified LDL (MDA-LDL) and maleyl-bovine serum albumin were prepared according to a previously described method (12,13). To quantify the amount of DiI Ac-LDL, cells were washed twice with medium A, with 10% fetal calf serum in PBS and then twice with PBS, solubilized with 0.1% Triton X-100, and the fluorescence intensity was measured at 590/35 nm with the excitation wavelength set at 530/25 nm employing a fluorescence multiplate reader (CytoFluorII, Perspective Biosystems).

RESULTS AND DISCUSSION
The cloned SREC cDNA is approximately 3.4 kb long, and nucleotide sequence analysis revealed that this cDNA contains an open reading frame of 2,490 base pairs. Fig. 1A shows the deduced amino acid sequence of the cDNA. The cDNA encodes a protein of 830 amino acids with a calculated molecular mass of 85,735 Da (mature peptide). Hydropathy analysis using the Kyte and Doolittle algorithm (14) shows that the molecule has two hydrophobic regions, in the N terminus and the middle of the molecule, which could serve as a signal sequence and a transmembrane domain, respectively. Since the N-terminal half of the molecule contains three potential N-linked glycosylation sites, we speculate at present that the molecule is a type I transmembrane protein, having an unusually long C-terminal cytoplasmic domain relative to the N-terminal extracellular domain.
The predicted SREC has no significant homology to other types of scavenger receptors but has several characteristic domain structures (Fig. 1B). There are 72 cysteine residues distributed throughout the putative extracellular domain, which is made up of 406 amino acids. As shown in Fig. 1C, the spacing of these residues forms 10 repeats. There are five repeats that exactly fit the consensus sequence for an EGF-like domain cysteine pattern signature (CXCXXXXXGXXC) (Fig. 1C) (15,16). Two repeats (shown as segments 7 and 8 in the figure) have one amino acid inserted into the motif. Since certain EGF-like repeats have been shown to mediate homophilic or heterophilic protein-protein interactions, the motifs detected in the SREC may contribute to the oligomerization of the molecule. Alternatively, as is the case with an LDL receptor, which contains EGF precursor homology domains in its N terminus, some of these motifs may also serve as the ligand binding domain (17,18). The putative transmembrane domain is followed by a serine-and proline-rich region (Ser/Pro-rich region) located in the N-terminal half of the cytoplasmic domain. It is difficult to speculate on the biological significance of this region at present; however, this domain contains several serine and threonine residues that could potentially be phosphorylated by various protein kinases, suggesting that some biological signals may be delivered via the domain. Downstream of the Ser/Prorich region is a glycine-rich region (Gly-rich region). There are 26 glycine residues distributed throughout a domain of 177 amino acids. In the C-terminal end of the domain, there is a tyrosine residue (Tyr 822 ) fitting the consensus sequence (RXX-EXXY) for a site phosphorylated by tyrosine kinases (19), suggesting that it may play a role in some signal transduction processes.
We compared the expression level of the message in HUVECs, CAEs, and CASMs. The 3.5-kb transcript was clearly detected in HUVECs and CAEs, whereas we did not detect any bands in CASMs (Fig. 2).
The expression plasmid containing SREC DNA was expressed in CHO cells (CHO-SREC). The binding of 125 I-Ac-LDL was assessed following incubation of the transformed cells with 125 I-Ac-LDL. Saturation binding of 125 I-Ac-LDL was observed at 4°C (Fig. 3A). Scatchard analysis (Fig. 3A, inset) showed the presence of a single class of receptors and the calculated K d was 3.0 g/ml 125 I-Ac-LDL (approximately 1.7 nM 125 I-Ac-LDL), which is comparable with the value of 2.7 nM for scavenger receptors on HUVECs (20). We have also performed binding experiments employing DiI Ac-LDL and obtained the same result (K d is 3.2 nM). Incubation of the transfectants at 37°C with DiI Ac-LDL led to the uptake of fluorescent lipid (DiI) into the cytoplasm, producing a punctate pattern (Fig. 4, A and B). Diffuse labeling, most likely of the cell surface, was also observed. When CHO-SREC cells were incubated with 125 I-Ac-LDL at 37°C for various lengths of time, chloroquine-sensitive cellular association of radioactivity and acid-soluble radioactivity in the medium continued to appear at a linear rate, reflecting the continuous uptake and degradation of 125 I-Ac-LDL (Fig. 3B).
To further characterize the binding properties of the SREC, the receptors activity was measured in the presence of various materials known as type I and type II macrophage scavenger receptor inhibitors (Fig. 5). As is the case with type I macrophage scavenger receptors, unlabeled Ac-LDL as well as poly I and dextran sulfate, but not native lipoproteins such as LDL and HDL, reduced the binding of DiI Ac-LDL on the cells to the  D) were seeded on day 0 at 1 ϫ 10 5 cells/well in medium A. On day 2, medium containing 2 g/ml protein of DiI Ac-LDL was added to the monolayers. After incubation at 37°C for 3 h, the cells were washed, fixed in 3% formaldehyde in PBS, and the uptake of DiI Ac-LDL was observed using fluorescence microscopy. Phase-contrast light micrographs of CHO monolayers (A, C) and the fluorescence of cell-associated DiI Ac-LDL (B, D) are shown. basal level. Ox-LDL, maleyl-bovine serum albumin, and MDA-LDL partially reduced the binding of DiI Ac-LDL on the cells. These results show that the SREC has a binding specificity similar to those of the type I and type II macrophage scavenger receptors.
In this study, we cloned the cDNA encoding a new type of scavenger receptor that is expressed by endothelial cells. Interestingly, the SREC has an unusually long C-terminal cytoplasmic domain that contains a Ser/Pro-rich domain and a Gly-rich domain. Together with the fact that the molecule also contains a tyrosine phosphorylation site in the C-terminal end, these characteristics tempt us to speculate that the cytoplasmic domain may mediate some biological signals. It has been demonstrated by several investigators that stimulation of scavenger receptors of endothelial cells induces the expression of proteins related to vascular functions. For example, Ac-LDL and Ox-LDL stimulate the release of endothelin, a potent vasoconstrictor peptide (21). Indeed, the circulating levels of endothelin are increased in patients with hyperlipidemia or atherosclerosis (22,23). Alternatively, poly I, a scavenger receptor agonist significantly induces the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin by a human endothelial cell line, EA hy.926 (24). Adherence of circulating monocytes to the arterial endothelium is first seen in high cholesterol diet-induced experimental atherosclerosis in rabbits (25). The molecular mechanism underlying such physiological changes of vascular endothelial cells in the pathogenesis of atherosclerosis is not well understood. The present report of the isolation of cDNA for endothelial cell scavenger receptor should help our understanding of the molecular mechanisms involved in a development of atherosclerosis.