Molecular Cloning of a Novel Scavenger Receptor for Oxidized Low Density Lipoprotein, SR-PSOX, on Macrophages*

Receptor-mediated endocytosis of oxidized low density lipoprotein (OxLDL) by macrophages has been implicated in foam cell transformation in the process of atherogenesis. Although several scavenger receptor molecules, including class A scavenger receptors and CD36, have been identified as OxLDL receptors on macrophages, additional molecules on macrophages may also be involved in the recognition of OxLDL. From a cDNA library of phorbol 12-myristate 13-acetate-stimulated THP-1 cells, we isolated a cDNA encoding a novel protein designated SR-PSOX (scavenger receptor that bindsphosphatidylserine and oxidized lipoprotein), which acts as a receptor for OxLDL. SR-PSOX was a type I membrane protein consisting of 254 amino acids, expression of which was shown on human and murine macrophages with a molecular mass of 30 kDa. SR-PSOX could specifically bind with high affinity, internalize, and degrade OxLDL. The recognition of OxLDL was blocked by polyinosinic acid and dextran sulfate but not by acetylated low density lipoprotein. Taken together, SR-PSOX is a novel class of molecule belonging to the scavenger receptor family, which may play important roles in pathophysiology including atherogenesis.

Receptor-mediated endocytosis of oxidized low density lipoprotein (OxLDL) 1 by macrophages has been implicated in foam cell transformation in the process of atherogenesis (1,2). Several scavenger receptor family members including macrophage class A scavenger receptors (SR-A) (3) and CD36 (4) have been identified as receptors for OxLDL on macrophages (5). Studies with SR-A or CD36 knockout mice showed that disruption of SR-A or CD36 partially inhibits OxLDL uptake in macrophages and prevents atherosclerotic progression in hypercholesterolemic mice (6,7). The roles of other scavenger receptors on macrophages such as CD68 (8) and LOX-1 (9) in atherogenesis remain to be determined. Furthermore, an additional scavenger receptor(s) may also be involved in OxLDL uptake by macrophages.
In the current study, by expression cloning from a cDNA library of phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells, we isolated a cDNA encoding a novel class of scavenger receptor designated SR-PSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein) that can act as a receptor for OxLDL.

EXPERIMENTAL PROCEDURES
Cells-THP-1 cells were differentiated into macrophages by treatment with 160 nM PMA for 3 days. Human monocyte-derived macrophages (HMDM) were prepared from human peripheral blood mononuclear cells by cultivation as adherent cells for 2 h or 10 days.
Expression Cloning of Human SR-PSOX-A cDNA library was constructed in pME18S (10) by use of a Time Saver cDNA synthesis kit (Amersham Pharmacia Biotech) from poly(A) ϩ RNA of THP-1 cells treated with PMA and was transfected into COS-7 cells. Binding of the transfected COS-7 cells to phosphatidylserine (PS)-coated plates was performed as follows. PS (bovine brain sodium salt; Avanti Polar Lipids) in 1 ml/dish ice-cold methanol (8 g/ml) was added to 60-mm nontreated dishes. The dishes (1 ml/dish) were kept at room temperature for 5 h to evaporate the methanol. Nonspecific binding was minimized by preblocking the plates with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.5% gelatin for 30 min at 37°C, and the plates were washed twice with phosphate-buffered saline containing 5 mM EDTA and 0.5% gelatin (Buffer A). Transfected COS cells (1 ϫ 10 6 cells in 2 ml of Buffer A) were loaded on a PS-coated plate for 30 min to allow cells to adhere. The plates were washed three times with Buffer A. Episomal DNA was recovered from COS-7 cells adherent to PScoated plates and was subjected to another round of transfection into COS-7 cells. After 5 rounds of transfection and selection, single clones of the plasmid DNA were isolated.
Cloning of Murine and Porcine SR-PSOX-We searched the EST data base for mouse and porcine homologues of human SR-PSOX and found EST clones encoding fragment cDNA of the mouse homologue (GenBank TM accession numbers AI019535 and AW261653) and the porcine homologue (GenBank TM accession numbers F23105 and AW347794). A mouse full-length cDNA of SR-PSOX was cloned by RT-PCR with total RNA isolated from mouse spleen and thymus using the forward primer, 5Ј-TCAGAATTCCCAACAAGCTCCGCAGAA-3Ј, and the reverse primer, 5Ј-TCAGGATCCTGTAGGCGCTAGGGTCTT-G-3Ј. A porcine homologue of SR-PSOX was cloned by PCR with a porcine fetal cDNA library using the forward primer, 5Ј-TCAGAATTC-GAGGCCGCCGGGAGAAGATG-3Ј, and the reverse primer, 5Ј-TCAC-TCGAGCTTTCATCCTTAGCTCAGGTG-3Ј.
Binding, Uptake, and Degradation of Lipoproteins-Cells were incubated with DiI-labeled lipoproteins (5 g/ml), with or without compet-* This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan and was performed in part through Special Coordination Funds of the Science and Technology Agency of the Japanese Government. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF275260, AF277001, and AF277000 for human SR-PSOX, murine SR-PSOX, and porcine SR-PSOX, respectively.
itors for 2 h, and washed three times with the culture medium. DiIlabeled lipoproteins internalized into the cells were detected using fluorescence microscopy. Binding of 125 I-labeled lipoproteins to cells was measured as described previously (14). Proteolytic degradation of 125 I-labeled lipoproteins by cells was carried out as described previously (15). The amount of the degraded lipoprotein was shown as the difference in radioactivity obtained from 125 I-labeled lipoprotein-containing medium incubated with and without cells.
Antibodies-Synthetic peptides corresponding to the amino acid residues 181-200 and 235-254 of human SR-PSOX and the amino acid residues 171-190 and 227-246 of murine SR-PSOX were conjugated to imject maleimide-activated mcKLH (Pierce). Rabbit polyclonal antiserum against human or murine SR-PSOX was raised by immunization with each of the conjugates according to the manufacturer's instructions.

