Abstract
There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.
Highlights
Self-aggregation of ApoE Proteins Determined by Size Distribution Analysis—ApoE3, apoE4, and their truncated proteins were chosen for this study
From the continuous size distribution analysis, we found that the average f/f0 values of apolipoprotein E (apoE) with lipid (1.9 –2.2) is always higher than that of apoE in PBS (1.2–1.6)
When apoE proteins exist in a lipid-free environment, most of apoE proteins appear as oligomers
Summary
Plasmids—The pET-29a(ϩ) (Novagen) vectors with a C-terminal His tag sequence (Ser2–His6) were used. The NdeI-XhoI-digested apoE3-(72–166) cDNA was ligated to the 5.2-kb NdeI-XhoI fragment from pET-29a(ϩ) This resulted in a 5.5-kb pETapoE3-(72–166) vector. Multiple scans at different time points were fitted to a continuous size distribution model by using the SEDFIT (26, 27) program as described previously (22). The cells were cooled on ice for 30 min, washed twice with PBS, and incubated with DMEM containing 50 g/ml 3H-LDL, and different receptor-binding competitors (apoE) at 4 °C for 2 h. Because the original concentration of cholesterol associated with VLDL ϩ LDL was high and the concentration of high density lipoprotein cholesterol in apoE(Ϫ) mice remained approximately constant (about 50 mg/dl) (35), the plasma cholesterol-lowering effect was mainly contributed by apoE proteins to accelerate VLDL clearance in the plasma
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