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INTRODUCTION |
Apolipoprotein E (apoE)1
is a 299-amino acid-long protein. ApoE, as a constituent of different
lipoprotein particles, serves as a ligand for several cell receptors
(apoER2, LDL receptor, LDL receptor-related protein, and VLDL receptor)
and promotes their catabolism by the liver (1-5). ApoE may also be
involved in cellular cholesterol efflux (6-9). These functions of apoE contribute to cell and tissue cholesterol homeostasis (6-9) and may
explain why, when expressed locally in macrophages or endothelial cells, apoE protects from atherosclerosis (9-11). Mutations in apoE
that prevent binding of apoE-containing lipoprotein remnants to the LDL
receptor and possibly other receptors, as well as to heparan sulfate
proteoglycans, are associated with type III hyperlipoproteinemia (12-18) and premature atherosclerosis (17, 19, 20).
SR-BI binds with high affinity HDL and LDL and discoidal reconstituted
particles containing apoA-I (21-24). On binding lipoproteins, SR-BI
mediates both selective cholesteryl ester uptake from the lipoprotein
to the cells (24-28) and bi-directional unesterified cholesterol
movement (28-32). In addition to cholesteryl esters (25), SR-BI can
mediate cellular uptake from HDL of free cholesterol (31, 32),
triglycerides (32, 33), phospholipids (34), and vitamin E (35-39). HDL
and reconstituted discoidal HDL particles also promote cholesterol
efflux from SR-BI-expressing cells (28-30, 40). Inactivation of the
SR-BI gene reduces cholesterol levels in the steroidogenic tissues and
bile and can promote atherosclerosis despite an associated increase in
plasma HDL cholesterol levels (41, 42), thus demonstrating the
beneficial physiological functions of this receptor (41, 42). Studies
with transgenic mice also corroborated the role of SR-BI in cholesterol
and bile acid homeostasis and in the protection against atherosclerosis (43-47).
In the current study, we have focused on the SR-BI binding of discoidal
POPC-apoE particles containing the natural apoE isoforms and truncated
apoE forms. The carboxyl-terminal mutants of apoE were generated to
assess the contribution of the carboxyl-terminal domain of apoE to
SR-BI binding. Recent studies by us established that the 1-185-region
of apoE suffices for the clearance of apoE-containing lipoprotein
remnants in vivo implying that this amino-terminal domain of
apoE contains the necessary determinants for binding to at least some
of the apoE receptors (48, 49). The current study establishes that apoE
is a ligand for SR-BI and that its receptor binding domain is found
within the amino-terminal 1-165 residues.
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EXPERIMENTAL PROCEDURES |
Materials
The Klenow fragment of DNA polymerase I, T4 ligase,
polynucleotide kinase, and restriction enzymes were purchased from New England Biolabs. Calf intestinal alkaline phosphatase was purchased from Stratagene (La Jolla, CA), and Vent polymerase from Promega. Other
materials for the polymerase chain reaction were obtained from
PerkinElmer Life Sciences. The Sequenase sequencing kit was purchased from U. S. Biochemical Corp. The oligonucleotides were purchased from GIBCO Life Technologies, Inc. Bacto-tryptone and bacto-yeast extract were obtained from VWR (Pittsburgh, PA).
Dulbecco's modified Eagle's medium (DMEM) was provided by Invitrogen.
Dextran sulfate and epoxy-activated Sepharose 6B were purchased from
Pharmacia, the column was from BioRad, iodo-beads iodination reagent
and the D-salt dextran plastic desalting columns were purchased from Pierce. The sodium 125I was purchased from PerkinElmer Life
Sciences. The bicinchoninic acid assay kit was purchased from
Pierce. Other reagents (and sources) were: fatty acid free bovine serum
albumin, cholesterol, sodium cholate, and
1-palmitoyl-2-oleoyl-L-phosphatidylcholine (POPC),
aprotinin, benzamidine, leupeptin, and phenylmethylsulfonyl fluoride
(Sigma); dialysis tubing (Spectrum Medical Industries, Inc., Los
Angeles, CA); Ham's F-12 medium, fetal bovine serum, and trypsin/EDTA
(JRH Biosciences, Lenexa, KS); and penicillin/streptomycin, glutamine,
and G418 sulfate (GIBCO Life Technologies, Inc.). All other reagents
were purchased from Sigma, Bio-Rad, or other standard commercial
sources as previously described (22). The SR-BI receptor blocking
monoclonal antibody KKB-1 was a gift of Karen Kozarsky.
Methods
Plasmid Construction and Generation of Expression Vector Carrying
ApoE Gene Derivatives--
Full-length apoE cDNA derivatives
cloned into the HindIII and BamHI site of the
pUC-19 plasmid (43) were used to generate three new sets of plasmids
(I, II, and III) containing WT or mutated apoE gene sequences (Fig.
