©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of LpA-I Composition and Structure on Cholesterol Transfer between Lipoproteins (*)

(Received for publication, May 20, 1994; and in revised form, December 12, 1994)

Qiang-Hua Meng Daniel L. Sparks Yves L. Marcel

From the Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Institute and the Departments of Pathology and Laboratory Medicine and of Biochemistry, University of Ottawa, Ottawa, Ontario, K1Y 4E9, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The effect of high density lipoprotein composition on the rates of unesterified cholesterol exchange between low density lipoproteins (LDL) and well-defined homogeneous discoidal lipoproteins (LpA-I) reconstituted with phosphatidylcholine, cholesterol, and apolipoprotein A-I (apoA-I) has been investigated. LpA-I containing cholesterol and 2, 3, and 4 apoA-I molecules per particle differed in their ability to accept or donate cholesterol. A significant cholesterol exchange occurs between LDL and Lp2A-I (7.8 and 9.6 nm), while there is little or no cholesterol exchange detectable between LDL and Lp3A-I (10.8 and 13.4 nm) and Lp4A-I (17.0 nm) complexes. The cholesterol transfer from LDL to the cholesterol-free Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm) particles also shows significant cholesterol transfer to Lp2A-I, while there is no detectable transfer to Lp3- and 4A-I particles. The rates of cholesterol transfer to cholesterol-free and cholesterol-containing Lp2A-I appear to differ significantly. Cholesterol transfer from LDL to cholesterol-free Lp2A-I is zero order with respect to acceptor concentrations when the Lp2A-I/LDL ratio is above 10. Transfer rates from LDL to cholesterol-free Lp2A-I are faster for the smaller Lp2A-I (8.5 nm) than to the larger Lp2A-I (9.7 nm) and exhibit half-times (t) at 25 °C of 4.0 and 5.3 h, respectively. In contrast, cholesterol transfer from LDL to cholesterol-containing Lp2A-I remains dependent upon acceptor concentrations to an acceptor/donor particle ratio of 80. In addition, transfer from LDL to cholesterol-containing Lp2A-I is faster to the 9.6 nm than to 7.8 nm particles, with t of 1.4 and 2.3 h, respectively. The rates of cholesterol transfer from Lp2A-I to LDL are higher than in the opposite direction, in particular for the small Lp2A-I (7.8 nm), which has a t of approximately 50 min. The results show that changes in the composition and structure of apoA-I-containing particles have a significant effect on inter-lipoprotein exchange of cholesterol. This suggests that the kinetics of cholesterol transfer to and from reconstituted discoidal LpA-I particles cannot be fully explained by passive aqueous diffusion.


INTRODUCTION

HDL (^1)is involved in the reverse transport of cholesterol from peripheral tissues to liver, and apoA-I containing lipoproteins have been shown to be preferred acceptors of cellular cholesterol efflux. Cholesterol present in different HDL subfractions originates from the de novo secretion of lipoproteins as well as from transfer from cell membranes and from apoB-containing lipoproteins (LDL)(1, 2, 3, 4, 5) . Recent studies by Fielding and colleagues indicate that cellular cholesterol may first transfer to a small pre-beta(1)-HDL and then be transferred to larger pre-beta(2)- and pre-beta(3)-HDL, where the esterification of cholesterol occurs(6, 7) . The same group has also reported that LDL-cholesterol is transferred to and esterified in alpha-HDL(3)(8, 9) . A recent study by another group showed that only a fraction of the cellular cholesterol transferred to the pre-beta-HDL pathway is esterified while the majority recycles through alpha-HDL, LDL, and pre-beta-HDL(10) .

Cholesterol exchange between lipoproteins and cells is an important mechanism involved in cholesterol redistribution, transport, and metabolism in vivo (reviewed in (11) ). A bidirectional flux of cholesterol molecules occurs between HDL and LDL particles when they are incubated in vitro(12) . Studies done by Phillips and co-workers (13, 14, 15) have shown that the physical state of cholesterol molecules in the phospholipid/water interface of serum lipoprotein particles differs on LDL, HDL, and small unilamellar vesicles (SUV) in a manner that parallels differences in cholesterol flux between these particles(16) . In contrast, the kinetics of cholesterol exchange between HDL and LDL (12) and between SUV (16) are similar and appear to be consistent with a passive aqueous diffusion mechanism(15, 16, 17, 18) . In addition, the presence of apolipoproteins A-I, A-II, and B-100 in lipid vesicles enhances the rate of cholesterol exchange from SUV(19) . These observations indicate that cholesterol transfer between two lipid-containing structures is affected by the presence of lipophilic surface proteins. In the case of lipoproteins, interfacial surfaces reflect both the intrinsic structure of the apolipoproteins and that of the amphipathic surface lipids. Therefore, the transfer of cellular cholesterol or LDL cholesterol to specific plasma lipoprotein subclasses that contain apoA-I appears to be guided by the molecular properties of these particles.

