Kinetic and Thermodynamic Analysis of Cholesterol Transfer between

Jul 17, 2015 - Phospholipase D (PLD) from Streptomyces chromofuscus was purchased from Biomol (Plymouth, PA). A series of n-(9-anthroyloxy)stearic ...
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Kinetic and Thermodynamic Analysis of Cholesterol Transfer between Phospholipid Vesicles and Nanodiscs Naoya Matsuzaki,† Tetsurou Handa,‡ and Minoru Nakano*,§ †

Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minami-Tamagaki-cho, Suzuka, Mie 513-8670, Japan § Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan ‡

S Supporting Information *

ABSTRACT: We investigated interparticle transfer of cholesterol (Chol) between large unilamellar vesicles (LUVs) and phospholipid bilayer nanodiscs. The Chol transfer rate from LUVs to nanodiscs was decreased by an increase in the Chol content or incorporation of sphingomyelin in donor phosphatidylcholine/Chol LUVs but was not influenced by the lipid composition of acceptor particles. These results suggest that Chol dissociation from the lipid bilayer into aqueous phase is the rate-limiting step of the transfer and that the process depends on the fluidity of the donor membranes. The Chol dissociation rate from nanodiscs was faster than that from LUVs with similar lipid composition. Chol preferably partitioned to LUVs rather than nanodiscs, which is consistent with the faster dissociation rate from nanodiscs. The activation energy of Chol dissociation from nanodiscs was 1.7 kJ/mol lower than that from LUV, which was brought by increased (less negative) activation entropy and enthalpy. In addition, fluorescence lifetime and anisotropy data revealed that the lipid bilayer of nanodiscs is more tightly packed than that of LUVs. These results suggest that the tighter lipid packing in nanodiscs destabilizes the Cholcontaining bilayer by reducing the entropy, which facilitates Chol dissociation.

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INTRODUCTION Phospholipid bilayer nanodiscs are discoidal complexes consisting of lipids and amphipathic apolipoproteins. Many researchers are interested in the application of these nanoparticles, partly because they have a simple and efficient configuration that minimizes lipid bilayer structure (normally two apolipoprotein A-I (apoA-I) molecules surround the hydrophobic edge of the bilayer of approximately one hundred phospholipids in a belt-like fashion1,2) and because they are fully biocompatible (apolipoprotein−lipid discoidal particles are seen in the early events of the generation of high-density lipoproteins (HDLs)3−5). Cholesterol (Chol) is one of the major and essential lipid components in mammalian cells. This water-insoluble compound has two different aspects; it is indispensable as a component of biomembranes, but accumulation of excess Chol results in the progression of atherosclerosis.6−8 Therefore, Chol transport between tissues via several classes of lipoproteins is important for maintaining homeostasis. Among these lipoproteins, HDLs play a role in reverse Chol transport, by which excess Chol in the peripheral cells is exported to the liver.9 There are a variety of mechanisms by which cellular Chol is transferred to HDLs, including physicochemical passive diffusion.10,11 In the diffusion process, Chol transfers between plasma membranes and HDLs bidirectionally according to the concentration gradient.11 Over the last few decades, many studies have aimed to elucidate the Chol diffusion mechanism. © 2015 American Chemical Society

Lipid particles containing phospholipids (PLs) behave as Chol acceptors in the passive diffusion pathway from cells, and the efficiency of this pathway depends on the concentration and size of the acceptor particles.12 Chol transfer has been also examined by using vesicles, HDLs, and low-density lipoproteins (LDLs).13,14 Although the relationship between the surface conditions of liposomes and the Chol exchange rate has been examined,15,16 the association of lipoprotein structure and Chol environment with Chol dissociation from lipoproteins has not been well-studied. In this study, we investigated the rate of Chol transfer from Chol-containing large unilamellar vesicles (LUVs) or nanodiscs to Chol-free nanodiscs or LUVs, respectively. LUVs and nanodiscs are different in size and density, so they can be separated by ultracentrifugation or gel filtration chromatography. It was revealed that Chol dissociation from nanodiscs is more frequent than dissociation from LUVs and that reduced entropy due to the closer membrane packing in nanodiscs is responsible for this enhanced Chol dissociation.