Molecular
Cloning of SR-PSOX-We observed that PMAstimulated THP-1 cells, but not unstimulated THP-1 cells, were bound to PS-coated plastic plates, and the binding was completely inhibited by OxLDL but not significantly by AcLDL (data not shown), suggesting that OxLDL and PS may share the same epitopes of macrophage OxLDL receptors. Therefore, to isolate cDNA for the receptor(s) on PMA-stimulated THP-1 cells, a cDNA library of the PMA-stimulated THP-1 cells was transfected into COS-7 cells, and episomal DNA was recovered from the cells bound to PS-coated plates. Finally, COS-7 cells transfected with a single clone, pSR-PS, showed prominent binding activity to PS-coated plates. Because the propagation of plasmid DNA in COS cells may cause the mutation or rearrangement of cDNA (16), we searched the EST data base and found that an EST clone (GenBank TM accession number AA290712) encoded an essentially similar cDNA to SR-PS. We designated the EST clone in the expression vector as phSR-PSOX. Comparison of SR-PS and hSR-PSOX showed that Ala 181 of hSR-PSOX was replaced by Val. hSR-PSOX encodes a novel type I membrane protein of 254 amino acids, without any structural homology to other established proteins including approved receptors for PS and/or OxLDL (Fig. 1).
We further isolated the cDNA clones encoding murine and porcine homologues of SR-PSOX. Murine and porcine SR-PSOX consisted of 246 and 250 amino acids with 44 and 51% identical amino acids to human SR-PSOX, respectively (Fig. 1).
SR-PSOX Is an Endocytosis Receptor for OxLDL-As expected, binding of human SR-PSOX-transfected COS cells to PS-coated plates was significantly inhibited by OxLDL (data not shown). Then, the uptake of DiI-OxLDL into SR-PSOXintroduced COS-7 cells was examined by fluorescence microscopy. COS-7 cells transfected human, murine, and porcine SR-PSOX (COS-hSR-PSOX, COS-mSR-PSOX, and COS-pSR-PSOX cells, respectively), but not control COS-7 cells, took up DiI-OxLDL, and the uptake was completely suppressed by polyinosinic acid (poly I), dextran sulfate, and excess amounts of unlabeled OxLDL but not by AcLDL, native LDL, polycyti-dylic acid, or chondroitin sulfate ( Fig. 2 and data not shown). We also examined the binding and proteolytic degradation of 125 I-OxLDL in COS-hSR-PSOX cells. COS-hSR-PSOX cells were shown to bind 125 I-OxLDL, which was inhibited by excess amounts of unlabeled OxLDL but not by AcLDL or native LDL (Fig. 3A). Thus, SR-PSOX was shown to be an OxLDL-specific receptor.
Scatchard analysis indicated that COS-hSR-PSOX cells specifically bound 125 I-OxLDL with an approximate dissociation constant (K d ) of 5.3 g/ml (Fig. 3B), which is comparable with that of other scavenger receptors (3,4,9). COS-hSR-PSOX cells were shown to proteolytically degrade 125 I-OxLDL, which was blocked by excess amounts of unlabeled OxLDL but not by unlabeled AcLDL or native LDL (Fig. 3C). The amounts of bound and degraded 125 I-OxLDL in COS-hSR-PSOX cells appeared to be comparable with those in CHO cells stably transfected with SR-A (CHO-SR-A cells; data not shown). In addition, the AcLDL used in the present study was shown to similarly inhibit uptake of DiI-OxLDL in CHO-SR-A (data not
shown) as described previously (17). These findings indicate that SR-PSOX is a specific receptor for OxLDL, whereas neither AcLDL nor native LDL is a ligand for SR-PSOX with high affinity.

