Abstract 117: Structure Function Analysis Of Guinea Pig And Human Apolipoprotein E To Obtain Insight Into Their Role In Cardiovascular Disease

Abstract

Apolipoprotein (apo) E3 (299 amino acids) is a major cholesterol transport protein in the plasma and brain of humans, with the isoform apoE4 considered a risk factor for cardiovascular and Alzheimer’s diseases (CVD and AD). The molecular basis underlying the link between apoE4, and CVD/AD is poorly understood. Previous studies have identified the C-terminal domain (CTD) in promoting protein-lipid, protein-protein and protein-amyloid peptide interactions. In the current study, apoE from Cavia porcellu s (guinea pig, GP) was used as a non-transgenic model to further understand the role of apoE4, since it lacks residues 193-197 and 246-252 of apoE4. We hypothesize that apoE4 CTD plays a critical role in lipid binding, protein folding and amyloid peptide interaction. Recombinant wildtype (WT) GP apoE and insertion mutants GP apoE/Ins193- 197/Ins246-252, and apoE4, and the corresponding deletion mutants apoE4/D193-197/D246-252 were generated. Western blot with anti-apoE4 antibody revealed cross reactivity with GP apoE. Circular dichroism spectroscopy of the variants revealed troughs at 208 and 222 nm indicting that they retained the helical structure observed in WT apoE4. WT GP apoE solubilizes lipids more efficiently than apoE4. Similarly, apoE4/D193-197/D246-252 displayed increased, while GP apoE/Ins193- 197/Ins246-252 showed decreased lipid solubilization ability compared to their WT counterparts. Limited proteolysis analysis indicates that GP apoE was the least susceptible to degradation by trypsin compared to apoE3 and apoE4. Fluorescence analysis using the extrinsic fluorophore anilinonaphthalene sulfonic acid suggests that GP apoE possesses a greater protein surface hydrophobicity than apoE3 and apoE4. Future studies will assess the ability of these variants to promote cholesterol efflux and Aβ binding to gain a better understanding of the role apoE4 in CVD and AD.