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EXPERIMENTAL SECTION Materials. Egg yolk phosphatidylcholine (PC), egg yolk sphingomyelin (SM), Chol, and horseradish peroxidase were Received: April 17, 2015 Revised: July 2, 2015 Published: July 17, 2015 9764

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by the manufacturers. After incubation, the fluorescence intensity of resorufin was measured with excitation/emission wavelengths of 566/590 nm on a FlexStation 3 Microplate Reader (Molecular Devices, Sunnyvale, CA). Chol solubilized by a surfactant in buffer was used as a Chol standard. A suspension of PC nanodiscs, whose concentration was determined with enzymatic assay kits from Wako Pure Chemicals, was used as a standard for PLs. For quantification of samples containing NaBr (see below), which can act as a fluorescent quencher, calibration was done with a standard that contained the same concentration of NaBr. Calibration curves for Chol and PL demonstrated a linear relationship between the concentration (0−20 μM) and the fluorescence intensity. Chol Transfer Measurements. Chol donor particles (Chol-containing LUVs or nanodiscs) and acceptor particles (Chol-free nanodiscs or LUVs) were mixed in Tris buffer, at a molar ratio of PLs in donor and acceptor of 1:10 and a total volume of 400 μL. After incubation for varying periods of time, the samples were mixed with 100 μL of NaBr solution with a density of 1.43 g/mL and ultracentrifuged on a Hitachi CS100EX with a S120AT3 rotor (Hitachi, Tokyo, Japan) at 100 000 rpm (370 000 × g) for 30 min. The nanodisc fraction (bottom, 200 μL) was collected and quantified for Chol and PLs using Amplex Red as described above. Assuming that dissociation from the particles (nanodisc and LUV) is the rate-limiting step of the interparticle Chol transfer, the change in Chol concentration is given by the following equation (see Supporting Information for details):

purchased from Sigma-Aldrich (St. Louis, MO). Ampex Red Reagent was obtained from Invitrogen (Eugene, OR). Cholesterol oxidase from Streptomyces sp. and choline oxidase from Alcaligene sp. were obtained from Funakoshi (Tokyo, Japan) and TOYOBO (Osaka, Japan), respectively. Phospholipase D (PLD) from Streptomyces chromofuscus was purchased from Biomol (Plymouth, PA). A series of n-(9-anthroyloxy)stearic acids (n-AS, n = 2, 6, and 12) were obtained from Invitrogen (Eugene, OR, for 2-AS and 12-AS) and Wako Pure Chemicals (Osaka, Japan, for 6-AS). ApoA-I was isolated from porcine plasma.17−19 Its purity was confirmed by SDS-PAGE, and its ability to solubilize dimyristoylphosphatidylcholine (DMPC) membranes was ascertained by clearance assay.20 All other chemicals were of the highest reagent grade. Preparation of LUVs. LUVs (d ∼ 120 nm) were obtained by the extrusion method. The dried lipid film was hydrated with Tris buffer (10 mM Tris−HCl, 150 mM NaCl, 0.01% NaN3, pH 7.4) and vortexed to form multilamellar vesicles. After seven freeze−thaw cycles, the suspension was extruded through a 100 nm pore polycarbonate filter (Avestin, Ottawa, Canada). Vesicle diameters were determined by dynamic light scattering using FPAR-1000 (Otsuka Electronic Co., Osaka, Japan). The concentrations of PLs (PC + SM) and Chol were determined using enzymatic assay kits purchased from Wako Pure Chemicals. Preparation of Nanodiscs. Nanodiscs were prepared by the cholate dialysis procedure.21 Starting molar ratios of PLs/ Chol/apoA-I/sodium deoxycholate were set at 80:0:1:260 or 80:20:1:260. Lipids (PLs and Chol) were hydrated with Tris− EDTA buffer (10 mM Tris−HCl, 150 mM NaCl, 1 mM EDTA, and 0.01% NaN3, pH 7.4) and mixed with apoA-I and sodium deoxycholate. The solution was incubated at 4 °C for 8 h and then dialyzed against Tris−EDTA buffer to remove sodium deoxycholate. The solution (500 μL) was mixed in a centrifuge tube with 2 mL of NaBr (aq) solution (density: 1.32 g/mL) to adjust the density to ∼1.26 g/mL, and subsequently gently overlaid with 2 mL of NaBr (aq) solution (density: 1.06 g/mL). After ultracentrifugation at 40 000 rpm for 16 h in a Beckman SW50.1 rotor (150 000 × g) to remove remaining free proteins and lipid vesicles, the middle layer was recovered and dialyzed against Tris buffer to obtain the nanodisc dispersion. Concentrations of lipids and apoA-I were determined by using assay kits for Chol, PC (Wako Pure Chemicals) and proteins (Bio-Rad, Hercules, CA), and the molar ratio of PLs:apoA-I was determined to be 80:1−100:1. The presence of nanodiscs with a hydrodynamic diameter of ca. 10 nm was confirmed by gel filtration chromatography with Superdex 200 (GE Healthcare, Tokyo, Japan). Lipid Assays with Amplex Red. To measure the relatively low concentrations (0−20 μM) of Chol and PLs, we used a fluorescence assay with Amplex Red, which reacts with hydrogen peroxide to produce the highly fluorescent compound resorufin. For Chol quantification, the sample solution was incubated at 37 °C for 30 min with an equal volume of working solution (2 U/mL cholesterol oxidase, 2 U/ mL peroxidase, 300 μM Amplex Red in phosphate buffer (100 mM phosphate, 50 mM NaCl, 5 mM cholic acid, 0.1% Triton X-100, pH 7.4)). For quantification of PLs (PC + SM), the sample solution was incubated at 37 °C for 30 min with an equal volume of working solution (20 U/mL PLD, 4 U/mL choline oxidase, 4 U/mL peroxidase, 300 μM Amplex Red in HEPES buffer (20 mM HEPES, 150 mM NaCl, 2 mM CaCl2, 0.2% Triton X-100, pH 7.4)). Enzyme units used were defined

