Apolipoprotein A-II Modulates the Binding and Selective Lipid
Uptake of Reconstituted High Density Lipoprotein by Scavenger Receptor
BI*
Maria C.
de Beer
,
Diane M.
Durbin§,
Lei
Cai
,
Nichole
Mirocha§,
Ana
Jonas§,
Nancy R.
Webb
,
Frederick C.
de Beer
, and
Deneys R.
van der Westhuyzen
¶
From the
Department of Internal Medicine, University
of Kentucky Medical Center, Lexington, Kentucky 40536, the Department
of Veterans Affair Medical Center, Lexington, Kentucky 40511, and the
§ Department of Biochemistry, College of Medicine at
Urbana-Champaign, University of Illinois, Urbana, Illinois 61801
Received for publication, January 10, 2001, and in revised form, February 9, 2001
 |
ABSTRACT |
High density lipoprotein (HDL) represents a
mixture of particles containing either apoA-I and apoA-II
(LpA-I/A-II) or apoA-I without apoA-II (LpA-I). Differences in the
function and metabolism of LpA-I and LpA-I/A-II have been reported,
and studies in transgenic mice have suggested that apoA-II is
pro-atherogenic in contrast to anti-atherogenic apoA-I. The molecular
basis for these observations is unclear. The scavenger receptor BI
(SR-BI) is an HDL receptor that plays a key role in HDL metabolism. In
this study we investigated the abilities of apoA-I and apoA-II to
mediate SR-BI-specific binding and selective uptake of cholesterol
ester using reconstituted HDLs (rHDLs) that were homogeneous in size
and apolipoprotein content. Particles were labeled in the protein (with
125I) and in the lipid (with
[3H]cholesterol ether) components and SR-BI-specific
events were analyzed in SR-BI-transfected Chinese hamster ovary cells.
At 1 µg/ml apolipoprotein, SR-BI-mediated cell association of
palmitoyloleoylphosphatidylcholine-containing AI-rHDL was significantly
greater (3-fold) than that of AI/AII-rHDL, with a lower
Kd and a higher Bmax for
AI-rHDL as compared with AI/AII-rHDL. Unexpectedly, selective
cholesterol ester uptake from AI/AII-rHDL was not compromised compared
with AI-rHDL, despite decreased binding. The efficiency of
selective cholesterol ester uptake in terms of SR-BI-associated
rHDL was 4-5-fold greater for AI/AII-rHDL than AI-rHDL. These results
are consistent with a two-step mechanism in which SR-BI binds ligand
and then mediates selective cholesterol ester uptake with an efficiency
dependent on the composition of the ligand. ApoA-II decreases binding
but increases selective uptake. These findings show that apoA-II can exert a significant influence on selective cholesterol ester uptake by
SR-BI and may consequently influence the metabolism and function of
HDL, as well as the pathway of reverse cholesterol transport.
 |
INTRODUCTION |
HDL1 levels are
inversely related to the risk of atherosclerotic disease (1, 2). One
protective function of HDL is its role in reverse cholesterol
transport, a pathway by which unesterified cholesterol from peripheral
cells is taken up by HDL, esterified by the enzyme lecithin
cholesterol acyltransferase on HDL, and then transported as cholesteryl
ester (CE) to liver and steroidogenic cells by a process of selective
lipid uptake in which cholesteryl esters of HDL are taken up without
concomitant apolipoprotein uptake (3, 4). HDL is a mixture of different
types of particles that vary in size, density, and composition (5) and
consists of particles containing both apoA-I and apoA-II (LpA-I/A-II)
and those containing apoA-I but not apoA-II (LpA-I). ApoA-II is the second most abundant protein in HDL, but its physiological function is
unclear. Studies have indicated that the levels of HDL cholesterol and
apoA-I are affected by the rate of synthesis of apoA-II in both humans
and mice (6, 7). ApoA-II-deficient mice have markedly reduced HDL
levels and increased HDL cholesterol and apoA-I fractional catabolic
rates (8). Most interestingly, some studies suggest that apoA-II
functions in a "pro-atherogenic manner," in contrast to
"anti-atherogenic" apoA-I. Transgenic mice overexpressing either
human (9) or mouse (10) apoA-II were found to be more susceptible to
atherosclerosis. In contrast, however, a recent report showed decreased
susceptibility to diet-induced atherosclerosis in human apoA-II
transgenic mice (11). Certain evidence has demonstrated differences in
the metabolism and function of LpA-I and LpA-I/A-II. Turnover studies
in humans revealed that apoA-I in LpA-I is more rapidly catabolized
than apoA-I in LpA-I/A-II (12), and in rats cholesteryl esters were
more efficiently secreted into the bile from LpA-I than LpA-I/A-II
(13). Studies in vitro have reported that LpA-I is more
efficient in the delivery of cholesteryl ester to cells (14) and, in
some (15, 16) but not all systems (17-19), in mediating cholesterol
efflux from cells. However, the molecular basis for these observations
is not understood. ApoA-II has been reported to negatively affect a
number of processes involved in HDL metabolism and reverse cholesterol
transport, including the activities of hepatic lipase (20),
cholesterylester transfer protein (21), and lecithin
cholesterol acyltransferase (22) and may therefore exert some effects
through the modulation of HDL lipid composition and/or apoA-I conformation.
