C-Terminus of Apolipoprotein A-I Removes Phospholipids from a Triolein/Phospholipids/Water Interface, but the N-Terminus Does Not: A Possible Mechanism for Nascent HDL Assembly

Abstract

Apolipoprotein A-I (ApoA-I) is the principle protein component of HDL, also known as “good cholesterol,” which is an inverse marker for cardiovascular disease. The N-terminal 44 amino acids of ApoA-I (N44) are predicted to be responsible for stabilization of soluble ApoA-I, whereas the C-terminal 46 amino acids (C46) are predicted to initiate lipid binding and oligomerization. In this work, we apply what we believe to be a novel application of drop tensiometry to study the adsorption and desorption of N44 and C46 at a triolein/POPC/water (TO/POPC/W) interface. The amount of peptide that adsorbed to the surface was dependent on the surface concentration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and pressure (Π) before adsorption. At a TO/POPC/W interface, the exclusion pressure (Π_EX) of C46 was 25.8 mN/m, and was 19.3 mN/m for N44. Once adsorbed, both peptides formed a homogeneous surface with POPC but were progressively ejected from the surface by compression. During a compression, C46 removed POPC from the surface whereas N44 did not. Repeated compressions caused C46 to deplete entirely the surface of phospholipid. If full-length ApoA-I could also remove phospholipid, this could provide a mechanism for the transfer of surface components of chylomicrons and very low density lipoprotein to high density lipoprotein with the assistance of phospholipid transfer protein.

Introduction

High density lipoprotein (HDL), also known as “good cholesterol,” is an inverse clinical marker of coronary heart disease and is responsible for reverse cholesterol transport. Apolipoprotein A-I (ApoA-I) is the principal protein component of HDL and is central to HDL assembly, remodeling, and metabolism (1). ApoA-I is an amphipathic protein that stabilizes plasma lipoprotein particles and binds to LCAT and ABC receptors to mediate the transport of cholesterol in and out of cells (2). ApoA-I has at least three in vivo conformational states:

• 1.Free or minimally lipidated in blood plasma.
• 2.A homodimer-stabilizing discoidal PC-rich HDL.
• 3.Surface-associated with numerous spherical lipoprotein particles including mature HDL, triacylglyceride-rich, very low density lipoproteins (VLDL), and chylomicrons (3).

To accommodate these various environments, ApoA-I must be intrinsically flexible.

ApoA-I is a 243-amino-acid (aa) protein. The core of ApoA-I is composed of 11/22-mer tandem repeats of amphipathic α-helices, which are predicted to interact and stabilize the hydrophobic lipid surface of a lipoprotein particle (4). The C-terminal 46 residues (aa 198–243) of ApoA-I (C46) are predicted to be the most lipophilic portion of the peptide and have the highest lipid affinity within the ApoA-I sequence (5). At low concentration (<0.2 mg/mL), C46 forms an unstructured monomer (6). Upon lipid (or lipid mimic) binding, the helical content of C46 increased to ∼60%. At higher concentrations, C46 self-associated, forming tetramers or pentamers with ∼50% helical content (6). When bound to lipid, C46 is predicted to form a helix-loop-helix structure (7–9).

Some potential structural models of C46 are illustrated in Fig. S1 in the Supporting Material and the interfacial hydrophobicity of each model is calculated in Table S1 in the Supporting Material using the GES scale (10) and White-Wimley octanol (11) and interfacial POPC scales (12). The N-terminal helix of C46 is predicted to have a lower lipid affinity than the C-terminal helix (13–16). C46 adsorbs to a triolein/water (TO/W) interface and exerts 17 mN/m of surface pressure (Π), forming a viscoelastic interface (17). Removing the peptide from the surrounding solution caused Π to fall by 3.1 mN/m, and repeated compressions of the surface to Π > 17 mN/m caused Π to approach the Π of a peptide-free interface showing that high Π can push the peptide off the surface (18).

The N-terminal 44 residues (aa 1–44) of ApoA-I (N44) is predicted to form a G^∗ type amphipathic helix spanning residues 8–33 upon lipid binding (4,7,9,19). Far-ultraviolet circular dichroism measurements indicated that N44 is unfolded in solution but upon lipid binding, ∼60% helical structure was induced. N44 has a lower affinity for lipids than C46, but can interact with DMPC to form discoidal complexes (20). At near-saturated protein conditions, only the C-terminus of ApoA-I bound to egg-PC vesicles and the N-terminus (aa 1–198) did not interact with lipid but when excess phospholipid surface was present, the entire peptide appeared to interact (21). The sequence of N44 and interfacial hydrophobicity are compared to C46 in Fig. S1 and Table S1 (4,9–12,17). At a TO/W interface, N44 was less surface-active than C46 and exerted only 15 mN/m of Π (17). Removing the peptide from the surrounding solution caused Π to fall by 3.8 mN/m and, like C46, it was pushed off the surface by compression (18).