We have previously reported that cholesterol transfer from cells to discoidal LpA-I and from discoidal LpA-I to other plasma lipoproteins varies with particle size and number of apoA-I molecules(20) . To understand the factors that control transfer of cholesterol to apoA-I-containing lipoproteins, we have systematically measured cholesterol exchange between LDL and several well defined, reconstituted discoidal LpA-I particles. The results show that a significant time- and acceptor concentration-dependent cholesterol exchange occurs between LDL and several different Lp2A-I discoidal particles, while there is little or no cholesterol transfer occurring between LDL and Lp3A-I and Lp4A-I particles.


EXPERIMENTAL PROCEDURES

Preparation of Plasma LDL, ApoA-I, and LpA-I Particles

Plasma LDL and apoA-I were prepared from pooled plasma from normolipidemic volunteers by sequential flotation ultracentrifugation as described(21) . The LDL fraction (KBr density = 1.020-1.063 g/ml) of plasma was dialyzed against TBS-EDTA buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 1 mM NaN(3) and stored at 4 °C under N(2) for up to 2 weeks.

Pure apoA-I was prepared as described earlier(22) . Reconstituted discoidal LpA-I particles containing cholesterol (LpA-I(C)) were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) (Sigma), cholesterol (Sigma), and apoA-I at an initial molar ratio of 120:6:1 or 88:44:1 by cholate dialysis following previously published procedures(23, 24, 25) . Homogeneous lipoprotein containing 2, 3, or 4 apoA-I molecules per particle (Lp2-, Lp3-, or Lp4A-I(C)) were purified by gel filtration on two serial agarose Bio-Gel 5M columns (95 times 2.5 cm) (Bio-Rad Laboratories) equilibrated with TBS-EDTA buffer. The cholesterol-containing Lp2A-I(C) (9.6 nm), Lp3A-I(C) (13.4 nm), and Lp4A-I(C) (17.0 nm) particles were purified from the lipoprotein population obtained with the initial molar ratio of 120:6:1 and Lp2A-I(C) (7.8 nm) and Lp3A-I(C)(10.8 nm) particles were purified from the lipoprotein population obtained with the initial molar ratio of 88:44:1. The chemical compositions of these particles were similar to those reported in previous studies(25) . Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm) without cholesterol were prepared at an initial POPC/apoA-I molar ratio of 120:1 as described earlier(20) . Lp2A-I with different POPC/apoA-I molar ratios without cholesterol were prepared by a cholate dispersion method of Sparks et al.(26) . The homogeneous particles produced by this method were centrifuged at KBr density (1.063 g/ml) to remove free lipids. Size characterization of LpA-I particles was as described(27) . The number of apoA-I molecules per LpA-I particle was determined by chemical cross-linking with dimethyl suberimidate(28) . The concentration of protein was measured by Lowry assay (29) and cholesterol and POPC by enzymatic kits (Boehringer Mannheim GmbH, Mannheim, Germany).

Cholesterol Transfer Assay between LDL and LpA-I

To measure cholesterol transfer from LDL to LpA-I, LDL was prelabeled with [^3H]cholesterol. Briefly, 10 µCi of [^3H]cholesterol solution in toluene was dried to completion under N(2). LDL (100 mg of protein) was diluted to 500 µl in 1% BSA, added into the [^3H]cholesterol tube, and incubated at 37 °C for 30 min. The incubation mixture was dialyzed against TBS containing 20 mM Tris, pH 7.7, 150 mM NaCl. All other components for this assay were also dialyzed against the same buffer at 4 °C overnight immediately prior to the experiment. Each assay mixture contained 60 µl of 2% defatted BSA, 1 µg of [^3H]cholesterol-labeled LDL in 100 µl of 1% BSA, different amounts of LpA-I particles diluted in 100 µl of 1% BSA, and TBS was added to a final volume of 460 µl. The mixtures were incubated at room temperature for different periods of time. The transfer reactions were stopped in an ice-bath. A heparin-Mn precipitation procedure was used to separate LDL and LpA-I (12, 16, 30) and was shown to precipitate more than 99% of LDL from the supernatant. Briefly, 25 µg of carrier LDL in 50 µl of TBS, 100 µl of 0.14% heparin, and 25 µl of 1 M MnCl(2) were added into each tube. The tubes were vortexed, kept on ice for 10 min, and then centrifuged in a Microfuge 5415C at 15,000 rpm for 10 min. The LDL precipitates and the supernatants containing LpA-I were subsequently counted in a scintillation counter.

The determination of cholesterol transfer from LpA-I particles to LDL was essentially the same as above, except that LpA-I rather than LDL was labeled with [^3H]cholesterol using the same procedure. The amount of LpA-I as cholesterol donor was constant at 1 µg per tube, and the amounts of LDL as cholesterol acceptor were varied.

