Structural and Functional Variation in Human Apolipoprotein E3 and E4

• Main Authors: chi-yuan Chou, 周記源
• Other Authors: gu-gang Chang
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/52330844874912331411

博士 === 國防醫學院 === 生命科學研究所 === 93 === Human apolipoprotein E (apoE) is a 34 kDa protein containing 299 amino acids that mediates the metabolism of various plasma lipoproteins including the catabolism of VLDL to LDL. There are three apoE isoforms, namely E2, E3, and E4, whose differences are single residue substitution involving Cys-Arg replacement at residues 112 and 158. ApoE4 (Arg-112/Arg158) is associated with late-onset and sporadic Alzheimer’s disease (AD) and atherosclerosis. ApoE4 and its C-terminal truncated fragments have been found in the senile plaques and neurofibrillary tangles in the brain of AD patients. The preferential distribution of apoE4 to VLDL and apoE3 to HDL may also enhance the catabolism of E4-bearing lipoproteins, leading to alterations in lipoprotein metabolism such as elevated LDL-C and possibly atherosclerosis. Detail structural information regarding isoform and domain interaction remains poorly understood. To elucidate the structural differences and functional disturbances between apoE3 and apoE4 isoforms, especially with respect to N-terminal and C-terminal truncation, I prepared full-length, N-, and C-terminal truncated apoE3 and E4 proteins and compared their structural variation. Sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation revealed that apoE372-299 consisted of a major species with a sedimentation coefficient of 5.9. ApoE472-299 displayed a wider and more complicated species distribution. The apoE3 and apoE4 N-terminal domain (apoE1-191) existed with their monomers as the major species together with some tetramers. The oligomerization and aggregation of apoE protein increased when the C-terminal domain (residue 192-271) was incorporated. It suggests that the structural influence of the C-terminal domain on apoE is to assist self-association with no obvious isoform preference. Circular dichroism and ANS-fluorescent spectroscopic studies demonstrated that apoE472-299 possessed a more predominant α-helical structure with more extensive exposure of its hydrophobic residue. These findings provide conclusive information to explain the more readily aggregating feature of E4 isoform. In vitro and in vivo functional studies were also conducted for the full-length and truncated apoE proteins. To elucidate the effects of various N- or C-terminal truncated apoE proteins on the lipid-binding abilities, dimyristoylphosphatidylcholine (DMPC) turbidity clearance assay was used. Results indicated that apoE41-299 and apoE72-299 retained the lipid-clearing abilities but those C-terminal truncated apoE1-191 and apoE1-231 proteins lost this ability extensively. The dependence and contribution of the C-terminal residue 232-270 for lipid binding of apoE protein were clearly demonstrated. Furthermore, the LDL receptor-binding abilities of apoE proteins were evaluated by using a competition assay with the binding of 3H-LDL to LDL receptor in cultured HepG2 cells. The results showed that truncated apoE4 proteins still maintained their LDL receptor binding abilities when the N- (residue 1-71) or C-terminal (residue 232-299) domain were truncated. With complete C-terminal truncation, apoE1-191 yet maintained half or more of LDL-receptor binding ability indicating that C-terminus might assist LDL-receptor binding. Another interesting finding was that apoE72-166 still maintained significant apoE function, especially apoE472-166. The DMPC clearance by apoE472-266 was very efficient and the LDL-receptor binding ability of apoE472-166 was about equal to that of full-length apoE4. The flexible, characteristic property of the two remaining helices in residues 72-166 may help apoE472-166 adsorb into DMPC lattice defects rapidly. This feature may also assist the exposure of the 141-150 basic, LDL-receptor binding region and lead to high efficiency in LDL-receptor binding. ApoE(-) mice display disturbed VLDL catabolism to LDL and elevated plasma cholesterol. This pathological animal model also develops atherosclerosis spontaneously. To elucidate the cholesterol-lowering functions of apoE3 and apoE4 with various domain truncations, the full-length and truncated apoE proteins were injected into the tail vein of apoE(-) mice and determined their abilities to lower plasma cholesterol in vivo. This animal study demonstrated that both apoE472-299 and apoE41-231 proteins were functional in lowering plasma cholesterol with potency equal to that of full-length apoE4 protein. In contrast, apoE372-299 and apoE31-231 lost their cholesterol-lowering abilities significantly. Based on the structural and functional elucidation, this study concludes that the potential structural stability of apoE4 protein to remain functional in solution, and possibly also in vivo, may explain the enhanced opportunity of apoE4 to display its pathophysiologic functions. The speculation can be extended to the higher tendency of apoE4-isoform to aggregate and co-aggregate with β-amyloid peptide in the molecular pathogenesis of Alzheimer’s disease. This structural information and related functional disturbances may also shed light on future drug development for the treatment of AD and atherosclerosis.