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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2341-2349.)
© 1997 American Heart Association, Inc.

Articles

CLA-1 Is an 85-kD Plasma Membrane Glycoprotein That Acts as a High-Affinity Receptor for Both Native (HDL, LDL, and VLDL) and Modified (OxLDL and AcLDL) Lipoproteins

Dominica Calvo; Diego Gómez-Coronado; Miguel A. Lasunción; ; Miguel A. Vega

From Hospital de la Princesa (D.C.), Madrid; Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal y Universidad de Alcalá de Henares (D.G.-C., M.A.L.), Madrid; and Instituto de Parasitología y Biomedicina (M.A.V.), CSIC, Granada, Spain.

Correspondence to Dr Miguel A. Vega, Servicio de Bioquímica-Investigacíon, Hospital Ramón y Cajal, Ctra. Colmenar Viejo km 9.1, 28034 Madrid, Spain.

	Abstract

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Abstract Lipoprotein metabolism is regulated by the functional interplay between lipoprotein components and the receptors and enzymes with which they interact. Recent evidence indicates that the structurally related glycoproteins CD36 and SR-BI act as cell surface receptors for some lipoproteins. Thus, CD36 has been reported to bind oxidized LDL (OxLDL) and acetylated LDL (AcLDL), while SR-BI also binds native LDL and HDL. The cDNA of human CLA-1 predicts a protein 509 amino acids long that displays a 30% and an 80% amino acid identity with CD36 and mouse or hamster SR-BI, respectively. In this report, we describe the structural characterization of CLA-1 as an 85-kD plasma membrane protein enriched in N-linked carbohydrates. The expression of CLA-1 on mammalian and insect cells has been used to demonstrate that CLA-1 is a high-affinity specific receptor for the lipoproteins HDL, LDL, VLDL, OxLDL, and AcLDL. Northern blot analysis of the tissue distribution of CLA-1 in humans indicated that its expression is mostly restricted to tissues performing very active cholesterol metabolism (liver and steroidogenic tissues). This finding, in the context of the capability of this receptor to bind to both native and modified lipoproteins, strongly suggests that the CLA-1 receptor contributes to lipid metabolism and atherogenesis.

Key Words: lipoproteins • scavenger receptor • fatty acids • cholesterol • atherosclerosis

	Introduction

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Alterations in lipoprotein metabolism are associated with a variety of diseases, of which atherosclerotic coronary heart disease is particularly relevant for its high incidence and associated mortality in westernized countries.¹ Multiple epidemiological, experimental, and genetic studies have demonstrated a correlation between elevated serum LDL cholesterol levels and increased risk of atherosclerosis.² By contrast, elevated HDL cholesterol levels appear to have a protective effect.³ The delineation of the cellular and molecular mechanisms that dictate the metabolism of the distinct plasma lipoproteins is therefore of major interest in reaching a clear understanding of the processes underlying the development of atherogenic lesions.

CD36⁴ together with the structurally related glycoproteins CLA-1⁵ and SR-BI⁶ and the lysosomal membrane glycoprotein LIMPII⁷ constitute a novel gene family.⁸ CD36 acts as a receptor for thrombospondin,⁹ collagens type I¹⁰ and IV,¹¹ Plasmodium falciparum–infected erythrocytes,¹² and fatty acids.¹³ In addition, both SR-BI and CD36 are able to bind some lipoproteins^{6 14 15 16} and phospholipids¹⁷ and to interact with apoptotic cells.^{18 19} On the basis of such a broad ligand-binding specificity, CD36 and SR-BI have been incorporated into the group of cell surface glycoproteins collectively designated as scavenger receptors.^{6 15 20} With respect to their interaction with lipoproteins, CD36 is able to bind OxLDL and AcLDL,^{6 15 16} while SR-BI, besides interacting with modified LDL,⁶ also recognizes native LDL⁶ and HDL.¹⁴ Through binding HDL, SR-BI participates in selective cellular cholesterol ester uptake, a role consistent with its restricted expression in steroidogenic tissues and liver in mouse and rat.^{14 21 22}

Human CLA-1⁵ cDNA predicts a protein 509 amino acids long that presents a 30% and an 80% amino acid identity with CD36 and SR-BI, respectively. Thus, it is likely, although it has not been confirmed, that CLA-1 and SR-BI represent the same gene from different species. So far, neither biochemical nor functional studies have been performed on human CLA-1.

In the present report, we have investigated the biochemical nature, subcellular location, and tissue distribution of human CLA-1 and examined the ability of CLA-1 to interact with native and in vitro modified lipoproteins.

	Methods

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Reagents and Cell Lines
The fluorescence probe DiI, BODIPY-LDL, and BODIPY-AcLDL were obtained from Molecular Probes Inc. N- and O-glycanases were purchased from Boëhringer Mannheim. The monoclonal antibodies reacting with CD36 and CD69 were FA6-152 and TP1/33, respectively.^{23 24} The cells lines used in the present study were C32 (human melanoma), COS-7 (kidney cells derived from monkey), and Sf9 (ovarian cells derived from the lepidopteron insect Spodoptera frugiperda).