The scavenger receptor, SR-BI, plays a major role in HDL metabolism,
binding HDL and mediating selective lipid uptake from HDL into the
liver and steroidogenic cells (23-26). SR-BI also facilitates the
efflux of cellular free cholesterol to HDL (27, 28). The receptor
exhibits a broad ligand specificity and binds native LDL, oxidized LDL,
and VLDL in addition to HDL (23, 29). Anionic phospholipids also
reportedly bind to SR-BI, as do the apolipoproteins A-I, A-II, and
C-III, either as lipid-bound molecules or as free apolipoproteins (30).
We report here the finding that the binding of rHDL by SR-BI is
significantly greater for particles containing only apoA-I (AI-rHDL)
compared with particles containing both apoA-I and apoA-II
(AI/AII-rHDL). The ability of apoAI/AII-rHDL to deliver cholesterol
ester via selective lipid uptake is, however, not compromised and is
actually increased. ApoA-II modulation of SR-BI activity, therefore,
may contribute to the reported differences in function and metabolism
between LpA-I and LpA-I/A-II.
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EXPERIMENTAL PROCEDURES |
Materials--
Human apoA-I and apoA-II were prepared from blood
plasma purchased from the Champaign County Blood Bank, Regional Health
Resource Center, as described previously (31, 32). Human
HDL3 (d = 1.13-1.18 g/ml) was
isolated by density gradient ultracentrifugation as previously
described (33). L-
-Palmitoyloleoylphosphatidylcholine (POPC), L-
-dipalmitoylphosphatidylcholine
(DPPC), crystalline cholesterol, sodium cholate, and fatty acid-free
bovine serum albumin (BSA) were purchased from Sigma. Radiolabeled DPPC
2-palmitoyl-9,10-3[H] came from PerkinElmer Life
Sciences; and [1
, 2
(n)3H]cholesteryl oleoyl ether
and sodium [125I]iodide were from Amersham Pharmacia Biotech.
Preparation of rHDL--
Reconstituted HDL containing POPC and
DPPC were prepared by the sodium cholate dialysis method as described
(34, 35). Briefly, rHDL containing human apoA-I were prepared using
molar ratios of 1/5/95, apoA-I/free
cholesterol/palmitoyloleoylphosphatidylcholine (POPC) or 1/5/100,
apoA-I/free cholesterol/dipalmitoylphosphatidylcholine (DPPC). The
dimeric apo-A-II (Mr 17,400) particles contained
the same mass of phospholipid and free cholesterol per mg of apoA-II protein as rHDL prepared with apoA-I. rHDL-containing cholesteryl esters were prepared by using molar ratios of 1/5/3/95 of apoA-I/free cholesterol/cholesteryl oleate/phospholipid. The purity and size of
rHDL were examined on 8-25% gradient gels under non-denaturing conditions using the Amersham Pharmacia Biotech Phast System. The rHDL
particles were ~100 Å in diameter and particles were >95%
homogeneous in size. Chemical cross-linking with
bis(sulfosuccinimidyl)suberate determined that AI-rHDL contained two
molecules of apoA-I, and AII-rHDL contained four molecules of dimeric
apoA-II with less than 5% lipid-free apoprotein. Protein
concentrations of lipid-free or lipid-bound apoA-I and apoA-II were
determined as the average of concentrations measured by the Lowry assay
(36) and absorbance at 280 nm, using an extinction coefficient of 1.13 ml/mg·cm for apoA-I, or at 276 nm, using an extinction coefficient of
0.69 ml/mg·cm for apoA-II (37). Experiments were performed within 20 days of particle preparation to avoid time-dependent size
rearrangement of particles. Hybrid AI/AII rHDL was prepared by
incubating lipid-free apo-AII with AI-rHDL at a molar ratio of 2:1
AI/AII for at least 20 min at room temperature. This resulted in one
dimeric apoA-II molecule on each rHDL. These particles were used within
24 h. For cell association studies, the different rHDL
preparations were added on the basis of equivalent particle numbers
(expressed as apoA-I equivalents).