In the past, phospholipids monolayers at an air/water interface have been used to model adsorption of apolipoproteins to a lipoprotein surface (22–25). The amount of protein that adsorbed to the surface was dependent on the initial surface concentration (Г) of phospholipids (PLs). At low Г_PL, a larger amount of peptide bound to the surface, causing a larger change in Π. As Г_PL increased, less peptide penetrated the surface until a specific Г_PL is reached where no protein adsorbed to the surface. The Π where no peptide adsorbed to the surface is defined as the exclusion Π (Π_EX) (25). The Π_EX of ApoA-I at an air/water interface was 30 mN/m, with other peptides varying between Π_EX of 25–30 mN/m (24–26).

In another approach, the adsorption of apolipoproteins to a triglyceride (TAG)/W interface was also been investigated using drop tensiometry (17,23,27–31), and demonstrated the differences in adsorption and surface behavior of different structural motifs and apolipoproteins as were shown in this work (17,18,29,30,32). For a review of this work, see Small et al. (18). At the surface of a TAG/PL emulsion particle, a majority of the surface is covered by PL but a small fraction (estimated by different methods to be between 3 and 7%) of the surface is occupied by TAG (33–35). To have a physiological understanding of the adsorption and desorption of apolipoproteins to a lipoprotein particle, a mixed layer of TAG and PL should be investigated. There is a very limited body of work using this interface (31), and, to date, no in-depth work has been published.

In this study, we introduce what we regard as a novel technique using drop tensiometry to examine the adsorption and behavior of apolipoprotein-derived peptides adsorbed to a triolein/palmitoyl-oleoyl- phosphatidylcholine/water (TO/POPC/W) interface. A TO/POPC/W interface is more physiologically relevant than a TO/W or Air/PC/W interface because it more closely resembles a lipoprotein surface. In addition, the change in the lipid monolayer during compression of a TO/PC/W interface more closely resembles lipoprotein remodeling.

When a PC/W interface is compressed, PC changes its chain alignment to adapt to a smaller area. When a TO/PC/W interface is compressed, TO is progressively expelled from the surface and the ratio of PC/TO increases to accommodate a smaller area, presumably without major changes to PC conformation (33). These considerations make drop tensiometry on a TO/PC/W interface a more realistic, and thus superior, system for studying apolipoprotein behavior at a lipid interface. In this study, we adsorbed N44 and C46 to a TO/POPC/W interface to determine if their difference in lipid affinity determined from vesicle and TO/W interface binding is reflected in their behavior at a TO/POPC/W interface. Surprisingly, we demonstrate that C46 can remove phospholipids from a TAG/W interface when the surface was compressed whereas N44 did not.

Materials and Methods

Materials

The N-terminal (1–44) and C-terminal (198–243) peptides of Apolipoprotein A-1 were synthesized and purified as described earlier (6,17,20). A stock solution of peptide was made by adding 2 mM sodium phosphate buffer, pH 7.4 (PB), to a final peptide concentration of 1 mg/mL. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was purchased from Avanti Polar Lipids (Alabaster, Alabama) dissolved in chloroform to a concentration of 21.3 mg/mL. Triolein (TO), which was >99% pure, was purchased from Nu-Chek Prep (Elysian, MN). Purity of both lipids was checked using high-dose thin-layer chromatography. The bulk buffer for all experiments was 2 mM PB, pH 7.4. All other reagents were analytical grade.

Small unilamellar vesicles (SUVs) were prepared by first drying the POPC by evaporating the chloroform. The lipid was suspended in 2 mM PB, pH 7.4, to a concentration of 1 mg/mL and sonicated for 60 min with a pulsed duty cycle of 30% (33,36). The purity of the SUVs was checked using high-dose, thin-layer chromatography. Negative stain electron microscopy gave a diameter of the POPC SUVs of 238 ± 34 Å.

