 |
INTRODUCTION |
It is well established that the risk of developing coronary
heart disease is inversely proportional to plasma
HDL1 cholesterol levels (1).
Decreased levels of apoA-I, the major protein of HDL, are also
associated with an increased risk for coronary heart disease (2). The
most widely accepted model explaining the anti-atherogenic properties
of apoA-I is reverse cholesterol transport (3). In this process, poorly
lipidated apoA-I first removes excess free cholesterol (FC) from
peripheral cells through a mechanism dependent on ABCA1 (4). The apoA-I then acts as a co-factor for LCAT, which transforms the FC to cholesteryl ester (CE). This event initiates the conversion of the
poorly lipidated apoA-I to a spherical HDL particle. After remodeling
by plasma enzymes including cholesteryl ester transfer protein, hepatic
lipase, and phospholipid transfer protein, the HDL finally delivers its
CE either to the liver, where it can be excreted or repackaged into new
lipoproteins, or to ovaries, testes, and adrenal glands, where it can
be used in the production of steroid hormones.
ApoA-I has a number of important roles in HDL metabolism including
activation of LCAT, determination of plasma HDL cholesterol levels, and
interaction with the ABCA1 transporter and the HDL receptor, SR-BI.
Mice deficient in apoA-I have a 70% reduction in total plasma
cholesterol and HDL cholesterol (5-7), a 75% reduction in LCAT
activity (8), and a severe depletion of cholesteryl ester stores in
steroidogenic tissues (9). In these animals, the cortical
cells of the adrenal gland, the luteal and interstitial cells of the
ovary, and the Leydig cells of the testis all display diminished CE
content, indicating that apoA-I is important for the SR-BI-mediated HDL
CE selective uptake process (10, 11). This deficiency appears to be
directly attributable to the absence of apoA-I because
apoA-II-deficient mice have a similar reduction in HDL cholesterol
but do not show reduced CE reserves in their adrenals, ovaries, and
testes (9).
In a recent study we addressed the role of apoA-I in HDL CE selective
uptake by analyzing the structural, chemical, and functional properties
of apoA-I+/+ and
apoA-I
/
HDL. Compared with the
apoA-I+/+ HDL, apoA-I
/
particles
were larger, more heterogeneous in size, and enriched in apoA-II,
apoCs, apoE, FC, and CE (12). Compared with
apoA-I+/+ HDL, CE selective uptake from
apoA-I
/
HDL was significantly reduced into
Y1-BS1 adrenal cells and Fu5AH hepatoma cells, which naturally express
SR-BI, and into ldlA[SR-BI] cells, a Chinese hamster ovary cell line
expressing SR-BI from a transfected cDNA. In Y1-BS1 and
ldlA[SR-BI] cells, the reduction in HDL CE selective uptake was
attributed to a reduced Vmax for CE transfer to
the cell. Interestingly, in both cell types,
apoA-I
/
HDL showed a lower
KD for HDL cell association, indicating that the
absence of apoA-I did not reduce the affinity of HDL for SR-BI. These
findings illustrate that HDL properties necessary for HDL binding to
SR-BI are distinct from those properties necessary for the transfer of
HDL CE to the cell membrane. Additionally, the
Vmax for endocytic uptake
and degradation of HDL did not differ between
apoA-I+/+ and apoA-I
/
HDL in either cell type. Thus, the absence of apoA-I on HDL particles selectively affected the SR-BI-mediated HDL CE selective uptake pathway.
In the current report we have explored the basis for the reduced
selective uptake of CE from apoA-I
/
HDL.
Variation in HDL particle size, cholesterol to phospholipid ratios, and
apolipoprotein compositions had little effect on HDL CE selective
uptake into Y1-BS1 adrenal cells. Addition of apoA-I to
apoA-I
/
HDL also had little effect. However,
addition of apoA-I to apoA-I
/
HDL in the
presence of LCAT reorganized HDL structure and produced an HDL particle
with enhanced CE selective uptake activity. These data suggest that the
conformation of apoA-I at the HDL surface is important for the
efficient transfer of CE to the cell.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The following reagents used for culturing Y1-BS1
cells were purchased from the listed vendors: poly-D-lysine
(Becton Dickinson), heat-denatured fetal bovine serum (Atlanta
Biologicals), 100× penicillin/streptomycin/glutamine (Invitrogen),
six-well plates (Costar), Cortrosyn (Organon), Ham's F-10 medium, and
heat-denatured horse serum (Sigma). Sodium[125I]iodide
and [3H]cholesteryl oleoyl ether were acquired from
PerkinElmer Life Sciences and Amersham Biosciences, respectively.
Animals--
apoA-I
/
C57BL/6J-Apoa1tm1Unc (6) and
apoA-I+/+ C57BL/6J mice, and
apoA-I
/
and apoA-I+/+
mice on an 8:1 FVB/N:C57BL/6 background, were obtained and maintained on a 12-h light/12-h dark cycle with standard rodent chow and water
ad libitum (12). Housing and experimental procedures were approved by the State University of New York at Stony Brook Committee on Laboratory Animal Resources and the Scripps Research Institute Institutional Animal Care and Use Committee.
