Lipid exchange of apolipoprotein A-I amyloidogenic variants in reconstituted high-density lipoprotein with artificial membranes

Abstract

High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.

Keywords: amyloidogenic variants; apolipoprotein A‐I; high‐density lipoprotein; reconstituted HDL.

FIGURE 1

rHDL ApoA‐I variants ability to remove lipids from fluid‐saturated membranes made of d‐DMPC (a and b) and monounsaturated membranes made of d‐POPC (c and d), in the absence of cholesterol depends on the ApoA‐I variant and the lipid composition. rHDL (0.132 mg/mL) particles made of POPC (b and d) or DMPC (a and c) were incubated for 3 h on the SLBs in h‐TBS at 37°C. The data represent the decrease in the signal of the C–D asymmetric peak at ~2100 cm⁻¹ and the means ± SE of at least two independent measurements. ApoA‐I, apolipoprotein A‐I; d‐DMPC, perdeuterated 1,2‐dimyristoyl‐d₅₄‐3‐sn‐glycerophosphatidylcholine; d‐POPC, perdeuterated 1‐palmitoyl‐oleoyl‐d₆₄‐3‐sn‐glycerophosphatidylcholine; h‐TBS, 50 mM Tris buffer, 150 mM NaCl, pH 7.4 in H₂O; rHDL, reconstituted high‐density lipoprotein; SLBs, supported lipid bilayer.

FIGURE 2

Ability of rHDLs ApoA‐I variants to remove lipids in fluid‐saturated membranes made of d‐DMPC in the presence of cholesterol. rHDL (0.132 mg/mL of the ApoA‐I proteins) samples made of DMPC (a) or POPC (b) were incubated for 3 hours on the SLBs in h‐TBS at 37°C. The data represent the decrease in the signal of the C‐D asymmetric peak at ~2100 cm⁻¹ and represent the means ± SE of at least two independent measurements. ApoA‐I, apolipoprotein A‐I; d‐DMPC, perdeuterated 1,2‐dimyristoyl‐d₅₄‐3‐sn‐glycerophosphatidylcholine; h‐TBS, 50 mM Tris buffer, 150 mM NaCl, pH 7.4 in H₂O; POPC, 1‐palmitoyl‐oleoyl‐d₆₄‐3‐sn‐glycerophosphatidylcholine; rHDL, reconstituted high‐density lipoprotein; SLBs, supported lipid bilayer.

FIGURE 3

Ability of the POPC and DMPC nanodiscs containing WT or L174S ApoA‐I proteins to remove and deposit PLs. Right panel: Neutron reflection profiles including best fits for supported lipid bilayers composed of d‐POPC, before (, CmSI; , d‐TBS; , h‐TBS) and after (, CmSI; , d‐TBS; , h‐TBS) incubation with 0.132 mg/mL rHDL containing the (a) POPC WT, (b) POPC L174S, (c) DMPC WT, and (d) DMPC L174S, measured at 37°C in d‐TBS, h‐TBS, and CmSi‐Tris. Left panel: SLD profiles of the SLB structures before (short dash) and after (solid line) incubation. Illustrations correspond to the SLB structure after incubation and rinse. ApoA‐I, apolipoprotein A‐I; d‐POPC, perdeuterated 1‐palmitoyl‐oleoyl‐d₆₄‐3‐sn‐glycerophosphatidylcholine; DMPC, 1,2‐dimyristoyl‐d₅₄‐3‐sn‐glycerophosphatidylcholine; h‐TBS, 50 mM Tris buffer, 150 mM NaCl, pH 7.4 in H₂O; PLs, phospholipids; rHDL, reconstituted high‐density lipoprotein; SLB, supported lipid bilayer; SLD, scattering length density; WT, wild‐type.

FIGURE 4

rHDL capacity to remove and deposit PLs in unsaturated membranes. The reflectivity profiles and best fits for the kinetic are shown in Figure S7 in Data S1. Errors are derived from Bayesian MCMC analysis. PLs, phospholipids.

FIGURE 5

SAXS profiles including best fits obtained by using a core‐shell‐bicelle‐elliptical‐belt‐rough model for POPC rHDL WT (a), POPC rHDL L174S (c), DMPC rHDL WT (b), and DMPC rHDL L174S (d) at 37°C. DMPC, 1,2‐dimyristoyl‐d₅₄‐3‐sn‐glycerophosphatidylcholine; POPC, 1‐palmitoyl‐oleoyl‐d₆₄‐3‐sn‐glycerophosphatidylcholine; rHDL, reconstituted high‐density lipoprotein; SAXS, small‐angle x‐ray scattering; WT, wild‐type.

FIGURE 6

Structural features of the POPC and DMPC nanodiscs containing WT or L174S ApoA‐I proteins. (a) Schematic representation of the model used in SASview to fit the SAXS data. (b, d, e, and g) Parameters obtained from the best‐fit curves are shown in Figure 5. Table S5 in Data S1 provide all the parameters obtained after fitting. (c and f) Calculated values. ApoA‐I, apolipoprotein A‐I; DMPC, 1,2‐dimyristoyl‐d₅₄‐3‐sn‐glycerophosphatidylcholine; POPC, 1‐palmitoyl‐oleoyl‐d₆₄‐3‐sn‐glycerophosphatidylcholine; SAXS, small‐angle x‐ray scattering.