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J Biol Chem, Vol. 275, Issue 13, 9163-9169, March 31, 2000
From the Departments of Medicine and There is growing evidence that CD36
has an important physiological function in the uptake of oxidized low
density lipoprotein (OxLDL) by macrophages. However, the ligand
specificity and the nature of the ligands on OxLDL that mediate the
binding to CD36 remain ill defined. Results from recent studies
suggested that some of the macrophage scavenger receptors involved in
the uptake of OxLDL recognized both the lipid and the
protein moieties of OxLDL, but there was no conclusive direct evidence
for this. The present studies were undertaken to test whether a single,
well characterized OxLDL receptor, CD36, could bind both the lipid and
protein moieties of OxLDL. COS-7 cells transiently transfected with
mouse CD36 cDNA bound intact OxLDL with high affinity. This binding
was very effectively inhibited (~50%) both by the reconstituted apoB
from OxLDL and by microemulsions prepared from OxLDL lipids. The
specific binding of both moieties to CD36 was further confirmed by
direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of
the OxLDL lipids and vice versa. Furthermore, a monoclonal
mouse antibody that recognizes oxidation-specific epitopes in OxLDL
inhibited the binding of intact OxLDL and also that of its purified
protein and lipid moieties to CD36. This antibody recognizes the
phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This
model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve
as ligands for recognition by CD36 and, at least in part, mediate the
high affinity binding of OxLDL to macrophages.
The oxidative modification of low density lipoprotein
(LDL)1 and the subsequent
uptake of oxidized LDL (OxLDL) by macrophages, leading to foam cell
formation, is an important pathway in atherogenesis (1, 2). OxLDL
interacts with macrophages via scavenger receptors, a family of
receptors characterized by broad ligand binding specificity. Macrophages express a number of scavenger receptors that bind OxLDL,
including SRA-1, SRA-2, SRA-3, MARCO, CD36, SR-B1, CD68/macrosialin, and LOX-1 (3-6). The nature of the ligand(s) on OxLDL recognized by
these receptors has not been clearly defined. Initially, it was assumed
that modifications of the protein structure were centrally important
because LDL could be converted to a high affinity ligand for macrophage
scavenger receptors by conjugating it with acetic anhydride or with
malondialdehyde or other reagents known to react with amino groups of
proteins (7, 8). Moreover, it was directly demonstrated that the apoB
isolated from OxLDL after exhaustive extraction of the lipids could
bind in a specific fashion to macrophages and compete for the binding
of intact OxLDL to these macrophages (9). However, the possible binding
of lipid moieties was not tested in those experiments, and recent
studies have demonstrated that indeed both the lipid and the protein
moieties of OxLDL can mediate the binding of intact OxLDL to
macrophages (10). Products derived by oxidation of pure phospholipids
(e.g. 1-palmitoyl 2-arachidonoyl phosphatidylcholine) have
also been shown to inhibit the binding of intact OxLDL (11) and also
that of isolated apoB and microemulsions of OxLDL lipids (10, 11).
Finally, a mouse monoclonal antibody (EO6), selected on the basis of
its recognition of OxLDL and specifically of the oxidized phospholipid,
1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine (POVPC), reacts with
both the isolated apoprotein of OxLDL and with the lipids from OxLDL,
but not with native apoB nor with lipids derived from native LDL (11,
12). This antibody strongly inhibits the macrophage binding and
degradation of intact OxLDL, as well as that of apoB and lipids derived
from OxLDL, presumably by masking the epitopes of OxLDL that are
recognized by the scavenger receptors.
Most of the studies summarized above were done by measuring binding to
intact mouse peritoneal macrophages and did not identify which of the
several scavenger receptors on the macrophage were involved. For a
number of reasons, it seemed possible that at least some of the
receptors involved were individually binding both the lipid moiety and
the modified protein moiety, but that could not be concluded with any
certainty. Therefore, we have undertaken to explore the ligand binding
specificity of individual scavenger receptors.
Recent studies provided direct evidence that CD36 is an important
physiological receptor involved in the uptake of OxLDL by macrophages
and may, therefore, play a role in foam cell formation in
vivo. Macrophages from subjects with CD36 deficiency are less efficient in binding and uptake of OxLDL compared with macrophages from
normal controls (13). Macrophages isolated from CD36-deficient mice
show a similar reduction in OxLDL binding and uptake (14), suggesting
an important role in macrophage function.
This report deals with the ligand binding specificity of CD36. It has
been established that cells transfected with CD36 show a large increase
in binding of intact OxLDL compared with nontransfected control cells
(15). The studies reported below show that CD36, which is transiently
expressed in COS-7 cells, exhibits ligand binding characteristics
similar to those reported for intact macrophages, i.e. it
can directly bind either apoB or the reconstituted lipids derived from
OxLDL. Cross-competition experiments suggest that the same
oxidation-specific epitope mediating the binding of OxLDL to CD36 is
present in both fractions. As in the case of intact macrophages,
monoclonal antibody EO6 inhibited the binding of OxLDL to CD36.