1A). Plasmid I (pBlueEexIV) contains the
EcoRI-EcoRI apoE gene region consisting of exon 4 and part of the flanking introns cloned in the pBlueScript II
KS± vector. Plasmid II (pBlueXapoEg) contains the entire
apoE gene between BamHI and HindIII flanked by
two bioengineered XhoI sites cloned in the pBlueScript II
KS± vector. Plasmid III (pBMT3XapoEg) contains the entire
apoE gene cloned in the XhoI site of the pBMT3X bovine
papilloma-based vector in front of the mouse metallothionein I
promoter. Each of the three plasmids was constructed with the apoE2,
apoE3, or apoE4 exon IV sequences or with mutated sequences. The
following protocol was utilized for generation of mutations.
(a) Mutations are generated in exon IV by PCR amplification
and mutagenesis using the pUCexIV apoE derivatives (50) as a template
and a set of two external primers spanning, for instance the
Sty-BbsI region, and mutagenic primers covering
the region that needs to be mutagenized. The primers used are shown in
Table I. (b) The amplified
mutant sequence was digested with StyI and BbsI
and was used to replace the corresponding apoE sequence in the
pBlueEexIV derivative (plasmid I). The mutated apoE sequence was
excised from the pBlueEexIV derivative by EcoRI digestion
and was used to replace the corresponding region in the pBluXapoEg
derivative (plasmid II). (c) The entire mutated apoE gene
sequence was excised by XhoI digestion and was cloned into
the XhoI site of the pBMT3X vector to generate the
pBMT3XapoEg derivatives. Plasmids with the correct orientation were
selected, sequenced to verify the mutations, and utilized further.
Generation and Characterization of Permanent Cell Lines
Expressing WT and Variant ApoE Forms--
To generate stable cell
lines expressing the mutant genes, C127 cells derived from a mouse
mammary tumor (51) were transfected with the different pBMT3XEg
derivatives as described previously (18). Cell clones expressing
apoE2/apoE3/apoE4 were labeled with a [35S]Met/Cys
mixture, and the secreted protein was immunoprecipitated and analyzed
by two-dimensional gel electrophoresis and autoradiography to verify
the known isoelectric point differences among the apoE isoforms
(18).
ApoE Production by Eukaryotic Expression Systems Using Permanent
ApoE-expressing Cell Lines--
To generate media for isolation of
full-length apoE isoforms, the cell clones having the highest
efficiency of production were thawed and placed in two T-75 flasks with
DMEM containing 10% fetal calf serum and 10 µM
CdCl2. Within 5-7 days the confluent flasks were
trypsinized, and the cells were placed into an 850-cm2
roller bottle. The bottle was placed on a roller rack turning at 1 rpm
to allow the cells to attach to the surface of the bottle. When the
cells grew to 80-85% confluence, 10 ml of packed conditioned microcarriers (beads) were added to the roller bottle and the speed of
the roller rack was increased to 7 rpm. The beads were prepared by
first autoclaving in phosphate-buffered saline solution and
subsequently incubating in media containing 1% fetal calf serum
overnight so that they could absorb nutrients. The cells were fed twice
a week with 300 ml of DMEM media containing 5% fetal calf serum and 10 µM CdCl2. For collection of media for protein
purification, the cells were rinsed twice with serum-free media, and
pre-incubated in the same media for 2 h. After one additional
rinse, the cells were incubated in serum-free media overnight. The
media were collected and stored in 1 mM EDTA, 0.01% NaN3, 10 mM aprotinin, and 10 mM
benzamidine. The media were collected twice a week for a period of one
month. Density gradient ultracentrifugation of the media and analysis
of the resulting fractions established that over 95% of apoE2, apoE3,
or apoE4 secreted by C127 cells is found in the d < 1.27 g/ml
fraction, which is conventionally defined as lipid-free/lipid-poor
fraction. Following further purification, this apoE was complexed with
POPC and used for receptor binding experiments.
ApoE Production by Eukaryotic Expression Systems Using Adenoviral
Infection of Cells--
To generate media for isolation of truncated
apoE forms, human astrocytoma HTB-13 cells, which do not express apoE,
were infected with recombinant adenoviruses expressing apoE4-259,
apoE4-229, apoE4-202, or apoE2-202 (48, 49), and the serum-free
medium was collected as described previously.
