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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1033-1039

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1033.)
© 1996 American Heart Association, Inc.

Articles

Identification on Human CD36 of a Domain (155-183) Implicated in Binding Oxidized Low-Density Lipoproteins (Ox-LDL)

Marta D. Puente Navazo; Laurent Daviet; Ewa Ninio; John L. McGregor

INSERM Unit 331 (M.D.P.N., L.D., J.L.M.), Faculté de Medicine René Laënnec, Lyon, France; INSERM Unit 321 (E.N.), Hpital de la Pitié, Paris, France; and Stanford (Calif) Medical School, Division of Hematology (J.L.M.).

Correspondence to Marta D. Puente Navazo, PhD, INSERM Unit 331, Faculté de Medicine René Laënnec, F-36372 Lyon, France. E-mail puente@cimac-res.univ-lyon1.fr.

Abstract

Uptake of oxidized LDL (oxLDL) by macrophages is one of the key events implicated in the initiation and perpetuation of atherosclerotic lesions. One of the major scavenging receptors, which binds modified LDL, on macrophages is CD36. The domain on CD36 implicated in the binding of oxLDL remains to be elucidated. In this study, COS cells transfected with human CD36 cDNA bound FITC-oxidized human LDL in a dose-dependent, saturable manner. This binding was inhibited by an excess of oxLDL but not by native LDL. Anti-CD36 monoclonal antibodies (mAbs) 10/5, FA6-152, and 8A6 (directed against domain 155-183), but not mAb 13/10 (directed against domain 30-76), completely inhibited oxLDL binding to human CD36-transfected COS cells. Cells transfected with a chimeric human CD36 construct (hmh 155-183), resulting from the swapping of human domain 155-183 with its murine counterpart, resulted in low binding of oxLDL. In contrast, cells transfected with a chimeric murine CD36 construct (mhm 155-183), resulting from the swapping of murine domain 155-183 with its human counterpart, resulted in high binding of oxidized human LDL. Binding of oxLDL to cells transfected by chimeric construct mhm 155-183 were only partially blocked by mAbs 10/5, FA6-152, and 8A6. In the present study we have identified, for the first time, an important functional domain (encompassing amino acids 155-183) on CD36 involved in the binding of oxLDL. In addition, the binding site for oxidized human LDL on murine CD36 seems to differ from its human counterpart.


Key Words: oxidized LDL • CD36 • foam cell formation

An early event in the initiation and perpetuation of atherosclerotic lesions is the scavenging or accumulation of lipids by subendothelial macrophages.1 Different receptors on macrophages are thought to mediate the scavenging of native or modified lipoproteins (acetylated LDL or oxLDL).2 However, in vivo and in vitro studies indicate that the massive accumulation of cholesterol by macrophages cannot be entirely due to the activity of the above-mentioned receptors.2 Thus, other receptors for LDL may play a predominant role in the endocytosis of lipoproteins by macrophages.

CD36 has recently been identified as an important receptor in scavenging oxLDL.3 4 5 mAbs directed against CD36 inhibit oxLDL uptake by macrophages.5 Moreover, macrophages from CD36-deficient individuals show a decreased capacity to take up oxLDL.6 CD36 is an 88-kD multifunctional adhesive glycoprotein expressed by platelets, monocytes, microvascular endothelial cells, mammary epithelial cells, erythroblasts, and several tumor cell lines.7 It is one of the receptors of collagen type I8 and thrombospondin.9 10 It mediates the cytoadhesion of Plasmodium falciparum–infected erythrocytes to brain postcapillary venular endothelium, a factor that contributes to the pathogenicity of P falciparum malaria.11 CD36 has also been reported to act as a signaling molecule capable of mediating an oxidative burst in monocytes12 and activating platelets13 by a process that seems to be triggered by tyrosine phosphorylation.14 Recent observations show that E-selectin, expressed by activated endothelial or transfected L cells, can increase the number of CD36 molecules expressed on the monocyte surface.15 CD36, together with {alpha}vß3 integrin, has been identified as one of the adhesion molecules on the surface of macrophages implicated in the clearance of polymorphonuclear leukocytes.16 Very little is known, at this stage, concerning the functional domain on CD36 involved in the recognition of oxLDL. The use of anti-CD36 mAbs, with well-defined epitopes in tandem with human/murine CD36 chimeric constructs, in COS transfection studies has allowed the identification of a domain on CD36 that is implicated in the binding to oxLDL.