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d[Chol(t )]ND = kND[Chol(t )]ND dt [PL]LUV /2 1 · − kLUV[Chol(t )]LUV · [PL]ND + [PL]LUV /2 2 [PL]ND [PL]ND + [PL]LUV /2

(1)

where [Chol(t)] and [PL] are the concentrations of Chol and PL, respectively, and k is the dissociation rate constant of Chol. Subscripts ND and LUV denote nanodisc and LUV, respectively. Because flip-flop of Chol in PL membranes is known to occur extremely faster than the dissociation (on the order of seconds or less),22,23 the concentration gradient of Chol in LUVs created by the dissociation from outer leaflet is immediately counteracted by the flop from inner leaflet. Hence, it can be assumed that Chol concentration in LUV is always the same between inner and outer leaflets. Fractional expressions for [PL] in the first and second term of eq 1 denote the probability of Chol to be incorporated into LUVs and nanodiscs, respectively. Notice the multiplication by 1/2, which represents that transfer occurs only at the outer leaflet of LUVs. When LUVs are used as the Chol donor and mixed with nanodiscs at 1:10 ϕND(t ) =

ϕLUV (0)kLUV kND + 10kLUV −

⎛ k + 10kLUV ⎞ exp⎜ − ND t⎟ ⎝ ⎠ kND + 10kLUV 21 ϕLUV (0)kLUV

(2)

where ϕ ND (t) = [Chol(t)] ND /[PL] ND and ϕ LUV (0) = [Chol(0)] LUV /[PL] LUV . Equation 2 indicates that k LUV predominantly determines the increasing rate of ϕND(t). Thus, only kLUV was determined from the data from the 9765

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Figure 1. Chol transfer from LUVs to nanodiscs at 37 °C. (A) Dependence of particle concentrations on Chol transfer from LUVs (PC/Chol = 100/76) to PC nanodiscs. PC Concentrations of LUVs and nanodiscs were 10 μM and 100 μM (red ●), 20 μM and 200 μM (blue ▲), and 40 μM and 400 μM (green ■), respectively. (B) Dependence of Chol content in LUVs (20 μM PC) on Chol transfer to PC nanodiscs (200 μM PC). The ratios of PC/Chol in LUVs are 100/9 (red ●), 100/38 (blue ▲), and 100/70 (green ■). (C) Effect of SM in LUV (20 μM PLs) on Chol transfer to PC nanodiscs (200 μM PC). The ratios of PC/SM/Chol in LUVs are 100/0/76 (red ●), 70/30/74(blue ▲), and 30/70/86 (green ■). (D) Effect of SM in nanodiscs (200 μM PLs) on Chol transfer from LUVs (PC/Chol = 100/76, 20 μM PC). The ratios of PC/SM in nanodiscs are 100/0 (red ●), 80/20 (blue ▲), and 60/40 (green ■). Data represent mean ± SD from triplicate analyses. Solid lines are curves fitted to the data with eq 3 and kLUV values indicated are those obtained from eq 4. Errors for kLUV were estimated from regression analysis.

“transfer from LUV to nanodisc” experiment. The data was fitted by the following equation with fitting variables A and k ϕND(t ) = A − A exp( −kt )

ϕND(t ) ϕND(0)

(3)

kLUV

kND =

ϕND(0)

=

[Chol(t )]ND [Chol(0)]ND

=

kLUV kLUV + 10kND +

⎛ k + 10kND ⎞ 10kND t⎟ exp⎜ − LUV ⎠ kLUV + 10kND ⎝ 12

6Ak 5

(7)

Chol Affinity for LUV and Nanodisc. Chol-containing LUVs (500 μM PC) and PC nanodiscs (500 μM PC) were incubated at 37 °C for 24 h to equilibrate the Chol distribution. After incubation, LUVs and nanodiscs were separated by gel filtration chromatography on a Sepharose CL-6B column (GE Healthcare, Tokyo, Japan) using Tris buffer as the elution buffer at a flow rate of 0.63 mL/min. Two fractions corresponding to LUVs and nanodiscs were collected, and their Chol and PC concentrations were quantified using enzymatic assay kits from Wako Pure Chemicals (Osaka, Japan). Fluorescence Measurements. For the fluorescence measurements, LUVs and nanodiscs that contain n-AS were prepared at a PC/Chol/n-AS molar ratio of 100:20:1. The mean fluorescence lifetimes ⟨τ⟩ of n-AS were measured on a Horiba NAES-550 Nanosecond Fluorometer (Kyoto, Japan) as

