From the
The specific recognition of anionic phospholipids in the outer
leaflets of cell membranes and lipoproteins by cell surface receptors
may play an important role in a variety of physiologic and
pathophysiologic processes (e.g. recognition of damaged or
senescent cells by the reticuloendothelial system or lipoprotein
homeostasis). Several investigators have described anionic phospholipid
binding to cells, and phosphatidylserine (PS) binding to a partially
purified
Phospholipids are key structural components of cell membranes
and lipoproteins. Based on studies of the red blood cell's and
other membranes (reviewed in Schroit and Zwaal, 1991), it is generally
assumed that there is an asymmetric distribution of phospholipids in
the plasma membranes of eukaryotic cells (Roelofsen and Op den Kamp,
1994). The outer leaflet appears to be composed predominantly of
neutral zwitterionic phospholipids, e.g. phosphatidylcholine
(PC)
Several investigators have described specific anionic phospholipid
binding to cells, especially to macrophages, using either direct
binding or indirect ligand-competition assays (Schroit and Fidler,
1982; Ratner et al., 1986; Allen et al., 1988;
Nishikawa et al., 1990; Lee et al., 1992a; Fukasawa et al., 1995; Sambrano and Steinberg, 1995). Several studies
have suggested that scavenger receptors which bind modified
lipoproteins, such as oxidized low density lipoprotein (OxLDL) or
acetylated LDL (AcLDL), may in some cases function as anionic
phospholipid receptors (Nishikawa et al., 1990; Fukasawa et al., 1995; for a review of scavenger receptors see Krieger
and Herz, 1994). The only receptor for anionic phospholipids to be
identified to date is a partially purified
In the current study,
we have examined the binding of phospholipids to class B scavenger
receptors (SR-B). SR-Bs are members of the CD36 superfamily of
proteins, including CD36 itself and SR-BI (Acton et al.,
1994). SR-Bs are expressed in a variety of cells and tissues, including
macrophages and endothelial cells (Abumrad et al., 1993; and
see Greenwalt et al., 1992 for review). The highest levels of
expression have been reported to be in adipose tissue (Abumrad et
al., 1993; Acton et al., 1994). In addition to binding
modified LDL (Endemann et al., 1993; Acton et al.,
1994; Nicholson et al., 1995), CD36 binds M-BSA (Acton et
al., 1994), thrombospondin (Asch et al., 1987), collagen
(Tandon et al., 1989), long chain fatty acids (Abumrad et
al., 1993; Nicholson et al., 1995), and P.
falciparum-infected erythrocytes (Oquendo et al., 1989).
Although its physiologic functions are unknown, CD36 may serve as an
adhesion molecule, a component in fatty acid transport (Abumrad et
al., 1993), a signal transduction molecule (Ockenhouse et
al., 1989; Huang et al., 1991), and a receptor for
senescent neutrophils (Savill et al., 1993). A striking
feature of SR-BI, not shared by CD36 or other well-defined scavenger
receptors, is its ability to bind native LDL with high affinity (Acton et al., 1994). Thus, SR-BI may play an important role in lipid
metabolism. Here, we show that SR-BI and CD-36 specifically bind
anionic PS and PI liposomes with high affinity, and are, therefore, the
first molecularly well-defined cell surface receptors for anionic
phospholipids to be described.
COS cells were transiently
transfected with plasmids encoding either haSR-BI (phaSR-BI; Acton et al., 1994) or huCD36 (phuCD36; Oquendo et al.,
1989) as described previously (Acton et al., 1994). Cells were
plated on day 0, transfected on day 1, and replated on day 2 (1
To determine if phospholipids could bind to haSR-BI, we
prepared 105 nm diameter PS liposomes (PS/
phosphatidylcholine/cholesterol, ratio 1:1:1) radiolabeled with trace
amounts of [
These results indicated that phospholipids
can bind to haSR-BI and that this binding might depend on the charge of
the phospholipid head group. The specificity of the binding was further
assessed by determining the competition for [
We previously established that haSR-BI can bind both native LDL and
AcLDL with high affinity (Acton et al., 1994). Fig. 2A shows that PS and PI liposomes inhibited
virtually all of the binding of
The
phospholipid and modified lipoprotein binding specificities of the
class B scavenger receptors (Endemann et al., 1993; Acton et al., 1994; Nicholson et al., 1995), the
ldlA[haSR-BI] cells
were plated on day 0. On day 2, the binding of
[
We thank Shangzhe Xu for expert technical assistance,
D. Alford and C. Larsen for generously providing equipment and advice
regarding liposome preparation during the initial stages of this work,
B. Seed for his generous gift of the plasmid encoding CD36, and A.
Pearson and D. Resnick for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
95-kDa membrane protein has recently been reported
(Sambrano, G. R., and Steinberg, D.(1995) Proc. Natl. Acad. Sci. U.
