Abstract 20984: Transfer of Functional Properties From the Two-Domain Apolipoprotein E3 to a Single Domain 5-Helix Bundle Apolipoprotein

Apolipoprotein E3 (apoE3) is a 34 kDa exchangeable apolipoprotein critical for lipid transport and associates with chylomicrons, VLDL, and a subclass of HDL. The protein contains two independently folded domains: a 22 kDa N-terminal domain and a 10 kDa C-terminal domain. The N-terminal domain contains LDL receptor binding sites and is tightly folded into a 4-helix bundle. The smaller C-terminal (CT) domain contains helical segments that: (i) promote protein-protein interaction and apoE3 self-association, (ii) bear high lipid binding affinity, and, (iii) facilitate cholesterol efflux from macrophages. In contrast, insect apolipophorin III (apoLp-III) is a one-domain protein bearing a 5-helix bundle, does not self-associate and binds to diacylglycerol enriched lipoproteins. To better understand the domain organization of apolipoproteins, a chimeric protein was constructed of apoLp-III and the CT domain of apoE3 (residues 201-299). The structure and function of the 28 kDa apoLp-III/CT-apoE3 chimera was determined and compared to that of parent proteins apoLp-III and apoE3. The novel chimeric construct was expressed in E. coli and purified using an N-terminal poly-histidine tag. Electrophoresis and Western blot analysis showed that the apoLp-III/CT-apoE3 chimera was of the expected size and reacted with apoE3 specific antibodies. Circular dichroism demonstrated that the chimera was α-helical, similar to both parent proteins. While apoLp-III was monomeric, the chimera formed tetramers similar to apoE3. Probing for hydrophobic pockets on the protein surface with anilinonaphthalene-8-sulfonic acid demonstrated that the CT domain provided increased binding sites for the probe. Addition of the CT domain to apoLp-III conferred an increased ability to promote cholesterol efflux from J774 macrophages, comparable to that of parent apoE3. Thus, the fold of apoLp-III/CT-apoE3 chimera was similar to that of apoE3, promoted self-association and cholesterol efflux. This demonstrated that functional characteristics of CT domain of apoE3 can be transferred independently to another apolipoprotein, allowing us to further increase our understanding of apolipoprotein domains.