(4)

Similarly, when nanodiscs are used as the Chol donor and mixed with LUVs at the PL molar ratio of 1:10 ϕND(t )

(6)

and comparing eqs 5 and 6 provides kND as

By comparing eqs 2 and 3, kLUV is given as 21Ak = ϕLUV (0)

= A exp( −kt ) + (1 − A)

(5)

The decay in ϕND(t)/ϕND(0) obtained from the “transfer from nanodisc to LUV” experiment was fitted by the following equation 9766

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The Journal of Physical Chemistry B described elsewhere.24−29 The steady-state fluorescence anisotropy rs of n-AS was measured on a Hitachi F-4500 spectrofluorometer (Tokyo, Japan).25

by the composition of the acceptor nanodiscs (Figure 1D). The difference in the Chol amount transferred into nanodiscs with different PC/SM compositions was within the experimental error. Taken together, these data indicate that under these experimental conditions, Chol dissociation from the surface of the donor LUVs was the rate-limiting process and was dependent on the composition of LUVs. Chol Transfer from Nanodisc to LUV. We also examined Chol transfer from Chol-containing nanodiscs to Chol-free LUVs. Nanodiscs (PC/Chol = 00/17, 100 μM PC) and PC LUVs (1 mM) were mixed and after varying time periods, the amount of Chol remaining in the nanodiscs was determined. As shown in Figure 2, Chol transfer occurred more rapidly

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RESULTS Chol Transfer from LUV to Nanodisc. First, we investigated Chol transfer from Chol-containing LUVs to Chol-free nanodiscs at 37 °C. LUVs (10, 20, or 40 μM PC) and nanodiscs (100, 200, or 400 μM PC) were mixed at a PC molar ratio of 1:10 and changes in the Chol/PC molar ratio in nanodiscs were determined. Because of high reactivity of Amplex Red, working solutions for the lipid assays increase their intrinsic fluorescence intensity in storage. Therefore, it was necessary to prepare working solution just before each assay. Chol transfer experiments were conducted with shifting the starting time point of the sample mixing, so that a series of the samples could be collected at the same time and assayed with an identical working solution. After mixing with LUVs (PC/Chol = 100/76), Chol amount in nanodiscs increased in a time-dependent manner and reached a plateau in ca. 24 h, as shown in Figure 1A. Time courses of the Chol/PC molar ratios in nanodiscs were essentially the same at the three different particle concentrations examined, suggesting that Chol transfer is not brought about by collisions of particles but by passive molecular diffusion through aqueous phase. Slight differences in the plateau values are ascribed to the interassay variations with different working solutions. Because the association of Chol into membranes should be a process dependent on the particle concentration, the result described above also suggests that the rate-limiting step of Chol transfer is the dissociation process from the particles. Therefore, net Chol transfer between the two kinds of particles can be represented as eq 1 with two dissociation rate constants, that is, for LUV (kLUV) and nanodisc (kND). When the initial Chol fraction in LUV was decreased, the kLUV values increased (Figure 1B). Furthermore, kLUV values were also shown to decrease with increasing SM content in LUVs (Figure 1C). Pure PC forms a liquid disordered phase (Ld phase), whereas both Chol and SM are known to increase membrane packing order and to promote the formation of the liquid ordered phase (Lo phase).30 Therefore, these results suggest that higher lipid packing order of bilayers represses Chol dissociation. Although we assumed that the parameter kLUV is independent of time in the curve fitting, following the dissociation of Chol, membranes are considered to reduce their Chol content and packing order, and thereby promote the Chol dissociation. Consideration of such a positive feedback in the Chol transfer would provide theoretical curves where the Chol/ PC molar ratios in nanodiscs increase more straightforwardly before they reach to the plateau. In any case, the initial slope of the curves should give the kLUV value for LUVs with the initial compositions. As a simple estimation, we compared the initial slopes in Figure 1A and found that the slopes calculated from two data points (t = 0 and 2 h) were 18% smaller than those of the fitting curves, that is, ϕND ′ (0). This implies that the assumption of the constant kLUV might overestimate this value with the largest overestimation of 18%. It was also shown that Chol tended to persist in LUVs containing a larger amount of SM (Figure 1C): PC/SM/Chol (30:70:86) LUV, which is expected to consist exclusively of the Lo phase from the phase diagram,30 hardly worked as Chol donor. This is due to preferential interaction of Chol with SM.31 On the other hand, the Chol transfer rate was unaffected