Drop tensiometer

The interfacial tension (γ), area, and volume of a TO drop was measured using an I.T. Concept (Longessaigne, France) Tracker oil-drop tensiometer (37). The interfacial pressure (Π) is equal to the tension of a clean TO/W surface (32 mN/m) minus the measured tension (Π = 32-γ). A TO drop was formed at the tip of a needle submerged in 2 mM PB, pH 7.4. The drop volume and area were manipulated by a motor attached to a syringe filled with TO. The value Π was automatically calculated based on LaPlace analysis of the bubble shape (37,38). Surface-active molecules (either POPC, N44, or C46) were washed-out of the cuvette by flowing 250 mL of 2 mM PB, pH 7.4, through the cuvette and sucking the buffer off the surface of the cuvette using a vacuum to keep the buffer volume constant. This protocol reduced the bulk concentration by >99.9% (30,33,39,40). All experiments were carried out at 24 ± 0.3°C.

Peptides adsorbed to a TO/POPC/W interface

POPC was deposited on a TO/W interface by injecting 500 μg of POPC SUVs into a 6 mL cuvette containing bulk buffer and a TO drop attached to a syringe (33). The starting area of the drop was varied between experiments depending on the desired initial Π (Π_I). If a high Π_I was desired (i.e., corresponding to a high Г_POPC), the area was large and vice versa for a low Π_I. The starting area varied between 6 and 45 mm². After the addition of the POPC SUVs, Π rose (Fig. 1, item 1). After >5000 s, the nonadsorbed POPC SUVs were washed-out of the cuvette by flowing 250 mL of 2 mM PB, pH 7.4, through the cuvette (Fig. 1, item 2).

Figure 1.

The protocol for determining the exclusion pressure of a peptide adsorbed to a TO/POPC/W interface is shown. A drop of TO was formed in a solution containing POPC SUVs (1). After the POPC adsorbed to the surface, then the excess POPC was washed out of the solution (2, solid bar) and the area was adjusted (3) to achieve the desired Π (Π_I). Either N44 or C46 was added to the solution (4), which caused Π to rise to a new equilibrium Π (Π_P) (in this example, C46 was added). ΔΠ = Π_P–Π_I. After equilibrium, the excess peptide was washed-out of the bulk (5, solid bar) and the drop area was linearly expanded (6) and then compressed (7). During the expansion, the area that corresponded to Π = Π_I is equal to A_w. The difference between A_w and the area at Π_I equals ΔA.

The surface concentration of POPC (Γ_POPC) was estimated by overlaying a drop tensiometry isotherm of a TO/POPC/W interface with a Langmuir trough isotherm of mixtures of POPC and TO above the envelope point as described in detail recently by Mitsche et al. (33). The area per POPC molecule at the collapse point is ∼65 Å²/molecule (33). By assuming that POPC occupies 65 Å²/molecule at a TO/W interface, the percentage of the surface occupied by POPC can be calculated (Fig. S2). Using the same assumption, the average area per POPC molecule (APM) can be calculated at a known Γ_POPC according to the equation

$$APM\left[{\mathit{\AA }}^2\right]=\frac{1}{{\Gamma }_{POPC}}\times \left(\frac{{10}^6\times {10}^{20}}{6.02\times {10}^{23}}\right).$$ (1)

After the washout, the area was adjusted to 30 mm² (Fig. 1, item 3). After Π reached an equilibrium value (which varied between 2 and 22 mN/m), 25 μg of either N44 of C46 were added to 6 mL of bulk buffer to a final concentration of ∼8 × 10⁻⁷ M. Adding peptide caused Π to rise to a new equilibrium value (Fig. 1, item 4). After Π reached equilibrium, one of three protocols were performed before a second washout, which removed the peptide from the aqueous phase:

• Protocol 1. The drop area remained constant at 30 mm² until a second buffer exchange (Fig. 1, items 3–5).

• Protocol 2. The drop area was increased at a rate of 0.036 mm²/s until the area reached ∼45 mm². The area was held constant for at least 10 min, then was compressed at a rate of 0.036 mm²/s until Π was >25 mN/m and then the area was held constant for at least 2 h. The area was then increased at a rate of 0.036 mm²/s until the original area of 30 mm² (Fig. S5, items 1–6).

• Protocol 3. The drop was rapidly compressed to a smaller area and held at that area for ∼15 min. Then it was reexpanded back to the original area. This was repeated with progressively larger compressions ranging from a 7–53% reduction in area all with peptide continuously present in the buffer (Fig. S6).