Radiolabeling of Lipoproteins--
Human HDL and rHDL
preparations were labeled in the protein moiety with sodium
[125I]iodide using the iodine monochloride method (38).
The specific activity of the HDL ranged from 400 to 600 cpm/ng protein.
The phospholipid component of the rHDL was also labeled with
3H by adding [3H]DPPC
(2-palmitoyl-9,10-3H)(40-50 cpm/ng apoA-I) to the bulk
lipids prior to preparation of the complexes. For studies involving the
selective uptake of cholesterol ester, the desired amount of CE as well
as [1
,2
(n)3H]cholesteryl oleoyl ether (40-50
cpm/ng apoA-I) were added to the lipids prior to preparation of the
complexes. The apolipoprotein component of these particles was labeled
with sodium [125I]iodide to a specific activity of less
than 100 cpm/ng protein.
Ligand Binding and Uptake Assays--
Human SR-BI cDNA was
amplified by PCR and cloned into the expression vector pCMV5 (39). CHO
cells (clone ldlA7) stably transfected with human SR-BI (CHO-SRBI) were
produced and maintained as described previously (33). Ligand binding to
CHO-SRBI cells was carried out in 12-well plates essentially as
described by Acton et al. (23, 40). Cell association at
37 °C was performed in Ham'-s F12 medium containing 100 units/ml
penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 0.5%
fatty acid-free BSA, and HDL labeled with 125I- or
[3H]DPPC. Selective uptake studies were performed with
HDL double-labeled with 125I- and
[3H]cholesteryl oleoyl ether. For binding at 4 °C,
cells were preincubated in the above medium at 37 °C for 1 h,
washed twice with ice-cold 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, containing 0.2% fatty acid-free BSA, and
then incubated at 4 °C for 2 h with the indicated ligand in
Ham's F-12 buffered with 20 mM Hepes, pH 7.4, and
containing 0.5% fatty acid-free BSA. After incubation for the required
time, cells were washed four times with 50 mM Tris-HCl, pH
7.4, containing 150 mM NaCl and 0.2% fatty acid-free BSA,
followed by two washes with 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and then dissolved in 0.1 N
NaOH for radioactivity measurement and protein determination. Non-iodide trichloroacetic acid-soluble degradation products were measured in the culture medium, and in all cases were <15% of the
cell-associated material. SR-BI-specific values were calculated as the
difference between the values obtained in CHO-SRBI cells and
untransfected control CHO cells. Values in untransfected cells were in
all cases less than 20% of the values in CHO-SRBI cells. The selective
uptake of ligand was determined by subtracting the amount of bound
ligand (calculated from the 125I cell-associated
radioactivity) from the total amount of cell-associated ligand
(calculated from the cell-associated 3H label). Apparent
Kd and Bmax values for
binding were determined by non-linear regression analysis of the
SR-BI-specific cell-associated values using Prism software.
 |
RESULTS |
To investigate the influence of apoA-II on the binding of
HDL to SR-BI, rHDL particles containing defined apolipoprotein
compositions were prepared. Each of these particles, in the form of
discs, were >95% homogeneous in size (~100 Å in diameter) and
contained either two molecules of apoA-I (AI-rHDL), four
molecules of apoA-II (AII-rHDL), or two molecules of apoA-I plus
one molecule of dimeric apoA-II (AI/AII-rHDL). Each preparation
contained less than 5% lipid-free apolipoprotein. To assess rHDL
binding to SR-BI, DPPC-containing particles were first iodinated with
125I and then incubated with CHO cells transfected with
human SR-BI (CHO-SRBI). Cell association at 37 °C of rHDL to
CHO-SRBI cells reached a maximum within 30 min (data not shown). Marked
differences in the association of the three types of particles were
observed (Fig. 1). In these experiments,
ligands were added on the basis of equivalent particle numbers
(expressed as A-I equivalents). Cell association is expressed in terms
of apoA-I binding and, in the case of AI/AII- and AII-rHDL, in terms of
apoA-I equivalents. This was calculated from the known apolipoprotein
content of each of the rHDL preparations. SR-BI-specific values are
shown and represent the difference between the values obtained in
CHO-SRBI cells and untransfected control CHO cells. SR-BI mediates the selective uptake of CE from HDL without uptake and subsequent degradation of HDL apolipoproteins (23). In line with this, relatively
low degradation of HDL protein was observed in all experiments
(degradation <15% of cell-associated protein). For this reason,
cell-associated values were taken to represent cell surface binding.