In the validation of the assay using I-labeled LpA-I, we observed that as reported previously for HDL(3)(12) , 60% of Lp4A-I co-precipitated with LDL. Co-precipitation of other LpA-I was substantially less than that for Lp4A-I: 5% for Lp2A-I (7.8 nm), 33% for Lp2A-I (9.6 nm), 20% for Lp3A-I (10.8 nm), and 47% for Lp3A-I (13.4 nm). These values are constant and reproducible between assays (S.D. < 1%), are not time or acceptor dose-dependent, and were therefore used to correct the rates (x) of cholesterol transfer in all assays. In addition, all transfer rates were also corrected by subtraction of the background values obtained in the absence of acceptor lipoproteins.

In some cholesterol transfer assays, LDL and LpA-I were also separated by KBr gradient ultracentrifugation. At the end of the incubation, the mixtures were adjusted to the final density of 1.063 g/ml and transferred into mini tubes for Beckman TLA-100 rotor with a total volume of 250 µl per tube and ultracentrifuged at 60,000 rpm for 200 min. The contents in these tubes were fractionated every 50 µl from the top to the bottom and counted in a beta-counter to monitor the movement of [^3H]cholesterol. LDL was recovered in the top 50-µl fraction, and LpA-I was recovered in the bottom 100 µl.

The kinetics of cholesterol transfer between LDL and LpA-I were calculated as described by Lund-Katz and colleagues(12, 31) .


RESULTS

Characteristics of Reconstituted LpA-I

The characteristics of reconstituted LpA-I particles used in the present studies are summarized in Table 1. The cholesterol-containing lipoproteins include three large LpA-I particles, Lp2A-I(C) (9.6 nm), Lp3A-I(C) (13.4 nm), and Lp4A-I(C) (17.0 nm), prepared from the initial molar ratio of POPC/cholesterol/A-I at 120:6:1(23) , and two small LpA-I, Lp2A-I(C) (7.8 nm) and Lp3A-I(C) (10.8 nm), prepared from the POPC/cholesterol/A-I molar ratio of 88:44:1(24) . The cholesterol-free lipoproteins include three large Lp2-, Lp3-, and Lp4A-I particles prepared from a POPC/A-I initial molar ratio of 120:1 which showed similar particle sizes and POPC/A-I compositions as their cholesterol-containing counterparts described earlier(20) . Other cholesterol-free lipoproteins are the Lp2A-I (8.5 nm), and Lp2A-I (9.7 nm) without cholesterol were prepared from an initial POPC/A-I molar ratio of 70:1 and 130:1, respectively, following the method of Sparks et al.(26) .



Bidirectional Cholesterol Transfer between LDL and LpA-I Particles Containing Cholesterol

Since the particle ratio of HDL to LDL is approximately 10 in normolipidemic plasma and increases further in lymph and interstitial fluid, all experiments were designed to measure the cholesterol flux from a less than normal physiological ratio to at least a 16-fold over normal concentration of HDL or LDL (i.e. LpA-I(C)/LDL ratios from 0.1 to 160 for assays of cholesterol transfer from LDL to LpA-I(C) and LpA-I(C)/LDL ratios from 200 to 0.6 for assays of cholesterol transfer from LpA-I(C) to LDL). In order to demonstrate that there is no association of apoA-I into LDL during cholesterol transfer assays, apoA-I in Lp2-, Lp3-, and Lp4A-I(C) was iodinated and then incubated with LDL under the normal conditions of the transfer assay. The lipoproteins in the incubation mixtures were then separated by 4-20% native gradient gel electrophoresis, and the repartition of radioactivity was analyzed. No apoA-I radioactivity could be seen in the LDL migration zone after incubation with any of the three LpA-I(C) particles (data not shown).

To measure cholesterol transfer from LDL to LpA-I(C) particles, donor LDL labeled with [^3H]cholesterol were incubated with acceptors Lp2A-I(C) (9.6 nm), Lp3A-I(C) (13.4 nm), and Lp4A-I(C) (17.0 nm) at different LpA-I(C)/LDL particle ratios and incubated for the period up to 90 min. Transfer of cholesterol from LDL to these LpA-I(C) particles is expressed by the rates of [^3H]cholesterol moving into LpA-I(C), and the representative data are shown in Fig. 1A. The cholesterol transfer from LDL to Lp2A-I(C) (9.6 nm) increases with increasing incubation time, and the maximum transfer is observed at 60 min. The rate of cholesterol transfer at 60 min at a ratio of 80 particles of Lp2A-I(C) (9.6 nm) to 1 LDL particle are 23%. Surprisingly, under the same experimental conditions, the cholesterol transfers from LDL to Lp3A-I(C) (13.4 nm) and Lp4A-I(C) (17.0 nm) are not significant and not time- and acceptor concentration-dependent.