In different experiments, one of these three protocols was performed. After the protocol was complete, the peptide was washed out of solution by flowing 250 mL of peptide free 2 mM PB, pH 7.4, through the cuvette. After the washout, the drop was expanded at a rate of 0.036 mm²/s, held at a large area for at least 10 min, and then compressed at a rate of 0.036 mm²/s until Π was above 25 mN/m to obtain a Π/area curve.

Based on these protocols, we defined the following parameters to compare peptide adsorption at different surface concentrations of POPC:

• Π_I, Initial pressure of a TO/POPC/W interface before peptide was added.

• Π_P, Pressure after peptide was added.

• ΔΠ, Change in pressure from adding peptide (ΔΠ = Π_P–Π_I).

• Π_EX, Pressure where peptide is excluded from the interface (ΔΠ = 0).

• ΔΠ_W, Change in pressure during a washout (solid bar).

• A_I, Area at Π_I before peptide was added.

• A_W, Area where Π = Π_I during a slow expansion after peptide was added and washed-out.

• ΔA, Change in area at the same pressure from adding peptide ((A_w–A_I)/A_W).

• APM_EX, area per molecule at the exclusion pressure (Π_EX).

Many of these parameters are graphically defined in Fig. 1 and are reported in Fig. 2 and Fig. S3. These parameters are useful for defining and comparing a peptide's interfacial affinity (ΔΠ), exchangeability (ΔΠ_W), and surface area (ΔA) at a TO/POPC/W interface.

Figure 2.

By varying Π_I, the relationship between Π_I and ΔΠ was established. When C46 was added (solid diamonds), ΔΠ was linearly related to Π_I (ΔΠ = −0.6481 Π_I + 16.646; R² = 0.9966) with an extrapolated exclusion Π (Π_EX) of 25.8 mN/m. When N44 was added (shaded squares), ΔΠ was linearly related to Π_I when Π_I was <18 mN/m (ΔΠ = −0.6258 Π_I +12.929; R² = 0.9892) and Π_EX = 19.3 mN/m. Above Π_I = 18, ΔΠ was constant at 1.9 ± 0.3 mN/m regardless of Π_I.

Results

Adsorption of N44 and C46 to a TO/POPC/W interface

Both N44 and C46 adsorbed to a TO/POPC/W interface causing Π to increase to a new equilibrium level (Π_P, Fig. 1). The change in pressure caused by peptide adsorption (ΔΠ) was linearly dependent on the pressure of the surface before peptide was added (Π_I). At a low Π_I, ΔΠ was larger than at a higher Π_I for both peptides (Fig. 2). For both peptides, the absolute Π after peptide addition (ΔΠ + Π_I) was larger when Π_I was larger. When C46 adsorbed to a TO/POPC/W interface, the relationship between ΔΠ and Π_I was linear (ΔΠ = −0.6481 Π_I + 16.646; R² = 0.9966).

The exclusion pressure (Π_EX) is defined as the Π_I where peptide does not adsorb to the surface and ΔΠ equals zero. The Π_EX of C46 was 25.8 mN/m, meaning that C46 cannot adsorb to a TO/POPC/W interface at a Π > 25.8 mN/m. When N44 adsorbed to a TO/POPC/W interface and Π_I was <18 mN/m, the relationship between ΔΠ and Π_I was linear (ΔΠ = −0.6258 Π_I +12.929; R² = 0.9892). However at high Π_I (Π_I >18 mN/m), N44 adsorption resulted in a constant ΔΠ, regardless of Π_I, of 1.9 ± 0.3 mN/m. At low Π_I the apparent Π_EX of N44 was 19.3 mN/m, but at Π_I > Π_EX ΔΠ does not equal zero, indicating that peptide can still penetrate the surface.

The relationship between Π_I and surface concentration of POPC (Γ_POPC) is known (see Materials and Methods, and Fig. S3), thus the relationship between area per POPC molecule (APM) and ΔΠ can be calculated (Fig. S3 A). At a low Γ_POPC, ΔΠ was larger than at a high Γ_POPC. C46 caused a larger ΔΠ than N44 at the same APM. A higher Γ_POPC prevents the adsorption of more peptide to the interface. To extrapolate the area per POPC molecule where the peptide was excluded from the surface (APM_EX), APM must be correlated to ΔΠ. APM is logarithmically related to Π_I (see Eq. 1), and Π_I is linearly related to ΔΠ; thus, although APM may appear linearly related at ΔΠ, a logarithmic fit is more realistic. The APM_EX for C46 and N44 were 120 and 154 Å²/POPC molecules, respectively. For both peptides the APM_EX was larger than the area per POPC molecule of a fully compressed TO/POPC interface, which is ∼95 Å²/POPC molecule (33).