S. A. 92, 1396-1400). Using both direct binding and ligand
competition assays in transfected cells, we have found that two class B
scavenger receptors, SR-BI and CD36, can tightly bind PS and
phosphatidylinositol (PI)-containing liposomes (K
for PS liposome binding to SR-BI is
15 µg
phospholipid/ml or 0.18 nM (mol PS liposomes/l)), but not
phosphatidylcholine, phosphatidylethanolamine, or sphingomyelin
liposomes. PS and PI liposomes, but not the others, could effectively
compete with PS liposomes and modified or native lipoproteins for
binding to these receptors. Phosphatidic acid, another anionic
phospholipid, could also compete, but was not as effective as PS or PI.
Class B scavenger receptors are the first molecularly well-defined,
specific cell surface receptors for anionic phospholipids to be
described.
(
)and sphingomyelin (SM), while the inner
leaflet is greatly enriched in negatively charged phospholipids, such
as phosphatidylserine (PS) and phosphatidylinositol (PI). Breakdown of
this asymmetry, especially the exposure of increased levels of PS on
the outer cell surface, occurs in a variety of physiologic and
pathologic states. These include platelet activation (Bevers et
al., 1983), cell aging (Shukla and Hanahan, 1982), programmed cell
death (apoptosis) (Fadok et al., 1992a, 1992b), sickle cell
anemia (Kuypers et al., 1994), and Plasmodium falciparum infection of erythrocytes (Joshi and Gupta, 1988). Exposure of
anionic phospholipids at the external surface of cells can stimulate
blood coagulation (Schroit and Zwaal, 1991) and has been proposed to
play a critical role in the recognition of damaged or senescent cells
by the reticuloendothelial system (Savill et al., 1993).
95-kDa membrane protein
from macrophages that was shown to directly bind PS and OxLDL using a
ligand blotting assay (de Rijke and van Berkel, 1994; Ottnad et
al., 1995; Sambrano and Steinberg, 1995). The primary sequence of
this protein has not been reported; thus, it is not known if it is a
member of one of the three classes of scavenger receptors, A, B, and C,
which have been previously defined based on their distinctive binding
activities and primary sequences (Acton et al., 1994; Pearson et al., 1995). Two of these, class A and class C scavenger
receptors (SR-A, SR-C), are expressed almost exclusively on mammalian
and embryonic Drosophila melanogaster macrophages,
respectively (Naito et al., 1991; Elomaa et al.,
1995; Bell et al., 1994; Pearson et al., 1995). Lee et al. (1992b) and Fukasawa et al.(1995) have shown
that at least two types of class A receptors are unlikely to be
involved in the cellular uptake of anionic phospholipid liposomes, and
we have recently obtained data suggesting that the Drosophila SR-C (Pearson et al., 1995) may not be able to recognize
anionic phospholipids.
(
)
Materials
Reagents (and sources) were: acetic
anhydride (Mallinckrodt, Inc., Paris, KY); egg phosphatidylcholine, egg
phosphatidic acid, liver phosphatidylinositol, brain
phosphatidylserine, egg phosphatidylethanolamine, and brain
sphingomyelin (Avanti Polar Lipids, Inc., Alabaster, AL); polycarbonate
membrane filters (Poretics Corp., Livermore, CA); sodium
[I]iodide and
1,2-dipalmitoyl-L-3-phosphatidyl[N-methyl-
H]choline
([
H]DPPC) (Amersham Corp.); DEAE-dextran
(Pharmacia Biotech Inc.); Ham's F-12 medium, Dulbecco's
modified Eagle's medium, fetal bovine serum, and trypsin/EDTA
(JRH Biosciences, Lenexa, KS); and penicillin/streptomycin, glutamine,
and G418 sulfate (Life Technologies, Inc.). All other reagents and
supplies were purchased from Sigma or were obtained as described
previously (Krieger, 1983). Human LDL, AcLDL,
I-labeled
LDL, and
I-labeled AcLDL (90-300 cpm/ng protein)
were prepared essentially as described previously (Goldstein et
al., 1983; Krieger, 1983; Acton et al., 1994).