Isolation and Analysis of Wild Type and ApoA-I-deficient
Lipoproteins--
After an overnight fast, mice were anesthetized and
exsanguinated by heart puncture, and blood cells were removed by
centrifugation at 2,000 × g for 30 min at 4 °C. If
not used immediately, plasma was frozen at
80 °C after adding
sucrose to a final concentration of 10%. Plasma was adjusted to 0.05%
NaN3, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 mM EDTA, and lipoproteins in the 1.02-1.21
g/ml density range were isolated by sequential density
ultracentrifugation at 1.02 and 1.21 g/ml, respectively. Following
dialysis against PBS-E (150 mM NaCl, 10 mM
potassium phosphate, 1 mM EDTA, pH 7.4), lipoproteins were
overlaid with argon and stored at 4 °C. Where specified,
d 1.02-1.21 g/ml lipoproteins were fractionated on a
Superose 6 size exclusion column (Amersham Biosciences) to obtain
apoA-I+/+ and apoA-I
/
HDL as described (12). Apolipoproteins were examined by separating 7.5 µg of protein on a 4-20% SDS-polyacrylamide gradient gel (Bio-Rad) and visualizing the proteins with 0.1% Coomassie Brilliant Blue R-250.
Total cholesterol, free cholesterol, and phospholipid concentrations were measured using commercially available enzymatic kits (Wako). A
modified Lowry assay using horse IgG as a standard (Pierce) was
employed to determine HDL protein concentration (13). HDL was double
radiolabeled with [3H]cholesteryl oleoyl ether
([3H]COE) and [125I]dilactitol tyramine
([125I]DLT) (14) to yield specific activities of 4-20
cpm/ng of protein for [3H] and 90-200 cpm/ng of protein
for [125I] as described (12).
Nondenaturing Gradient Gel Electrophoresis of HDL--
HDL was
analyzed by nondenaturing 4-25% polyacrylamide gradient gel
electrophoresis followed by staining with 0.1% Coomassie Brilliant
Blue R-250 (12). Alternatively, HDL apolipoproteins were transferred to
a nitrocellulose membrane using a Trans-Blot Cell (Bio-Rad) run at 100 V for 90 min. The membrane was blocked for 1 h at room temperature
with 5% nonfat dried milk in 150 mM NaCl, 20 mM Tris, pH 7.4, 0.05% Tween 20, and incubated overnight at 4 °C with either rabbit anti-mouse apoA-I (Biodesign) or rabbit anti-mouse apoE antiserum (15) diluted 1:5,000 in 150 mM
NaCl, 20 mM Tris, pH 7.4, 0.2% Tween 20. The blot was
washed with 150 mM NaCl, 20 mM Tris, pH 7.4, 0.05% Tween 20, probed with 1:10,000 goat anti-rabbit IgG conjugated
to horseradish peroxidase (Amersham Biosciences) for 1 h at room
temperature, and washed again. Immunoreactive proteins were visualized
by exposing X-Omat Blue XB-1 film (Eastman Kodak Co.) to the membrane,
which had been treated with SuperSignal-West Pico chemiluminescent
substrate (Pierce).
Isolation of Lipid-free Mouse ApoA-I--
Lipid-free mouse
apoA-I was isolated with a modification of a previously published
protocol (16). HDL (1.063-1.21 g/ml) was isolated from
apoA-I+/+ FVB/N mouse plasma using sequential
density ultracentrifugation. Following dialysis against PBS-E, HDL was
incubated in 3 M guanidine HCl for 2 h at 37 °C,
dialyzed against PBS-E, and subjected to ultracentrifugation (39,000 rpm, 19 h, 15 °C) at d = 1.21 g/ml in a SW60 Ti
rotor. HDL remnants (d < 1.21 g/ml) were separated from the lipid-free apoA-I (d > 1.21) using a tube
slicer. ApoA-I was dialyzed against PBS-E and stored under argon at
4 °C. Because of the tendency of the protein to form multimers at
concentrations above 1 mg/ml, the apoA-I was not concentrated after the
final dialysis step (17).
Size Fractionation of ApoA-I+/+ and
ApoA-I
/
HDL--
Lipoproteins (1.02-1.21 g/ml)
radiolabeled with [3H]COE were separated using a Superose
6 column run at a flow rate of 0.4 ml/min. Following the initial
injection of the sample, fractions were collected from 33-35.6,
35.6-37, and 37-40 min for apoA-I+/+ HDL and
from 31-33.3, 33.3-35.3, and 35.3-40 min for
apoA-I
/
HDL. Each fraction (10 µg of total
cholesterol) was analyzed on a nondenaturing 4-25% polyacrylamide
gradient gel as described above. The HDL apolipoproteins were then
visualized using 0.1% Coomassie Brilliant Blue R-250. After
concentrating the HDL fractions using a Centricon 50 (Millipore), HDL
apolipoproteins were radiolabeled using [125I]DLT as
described above. Using 12% SDS-PAGE, the apolipoprotein complement of
the [3H]COE-[125I]DLT HDL was analyzed by
separating an equal number of [125I] counts from each
fraction. Following fixation of the gel with 40% methanol, 10% acetic
acid, the [125I]DLT-labeled apolipoproteins were
visualized by PhosphorImager analysis (Amersham Biosciences).