Figure 1: Cholesterol exchange between LDL and LpA-I(C). Data presented in this figure are means from three independent assays. Error bars indicate S.D. A, cholesterol transfer from LDL to LpA-I(C). Data represent cholesterol transfer rates from LDL to Lp2A-I(C) (9.6 nm) (box), Lp3A-I(C) (13.4 nm) (), and Lp4A-I(C) (17.0 nm) (Delta) at LpA-I(C)/LDL particle ratios of 80 and as a function of time. B, cholesterol transfer from LpA-I(C) to LDL. Data represent cholesterol transfer rates from Lp2A-I(C) (9.6 nm) (box), Lp3A-I(C) (13.4 nm) (), and Lp4A-I(C) (17.0 nm) (Delta) at a LpA-(C)/LDL particle ratio of 0.6 and as a function of time.



Cholesterol transfer in the opposite direction from LpA-I(C) particles to LDL were determined in similar experiments. LpA-I(C) particles were labeled with [^3H]cholesterol and incubated with acceptor LDL. Significant transfer of cholesterol from Lp2A-I(C) (9.6 nm) to LDL has been observed. The maximum transfers are about 36% at a Lp2A-I(C)/LDL particle ratio of 0.6 at 90 min of incubation. There is no further increase of transfer with the incubation extended to 120 min. Interestingly, cholesterol transfer from Lp3A-I(C) (13.4 nm) and Lp4A-I(C) (17.0 nm) to LDL is not significant and is not acceptor concentration-dependent (Fig. 1B). Therefore, the same differences are observed in the transfers of cholesterol between LDL and Lp2A-I(C) (9.6 nm) and Lp3- and 4A-I(C) in both directions.

Cholesterol transfer from LDL to Lp2A-I(C) (9.6 nm) has been studied in detail by varying LpA-I(C)/LDL particle ratios and as a function of time between 0 and 90 min. The transfer of cholesterol from LDL to Lp2A-I(C) (9.6 nm) is both time- and acceptor (LDL) dose-dependent when the acceptor/donor ratio is above 5 (Fig. 2A). There is no detectable cholesterol transfer when the Lp2A-I(C) (9.6 nm)/LDL particle ratio is below 2.5. No significant cholesterol transfer from LDL to either Lp3A-I(C) (13.4 nm) or Lp4A-I(C) (17.0 nm) has been observed under all of the conditions tested above.


Figure 2: Cholesterol transfer from LDL to cholesterol-containing Lp2A-I(C) (9.6 and 7.8 nm). For each data point, S.D. ranges from 0.1 to 2.9% which were generated from 4 independent assays. A, cholesterol transfer from LDL to Lp2A-I(C) (9.6 nm). Data shown in the figure and the inset are the transfer rates at acceptor/donor particle ratios of 80 (bullet), 40 (), 20 (), and 10 (), respectively. B, cholesterol transfer from LDL to Lp2A-I(C) (7.8 nm). Data shown in the figure and inset are the transfer rates at acceptor/donor particle ratios of 80 (bullet), 40 (), 20 (), and 10 (). All data are representatives of three independent assays using the Lp2A-I(C) particles prepared from initial POPC/cholesterol/AI molar ratio of 120:6:1 and 88:44:1 as described in the text. Insets, linear regression plots of the percentage of [^3H]cholesterol radioactivity which remains in LDL as a function of time are shown. The correlation coefficients (r) are >0.93 in all instances.



Further experiments were done to measure cholesterol transfer between LDL and smaller Lp2- and Lp3A-I(C) particles, that is Lp2A-I(C) (7.8 nm) and Lp3A-I (10.8 nm). The rates of cholesterol transfer from LDL to Lp2A-I(C) (7.8 nm) are lower than those to larger Lp2A-I(C) (9.6 nm), but are significant and time- and acceptor concentration-dependent. The maximum transfer rate of cholesterol from LDL to Lp2A-I(C) (7.8 nm) is approximately 16% at an acceptor/donor ratio of 80 and at 60 min (Fig. 2B). The transfer of cholesterol from LDL to Lp2A-I(C) (7.8 nm) reaches saturation between 90 and 120 min of incubation (data not shown). As seen with Lp3A-I(C) (13.4 nm), there is no detectable cholesterol transfer from LDL to Lp3A-I(C) (10.8 nm) at all conditions tested (data not shown).

The time curves of cholesterol transfer from Lp2A-I(C) (7.8 nm and 9.6 nm) to LDL are shown in Fig. 3. Cholesterol transfer was measured at LpA-I(C)/LDL particle ratios between 0.6 and 200 and at different time intervals up to 90 min. In contrast to the transfer out of LDL, the reverse transfer to LDL is faster from Lp2A-I(C) (7.8 nm) than that from Lp2A-I(C) (9.6 nm) to LDL. There is no detectable cholesterol transfer from Lp2A-I(C) (9.6 nm) to LDL when the Lp2A-I(C)/LDL ratio is above 5 (Fig. 3A). However, the cholesterol flux from Lp2A-I(C) (7.8 nm) to LDL is still significant at an acceptor/donor ratio of 20 and at 60 min of incubation (Fig. 3B).