Purification of Lipid-free ApoE by Ion Exchange Chromatography
Using Dextran Sulfate-Sepharose Column--
ApoE was purified from the
culture medium of C127 cells expressing the apoE2, apoE3, or apoE4
genes or from the media of HTB-13 cells expressing the truncated apoE
forms. The purification scheme involved dextran sulfate-Sepharose
column fractionation. Briefly, 30 ml of dextran sulfate-Sepharose
column was equilibrated with 20 mM Tris-HCl, and 0.2 M NaCl, pH 7 4. A total of 2 liters of apoE containing
culture medium was concentrated to 75 ml in an Amicon concentrator
using a membrane with a cutting molecular mass of 10 kDa, and
the NaCl concentration was adjusted to 0.2 M and loaded
over the column at a flow rate of ~80 ml/h. The column was eluted
with a 120-ml 0.2-1.0 M NaCl gradient in 20 mM
Tris-HCl, pH 7.4, at the same flow rate and fractions of 3 ml were
collected. The pure protein fractions were pooled and were dialyzed
extensively against 0.05 M NH4HCO3
and lyophilized. The protein yield was 10-20 mg/liter depending on the
apoE variant.
Preparation of Discoidal Reconstituted HDL Particles Containing
ApoE [POPC-ApoE]--
POPC was used to prepare the reconstituted
discoidal POPC-apoE particles employing the sodium cholate dialysis
method with only minor modifications (52). POPC-ApoE particles were
prepared from a molar ratio of 100:10:1:100 of
POPC:cholesterol:apoE:sodium cholate. In a typical experiment, 0.14 mg
of cholesterol and 2.71 mg of POPC were placed in glass tubes, vortexed
gently, and dried under nitrogen. The dried lipid was dissolved in a 10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, and 0.01% EDTA buffer by vortexing for ~30 s, followed by storage on ice. The process was repeated until
the phospholipid was completely suspended in the buffer. This required
~2 h. The sodium cholate was added, and the solution was placed on
ice for one more hour. The apoE (usually 1 mg) was then added, and the
incubation on ice continued for another hour. To remove the sodium
cholate the solution was dialyzed against 5-6 liters of the 10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, and 0.01% EDTA buffer at 4 °C
using tubing with a molecular mass cut-off of 12-14 kDa.
Finally a gradient gel (8-25%) was run under native conditions at
15 °C on a Pharmacia Phast-Gel system to ascertain the size of the
particle or used for electron microscopy analysis described below. The
POPC-apoE particles were stored at 4 °C under nitrogen to prevent
the oxidation of lipids.
Electron Microscopy (EM)--
POPC-apoE particles prepared with
various mutant apoE forms as described above were analyzed by EM. To
prepare the sample for EM, 50 µg of POPC-apoE were desalted three
times using Amicon centrifugal filter devices (Microcon). The final
concentration was ~1 mg/ml in deionized water. A 5-µl aliquot
suspension of each vesicle was applied for 10 s to a Formvar
carbon-coated 300-mesh copper grid. The carbon film surface was made
hydrophilic by glow discharge in a Balzers Union CTA 010 glow discharge
apparatus and used immediately. Excess POPC-apoE suspension was removed by blotting with filter paper and immediately replaced with a 5-µl
droplet of 1% sodium phosphotungstate, pH 7.4. After a few seconds,
excess stain was removed, and the grid was air dried. Fields of
particles were photographed with a Philips CM12 electron microscope
(Philips Electron Optics, Eindhoven, The Netherlands).
Iodination of ApoE--
ApoE was labeled by 125I
using the Iodo-Beads (53) iodination reagent and Na125I
(PerkinElmer Life Sciences). Each reaction used one mCi of
125I and three beads and 1 mg of apoE. The reaction was
carried out in Tris-HCl buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.01% EDTA). An aliquot of 100 µl of
Tris-HCl buffer was added to the 125I container and mixed.
The diluted radioactive 125I solution was transferred to an
Eppendorf tube containing 1-2 mg of apoE in the form of POPC-apoE
particles in Tris-HCl salt buffer. The final volume was adjusted to 900 µl using Tris-HCl salt buffer. The sample was placed in a lead pig.
Just prior to use, beads were washed with 500 µl of Tris HCl salt
buffer per bead and dried on a filter paper. This washing step removes
any loose particles and reagent from the beads. The beads were added to
the reaction solution and kept at room temperature for 45 min with
mixing every 5-10 min. The reaction was terminated by removing the
solution from the reaction vessel. The 125I-labeled apoE
was separated from the unincorparated Na125I by gel
filtration using Pierce's Presto Desalting Columns (Pierce, Inc.). Ten
fractions (0.5 ml each) were collected, and 1 µl of each fraction was
used for determination of the 125I counts. Ten µl of each
fraction were used to measure the protein concentration by
bicinchoninic acid protein assay (54). Another 10 µl of each
fraction were used for PAGE gel analysis of the sample. The specific
activity was calculated based on the protein concentration and the
125I counts and expressed as cpm/ng protein. Specific
activities of 1000-1500 cpm/ng protein were obtained.