Treatment of ApoA-I+/+ and ApoA-I
/
HDL with d > 1.21 g/ml Plasma--
Plasma was
isolated from FVB/N mice as described above and subjected to
ultracentrifugation (39,000 rpm, 24 h, 15 °C) at
d = 1.21 g/ml in a SW41 Ti rotor. The d > 1.21 fraction was washed by ultracentrifugation (39,000 rpm, 24 h, 15 °C) at d = 1.21 g/ml and dialyzed against
PBS-E supplemented with 0.02% NaN3.
[3H]COE apoA-I+/+ and
apoA-I
/
HDL (1.5 mg of protein) were
incubated at 37 °C for 16 h in 5.5 ml of PBS-E containing 50%
by volume d > 1.21 g/ml plasma or d > 1.21 g/ml plasma plus 0.3 mg/ml lipid-free apoA-I, respectively. For
controls, HDL (1.5 mg of protein) was incubated under similar
conditions in PBS-E alone. The density of the samples was adjusted to
1.21 g/ml with KBr, and HDL was isolated by ultracentrifugation (39,000 rpm, 19 h, 15 °C) in a SW41 Ti rotor. The HDL was dialyzed
against PBS-E, and apolipoproteins were radiolabeled using
[125I]DLT as described above.
LCAT Treatment of [3H]COE-labeled
ApoA-I+/+ and ApoA-I
/
HDL--
Recombinant
human LCAT was purified as previously described and stored at
70 °C in 50 mM imidazole, 10% glycerol (18). LCAT in
the presence of fatty acid-free bovine serum albumin (BSA; Intergen)
was concentrated at 4 °C using an Ultrafree-15 centrifugal filter
device with a 50,000 Mr cut-off (Millipore). The
final concentrations of the LCAT and the BSA were 1.1-1.4 × 105 units (nmol of CE formed/h/µg of enzyme)/ml and 60 mg/ml, respectively. [3H]COE
apoA-I
/
HDL (1.2 mg of protein) was
incubated at 37 °C for 24 h in 2.3 ml of reaction buffer (150 mM NaCl, 10 mM potassium phosphate, 1 mM EDTA, 0.05% NaN3, 5 mM
imidazole, 1% glycerol, pH 7.4) containing 12,000 LCAT units/ml plus
or minus 0.45 mg/ml lipid-free apoA-I. For controls,
[3H]COE apoA-I+/+ and
apoA-I
/
HDL (1.2 mg of protein) were
incubated under the same conditions in reaction buffer supplemented
with 6 mg/ml BSA. The sample was adjusted to 1.21 g/ml with KBr, and
HDL was isolated by ultracentrifugation (39,000 rpm, 19 h,
15 °C) in a SW60 Ti rotor. Following dialysis against PBS-E, HDL
were then radiolabeled using [125I]DLT as described above.
Using particles that were only [3H]COE-radiolabeled, the
total cholesterol concentrations of the HDL samples were determined using the Cholesterol CII enzymatic assay (Wako). An equal amount of
each sample was analyzed by 12% SDS-polyacrylamide electrophoresis (5 µg of total cholesterol) and nondenaturing 4-25% polyacrylamide gradient gels (5 and 10 µg of total cholesterol) as described above.
Gels were stained with 0.1% Coomassie Brilliant Blue R-250 or
evaluated by Western blot analysis as described above. Similar conditions were used to modify and analyze other HDL particles with the
following exceptions. For one study, the [3H]COE
apoA-I
/
HDL were incubated with 0.45 mg/ml lipid-free
apoA-I in the absence or presence of 12,000 LCAT units/ml.
Culturing Conditions for Y1-BS1 Cells--
Y1-BS1 cells were
maintained in a 37 °C humidified 95% air, 5% CO2
atmosphere in Ham's F-10 complete medium (12.5% heat-denatured horse
serum, 2.5% heat-denatured fetal bovine serum, 2 mM
glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin). For
experiments, the cells were seeded at a density of 1.5 × 106 cells/well into six-well plates, which had been treated
with 100 µg/ml poly-D-lysine. After 48 h, medium was
replaced with Ham's F-10 complete medium plus 100 nM
Cortrosyn, a 1-24ACTH synthetic analogue. All studies were
conducted following a 24-h exposure to ACTH.
Determination of Cell Association, Degradation, and Selective
Uptake of HDL-CE--
After being seeded and treated as listed above,
Y1-BS1 cells were washed and the medium replaced with serum-free Ham's
F-10 medium. [3H]COE-[125I]DLT
apoA-I+/+ or apoA-I
/
HDL was added to the final concentration specified in the figure legends. Following a 4-h incubation at 37 °C, the cells were washed three times with phosphate-buffered saline plus 0.1% BSA, pH 7.4; one
time with phosphate-buffered saline, pH 7.4; lysed with 1.25 ml of 0.1 N NaOH; and passed five times through a 28.5-gauge needle. The lysate was then processed to determine trichloroacetic acid-soluble and -insoluble 125I radioactivity and organic
solvent-extractable 3H radioactivity as described (14, 19).