Figure 3: Cholesterol transfer from cholesterol-containing Lp2A-I(C) (9.6 and 7.8 nm) to LDL. All data are the means from three independent assays where the concentration of donor (LpA-I(C) containing [^3H]cholesterol) was constant and the concentration of acceptor LDL varied (LpA-I(C)/LDL particle ratios of 0.6 to 200). For each data point, S.D. ranges from 0.05 to 3.8%. A, cholesterol transfer from Lp2A-I(C) (9.6 nm) to LDL. Data presented here are cholesterol transfer rates at LpA-I(C)/LDL ratios of 0.6 (), 1.2 (), and 2.4 (). B, cholesterol transfer from Lp2A-I(C) (7.8 nm) to LDL. Data are the cholesterol transfer rates at a LpA-I(C)/LDL particle ratio of 0.6 (), 2.4 (), 5 (), and 20 (). The transfer of cholesterol from LDL to Lp2A-I(C) (10.8 nm) reaches saturation between 60 and 90 min of incubation. Insets, linear regression plots of the percentage of [^3H]cholesterol radioactivity which remains in Lp2A-I(C) as a function of time are shown. The correlation coefficients (r) are >0.96 for Lp2A-I(C) (7.8 nm) and >0.93 for Lp2A-I(C) (9.6 nm).



Effect of LpA-I Lipid Composition on Cholesterol Transfer from LDL to LpA-I

Since the large and small Lp2-, Lp3-, and Lp4A-I(C) particles cited above differ not only in the numbers of apoA-I molecules per particle, but also in POPC and cholesterol contents, we have repeated the cholesterol transfer experiments with cholesterol-free Lp2-, Lp3-, and Lp4A-I particles with sizes similar to their cholesterol-containing counterparts, e.g. Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm) as described in Table 1. The transfer assays were done at LpA-I/LDL particle ratios ranging from 80 to 2.5 with incubation periods of up to 90 min. When cholesterol-free Lp2A-I (9.6 nm) is used as cholesterol acceptor from LDL, a time- and concentration-dependent cholesterol transfer from LDL is observed. However, as seen above with cholesterol-containing Lp3- and 4A-I(C), there is little or no significant cholesterol transfer from LDL to either Lp3A-I (13.4 nm) or Lp4A-I (17.0 nm) containing no cholesterol (data not illustrated). Therefore, the impaired cholesterol transfer observed to Lp3- and Lp4A-I with or without cholesterol is an intrinsic characteristic of these lipoproteins independent of their cholesterol content.

To determine whether the POPC content and size of LpA-I affect their ability to accept cholesterol from LDL, two Lp2A-I particles without cholesterol and with different POPC contents, Lp2A-I (9.7 nm, PC/A-I = 95) and Lp2A-I (8.5 nm, PC/A-I = 67), were used as acceptors for LDL cholesterol. Time- and acceptor concentration-dependent increases in cholesterol transfer from LDL to both of these two Lp2A-I particles were, again, observed (Fig. 4). However, there is some difference between these two acceptors with different POPC contents. Cholesterol transfer from LDL to the larger Lp2A-I (9.7 nm, PC/A-I = 95) is slower and does not reach saturation at most acceptor concentrations tested up to 90 min of incubation (Fig. 4A), whereas transfers to Lp2A-I (8.5 nm, PC/A-I = 67) are faster at most acceptor/donor ratios tested, and the difference is most significant for the shorter time (15 min) (Fig. 4B).


Figure 4: Comparison of cholesterol transfer from LDL to cholesterol-free Lp2A-I (9.7 and 8.5 nm) with different PC/apoAI molar ratios. Homogeneous Lp2A-I particles without cholesterol and with different PC/A-I molar ratios were prepared using a method (26) different from that used (23, 24) for the experiments in Fig. 1to 3. The final preparations of Lp2A-I (9.7 nm) and Lp2A-I (8.5 nm) have POPC/apoA-I molar ratios of 95:1 and 67:1, respectively. Data in this figure are means of three independent assays using LDL and LpA-I particles from different preparations. The S.D. values of each data point range between ±0.01-3.5%. The cholesterol transfer rates are presented at LDL/Lp2A-I particle ratios of 80 (bullet), 40 (), 10 (), and 2.5 (). A, cholesterol transfer from LDL to Lp2A-I (9.7 nm, PC/apoA-I = 95). B, cholesterol transfer from LDL to Lp2A-I (8.5 nm, PC/apoAI = 67). Insets, linear regression plots of the percentage of [^3H]cholesterol radioactivity which remains in LDL as a function of time are shown. The correlation coefficients (r) are >0.96 for Lp2A-I (9.7 nm) and >0.98 for Lp2A-I (8.5 nm).