Generation of Polyclonal Antibodies Reactive With Human CLA-1 Glycoprotein
A BamHI-EcoRI fragment from CLA-1 cDNA, encoding amino acid residues 230 to 328 of the longest CLA-1 protein form,⁵ was subcloned into the bacterial expression vector pGEX-3X.²⁵ The encoded glutathione S-transferase-CLA-1 fusion protein was produced in the bacterial strain JM109 as described²⁵ and purified by affinity chromatography on immobilized glutathione (Pharmacia) followed by gel electrophoresis and electroelution. New Zealand White rabbits were immunized several times at intervals of 1 month with 125 µg of the purified fusion protein. The specificity and titer of the sera collected from the immunized rabbits were tested by slot blot and ELISA assays using purified fusion protein and glutathione S-transferase as control.

Iodination of Cells, Immunoprecipitation, and Treatments With N- and O-Glycanases
Four million C32 melanoma cells were cell surface radioiodinated on ice, lysed, and immunoprecipitated basically as described previously.²⁶ Briefly, cells were lysed with Triton X-100, and the lysates were first precleared with protein-A sepharose (Pharmacia). Aliquots of cell lysates to be immunoprecipitated by the CLA-1 specific antiserum or the anti-CD36 monoclonal antibody were precleared with an excess of the rabbit preimmune serum or the control monoclonal antibody P3X63, respectively, followed by incubation with protein-A sepharose. After the sepharose beads were removed by means of centrifugation, CLA-1 was immunoprecipitated with 5 µL of the antiserum generated and protein-A sepharose, and the CD36 glycoprotein (used as a positive control) was immunoprecipitated with 0.2 µg of purified anti-CD36 monoclonal antibody FA6-152 and protein-A sepharose. For N- and O-glycanase treatments, the immunoprecipitates were resuspended in 30 µL of 0.1 mol/L sodium phosphate buffer, pH 6.5, 0.1% SDS, 1% NP40, 2 mmol/L 2-mercaptoethanol, 10 mmol/L EDTA, 0.1 mmol/L PMSF, and 2 µg/mL leupeptin, boiled 5 minutes, and incubated overnight at 37°C with either 0.4 U N-glycanase or 1.5 mU O-glycanase. Finally, glycanase-treated and -untreated samples were run on 7.5% polyacrylamide SDS-PAGE under reducing conditions and analyzed by use of autoradiography. Radioiodination of Sf9 cells and immunoprecipitation of CLA-1 from Sf9 cell lysates were performed similarly as described for the C32 cells.

DNA Constructs
The construct containing the large form of CLA-1 was generated as follows. First, the short CLA-1 form was ligated to the EcoRI site of the pCEXV-3 vector.⁵ This construct was independently digested with either Afl II-Bcl I or Sca I-Bcl I, and fragments corresponding to sizes of 5 kbp and 180 bp were respectively isolated (fragments 1 and 2). A PCR fragment containing the CLA-1 region absent in the short CLA-1 form was generated by amplification of single-stranded cDNA synthesized from PMA-treated HL60 cells, with oligonucleotides spanning the regions 150 to 171 and 343 to 363 (numbers refer to the short cDNA form⁵ ). This PCR-derived fragment was digested with Afl II and Sca I enzymes, and the resulting 460-bp fragment was purified (fragment 3). Finally, fragments 1 to 3 were ligated together to yield the large CLA-1 form in vector pCEXV-3.

To generate the pBacPAK-CLA-1 construct, a Sac II-Bsu36I CLA-1 cDNA fragment containing nucleotides 46 to 1604 was blunt ended with T4 DNA polymerase, ligated to Bgl II linkers, and subcloned into the Bgl II site of the vector pBacPAK9(Clontech). To generate the pBacPAK-CD36 construct, an EcoRI-Xba I fragment from the CD36 cDNA that spans nucleotides 1 to 1725 was subcloned into the pBacPAK9 vector digested with EcoRI and Xba I.

Transfection of COS7 Cells
cDNA of CLA-1 subcloned into the expression plasmid pCEXV-3 and of ICAM-3 subcloned into the vector pCR3 (kindly provided by Dr F. Sánchez-Madrid, Hospital de la Princesa) were transfected into COS-7 cells by the DEAE-dextran procedure as previously described.⁵ Transfected cells were used for experiments 48 hours after transfection.

Expression of Human CLA-1 on the Surface of Insect Sf9 Cells
Procedures for the generation and handling of recombinant baculoviruses containing the cDNAs of CLA-1 (BP-CLA-1) and CD36 (BP-CD36) were performed following the instructions of the BacPAK Baculovirus Expression System (Clontech). Sf9 cells were infected with viruses BacPAK6, BP-CD69 (a baculovirus containing the cDNA of CD69, kindly provided by Dr F. Sánchez-Madrid, Hospital de la Princesa), BP-CLA-1, or BP-CD36 using a moi of 10 viruses per cell. Infected cells were used for binding assays 36 to 48 hours after infection.