Trichloroacetic acid-insoluble 125I radioactivity
represents cell-associated HDL apolipoprotein that is the sum of cell
surface-bound apolipoprotein and endocytosed apolipoprotein that is not
yet degraded. Trichloroacetic acid-soluble 125I
radioactivity represents endocytosed and degraded apolipoprotein that
is trapped in lysosomes as a result of the dilactitol tyramine label
(14, 20). The sum of the 125I-degraded and
125I-cell-associated undegraded apolipoprotein expressed as
CE equivalents was subtracted from the CE measured as extractable
3H radioactivity to give the selective uptake of HDL-CE
(14, 19). Values for these parameters are expressed as nanograms of
HDL-CE/mg of cell protein.
 |
RESULTS |
Role of Apolipoprotein Content and Particle Size in Reduced CE
Selective Uptake Activity of ApoA-I
/
HDL--
Previous
studies with reconstituted or modified HDL suggest that specific
apolipoproteins, particularly apoA-II and apoE, may alter the
efficiency of SR-BI-mediated HDL CE selective uptake. However, there is
no clear consensus in the literature as to whether these proteins have
inhibitory or stimulatory effects (21-25). To compare
apoA-I
/
HDL with different apoA-II and apoE
contents, HDL were isolated from mice on FVB/N or C57BL/6 genetic
backgrounds. Previous studies showed that
apoA-I
/
HDL from C57BL/6 mice are enriched in
apoE (5, 7), and we noted that HDL from FVB/N mice are enriched in
apoA-II. The SDS-PAGE analysis in Fig.
1A shows the relative
enrichment of FVB/N apoA-I
/
HDL in apoA-II
in comparison to the enrichment of C57BL/6
apoA-I
/
HDL in apoE. Each of these HDLs
along with the respective apoA-I+/+ HDLs were
labeled with [3H]COE and [125I]DLT and
tested in a standard selective uptake assay using ACTH-treated Y1-BS1
adrenocortical cells in which HDL CE selective uptake is primarily the
result of SR-BI (26). Comparing particles isolated from mice of the
same genetic strain, ~2-fold more selective CE uptake was observed
from apoA-I+/+ than from
apoA-I
/
HDL (Fig. 1D).
Additionally, HDL CE selective uptake was similar when comparing
apoA-I
/
HDL of both strains. This result
indicates that apoA-I
/
HDL, regardless of
its apoA-II or apoE content, is less efficient than wild type HDL at
selectively transferring its CE to the Y1-BS1 cells. Interestingly, the
Y1-BS1 cells displayed similar HDL-CE cell association and degradation
of the different particles with the exception of the C57BL/6
apoA-I
/
HDL (Fig. 1, B and
C). The 2-fold increase in these parameters for the C57BL/6
apoA-I
/
HDL is likely a result of this
apoE-rich HDL being bound and internalized by proteoglycans or members
of the LDL receptor family (27). In contrast, differences in the
apoA-II and apoE content of the apoA-I
/
HDL
did not alter the ability of the Y1-BS1 cells to internalize CE via
SR-BI-dependent selective uptake.

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Fig. 1.
Compositional and functional properties of
apoA-I+/+ and
apoA-I /
HDL isolated from FVB/N and C57BL/6 mice. A, an equal amount
of FVB/N and C57BL/6 apoA-I+/+ and
apoA-I / HDL (7.5 µg of protein) was
separated on a 4-20% SDS-polyacrylamide gradient gel. The proteins
were visualized using 0.1% Coomassie Brilliant Blue R-250.
ACTH-treated Y1-BS1 cells were exposed to 25 µg of protein/ml of
[3H]COE-[125I]DLT
apoA-I+/+ and apoA-I /
HDL for 4 h at 37 °C. The amounts of cell association
(B), degradation (C), and selective uptake
(D) of HDL-CE were determined as described under
"Experimental Procedures." Each column represents the
mean of four samples (± S.E.) from two experiments. Note that the
scale of the y axis for panels B and
C is different from that in panel
D.
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|
Several studies have shown that HDL particle size affects the ability
of cells to selectively internalize HDL-CE (28-30). To test whether
the larger size and heterogeneity of the
apoA-I
/
HDL (5, 12) may explain its reduced
selective uptake activity, [3H]COE-labeled
apoA-I+/+ and apoA-I
/
HDL were separated into three size fractions by gel exclusion chromatography (Fig. 2A).
Analysis by nondenaturing gradient gel electrophoresis indicated that
the HDL was separated into fractions with different mean particle
diameters (Fig. 2B). Following radiolabeling of the HDL with
[125I]DLT, the apolipoprotein complement of each fraction
was determined by SDS-PAGE (Fig. 2C). As observed previously
(12), apoA-II was enriched on smaller particles and apoE on larger
particles of apoA-I
/
HDL.

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Fig. 2.