The binding of AI-rHDL was found to be significantly higher than that
of the apoA-II-containing particles at all concentrations tested. At a
ligand concentration of 10 µg/ml, binding of AI-rHDL was ~3- and
12-fold greater than the binding of AI/AII-rHDL and AII-rHDL,
respectively. The affinity of binding of AI-rHDL was greater than that
of AI/AII-rHDL or AII-rHDL (apparent Kd = 3.2 ± 0.9 µg/ml, 5.6 ± 0.8 µg/ml and 12.5 ± 5.1 µg/ml,
respectively). The maximum binding values for the three ligands also
varied (Bmax = 2.3 ± 0.2, 0.96 ± 0.05 and 0.33 ± 0.07 µg/mg cell protein) for AI-rHDL,
AI/AII-rHDL, and AII-rHDL, respectively). The affinity of binding of
these particles was relatively high compared with that of HDL that has
been reported to bind SR-BI with a Kd value between
15 and 30 µg/ml (23, 33, 41).

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Fig. 1.
Concentration-dependent binding
of DPPC-containing rHDL to SR-BI. CHO cells transfected with human
SR-BI (CHO-SRBI) were incubated for 1 h at 37 °C with
increasing concentrations of 125I-labeled DPPC-containing
AI-rHDL, AII-rHDL, and AI/AII-rHDL. Equivalent moles of each rHDL were
added at each ligand concentration. Cell-associated rHDL was quantified
as described under "Experimental Procedures." SR-BI-specific cell
association is shown, which was calculated as the difference between
values for CHO-SRBI cells and non-transfected CHO cells. Values are the
mean of duplicate determinations. Similar results were obtained in
three separate experiments using two batches of ligand.
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As an alternative type of ligand, rHDL were prepared using POPC
in place of DPPC. These phospholipids exhibit a lower phase transition
more typical of naturally occurring phospholipids in HDL.
POPC-containing rHDL particles were prepared by the sodium cholate
dialysis method and fractionated by gel filtration chromatography to a
homogeneous 96-Å-sized population before use. Fig.
2 shows the cell association of
POPC-containing particles labeled with the phospholipid
[3H]DPPC as an alternative approach to 125I
labeling. SR-BI-specific association of AI-rHDL was greater than that
of the two other ligands. The affinity of binding of POPC-containing
AI-rHDL was greater than the affinities of POPC-containing AI/AII-rHDL
and AII-rHDL (apparent Kd = 1.2 ± 0.1, 2.6 ± 0.6, and 2.8 ± 0.3 µg/ml, respectively).
Bmax values for AI-rHDL, AI/AII-rHDL, and
AII-rHDL were 0.83 ± 0.02, 0.56 ± 0.04, and 0.78 ± 0.02 µg/ml, respectively. At a ligand concentration of 1 µg/ml, AI-rHDL binding was 1.8- and 3-fold greater than the binding of AII-rHDL and AI/AII-rHDL, respectively. It is possible that the values
obtained in this experiment (Fig. 2) may in part represent cellular
uptake of the phospholipid label, in addition to cell-surface binding,
since SR-BI has been reported to exhibit phospholipid uptake activity
(42). The three POPC-containing rHDLs were also studied using particles
labeled by 125I iodination. Relative binding of the
three 125I-labeled rHDLs at 37 °C was similar to that
obtained using [3H]DPPC as the label (Fig.
3). In the case of AI-rHDL and
AI/AII-rHDL, binding levels were similar at 4 and 37 °C (Fig. 3),
consistent with the conclusion that 125I-ligand association
reflected binding of particles at the cell surface. The results (Figs.
1-3) indicate that apoA-II serves as a high -affinity ligand for SR-BI
but binds somewhat less effectively than apoA-I. This finding is in
agreement with previous reports by Xu et al. (30) and Pilon
et al. (43). However, in those studies the rHDL preparations
were heterogeneous in size, and their apolipoprotein content, in terms
of number of apolipoprotein molecules per particle, was not defined.
The level of binding at 37 °C of 125I-labeled
AII-rHDL, relative to the other rHDLs, was greater in POPC-containing
particles than in DPPC-containing particles (Fig. 1). Incubation at
4 °C (Fig. 3) resulted in relative levels of binding of the three
ligands that were similar to those observed with DPPC-containing
particles (Fig. 1), with AII-rHDL exhibiting the lowest binding of the
three rHDL types. These results indicate that the relative abilities of
the rHDLs to bind SR-BI can be modulated by the phospholipid
composition of the particle and temperature. For particles containing
both apoA-I and apoA-II, the presence of apoA-II consistently exerted
an inhibitory effect on SR-BI association.

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Fig. 2.