For immunofluorescence, Sf9 cells were infected with an moi of 1 to 10. An moi of 1 was used when indicated to guarantee the existence of noninfected cells in the preparation. Forty hours after infection, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature and immunostained by the anti–CLA-1 serum followed by fluorescein-labeled second antibodies, essentially as described previously.⁵

Isolation and Labeling of Lipoproteins
Native plasma lipoproteins VLDL, LDL, and HDL were isolated by preparative sequential ultracentrifugation of pooled normolipidemic sera from humans after an overnight fast. Na₂ EDTA (0.5 g/L) and Trasylol (300 IU/mL) were added as preservatives. Purified lipoproteins were extensively dialyzed at 4°C against 0.15 mol/L NaCl, 0.01% (wt/vol) Na₂ EDTA, pH 7.4. When required, HDL was subfractionated into particles containing and not containing apolipoprotein E (apo E+ and apo E- fractions, respectively) by heparin-sepharose affinity chromatography as previously described.²⁷ AcLDL was prepared from LDL by the addition of acetic anhydride.²⁸ OxLDL was prepared by incubating 3 mg of LDL protein/mL in 20 µmol/L CuSO₄ for 20 hours at room temperature. All lipoproteins were filtered through 0.4-µm filters and stored in 150 mmol/L NaCl, 0.01% Na₂ EDTA, pH 7.4 at 4°C. Oxidation and acetylation of LDL were checked by agarose gel electrophoresis.

Labeling of lipoproteins with the fluorescence probe DiI was carried out according to Via and Smith,²⁹ with some minor modifications. Briefly, lipoproteins were incubated with the DiI probe in lipoprotein deficient serum for 12 hours at 37°C, using the following relative amounts: 300 µg of DiI/3 mg of lipoprotein lipid/2 mL of lipoprotein-deficient serum. After the labeling reaction, the labeled lipoproteins were reisolated by ultracentrifugation, stored in the dark, and used within 2 weeks after their preparation.

Lipoprotein Binding Assays
Cell association of DiI-labeled lipoproteins to COS-7 cells was performed by incubating 5x10⁴ cells grown on coverslips with 5 µg/mL of DiI-labeled lipoproteins in PBS containing 1 mmol/L CaCl₂, 1 mmol/L MgCl₂ for 1 hour at 37°C. Binding of DiI-labeled lipoproteins to Sf9 cells was carried out by incubating cells in 100 to 200 µL of PBS containing 1 mmol/L CaCl₂, 1 mmol/L MgCl₂ with DiI-labeled lipoproteins at 5 µg/mL for 2 hours at 4°C or 1 hour at room temperature. After incubation, cells were washed with cold PBS, fixed with 3% formaldehyde in PBS for 10 minutes at room temperature, and analyzed under fluorescence microscopy or by fluorescence flow cytometry using a FACScan from Becton Dickinson. For flow cytometry analysis, forward-angle light-scatter gates were established to exclude cellular debris and cellular aggregation. At least 5000 cells were analyzed for each sample. Cell-associated lipoprotein binding was expressed as MIF assessed using fluorescence windows (channel numbers). Fluorescence emitted by cells alone (autofluorescence) was subtracted. To determine binding dissociation constants (K_d), the cells and the DiI-labeled lipoproteins were incubated for 2 hours at 4°C in the presence or absence of a 50-fold excess of the corresponding unlabeled lipoprotein to determine nonspecific binding. Experiments were done several times using different lipoproteins isolates. K_d values, expressed as milligrams of lipoprotein per milliliter, were obtained by analyzing the data with the InPlot-4 software (GraphPad). For inhibition experiments, the inhibitor and the labeled lipoprotein were added simultaneously to the cells.

Northern Blot Analysis
Blots containing poly A+ RNA isolated from a panel of human tissues were purchased from Clontech. Hybridizations were carried out as previously described⁵ using isolated CD36, CLA-1, and ß-actin cDNAs as probes.

	Results

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Human CLA-1 Gene Product Is Expressed on the Plasma Membrane as an 85-kD Glycoprotein Enriched in N-Linked Carbohydrates
To biochemically characterize and determine the subcellular location of the human CLA-1 gene product, we first generated a polyclonal antibody specific for the CLA-1 protein by immunizing rabbits with a fusion protein produced in bacteria, which contains amino acids 230 to 328 of CLA-1 (see "Methods" for details). C32 melanoma cells, which display high mRNA levels for CLA-1⁵ and high protein expression levels for CD36,¹² were radioiodinated on ice at the cell surface, lysed, and subjected to immunoprecipitation with the rabbit anti-CLA-1 antiserum or with the anti-CD36 monoclonal antibody FA6-152 (used as control). Gel electrophoresis analysis of the immunoprecipitates revealed for CLA-1 a single band with an apparent molecular weight of 85 kD and, as expected, an 88-kD band for CD36 (Fig 1, lanes 1 and 4). Moreover, similar electrophoretic patterns were obtained when CLA-1 was immunoprecipitated from lysates derived from the human monocytic cell line U937, the human epitheloid carcinoma HeLa, and COS-7 cells transiently transfected with the cDNA of CLA-1, while no products were immunoprecipitated with either serum from preimmune rabbits or with the control antibody P3X63 (unpublished data, 1997). Furthermore, COS-7 cells transiently transfected with the cDNA of CLA-1 were stained on the plasma membrane with the CLA-1 specific antiserum (unpublished data, 1997). Taken together, these results demonstrate that the CLA-1 gene encodes a plasma membrane protein with an apparent molecular weight of 85 kD.