Phospholipid Liposome Preparation
Unilamellar
liposomes were made by extrusion through polycarbonate membranes (Szoka et al., 1980). Phospholipid liposomes were prepared containing
the indicated phospholipid, phosphatidylcholine, and free cholesterol
in a molar ratio of 1:1:1. The lipids were mixed in chloroform and
dried by rotary evaporation for 30 min. For preparation of radiolabeled
liposomes, 50-75 µCi of [H]DPPC (62
Ci/mmol) were added to the lipid mixtures before drying. The dried
lipids were resuspended in 150 mM NaCl, 0.1 mM EDTA,
10 mM HEPES, pH 7.5 (Buffer A). Once the samples were fully
hydrated, they were extruded through 0.1-µm pore size polycarbonate
membranes using a mini-extruder device (Avanti Polar Lipids, Inc.,
Alabaster, AL). After extrusion, liposomes were dialyzed against Buffer
A and then stored under nitrogen at 4 °C until use. Liposomes were
used within 2 weeks of preparation. The final phospholipid
concentration was determined by the method of Bartlett(1959). The
average diameters of unlabeled liposomes, which were determined from
either two or three independent preparations using light scattering
with a Coulter N4 plus light scatterer apparatus (Coulter Electronics
Inc., Hialeah, FL), were: PS, 105; PC, 114; PA, 125; PE, 129; PI, 113;
and SM, 131 nm. The number of phospholipid molecules/PS liposome was
calculated as follows. Cross-sectional areas for cholesterol and
phospholipid molecules in hydrated bilayers are assumed to be 0.35
nm
and 0.47 nm
, respectively (Levine and
Wilkins, 1971); assuming an homogenous distribution of the components
throughout the PS/PC/cholesterol (1:1:1) liposomes, 73% of the surface
area (4
r
2 (bilayer)
0.73 =
50477 nm
) was phospholipid, or 107,398 phospholipid
molecules/liposome (50477 nm
/0.47 nm
). Based on
an average phospholipid mass of 785 g/mol, a liposome concentration of
10 µg phospholipid/ml converts to 0.12 nM in liposome
particles.
Cell Culture and Transfections
CHO, ldlA (clone
7), ldlA[haSR-BI], and COS M6 cells were grown in culture as
described previously (Krieger et al., 1983; Acton et
al., 1993, 1994). ldlA (clone 7) is a CHO cell mutant clone whose
defective LDL receptor gene results in an essentially LDL
receptor-negative phenotype (Kingsley and Krieger, 1984; Sege et
al., 1986; Kingsley et al., 1986). CHO and ldlA cells
were maintained in monolayer culture with Ham's F-12 medium
containing 5% fetal bovine serum, 100 units/ml penicillin, 100
µg/ml streptomycin, and 2 mM glutamine (medium A).
ldlA[haSR-BI] cells are ldlA cells which express hamster
SR-BI (Acton et al., 1994) and were cultured in medium A
supplemented with 0.25 mg/ml G418 (medium B). COS M6 cells were grown
in Dulbecco's modified Eagle's medium with 10% fetal bovine
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2
mM glutamine (medium C). All incubations with cells were
performed at 37 °C in a humidified 5% CO, 95% air
incubator unless otherwise noted.
10
cells/well in six-well dishes) in medium C supplemented
with 1 mM sodium butyrate. Ligand binding assays were
performed on day 3.
Ligand Binding Assays
On day 0, ldlA and
ldlA[haSR-BI] cells were plated (2.5 10
cells/well in six-well dishes) in medium A or B, respectively,
and the assay was performed on day 2. Transfected COS cells were
prepared as described above. Binding assays were performed as described
previously (Acton et al., 1994), with the following minor
modifications. Cells were prechilled on ice for 30 min, incubated with
the indicated radiolabeled ligands (
I-LDL,
I-AcLDL, or
H-labeled liposomes) in ice-cold
medium D (Ham's F-12 containing 0.5% (w/v) fatty acid free bovine
serum albumin (FAF-BSA) and 10 mM HEPES, pH 7.4), with or
without unlabeled competitors, for 2 h at 4 °C with gentle shaking.
