Apolipoprotein A-II Modulates the Binding and Selective Lipid Uptake of Reconstituted High Density Lipoprotein by Scavenger Receptor BI*

Maria C. de BeerDagger , Diane M. Durbin§, Lei CaiDagger , Nichole Mirocha§, Ana Jonas§, Nancy R. WebbDagger , Frederick C. de BeerDagger , and Deneys R. van der WesthuyzenDagger

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-alpha -Palmitoyloleoylphosphatidylcholine (POPC), L-alpha -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 [1alpha , 2alpha (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 [1alpha ,2alpha (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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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.

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.

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.

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.

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 open circle ) was quantified as described under "Experimental Procedures." The selective uptake of ligand (solid symbols, black-square 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.

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 open circle ) was quantified as described under "Experimental Procedures." The selective uptake of ligand (solid symbols, black-square 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 open circle ) was quantified as described under "Experimental Procedures." The selective uptake of ligand (solid symbols, black-square 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -helix was shown to bind SR-BI with high affinity, suggesting that multiple amphipathic alpha -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-alpha -palmitoyloleolyl-phosphatidylcholine; CE, cholesterol ester.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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