Apolipoprotein E associated with reconstituted high-density lipoprotein-like particles is protected from aggregation

Abstract

Apolipoprotein E (APOE) genotype determines Alzheimer's disease (AD) susceptibility, with the APOE ε4 allele being an established risk factor for late-onset AD. The ApoE lipidation status has been reported to impact amyloid-beta (Aβ) peptide metabolism. The details of how lipidation affects ApoE behavior remain to be elucidated. In this study, we prepared lipid-free and lipid-bound ApoE particles, mimicking the high-density lipoprotein particles found in vivo, for all three isoforms (ApoE2, ApoE3, and ApoE4) and biophysically characterized them. We find that lipid-free ApoE in solution has the tendency to aggregate in vitro in an isoform-dependent manner under near-physiological conditions and that aggregation is impeded by lipidation of ApoE.

Keywords: Alzheimer's disease; aggregation; apolipoprotein E; high-density lipoprotein; isoform; lipidation.

Figure 1

Assessment of the formation of HDL‐like discoidal ApoE particles with TEM. The majority of the discoidal ApoE particles are visualized from their top/bottom, but some can also be seen from a lateral perspective (indicated by arrows). The scale bars represent 200 nm. The image is representative of at least three independently prepared samples.

Figure 2

The heterogeneous composition of HDL‐like ApoE particles. Lipid‐free and HDL‐like ApoE particles (0.1 mg·mL⁻¹ in PBS) were separated by FFF and their composition was compared by their (A) differential refractive index, (B) intensity of differential light scattering, and (C) UV absorbance at 215 nm. Obtained spectra are representative of two independently prepared ApoE isoform samples.

Figure 3

Lipidation impedes aggregation of ApoE. Migration patterns and size distributions of lipid‐free and HDL‐like ApoE particles (0.1 mg·mL⁻¹ in PBS) were obtained by native PAGE and DLS, respectively. (A) Lipidated ApoE migrates further in a 4–20% Tris‐glycine gel compared to lipid‐free ApoE (M: NativeMarkTM Unstained protein standard). (B) The hydrodynamic radius of lipidated ApoE is smaller than that of lipid‐free ApoE. Obtained data are representative of two independently prepared ApoE isoform samples.

Figure 4

Lipid‐free ApoE4 self‐assembles into amorphous aggregates. (A) Lipid‐free ApoE aggregates displayed an amorphous morphology, similar for all three isoforms, as assessed by TEM. Lipid‐free ApoE4 aggregates are depicted. (B) An enlarged image of lipid‐free ApoE4 aggregates. The scale bars represent 200 nm. Images are representative of at least three independently prepared samples.

Figure 5

Effect of lipidation on the secondary structure of ApoE. The secondary structure content of lipid‐free and HDL‐like ApoE particles (0.1 mg·mL⁻¹ in PBS) was studied by CD. (A) CD reveals a predominant α‐helical structural signature for all samples characterized by double minima around 208 and 222 nm. (B) Secondary structure content of each sample was estimated using cdsstr software 41, 42. The goodness of fit of the experimental CD data with the reference data is indicated by the NRMSD value. Spectra are averages of two independently prepared duplicates.

Figure 6

Effect of lipidation on the tertiary structure of ApoE. (A) Intrinsic Trp fluorescence emission spectra (λex = 280 nm) corresponding to lipid‐free and HDL‐like ApoE particles (0.1 mg·mL⁻¹ in PBS). (B) The maximum of the Trp fluorescence emission spectrum of lipidated ApoE is blue shifted compared to that of lipid‐free ApoE. Statistical significance of the results was established by P‐values using unpaired two‐tailed t‐tests, with *P < 0.05. Spectra are averages of two independently prepared duplicates.