View larger version (91K): [in this window] [in a new window]   Figure 1. Biochemical characterization of human CLA-1. 125I-labeled C32 melanoma cells were lysed, and the CLA-1 (lanes 1-3) and CD36 (lanes 4-6) species were immunoprecipitated by the anti–CLA-1 antiserum and by the antibody FA6-152, respectively, as described in "Methods." Immunoprecipitates were nontreated (lanes 1 and 4), treated with N-glycanase (lanes 2 and 5), or treated with O-glycanase (lanes 3 and 6) and analyzed by SDS-PAGE. Molecular sizes of some markers are indicated on the left in kilodaltons.

To study the chemical nature of the carbohydrates attached to the CLA-1 polypeptide chain, CLA-1 and CD36 (used as control) immunoprecipitates derived from C32 cells were subjected to treatments with N- and O-glycanases. As expected, SDS-PAGE analysis of the CD36 immunoprecipitate treated with N-glycanase revealed a change in gel mobility with respect to the nontreated sample (Fig 1, lanes 4 and 5), whose size is consistent with the reported presence of both N- and O-linked carbohydrate types in the CD36 mature protein³⁰ (see below). However, a higher relative decrease in the apparent molecular weight of CLA-1 was observed on treatment of the CLA-1 immunoprecipitates with N-glycanase (Fig 1, lanes 1 and 2). The size of the band corresponding to CLA-1 treated with N-glycanase agrees with the size predicted for the deglycosylated CLA-1 polypeptide chain, and the migration change is consistent with the occupancy of the 10 potential N-glycosylation sites found along its amino acid sequence.⁵ Treatment of CD36 and CLA-1 immunoprecipitates with O-glycanase revealed no changes (for CLA-1) or minor although detectable changes (for CD36) in gel mobility with respect to the nontreated samples (Fig 1, lanes 3 and 6). Thus, like the structurally related lysosomal membrane protein LIMPII,^{7 31} the mature polypeptide chain of CLA-1 is either mainly or only N-glycosylated.

Cell Association of the Native Plasma Lipoproteins HDL, LDL, and VLDL and In Vitro Oxidized or Acetylated LDL With COS-7 Cells Expressing Human CLA-1
To examine the binding of native plasma lipoproteins HDL, LDL, and VLDL and the in vitro modified lipoproteins OxLDL and AcLDL to CLA-1, purified lipoproteins were labeled with the fluorescent probe DiI, a technique extensively used in binding and cell association studies.^{14 32 33}

The capacity of CLA-1 to bind OxLDL, a previously described ligand for hamster SR-BI and human CD36,^{6 15} was examined under fluorescence microscopy after incubating DiI-labeled OxLDL with COS-7 cells transiently transfected with expression vectors carrying either CLA-1 (COS-CLA-1) or ICAM-3 (COS-ICAM-3). Untransfected COS-7 cells (COS), mock cells (COS-mock), and COS-ICAM-3 cells appeared weakly labeled in the cytoplasm (Fig 2A and 2B), while a subset of COS-CLA-1 cells (Fig 2C) exhibited an intense fluorescence at the cell surface and some minor, small, fluorescence-punctuated foci, possibly representing internal vesicles. Staining of COS-CLA-1 cells was inhibited by the addition of a 50-fold excess of unlabeled OxLDL (Fig 2D). Identical results were obtained when AcLDL was used instead of OxLDL (unpublished data, 1997). These results demonstrate that human CLA-1 interacts with both OxLDL and AcLDL. A similar set of experiments were carried out to investigate the binding of the plasma lipoproteins HDL, LDL, and VLDL to COS-7 cells expressing CLA-1. An internal and punctuated staining pattern, which reflects the endosomes and lysosomes containing internalized lipoproteins particles, was observed when COS, COS-mock, and COS-ICAM-3 cells were assayed with DiI-labeled HDL, LDL, and VLDL (Fig 2E, 2F, 2I, 2J, 2M, and 2N). Lipoprotein internalization possibly occurred as a consequence of the interaction of the endogenous LDL receptors with apo B and/or apo E present in lipoproteins. However, when COS-CLA-1 cells were incubated with either HDL (Fig 2G), LDL (Fig 2K), or VLDL (Fig 2O), a subset of cells exhibited an intense plasma membrane staining that overlapped the punctuated staining pattern observed for the COS, COS-mock, and COS-ICAM-3 cells (Fig 2E, 2F, 2I, 2J, 2M, and 2N). The number of cells in each preparation displaying staining in the plasma membrane matched the number of cells expressing CLA-1 as calculated by staining with the CLA-1 specific antisera. This observation strongly suggests that only the subset of cells expressing CLA-1 is responsible for the plasma membrane-staining pattern. In addition, as shown for the binding of OxLDL, a 50-fold excess of unlabeled lipoproteins inhibited the DiI-lipoprotein induced cell staining (Fig 2H, 2L, and 2P), demonstrating that the binding was specific and therefore indicating that the binding of DiI-labeled lipoproteins to the CLA-1 receptor mirrors the binding of the unlabeled lipoproteins. This point was further confirmed by the appearance of identical staining patterns when LDL and AcLDL labeled with DiI and LDL and AcLDL labeled with the structurally unrelated fluorescence probe BODIPY were used (unpublished data, 1997). Altogether, these results enable us to conclude that CLA-1, when expressed on COS-7 cells, mediates the binding of the native plasma lipoproteins HDL, LDL, and VLDL, in addition to interacting with the in vitro modified lipoproteins AcLDL and OxLDL.