Measurement of Cholesterol Transfer from LDL to LpA-I by KBr Density Separation of Lipoproteins

To confirm the results obtained by heparin-Mn precipitation, the transfer of [^3H]cholesterol from LDL to the three large Lp2-, Lp3-, and Lp4A-I(C) was measured after separation of LDL from LpA-I(C) by ultracentrifugation at KBr density of 1.063 g/ml. Compared to background (tubes without acceptor LpA-I(C) or apoA-I), cholesterol transfer from LDL to Lp2A-I(C) (9.6 nm) is significantly increased. However, there is no significant difference observed in cholesterol transfer to free apoA-I, Lp3A-I(C), and Lp4A-I(C) (Fig. 5) in comparison to control incubations.


Figure 5: Measurement of cholesterol transfer from LDL to LpA-I(C) separated by ultracentrifugation. Lp2A-I(C) (9.6 nm), Lp3A-I(C) (13.4 nm), and Lp4A-I(C) (17.0 nm) were incubated with [^3H]cholesterol-labeled LDL at an acceptor/donor particle ratio of 40 for 30 min. The incubation mixtures were adjusted to KBr density of 1.063 g/ml, transferred into microtubes, and centrifuged in a Beckman Optima TL ultracentrifuge using a TLK-100 rotor at 4 °C, 60,000 rpm for 200 min. The contents in the tubes were collected in 50-µl fractions from the top to the bottom and counted in a beta-counter. Data in this figure represent means of quadruplicates, and the error bars indicate S.D. values of quadruplicates.



Kinetic Analysis of Cholesterol Transfer between LDL and Lp2A-I

The kinetics of cholesterol transfer from LDL to Lp2A-I(C) were investigated at 25 °C with constant LDL concentrations and variable Lp2A-I(C) concentrations. Consistent with studies at 37 °C(12) , cholesterol exchange at 25 °C is first order with respect to cholesterol concentration in the LDL particles (Fig. 2, insets). The dependence of cholesterol exchange on acceptor concentrations was investigated over Lp2A-I(C) concentrations that varied from 1 to 80 acceptor particles per donor particle. Kinetic parameters for cholesterol transfer from LDL to two cholesterol-free Lp2A-I (8.5 and 9.7 nm) and two cholesterol-containing Lp2A-I(C) (7.8 and 9.6 nm) are summarized in Table 2and Fig. 6. For cholesterol-free Lp2A-I, the cholesterol transfer rates from LDL are strongly acceptor concentration-dependent at low acceptor/donor ratios, while rate constants, k, plateau at acceptor/donor ratios above 10. Initial cholesterol transfer rates are slower and appear to reach a reduced maximum rate of cholesterol exchange for the larger Lp2A-I (9.7 nm) than for the small Lp2A-I (8.5 nm).




Figure 6: Kinetics of cholesterol exchange from LDL to Lp2A-I(C) and Lp2A-I. The rate constants (k) of cholesterol transfer from LDL to different Lp2A-I particles were calculated by k = -(slope)x, where the slopes were obtained by least squares linear regression analysis from ln(1 [[[- x/x) versus t at different acceptor/donor particle ratios as presented in Fig. 2and Fig. 4and described in the text(12) . Data represent k values of cholesterol transfer from LDL to cholesterol containing Lp2A-I(C), 7.8 nm () and 9.6 nm (bullet), and to cholesterol-free Lp2A-I, 8.5 nm (box) and 9.7 nm (circle), at different Lp2A-I/LDL particle ratios. The k values were calculated from 3 to 4 assays and S.D. ranged between ±0.05-0.39 times 10.



The ability of the cholesterol-containing Lp2A-I(C) (9.6 nm) to accept cholesterol from LDL differs substantially from the cholesterol-free Lp2A-I particles ( Fig. 6and Table 2). While initial cholesterol transfer rates for Lp2A-I(C) are substantially less than that shown for the cholesterol-free particles, transfer rates are dependent on acceptor concentrations up to an acceptor/donor ratio of 80 and do not reach saturation for either Lp2A-I(C) (9.6 nm) or Lp2A-I(C) (7.8 nm). This appears to correspond to an almost 4-fold increased rate of cholesterol transfer to the Lp2A-I(C) relative to that for the cholesterol-free Lp2A-I. For a small Lp2A-I(C) (7.8 nm), the k values are 5.0 times 10 for an acceptor/donor ratio of 80 and correspond to t of 140 min. The t values obtained here are similar to those obtained by Lund-Katz and Phillips(12) .

Cholesterol transfer from Lp2A-I(C) (7.8 nm and 9.6 nm) to LDL was also studied at 25 °C with constant Lp2A-I(C) concentrations but with varying acceptor LDL concentrations (Lp2A-I(C)/LDL ratios from 0.6 to 100). The rate of cholesterol transfer to LDL is dependent upon acceptor concentration for both donor Lp2A-I(C)s; however, the kinetics of cholesterol transfer from Lp2A-I(C) (7.8 nm) and Lp2A-I(C) (9.6 nm) to LDL differ significantly. With the small Lp2A-I(C) (7.8 nm) as cholesterol donor particles, rate constants, k, increase sharply at a Lp2A-I(C)/LDL ratio of 2.5 and then plateau. With the larger Lp2A-I(C) (9.6 nm), the rate constants are slower and do not plateau. The rate constants and t are generally constant with the studies of Letizia and Phillips(19) .