Moreover, a well characterized synthetic oxidized phospholipid POVPC,
the epitope of EO6 found in OxLDL (16) and previously shown to prevent
the binding of OxLDL to macrophages (11), also inhibited very
effectively the binding of OxLDL to CD36-transfected cells.
Materials--
COS-7 cells were purchased from American Type
Culture Collection. Dulbecco's modified Eagle's medium with 4.5 g/liter glucose was from Bio-Whittaker; fetal bovine serum was from
Gemini Bioproducts Inc. Penicillin-streptomycin,
L-glutamine, and trypsin-EDTA were from Irvine Scientific.
FuGene6 and D-octyl glucoside were purchased from Roche
Molecular Biochemicals. Polycarbonate membranes were from Poretics, and
1-palmitoyl 2-arachidonoyl phosphatidylcholine was from Avanti Polar
Lipids. Na125I (2000Ci/mmol) was from ICN, and
3,3'-dihexadecylooxacarbocyanine perchlorate (DiO) was from Molecular
Probes. The monoclonal antibodies EO6 and EO11 were provided by Dr.
J. L. Witztum (University of California, San Diego).
Lipoproteins--
LDL (density = 1.019-1.063 g/ml) was isolated
from normolipemic human plasma by ultracentrifugation (17) and dialyzed
against phosphate-buffered saline (PBS) containing 0.3 mM
EDTA, and the protein concentration was determined (18). Native LDL was
iodinated by the method of Salacinski et al. (19). After
extensive dialysis against PBS to remove free 125I and
EDTA, the LDL was adjusted to 100 µg/ml and oxidized with 10 µM CuSO4 for 18 h at 37 °C. The
degree of oxidation was determined by measuring the amount of
thiobarbituric acid-reactive substances (20). Butylated hydroxytoluene
(20 µM) and EDTA (0.1 mM) were added to
prevent further oxidation, and the OxLDL was then concentrated to
approximately 1 mg/ml and stored at 4 oC.
Isolation of ApoB and Lipids from OxLDL and Preparation of
Liposomes--
ApoB was isolated from OxLDL by extracting the lipids
with ice-cold methanol:chloroform (1:1) as described (9). The residual protein was washed with water and acetone and solubilized in
octylglucoside (octylglucoside:protein = 30:1 (w/w)). The
detergent was removed by dialysis, and the protein concentration was
determined (18). The apoB isolated from OxLDL was iodinated as
described above for OxLDL.
The lipids from native and oxidized LDL were isolated essentially as
described previously (21). Briefly, HCl was added to the lipoprotein to
a final concentration of 10 mM, and the lipids were
extracted with chloroform:methanol (1:1) (v/v) and separated by
centrifugation at 800 × g. The choloroform phase was
removed and dried under N2, and the dried lipids were
suspended in 10 mM Tris buffer containing 1 mM
EDTA and 150 mM NaCl, pH 7.4 (Buffer A). The suspension was
extruded 8 to 10 times at 37 °C through 0.1 µm polycarbonate
membranes under N2, yielding microemulsions with particle
sizes of 80-120 nm. For the fluorescence labeling of lipids, DiO in an
amount equal to 0.2% of the weight of total lipids was added to the
chloroform phase before drying and resuspending the lipids in Buffer A. Unincorporated DiO was removed by dialysis against Buffer A. The
concentration of the lipid microemulsions was expressed in terms of
phospholipid, determined by phosphorus assay (22). POVPC was
synthesized by ozone-mediated oxidation of 1-palmitoyl 2-arachidonoyl
phosphatidylcholine and a stable, covalent adduct of POVPC, and BSA was
prepared as described previously (10, 11).
DNA Constructs and Transfection--
The cDNA of murine CD36
was amplified from reverse-transcribed mRNA obtained from mouse
macrophages by the polymerase chain reaction, using primers selected on
the basis of the published sequence of mCD36 (15). The sense primer,
5'-GAATTCGCCGCCACCATGGGCTGTGATCGGAACTGTGGGC-3', contained
an EcoRI recognition site and a consensus sequence for initiation of translation, added immediately upstream from the start
codon (23). The antisense primer, 5'-GGATCCTTATTTTCCATTCTTGGATTTG-3', contained the stop codon and a BamHI recognition site. The
amplified mCD36 cDNA was subcloned into pSG5 (Stratagene),
sequenced, and transiently transfected into COS-7 cells using FuGene6.