Radioreceptor Binding Assay--
ldlA-7 is an LDL
receptor-deficient Chinese hamster ovary cell mutant (55, 56), which
expresses very little SR-BI protein or HDL binding/selective uptake
activity (25, 57). The ldlA-7 cells and the ldlA[mSR-BI] cells, which
are ldlA-7 cells stably transfected with murine SR-BI cDNA (25,
57), were maintained in monolayer culture in Ham's F12 medium
containing 5% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 2 mM glutamine. All incubations
with cells were performed at 37 °C in a humidified 5%
CO2, 95% air incubator.
SR-BI activity at 37 or 4 °C was assessed by measuring cell
association of radiolabeled ligands. Briefly, on day 0 cells (both ldlA-7 and ldlA [mSR-BI] or ldlA-7 and ldlA [Q402R/Q418R] (28) were
plated at concentrations of 4.5-5 × 104 cells/well
in 24-well dishes in complete F12 medium. On day 2, the monolayers were
washed twice with Ham's F12 medium and then refed with 0.4-0.5 ml of
medium (Ham's F-12 containing 0.5% (w/v) fatty acid-free bovine serum
albumin, 100 units/ml penicillin, 100 units/ml streptomycin, 2 mM glutamine) with the indicated radiolabeled ligands
(125I-POPC-apoE). Eight different concentrations,
ranging from 0.5-100 µg/ml, were used, and the experiments were
performed in duplicate. After a 1.5-h incubation at either 37 or
4 °C, the cells were washed twice at 4 °C with buffer B (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl) containing 2 mg/ml fatty acid-free bovine serum albumin, followed by one rapid wash
with buffer B alone. The cells were then solubilized with 0.1 N NaOH (300 µl each well). Aliquots of 200 µl were used
for radioactivity determinations, and 25 µl were used for
determination of the protein concentration using the
bicinchoninic acid assay. The specific binding was obtained by
subtracting the binding of the untransfected cells (ldlA-7) from the
binding of the receptor-expressing cell lines ldlA[mSR-BI] or ldlA
[Q402R/Q418R] (28). Receptor binding studies to the untransfected and
transfected cells were also performed in the presence and absence of
the SR-BI blocking monoclonal antibody KKB-1 (28). Receptor blocking
experiments were performed as described previously (28). Binding
parameters Kd and Bmax were
determined on the basis of the specific binding curve using the Prism
program (GraphPad Software, Inc.). The specific binding (cell
association) values of the saturation curves is expressed as ng of apoE
in the complex associated with the cells per mg of total cell protein.
Immunoreceptor Assay Using ELISA--
For the
immunoreceptor association experiments, the
Maxisorb 96-well plates were coated with anti-human apoE polyclonal
antibody (1 µg/ml) (Biodesign) diluted 1:800 in phosphate-buffered
saline at 100 µl/well and stored at 4 °C overnight. The coating
solution was aspirated, and the well was washed three times with
washing buffer (0.05% Tween 20 in phosphate-buffered saline) at 200 µl/well. Blocking buffer (10% nonfat dry milk in washing buffer) was
then added at 200 µl/well. The plates were incubated at room
temperature for 1.5 h and washed three times with washing buffer.
Cell lysates were diluted in phosphate-buffered saline to a total
volume of 100 µl, added in each well, and then incubated at room
temperature for 1 h. To obtain a standard curve, POPC-apoE
particles containing different amounts of apoE, mixed with 25 µg of
the lysate of blank cells (ldlA-7 or ldlA[apoER-2]) that were not
treated with rHDL particles or any lipoprotein, were adjusted to a
total volume of 100 µl and were added to different wells. After the
plates were washed with washing buffer three times, the secondary
antibody (rabbit anti-human IgG, peroxidase-conjugated) (Biodesign)
diluted 1:500 in blocking buffer was added at 100 µl/well. The plates were incubated at room temperature for 1 h and washed with washing buffer three times. An aliquot of 200 µl of
o-phenylenediamine dihydrochloride substrate
(o-phenylenediamine dihydrochloride, 0.4 mg/ml; urea
hydrogen peroxide, 0.4 mg/ml; phosphate-citrate buffer 0.05 M) was added to each well. After 30 min of incubation at
room temperature, the reaction was terminated by adding 50 µl of 2 N H2SO4 per well. The absorbance was
measured at 490 nm using a microtiter plate reader. A polynomial second
order regression was used as the best fit for the standard curve. The
specific binding was obtained by subtracting the binding of the ldlA-7 cells from the binding of the apoER-2-expressing cells from each experimental point, and it is expressed as ng of cell-associated POPC-apoE particles per mg of total cell protein. The cell-associated ligands were analyzed as a function of concentration by nonlinear regression by the program Prism using a one-site binding isotherm.
Competition Assays--
Competition assays were carried out as
described above for direct association experiments, except that on day
2, unlabeled competitor protein in the concentration range 0.5-100
µg/ml was added in addition to the labeled ligand. The average
percent cell association for the competition assays was calculated
relative to the control values. The 100% value corresponds to binding
of 5 µg of 125I-labeled HDL or 5 µg of
125I-POPC-apoE and in the absence of competitor. Two
independent competition experiments were performed in duplicate.