Concentration-dependent binding
of POPC-containing rHDL to SR-BI. CHO-SRBI cells were incubated
for 30 min at 37 °C with increasing concentrations of
POPC-containing AI-rHDL, AII-rHDL, and AI/AII-rHDL. POPC-rHDL particles
were labeled with trace amounts of [3H]DPPC. Equivalent
moles of each rHDL were added at each ligand concentration.
Cell-associated ligand was quantified as described under
"Experimental Procedures." SR-BI-specific cell association is shown
which was calculated as the difference between values for CHO-SRBI
cells and non-transfected CHO cells. Values are the average of
duplicate determinations. Similar results were obtained in three
additional experiments using two preparations of ligand.
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Fig. 3.
Comparative binding of POPC-containing rHDL
to SR-BI at 37 and 4 °C. CHO-SRBI cells
were incubated for 1 h at 37 °C or for 2 h at 4 °C with
POPC-containing 125I-labeled rHDL. Equivalent moles of each
rHDL were added (5 µg/ml apoA-I equivalents).
Cell-associated label was quantified as described
under "Experimental Procedures." SR-BI-specific cell association is
shown that was calculated as the difference between values for CHO-SRBI
cells and non-transfected CHO cells. Values are the means (±S.D.) of
triplicate determinations. Similar results were obtained in an
additional experiment.
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SR-BI binding of the different rHDL preparations was also assessed by
competitive ligand binding using 125I-HDL3 as
the labeled ligand and POPC-containing rHDL preparations as competitors
(Fig. 4). AI-rHDL was the most effective
competitor against HDL3 binding, whereas AII-rHDL and
AI/AII-rHDL competed significantly less effectively for SR-BI binding.
Unlabeled HDL3 inhibited binding the least effectively, in
line with its lower affinity for SR-BI compared with rHDL. These
results are therefore consistent with the direct binding studies.

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Fig. 4.
Inhibition of HDL3 binding to
SR-BI by POPC-containing rHDL. CHO-SRBI cells were incubated for
1 h at 37 °C with 5 µg/ml 125I-labeled
HDL3 and the indicated concentrations of POPC-containing
AI-rHDL, AII-rHDL, and AI/AII-rHDL. Equivalent moles of each rHDL were
added at each ligand concentration. The cell-associated label was
quantified as described under "Experimental Procedures."
SR-BI-specific cell association is shown which was calculated as the
difference between values for CHO-SRBI cells and non-transfected CHO
cells. Values are the average of duplicate determinations.
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One possible explanation for the differences in binding of the three
rHDL types is that their rates of dissociation from SR-BI during the
washing of cells at 4 °C, following incubation with the ligand, vary
significantly. Rates of ligand dissociation at 4 °C were measured in
Fig. 5. POPC-containing ligands were
first bound at 4 °C to CHO-SRBI cells, rapidly washed free of
unbound ligand within 5 min, and then incubated for increasing periods before determining the cell-associated label. During the subsequent 4 °C incubation of the washed cells, there was a significant but relatively slow dissociation of each of the bound ligands (2-12% dissociation of cell-bound ligand/h). However, the relative levels of
binding of the three ligands remained similar throughout the incubation
period, indicating that the observed binding differences between the
ligands do not result from differences in the rates of ligand
dissociation during the washing step that follows incubation with
ligand.

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Fig. 5.
Dissociation of POPC-containing rHDL from
SR-BI. CHO-SRBI cells were incubated for 1.5 h at 4 °C
with POPC-containing 3H-labeled AI-rHDL, AII-rHDL, and
AI/AII-rHDL. Equivalent moles of each rHDL were added (1 µg/ml apoA-I
equivalents). After rapid washing at 4 °C, cells were chased for up
to 2 h at 4 °C, and cell-associated label was then quantified
as described under "Experimental Procedures". SR-BI-specific cell
association is shown that was calculated as the difference between
values for CHO-SRBI cells and non-transfected CHO cells. Values are the
average of duplicate determinations.
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We next assessed the influence of apoA-II on the ability of
apoA-I-containing particles to deliver CE to cells by SR-BI. AI-rHDL and AI/AII-rHDL were compared as ligands for SR-BI-mediated CE uptake
using rHDL ligands that were double-labeled with 125I- and
[3H]CE. These particles contained 3 mol % of CE. Fig.
6 shows the binding of and selective CE
uptake from double-labeled DPPC-containing particles (1 µg/ml) as a
function of time. In these experiments, selective CE uptake is
calculated from the amount of cell-associated [3H]CE that
was not accounted for by whole particle binding (quantified from cell
association of 125I-rHDL protein). Selective CE uptake is
expressed in terms of apparent rHDL protein uptake (apoA-I equivalents)
assuming uptake occurred only through the uptake of whole particles
(44). As expected, SR-BI-specific association of 125I-label
was significantly greater for AI-rHDL than for AI/AII-rHDL. For both
ligands, total [3H]CE uptake over the 2-h incubation
exceeded the corresponding amount of 125I cell association.