Size fractionation of
apoA-I+/+ and
apoA-I /
HDL. A, using a Superose 6 gel filtration chromatography
column, [3H]COE apoA-I+/+ and
apoA-I / 1.02-1.21 g/ml lipoproteins were
separated into three HDL fractions. B, an equal amount of
total (T) and fractionated HDL (10 µg of total
cholesterol) was analyzed on a 4-25% nondenaturing gradient gel. The
proteins were visualized using 0.1% Coomassie Brilliant Blue R-250.
C, total and fractionated HDL, containing an equivalent
number of [125I] counts (5 × 105), were
analyzed by 12% SDS-PAGE. After fixing with 40% methanol plus 10%
acetic acid, the proteins were visualized by PhosphorImager
analysis.
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The functional properties of the size-fractionated HDL were tested on
ACTH-treated Y1-BS1 cells. Although the two types of unfractionated
particles were bound and degraded to similar extents (Fig.
3, A and B, T
columns), more selective CE uptake was seen with
apoA-I+/+ compared with the
apoA-I
/
total HDL fraction (Fig.
3C, T columns)). In contrast, cell association and degradation of the fractionated particles increased in proportion to their diameter for both apoA-I+/+ and
apoA-I
/
HDL (Fig. 3, A and
B). These differences among the size-fractionated particles
likely reflect the larger, apoE-rich HDL interacting with LDL receptor
family members or proteoglycans. In contrast, little difference was
seen for HDL CE selective uptake among the size-fractionated particles
for either apoA-I+/+ or
apoA-I
/
HDL (Fig. 3C).
Additionally, more selective CE uptake was seen from
apoA-I+/+ than
apoA-I
/
HDL when particles of similar
diameter were compared. For instance, the Y1-BS1 cells selectively
internalized more HDL CE from apoA-I+/+ fraction
2 than from the similar sized apoA-I
/
fraction 3. Similar results were obtained with size-fractionated particles from two independent HDL preparations. Several conclusions can be drawn. First, size subpopulations within the
apoA-I+/+ and apoA-I
/
HDL do not differentially transfer CE to the Y1-BS1 cells by selective
uptake. Second, in agreement with the experiments comparing FVB/N and
C57BL/6 apoA-I
/
HDL (Fig. 1), selective CE
uptake from apoA-I
/
HDL is not significantly
affected by the apoA-II and apoE content. Third, the larger size and
heterogeneity of apoA-I
/
HDL are not
responsible for the diminished SR-BI-mediated selective CE uptake.

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Fig. 3.
Functional analysis of size-fractionated
apoA-I+/+ and
apoA-I /
HDL. ACTH-treated Y1-BS1 cells were incubated for 4 h at
37 °C with 10 µg of protein/ml of total (T) or
size-fractionated apoA-I+/+ and
apoA-I / HDL that had been radiolabeled with
[3H]COE and [125I]DLT. The amounts of cell
association (A), degradation (B), and selective
uptake (C) of HDL-CE were determined as described under
"Experimental Procedures." Each column represents the
mean of three samples (± S.E.). Note that the scale of the
y axis for panels A and B
is different from that in panel C. Similar
results were seen using a separately isolated and radiolabeled batch of
particles.
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Role of Free Cholesterol Content in Reduced CE Selective Uptake
Activity of ApoA-I
/
HDL--
In a previous analysis,
we noted that apoA-I
/
HDL has a
significantly higher FC content than apoA-I+/+
HDL, a factor that may reduce the fluidity of the PL monolayer and
hinder SR-BI-mediated transfer of CE from the HDL core (12). To test
the importance of the HDL FC content for CE selective uptake,
[3H]COE apoA-I
/
HDL was
incubated with apoA-I+/+ d > 1.21 g/ml plasma, which acted as a source of LCAT, and lipid-free mouse
apoA-I. Radiolabeled apoA-I+/+ HDL was treated
in a similar fashion but without the addition of apoA-I. Incubation
with d > 1.21 g/ml plasma resulted in significant reductions in the FC content of both apoA-I+/+
and apoA-I
/
HDL (Table
I). In addition, apoA-I associated with
the apoA-I
/
particles (data not shown).
After radiolabeling with [125I]DLT, the HDL were
incubated with ACTH-treated Y1-BS1 cells. The selective CE uptake from
the particles exposed to the d > 1.21 g/ml plasma was
significantly increased compared with their respective mock-treated
controls (Table I). This result suggested that the high FC content of
the apoA-I
/
HDL may impede selective
transfer of CE to Y1-BS1 cells. However, the functional properties of
the apoA-I
/
HDL may also may have been
altered by the acquisition of apoA-I or modification by enzymes other
than LCAT in the d > 1.21 g/ml plasma.
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Table I
Impact of d > 1.21 g/ml mouse plasma on
apoA-I+/+ and apoA-I / HDL
ACTH-treated Y1-BS1 cells were incubated with 25 µg of CE/ml of
[3H]COE-[125I]DLT apoA-I+/+ and
apoA-I / HDL for 4 h at 37 °C, and the
amount of selective CE uptake was determined as described under
"Experimental Procedures." The results represent the mean of six
values (± S.E.) from two experiments. PT, protein; FC, free
cholesterol.