Postwashout expansions and compressions

After the peptide adsorbed to the interface, the excess peptide was washed-out of the bulk by flowing peptide-free buffer through the cuvette. During the exchange, Π fell to a new steady-state value. The change in Π during washout (ΔΠ_W) for C46 was 3.1 ± 0.2 mN/m and was 3.8 ± 0.4 mN/m for N44. ΔΠ_W was independent of Π_I, except when ΔΠ < ΔΠ_w, in which case Π returned to Π_I after washout. In other words, for N44 when ΔΠ was <3.8 mN/m, washout caused Π to return to the same value as before the peptide was added.

After the washout, the surface was expanded then compressed at a slow linear rate. As the surface was expanded, Π fell until at a specific area (A_W) where Π equaled Π_I, at which the surface had the same energy as it had before the peptide was added. The difference in area between the area at Π_I and A_W (ΔA, see Fig. S3 B) is the amount of surface covered by peptide. When ΔA is normalized to A_w, the percentage of the surface covered by peptide can be estimated as a function of Π_I (Fig. S3 B). For both peptides as Π_I decreased, less of the surface was occupied by POPC and more by peptide. At the same Π_I, C46 occupied a higher percentage of the surface than N44.

As the surfaces were compressed, Π increased at an increasing rate until a discontinuity caused the slope to decrease, then the slopes gradually increased again (Fig. 3, left). The discontinuity in the curve is commonly referred to as the “envelope point”, and is indicative of a change in the monolayer. Below the envelope point, the isobaric APMs were larger when Π_I was smaller, meaning that POPC was less concentrated at the same Π when Π_I was smaller and more peptide went onto the surface (Fig. S4). The area per POPC molecule at the envelope point (APM_ENV) and the pressure at the envelope point (Π_ENV) were also dependent on Π_I (Fig. 3, middle and right).

Figure 3.

Postwashout Π-A compression isotherms of either N44 or C46 at a TO/POPC/W (Left panel solid curve) have a discontinuity in the slope called the envelope point (solid arrow). A TO/POPC/W interface without any peptide had no discontinuities in the slope and has a smaller APM at the same Π (left panel shaded curve). For both N44 (middle panel) and C46 (right panel), the area per POPC molecule (APM, solid squares) and pressure (Π, shaded circles) at the envelope were dependent on Π_I. This indicates that both peptides form a homogeneous surface with POPC and TO.

At a lower Π_I, APM_ENV was larger and Π_ENV was smaller. Therefore, the APM and Π at the envelope point are inversely related to one another. The area per POPC molecule at the envelope point was smaller for C46 than N44 and Π_ENV was higher for C46. Above the envelope points, the N44 isotherms roughly overlay with one another where there is a unique APM at a given Π, regardless of Π_I, indicating only 1° of freedom to the surface (Fig. S4). Above the envelope point when Π_I was >11 mN/m, the isotherms overlay with one another, but below Π_I = 11 mN/m the isotherms have a higher Π at the same APM. When Π_I was smaller, the Π was higher at the same APM. In all cases, Π was greater than a TO/POPC/W interface without any peptide at all APMs.

Prewashout expansions and compressions

In some experiments, after the peptide was adsorbed the surface area was linearly expanded, compressed, held at a small area, and then reexpanded to the original area (see Protocol 2 in Materials and Methods). The peptide was then washed-out of the cuvette and the surface was expanded and compressed (Fig. S5). During the prewashout expansion (Fig. 4, A and B, curve 1), Π moderately declined but to a lesser extent than during a postwashout compression (Fig. 3, left). When the surface was compressed (Fig. 4, A and B, curve 2), the isotherms were slightly higher than the expansion isotherm (<1 mN/m).

Figure 4.