COS-7 cells were maintained in glucose-containing Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml
streptomycin, 100 units/ml penicillin, and 2 mM
L-glutamine. For transfection, the cells were grown in
6-well plates to about 60% confluence and transfected with a mixture
of 2 µg of DNA and 6 µl of FuGene6. After 24 h, the cells were
harvested with EDTA and plated at a density of 0.2 × 106 cells/well in 24-well plates, and 48 h after
transfection, the cells were used for the assays. The expression of
CD36 on the plasma membrane was examined by flow cytometry using an
anti-mouse CD36 antiserum generated against a fusion protein (amino
acid residues 169-244 of CD36) in guinea pigs (24). Cells (2 × 105) were harvested with EDTA, washed in ice-cold PBS
containing 0.1% BSA and 0.001% NaN3, incubated with
guinea pig preimmune serum or anti-mouse CD36 antiserum (1:20) at
4 °C for 30 min, washed, and then incubated for another 30 min in
the dark with a fluorescein isothiocyanate-labeled rabbit anti-guinea
pig IgG (Sigma; 1:100). Cells were washed and analyzed using a FACScan instrument, and the data were analyzed using Cell Quest software (Becton Dickinson).
125I-OxLDL and 125I-ApoB Binding
Assay--
The ligand binding assays were carried out 48 h after
transfection. The cells were cooled on ice for 10 min, washed twice with ice-cold PBS, and incubated for 2 h at 4 °C with various concentrations of 125I-labeled OxLDL in the absence or
presence of a 20-fold excess of unlabeled OxLDL. After the incubation,
cells were first washed twice with cold PBS containing 1% BSA and then
twice with cold PBS. Cells were then lysed by addition of 0.2 N NaOH (0.5 ml/well). Aliquots were taken to measure the
cell-associated radioactivity and to measure the protein content by the
method of Lowry et al. (18). The specific binding was
determined by subtracting nonspecific binding (binding in the presence
of excess unlabeled OxLDL) from the total binding. All assays were done
in triplicate, and the binding isotherms were determined using the
Ligand program (25). The binding of 125I-apoB from OxLDL
was determined by the same procedure. In the various competition
experiments, 125I-apoB was added to the cells
simultaneously with the unlabeled competitors, except when lipids were
the competitor. To prevent adsorption of 125I-apoB onto
lipids, the cells were preincubated with microemulsions of lipids from
LDL or OxLDL for 1 h at 4 °C, washed twice with PBS, and then
incubated with 125I-apoB for 1 h at 4 °C.
Lipid Binding Analysis--
The day after transfection, the
cells were harvested with EDTA and plated in 12-well plates at a
density of 0.4 × 106 cells/well. 48 h after
transfection, the cells were cooled on ice for 10 min and washed twice
with cold PBS. The cells were incubated with DiO-labeled microemulsions
prepared from lipids from OxLDL (5 µg/ml phospholipids) for 2 h
at 4 °C with or without various competitors. After the incubation,
the cells were washed twice with PBS and scraped into 1 ml of PBS
containing 0.1% BSA and 0.01% NaN3. After centrifugation,
the cells were resuspended in the same buffer. The binding of OxLDL
lipid microemulsions was measured by flow cytometry using a FACScan
instrument and analyzed using Cell Quest software (Becton Dickinson).
Binding of OxLDL to mCD36-transfected Cells--
Analysis
of the transiently transfected COS-7 cells by flow cytometry
demonstrated that, on average, at least 40% of the cell population was
successfully transfected and expressed CD36 on the cell surface (Fig.
1). As expected, the transfected cells bound OxLDL with high affinity, and the binding was both specific and
saturable (Fig. 2). Analysis of the
equilibrium binding data revealed a binding affinity of 4.1 ± 0.6 µg of OxLDL protein/ml, comparable to values reported by others (15),
and a maximal binding of 1.1 µg of OxLDL protein/mg of cell protein.
The control-transfected cells also displayed some saturable OxLDL
binding, but compared with the CD36 transfectants, the maximal binding
was much lower (~5-fold).
Binding of the Lipid Moiety and ApoB from OxLDL to mCD36--
As
shown in Fig. 3, both the reconstituted
lipid and the apoB from OxLDL competed for the binding of intact
125I-OxLDL to the CD36-transfected cells significantly
The recognition of 125I-apoB from OxLDL was further
analyzed in direct ligand binding experiments. The binding of the
delipidated and resolubilized apolipoprotein was about 10-fold greater
to mCD36-transfected cells than to nontransfected cells. The binding was specific and was almost completely inhibited by unlabeled apoB from
OxLDL and also by intact unlabeled OxLDL (Fig.
4A). As shown above (Fig. 3),
the binding of intact 125I-OxLDL was inhibited by unlabeled
apoB from OxLDL, i.e. the competition was reciprocal. The
microemulsions of lipids derived from OxLDL also competed with the
binding of oxidized apoB by at least 50%. Intact native LDL had no
effect (data not shown).
DiO-labeled microemulsions prepared from OxLDL lipids also bound to
CD36-transfected cells. As with oxidized apoB, the binding was specific
and was inhibited both by unlabeled microemulsions of OxLDL lipids
(about 55%) and by intact OxLDL (up to 80%) (Fig. 4B). We
also tested for reciprocal competition and found that apoB from OxLDL
was an effective competitor, reducing the binding of oxidized lipids by
over 70%. In contrast, neither intact native LDL nor lipids from
native LDL exhibited any inhibitory effects (data not shown). Together,
these data suggested that both the lipid and the protein fractions from
OxLDL contain structurally similar ligands that mediate the recognition
by CD36.