View larger version (151K): [in this window] [in a new window]   Figure 2. Cell association of Di-I–labeled lipoproteins with COS-7 cells expressing CLA-1. COS-7 cells mock transfected or transfected with the indicated cDNAs were grown on coverslips and incubated for 1 hour at 37°C with 5 µg/mL of the indicated DiI-labeled lipoproteins. Panels D, H, L, and P correspond to COS-CLA-1 cells incubated with the indicated DiI-labeled lipoproteins in the presence of a 50-fold excess of the corresponding unlabeled lipoprotein. Identical staining patterns to those displayed by COS-mock and COS-ICAM-3 cells were obtained with untransfected COS cells. Bar=30 µm.

Expression of CLA-1 on the Plasma Membrane of Insect Sf9 Cells
The expression of endogenous lipoprotein receptors in most cultured mammalian cell lines impairs the performance of accurate binding experiments between CLA-1 and lipoproteins. To facilitate such study, we considered the possibility of expressing CLA-1 on the plasma membrane of insect Sf9 cells. In general, the biological activities and immunologic reactivities of mammalian glycoproteins expressed by insect cells are similar to those obtained using mammalian expression systems. Moreover, insect cells recognize a different set of lipoproteins than mammalian cells,³⁴ a property that makes these cells suitable for overcoming the technical drawbacks indicated above when mammalian cells are used. In fact, and as noted below, Sf9 cells exhibited a negligible binding of human lipoproteins.

Infection of Sf9 cells by baculoviruses carrying the CLA-1 cDNA (Sf9-CLA-1) resulted in expression of this molecule on the plasma membrane as assayed by cell surface staining by the anti-CLA-1 antisera (Fig 3A and B) and by SDS-PAGE analysis of the CLA-1 immunoprecipitated from lysates from ¹²⁵I–surface-labeled Sf9-CLA-1 cells (Fig 3C). The size of the CLA-1 protein form immunoprecipitated from the Sf9-CLA-1 cells was slightly shorter than the CLA-1 form immunoprecipitated from the human melanoma cell line C32 (Fig 3C), a feature consistent with the limited processing of the core oligosaccharides by the Sf9 cells.³⁵

View larger version (55K): [in this window] [in a new window]   Figure 3. Expression of CLA-1 on the plasma membrane of Sf9 cells. Sf9 cells were infected with the recombinant viruses BP-CD36 (A) and BP-CLA-1 (B), and 36 to 48 hours later were fixed with paraformaldehyde and immunostained with the anti–CLA-1 antiserum. An moi of 1 was used to avoid infection of all cells in the preparation and therefore to allow the comparison of the staining between infected versus noninfected cells. When an moi of 10 was used, all cells appeared stained (unpublished data, 1997) (bar=10 µm). C, SDS-PAGE analysis of CLA-1 inmunoprecipitated from lysates from cell surface iodinated human melanoma cells C32 and from insect Sf9-CLA-1 cells. Molecular sizes of some markers are indicated on the left in kilodaltons.

CLA-1 Confers on Sf9 Cells the Ability to Bind HDL, LDL, VLDL and the In Vitro Modified Forms of OxLDL and AcLDL
To investigate whether OxLDL, HDL, LDL, and VLDL were able to bind to Sf9-CLA-1 cells, Sf9 cells were infected with the BacPAK6 baculovirus or with the baculovirus BP-CD69, which carries the cDNA of the cell surface molecule CD69 (Sf9-CD69) (used as negative controls), and incubated with DiI-labeled lipoproteins before examination by fluorescence microscopy (Fig 4) and by fluorescence-activated cell sorter analysis (see below). In all cases, cells exhibited no staining or a barely detectable staining that revealed a negligible binding activity (Fig 4A, 4B, 4D, 4E, 4G, 4H, 4J, and 4K). In contrast, Sf9-CLA-1 incubated with the same set of DiI-labeled lipoproteins displayed an intense fluorescence at the plasma membrane consistent with a binding activity (Fig 4C, 4F, 4I, and 4L). Lipoprotein binding increased when the temperature was raised from 4°C to 37°C and in a linear fashion with the multiplicity of infection, which directly correlates with the cell surface expression levels (unpublished data, 1997). Similar results were obtained when DiI-labeled AcLDL was used (unpublished data, 1997). Therefore, CLA-1 conferred to Sf9 cells the capability to bind HDL, LDL, VLDL, AcLDL, and OxLDL.