Thus, uptake of [3H]CE from both types of rHDL occurred
in a selective manner. Whereas binding was 2-fold greater for AI-rHDL
than AI/AII-rHDL at 2 h, selective uptake of CE from AI-rHDL was
only 40% higher. Thus, the efficiency of selective uptake from bound
particles was greater in the case of AI/AII-rHDL. The efficiency of
selective uptake can be defined as selective uptake relative to the
amount of surface-bound ligand (calculated from 125I cell
association). In five experiments, the efficiency of selective uptake
from AI/AII-rHDL was ~2-fold higher than from AI-rHDL.

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Fig. 6.
Time-dependent association of
DPPC-containing rHDL with SR-BI. CHO-SRBI cells were incubated
for the indicated times at 37 °C with DPPC-containing
125I- and [3H]CE-labeled AI-rHDL and
AI/AII-rHDL. Equivalent moles of each rHDL (1 µg/ml apoA-I
equivalents) were added. The cell-associated 125I label
(open symbols, and ) was quantified as described
under "Experimental Procedures." The selective uptake of ligand
(solid symbols, and , expressed as nanograms of
apoA-I equivalents) was determined by subtracting the
nanograms of bound ligand (calculated from the 125I
cell-associated radioactivity) from the total nanograms of
cell-associated ligand (calculated from the cell-associated
3H label). Shown is SR-BI-specific cell association that
was calculated as the difference between values for CHO-SRBI cells and
non-transfected CHO cells. Values represent the mean of duplicate
determinations. Similar results were obtained in four separate
experiments.
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SR-BI-specific selective uptake from AI-rHDL and AI/AII rHDL
reconstituted with POPC was also compared (Figs.
7 and 8). 125I-rHDL
association reached a maximum value within 30 min and then remained
nearly constant for the remainder of the incubation period (Fig. 7). As
in the case of DPPC particles, selective uptake of [3H]CE
was observed for both ligands at rates that remained nearly constant
during the 2-h incubation period. Unexpectedly and in contrast to the
relative binding of the two rHDLs, selective uptake was significantly
and consistently higher (~1.5-fold) for AI/AII-rHDL than for AI-rHDL,
despite lower binding in the case of AI/AII-rHDL. As for
DPPC-containing rHDLs, this indicated a greater efficiency of selective
uptake for AI/AII-rHDL than for AI-rHDL. A difference in selective
uptake efficiency from the two rHDL preparations was observed for a
range of ligand concentrations (Fig. 8).
In five separate experiments the efficiency of selective uptake from AI/AII-rHDL was ~4-5-fold greater than from AI-rHDL.

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Fig. 7.
Time-dependent association of
POPC-containing rHDL with SR-BI. CHO-SRBI cells were incubated for
the indicated times at 37 °C with 125I- and
[3H]CE-labeled AI-rHDL and AI/AII-rHDL. Equivalent moles
of each rHDL (20 µg/ml apoA-I equivalents) were added. The
cell-associated 125I label (open symbols, and ) was quantified as described under "Experimental
Procedures." The selective uptake of ligand (solid
symbols, and , expressed as nanograms of apoA-I
equivalents) was determined by subtracting the nanograms of bound
ligand (calculated from the 125I cell-associated
radioactivity) from the total nanograms of cell-associated ligand
(calculated from the cell-associated 3H label).
SR-BI-specific cell association is shown that was calculated as the
difference between values for CHO-SRBI cells and non-transfected CHO
cells. Values represent the mean of duplicate determinations. Similar
results were obtained in three separate experiments using two batches
of ligand.
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Fig. 8.
Concentration-dependent
association of POPC-containing rHDL with SR-BI. CHO-SRBI cells
were incubated for 1 h at 37 °C with increasing concentrations
of 125I- and [3H]CE-labeled AI-rHDL, and
AI/AII-rHDL. Equivalent moles of each rHDL were added at each ligand
concentration. The cell-associated 125I label (open
symbols, and ) was quantified as described under
"Experimental Procedures." The selective uptake of ligand
(solid symbols, and , expressed as nanograms of
apoA-I equivalents) was determined by subtracting the nanograms of
bound ligand (calculated from the 125I cell-associated
radioactivity) from the total nanograms of cell-associated ligand
(calculated from the cell-associated 3H label).
SR-BI-specific cell association is shown that was calculated as the
difference between values for CHO-SR-BI cells and non-transfected CHO
cells. Values represent the mean of duplicate determinations. Similar
results were obtained in five separate experiments using two different
batches of ligand.