Identification of a Ligand Present on the Lipid and Protein
Moieties That Mediates Binding of OxLDL by CD36--
A series of
monoclonal antibodies against various epitopes of OxLDL was
identified recently in hypercholesterolemic apolipoprotein E-deficient
mice (12, 26). One of these autoantibodies, designated EO6, which
recognizes POVPC, bound to the protein as well as to the lipid fraction
of intact OxLDL and prevented the macrophage binding of both apoB and
lipids from oxidized LDL (10, 11).
To test whether the epitopes recognized by EO6 also played a role in
the binding of OxLDL to CD36, we included the antibody in ligand
binding experiments. As shown in Fig. 5,
EO6 reduced the binding of intact OxLDL to CD36 in a
dose-dependent manner. To determine whether similar
epitopes are responsible for the binding of apoB and lipids from OxLDL,
we examined in separate experiments the binding of
125I-apoB derived from OxLDL and DiO-labeled microemulsions
of lipids isolated from OxLDL to CD36-transfected cells in the presence and absence of EO6. The antibody inhibited the binding of the OxLDL
lipids very strongly and also, although to a lesser extent, that of
apoB from OxLDL (Fig. 6). The control
IgM, another autoantibody from apoE-deficient mice that recognized
neither OxLDL nor POVPC (11), had only minor inhibitory effects. These
results suggested that both the apoB and the lipid fractions of OxLDL
contain a common epitope(s) that is recognized by EO6 and are involved
in the binding to CD36.
Although the exact nature of the ligands of OxLDL that are recognized
by macrophage scavenger receptors is still unknown, POVPC-like
structures are likely candidates (10, 11). To determine whether CD36
can bind similar ligands, we tested the inhibitory potential of a
covalent adduct of POVPC, a specific oxidized phospholipid, and BSA.
Similar to the results seen with EO6, the POVPC-BSA adduct inhibited in
a dose-dependent fashion the binding of intact OxLDL to the
CD36-transfected cells (Fig. 7).
Moreover, the POVPC-BSA adduct also inhibited the binding of
resolubilized apoB from OxLDL and DiO-labeled microemulsions of lipids
isolated from OxLDL to CD36 by about 50% (Fig.
8), suggesting that oxidized
phospholipids covalently attached to apoB mediate in part the binding
of intact OxLDL to CD36. BSA, which was used as a control, had no
significant inhibitory effect.
The data presented show conclusively that a single receptor, CD36,
can bind both a ligand or ligands associated with the lipid moiety of OxLDL and also a ligand or ligands associated with the delipidated apoprotein B from OxLDL. Other investigators have suggested
that CD36 recognition of OxLDL might depend primarily on the lipid
moiety because they found no difference, or very little difference, in
the binding of the isolated apoprotein to CD36-transfected cells and
mock-transfected cells (27). However, in those studies, the binding of
the lipid moiety was not directly tested. The pattern of our results
using CD36-transfected cells qualitatively matches that of our previous
results using resident mouse peritoneal macrophages, where several
different scavenger receptors are undoubtedly involved (10). The
concordance of those results with the present results suggests that
CD36 is a major contributor to macrophage binding of OxLDL or that
other scavenger receptors also can bind the lipid and protein moieties of OxLDL.
Several receptor segments of CD36 have been implicated in the binding
of intact OxLDL (28, 29). The fact that the isolated apoprotein and the
reconstituted lipids showed a highly significant degree of reciprocal
competition indicates that at least part of the binding is to a common
region of the receptor. However, the competition was incomplete, and
some portion of the binding may well be to different sites on the
receptor. CD36 binds a number of different ligands, and there is reason
to believe that it functions differently in respect to these ligands,
suggesting that different segments are involved in ligand binding. For
example, the binding of thrombospondin depends upon an interaction
between CD36 and the vitronectin receptor
At first glance, it seems paradoxical that the apoprotein moiety and
the lipid moieties would bind to the same site on a receptor. The
probable explanation is that some fraction of oxidized phospholipids becomes covalently bonded to the apoprotein during the oxidation of LDL
and remains associated with it despite exhaustive extraction of the
noncovalently bound lipids. Indirect evidence for this was presented in
our previous studies showing that a monoclonal antibody against
oxidized phospholipids (EO6) reacted not only with intact OxLDL but
also with the separated apoprotein and lipid moieties (11) and
inhibited their binding to mouse macrophages (10-12). Moreover,
current studies in this
laboratory2 show directly
that during oxidation of LDL, there is a progressive increase in the
amount of phosphorus covalently linked to apoB, reaching a maximum of
about 70 mol per mol of protein.