(A and B) Pre- and postwashout compression isotherms of N44 (A) and C46 (B) adsorbed to a TO/POPC/W interface. After N44 was adsorbed to the interface, the surface was expanded (1, blue), compressed (2, red), then reexpanded back to the original area (3, green). After compression and reexpansion of N44, the Π remained the same at the same area, whereas C46 had a smaller Π at the same area, i.e., the difference between curve 1 and 3. A washout caused Π to fall (solid arrows). The surface was then expanded (4, purple) and compressed (5, cyan). (C and D) The Π-A relationship of a postwashout compression isotherm when there was a compression before the washout (red, Fig. S2, item 9) or no compression before the washout (blue, Fig. 1, item 7) of N44 (C) and C46 (D) at the same Π_I. When N44 was compressed at a Π_I equal to 11.7 and 11.5 mN/m with and without a prewashout compression, respectively, the isotherms aligned and behaved similarly. When C46 was compressed at a Π_I equal to 17.2 and 17.5 mN/m with (red) and without (blue) a prewashout compression, respectively, the isotherm with a prewashout compression had a lower Π_ENV, and the Π-area relationship was similar to an isotherm with a lower Π_I. This indicates that during the prewashout compression some of the phospholipid was removed from the surface by C46.

After the compression, the surface was left at a small area for >2 h. During the time at the small area, the Π fell; however, the behavior was different for N44 and C46. The N44 surface fell 3–4 mN/m over the first 10 min and then maintained a new steady-state Π value. When the area was held constant after the compression, the C46 surface continuously fell in Π to a value 5–10 mN/m lower than the initial value, depending on how large the compression was, the concentration of POPC, and the length of wait time. When the surface was reexpanded to the original area (Fig. 4, A and B, curve 3), the N44 surface returned to the original Π whereas the C46 surface returned to a smaller Π at the same area than before the compression.

During the washout, the change in pressure (shown by the solid arrow in Fig. 4, A and B) was consistent with other washout experiments (see above). When the surface was reexpanded after the washout (Fig. 4, A and B, curve 4), Π fell at a faster rate than the prewashout expansion (Fig. 4, A and B, curve 1) because there is no peptide to adsorb to the surface. When the surface was compressed, the isotherm followed the postwashout expansion isotherm, then exhibited an envelope point. After the envelope point, the isotherm followed the second preexchange expansions (curve 3) until the area was smaller than the minimum area of the expansion and then as the surface was compressed Π continued to rise. During the N44 compression, the APM remained in the same pressure range as the postexchange compressions (Fig. S4). The C46 compression isotherm occurred at a smaller APM and during the high Π compression (Fig. 4 B, far left) the APM was <40 Å², which is much less than the minimum area of POPC of ∼65 Å². This indicates that some of the POPC must have been removed from the surface by C46.

By comparing postexchange compression isotherms at the same Π_I when there was a compression before washout (Fig. S5, item 9) and when there was not a compression before washout (Fig. 1, item 7), the effect of a prewashout compression can be deduced. The postwashout compression isotherm of N44 with and without a compression before washout behaved similarly starting at the same Π_I (Fig. 4 C). At a given APM, the Π for both isotherms was within 1 mN/m throughout the compression and the envelope point occurred at the same Π and APM. The postwashout compression isotherm of C46 with a compression before washout had different characteristics than a postwashout compression isotherm without a compression before washout. The isotherm after the prewashout compression had a lower APM at the same Π, a lower envelope Π, and had a slope characteristic of a surface with a lower Π_I. Note that the APM assumes that all the POPC that was on the surface before peptide addition was still on the surface.

In a different set of experiments (see Protocol 3 in Materials and Methods), after each peptide was adsorbed to a TO/POPC/W interface and reached an equilibrium Π, the surface was rapidly compressed and reexpanded back to the original area (Fig. S6). When the N44 surface was compressed, Π rapidly rose followed by a relaxation to a lower Π, which was higher than the equilibrium Π before the compression. Larger compressions yielded a larger difference. When the surface was reexpanded, Π immediately fell but quickly returned to the Π before the compression. When the C46 surface was compressed, Π immediately rose, but was followed by a gradual lowering of Π above the precompression equilibrium. When the surface was reexpanded to the same area, Π was lower than it was before the compression. Repeated compressions caused Π to approach the Π of C46 at a TO/W interface without any POPC present (17).

Discussion

N44 and C46 are similar peptides. Both have comparable molecular weights, are unfolded in solution, and upon lipid binding adopt an amphipathic α-helical conformation (6,20). We estimated ΔG from water to oil (ΔG_W→O) of C46 is −2.55 kcal/mol whereas ΔG_W→O of N44 is −1.47 kcal/mol, indicating that C46 has a higher affinity for a hydrophobic interface than N44 (17). The higher affinity for a hydrophobic surface was reflected in the fact that C46 exerted 2.5 mN/m of Π more than N44 at a TO/W interface.