Cells were then washed twice with Tris wash buffer (50 mM Tris-HCl, 0.15 M NaCl, pH 7.4) containing 2 mg/ml
FAF-BSA, followed by one rapid wash with Tris wash buffer without
FAF-BSA. The cells were then solubilized with 0.1 N NaOH, and
radioactivity and protein determinations were made as described
previously (Acton et al., 1994). The specific, high affinity
ligand binding activities shown represent the differences between
values obtained in the absence (total binding) and presence
(nonspecific binding) of an excess of the indicated unlabeled ligands.
Nonspecific binding of [
H]PS liposomes to cells
was generally low. For example, for the experiment shown in Fig. 1, nonspecific binding ranged from 10% (at 2.5 µg
phospholipid/ml) to 28% (at 100 µg phospholipid/ml) of the total
binding. For [
H]PC liposomes, nonspecific binding
ranged from 21% (at 2.5 µg phospholipid/ml) to 58% (at 100 µg
phospholipid/ml) of the total binding. The binding values are expressed
as nanograms of bound
I-labeled protein or ng of total
phospholipids from
H-labeled liposomes/milligram of cell
protein.
Figure 1:
Concentration dependence of
[H]PS and [
H]PC liposome
binding to transfected ldlA cells expressing haSR-BI
(ldlA[haSR-BI]) or to untransfected ldlA control cells.
Transfected ldlA[haSR-BI] cells (circles) or
untransfected ldlA cells (squares) were plated on day 0 and
[
H]PS (filled symbols) or
[
H]PC (open symbols) (25-50 cpm/ng
total phospholipid) binding at 4 °C was measured on day 2 as
described under ``Experimental Procedures.'' The high
affinity values shown represent the differences between measurements
made with the indicated concentrations of radiolabeled liposomes in the
absence (duplicate incubations) and presence (single incubations) of an
excess of the corresponding unlabeled liposomes (500 µg of total
phospholipid/ml). Data are from two independent experiments in which
the data for [
H]PS binding to
ldlA[haSR-BI] cells were essentially
identical.
H]dipalmitoyl phosphatidylcholine (62
Ci/mmol) and examined their binding at 4 °C to untransfected cells
(ldlA) and transfected cells which express haSR-BI
(ldlA[haSR-BI]). Fig. 1shows that there was
substantial, high affinity (K
15
µg phospholipid/ml) and saturable binding to the transfected cells (filled circles), but relatively little binding to the
untransfected cells (filled squares). Assuming that the
phospholipid and cholesterol were uniformly distributed in homogenous
liposomes containing approximately 107,400 molecules of
phospholipid/liposome (see ``Experimental Procedures''), we
estimate the K
(mol of PS
liposomes/liter) to be
0.18 nM. PS binding was apparently
independent of divalent cations because it was not inhibited by EDTA
(1-10 mM, data not shown). Binding depended on the
phospholipid composition of the liposomes. In contrast to that of
[
H]PS liposomes, the binding of radiolabeled PC
liposomes (PC/cholesterol, 2:1, Fig. 1, open circles)
was very low and similar to [
H]PS binding to
untransfected ldlA cells.
H]PS
binding by unlabeled liposomes of various compositions (indicated
phospholipid/PC/cholesterol, ratio 1:1:1). shows that the
anionic phospholipids PS and PI were effective inhibitors while the
zwitterionic PC and PE as well as SM were not. PA, another anionic
phospholipid, was able to compete, but not as effectively as PS and PI.
I-AcLDL to haSR-BI in
transiently transfected COS cells (>50% inhibition at concentrations
>10 µg phospholipid/ml), while PC had virtually no effect at
concentrations as high as 250 µg phospholipid/ml. We also observed
that PS, but not PC, liposomes inhibited most of the binding of native
I-LDL to haSR-BI in ldlA[haSR-BI] cells.