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Role of ApoA-I and LCAT in Reduced CE Selective Uptake Activity of
ApoA-I
/
HDL--
To test more directly the impact of
the FC content of apoA-I
/
HDL on selective
CE uptake, apoA-I
/
particles were incubated
with purified recombinant human LCAT. Regardless of the absence or
presence of lipid-free mouse apoA-I, LCAT treatment significantly
decreased the FC content of the apoA-I
/
HDL
resulting in FC/protein and FC/PL ratios that were lower than those of
apoA-I+/+ HDL (Table
II). In addition, the size and the
apolipoprotein composition of the apoA-I
/
particles were modified by exposure to LCAT. SDS-PAGE showed that
apoA-I
/
HDL incubated with LCAT and apoA-I
acquired apoA-I that was stable to re-isolation of the particles (Fig.
4A, lane
4). NDGGE of apoA-I
/
HDL that had
been incubated with LCAT alone showed a minor reduction in particle
mobility with no change in heterogeneity of the particles (Fig.
4B, lane 3). Western blot analysis
showed that LCAT treatment released most of the apoE from
apoA-I
/
HDL (Fig. 4B,
lane 3). Incubation of
apoA-I
/
HDL with LCAT plus apoA-I generated
a smaller, more distinct particle population, the diameter of which was
only slightly larger than that of apoA-I+/+ HDL
(Fig. 4B, lane 4). Western blot
analysis of the HDL separated by NDGGE revealed that apoA-I was present
on these particles, whereas apoE resided on particles with slightly
larger diameters (Fig. 4B, lanes 2 and
4).
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Table II
Lipid composition of apoA-I / HDL treated with LCAT ± apoA-I
CE, cholesteryl ester; PT, protein; PL, phospholipid; FC, free
cholesterol.
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Fig. 4.
Structural and functional modifications of
apoA-I /
HDL treated with LCAT in the presence or absence of lipid-free
apoA-I. Following either mock treatment or exposure to LCAT plus
or minus lipid-free apoA-I, an equal amount of each HDL was analyzed by
12% SDS-PAGE (A) and 4-25% NDGGE (B). As
described under "Experimental Procedures," HDL samples (5 µg of
TC) that had been subjected to NDGGE were evaluated for apoA-I and apoE
content by Western blot analysis. The protein of the other HDL samples
that had been separated by SDS-PAGE (5 µg of TC) and NDGGE (10 µg
of TC) were directly visualized using 0.1% Coomassie Brilliant Blue
R-250. * designates LCAT in B. ACTH-treated
Y1-BS1 cells were incubated for 4 h at 37 °C with 10 µg of
protein/ml of mock-treated apoA-I+/+ and
apoA-I / HDL that had been radiolabeled with
[3H]COE and [125I]DLT. Because of the
increased CE content, the amount of LCAT ± apoA-I-treated
apoA-I / HDL incubated with the cells was normalized to
the level of CE in the mock-treated apoA-I / HDL. The
amounts of cell association (C), degradation (D),
and selective uptake (E) of HDL-CE were determined as
described under "Experimental Procedures." Each column
represents the mean of four samples (± S.E.) from two experiments.
Note that the scale of the y axis for panels
C and D is different from that in
panel E. Similar results were seen using a
separately treated and radiolabeled batch of particles.
|
|
The functional properties of the HDL were then tested on ACTH-treated
Y1-BS1 cells. Compared with the two mock-treated particles, more HDL-CE
cell association and degradation was observed from the
apoA-I
/
HDL that had been exposed to LCAT
(Fig. 4, C and D, bars 3 and 4). SDS-PAGE showed that all the samples contained BSA
that had not been completely removed during re-isolation of the HDL by density ultracentrifugation (Fig. 4A). However, NDGGE
revealed that LCAT, which has a molecular weight similar to BSA on
SDS-PAGE, was also present in the treated samples (Fig. 4B,
lanes 3 and 4, *).
Therefore, it was concluded that the Y1-BS1 cells bound and degraded
the LCAT-treated apoA-I
/
HDL to a greater extent as a
result of the presence of this enzyme.
An enhancement in selective CE uptake was not observed from
apoA-I
/
HDL treated with LCAT alone (Fig.
4E, compare bars 2 and 3), even though these particles had a lower FC/PL ratio than
apoA-I+/+ HDL and a PL/PT ratio nearly
the same as apoA-I+/+ HDL (Table II). However,
the modifications imparted by incubation with apoA-I plus LCAT resulted
in increased CE selective uptake from
apoA-I
/
HDL (Fig. 4E, compare
bars 2 and 4). Several conclusions can be drawn from these data. First, the failure of LCAT treatment alone to
increase HDL CE selective uptake from
apoA-I
/
HDL, despite decreasing the FC and
PL content and the FC/PL ratio, indicates that these parameters are not
key factors in the reduced efficiency of CE transfer from
apoA-I
/
HDL. Second, enhanced cell
association of apoA-I
/
HDL treated with LCAT
alone (Fig. 4C, compare bars 2 and
3) was accompanied by increased HDL processing by the
endocytic uptake pathway (Fig. 4D, compare bars
2 and 3), but not by SR-BI-mediated selective
uptake (Fig. 4E, compare bars 2 and
3). Third, the increased CE selective uptake resulting from
treatment of apoA-I
/
HDL with LCAT plus
apoA-I was accompanied by a reorganization of the HDL to yield a more
discrete size population similar in diameter to
apoA-I+/+ HDL.