View larger version (153K): [in this window] [in a new window]   Figure 4. Binding of DiI-labeled lipoproteins to Sf9 cells expressing CLA-1. Sf9 cells infected with the baculoviruses containing the cDNAs indicated were grown on coverslips and incubated for 1 hour at room temperature with 5 µg/mL of the indicated DiI-labeled lipoproteins. After washing, cell association of DiI-labeled lipoproteins was observed under fluorescence microscopy. Bar=30 µm.

Binding activity of increasing concentrations of OxLDL, LDL, HDL, and VLDL to Sf9-CLA-1 cells increased in a saturable fashion (Fig 5 and Table). The dissociation constant (K_d) calculated for the binding of OxLDL at 4°C to Sf9-CD36 cells (1.7 µg/mL) (unpublished data, 1997) agreed with the one reported for the binding of OxLDL to human CD36 when expressed on mammalian cells (1.5 µg/mL),¹⁵ a finding that suggests that CD36 when expressed on insect cells maintains its lipoprotein binding properties without modification and supports the validity of the binding constants determined using this expression system. All the dissociation constants determined from these experiments (Table 1) lie within the range of dissociation constants of other lipoprotein receptors and indicate high-affinity interactions.

View larger version (20K): [in this window] [in a new window]   Figure 5. Fig 5. Saturation curves of OxLDL, LDL, HDL, and VLDL binding to Sf9-CLA-1 cells. BacPAK6- and CLA-1-infected Sf9 cells (infected with an moi of 10) were incubated for 2 hours at 4°C with increasing amounts of the DiI-labeled lipoproteins OxLDL, LDL, HDL, and VLDL. After washing, cell-associated fluorescence was expressed as MIF values of bound lipoprotein (Lp). The specific binding values () of Sf9-CLA-1 cells to each lipoprotein represent the differences in the median fluorescence values between the total binding () and the nonspecific binding () obtained in the presence of a 50-fold excess of the corresponding unlabeled lipoprotein (which in all cases was not saturable and increased in a linear fashion with the lipoprotein concentration). For Sf9-BacPAK6 cells, only the specific binding, calculated as indicated above, is shown (). Sf9-CD69 cells exhibited similar binding profiles to those obtained for the Sf9-BacPAK6 cells. Duplicate determinations were done for each condition. Data shown correspond to a representative experiment. At least two different experiments using two different lipoprotein preparations were done.

View this table: [in this window] [in a new window]   Table 1. Dissociation Constant (Kd) Values, Expressed in Micrograms of Lipoprotein Per Milliliter, Calculated for the Binding of the Lipoproteins OxLDL, LDL, HDL, and VLDL to CLA-1

The binding specificity of CLA-1 to VLDL and HDL was additionally explored by competition experiments with unlabeled lipoproteins. As expected, competition was observed in all cases, although to different degrees (Fig 6). Except for VLDL (see below), the inhibitory capacity of each lipoprotein was consistent with its respective K_d value. Thus, unlabeled HDL and OxLDL displayed a higher inhibitory capacity than LDL for the binding of HDL and VLDL to Sf9 cells expressing CLA-1. The low inhibitory capacity of VLDL may be a consequence of a nonreciprocal cross-competition effect with respect to HDL and OxLDL, a phenomenon extensively documented for the binding of AcLDL and OxLDL to the scavenger receptors SR-AI and SR-AII.³⁶ An alternative explanation we favor relates to a lattice-model–like effect.³⁷ According to this model, the large size of a given VLDL particle bound to CLA-1 may impede, by steric hindrance, the binding of other VLDL particles to nearby unoccupied receptors but not the binding of the much smaller particles such as HDL.

View larger version (16K): [in this window] [in a new window]   Figure 6. Lipoprotein displacement of HDL (A) and VLDL (B) binding to Sf9-CLA-1 cells. Sf9-CLA-1 cells were incubated in duplicate with 10 µg/mL of DiI-labeled HDL or 10 µg/mL of DiI-labeled VLDL for 2 hours at 4°C in the presence of increasing concentrations of the following unlabeled inhibitors: HDL, LDL, VLDL, and OxLDL. After washing, binding of DiI-labeled HDL or DiI-labeled VLDL to cells was determined by fluorescence flow cytometry. Depicted values represent the percentages of binding with respect to the experiment carried out in the absence of any competitor. Data shown were representative of two independent experiments.