Methods

Materials
The cDNAs coding for human (h) CD36 and murine (m) CD36 were generously given by Dr Brian Seed (Harvard Medical School) and Dr Gerda Endemann (Scios Nova Inc), respectively. LipofectAmine reagent was from GIBCO-BRL. Restriction endonucleases were from New England Biolabs or GIBCO-BRL. The COS-7 cell line was obtained from the European Collection of Animal Cell Cultures. Culture media and supplements were from GIBCO-BRL. Other reagents were from different sources and were of the highest available purity.

mAbs
mAbs anti-CD36 10/5 and 13/10 were produced in our laboratory and used for immunization of a CD36-expressing recombinant vaccinia virus. Culture supernatants were tested by an ELISA with mouse L cells stably transfected with hCD36 cDNA and control nontransfected L cells as targets. These antibodies were further characterized by immunoprecipitation, Western blots, and FACS analysis of human platelets. mAbs 10/5 and 13/10 were found to be IgG2a and IgG2b, respectively, as determined by the Amersham isotyping kit (Amersham International).17 These antibodies were used as immunoglobulins by purification on a Protein-A Sepharose column (Pharmacia). FA6-152 was a generous gift from A. Aghtoven (Immunotech, Luminy, France). mAb 8A6 was kindly provided by Dr J. Barnwell (New York University Medical Center). As control, different irrelevant immunoglobulins were used: a nonimmune IgG2a (The Binding Site) and LYP20, an IgG1 directed against human P-selectin.18 A polyclonal anti-CD36 human binding serum (anti-Naka) was a generous gift from Prof Y. Shibata (Toranomon Hospital, Tokyo, Japan).

CD36 Constructs
Constructs were generated by the introduction of endonuclease sites into the hCD36 and mCD36 cDNA without altering the coding sequence as described previously.19 They were subsequently cloned in the eukaryotic expression vector pCDM8, digested with the appropriate restriction endonuclease to allow exchange of sequences between hCD36 and mCD36 cDNA, and the resulting ligation products transformed into E. coli MC1061/p3. The absence of mutations other than the substitution of the murine/human region was confirmed by DNA sequencing.

COS Cell Transfection
COS cell monolayers cultured in 35-mm-diameter dishes at 70% confluence were transfected with 2 µg DNA and 6 µL LipofectAmine, in DMEM, according to manufacturer's instructions. After 48 hours' transfection, CD36 expression on the surface of COS cells was tested. Cells were detached with trypsin-EDTA, washed in DMEM/10% fetal calf serum, and resuspended to a cell density of 1x106 cells/mL in PBS/1% BSA. Cells were then incubated for 1 hour at 4°C with mAb 10/5, mAb 13/10, or irrelevant antibodies, at a final concentration of 5 µg/mL. After two washes in PBS, cells were incubated for a further period of 1 hour at 4°C, with goat anti-mouse IgG and IgM antibody, FITC conjugated to a final concentration of 10 µg/mL. After another series of washings, the expression was immediately assessed by flow cytometry with the FACScan system (Becton Dickinson), with LYSIS II software.

