Effects of protein oxidation on the structure and stability of model discoidal high-density lipoproteins

Abstract

High-density lipoproteins (HDLs) prevent atherosclerosis by removing cholesterol from macrophages and by providing antioxidants for low-density lipoproteins. Oxidation of HDLs affects their functions via the complex mechanisms that involve multiple protein and lipid modifications. To differentiate between the roles of oxidative modifications in HDL proteins and lipids, we analyzed the effects of selective protein oxidation by hypochlorite (HOCl) on the structure, stability, and remodeling of discoidal HDLs reconstituted from human apolipoproteins (A-I, A-II, or C-I) and phosphatidylcholines. Gel electrophoresis and electron microscopy revealed that, at ambient temperatures, protein oxidation in discoidal complexes promotes their remodeling into larger and smaller particles. Thermal denaturation monitored by far-UV circular dichroism and light scattering in melting and kinetic experiments shows that protein oxidation destabilizes discoidal lipoproteins and accelerates protein unfolding, dissociation, and lipoprotein fusion. This is likely due to the reduced affinity of the protein for lipid resulting from oxidation of Met and aromatic residues in the lipid-binding faces of amphipathic alpha-helices and to apolipoprotein cross-linking into dimers and trimers on the particle surface. We conclude that protein oxidation destabilizes HDL disk assembly and accelerates its remodeling and fusion. This result, which is not limited to model discoidal but also extends to plasma spherical HDL, helps explain the complex effects of oxidation on plasma lipoproteins.

Figure 1

Effects of apoA-I oxidation to various stages on protein cross-linking analyzed by SDS PAGE. Lipid-free apoA-I (lanes 0, 1′, 2′) or A-I:DMPC disks (lanes 1-4) containing 1 mg/mL protein in standard buffer (10 mM Na phosphate, 100 μM DTPA, pH 7.5), were incubated with various concentrations of HOCl as described in Methods. Lane numbers correspond to oxidant:protein molar ratios of 0:1 (0), 5:1 (1, 1′), 10:1 (2, 2′), 25:1 (3), 50:1 (4). Gel was stained with Imperial protein stain.

Figure 2

Effects of protein oxidation on the size of apoA-I:DMPC complexes at 25 °C analyzed by non-denaturing PAGE. Ten μl of 1 mg/mL apoA-I solution was applied to each lane; at this concentration, lipid-free apoA-I aggregates and runs at about 7 - 8 nm (lane A-I, (1)). Lanes 0-4 show discoidal complexes reconstituted from intact apoA-I and DMPC that were oxidized using oxidant:protein molar ratios of 0:1 (0), 5:1 (1), 10:1 (2), 25:1 (3), or 50:1 (4). Lanes 1′ and 2′ show complexes reconstituted from oxidized apoA-I and DMPC; the oxidant:protein ratios are 5:1 (1′) or 10:1 (2′). Molecular size standards (st) in nm are indicated.

Figure 3

Effects of protein oxidation on the size and morphology of A-I:DMPC complexes analyzed by negative staining EM. Upper row shows electron micrographs and lower row shows the corresponding particle size distributions. Numbers in panels A-E correspond to those in Fig. 2 and show intact A-I:DMPC disks (0) and disks that were treated with oxidant:protein ratios of 5:1 (1), 10:1 (2), 25:1 (3), 50:1 (4). Panels F, G show disks reconstituted from oxidized apoA-I and DMPC; HOCl:A-I ratios are 5:1 (1′) or 10:1 (2′). Average particle diameters with standard deviations are indicated in selected panels.

Figure 4

Effect of oxidation on the secondary structure of apoA-I in DMPC complexes at 22 °C. Far-UV CD spectra were recorded from the ox(A-I:DMPC) disks (20 μg/mL protein) that were incubated with HOCl using oxidant:protein ratios of 0:1 (0), 5:1 (1), 10:1 (2), 25:1 (3), 50:1 (4).

Figure 5

Effects of oxidation on thermal denaturation of A-I:DMPC disks. Disk samples, which were prepared as in Fig. 4, were heated and cooled from 10–98 °C at a constant rate of 11 °C/h, and the CD (A) and light scattering (B) melting data were recorded simultaneously at 222 nm to monitor changes in the protein helical content and in the particle size, respectively; the negative slopes in the light scattering data result from an optical artifact in the CD instrument and from the temperature dependence of the refractive index (47). The heating curves of non-oxidized disks (0, black lines in panels A, B) show a transition centered at 82 °C that corresponds to protein unfolding and concomitant lipoprotein fusion (38). The apparent transition temperature decreases upon oxidation; the heating curves of highly oxidized disks (4, light grey) show a similar transition centered at 60 °C followed by an additional fusion transition observed by light scattering at higher temperatures (panel B). Panel C shows CD melting data recorded at 222 nm, 11 °C/h scan rate of ox(A-I:DMPC) (which were reconstituted from intact apoA-1 and then oxidized) and (oxA-I):DMPC (which were reconstituted from oxidized apoA-I); oxidant:protein ratio was 10:1.

Figure 6

Effects of oxidation on the protein unfolding kinetics in DMPC complexes. Intact A-I:DMPC (0), ox(A-I:DMPC) (1-4), and ox(A-I):DMPC complexes (1′, 2′), which were treated using HOCl:A-I molar ratios of 5:1 (1, 1′), 10:1 (2, 2′), 25:1 (3), or 50:1 (4), were subjected to T-jumps from 25 to 80 °C. The time course of the protein unfolding was monitored by CD at 222 nm. The 2, 2′ (not shown) and 1, 1′ data partially overlap.

Figure 7

Effects of protein oxidation on the kinetics of DMPC clearance and on Trp modifications in apoA-I:DMPC disks. (A) DMPC multilamellar vesicles in standard buffer were incubated at 24 °C. The clearance was triggered at time t=0 by adding the protein; the final concentration was 20 μg/mL apoA-I and 80 μg/mL DMPC. ApoA-I was non-oxidized (0) or oxidized by using HOCl:apoA-I molar ratio of 5:1 (1′), 10:1 (2′), 25:1 (3′), or 50:1 (4′). (B) Trp emission spectra of normal A-I:DMPC disks (0) or of similar disks that were oxidized by using HOCl:A-I molar ratios of 5:1 (1), 10:1 (2), 25:1 (3), or 50:1 (4).

Figure 8

Effect of oxidation on the thermal denaturation of A-I:DMPC:FC disks. Disk samples containing 10% cholesterol were prepared as described in Materials and Methods. The disks were heated and cooled from 10-98 °C at a constant rate of 11 °C/h. The protein unfolding was monitored by CD at 222 nm. Numbers correspond to those in Figs 4 and 5 and show intact disks (0) and disks that were oxidized using HOCl:apoA-I molar ratio of 5:1 (1), 10:1 (2), 25:1 (3), or 50:1 (4).