Estimation of the interfacial flux of cholesterol from the surface of LDL particles into the aqueous phase varies in incubations with different acceptor particles (Table 2). For acceptor particles devoid of cholesterol, maximum interfacial cholesterol flux ranged from 0.5 to 0.4 mol/10 nm^2 h for the small and large Lp2A-I (8.5 and 9.7 nm), respectively. Interfacial flux values for cholesterol-containing Lp2A-I(C) acceptor particles reach maximum values of 0.9 molecule/10 nm^2/h for small Lp2A-I(C) (7.8 nm) and 2.45 molecules/10 nm^2/h for the larger Lp2A-I(C) (9.6 nm). This sensitivity of interfacial flux to acceptor particle structure may explain why these values are slightly less than that previously shown for exchanges between LDL and native HDL acceptor particles (4 mol/10 nm^2/h)(12) . Interfacial cholesterol flux from Lp2A-I(C) is considerably less than that from LDL and is 0.27 molecule/10 nm^2/h from the smaller Lp2A-I(C) (7.8 nm) and 0.04 mol/10 nm^2/h from the larger Lp2A-I(C) (9.6 nm) (Table 3).




DISCUSSION

This study shows that changes in the composition of various kinds of reconstituted discoidal LpA-I particles has a direct effect on the exchange of cholesterol with LDL particles. Previous studies have also shown that different native (12) or reconstituted LpA-I (19) vary in their ability to allow for the desorption of cholesterol; however, the mechanism that regulates this phenomenon is unclear. Investigations with small unilamellar vesicles (SUV) have shown that increasing the number of apoA-I molecules on their surfaces will increase the rate of cholesterol desorption(19) . This prompted the authors to propose that apoA-I may perturb the interactions between cholesterol and phospholipid and promote a transition state that allows for cholesterol desorption(19) . In the same study, discoidal LpA-I particles exhibited an almost 6-fold increased rate of cholesterol desorption as compared to SUV and also showed a similar relationship between phospholipid:apoA-I ratio and cholesterol desorption. An increased propensity for cholesterol desorption was associated with a reduced phospholipid:apoA-I ratio in LpA-I discoidal complexes. This is consistent with that observed in the present study for Lp2A-I(C) particles: a decrease in phospholipid:apoA-I ratio in Lp2A-I(C) is associated with an increased rate of cholesterol desorption. Similarly, Lp3A-I(C) and Lp4A-I(C) generally exhibit high phospholipid:apoA-I ratios and very low rates of cholesterol transfer from LDL to LpA-I(C). An exception, however, is the cholesterol-containing Lp3A-I(C) (10.8 nm) which does not exhibit a high phospholipid:apoA-I ratio but which is a very poor acceptor of cholesterol. Low rates of cholesterol transfer to this complex may be related to its very high content of cholesterol(34) . A recent study by Jonas et al.(35) is also consistent with a cholesterol transfer that is faster to Lp2A-I(C) (9.6 nm), slower to Lp2A-I(C) (7.8 nm), and slowest to Lp3A-I(C) (10.8 nm) after correction of the background at zero time.

The increased desorption of cholesterol from smaller discoidal particles can result from perturbations in phospholipid-cholesterol interactions that arise from changes in the order of phospholipid acyl chains(36, 37) . A low phospholipid:apoA-I ratio in discoidal Lp2A-I is also associated with a reduction in the content and stability of apoA-I alpha-helices and with an increase in the density of negative surface charge(26) . Such changes in apoA-I conformation may also affect cholesterol desorption. An increased thermodynamic instability that results from a particular apoA-I conformation may affect the association of apoA-I, phospholipid, and cholesterol in boundary regions immediately adjacent to apoA-I molecules(38) .