What is the precise nature of the lipid ligand or ligands involved and
how many different ligands are there on OxLDL? It seems likely that the
heterogeneous mixture of oxidized products formed during LDL oxidation
includes several different molecules that can be involved in receptor
recognition. However, there is now evidence that oxidized phospholipids
play a major role in the binding of OxLDL to CD36. In previous studies,
we have shown that the monoclonal antibody EO6 can inhibit the binding
of intact OxLDL by as much as 90%, implying that oxidized
phospholipids recognized by this antibody account for much of the
binding of OxLDL to macrophage scavenger receptors (11). In the present study, this monoclonal antibody was able to substantially inhibit the
CD36 binding of intact OxLDL (70%), of the isolated oxidized lipid
fraction (85%), and, to a lesser degree, of apoB from OxLDL (35%).
The monoclonal antibody EO6 was cloned from spleens of apolipoprotein
E-deficient mice (26) and specifically recognizes oxidized
phospholipids, including POVPC, when presented either as pure lipid or
as lipid-protein adduct (11). POVPC is an oxidation product of
1-palmitoyl 2-arachidonoyl phosphatidylcholine and is thought to be one
of the biologically active components of minimally modified LDL (16).
POVPC competed for the binding of OxLDL and its isolated fractions to a
degree that was similar to that exhibited by EO6. Taken together, these
binding data suggest that more than 50% of the binding of OxLDL to
CD36 is mediated by oxidized phospholipids, present either in the lipid
phase or covalently attached to the apoprotein. It should be stressed
that the studies reported here deal exclusively with the binding of ligands to CD36. However, other studies have demonstrated that CD36
fulfills many of the criteria for classification as a scavenger receptor contributing to uptake and degradation of OxLDL (13, 27).
OxLDL is known to bind to several members of the rapidly growing family
of macrophage scavenger receptors that now includes scavenger receptor
A (32), CD36 (15), macrosialin/CD68 (33), scavenger receptor BI (34,
35), and LOX-1 (6, 36). Although their relative importance in
macrophage function is difficult to estimate given the redundancy that
is built into an essential biological system, such as host defense,
CD36 appears to play a prominent role in OxLDL uptake by macrophages
and possibly foam cell formation. Consistent with an important
function, CD36 expression was up-regulated in an autocrine or paracrine
fashion when macrophages were exposed to OxLDL (37), involving
mechanisms that included activation of peroxisome
proliferator-activated receptor Circulating monocytes and tissue macrophages mediate many of the innate
immune responses that included recognition and phagocytosis of
apoptotic cells. A common characteristic of apoptotic cells is the cell
surface expression of molecules that are not found on normal cells and
that are recognized by scavenger receptors. Although the molecular
structures of apoptotic cells that mediate the interaction remain
ill-defined, recent studies suggested that they might be similar to
some of the epitopes found on OxLDL (41), including oxidized
phospholipids, such as POVPC (42). CD36 appears to be directly involved
in the uptake of apoptotic cells (43), and future experiments in this
laboratory will be aimed at the analysis of molecular structures that
mediate their recognition by CD36.
We thank Jennifer Pattison and Nonna
Kondratenko for expert technical assistance.
*
This work was supported by National Institutes of Health
Grant HL56989 (Specialized Center of Research in Molecular Medicine and
Atherosclerosis, La Jolla and by a grant from the American Heart
Association) (to O. Q.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by National Institutes of Health Training Grant DK07044.
¶
Supported by fellowships from the American Heart Association.
**
Established Investigator of the American Heart Association. To whom
correspondence should be addressed: Dept. of Medicine, 0682,
University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0682. Tel.: 858-534-4401; Fax: 858-534-2005; E-mail: oquehenberger@ucsd.edu.
2
K. L. Gillotte, S. Hörkkö, J. L. Witztum, and D. Steinberg, unpublished data.
The abbreviations used are:
LDL, low density
lipoprotein;
OxLDL, oxidized LDL;
POVPC, 1-palmitoyl 2-(5'-oxovaleroyl)
phosphatidylcholine;
DiO, 3,3'-dihexadecylooxacarbocyanine perchlorate;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin.
The Binding of Oxidized Low Density Lipoprotein to Mouse CD36 Is
Mediated in Part by Oxidized Phospholipids That Are Associated with
Both the Lipid and Protein Moieties of the Lipoprotein*
,
, Chemistry and
Biochemistry, University of California, San Diego,
La Jolla, California 92093-0682
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD36 expression in transiently transfected
COS-7 cells. The transient expression of CD36 in COS-7 cells was
determined by flow cytometry. Control (fine line) and mouse
CD36-transfected (bold line) cells were incubated with
guinea pig anti-mouse CD36 antiserum (1:20) followed by fluorescein
isothiocyanate-labeled rabbit anti-guinea pig IgG and analyzed.
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Fig. 2.
Specific binding of 125I-OxLDL to
mouse CD36-transfected COS-7 cells. Cells were incubated with
various concentrations of 125I-OxLDL for 2 h at
4 °C in the absence or presence of a 20-fold excess of unlabeled
OxLDL. The specific binding of 125I-OxLDL to
CD36-transfected cells ( 
) and control cells (
) was calculated by
subtracting nonspecific binding, determined in the presence of a
20-fold excess of unlabeled OxLDL, from total binding. The values
represent the means ± S.D. (n = 3). The Scatchard
plot analysis of the binding data is shown in the inset. A
binding affinity of 4.1 µg/ml for OxLDL was calculated.
by
about 50%. Neither intact native LDL nor microemulsions prepared from
lipids of native LDL exhibited any inhibitory effects.