Figure 2. Chol transfer from nanodisc (PC/Chol = 100/17, 100 μM PC) to PC LUV (1 mM PC) at 37 °C. Data represent mean ± SD from triplicate analyses. Solid lines are curves fitted to the data with eq 6 and kND values indicated are those obtained from eq 7. Errors for kND were estimated from regression analysis.

compared with that from LUVs to nanodiscs (Figure 1), and the Chol amount in nanodiscs reached a plateau in ca. 2 h. The faster transfer is partly because the transfer from nanodiscs occurs from both bilayer leaflets, whereas the transfer from LUVs occurs only from the one leaflet that is exposed to outer aqueous media. eq 5, which is used to fit the data, takes this into account. Nevertheless, the dissociation rate constant obtained (kND = 2.14 h−1) was still higher than kLUV, suggesting faster dissociation from nanodiscs. Temperature Dependence of Chol Transfer. We next examined the temperature dependence of Chol transfer. To determine kLUV, Chol transfer assays from LUVs to nanodiscs were conducted with Chol-containing LUVs (PC/Chol = 100/ 19, 40 μM PC) and PC nanodiscs (400 μM) at five different temperatures between 22 and 42 °C. To assess the temperature dependence of Chol transfer from nanodiscs to LUVs (kND), nanodiscs (PC/Chol = 100/17, 100 μM PC) and PC LUV (1 mM) were mixed at five different temperatures between 17 and 37 °C. The Arrhenius plots of the dissociation constants revealed that kND was always larger than kLUV at the temperatures examined (Figure 3). The activation enthalpy (ΔH⧧), entropy (ΔS⧧), and free energy (ΔG⧧) of Chol dissociation were determined from the plots according to the absolute rate theory.32 As shown in Table 1, ΔG⧧ of Chol dissociation from nanodiscs was 1.7 kJ/mol lower than that 9767

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of ca. 10%, we can infer from the data that dissociation of Chol from nanodiscs is a more entropically favorable (and enthalpically unfavorable) process than that from LUVs. Chol Affinity for LUVs and Nanodiscs. The difference in the dissociation rate constants should directly reflect differences in the Chol affinity for LUVs and nanodiscs. To determine the Chol affinity, LUVs (PC/Chol = 100/20, 500 μM PC) and PC nanodiscs (500 μM PC) were equilibrated by incubating for 24 h at 37 °C, and were subsequently separated by gel filtration. The gel filtration profile of the incubated mixture demonstrates that LUVs and nanodiscs can be distinctly separated (Figure S1 in the Supporting Information). Chol and PC concentrations of each fraction were quantified, and the Chol/PC molar ratios were determined to be 0.117 ± 0.003 and 0.073 ± 0.002 for LUVs and nanodiscs, respectively. This result suggests higher affinity of Chol for LUVs than for nanodiscs. Chol distribution at equilibrium state (t = ∞) is expressed by d[Chol(t)]ND/dt = 0 in eq 1, so that the partition coefficient PLUV/ND is Figure 3. Arrhenius plots of Chol transfer rate. The kLUV values (red ●) were determined by Chol transfer assays from LUVs (PC/Chol = 100/19, 40 μM PC) to PC nanodiscs (400 μM PC) at temperatures between 22 and 42 °C, and the kND values (blue ▲) were determined by transfer assays from nanodiscs (PC/Chol = 100/17, 100 μM PC) to PC LUV (1 mM) at temperatures between 17 and 37 °C. Errors for kLUV and kND were estimated from regression analysis.

PLUV/ND =

LUV nanodisc a

ΔG⧧ (kJ/mol)

ΔH⧧ (kJ/mol)

TΔS⧧ (kJ/mol)

1.11 ± 0.09 2.14 ± 0.16

96.9 ± 0.2 95.2 ± 0.2

88.7 94.8

−8.2 −0.4

ϕND(∞)

=

kND kLUV

(8)

The value of PLUV/ND calculated from the dissociation rate constants (Table 1) was 1.93, and the difference in the activation free energy of the Chol transfer from nanodiscs and LUVs (ΔΔG⧧ ≡ ΔG⧧LUV − ΔG⧧ND) was 1.7 kJ/mol, as described before. These values were close to the partition coefficient and the absolute value of the difference in the standard free energy obtained from the Chol/PC molar ratios (PLUV/ND = 0.117/ ° − ΔGND ° = −RTlnPLUV/ND = 0.073 = 1.60, ΔΔG° ≡ ΔGLUV −1.2 kJ/mol), respectively. Hence, it is concluded that Chol in nanodiscs possesses higher free energy and, thus, exhibits a higher dissociation rate than that in LUVs. Differences in the Membrane Properties between LUVs and Nanodiscs. Differences in the membrane properties of Chol-containing bilayers between LUVs and nanodiscs were examined by evaluating the fluorescence lifetimes and steady-state fluorescence anisotropy of n-AS. The fluorophores, which are the anthroyloxyl groups of n-AS, are located in the hydrocarbon region of the membranes, depending on the