It is evident that variations in the structural properties of different LpA-I may directly affect the ability of these lipoproteins to receive cholesterol. Changes in the structure of reconstituted LpA-I not only affect the desorption of cholesterol from these particles, but also indirectly affect the adsorption of cholesterol transferred from donor LDL particles. This study shows that the presence of a small amount of cholesterol in Lp2A-I(C) promotes a 2- to 3-fold increase in the rate of cholesterol transfer from LDL compared to cholesterol-free LpA-I. The specificity of cholesterol transfer is also made more complex by the effect of particle size which has inverse effects in cholesterol-free or -containing particles: with cholesterol containing Lp2A-I(C), smaller particles are poorer acceptors of cholesterol than larger ones, while with cholesterol-free Lp2A-I, small particles are better than the larger ones. This differential effect may be a result of unique structures of the different LpA-Is or may be due to a distinct effect of cholesterol on the different particles. Previous studies have shown that addition of cholesterol to a reconstituted discoidal LpA-I(C) directly affects both the conformation and charge of apoA-I and thereby significantly modifies the physical properties of the LpA-I particles(39) . As such, it is possible that cholesterol-containing Lp2A-I(C) may have unique molecular properties that may stimulate cholesterol desorption from LDL by changing the interfacial interactions between LpA-I(C) and LDL. It is of note that both a reduction in phospholipid:apoA-I ratio and an increase in cholesterol content increase the negative surface potential on LpA-I(C)(26, 39) . A change in the surface charge on an LpA-I particle may affect the characteristics of the unstirred water layer and/or the ionic double layer around the particle and may thereby indirectly affect interfacial interactions on the particle surface such as cholesterol exchange. Further, it is possible that changes in specific interactions between apoA-I and cholesterol in an LpA-I(C) particle may also affect cholesterol exchange. Addition of cholesterol to an LpA-I(C) complex causes a significant reduction in the amount of alpha-helical structure in apoA-I, but increases the stability of the remaining helices(39) . Studies (38, 39) have shown that cholesterol may exist in two domains in a LpA-I(C) particle: 1) in close contact with apoA-I and 2) in the bulk phase of phospholipid. It appears that a small amount of cholesterol will preferentially associate with apoA-I and will reduce the helix-phospholipid interactions in the LpA-I(C) particle. This structural change may decrease the hydrophobic solvation of surface phospholipid by apoA-I and may allow for an increased accessibility of the bulk phospholipid to cholesterol. A reduction in phospholipid-apoA-I interactions may therefore increase the ability of the Lp2A-I particles to receive cholesterol from LDL by reducing a competition between apoA-I and cholesterol for the solvation of phospholipid(38, 39) . The increased transfer of LDL cholesterol to Lp2A-I(C) compared with that to cholesterol-free Lp2A-I observed here may be explained by the two cholesterol domain hypothesis. Future studies should aim to test this hypothesis.

Studies by several groups (12, 16, 17, 40) have provided evidence that the kinetics of cholesterol exchange between lipoproteins are consistent with a mechanism of exchange in which cholesterol molecules must diffuse freely through an aqueous phase. The only activation energy required in this process appears to be the desorption of cholesterol from the interfacial lipid phase. In the present study centered on transfers between LDL and model discoidal LpA-I, the rate of cholesterol flux to cholesterol-free Lp2A-I is independent of acceptor concentrations above acceptor:donor ratios of 10. A zero order rate of exchange with respect to acceptor concentration suggests that the frequency of collisions between donor and acceptor particles is not rate-determining for cholesterol transfer to cholesterol-free Lp2A-I. This is similar to that observed in other studies and appears to be consistent with the aqueous diffusion model proposed by Phillips et al.(11, 12, 16, 17) . Cholesterol transfer to Lp2A-I(C), however, is first order with respect to acceptor Lp2A-I(C) concentrations, even when acceptor concentrations are increased 80-fold. It therefore appears that the frequency of collisions between donor and acceptor may be rate-limiting for cholesterol transfer from LDL to the cholesterol-containing Lp2A-I(C). This experimental data appear to be inconsistent with an aqueous diffusion mechanism of cholesterol exchange and instead is supportive of a mechanism that may involve the transient fusion of surface monolayers following the collision of two particles(41) . It is apparent that an aqueous diffusion mechanism may not adequately describe all of the factors that are involved in the transfer of cholesterol between plasma lipoproteins at least for a number of model discoidal particles studied here. Consolidating these mechanistic interpretations into one comprehensive model will require further experiments that need to address how changes in the structure and perhaps charge of different kinds of lipoprotein particles may modulate both lipoprotein-lipoprotein and lipoprotein-cell interactions.

In conclusion, studies of cholesterol exchange between plasma LDL and several structurally well-defined reconstituted LpA-I particles containing different numbers of apoA-I molecules show that unesterified cholesterol exchange is highly sensitive to changes in the compositional and thereby structural properties of the different lipoproteins. Increases in the number of apoA-I molecules in LpA-I significantly inhibit their ability to exchange cholesterol with LDL. In addition, significant differences in cholesterol exchange kinetics between cholesterol-free and cholesterol-containing Lp2A-I acceptors indicate that the presence of cholesterol in Lp2A-I(C) particles also directly influences the function of these particles. Since other studies have shown that variations in LpA-I phospholipid:apoA-I ratio or cholesterol content have direct effects on the conformation and charge of apoA-I(26, 38) , it appears that the physical properties of apoA-I may be critical to the function of HDL and metabolism of cholesterol.


FOOTNOTES

*
This work was supported by a Group Grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: HDL, high density lipoprotein(s); apo, apolipoprotein; LDL, low density lipoprotein(s); LpA-I, lipoprotein containing apoA-I (reconstituted HDL); LpA-I(C), lipoprotein containing apoA-I and cholesterol; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; SUV, small unilamellar vesicles.


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