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Fig. 3.
Inhibition of 125I-OxLDL binding
to CD36 by unlabeled apoB and by unlabeled microemulsions of the lipids
from OxLDL. Transfected COS-7 cells expressing CD36 were incubated
with 2.5 µg of protein/ml of 125I-OxLDL for 2 h at
4 °C in the absence and presence of the competitors. Unlabeled
intact OxLDL (OxLDL), intact native LDL (nLDL),
and apoB from OxLDL (OxapoB) were added at concentrations of
50 µg of protein/ml. Microemulsions of lipids from OxLDL (OxLDL
lipids) and from native LDL (nLDL lipids) were added at
concentrations of 50 µg phospholipid/ml. The cells were washed and
lysed, and the cell-associated radioactivity was determined as
described under "Experimental Procedures." Shown is the specific
binding calculated by subtracting the binding to mock-transfected
control cells from that to the CD36-transfected cells. The specific
binding in the absence of competitor was taken as 100%. Each value
represents the mean ± S.D. of three independent
experiments.
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Fig. 4.
Specific binding of 125I-apoB and
DiO-labeled lipids isolated from OxLDL to COS-7 cells expressing mouse
CD36. A, binding of apoB from OxLDL to CD36.
Transfected COS-7 cells were incubated with 5 µg/ml of
125I-apoB isolated from OxLDL for 2 h at 4 °C
either in the absence of competitors (none) or in the
presence of 50 µg/ml unlabeled apoB from OxLDL (OxapoB),
50 µg of protein/ml of intact OxLDL (OxLDL), or
microemulsions of lipids from OxLDL at 50 µg of phospholipid/ml
(OxLDL lipid). At the end of the incubation, the cells were
washed and lysed, and the binding of 125I-apoB from OxLDL
was determined as described under "Experimental Procedures." Shown
is the specific binding calculated by subtracting the binding to
mock-transfected control cells from that to the CD36-transfected cells.
The specific binding in the absence of competitor was taken as 100%.
B, binding of OxLDL lipids to CD36. CD36-transfected cells
were incubated with DiO-labeled microemulsions of lipids from OxLDL (5 µg of phospholipids/ml) for 2 h at 4 °C in the absence
(none) or presence of unlabeled microemulsions of OxLDL
lipids (100 µg of phospholipids/ml), intact OxLDL (100 µg of
protein/ml), or OxapoB (100 µg of protein/ml). The binding was
estimated by flow cytometry. Shown is the specific binding calculated
as described above. All data represent the mean ± S.D. of three
independent experiments.
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Fig. 5.
Inhibition of intact 125I-OxLDL
binding to mouse CD36 by the monoclonal antibody EO6.
CD36-transfected ( ) and control () cells were incubated with
125I-OxLDL (2.5 µg of protein/ml) in the presence of the
indicated concentrations of the IgM monoclonal antibody EO6 for 2 h at 4 oC. At the end of the incubation period, OxLDL
binding was determined. The values represent the means ± S.D. of
two independent experiments.
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Fig. 6.
Inhibition of the binding of
125I-apoB and DiO-labeled lipids from OxLDL by the
monoclonal antibody EO6. The CD36-transfected cells were incubated
with 2.5 µg/ml of 125I-apo B from OxLDL or DiO-labeled
OxLDL lipids (5 µg of phospholipid/ml) in the presence of the
monoclonal antibody EO6 at a concentration of 150 µg/ml (closed
bars). EO11 (150 µg/ml), another IgM isolated from
hypercholesterolemic apolipoprotein E-deficient mice, was included in
control experiments (open bars). The binding assays were
performed for 2 h at 4 oC as described in Fig. 4.
The results were compared with the binding observed in the absence of
any antibody, which was taken as 100%. The values represent the
means ± S.D. (n = 3).
View larger version (15K):
[in a new window]
Fig. 7.
Inhibition of intact OxLDL binding to CD36 by
POVPC-BSA. CD36-transfected ( ) and control COS-7 () cells
were incubated with intact 125I-labeled OxLDL (2.5 µg of
protein/ml) in the presence of the indicated concentrations of
POVPC-BSA for 2 h at 4 °C. At the end of the incubation period,
the cells were washed and lysed, and OxLDL binding was estimated as
described in Fig. 2. The concentrations of POVPC-BSA are expressed as
µg of protein/ml. The data represent the means ± S.D. of three
independent experiments.
View larger version (30K):
[in a new window]
Fig. 8.