Table 1. Kinetic and Thermodynamic Parametersa of the Activated State of Chol Dissociation from LUVs and Nanodiscs at 37 °C kLUV, kND (h−1)

ϕLUV (∞)

Parameters were obtained from Figure 3.

from LUVs. It is interesting to note that the entropy loss accompanying the Chol dissociation was minimized for nanodiscs: TΔS⧧ was −0.4 kJ/mol for nanodiscs, which was less negative than that for LUVs (−8.2 kJ/mol). Although the slope of the Arrhenius plots (and thus ΔH⧧) includes an error

Figure 4. Fluorescence lifetime and steady-state fluorescence anisotropy of n-AS. (A) Mean fluorescence lifetimes ⟨τ⟩ of n-AS in LUVs (red ●) and in nanodiscs (blue ▲). (B) Steady-state fluorescence anisotropy of n-AS in LUVs (red ●) and in nanodiscs (blue ▲). The PC/Chol molar ratio was 100/17 in both nanodiscs and LUVs. The values are plotted against n. Data represent mean ± SD from triplicate analyses. 9768

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The Journal of Physical Chemistry B position number (n).33,34 Lifetimes are sensitive to the local environment and exposure to water molecules promotes fluorescence quenching.35,36 As shown in Figure 4A, the mean fluorescence lifetime (⟨τ⟩) of n-AS was significantly longer in nanodiscs than in LUVs for n = 2 and 6, suggesting a lesser degree of water penetration into the acyl chain region of the nanodiscs. The steady-state fluorescence anisotropy of n-AS reports on the acyl chain packing order in the lipid bilayer. The anisotropy values of n-AS in nanodiscs were significantly higher than those in LUVs (Figure 4B). These results demonstrated that lipid bilayers of nanodiscs were more tightly packed than those of LUVs, presumably due to the constriction by apoA-I molecules surrounding the edge of lipid bilayer.

dissociation rate. Although DMPC with a large polar headgroup seems less hydrophobic than cholesterol, two acylchains (C14 × 2) bring hydrophobic area comparable to or slightly larger than that of cholesterol, which contains 27 carbons. We have also applied TR-SANS technique to nanodiscs consisting of apoA-I and DMPC and demonstrated that nanodiscs enable a 20-fold higher lipid transfer via an entropically favorable process.26 It is predicted that not only phospholipid but also Chol experiences a different environment in nanodiscs versus in LUVs, and that the environment in nanodiscs promotes Chol dissociation. Fluorescence measurements suggested that lipid bilayer in nanodiscs is more tightly packed than that in LUVs, which is consistent with the report of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene.41 We have previously shown that although nanodisc formation is a thermodynamically favorable process primarily due to the helix formation of apoA-I, it causes an increase in the Gibbs energy of the bilayers, accompanied by a decrease in both entropy and enthalpy.29 Fluorescence data from the present study (Figure 4) also indicate that lipids in nanodiscs have lower enthalpy and entropy as compared with those in vesicles. Kinetic analysis demonstrated that the rapid transfer of Chol from nanodiscs was accompanied by an increase in the activation entropy ΔS⧧ (Table 1). The difference in the activation free energy ΔΔG⧧ was directly correlated with the difference in the standard free energy ΔΔG°. Therefore, the increased activation entropy could be ascribed to a decrease in the standard entropy of Chol in nanodiscs, which was caused by tighter membrane packing. It is interesting to note that the increased packing order altered the Chol dynamics differently in the two systems, that is, hindered the dissociation in LUVs, whereas accelerated it in nanodiscs. SM and Chol in LUVs reinforce van der Waals interaction between lipids and thereby strongly promote denser packing, whereas apoA-I molecules on the edge of nanodiscs constrict bilayers, which compels them to pack inward tightly. HDLs are generated by the interaction of apoA-I with ATP binding cassette transporter A1 (ABCA1).42,43 They are initially a discoidal complex consisting mainly of apoA-I, PLs, and Chol.44 Cellular Chol release involves several mechanisms, including protein-independent aqueous diffusion and proteinmediated transport by ABCA1, ATP binding cassette protein G1 (ABCG1), and scavenger receptor BI (SR-BI).10,11,45 Aqueous diffusion of Chol to HDLs is normally inefficient and requires the activation of particular proteins, such as lecithin:cholesterol acyltransferase (LCAT), which acts mainly on preβ-HDL to convert Chol to cholesteryl ester.3 A number of studies have attempted to elucidate the function of ABCA1, ABCG1, and SR-BI. Interestingly, there is experimental evidence that these proteins alter lipid distribution of the plasma membrane: ABCA1 functions to redistribute SM and Chol from lipid rafts to nonraft regions.46 It has also been reported that methyl-β-cyclodextrin, a nonphysiological Chol acceptor, extracts more Chol from ABCA1-expressing cells than from mock cells.47 Furthermore, Chol efflux by ABCG1 and SR-BI has been suggested to involve a passive diffusion process because ABCG1 mediates Chol efflux to HDLs without binding to HDLs and the protein increases the rate of Chol efflux and the size of Chol pool available for efflux.48 SR-BI facilitates Chol efflux under conditions at which binding of HDL to SR-BI is oversaturated, suggesting the existence of a pathway independent of binding to SR-BI.49 All of these proteins (i.e., ABCA1,50 ABCG1,51 and SR-BI52) increase the fraction of

