On the surface of lipoproteins, it is reasonably assumed that apolipoproteins, including apoC-I, interact with surface-located phospholipids (55
). It is also possible that apolipoproteins interact with core molecules, such as TAG or CE, notably when the number of surface lipid molecules is too small to adequately cover the core (55
). In this study, our goals were twofold. First, we sought to characterize the surface activity and reversible binding of human apoC-I at TO/W and the more physiological POPC/TO/W interfaces. Second, we sought to understand the effects of apoC-I interactions with POPC on peptide desorption from and retention on the lipoprotein surface.
The first objective of this study was to demonstrate the surface activity of apoC-I at TO/W and POPC/TO/W interfaces. demonstrates the surface activity of apoC-I at both TO/W and POPC/TO/W interfaces. An increase in the rate of peptide adsorption was observed as apoC-I concentration in the aqueous phase increased. The decrease in γ (or increase in Π) upon binding to each interface was comparable, equal to 16.5–18.0 mN/m and 15.5–16.0 mN/m, respectively. At a concentration of 1.2 × 10−6
M, similar to that of apoC-I used at a TO/W interface, [1–44] apoA-I and [198–243] apoA-I could increase Π by around 13.0 mN/m, respectively, at a TO/W interface (55
). This suggests a higher surface affinity for apoC-I than the C- and N-termini of apoA-I.
The observed surface affinity of apoC-I for either interface is imparted by its structure. Apolipoproteins exhibit strong lipid-binding properties due to their characteristic secondary structures, containing amphipathic α-helix (AαH) or amphipathic β-sheets (AβS) (29
). The lipid-binding motifs of apoC-I lie in its predicted N- and C-terminal AαHs (25
), as shown in . The hydrophobic face of each AαH (marked by a dotted line) is essential for interactions with apolar moieties on phospholipids (here, POPC) and neutral lipids (here, TO). According to the scale determined by Wimley and White (62
of partitioning for the hydrophobic face of the N-terminal AαH is 3.76 kcal/mol, or 0.42 kcal/mol per residue. ΔGb→w
for the hydrophobic face of the C-terminal AαH is 4.13 kcal/mol, or 0.69 kcal/mol per residue. Preferential lipid-binding is implicated in the unfavorable, positive ΔGb→w
of transfer from
a bilayer interface to
the aqueous phase for the hydrophobic faces of both AαHs.
Linked with its surface activity, we sought to determine the nature of apoC-I binding to TO/W and POPC/TO/W interfaces. AαH structures typically show reversible binding to the TO/W surface, desorbing from and readsorbing to the surface upon compression and re-expansion (49
). reveals that apoC-I reversibly
binds to both interfaces, desorbing when each interface is instantaneously compressed above ΠMAX
. ApoC-I’s complete peptide desorption is evident in the γ responses of both interfaces for compression-expansion protocols after buffer exchange (right side of ). With peptide present in the aqueous phase (), apoC-I readsorbs to both interfaces on re-expansion, decreasing γ to its initial equilibrium value (15.0 mN/m and 9.5 mN/m, respectively).
The left half of suggest conformational rearrangement on compression of the AαHs of apoC-I prior to complete peptide desorption at both interfaces, as the γe values reached after each compression are lower than the initial γe. The rise in γ at both interfaces due to the buffer exchange depicted in further suggests peptide conformational rearrangement at the surface, potentially followed by complete peptide desorption into the aqueous solution. To explain such conformational rearrangement, results of previous studies may suggest a two-step desorption model for apoC-I, with one AαH desorbing prior to complete peptide ejection.
Compression of apoC-I deposited on a DPPC monolayer at an A/W interface revealed two phase transitions (46
). The first occurs between Π = 24–27 mN/m and the second at Π ~ 49 mN/m. Brewster Angle Microscopy showed that only apoC-I was affected in each phase transition (46
) This led to the proposal that, at Πs from 24–27 mN/m, one AαH on apoC-I desorbs from the subphase, aligning itself towards the air, in a similar direction to that of the hydrophobic DPPC acyl-chains (46
). The entire peptide was ejected from the monolayer at Π ~ 49 mN/m.