The difference in hydrophobicity was also seen at a TO/POPC/W interface where at the same Π_I, ΔΠ was ∼2.5 mN/m higher for C46 than N44. At a high phospholipid Г, the energy of the surface is lower because the phospholipid headgroups shield the hydrophobic acyl-chains of TAG and POPC from water. When peptides adsorb to a lower energy surface there is less energy gained from adsorption, thus a smaller ΔΠ. When ΔΠ was equal to zero there is no energy gained from peptide adsorption. This defines the exclusion pressure (Π_EX). Therefore, at a higher concentration of POPC, less peptide adsorbed to the surface indicating that they compete for the same binding sites on the TO/W interface. Because the POPC and peptides occupy the same portion of the surface, there would be effectively fewer binding sites for C46 or N44 to interact with the surface at a higher concentration of POPC (thus higher Π_I). This is reflected in the dependence of ΔA/A_W on Π_I. Because less peptide adsorbed, ΔΠ was smaller.

Adsorption was driven by the peptide affinity for the lipids on the surface of the TO/POPC/W interface. At the interface, the lipophilic amino acids are exposed to oil and the hydrophilic amino acids are exposed to water, which minimizes the energy of the peptide. When the peptides bind to the interface, they can potentially bind to POPC chains and/or patches of interfacial TO which are displaced from the surface by peptide binding. At a low Π_I more TO is exposed to water, and more peptides bind probably to TO. At high Π_I, peptide must contact both POPC and TO. When Π_I > Π_EX, the POPC was too tightly packed and patches of TO were too small to accommodate C46. When Π_I > Π_EX, N44 was still able to penetrate the surface, lowering Π by ∼1.9 mN/m. The intrinsic flexibility of N44 may allow a small portion of the peptide to interact with the surface.

When a TO/POPC/peptide/W surface was expanded with peptide in the bulk (prewashout expansion, Fig. 4, curve 1) there was a moderate decline in Π. During the expansion, the peptide already adsorbed to the surface had more space to spread and during the expansion more TO became exposed to water. The exposed TOs formed new binding sites for peptide, allowing adsorption, and increasing the peptide/POPC in the surface. Both N44 and C46 can be pushed off the surface by compression (17,18). When the surface was compressed, the adsorbed peptide was ejected from the surface. When the surface was expanded without peptide in the bulk (postwashout expansion, Fig. 1, curve 7), Π fell more dramatically than with peptide in the bulk. This is due to peptide not being available to bind to the interface on expansion, allowing TO molecules to become exposed to water and adopt a trident conformation at the surface, where all three ester groups are in the interface facing the water (35). Because TO is less amphipathic than POPC or peptide, the Π was less. After an expansion, the surface was compressed, causing Π to increase. The least surface-active component, TO, was first progressively expelled from the interface into the TO bubble and later, at the envelope point, peptide began to be pushed into the water phase.

Below Π_ENV, Π was dependent on both APM and Π_I (Fig. S4). Above the envelope point, the isotherms roughly overlay one another, indicating that Π was directly related to APM and independent of Π_I. In other words, at the envelope point, the degrees of freedom of the system were reduced from 2 to 1. The area and Π where the envelope point occurred was dependent on Π_I. The envelope point represents the point in the compression where the peptide began to be expelled from the surface. Because the peptide is soluble in water, as it was expelled from the surface it dissolved in the aqueous buffer. Further compression caused more peptide to be expelled. A high Г_POPC allows some peptide to remain in the interface even at high Π.

The phases of a multicomponent monolayer are reflected in the value of Π_ENV as a function of the starting compositions. If the components form a heterogeneous monolayer with separate phases, the Π_ENV will occur at the same value regardless of the composition of the monolayer. This occurs because the less surface-active component, in this case peptide, will be ejected from the monolayer at its collapse point. Because the only interactions are at the phase boundaries, Π_EX will be unaffected by the other component. If the components form a homogeneous monolayer, Π_ENV will be dependent on the composition of the monolayer (41). When a homogeneous surface is enriched in the more surface-active component, in this case POPC, Π_ENV will be higher due to interactions between the surface molecules holding the less surface-active molecule in the interface. For both N44 and C46, Π_ENV was dependent on the composition of the surface. Therefore, the peptide and POPC form a homogeneous monolayer where both components interact with one another and rest on the TO core.