The existence of two HDL fractions differing in their apo E content provided us the opportunity to examine the requirement of apo E in the binding of HDL to CLA-1. To that end, HDL was fractionated in two subpopulations by heparin affinity chromatography, one with apo E and one lacking apo E. As shown in Fig 7, both HDL-apo E+ and HDL-apo E-, like total HDL, were effective competitors for the binding of total DiI-labeled HDL. This result indicates that binding of CLA-1 to HDL does not require the presence of apo E in the lipoprotein particle, a conclusion that could be also applied to other apo E–containing lipoproteins (eg, VLDL).

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of HDL fractions, HDL-ApoE+, and HDL-ApoE- on the association of DiI-HDL to Sf9-CLA-1 cells. Sf9-CLA-1 cells were incubated in duplicate with 5 µg/mL of DiI-HDL for 2 hours at 4°C in the presence of increasing concentrations (0, 5, 10, 20, 40, 100, and 250 µg/mL) of the following inhibitors: total HDL, HDL-ApoE-, and HDL-ApoE+. After washing, binding of DiI-labeled HDL to cells was determined by fluorescence flow cytometry. Depicted values represent the percentages of binding with respect to the experiment carried out in the absence of any competitor. Data shown correspond to a representative experiment. Two different experiments using different lipoprotein preparations were done.

Binding of CD36 and CLA-1 to Scavenger Receptor Polyanionic Ligands
The ability of CLA-1 to interact with modified lipoproteins prompted us to investigate the scavenger receptor activity of the CLA-1 receptor compared with that of CD36, as denoted by the ability of a panel of polyanionic compounds to inhibit the binding of AcLDL to Sf9-CLA-1 and Sf9-CD36 cells. As shown in Fig 8, whereas a 50-fold excess of unlabeled AcLDL abrogated the binding of the DiI-labeled AcLDL to both Sf9-CD36 and Sf9-CLA-1 cells, none of the other compounds assayed, including the macrophage scavenger receptor SR-A inhibitors fucoidin and polyinosinic acid,²⁰ were effective competitors. Thus, it could be concluded from these data that neither CD36 nor CLA-1 exhibited the ligand-binding specificity ascribed to the classic SR-A scavenger receptors.

View larger version (37K): [in this window] [in a new window]   Figure 8. Scavenger receptor properties of CLA-1 and CD36. Binding of DiI-labeled AcLDL (5 µg/mL) to Sf9-CLA-1 and Sf9-CD36 cells at room temperature for 1 hour was measured by fluorescence flow cytometry in the absence (none) or presence of the following competitors: unlabeled AcLDL (200 µg/mL), fucoidin (200 µg/mL), poly-I (500 µg/mL), poly-C (500 µg/mL), chondroitin sulfate A (CSA) (200 µg/mL), and chondroitin sulfate B (CSB) (200 µg/mL). For each cell line, the results of the experiments were represented as percentages of binding with respect to the experiment performed in the absence of any competitor. Data shown correspond to a representative experiment. Two different experiments using different lipoprotein preparations were done.

Tissular Distribution of Human CD36 and CLA-1
To explore the tissue distribution of human CLA-1, we examined its mRNA relative abundance in a panel of human tissues and compared it with the expression of the structurally related cell surface receptor CD36 (Fig 9). Whereas CLA-1 mRNA expression was mainly found in liver and in the steroidogenic tissues of the ovary, testis, and placenta, CD36 mRNA was more abundantly present in skeletal muscle, heart, colon, small intestine, peripheral blood leukocytes, spleen, and possibly in adipose tissue³⁸ and mammary gland,³⁹ as determined for mouse and bovine, respectively.

View larger version (80K): [in this window] [in a new window]   Figure 9. Northern blot analysis of CD36 and CLA-1 mRNA expression in a panel of human tissues. Two micrograms of poly A+ mRNA isolated from the indicated tissues were probed with the cDNAs of CD36, CLA-1, and ß-actin (used as a control for equal RNA loading). Sk. indicates skeletal; PBL, peripheral blood leukocytes; and S. intestine, small intestine.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this report, we demonstrate that CLA-1 is a promiscuous cell surface high-affinity receptor for the plasma lipoproteins HDL, LDL, and VLDL and for the in vitro LDL modified forms, OxLDL and AcLDL. Interestingly, the ability of CLA-1 to recognize VLDL constitutes the first description of the interaction of a glycoprotein of the CD36 family with VLDL.

Although recognition of modified lipoproteins is a hallmark of the scavenger receptors,²⁰ CLA-1 and CD36 do not exhibit the characteristic broad ligand specificity of most scavenger receptors (Fig 8). Although the contribution of CD36 expressed on monocyte/macrophages to OxLDL uptake and foam cell formation has been demonstrated,^{16 40} the expression level of CLA-1 in those cells is unknown. In addition to monocyte/macrophages, the liver is the organ responsible for most of the uptake of modified lipoproteins from blood.⁴¹ In this organ, the parenchymal and Kupffer cells internalize and degrade OxLDL by pathways that do not involve the scavenger receptor SR-A.⁴² Whether CLA-1 mediates uptake of modified lipoproteins in these cell types should be investigated.