Indicative of the order of apoC-I AαH desorption, the amide proton resonance line widths and deuterium exchange rates of the NMR structure of apoC-I:sodium dodecyl sulfate complexes suggest that the N-terminal AαH binds less tightly to the detergent than the C-terminal AαH (25
). In agreement with these results, James, et. al., used mass spectrometry to show that the C-terminal region was the portion of apoC-I most highly protected from proteolysis (36
). DMPC clearance assays and electron microscopy further revealed that single or double mutations in the apolar face of the C-terminal AαH of F42 and F46 to G or A result in a 2–3.5-fold reduction in apoC-I binding affinity for DMPC vesicles and apoC-I:DMPC complexes with atypical diameters (36
). However, CD studies of apoC-I:DMPC complexes show that interruptions in the apolar face of either
the N- (G15P) or C-terminal (T41P, T45P) AαH greatly reduce protein-lipid binding (20
).Of all explored mutations, these also show the lowest ability to reconstitute apoC-I:DMPC disks on thermal renaturation (20
). This indicates an essential cooperativity in binding of the α-helices to the phospholipid surface (20
).The thermal denaturation and reconstitution of apoC-I:DMPC complexes, even at low ramp rates and small temperature changes, failed to show multiphase kinetics indicative of a two-step peptide desorption from the lipid/water interface (20
If apoC-I follows a two-step desorption from the lipoprotein surface, the NMR structure of apoC-I and greater ΔGb->w values for the hydrophobic face of the C-terminal AαH predict that the N-terminal AαH desorbs prior to complete peptide desorption. Consequently, in the seconds following each instantaneous compression in , the N-terminal AαH desorbs from the surface. This conversion is followed by a longer period of desorption of the C-terminal AαH of apoC-I to reach a new peptide equilibrium with the bulk solution.
Having characterized the reversible binding of apoC-I to TO/W and POPC/TO/W interfaces, the second goal was to determine the effect of POPC on apoC-I surface binding and retention. To that end, the pressure ΠMAX
, at which the entire peptide begins to be ejected from TO/W and POPC/TO/W interfaces was determined. reveals a ΠMAX
value of 16.8 mN/m for apoC-I at a TO/W interface. This ΠMAX
is consistent with previous findings for AαHs at a TO/W interface, falling within the range of 13–19 mN/m (49
). Comparatively, the ΠMAX
values for [198–243] apoA-I and [1–44] apoA-I are 16.2 and 13.2 mN/m, respectively (55
for apoC-I at a POPC/TO/W depends on ΓPOPC
. reveals that apoC-I has a ΠMAX
of 20.7 mN/m when ~38% of the TO drop is covered by POPC. This higher ΠMAX
at a POPC/TO/W interface is likely due to AαH interactions with phospholipids and neutral lipids increasing peptide affinity for the surface, keeping it bound at higher Πs.
The increase of affinity for the interface induced by apoC-I/POPC interactions was further explored by sinusoidal oscillations of both interfaces following apoC-I adsorption. reveals the elastic properties of apoC-I at TO/W and POPC/TO/W interfaces. Previous studies have revealed that apoA-I, the major apolipoprotein constituent of HDL and primary activator of LCAT, binds TO/W interfaces via its AαH structural motifs and creates a highly flexible visco-elastic interface (52
). Similarly, analysis of large oscillations or small oscillations with a slow period in reveals apoC-I forms a visco-elastic surface at the TO/W interface, marked by a large hysteresis in compression and expansion Π-A isotherms, a low ε, and large phase angle (
ApoC-I at a TO/W interface exhibits a marked hysteresis in its Π-A oscillation isotherms only at compressions of larger amplitudes and longer periods. This supports the two-step model of apoC-I desorption from the interface, as ample time must be provided for the N-terminal AαH to desorb prior to complete peptide desorption. Oscillations of amplitude ± 2 µL over a range of increasing periods yield Π-A isotherms for apoC-I at a TO/W interface with
increasing by 12.9° from 10.6 to 23.5° and ε decreasing by 9.2 mN/m from 29.6 to 20.4 mN/m.