Because the prior experiments showed that selective CE uptake was
increased from apoA-I
/
HDL treated with
apoA-I plus LCAT, we next determined whether apoA-I alone could
generate similar changes in the particles. For this study,
apoA-I
/
HDL were incubated with lipid-free
apoA-I in the absence or presence of LCAT and were subsequently
analyzed for changes in apolipoprotein content and size by SDS-PAGE and
NDGGE. As seen previously, apoA-I
/
HDL
incubated with LCAT and apoA-I had a decreased level of apoE, had
acquired apoA-I (Fig. 5A,
lane 4), and had formed a distinct particle
population with a diameter similar to apoA-I+/+
HDL (Fig. 5B, lane 4). Incubation of
the apoA-I
/
HDL with apoA-I alone also
caused the gain of apoA-I (Fig. 5A, lane
3). However, the size distribution of these particles was not changed (Fig. 5B, lane 2 versus lane 3). Western blot analysis showed that apoA-I was equally distributed among these heterogeneously sized particles, whereas it was concentrated on the smaller distinct particle population formed by apoA-I plus LCAT (Fig. 5B,
lane 3 versus lane
4). In addition, the apoA-I
/
HDL
incubated with only apoA-I was found to have lost a significant amount
of its apoE but not nearly as much as the particles treated with apoA-I
plus LCAT.

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Fig. 5.
Structural and functional modifications of
apoA-I /
HDL exposed to lipid-free apoA-I in the presence or absence of
LCAT. Following either mock treatment or exposure to lipid-free
apoA-I plus or minus LCAT, the HDL particles were analyzed as described
in the legend of Fig. 4. Each column represents the mean of
four samples (± S.E.) from two experiments. Note that the scale of the
y axis for panels C and D
is different from that in panel E. Similar
results were seen using a separately treated and radiolabeled batch of
particles.
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|
Testing the functional properties of the HDL on ACTH-treated Y1-BS1
cells showed that addition of apoA-I did not enhance selective CE
uptake (Fig. 5E, compare bars 2 and
3). In contrast, selective CE uptake was enhanced following
incubation of the apoA-I
/
HDL with apoA-I
and LCAT (Fig. 5E, compare bars 2 and
4). Compared with their mock-treated equivalent, the lone
addition of apoA-I to the apoA-I
/
HDL caused
a slight reduction in HDL-CE cell association and degradation (Fig. 5,
C and D). The decrease in these parameters most
likely reflects the decreased apoE content (Fig. 5B, compare lanes 2 and 3). From these results it
can be concluded that apoA-I is necessary but not sufficient for the
enhancement of CE selective uptake and the conversion of the
apoA-I
/
HDL into smaller particles of a more
discrete size.
In the previous experiments (Figs. 4 and 5), some LCAT was
recovered in the LCAT-treated apoA-I
/
HDL
after re-isolation of the particles and, thus, was present during the
HDL CE selective uptake measurements with the Y1-BS1 cells. To test
whether LCAT activity might contribute to the selective uptake process
itself (and not only to the HDL particle reorganization), apoA-I
/
HDL were incubated with apoA-I and
LCAT to permit particle reorganization, and then treated with 2 mM DTNB to inactivate LCAT (31). These particles and the
mock-treated controls were then re-isolated by centrifugation, and
tested for HDL CE selective uptake activity with the Y1-BS1 cells. As
shown in Fig. 6,
apoA-I
/
HDL incubated with LCAT and apoA-I
showed the same increase in HDL CE selective uptake compared with
apoA-I
/
HDL whether or not the particles
were treated with DTNB (compare bars 3 and
4 with bar 2). Thus, LCAT activity
remaining after HDL re-isolation did not contribute to HDL CE selective
uptake activity. In other experiments we also have shown that treatment of LCAT with 2 mM DTNB prior to incubation with HDL and
apoA-I effectively blocked LCAT activity (data not shown).

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Fig. 6.
Effect of LCAT inactivation after HDL
reorganization on HDL CE selective uptake from
apoA-I /
HDL. ACTH-treated Y1-BS1 cells were incubated for 4 h at
37 °C with 25 µg of protein/ml of mock-treated
apoA-I+/+ and apoA-I /
HDL that had been radiolabeled with [3H]COE and
[125I]DLT. Because of the increased CE content, the
amount of LCAT + apoA-I-treated apoA-I / HDL incubated
with the cells was normalized to the level of CE in the mock-treated
apoA-I / HDL. For apoA-I / HDL
sample 4 (A-I / + A-I/LCAT/DTNB),
LCAT plus lipid-free apoA-I was incubated with
apoA-I / HDL for 24 h at 37 °C and
then the particles were treated for 30 min at 37 °C with 2 mM DTNB. The amount of selective uptake of HDL-CE was
determined as described under "Experimental Procedures."