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DISCUSSION In the present study, Chol transfer between LUVs and nanodiscs was observed. Toledo et al. proposed that Chol transfer between vesicles and discoidal particles involves the vesicles binding to the discoidal particles via a specific domain of apoA-I.14 This proposal is based on the finding that discoidal particles leak internal components of vesicles in a manner that is dependent on the lipid composition of the vesicles.37 In the present study, however, we confirmed that calcein encapsulated in LUVs was not leaked out by nanodiscs and was only leaked by lipid-free apoA-I (Supporting Information Figure S2). This implies a lack of strong attractive interaction between these particles. The inconsistency between our study and the report by Toledo et al. presumably results from a difference in the size of the vesicles studied. They used sonicated (and thus probably small) vesicles, whereas extruded LUVs were used in the present study. Hence, our study is considered to be an appropriate model system for Chol diffusion between biomembranes and HDLs. We demonstrated that the lipid composition of acceptor particles had little influence on the Chol transfer rate (Figure 1C). Chol transfer rate has also been reported to be insensitive to the size of acceptor vesicles.38 These findings suggest that Chol dissociation from donor particles is the rate-limiting step. Indeed, in the “transfer from LUV to nanodisc” experiment, Chol transfer was affected by the lipid composition of donor particles, and the most influential factor was the packing order of the layers. This is consistent with a previous report that the Chol exchange rate among liposomes decreases with increasing the amount of SM or PC having saturated acylchains.15 Another study has shown that small unilamellar vesicles, which have higher positive curvature on their outer leaflet, can donate more Chol than LUVs.38 Taken together, the data imply that vesicles with higher fluidity or curvature have lower activation energy for Chol dissociation. We also showed that Chol hardly dissociates from LUVs consisting of the Lo phase, presumably due to the preferential interaction of Chol with SM.31 This agrees with the observation in giant vesicles that Chol is removed from the Ld phase.39 Previously, we have succeeded in characterizing interbilayer and transbilayer lipid transfers of DMPC with time-resolved small-angle neutron scattering (TR-SANS).40 The transfer rate of DMPC in LUV was 0.276 h−1, which is slightly smaller than that of Chol obtained in the present study (1.11 h−1). The energy barrier for the lipid transfer is the exposure of hydrophobic region of the lipid into aqueous media (hydrophobic hydration) on the dissociation process. Therefore, not the hydrophobicity of the entire molecule but the surface area of the hydrophobic part predominantly determines its 9769