Reduction by POVPC-BSA of the binding of
125I-apoB and DiO-labeled lipids from OxLDL to
CD36. A, control cells (black bars) and
COS-7 cells expressing CD36 (hatched bars) were incubated
with 5 µg/ml of 125I-apoB from OxLDL in the presence of
excess (50 µg/ml) POVPC-BSA or BSA for 2 h at 4 °C. Binding
analysis was performed as described in Fig. 4. The values represent the
mean ± S.D. (n = 3). B, binding of
DiO-labeled OxLDL lipids to CD36. CD36-transfected cells were incubated
with DiO-labeled microemulsions of lipids from OxLDL (5 µg of
phospholipids/ml) for 2 h at 4 °C with either no competitor
(none) or in the presence of excess (100 µg/ml) POVPC-BSA
or BSA for 2 h at 4 °C. The binding was estimated by flow
cytometry. Shown is the specific binding calculated as described above.
All data represent the mean ± S.D. of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3 (30). In platelets, the binding of
thrombospondin is further controlled by the phosphorylation state.
Phosphorylation of a CD36 ectodomain switches the ligand specificity
and decreases thrombospondin binding, with a reciprocal increase in
platelet binding to collagen (31).
(38, 39) or other,
cholesterol-mediated, pathways (40).
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by fellowships from the Fondation pour la Recherche
Médicale and Arcol-Parke Davis, Paris, France.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Steinberg, D.,
Parthasarathy, S.,
Carew, T. E.,
Khoo, J. C.,
and Witztum, J. L.
(1989)
N. Engl. J. Med.
320,
915-924[Medline]
[Order article via Infotrieve]
2.
Witztum, J. L.,
and Steinberg, D.
(1991)
J. Clin. Invest.
88,
1785-1792
3.
Krieger, M.
(1997)
Curr. Opin. Lipidol.
8,
275-280[Medline]
[Order article via Infotrieve]
4.
Yamada, Y.,
Doi, T.,
Hamakubo, T.,
and Kodama, T.
(1998)
Cell Mol. Life Sci.
54,
628-640[CrossRef][Medline]
[Order article via Infotrieve]
5.
Steinberg, D.
(1997)
J. Biol. Chem.
272,
20963-20966 6.
Sawamura, T.,
Kume, N.,
Aoyama, T.,
Moriwaki, H.,
Hoshikawa, H.,
Aiba, Y.,
Tanaka, T.,
Miwa, S.,
Katsura, Y.,
Kita, T.,
and Masaki, T.
(1997)
Nature
386,
73-77[CrossRef][Medline]
[Order article via Infotrieve]
7.
Goldstein, J. L.,
Ho, Y. K.,
Basu, S. K.,
and Brown, M. S.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
333-337 8.
Fogelman, A. M.,
Shechter, I.,
Seager, J.,
Hokom, M.,
Child, J. S.,
and Edwards, P. A.
(1980)
Proc. Natl. Acad. Sci. U. S. A.
77,
2214-2218 9.
Parthasarathy, S.,
Fong, L. G.,
Otero, D.,
and Steinberg, D.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
537-540 10.
Bird, D. A.,
Gillotte, K. L.,
Hörkkö, S.,
Friedman, P.,
Dennis, E. A.,
Witztum, J. L.,
and Steinberg, D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6347-6352 11.
Hörkkö, S.,
Bird, D. A.,
Miller, E.,
Itabe, H.,
Leitinger, N.,
Subbanagounder, G.,
Berliner, J. A.,
Friedman, P.,
Dennis, E. A.,
Curtiss, L. K.,
Palinski, W.,
and Witztum, J. L.
(1999)
J. Clin. Invest.
103,
117-128[Medline]
[Order article via Infotrieve]
12.
Hörkkö, S.,
Miller, E.,
Dudl, E.,
Reaven, P.,
Curtiss, L. K.,
Zvaifler, N. J.,
Terkeltaub, R.,
Pierangeli, S. S.,
Branch, D. W.,
Palinski, W.,
and Witztum, J. L.
(1996)
J. Clin. Invest.
98,
815-825[Medline]
[Order article via Infotrieve]
13.
Nozaki, S.,
Kashiwagi, H.,
Yamashita, S.,
Nakagawa, T.,
Kostner, B.,
Tomiyama, Y.,
Nakata, A.,
Ishigami, M.,
Miyagawa, J. I.,
Kameda-Takemura, K.,
Kurata, Y.,
and Matsuzawa, Y.
(1995)
J. Clin. Invest.
96,
1859-1865
14.
Febbraio, M.,
Abumrad, N. A.,
Hajjar, D. P.,
Sharma, K.,
Cheng, W.,
Pearce, S. F.,
and Silverstein, R. L.
(1999)
J. Biol. Chem.
274,
19055-19062 15.
Endemann, G.,
Stanton, L. W.,
Madden, K. S.,
Bryant, C. M.,
White, R. T.,
and Protter, A. A.
(1993)
J. Biol. Chem.
268,
11811-11816 16.
Watson, A. D.,
Leitinger, N.,
Navab, M.,
Faull, K. F.,
Hörkkö, S.,
Witztum, J. L.,
Palinski, W.,
Schwenke, D.,
Salomon, R. G.,
Sha, W.,
Subbanagounder, G.,
Fogelman, A. M.,
and Berliner, J. A.