Purification and Oxidation of Lipoproteins
LDL was isolated from human normolipidemic plasma by sequential centrifugation in the 1.025- to 1.050-g/mL–density interval and exhaustively dialyzed against PBS containing 3 mmol/L EDTA, pH 7.4. The purity of the preparations was evaluated as previously described20 and their protein content determined as also previously described.21 Before oxidation, LDL was dialyzed against PBS, pH 7.4, to remove EDTA. OxLDL was prepared by incubating 500 µg LDL protein per milliliter in PBS containing 2.5 µmol/L CuCl2 for 48 hours at 37°C. OxLDL was then extensively dialyzed against PBS/EDTA and subsequently filtered though a 0.22-µm filter (Millipore). The time course of copper-induced oxidation of LDL was measured as conjugated diene formation. The net electrical charge of both native and modified LDL at pH 8.6 was estimated by electrophoresis in agarose gel.22 The degree of lipid peroxidation of modified LDL was compared with that of native LDL and measured by the amount of TBARS23 and by hydroperoxide formation assay.24 The electrophoretic mobility on agarose gel, and thus the negative charge of oxLDL, was compared with that of native LDL: relative electrophoretic mobility=4.8±0.2 (n=3). The generation of aldehydes, estimated as the content of TBARS, was 0.8±0.07 nmol/mg protein for native LDL and 39.5±3.3 nmol/mg protein for oxLDL (P<.0001, n=3). The level of endoperoxides was detected as hydroperoxide formation assay LPO and was 10.8±3.4 nmol/mg protein for native LDL and 293.4±86.2 nmol/mg protein for oxLDL (P<.003, n=3).

Labeling of OxLDL
OxLDL was labeled using FITC (Molecular Probes) according to Homburg et al.25 Briefly, oxLDL was dialyzed against coupling buffer (50 mmol/L sodium borate/NaOH, 150 mmol/L NaCl, and 1 mmol/L EDTA, pH 9) for 48 hours in darkness at 4°C. Lipoproteins (3 mg oxLDL or 10 nmol apoB) were then mixed with 500 nmol FITC and incubated for 2 hours at 37°C. The mixture was first dialyzed against 50 mmol/L Tris, 80 mmol/L NaCl, and 1 mmol/L EDTA, pH 8, for 24 hours at 4°C in the dark and then with PBS/EDTA for another 24-hour period. The FITC-oxLDL preparation was then analyzed for protein content and absorbance at 492 nm (for FITC, e492=78 000 L·mol-1·cm-1) to measure the coupling of FITC to oxLDL. In the experiments presented here, 10 molecules of FITC were coupled to 1 molecule of apoB.

Assay for Binding of OxLDL to Transfected COS Cells
Forty-eight hours after transfection, COS cells were detached with trypsin-EDTA, washed in DMEM/10% FCS, and resuspended in DMEM/1% BSA to a cell density of 1x106 cells/mL. A 200-µL aliquot of this cell suspension was placed in a FACS tube, washed once with DMEM/1% BSA, and centrifuged 5 minutes at 1500 rpm. The resulting pellet was then resuspended in 500 µL of a solution of 10 µg/mL FITC-oxLDL (or otherwise stated) in DMEM/1% BSA, and incubated for 2 hours at 4°C. Cells were then washed twice with DMEM/1% BSA and finally resuspended with 200 µL PBS. Cell association of oxLDL was immediately assessed by flow cytometry with the FACScan system (Becton Dickinson), with LYSIS II software. The FITC-oxLDL cell-binding data were analyzed by histograms of fluorescence, using mean fluorescent intensity of the positive cells.

Effects of mAbs
Forty-eight hours after transfection, COS cells were washed, and 200 µL of mAb at a final concentration of 25 µg/mL in DMEM was added to each tube. After an incubation period of 30 minutes at 4°C, cells were washed in 2 mL DMEM and resuspended in an FITC-oxLDL solution as indicated above.

Results

Expression of CD36 Constructs
mAbs directed against different regions of the CD36 molecule were used to monitor wild-type expression of human or chimeric human/murine CD36 constructs expressed by transfected COS cells. COS-7 cells transfected with mCD36 cDNA, in which the domain coding for aa 155-183 was replaced by its human counterpart (chimera mhm 155-183), expressed the epitope for the mAb 10/5 but not for mAb 13/10. In addition, mAb 10/5 binding to the mhm 155-183 construct was very similar to that observed for wild-type hCD36 (TableDown). In contrast, mAb 10/5 did not bind to hCD36 cDNA constructs, in which the region coding for the 155-183 domain was replaced by its murine counterpart (chimera designated as hmh 155-183). This hmh 155-183 chimera bound mAb 13/10 to the same extent as wild-type hCD36 (TableDown). The level of expression of mCD36 could not be assessed by the binding of the above antibodies, as they failed to recognize mCD36.19 The expression of mCD36 on the surface of COS cells on the corresponding cDNA transfection was tested by flow cytometry, with a human anti-Naka antiserum, which recognizes both hCD36 and mCD36.26 The level of expression of mCD36 on the surface of COS cells was similar to that observed for hCD36 (data not shown).