In vivo, the fate of lipoproteins is largely determined by the interaction between their constituent apolipoprotein moieties and the cellular receptors and enzymes involved in their metabolism.⁴³ This feature provides a rational molecular explanation for the specific metabolism of each lipoprotein type. The ability of CLA-1 to interact with the major lipoprotein classes highlights the interest in determining the structural nature of the ligand recognized on each lipoprotein type. Although we cannot answer this query thus far, the ability of some anionic phospholipids to interact with CD36 and SR-BI¹⁷ strongly suggests that the phospholipids may contribute to the structural moieties that are recognized by CLA-1 on lipoproteins. Our data demonstrating the apo E–independent recognition of HDL by CLA-1 (Fig 7) are compatible with the above proposal.

An examination of the tissue distribution of CLA-1 reveals that its expression is mostly restricted to tissues involved in cholesterol metabolism, either for storage, secretion, or transformation (liver) or for steroid hormone synthesis(ovary, testis, and placenta) (Fig 9). The CLA-1 tissue distribution contrasts with that displayed by its related product, CD36, which appears more prominently expressed on tissues performing very active fatty acid metabolism, such as skeletal muscle, heart, intestine, adipose tissue,³⁸ and mammary epithelia³⁹ (Fig 9), and possibly in the monocytes/macrophages present in peripheral blood and spleen (Fig 9). A major difference between the tissue distribution of human CLA-1 and that of mouse or rat SR-BI^{14 21} is the absence of a specific messenger for SR-BI in placenta.

The physiological role of CLA-1 is presently unknown. Mouse and rat SR-BI acts as an HDL receptor that mediates cholesteryl ester selective uptake, a cholesterol-delivery pathway that has been demonstrated to operate mainly in steroidogenic tissues and liver.^{21 22} The tissue distribution of human CLA-1 and its ability to bind HDL are consistent with this role. Moreover, it has been reported that HDL delivers cholesterol to cultured human placental trophoblasts and stimulates the secretion of progesterone through an LDL receptor–independent pathway.⁴⁴ The expression of mRNA for human CLA-1 in placenta allows us to suggest the participation of CLA-1 in the above process. Thus, binding to CLA-1 could be a recruitment mechanism for lipoproteins on the cell surface, which allows the subsequent uptake of the particle-carried lipids. The possibility exists that in this location a functional interplay with different molecules takes place, similar to the ligand-transfer model that has been proposed for proteoglycans or lipoprotein lipase and the LDL receptor–related protein.⁴⁵ In this regard, it is interesting to note that the lipoprotein binding and tissue distribution of CLA-1 largely overlap with those corresponding to hepatic lipase. This enzyme is secreted by hepatocytes to localize mainly in the sinusoids of the liver and to be transported to steroidogenic tissues that also display hepatic lipase activity.^{46 47} Hepatic lipase, besides mediating lipolysis of triglyceride-rich lipoproteins and conversion of HDL₂ to HDL₃, has been shown to enhance LDL and VLDL uptake^{48 49} and HDL cholesteryl ester delivery to cells.⁵⁰ Thus, we can speculate that cooperation exists between hepatic lipase and CLA-1 that would modulate the cholesterol/fatty acids flux between lipoproteins and cells. Such a proposal is consistent with the observed upregulation of SR-BI mRNA in the adrenal gland of hepatic lipase knockout mice as a response to depletion of cholesterol stores.²²

Altogether, our studies provide firm evidence for the lipoprotein-binding activity of the human glycoprotein CLA-1. Strikingly, the competence of CLA-1 to bind to the so-called atherogenic (LDL, OxLDL, and VLDL) and antiatherogenic lipoproteins (HDL) makes prediction of its role in atherogenesis uncertain. Mice overexpressing or lacking CLA-1 will constitute powerful tools in the quest to unravel the in vivo contribution of the CLA-1 receptor in lipid metabolism and to study its role in the formation and development of atherosclerotic lesions.

	Selected Abbreviations and Acronyms

 

AcLDL = acetylated LDL apo = apolipoprotein bp = base pair DiI = 1,1'-dioctadecyl-3-3-3'-3'-tetramethyllindocarbocyanine perchlorate kbp = kilobase pair MIF = median intensity of fluorescence moi = multiplicity of infection OxLDL = oxidized LDL PCR = polymerase chain reaction SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis

	Acknowledgments

 
This work was supported by grants from the Ministerio de Educación y Ciencia of Spain PM91/031 and PB93/1020 to Miguel Vega and F.I.S. 94/0540 to Diego Gómez-Coronado. Dominica Calvo is the recipient of a predoctoral fellowship from the Comunidad Autónoma de Madrid. We thank Drs F. Sánchez-Madrid and José L. Alonso for their generous gifts of reagents (ICAM-3 expression vector, the CD69 baculovirus, and the monoclonal antibody TP1/33). We also thank Dr Angel Corbí for critical reading of the manuscript.

Received December 31, 1996; accepted March 21, 1997.

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