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RESULTS |
Expression, Production, Purification, and Characterization of WT
and Variant ApoE Forms--
To characterize the apoE isoforms and the
mutant apoE forms, cell clones expressing WT and variant apoE genes
were labeled with [35S]Met/Cys, immunoprecipitated, and
analyzed by one- or two-dimensional polyacrylamide gel electrophoresis
and autoradiography using 10 µg of VLDL from a subject with apoE4/4
phenotype as an internal marker. The Coomassie Brilliant Blue-stained
gel obtained from this analysis showed the position of the apoE4 that
was included in the sample, and the autoradiogram showed the position
of the newly synthesized apoE. Superimposition of the gel on the
autoradiogram establishes the charge and size differences between the
apoE4 and the newly synthesized variant apoE forms as described
previously (data not shown). Fig.
1B shows SDS-PAGE analysis of
the full-length and truncated apoE forms. ApoE was purified from the
culture medium of apoE-expressing cells as described under
"Experimental Procedures." Fig. 1C shows SDS-PAGE
analysis of different purified apoE forms.
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Fig. 1.
A-C, generation, production, and
purification of WT and mutant apoE forms. A, apoE gene
plasmids used to express the apoE mutant forms. Construction of these
plasmids is described under "Experimental Procedures."
B, SDS-polyacrylamide gel electrophoresis of
media of cells expressing WT or the truncated apoE forms. C,
SDS-PAGE of apoE purified from the media of apoE-expressing cells by
ion exchange chromatography. The media containing full-length apoE was
obtained from permanent C127 cell lines expressing the different apoE
isoforms. Lane M on the left indicates protein standards of
known molecular mass. The media containing the truncated apoE forms was
obtained from human astrocytoma HTB-13 cells following infection with
recombinant adenoviruses expressing the corresponding apoE forms (48,
49). Lane M on the right indicates protein standards used in
the analysis of the truncated apoE forms.
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Generation, Characterization, and EM Analysis of Reconstituted
POPC-ApoE Particles--
The wild-type and different mutant apoE
proteins were reconstituted in particles containing POPC and
cholesterol at a ratio of 100:10:1 of POPC:cholesterol:apoE, using the
sodium cholate dialysis method (52). The sodium cholate dialysis method
allowed the formation of discoidal particles with all the WT and the
mutant apoE forms tested. The POPC-apoE particles were negatively
stained with potassium phosphotungstate, overlaid on carbon-coated
grids, and photographed with a Philips CM12 electron microscope. Fig. 2, A-G shows formation of the
[POPC-apoE] particles with all the WT and mutant apoE forms. Under
the negative staining conditions used, these particles form the typical
"rouleaux," indicating that they are discoidal and have the
thickness of a phospholipid bilayer. The number of rouleaux observed
depends on the concentration of the sample on the carbon grid. In less
concentrated samples, a large number of round particles that lie flat
on the grid were also observed. These particles did not seem to pack
hexagonally on the grid during aggregation (a characteristic of
spherical structures), providing further evidence that these particles
were, in fact, discoidal. To further characterize the size of
the discoidal POPC-apoE, cross-linking experiments were performed with
particles isolated by gel filtration using sodium suberimidate. This
analysis showed the presence of two apoE2 or apoE2-202 molecules per
particle. The average size of POPC particles, determined from the EM
pictures, was 174 ± 49 Å (data not shown).
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Fig. 2.
A-G, electron microscopy of the
discoidal POPC-apoE particles used as ligands for binding the SR-BI.
A, apoE2; B, poE3; C,
apoE4; D, apoE4-259; E, apoE4-229;
F, apoE4-202; G, apoE4-165.
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High Affinity Binding of Discoidal Reconstituted POPC-ApoE
Particles to SR-BI--
Binding is reduced in an SR-BI mutant. HDL and
reconstituted discoidal HDL particles containing apoA-I, apoA-II, or
apoCIII bind with high affinity to SR-BI, whereas lipid-free apoA-I and pre-
HDL are poor ligands (22, 23). In the current study, we have
examined the binding of reconstituted POPC-apoE particles to SR-BI. To
determine the specific binding of the POPC-apoE ligands to SR-BI,
binding studies were performed using both SR-BI-expressing (ldlA
[mSR-BI]) transfected cells and their control untransfected cells
(ldlA-7). Both approaches were necessary since the
SR-BI-dependent association of the particles with the
control cells was high, and high background was clearly manifest in the
transfected cells (see below). This is presumably due to the presence
in the cells of other apoE binding sites. The specific binding curve
and the binding parameters Kd and
Bmax for ldlA [mSR-BI] cells were determined
by subtracting the binding values for the untransfected cells from the
corresponding values from the transfected cells (Fig.