When a TO/POPC/N44/W surface was compressed and expanded the surface returned to the same Π, whereas a TO/POPC/C46/W surface returned to a lower Π at the same area (Fig. 4, A and B). Repeated compressions and expansions of a TO/POPC/C46/W interface caused Π to approach the Π of a TO/C46/W interface. During the compression, phospholipid was removed from the surface by C46, but not by N44. This is supported by the fact that the postwashout compression resembled a surface with a higher peptide/POPC and upon repeated compression the surface approached the Π of a TO/C46/W interface without POPC (Fig. 4 D and Fig. S6). Therefore, phospholipid was removed from the surface during the C46 compression but not during the N44 compression.

In summary, when N44 or C46 bind to a hydrophobic interface, the peptides adopt an amphipathic α-helical conformation. In this conformation, the hydrophobic amino acids interact directly with the acyl-chains of TO and/or POPC whereas the hydrophilic amino acids remain solvated with water and interact with the zwitterionic phospholipid headgroup. The peptides displace the triolein molecules from the interface, which diffuse into the core of the droplet. Because these peptides are more amphipathic than triolein, peptide binding lowers the energy of the interface (and thus raises the pressure). When C46 or N44 are compressed at a TO/W interface, the peptides are ejected from the surface and can readsorb when the surface is reexpanded.

At a TO/POPC/W interface, both peptides are also ejected from the interface by compression, but C46 removes phospholipids from the surface whereas N44 does not. When C46 was compressed it remained associated with POPC and was ejected from the surface as a peptide-lipid complex. This process is depicted in Fig. 5. C46 is generally more hydrophobic (Table S1) and structurally rigid than N44, which may be the reason why C46 remains associated with POPC whereas N44 does not. The phospholipid transfer activity of the C-terminus of ApoA-I could have a physiologically important role in lipoprotein metabolism and homeostasis.

Figure 5.

Schematic representation of C46 adsorption to a TO/POPC/W interface where peptide binding induces amphipathic α-helical formation (blue and red circles) and where the hydrophobic face of the helix (green arc) displaces triolein from the interface. Compression of the surface by decreasing the droplet area causes the peptide to be ejected from the surface and transfer phospholipid from the surface to the aqueous buffer. Compression of a peptide-free surface is shown below. N44 also adsorbed to a TO/POPC/W interface, but when the surface is compressed it is ejected without POPC (not shown).

In circulation, triglycerides in chylomicrons or VLDL are hydrolyzed by lipoprotein lipase yielding a monoacylglyceride and two free fatty acids. The fatty acids are ultimately transferred to albumin and peripheral tissues. This process decreases the lipoprotein core volume and acutely increases the amount of surface molecules which, in turn, increases the Π in the particle surface. As the Π increases, exchangeable lipoproteins, such as ApoA-I, are ejected from the lipoprotein surface along with other surface components.

In 1978, Tall and Small (42) proposed that some HDL was produced from the excess surface phase of chylomicrons and VLDL caused by depletion of the core phase. The excess surface phase either forms unstable lamellar bilayer fragments or pre-HDL-like particles directly from the surface. This model has corroborated the finding that radio-labeled phospholipids were transferred from the chylomicron to HDL fraction (43). Phospholipid transfer protein (PLTP) has an important role in the transfer of the surface components of triglyceride-rich lipoprotein to HDL. In a PLTP^−/− mouse there was a dramatic reduction in the transfer of radio-labeled phospholipids from vesicles to HDL versus a wild-type mouse. In addition, the PLTP^−/− mouse had a fourfold reduction in HDL and sevenfold reduction in plasma ApoA-I concentration (44). Thus, phospholipid transfer plays a major role in the formation of human HDL particle, and PLTP and ApoA-I may work in concert to facilitate phospholipid transfer.

The depletion of TAG from the core of lipoproteins by lipoprotein lipase is analogous to the experiments performed in this study. As the core is depleted (i.e., the drop is compressed), exchangeable apolipoproteins are pushed out of the surface because of the higher Π. This study demonstrated that the C-terminus of ApoA-I associates with phospholipids and removes them from the interface, presumably forming a phospholipid/C46 complex. This occurs without any PLTP in the system. This complex may form in vivo with the assistance of PLTP and be comparable to a pre-β-HDL particle, which is further lipidated by ABCA-I and ABCG-I (1). The formation of pre-HDL particles from the surface of VLDL or chylomicrons may serve as a form of feed-forward control on cholesterol homeostasis. Understanding this process is important to understanding the stability and formation of HDL particles and could be exploited to increase the quantity and quality of plasma HDL.