The Structure-Function Relationship of Atheroprotective ApoA-I Mutants in High-Density Lipoproteins

Lau, Sophie and Middleton, David and Anwar, Jamshed (2021) The Structure-Function Relationship of Atheroprotective ApoA-I Mutants in High-Density Lipoproteins. PhD thesis, Lancaster University.

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Abstract

In atherosclerosis cholesterol accumulation in arterial walls forms plaques which lead to associated cardiovascular diseases. High-density lipoprotein (HDL) nanoparticles known as “good cholesterol” transport cholesterol from arterial plaques to the liver for excretion. Therefore, HDL and its components have been targeted for tackling atherosclerosis treatment. Significantly, carriers of the R173C or L144R mutation in the major HDL protein apolipoprotein A-I (apoA-I) have low levels of HDL with a low atherosclerosis incidence. This suggests the mutations are atheroprotective as wild-type (WT) apoA-I carriers have a higher risk of atherosclerosis with low HDL levels. However, the mechanism for the mutants atheroprotective function is unknown. This work compares the molecular structure of reconstituted HDL (rHDL) nanoparticles containing the apoA-I variants using biophysical techniques. To determine if the rHDL structure affects the nanoparticle function and aid atherosclerosis treatment development. The expression and purification of unlabelled and uniformly [13C/15N]-labelled recombinant apoA-I variants was established. rHDL nanoparticles were prepared containing POPC, ±cholesterol, and apoA-I using detergent-mediated dialysis and density-based ultracentrifugation. The rHDL nanoparticle size and morphology were analysed with techniques such as NativePAGE gradient gel electrophoresis and transmission electron microscopy (TEM). A novel morphology characterisation was developed using 31P solid-state NMR spectroscopy of oriented rHDL nanoparticles on glass substrates. Three distinct rHDL nanoparticle morphologies were identified that were inaccessible by TEM. The rHDL-L144R nanoparticles showed a different surface curvature when compared to the rHDL-WT and R173C nanoparticles. Subtle differences in the protein secondary structure were identified by circular dichroism and high-resolution 2D 13C-13C NMR spectra of PEG-precipitated rHDL-WT and L144R nanoparticles. Consequently, the different rHDL nanoparticle morphologies detected could be connected to the atheroprotective functional properties of the rHDL-L144R nanoparticles. The morphology characterisation technique was applied to a plasma HDL sample which provides a framework for application to clinical samples.