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 |
DISCUSSION |
HDL represents a heterogeneous population of particles differing
in size, composition, and apolipoprotein content. The relative abilities of these different types of HDL to function as ligands for
SR-BI binding and selective lipid uptake are poorly understood. In this
study we have investigated the influence of apoA-II on HDL binding and
selective uptake by SR-BI. The approach was to use reconstituted HDL
particles as model ligands whose structure and composition could be
more specifically controlled than would be the case for naturally
occurring HDL. The major findings of the study are that apo A-II
functions as a high affinity ligand for SR-BI but that its presence on
apoA-I-containing particles reduces binding to SR-BI. The ability of
apoA-II-containing particles to deliver CE via selective uptake is,
however, increased.
A number of apolipoproteins, apoA-I (30, 41, 45), apoA-II (30, 43), and
apoC-III (30), have been shown to bind with high affinity to SR-BI.
Some evidence has also implicated apoE in HDL binding to SR-BI (46). A
model class A
-helix was shown to bind SR-BI with high affinity,
suggesting that multiple amphipathic
-helical sites on apoA-I or
other apolipoproteins may mediate binding to SR-BI (45). We and others
(41, 47) have previously found that HDL2 binds with greater
affinity than HDL3, and we also showed greater selective CE
uptake from HDL2 than from HDL3 (47). These
observations suggest that size may exert a significant influence on
SR-BI binding, with larger cholesterol ester-enriched HDL being a
preferred ligand for SR-BI. This conclusion was supported by the
finding that larger reconstituted particles (96 Å diameter) bound with
50-fold greater affinity than smaller 78-Å particles (47). These
studies provide strong evidence that binding of apoA-I by SR-BI is
markedly influenced by changes in particle size and more specifically
by changes in apoA-I conformation, most likely in the central region of
apoA-I.
Human HDL consists of LpAI and LpAI/AII particles with few, if any,
particles containing apoA-II only. The influence of apoA-II was
therefore investigated using AI/AII-rHDL that contained two molecules
of apoA-I and one molecule of dimeric apoA-II. Interestingly, the
addition of apoA-II to AI-rHDL significantly reduced binding to SR-BI.
This was observed when binding was carried out both at 4 and at
37 °C. A possible explanation is that apoA-II on rHDL may compete
with the apoA-I molecules for SR-BI binding. However, the binding of
POPC-containing AI/AII-rHDL is significantly lower than even that of
AII-rHDL. Alternatively, apoA-II may sterically interfere with apoA-I
binding. More likely is that the presence of apoA-II on rHDL results in
a conformational change in apoA-I that reduces its affinity for SR-BI.
As we have shown, the ability of apoA-I to bind SR-BI can be markedly
altered by changes in rHDL size, which probably changes apoA-I
conformation (47). The addition of apoA-II to discoidal AI-rHDL
containing DPPC has, in fact, been shown to alter the structure and
stability of a lipid-bound apoA-I molecule (32). In addition to having
different binding affinities for SR-BI, the three rHDL ligands also
showed differences in maximal binding (Bmax) to
cells, which contributed to the overall differences in ligand binding.
Thus AI-rHDL bound with higher affinity than AI/AII-rHDL and AII-rHDL
and also showed a greater Bmax value. Such
differences might reflect differences in the stoichiometry of
SR-BI/ligand binding. The presence of SR-BI dimers has been suggested
by cross-linking studies (45), but the stoichiometry of ligand binding
by SR-BI remains to be determined. Another possible explanation for the
Bmax variation is that SR-BI exists in different
functional forms, as proposed for the asialoglycoprotein receptor (48),
which differ in their ability to bind different ligands. It is also
possible that ligands may induce alterations in the cellular
distribution of SR-BI, although this is unlikely to account for the
observed differences between ligands in our studies, since similar
differences were found when binding was carried out at 4 °C.
Unexpectedly, selective uptake from AI/AII-rHDL was greater than from
AI-only particles, even though binding of AI/AII-rHDL was less than
AI-rHDL. Heterogeneity of particles in the ligand preparation could, in
principle, result in selective uptake that does not appear to correlate
directly with binding if there is preferential binding of a
subpopulation of particles that differ in protein and/or CE content
from that of the starting ligand population. This is unlikely in our
experiments because the rHDLs used were quite homogeneous in size as
well as in apolipoprotein content. Another factor that could influence
particle binding is the possible remodeling of surface-bound particles.
Although the cell-bound apolipoproteins appear to be associated with
lipoprotein particles, it is possible that their protein component
undergoes alterations, for example, losing certain apolipoproteins.