Indeed, the specificity of liposome inhibition of
I-LDL
binding was almost identical to that for the inhibition of
[
H]PS binding (). The extent of PS
inhibition of
I-LDL binding depended on the relative PS
content of the liposomes. Fig. 3shows that inhibition by 500
µg phospholipid/ml increased substantially as the amount of PS in
PS/PC mixed liposomes increased from 0 to 50 mol % of total
phospholipid, with greater than 50% inhibition occurring when the PS
mol % was >10. These competition experiments suggest that anionic
phospholipids bound to haSR-BI at a site close to or identical with the
site(s) of native and modified LDL binding and that interaction of the
liposomes with haSR-BI may involve polyvalent binding via multiple
anionic phospholipid molecules.
Figure 2:
Inhibition by PS, PI, and PC liposomes of I-AcLDL binding to either haSR-BI (A) or huCD36 (B) expressed by transfected COS cells. On day 1, COS cells
were transfected with expression vectors for haSR-BI (panel A)
or huCD36 (panel B) as described under ``Experimental
Procedures.'' On day 2, transfected cells were plated in six-well
dishes in medium C plus 1 mM sodium butyrate. On day 3, medium
D containing
I-AcLDL (5 µg protein/ml, 299 cpm/ng
protein) was added, and 4 °C binding was measured in the presence
of the indicated concentrations of phosphatidylserine (filled
circles), phosphatidylinositol (filled triangles), or
phosphatidylcholine (open circles) liposomes. The values
represent the means of duplicate determinations. The 100% control
values were 119 (panel A) or 86 (panel B) ng
I-AcLDL protein/mg cell
protein.
Figure 3:
Effect of the
phosphatidylserine/phosphatidylcholine molar ratio on liposome
inhibition of I-LDL binding to haSR-BI at 4 °C. On
day 2 of cell growth, the binding of
I-LDL (5 µg
protein/ml, 278 cpm/ng protein) to ldlA[haSR-BI] cells was
measured as described under ``Experimental Procedures'' in
the presence of PS/PC/cholesterol liposomes (500 µg
phospholipid/ml, total phospholipid/cholesterol ratio of 2:1) with the
indicated composition of phosphatidylserine (expressed as mole
percentage of total phospholipid). The binding values represent the
means of duplicate determinations. The 100% control value was 560 ng
I-LDL protein/mg cell protein.
haSR-BI is a member of the CD36
superfamily of proteins (Acton et al., 1994). Both haSR-BI and
human CD36 (huCD36, 32% amino acid sequence identity to haSR-BI) are
class B scavenger receptors which can bind a variety of modified
proteins (AcLDL, OxLDL, maleylated BSA); however, they cannot bind to
many of the other polyanions which are ligands of the class A and C
scavenger receptors (Acton et al., 1994). Fig. 2B shows that, as was the case for haSR-BI, the binding of I-AcLDL to huCD36 expressed in COS cells was inhibited by
PI and PS, but not by PC. PI was a significantly better inhibitor than
PS. Thus, CD36, as well as haSR-BI, apparently can serve as a receptor
for anionic phospholipids. Except for the apparent differences in the
ability to recognize PE, the receptor activity reported by Fukasawa et al.(1995) is remarkably similar to that of CD36.
95
kDa OxLDL receptor (Sambrano and Steinberg, 1995; de Rijke and van
Berkel, 1994), and the AcLDL receptor activity reported by Nishikawa et al.(1990) are not identical. Therefore, in addition to
SR-BI and CD36 there may be other receptors for phospholipids.
Establishing the structural and functional relationships of class B
receptors to previously described anionic phospholipid receptor
activities and determining the physiologic significance of anionic
phospholipid binding by class B scavenger receptors, particularly with
regard to the recognition of lipoproteins and damaged or senescent
cells, will require additional studies.
Table: Specificity of
[H]PS and
I-LDL binding to haSR-BI
expressed in transfected ldlA cells
H]PS liposomes (10 µg total phospholipid/ml,
28 cpm/ng total phospholipid) or
I-LDL (5 µg
protein/ml, 94 cpm/ng protein) at 4 °C was measured as described
under ``Experimental Procedures'' in the absence
(``none'') or presence of the indicated phospholipid
liposomes (150 µg total phospholipid/ml). The values represent the
average of two determinations. The 100% of control values for the
binding of [
H]PS liposomes (experiment 1) and
I-LDL (experiment 2) were 410 and 139 ng/mg cell protein,
respectively.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.