Analysis of the Molecular Organization of Lipoprotein-Associated Apolipoprotein E, an Anti-Atherogenic Protein

Abstract

Human apolipoprotein E3 (apoE3) is a 299 residue exchangeable apolipoprotein that has the ability to exist in lipid-free and lipoprotein-bound states. It is composed of an N-terminal domain bearing LDL-receptor binding sites and a C-terminal (CT) domain bearing high-affinity lipid-binding sites; the latter is also responsible for mediating cellular cholesterol efflux. ApoE plays a crucial role as an anti-atherogenic agent in atherosclerosis by virtue of its ability to mediate reverse cholesterol transport by promoting cholesterol efflux from macrophages, a process that leads to the initial formation of nascent discoidal high-density lipoproteins (nHDL). The objective of this study is to understand the molecular organization and conformation of apoE3 (1-299) and isolated CT domain (201-299) on reconstituted HDL (rHDL) and nHDL generated by macrophages. To accomplish this, we over-expressed and purified recombinant apoE3 and the CT domain bearing single Cys at selected sites. rHDL was prepared using unlabeled or pyrene-labeled single Cys variants and phospholipids, while nHDL was generated by exposing cholesterol-loaded J774 macrophages to unlabeled or labeled apoE bearing probes at defined sites. Lipid-associated apoE was obtained by density gradient ultracentrifugation. The relative organization of apoE molecules on rHDL and nHDL was followed by monitoring pyrene excimer fluorescence and by site-specific cross-linking studies, both complementary validators of spatial proximity at ∼10 A distance. The appearance of excimer emission and cross-linked dimers regardless of the location of the single Cys is indicative of pairs of apoE3 molecules oriented parallel to each other on the HDL to form a dimer. Based on our results, we propose that apoE molecules circumscribe a bilayer of lipids adopting a belt-like conformation. Our study offers mechanistic details of apoE interaction with lipids, which aids in understanding its role in cardiovascular disease.