Bars 1 and 2 represent the mean of 6 samples (± S.E.), whereas bars 3 and
4 represent the mean of 12 samples (± S.E.) from two
experiments. Bars 3 and 4 are not
statistically different (p = 0.44).
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 |
DISCUSSION |
The results from this study indicate that the reduced
efficiency of CE selective uptake from
apoA-I
/
HDL into adrenal cells is caused by
the physical absence of apoA-I. Within the range of properties
evaluated, variations in particle size, FC and PL content, the FC/PL
ratio, or the apoA-II and apoE contents had little influence on
SR-BI-mediated CE selective uptake from
apoA-I
/
HDL. Similarly, addition of apoA-I
to apoA-I
/
HDL or treatment of the particles
with LCAT had little effect. In contrast, addition of apoA-I in
combination with LCAT treatment reorganized the
apoA-I
/
HDL to a smaller and less
heterogeneous particle with increased HDL CE selective uptake activity.
The particle size analysis described above indicates that it is not the
smaller size per se of the reorganized HDL that is important
for enhanced selective uptake activity. These results suggest that not
only the presence but also the proper orientation of apoA-I is needed
for optimal SR-BI-mediated selective CE uptake from HDL. The inability
of LCAT treatment alone to alter the particle size distribution or the
CE selective uptake activity is supported by in vivo
findings as well. In the apoA-I
/
mouse, HDL
is exposed to sufficient LCAT activity to generate CE-rich particles,
but the particles remain large and heterogeneous and have reduced CE
selective uptake activity (8, 12). That observation as well as the
in vitro LCAT experiments reported here indicate that other
HDL apolipoproteins cannot substitute for apoA-I in these processes,
despite the fact that apoCI, apoE, and apoA-IV can activate LCAT
(32-34). Thus, the reorganization of
apoA-I
/
HDL particles and the enhancement of
HDL CE selective uptake appear to be unique properties of apoA-I.
The mechanism by which apoA-I enhances SR-BI-mediated HDL CE selective
uptake is unclear but most likely requires apoA-I to assume a specific
conformation on the HDL surface in a process requiring LCAT. The
presence of apoA-I on the surface of apoA-I
/
HDL had little effect on CE selective uptake until the HDL was reorganized by LCAT. We speculate that, during particle reorganization, apoA-I assumes a specific conformation that is important for efficient lipid transfer but is not important for the initial docking of HDL to
SR-BI. Several studies support this dissociation between HDL binding
and lipid transfer. For example, lipid-poor apoA-I does not support
SR-BI-mediated FC efflux from cells despite binding to SR-BI with high
affinity (35, 36). Similarly, a recent study identified apoA-I
mutations that disrupt FC efflux but not HDL binding to SR-BI (37).
Additionally, chimeric receptors formed with the transmembrane and
intracellular domains of SR-BI and the extracellular domain of the
closely related scavenger receptor CD36 bind HDL with high affinity but
fail to mediate efficient HDL CE selective uptake (38, 39) or increase
the bi-directional flux of FC (40). These data, as well as the present and previous studies with apoA-I
/
HDL (12),
support the hypothesis that lipid transfer via SR-BI is a two-step
process in which the lipid transfer step is distinct from the
apolipoprotein-mediated docking of HDL to SR-BI (38, 39).
Elucidation of the actual role that apoA-I plays in the lipid transfer
process requires further study. One possibility is that apoA-I
interacts with SR-BI in a unique manner to form a hydrophobic channel
through which lipids move from the HDL particle to the cell membrane
(41). Interestingly, Corsico et al. (42) recently showed
that two amphipathic
-helices of free or lipidated apoA-I can
penetrate the bilayer of a
1-palmitoyl-2-oleoyl-sn-glycerophosphocholine/FC vesicle. If
this characteristic is specific to apoA-I, it can be envisioned that,
when complexed to an HDL particle, apoA-I in combination with SR-BI
forms a hydrophobic channel by inserting these
-helices into the
plasma membrane, thereby facilitating lipid transfer. This process
would imply a specific register of apoA-I
-helical amphipathic
repeat units interacting with SR-BI to provide efficient lipid
transfer, although multiple and distinct repeat units are sufficient to
mediate binding to SR-BI (36).
Another possibility is that apoA-I modifies the structure
of the HDL surface thereby allowing the CE to be readily accessed by
SR-BI. Evidence to support this idea comes from a study in which the PL
acyl chain and head group packing order were analyzed in recombinant
apoA-I HDL or apoA-II HDL (43). The findings indicated that, compared
with apoA-II, apoA-I caused the PL head groups to have a less ordered
structure. If apoA-I has similar properties when bound to native HDL, a
less ordered PL monolayer may enable SR-BI to more readily access the
CE core of the particle. These possibilities are not mutually exclusive.
In summary, we have determined that the failure of adrenal cells to
efficiently internalize CE from apoA-I
/
HDL
via selective uptake is directly caused by the absence of apoA-I from
the particles. On apoA-I+/+ HDL and
apoA-I
/
HDL treated with apoA-I and LCAT,
apoA-I appears to be organized in such a manner that, through
interactions with SR-BI or via effects on HDL lipid organization, or
both, HDL CE selective uptake is enhanced.