Characterization of a novel apolipophorin‐III/apolipoprotein E C‐terminal domain chimera

Abstract

Human apolipoprotein E (apoE) is a two‐domain anti‐atherogenic lipid transport protein that mediates plasma cholesterol homeostasis by serving as a ligand for the low density lipoprotein (LDL) receptor. Of its three isoforms E3, E2, E4, the latter two have been associated with increased risk of cardiovascular and Alzheimer's disease respectively, yet all three contain an identical C‐terminal (CT) domain. The CT domain (residues 201–299) is responsible for tetramerization and has been found to initiate lipid binding that influences critical functional features of apoE. The N‐terminal (NT) domain of apoE has four α‐helices arranged in a bundle similar to apolipophorin III (apoLp‐III), a model insect apolipoprotein containing five α‐helices in one domain. To better understand the role apolipoprotein domains play in structure and function, a novel chimeric apolipoprotein was designed by attaching apoE‐CT to apoLp‐III. A disulfide bond was introduced to apoLp‐III to lock the helix bundle and eliminate its lipid binding. Recombinant apoLp‐III/apoE‐CT, apoE3, apoE‐CT, and apoLp‐III were expressed in bacterial cells, purified by affinity chromatography, and the purity verified by SDS‐PAGE. Western blot analysis using monoclonal apoE‐CT specific antibody confirmed the presence of apoE‐CT in the chimera. Crosslinking studies using dimethylsuberimidate revealed that the apoLp‐III/apoE‐CT chimera formed oligomers similar to apoE, while apoLp‐III was monomeric. Far UV circular dichroism showed increased α‐helical content, and incubation with 1‐anilinonaphthalene‐8‐sulfonic acid showed increased fluorescence intensity when apoE‐CT was added to apoLp‐III, consistent with the presence of water‐shielded dye binding exposed hydrophobic regions. Our results suggest that the chimeric protein has structural similarities to the parent proteins, and that self‐association properties of apoE can be transferred to apoLp‐III through the addition of the CT domain. Future studies will include measuring the ability of the chimera to solubilize phospholipid bilayers and binding to low density lipoproteins. The chimeric approach offers the potential to obtain insight into domain interactions and the structure‐function relationships in apolipoproteins.Support or Funding InformationThis research was supported by the National Institute of General Medical Sciences of the NIH (8UL1GM118979‐02, 8TL4GM118980‐02, 8RL5GM118978‐02, GM089564 and GM105561).