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  		Table 1. Binding of Anti-CD36 mAbs to COS Cells Expressing Wild-Type Human and Murine CD36 or Human-Mouse Chimeric Constructs (mhm 155-183 and hmh 155-183)

Recognition of OxLDL by CD36
Transfection of COS cells with hCD36 cDNA leads to the binding of FITC-oxLDL (Fig 1Down). However, no binding of FITC-oxLDL is observed to COS cells transfected with pCDM8 vector bearing no insert (mock-transfected cells). Moreover, FITC-oxLDL binding was completely blocked by an excess of unlabeled oxLDL but not by native LDL. These data indicate that FITC-oxLDL carefully mirrors the binding of oxLDL to expressed CD36.



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  		Figure 1. Specificity of the binding of FITC-oxLDL to CD36-transfected COS cells. Forty-eight hours after transfection, cells were incubated with 10 µg/mL FITC-oxLDL for 2 hours at 4°C. LDL binding was subsequently analyzed by flow cytometry. Data presented are typical fluorescence histograms. A, Cells transfected with the empty pCDM8 vector. B, Human CD36–transfected COS cells incubated with 10 µg/mL FITC-oxLDL. C, Human CD36–transfected COS cells incubated with 10 µg/mL FITC-oxLDL and 250 µg/mL oxLDL. D, Human CD36–transfected COS cells incubated with 10 µg/mL FITC-oxLDL and 250 µg/mL native LDL.

mAbs Directed Against Domain 155-183 Inhibit OxLDL Binding
Preincubation of hCD36-transfected COS cells with anti-CD36 mAbs (10/5, FA6-152, 8A6), whose epitopes are located within aa 155-183,19 completely inhibited FITC-oxLDL binding (Fig 2Down). In contrast, mAb 13/10, whose epitope is present within region aa 30-76,19 did not affect FITC-oxLDL binding (Fig 2Down). Moreover, an irrelevant mAb of the same isotype as 10/5 (IgG2a) or an anti–P-selectin mAb (LYP20, an IgG1) of the same isotype as FA6-152 and 8A6 did not affect FITC-oxLDL binding (Fig 2Down). The panel of anti-CD36 mAbs (10/5, 8A6, and FA6-152), but not mAb 13/10, inhibited FITC-oxLDL binding to ltk- cells stably transfected with hCD36 (data not shown). Thus, the functional effects of anti-CD36 mAb, with epitopes located on the 155-183 domain, were not exclusive to COS-7 cells transiently expressing hCD36. F(ab')2 fragments of 10/5, but not 13/10, completely inhibited FITC-oxLDL binding, in a manner that was similar to that observed for whole IgG (Fig 3Down). Thus, the effect of mAb 10/5 on FITC-oxLDL binding by hCD36-transfected COS-7 cells is independent of its Fc fragment. These results indicate that functional mAbs, directed against the 155-183 domain of hCD36, are capable of completely inhibiting oxLDL binding to CD36-transfected cells.



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  		Figure 2. Inhibition of oxLDL binding by CD36-transfected COS cells caused by mAbs directed against CD36 having epitopes located on domain 155-183. mAbs were used in the form of purified immunoglobulins at a final concentration of 25 µg/mL. Data are from six independent experiments. Error bars are SEMs.



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  		Figure 3. Inhibition of oxLDL binding by Fab fragments of anti-CD36 antibodies. F(ab')2 or Fab fragments were used at a final concentration of 25 µg/mL. Data are presented as the percentage of mean fluorescence intensity of mock-transfected cells. Data are from three independent experiments. **Significantly different from control at P<.02, as analyzed by Student's t test.

Binding of OxLDL to CD36 Chimeras
Binding of FITC-oxLDL to both hCD36 and mCD36, as measured by the mean fluorescence intensity of positive cells, was saturable and of high affinity (Fig 4Down). FITC-oxLDL, when added to COS cells transfected with the different CD36 variants in the presence of a 50-fold excess of unlabeled oxLDL, showed no binding.



