Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia

Abstract

Background: Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential.

Methodology/principal findings: In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent.

Conclusions/significance: Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.

Figure 1. Top, Cartoon representation of the N-terminal structure of human apoE3 (PDB code: 1LPE) indicating the location of the mutations; protein is depicted in side and top view.

Bottom, SDS-PAGE analysis of the recombinant apoE3 variants and wild-type protein. Molecular mass marker bands are indicated. Images were prepared using PyMol 1.3 (www.pymol.org).

Figure 2. Circular dichroism spectra of wild-type apoE3 and variants (wild-type in black, R145C in blue, K146E in red and R136S in green).

Spectra shown are the average of three separate measurements on different days with different protein batches. Inset: Helical content of WT apoE3 and variants calculated using the molar ellipticity at 222 nm as described in the materials and methods section. Error bars represent standard deviation based on three independent measurements.

Figure 3. Thermal denaturation profiles of wild-type apoE3 and apoE3 variants.

Each apoE3 variant (black dots) is presented in comparison with the wild-type protein (gray dots).

Figure 4. Reversibility of the thermal denaturation of WT apoE3 and variants.

ApoE3 was thermally unfolded while following the CD signal at 222 nm (red trace). After reaching 80°C, the protein was gradually refolded by cooling the sample chamber down to 20°C (blue trace). The protein sample was then thermally unfolded again (green trace).

Figure 5. Chemical denaturation profiles of wild-type apoE3 and apoE3 variants.

Each variant is presented in comparison with the wild-type protein (black circles). Solid lines represent non-linear regression fits to a three-state unfolding model as described in the materials and methods section.

Figure 6. ANS fluorescence spectra in the presence or absence of wild-type apoE3 and variants.

Spectra are averages of three independent experiments. Error bars indicate standard deviation based on three independent measurements. *p = 0.03.

Figure 7. Time course of remodeling of multilamellar DMPC vesicles by wild-type apoE3 and apoE3 variants.

Absorbance at 325 nm was followed for 1 hr after addition of apoE3 to DMPC vesicles as described in the materials and methods section. Experimental data (closed circles) were fit to a two-phase exponential decay model (solid lines).

Figure 8. Volume-normalized distribution of particle hydrodynamic diameters in 0.1 mg/ml samples of WT apoE3 and variants.

Peak analysis parameters are shown in Table 5.

Figure 9. Panel A, Location of amino-acid substitutions in the apoE3 variants under study in the recently determined structure of a monomeric variant of apoE3 (pdb code 2L7B).

Protein is shown in cartoon representation with the N-terminal domain colored in blue, the C-terminal domain colored in red and the hinge-domain colored in green (see reference [68]). Panel B, same as in panel A, but with the C-terminal and N-terminal domain shown in surface representation to demonstrate that positions 136, 145 and 146 are not solvent exposed in this structure. Panel C, interactions between amino acids R136, R145 and K146 and the C-terminal or N-terminal domain of the protein. All three amino acids are stabilized by specific interactions with either charged residues of the C-terminal domain (for K146 or R136) or with hydrogen bonding within the N-terminal domain . Images were prepared using PyMol 1.3 (www.pymol.org).