3A). To ensure that the
differences obtained reflected the overexpression of mSR-BI in the
transfected cells and not a fortuitous increase in expression of the
endogenous mouse SR-BI, we performed receptor binding studies in the
untransfected and transfected cells in the presence of SR-BI
receptor-blocking antibodies. Specific binding was observed only in the
transfected cells (Fig. 3, B and C). The binding
parameters obtained by using the SR-BI receptor-blocking antibodies and
by subtracting the binding curves of the untransfected from those of
mSR-BI-expressing cell line were similar (Fig. 3, A and
B). The ligand-receptor specificity was also assessed in cells expressing a mutant mSR-BI (double substitution of arginines for
glutamines at positions 402 and 418, designated ldlA [Q402R/Q418R]) (28). Even after correction for the lower level of mutant receptor expression in ldlA [Q402R/Q418R] cells relative to wild-type receptor expression in ldlA[mSR-BI] cells, the ability of this mutant to bind
native HDL was dramatically reduced relative to that of the wild-type
receptor (28). We have shown recently that POPC-apoA-I particles bind
more tightly to SR-BI than native HDL (22, 23) and that the binding of
these particles to this mutant receptor is more readily detected than
the binding of native HDL.2
Fig. 3D shows the specific binding of POPC-apoE4 to cells
expressing the WT and the mutant SR-BI. We have found that the binding
affinity of the POPC-apoE particles to this mutant was clearly reduced (Kd = 185 µg/ml) for the mutant SR-BI as compared
with wild-type SR-BI (38 µg/ml) (Table
II). To establish whether iodination of
apoE changes its binding properties to SR-BI, we have determined receptor binding using an ELISA assay. This control experiment showed
that the binding parameters for apoE2 and apoE2-202 were similar to
those obtained when iodinated apoE was used in binding studies
(Kd = 45.5 ± 8 and 35.5 ± 4.5 µl/ml)
(Fig. 3E and Table II). Similar results were obtained when
binding experiments of apoE2, apoE4, and apoE2-202 were performed at
4 °C (Fig. 3, F and G and Table II). It is
interesting that the nonspecific binding at 4 °C was reduced,
whereas the SR-BI-specific binding was not altered considerably.
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Fig. 3.
A-G,
concentration-dependent binding of
125I-POPC-apoE4 complexes to confluent monolayers of ldlA
[mSR-BI] and ldlA [Q402R/Q418R]. A, ldlA-7 cells
and ldlA [mSR-BI] cells in the microtiter wells were washed and
incubated with various concentrations of 125I-POPC-apoE4.
Total binding to ldlA [mSR-BI] cells and total binding to
untransfected ldlA-7 cells was obtained experimentally. The specific binding was determined by subtracting the values of binding of
the ldlA-7 cells from the corresponding values of binding to the ldlA
[mSR-BI] cells. B and C, ldlA-7 cells and ldlA
[mSR-BI] cells in the microtiter wells were washed and incubated with
various concentrations of 125I-POPC-apoE4in the presence
and absence of receptor-blocking monoclonal antibodies. Total binding
to ldlA [mSR-BI] and ldlA-7 cells was obtained experimentally. The
specific binding to the ldlA [mSR-BI] cells (B) or to
ldlA-7 cells (C) was determined by subtracting the values of
binding to each of the two cell lines in the presence of receptor
blocking monoclonal antibodies from the values obtained in the absence
of SR-BI receptor blocking monoclonal antibodies. Note that there is
specific binding in the ldlA [mSR-BI] cells (B) but
not in the ldlA cells (C). D,
concentration-dependent binding of
125I-POPC-apoE4 complexes to confluent monolayers of ldlA
cells expressing WT mouse SR-BI-designated ldlA [mSR-BI] or mutant
mouse SR-BI-designated ldlA [Q402R/Q418R] cells. Cells in the
microtiter wells were washed and incubated with various concentrations
of 125I-POPC-apoE4. Total binding to ldlA [mSR-BI] or to
ldlA [Q402R/Q418R] cells and total binding to untransfected ldlA-7
cells were obtained experimentally. The specific binding for the WT and
the mutant receptor was determined by subtracting the values of binding
to the ldlA-7 cells from the corresponding values of binding to the
ldlA [mSR-BI] or ldlA [Q402R/Q418R] cells. The specific activity of
125I-POPC-apoE4 was in the range of 1000-1500 cpm/ng
protein. The experiments in A-D were performed by the
radioreceptor assay at 37 °C. E, F,
and G represent control experiments. In
E, the binding at 37 °C was measured using ELISA assay as
described under "Experimental Procedures." Specific binding was
determined as explained. In F and G, the binding
at 4 °C was measured using the radioreceptor assay as described
above and in A and under "Experimental Procedures." Note
that the binding parameters obtained under different experimental
conditions are similar (Table II).