SR-PSOX Is a 30-kDa Glycoprotein Expressed on
Macrophages-To analyze SR-PSOX at the protein level, we developed polyclonal antibodies directed against human and murine SR-PSOX. Immunoblotting with these anti-human and antimurine SR-PSOX antibodies revealed that SR-PSOX expressed in COS-hSR-PSOX cells, PMA-stimulated THP-1 cells, HMDM, COS-mSR-PSOX cells, and murine thioglycollate-elicited peritoneal macrophages. The molecular masses of both human and murine SR-PSOX expressed in the cDNA-transfected COS-7 cells were 27 and 30 kDa, whereas we observed only 30-kDa forms in human and mouse macrophages (Fig. 4). The expression level of human SR-PSOX on HMDM pre-cultured for 10 days, which are differentiated macrophages, is higher than that on HMDM pre-cultured for only 2 h, which can be considered to be premature macrophages. Immunoblotting with antibodies against other amino acid residues of human and murine SR-PSOX showed similar findings (data not shown).
Northern blot analysis also showed that PMA-stimulated THP-1 cells and the 2 h-cultured HMDM produced only 2.5-kb mRNA for SR-PSOX, and the 10 day-cultured HMDM produced higher amounts of mRNA for SR-PSOX with both 1.8 and 2.5 kb (Fig. 5, A and B). Thus, Western blot analysis and Northern blot analysis suggest that expression of SR-PSOX was induced in monocytes during differentiation into macrophages. To explore SR-PSOX expression in vivo, we carried out Northern blot analysis with mRNA isolated from various human organs. As shown in Fig. 5C, SR-PSOX was expressed mainly as 2.5-kb mRNA in liver, lung, peripheral blood leukocytes, prostate, heart, kidney, and pancreas and mainly as 1.8-kb mRNA in spleen, thymus, and testis. SR-PSOX was shown to be expressed in various organs except for brain, skeletal muscle, or colon. DISCUSSION We have successfully isolated a cDNA clone encoding a novel scavenger receptor on macrophages designated SR-PSOX, which specifically bind to OxLDL. We also identified murine and porcine homologues of SR-PSOX, which specifically bind and internalize OxLDL, suggesting SR-PSOX plays a significant role in not only humans but also other mammals. Uptake of OxLDL by macrophages accumulated in the arterial intima appears to play a key role in foam cell transformation and subsequent atherosclerotic progression (1,2). Other cell surface molecules belonging to the scavenger receptor family such as SR-A (3), CD36 (4), CD68 (8), and LOX-1 (9) have been suggested to be involved in OxLDL uptake by macrophages. Macrophages in atherosclerotic lesions have been shown to express SR-A, CD36, and LOX-1 (18 -20). Double knockout mice of SR-A and apolipoprotein E, and those of CD36 and apolipoprotein E, showed less atherosclerotic lesions when compared with apolipoprotein E-deficient mice, suggesting the involvement of SR-A and CD36 in atherogenesis (6,7). Although the role of other scavenger receptors, including SR-PSOX, in atherogenesis remains to be clarified, it is suggested that uptake of OxLDL by SR-PSOX may be pathophysiologically relevant, because the ability of SR-PSOX-transfected COS cells to bind and take up OxLDL is comparable with that of CHO cells stably expressing SR-A. Further studies are in progress to elucidate the expression of SR-PSOX in atherosclerotic lesions and its pathophysiological relevance in vivo.
Northern blot analysis in Fig. 5C showed that SR-PSOX was expressed as 1.8-and 2.5-kb mRNA with different proportions in various organs. Almost 100 EST clones encoding human SR-PSOX showed essentially the same nucleotide sequence in the coding region, while showing different 3Ј-noncoding regions with different lengths. Therefore, the differences in the sizes of human SR-PSOX mRNA appears to result from the different lengths of 3Ј-noncoding regions. The present findings shown in Fig. 5C suggested that SR-PSOX may be expressed mainly by macrophages, because most of SR-PSOX-positive organs contain significant numbers of macrophages except for peripheral blood leukocytes. Currently, we are examining whether freshly isolated human peripheral blood monocytes or other types of leukocytes express SR-PSOX.
Human and murine SR-PSOX showed approximate molecular masses of 30 kDa, although the calculated molecular masses from the deduced amino acid sequences were 27 kDa. In fact, we observed both 27-and 30-kDa forms of human and murine SR-PSOX in the cDNA-transfected COS-7 cells (Fig. 4, A and C). These differences may result from the addition of carbohydrate chains at potential N-linked glycosylation sites and/or other unpredictable glycosylation sites, although we cannot exclude other possibilities such as degradation. In another class of the scavenger receptor LOX-1, N-glycosylation has been shown to affect its intracellular transport and binding affinity for OxLDL (21). Currently, we are investigating whether the 27-kDa form of SR-PSOX in COS cells is in fact a precursor protein with less carbohydrate modification.
SR-PSOX could recognize OxLDL but not AcLDL effectively (Figs. 1-3). Such ligand specificity of SR-PSOX is similar to that of LOX-1 (17), although it is different from that of SR-A, which binds AcLDL, as well as OxLDL. Scavenger receptor family molecules have been shown to have a variety of ligands (5), and it also remains to be determined whether SR-PSOX can act as a receptor for ligands other than OxLDL of scavenger receptor family molecules including PS on aged and apoptotic cells. Further studies would elucidate the roles of SR-PSOX in various physiological settings including atherogenesis.