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  		Figure 4. Binding of oxLDL to human (h) and murine (m) wild-type CD36 and human/murine chimeric constructs. COS cells were transfected with hCD36, mCD36, or the chimeric constructs [mhm (155-183) or hmh (155-183)] as described. Forty-eight hours after transfection, cells were incubated with different concentrations of FITC-oxLDL, and after a 2-hour incubation period, fluorescence was measured by flow cytometry. Data are presented as mean fluorescence intensity. Data are from four independent experiments. Error bars are SEMs. Hyperbolic curve fit was performed using Excel software.

Scatchard analysis of the binding of FITC-oxLDL to mCD36-transfected cells indicated that maximal binding of FITC-oxLDL to mCD36-transfected cells was higher (344.1±66.5) than that observed for hCD36-transfected cells (284.2±30.3). The Kd for FITC-oxLDL binding to hCD36- and mCD36-transfected cells was 0.50±0.11 and 0.69±0.14 µg/mL, respectively. Such differences in binding capacity between hCD36 and mCD36 were used to identify the functional domain on human cDNA CD36 implicated in binding human oxLDL. For that purpose, chimeric constructs of hCD36 and mCD36 were generated and subsequently transfected in COS-7 cells. Cells transfected with chimeric hCD36 (hmh 155-183), in which the region coding for domain 155-183 was replaced by its murine counterpart, showed a significantly lower FITC-oxLDL binding capacity compared with wild-type hCD36. The results of the Scatchard analysis revealed that the maximal mean fluorescence for this chimeric construct was 161.5±0.5 and its Kd was 0.375±0.08 µg/mL. In contrast, COS cells transfected with an mCD36 in which the region coding for domain 155-183 was replaced by its human counterpart (chimeric construct mhm 155-183) had a very high capacity to bind FITC-oxLDL, very similar to that observed for mCD36-transfected cells. The maximal mean fluorescence intensity for this chimeric construct was 343.5±77.0 and its Kd was 1.025±0.48 µg/mL, as calculated from the Scatchard plot from data displayed in Fig 4Up. These results indicate that swapping the 155-183 domain between hCD36 and mCD36 modifies the capacity of both to recognize and bind human oxLDL.

Effect of mAbs on FITC-OxLDL Binding to Chimeric Constructs
Binding of FITC-oxLDL to COS cells transfected with construct mhm 155-183 was partially inhibited by anti-CD36 mAbs 10/5 (by 36.7±7.2%), FA6-152 (by 50±3.4%), and 8A6 (by 58.5±12.8%) (domain encompassing aa 155-183) but not by mAb 13/10 (domain encompassing aa 30-76). None of the anti-CD36 mAbs tested (10/5, FA6-152, or 8A6) affected FITC-oxLDL binding to wild-type mCD36 or hmh 155-183 CD36-transfected COS cells (Fig 5Down).




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  		Figure 5. Effect of different anti-CD36 mAbs on oxLDL binding by COS cells transfected with human CD36, murine CD36, or chimeric CD36 constructs. mAbs were used in the form of purified immunoglobulins at a final concentration of 25 µg/mL. Data are presented as the percentage of mean fluorescence intensity of mock-transfected cells. Data are from six independent experiments. Error bars are SEMs.

Discussion

In the present study we have identified, for the first time, an important functional domain on CD36 involved in the binding of oxLDL. The role of this domain in mediating the binding of oxLDL is supported by a number of lines of evidence: (1) A panel of mAbs directed against domain 155-183 but not domain 30-76 completely inhibits oxLDL binding to CD36. (2) Swapping region 155-183 on hCD36 for its murine counterpart, in a chimeric construct, results in a much lower oxLDL binding. (3) Inserting the hCD36 155-183 domain in the mCD36 does not change its capacity to bind oxLDL. Moreover, this study also shows that hCD36 has a different binding for human oxLDL than does mCD36.