Such remodeling may differ between AI-rHDL and AI/AII-rHDL. However, this does not appear to explain the difference in binding between these
two ligands since similar differences were observed in binding studies
at 4 °C and also when binding was measured using particles labeled
in the phospholipid rather than the protein moiety.
Selective uptake efficiency, calculated as the rate of selective uptake
per receptor-bound particle, is considerably greater for AI/AII-rHDL
than for AI-rHDL. Thus, the apolipoprotein content of the particle
influences both the binding of ligand to SR-BI, as well as the ability
of receptor-bound ligand to donate cholesterol ester to cells via
SR-BI. Differences in selective uptake efficiency from the two types
might be due to differences in particle structure and composition which
in turn could influence the ease with which CE is extracted from
particles. The composition of HDL has been shown to affect selective CE
uptake in the case of triglyceride-rich HDL that acts as a less
efficient ligand than normal HDL (49). We have previously shown (50)
that HDL subjected to hydrolysis by secretory phospholipase
A2 acts as a more efficient SR-BI ligand than normal HDL.
An alternative explanation may relate to possible differences in SR-BI
binding stoichiometry between the two rHDL types. As discussed, the
binding of an AI/AII-rHDL particle might involve more than one receptor
molecule. This could, in turn, result in a greater efficiency of CE
uptake from that particle than from an AI-rHDL particle that may be
bound by only one receptor.
The influence of apoA-II on selective CE uptake has been
addressed in recent reports. Rinninger et al. (14) reported
that LpAI/AII was associated to a lesser extent to HepG2 cells and fibroblasts and promoted less selective uptake than LpAI. Pilon et al. (43) compared, in an adrenal cell line, selective
uptake from HDL and HDL that were enriched by the addition of purified apoA-II in vitro. ApoA-II enrichment of HDL was found to
actually increase cell association of HDL but, in an inverse manner, to decrease selective uptake. The positive correlation of apoA-II content
with cell association contrasts with our finding of reduced association, as does the decreased selective CE uptake from particles containing apoA-II (43). The explanation for this is not clear. Comparisons are difficult given the differences in the cell systems and
ligands in the three studies. In contrast to our studies, the studies
in HepG2 and adrenal cells did not determine SR-BI-specific events, and
other cellular HDL-binding sites and selective uptake processes may
contribute to the observed activities. The studies that show apoA-II
increases the association of HDL with cells were based primarily on
competition studies in which the different competing ligands were added
in excess (43). An alternative explanation may be that the addition of
highly apoA-II-enriched HDLs as competitive ligands leads to apoA-II
enrichment of the 125I-labeled ligand and thereby reduces
its binding in the same manner we have demonstrated with
AI/AII-rHDL. The comparison of naturally occurring HDL fractions is
also complicated by known heterogeneity of HDL fractions studied with
respect to composition and size. For example, the presence of apoE on
HDL appears to influence the ability of HDL to function in selective
uptake (46), and the concentration of this apolipoprotein likely
differed in the various fractions studied (43).
Our results demonstrate that the ability of apolipoproteins associated
with lipoprotein particles to bind to SR-BI is markedly influenced by
the apolipoprotein composition of the particle. ApoA-II appears to
exert independent effects on the processes of SR-BI binding and
selective lipid uptake. Whereas apoA-II has a marked inhibitory effect
on AI/AII-rHDL association with SR-BI, it facilitates the efficiency of
the uptake step whereby cholesterol ester is selectively transferred to
the cell. Given that CE uptake from the POPC rHDLs is actually
increased when apoA-II is present, apoA-II may be predicted to have a
positive effect on reverse cholesterol transport. This, in turn, could
contribute to an anti-atherogenic effect for apoA-II, as shown in at
least one study in a mouse model (11). Reported discrepancies in the
effects of apoA-II on atherogenesis and on selective lipid uptake may
relate to its independent effects on ligand binding and selective CE
uptake efficiency and the possibility that such effects may vary in
different types of HDL.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL-59376 and HL-63763 (to D. R. vd W.) and HL-16059 (to
A. J.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Internal
Medicine, University of Kentucky Medical Center MN520, 800 Rose St.,
Lexington, KY 40536. Tel: 859-233-4511 (Ext. 4580); Fax: 859-323-5707;
E-mail: dvwest1@pop.uky.edu.
Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M100228200
 |
ABBREVIATIONS |
The abbreviations used are:
HDL, high density
lipoprotein;
SR-BI, scavenger receptor class B, type I;
CHO, Chinese
hamster ovary;
BSA, bovine serum albumin;
rHDL, reconstituted HDL;
DPPC, dipalmitoylphosphatidyl choline;
POPC, L-
-palmitoyloleolyl-phosphatidylcholine;
CE, cholesterol
ester.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.