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microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I. J. Lipid Res. 2006, 47, 832−843. (5) Lee, J. Y.; Parks, J. S. ATP-binding cassette transporter Al and its role in HDL formation. Curr. Opin. Lipidol. 2005, 16, 19−25. (6) Williams, K. J.; Tabas, I. The response-to-retention hypothesis of early atherogenesis. Arterioscler., Thromb., Vasc. Biol. 1995, 15, 551− 561. (7) Srinivasan, S. R.; Radhakrishnamurthy, B.; Vijayagopal, P.; Berenson, G. S. Proteoglycans, lipoproteins, and atherosclerosis. Adv. Exp. Med. Biol. 1991, 285, 373−381. (8) Smith, E. B. The relationship between plasma and tissue lipids in human atherosclerosis. Adv. Lipid Res. 1974, 12, 1−49. (9) Fielding, C. J.; Fielding, P. E. Molecular physiology of reverse cholesterol transport. J. Lipid Res. 1995, 36, 211−228. (10) Rothblat, G. H.; de la Llera-Moya, M.; Atger, V.; Kellner-Weibel, G.; Williams, D. L.; Phillips, M. C. Cell cholesterol efflux: integration of old and new observations provides new insights. J. Lipid Res. 1999, 40, 781−796. (11) Yancey, P. G.; Bortnick, A. E.; Kellner-Weibel, G.; de la LleraMoya, M.; Phillips, M. C.; Rothblat, G. H. Importance of different pathways of cellular cholesterol efflux. Arterioscler., Thromb., Vasc. Biol. 2003, 23, 712−719. (12) Davidson, W. S.; Rodrigueza, W. V.; Lundkatz, S.; Johnson, W. J.; Rothblat, G. H.; Phillips, M. C. Effects of acceptor particle-size on the efflux of cellular free-cholesterol. J. Biol. Chem. 1995, 270, 17106− 17113. (13) Meng, Q. H.; Sparks, D. L.; Marcel, Y. L. Effect of LpA-I composition and structure on cholesterol transfer between lipoproteins. J. Biol. Chem. 1995, 270, 4280−4287. (14) Toledo, J. D.; Tricerri, M. A.; Corsico, B.; Garda, H. A. Cholesterol flux between lipid vesicles and apolipoprotein AI discs of variable size and composition. Arch. Biochem. Biophys. 2000, 380, 63− 70. (15) Lundkatz, S.; Laboda, H. M.; Mclean, L. R.; Phillips, M. C. Influence of molecular packing and phospholipid type on rates of cholesterol exchange. Biochemistry 1988, 27, 3416−3423. (16) Thomas, P. D.; Poznansky, M. J. Cholesterol transfer between lipid vesicles - effect of phospholipids and gangliosides. Biochem. J. 1988, 251, 55−61. (17) Saito, H.; Miyako, Y.; Handa, T.; Miyajima, K. Effect of cholesterol on apolipoprotein A-I binding to lipid bilayers and emulsions. J. Lipid Res. 1997, 38, 287−294. (18) Handa, T.; Komatsu, H.; Kakee, A.; Miyajima, K. Interactions of lecithin and pig apolipoproteins of high-density lipoproteins at the surface monolayer of reconstituted very small particles. Chem. Pharm. Bull. 1990, 38, 2079−2082. (19) Handa, T.; Saito, H.; Tanaka, I.; Kakee, A.; Tanaka, K.; Miyajima, K. Lateral interactions of pig apolipoprotein-A-I with eggyolk phosphatidylcholine and with cholesterol in mixed monolayers at the triolein saline interface. Biochemistry 1992, 31, 1415−1420. (20) Surewicz, W. K.; Epand, R. M.; Pownall, H. J.; Hui, S. W. Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids. J. Biol. Chem. 1986, 261, 6191−6197. (21) Matz, C. E.; Jonas, A. Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions. J. Biol. Chem. 1982, 257, 4535−4540. (22) Steck, T. L.; Ye, J.; Lange, Y. Probing red cell membrane cholesterol movement with cyclodextrin. Biophys. J. 2002, 83, 2118− 2125. (23) Hamilton, J. A. Fast flip-flop of cholesterol and fatty acids in membranes: implications for membrane transport proteins. Curr. Opin. Lipidol. 2003, 14, 263−271. (24) Kamo, T.; Handa, T.; Nakano, M. Lateral pressure change on phase transitions of phosphatidylcholine/diolein mixed membranes. Colloids Surf., B 2013, 104, 128−132. (25) Ozawa, M.; Handa, T.; Nakano, M. Effect of cholesterol on binding of amphipathic helices to lipid emulsions. J. Phys. Chem. B 2012, 116, 476−482.

cellular Chol accessible to Chol oxidase, which reacts preferentially to Chol in the Ld phase. In the present study, we demonstrated that Chol in the Ld phase dissociates more easily from LUVs, whereas Chol in the Lo phase hardly dissociates. The mechanism of redistribution of phospholipids and Chol by these proteins has not yet been clarified in detail. However, considering that an aqueous diffusion process might be involved in their enhancement of the Chol efflux, we can speculate that local modification of phospholipids and Chol by these proteins expands the region of loosely packed Ld phase.

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CONCLUSIONS In this study, we determined the interparticle Chol transfer and demonstrated that Chol dissociation from the lipid bilayer into aqueous phase is the rate-limiting step of the transfer, which depends on the fluidity of the bilayers. Kinetic and thermodynamic analysis of the Chol transfer, together with the fluorescence lifetime and anisotropy data, revealed that the tighter lipid packing in nanodiscs increases the free energy of Chol and, thus, facilitates the Chol dissociation. Newly synthesized preβ-HDLs are released into the extracellular medium and subsequently receive Chol by passive diffusion. Our data suggests that Chol in preβ-HDLs is less stable and hence more readily dissociates into aqueous medium, compared with that in plasma membranes. Instability of Chol may be linked to higher susceptibility to esterification by LCAT. Furthermore, promotion of Chol diffusion from preβ-HDL to other lipoproteins may contribute to a cooperative efflux effect of these particles.

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ASSOCIATED CONTENT

S Supporting Information *

Derivation of eq 1, Figure S1 (gel filtration profiles), and Figure S2 (calcein leakage assays). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b03682.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-76-434-7565. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by JSPS KAKENHI Grant Numbers 25610120 and 26287098. REFERENCES

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