(1997)
J. Biol. Chem.
272,
13597-13607 17.
Havel, R. J.,
Eder, H. A.,
and Bragdon, J. H.
(1955)
J. Clin. Invest.
34,
1345-1353
18.
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 19.
Salacinski, P. R.,
McLean, C.,
Sykes, J. E.,
Clement-Jones, V. V.,
and Lowry, P. J.
(1981)
Anal. Biochem.
117,
136-146[CrossRef][Medline]
[Order article via Infotrieve]
20.
Yagi, K.
(1976)
Biochem. Med.
15,
212-216[CrossRef][Medline]
[Order article via Infotrieve]
21.
Terpstra, V.,
Bird, D. A.,
and Steinberg, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1806-1811 22.
Marinetti, G.
(1962)
J. Lipid Res.
3,
1-20
23.
Kozak, M.
(1991)
J. Biol. Chem.
266,
19867-19870 24.
Ottnad, E.,
Parthasarathy, S.,
Sambrano, G. R.,
Ramprasad, M. P.,
Quehenberger, O.,
Kondratenko, N.,
Green, S.,
and Steinberg, D.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1391-1395 25.
Munson, P. J.,
and Rodbard, D.
(1980)
Anal. Biochem.
107,
220-239[CrossRef][Medline]
[Order article via Infotrieve]
26.
Palinski, W.,
Hörkkö, S.,
Miller, E.,
Steinbrecher, U. P.,
Powell, H. C.,
Curtiss, L. K.,
and Witztum, J. L.
(1996)
J. Clin. Invest.
98,
800-814[Medline]
[Order article via Infotrieve]
27.
Nicholson, A. C.,
Frieda, S.,
Pearce, A.,
and Silverstein, R. L.
(1995)
Arterioscler. Thromb. Vasc. Biol.
15,
269-275 28.
Puente Navazo, M. D.,
Daviet, L.,
Ninio, E.,
and McGregor, J. L.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
1033-1039 29.
Pearce, S. F.,
Roy, P.,
Nicholson, A. C.,
Hajjar, D. P.,
Febbraio, M.,
and Silverstein, R. L.
(1998)
J. Biol. Chem.
273,
34875-34881 30.
Savill, J.,
Hogg, N.,
Ren, Y.,
and Haslett, C.
(1992)
J. Clin. Invest.
90,
1513-1522
31.
Asch, A. S.,
Liu, I.,
Briccetti, F. M.,
Barnwell, J. W.,
Kwakye-Berko, F.,
Dokun, A.,
Goldberger, J.,
and Pernambuco, M.
(1993)
Science
262,
1436-1440 32.
Kodama, T.,
Reddy, P.,
Kishimoto, C.,
and Krieger, M.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
9238-9242 33.
Ramprasad, M. P.,
Fischer, W.,
Witztum, J. L.,
Sambrano, G. R.,
Quehenberger, O.,
and Steinberg, D.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
9580-9584 34.
Acton, S. L.,
Scherer, P. E.,
Lodish, H. F.,
and Krieger, M.
(1994)
J. Biol. Chem.
269,
21003-21009 35.
Murao, K.,
Terpstra, V.,
Green, S. R.,
Kondratenko, N.,
Steinberg, D.,
and Quehenberger, O.
(1997)
J. Biol. Chem.
272,
17551-17557 36.
Yoshida, H.,
Kondratenko, N.,
Green, S.,
Steinberg, D.,
and Quehenberger, O.
(1998)
Biochem. J.
334,
9-13
37.
Yoshida, H.,
Quehenberger, O.,
Kondratenko, N.,
Green, S.,
and Steinberg, D.
(1998)
Arterioscler. Thromb. Vasc. Biol.
18,
794-802 38.
Nagy, L.,
Tontonoz, P.,
Alvarez, J. G.,
Chen, H.,
and Evans, R. M.
(1998)
Cell
93,
229-240[CrossRef][Medline]
[Order article via Infotrieve]
39.
Huang, J. T.,
Welch, J. S.,
Ricote, M.,
Binder, C. J.,
Willson, T. M.,
Kelly, C.,
Witztum, J. L.,
Funk, C. D.,
Conrad, D.,
and Glass, C. K.
(1999)
Nature
400,
378-382[CrossRef][Medline]
[Order article via Infotrieve]
40.
Han, J. H.,
Hajjar, D. P.,
Tauras, J. M.,
and Nicholson, A. C.
(1999)
J. Lipid Res.
40,
830-838 41.
Sambrano, G. R.,
Parthasarathy, S.,
and Steinberg, D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
3265-3269 42.
Chang, M. K.,
Bergmark, C.,
Laurila, A.,
Hörkkö, S.,
Han, K. H.,
Friedman, P.,
Dennis, E. A.,
and Witztum, J. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6353-6358 43.
Ren, Y.,
Silverstein, R. L.,
and Savill, J.
(1995)
J. Exp. Med.
181,
1857-1862
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