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We have previously shown that under normal cell culture ligand-binding
conditions, LDL is a relatively poor inhibitor of HDL binding, possibly
because SR-BI exhibits multiple classes of ligand binding sites (24,
28). Fig. 4 shows that the binding of
125I-labeled HDL to SR-BI is effectively competed by HDL,
less effectively by POPC-apoE4, and much less effectively by LDL. The
findings are consistent with the Kd values of
POPC-apoE particles for SR-BI (22) (Table II) as well as with the
reduced binding of POPC-apoE4 to the ldlA [Q402R/Q418R] cells (Fig.
3D) (28).
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Fig. 4.
Competition of binding of
125I-HDL and 125I-POPC-apoE by excess of HDL,
LDL, and POPC-apoE. On day 2 after plating ldlA [mSR-BI] cells
were incubated with 5 µg/ml 125I-labeled HDL and 0.5-100
µg/ml of unlabeled LDL, POPC-apoE4, or HDL as explained under
"Experimental Procedures." The binding obtained in the absence of
competitor was set to 100%. The values represented are the average of
duplicate determinations. The specific activity of
125I-labeled HDL was in the range of 1000 to 1500 cpm/ng.
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The Natural ApoE Isoforms and Mutant ApoE Forms Have Similar
Affinities for SR-BI--
Receptor binding is maintained when the
carboxyl-terminal residues 166-299 are deleted. To address the
question of whether specific domains or residues of apoE are involved
in receptor binding, we have generated several carboxyl-terminally
truncated apoE forms extending to residues 259, 229, 202, and 165. The
purpose of the mutations was to assess the importance of the
carboxyl-terminal region of apoE to the SR-BI-specific binding.
Previous studies have shown that residue Arg-158 is important
for the binding of apoE containing lipoproteins to the LDL receptor
(58-61). Similarly in vitro experiments have shown that
residues in the 171-183 region of apoE contribute directly or
indirectly to LDL receptor binding (60). Analysis of the binding of the
truncated variant apoE forms to SR-BI showed the truncated apoE forms
bind with similar affinities (Kd, 35-45 µg/ml)
(Fig. 5, A-G and Table II). This indicates that the amino-terminal residues 1-165 of apoE contain
the determinant region for binding to SR-BI. Fig. 5H shows for comparison the binding of POPC-[apoA-I] particles to ldlA [mSR-BI] cells determined by an immunoreceptor assay as
described previously (22). The affinity of POPC-apoA-I particles
(3.8 ± 0.4 µg of protein/ml) is an order of magnitude greater
than that of the POPC-apoE particles.
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Fig. 5.
A-H,
concentration-dependent binding of
125I-POPC-apoE4 and POPC-apoA-I complexes containing
different apoE isoforms, mutant and truncated apoE forms, and apoA-I to
confluent monolayers of ldlA [mSR-BI] cells. A-G,
Cells in the microtiter wells were washed and incubated with
various concentrations of 125I-POPC-apoE4. Total binding to
ldlA [mSR-BI] cells and total binding to untransfected ldlA-7 cells
was obtained experimentally. The specific binding shown in this figure
was determined by subtracting the values of binding to the ldlA-7 cells
from the corresponding values of binding to the ldlA [mSR-BI] cells.
The specific activity of apoE was in the range of 1000 to 1500 cpm/ng.
Two to four independent experiments were performed in duplicate for
each apoE form. The average Kd and
Bmax values thus determined are shown in Table
II. The apoE forms used are A, apoE2; B, apoE3;
C, apoE4; D, apoE4-259; E,
apoE4-229; F, apoE4-202; G, apoE4-165.
H shows the specific binding curve of non-radiolabeled
POPC-apoA-I to ldlA [mSR-BI] cells determined by an immunoreceptor
assay as described (22). The apoA-I was obtained using a baculovirus
expression system (22).
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DISCUSSION |
Background--
In the current study, we have focused on
interactions of SR-BI with another potential ligand, the apolipoprotein
E, which plays an important role for cholesterol homeostasis in the
circulation and in the brain (6-9, 62). Apolipoprotein E has
remarkable structural similarities with apoA-I. Both proteins contain
11-or 22-amino acid-long repeats that are organized in amphipathic 
helices. ApoE is a ligand for several cell receptors that serve to
deliver cholesterol to cells (1-5). ApoE may also play an important
role in cholesterol efflux (6-9). It was shown initially that
apoE-containing lipoprotein particles with 
electrophoretic mobility
(Lp-E) are very effective in removing excess cholesterol from
cholesterol-loaded cell cultures (6). Plasma of mice lacking apoE has a
reduced capacity to promote cholesterol efflux from macrophages.
However, this defect was corrected by selective expression of apoE in
macrophages of the E
/ mice (7).