Comparatively, the Π-A isotherms for apoC-I at a POPC/TO/W interface, oscillated over a range of amplitudes and periods, exhibit a much smaller hysteresis, even at larger amplitudes and longer periods. This is indicative of a more elastic interface. Oscillations of amplitude ± 2 µL over a range of increasing periods yield Π-A isotherms for apoC-I at a POPC/TO/W interface with
increasing by only 6.2° from 6.4 to 12.6° and ε decreasing by only 3.1 mN/m from 51.5 to 48.4 mN/m. Compared to similar oscillations at a TO/W interface, these higher
ε and lower
values are more resistant to change at a POPC/TO/W interface, demonstrative of a more elastic interface. These data indicate that apoC-I AαH interactions with POPC increase its affinity for and retention on the surface, thereby increasing surface elasticity.
To elucidate the role of AαH/phospholipid interactions on peptide retention at the lipoprotein surface, TO drops with apoC-I bound and increasing ΓPOPC underwent slow expansions and subsequent compressions. and show the dependency of the expulsion of apoC-I from the POPC/TO/W interface on ΓPOPC of the TO drop. Increases in ΓPOPC of the interface yield increases in ΠE and decreases in AE, that is, the envelope pressure and area, respectively, at which apoC-I begins to be ejected from the interface. When POPC covers 32.8% of the TO drop surface (line (a) in ), ΠE = 21.2 ± 0.2 mN/m and AE = 37.3 ± 0.2 mm2, while at a POPC coverage of 51.8% (line (d)), ΠE = 26.4 ± 0.3 mN/m and AE = 22.2 ± 0.3 mm2. These results reveal that larger POPC:apoC-I ratios cause retention of apoC-I to higher Πs and more compact areas.
Based on the findings of this study, we propose possible mechanisms by which apoC-I may contribute to steps of lipoprotein remodeling, including CETP inhibition and prevention of hepatic uptake of TAG-rich lipoproteins. In order to inhibit CETP activity, the N-terminus of apoC-I could desorb from the lipoprotein surface, interacting with CETP and thereby interfering with its function (7
). With no discovered CETP-binding domain on apoC-I, a more likely mechanism of inhibition (66
) is that apoC-I has a higher affinity for the lipoprotein surface than CETP or its activators: apolipoproteins A-I, A-II, A-IV, and E (63
). ApoA-IV has the weakest lipid affinity of any apolipoprotein (68
) and Human C-apolipoproteins have been shown to displace apoA-IV from TAG-rich lipoprotein surfaces (69
). The C-terminus of apoA-I is predicted to be the most lipophilic portion of apoA-I, with the N-terminal helix bundle providing stability at the lipoprotein surface (70
). Yet, as shown, apoC-I causes greater Π changes and has a higher ΠMAX
than the C-terminal of apoA-I (55
). Under this model of inhibition, apoC-I, at a high enough plasma concentration, could bind TAG-rich lipoproteins or compact HDL particles and drastically increase Π. These Π modifications could displace bound CETP and CETP activators or prevent them from binding (66
). Structurally similar to apoA-I, apoE could also be displaced from β-VLDL by apoC-I-induced Π modifications, thereby inhibiting lipoprotein uptake (16
In summary, our interfacial studies reveal that apoC-I is surface active at both TO/W and POPC/TO/W interfaces, binding the interfaces via its two amphipathic α-helices and lowering interfacial tension, γ. ApoC-I exhibits properties of an exchangeable apolipoprotein, desorbing upon compression and readsorbing upon re-expansion of either interface. AαH interactions with POPC increase apoC-I affinity for the interface, demonstrated by a higher ΠMAX
when roughly 38% of the interface was covered with POPC. AαH interactions with POPC also increase surface elasticity, demonstrated by a higher elasticity modulus (ε), a lower phase angle (
), and higher Πs upon compression during interface oscillation. Finally, apoC-I affinity for the POPC/TO/W interface increases as ΓPOPC
increases, evident in higher envelope point Πs at lower envelope areas upon compression. We propose that the high affinity binding apoC-I to lipoprotein surfaces provides a possible model for inhibition of CETP activity by displacing or preventing the binding of its apolipoprotein activators. Similarly, the high Π changes on binding to triacylglycerol-rich lipoproteins could displace or prevent the binding of apoE, so as to inhibit their receptor-mediated uptake.