Results from this work are in support of the suggestion by Endemann et al4 that the OKM5 epitope is colocalized with the oxLDL binding site. The epitope of this mAb (OKM5) colocalizes with the mAbs used in this study (10/5, FA6-152, and 8A6) within the region encompassing aa 155-183.19 The 155-183 domain on CD36 is adjacent to a region encompassing aa 139-155 that has already been reported to represent a part of the OKM5 epitope and implicated in the initial binding to thrombospondin.27 It is important to note that domain 139-155 is identical in both the human and mouse proteins, with the exception of residues 146 and 152.19 Thus, binding of oxLDL is dependent on the 155-183 region, but residues in the adjacent 139-155 sequence could also be important, as observed for OKM5 binding.19 27 Human oxLDL may have its initial interaction with CD36 linked to a discontinuous site involving aa 139-155 and 155-183 domains. Replacing the 155-183 domain in hCD36 with its murine homologue may affect the stability of the oxLDL-CD36 interaction and/or the transmission of necessary outside-in signals14 to the cell and prevent the recognition of human oxLDL. Alternatively, any changes in the sequence of aa 155-183 from human to murine may affect the conformation of CD36 and its capacity to be implicated in the mechanism of binding to human oxLDL. Domain 155-183 is quite hydrophobic and is probably associated with the membrane.7 In view of the work of Nicholson et al5 suggesting that the lipid moiety of oxLDL is important in its interaction with CD36, one might speculate that a hydrophobic environment for CD36 within the region 155-183 could be advantageous for oxLDL binding. A recent report28 suggests that CD36 contains a hydrophobic pocket that could mediate CD36–fatty acid or CD36-oxLDL binding interactions. It is conceivable that region 155-183 might be present in this hydrophobic pocket.

Results from this study show that the domain on hCD36, implicated in binding oxidized human LDL, differs from the one present on its murine counterpart. When human domain 155-183 is swapped for its murine counterpart, the binding capacity of hCD36 for human oxLDL is considerably reduced. It is possible that human 155-183 bears a special tag, absent in the mouse homologous region, necessary for the recognition and binding of human oxLDL. In contrast, when murine domain 155-183 is swapped for its human counterpart, the capacity of mCD36 to bind oxLDL is unaltered. In addition, the binding of oxLDL to a murine chimera bearing the 155-183 human domain is only partially inhibited by a panel of mAbs known to completely block binding in wild-type hCD36. These results suggest that sites (other than those present on human domain 155-183) that are blocked by mAbs are present on mCD36 to bind human oxLDL.

Other functional domains have been described on CD36 by the work of Asch et al,29 which implicates aa 87-99 in the binding of thrombospondin and shows that this binding is linked to the phosphorylation of Thr92.29 Moreover, Pearce et al30 used recombinant proteins to show the role of the 93-120 domain in binding thrombospondin.

In conclusion, the data presented in this study indicate that the functional domain encompassing aa 155-183 plays a critical role in oxLDL binding. In addition, recent data from our laboratory suggest that region 155-183 is a multifunctional domain, implicated as well in the cytoadherence of P falciparum–infected erythrocytes as a signaling molecule involved in inducing platelet aggregation and secretion17 31 and in the recognition and phagocytosis of apoptotic neutrophils.32 The domain 155-183 is coded by a single exon (Exon VI),33 and it is likely that this exon delineates a structural and functional domain on hCD36 directly involved in CD36-ligand interactions.

Selected Abbreviations and Acronyms


aa = amino acids
DMEM = Dulbecco's modified Eagle's medium
FACS = fluorescence-activated cell sorter
FITC-oxLDL = FITC-coupled oxLDL
hCD36 = human CD36
mAb(s) = monoclonal antibody(ies)
mCD36 = mouse CD36
oxLDL = oxidized LDL
TBARS = thiobarbituric acid–reactive substances

Acknowledgments

This work was supported by MESR ACC-SV9, ARC (subvention 6586). Dr Puente Navazo is indebted to INSERM for a postdoctoral fellowship. We thank B. Seed (Harvard Medical School) for the human CD36 clone and G. Endemann (Scios Nova Inc) for the mouse CD36 clone. Dr E. Chignier and G. Panae are acknowledged for help and advice in flow cytometry.

Received October 16, 1995; revision received March 11, 1996; References

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