Cholesterol efflux may account for the protective role of apoE against
atherosclerosis when it is expressed locally in the arterial wall by
macrophages and endothelial cells (9-11). Numerous studies have also
implicated SR-BI in cholesterol efflux in vitro (28-30,
40). It has been shown that transiently transfected cells and permanent
cell lines overexpressing SR-BI promote net cholesterol efflux from
cells to the HDL and rHDL cholesterol acceptors, but not to lipid-free
apoA-I (28, 30). In a variety of cell lines studied, the rate of
cholesterol efflux correlates with the levels of SR-BI expression (30).
Potential interactions of apoE-containing lipoproteins with SR-BI could
contribute to cholesterol ester delivery as well as to cholesterol
efflux from cells that express SR-BI (63).
Specific Binding of Reconstituted POPC-ApoE Particles to
SR-BI--
Cells contain numerous apoE-recognizing receptors. The most
prominent are the LDL receptor (1, 2, 64), the LDL receptor-related protein (3), the VLDL receptor (5), and the apoE receptor-2 (4). ldlA-7
do not have the LDL receptor (55, 56) but do have other
apoE-recognizing receptors. We observed high background binding,
presumably due to the presence in these cells of other apoE-recognizing
receptors. To establish specific binding of apoE-containing lipoprotein
particles to SR-BI, it was necessary to subtract the background binding
of apoE to all these receptors in a Chinese hamster ovary cell line
that overexpresses the mouse SR-BI (ldlA [mSR-BI]). Binding
experiments using apoE4-established specific binding of POPC-apoE
particles to ldlA [mSR-BI] occurs with Kd = 38 µg/ml and Bmax = 1700 ng/mg cell protein.
To ensure that the specific values determined were valid, we measured
the SR-BI-dependent binding using SR-BI receptor-blocking antibodies. The specific binding curves and the binding parameters obtained by both procedures were essentially identical
(Kd = 39 µg/ml and Bmax = 1800 ng/mg cell protein). Similar binding parameters were obtained when
the binding experiments were performed at 4 °C or when receptor
binding was determined by ELISA.
The binding of POPC-apoE4 was affected by a double mutation in SR-BI
that eliminated most of the binding of HDL but not of LDL (28). This
observation suggests that there are similarities in the modes of
binding of apoA-I containing native HDL and POPC-apoE4. This conclusion
is supported by competition experiments, suggesting that the
recognition site for apoE on the SR-BI is more like that of the site
that binds HDL than the site that binds LDL.
Effects of ApoE Mutations on the Binding of POPC-ApoE Particles to
SR-BI--
Binding of apoE to the LDL receptor is affected by
mutations in residue 158 as well as by substitutions of charged
residues in the 140-150 region of apoE (58-60). In the current study,
we examined the binding of three naturally occurring apoE forms (apoE2 [C112/C158], apoE3 [C112/R158], and apoE4 [R118/R158]) and
several truncated apoE forms. These truncations produced the
amino-terminal segments of apoE, which extend to residues 259, 229, 202, or 165. In previous studies we have used the cell lines expressing
mouse SR-BI to determine the ligand specificity of the receptor using ligands containing human apoA-I (22). The use of this heterologous system in the present study offers the advantage of the availability of
the large number of mouse SR-BI and human apoE mutations, which are not
available for the human SR-BI and the mouse apoE. Although mouse and
human apoE, as well as mouse and human SR-BI, have considerable sequence similarities, one can not exclude the possibility for species
differences in the receptor ligand specificity due to the use of a
heterologous system.
As shown in Fig. 5, A-H and Table II the binding
parameters of POPC-apoE particles to SR-BI are similar for the WT apoE
and the truncated apoE forms (range of Kd 35-45
µg/ml). The findings suggest that the amino-terminal region 1-165 of
apoE contains the necessary determinants for its recognition by SR-BI and that Arg-158 is not critical for SR-BI receptor binding. Similar studies with the LDL receptor have shown previously that Arg-158 as
well as the carboxyl-terminal 171-183 apoE region are important for
the binding of apoE-containing liposomes to the LDL receptor (60). The
receptor-binding properties of POPC-apoE particles are similar to those
involving rHDL-apoA-I particles. In the case of apoA-I, deletion
of residues 186-243 did not affect binding of POPC-apoA-I to SR-BI
(22). The interactions of SR-BI with HDL and LDL are associated with
selective lipid uptake (24-28, 31-34) as well as cholesterol efflux
(28-30, 40). Recent studies indicated that SR-BI is expressed by
astrocytes but not by microglia in the brain (63), indicating that
interactions of apoE-containing lipoproteins with SR-BI may contribute
to brain cholesterol homeostasis. Reconstituted POPC/apoE particles
containing apoE have the ability to deliver cholesteryl esters to cells
(32). The role of apoE-containing lipoproteins in SR-BI-mediated
cholesterol efflux and selective lipid uptake in the brain and the
impact of apoE mutations on these processes remains to be determined.
,
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