Study of Domain Interaction of Human Apolipoprotein E

• Main Authors: Chi-Jen Lo, 駱啟仁
• Other Authors: Ta-Hsien Lin
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/87e7za

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 103 === Human apolipoprotein E (ApoE) is a polymorphic protein of 299 amino acids with a molecular mass of ~34 kDa. It is composed of two independently folded domains (NH2-terminal and COOH-terminal domain) separated by a hinge region. The 22-kDa NH2-terminal domain (residues 1-191) and the 10-kDa COOH-terminal domain (residues 216-299) of ApoE are responsible for LDL receptor and lipid binding, respectively. The APOE gene has three major alleles, ε2, ε3 and ε4, which encode three major isoforms, ApoE2, ApoE3, and ApoE4, respectively. The three isoforms differ from one another at residue 112 and/or 158, but have marked differences in their biological functions. ApoE has been known to play a key role in the transport of plasma cholesterol and lipoprotein metabolism. It is a major determinant of cardiovascular disease. ApoE is also highly associated with late-onset familial and sporadic Alzheimer’s disease (AD). The lipoprotein- and receptor-binding abilities of ApoE are isoform-specific. These phenomena are thought to be associated with the interaction between N-terminal domain and C-terminal domain of ApoE isoforms. The domain interaction of ApoE is also isoform-specific. However, the underlying molecular mechanism remains unclear. Studies suggested that structural characteristics of ApoE isoforms play important role in their domain interactions. Three-dimensional structures of ApoE isoforms may provide the information of their domain interactions and help us gain insight into the isoform-specific functions of ApoE. The goal of this study is to characterize the molecular mechanism of isoform-specific domain interaction of ApoE from the structural point of view. A straightforward approach to investigate the molecular mechanism of domain interaction of ApoE isoforms is to solve the three-dimensional structures of full-length ApoE isoforms in the presence or absence of lipid by X-ray crystallography and/or NMR spectroscopy. The three-dimensional structures of the NH2-terminal domain of ApoE isoforms have been solved by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, but the structural information of the COOH-terminal domain of ApoE and full-length ApoE isoforms are limited. Full-length ApoE isoforms tend to form oligomers in aqueous solution due to the self-association of COOH-terminal domain. This hindered the structural determination of full-length ApoE isoforms by X-ray crystallography or NMR spectroscopy. In this study, an alternative approach was applied to overcome the difficulty. First, characterize the structure of ApoE COOH-terminal domain (ApoE(195-299)) in the presence and absence of lipid by employing structural biology techniques, such as small angle X-ray scatting (SAXS), circular dichroism (CD), analytical ultracentrifugation (AUC) and NMR spectroscopy, then characterize the structure of ApoE COOH-terminal domain in complex with ApoE4 NH2-terminal domain (ApoE4(1-191)). The results of AUC and SAXS analyses suggested that ApoE(195-299) formed a tetramer with a cylindrical structure in aqueous solution. However, the monomeric form of ApoE(195-299) couldn't be detected in the aqueous solution. In the presence of DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphatidylcholine) micelles, ApoE(195-299) formed complex with DHPC micelle. AUC data suggested that the complex contained one ApoE(195-299) molecule and one DHPC micelle which contained ~60 DHPC molecules. Structural analysis of ApoE(195-299) in complex with DHPC micelles by NMR suggested that ApoE(195-299) adopted three α-helices located at residues 204-223, 228-265, and 268-286. The interaction between ApoE(195-299) and ApoE4(1-191) in the presence of DHPC micelles was also examined by using NMR spectroscopy. The results suggested that ApoE(195-299) did not interact with ApoE(1-191) in the presence of DHPC micelles. Perhaps, the domain interaction of ApoE4 in the presence of DHPC micelles cannot be detected when full-length ApoE4 was separated into two fragments. According to the structural information of ApoE(195-299) in the presences of DHPC, ApoE(222-271), which was the major lipid binding region and covered the second α-helical region of ApoE(195-299), was prepared by using molecular biology techniques for further studies of domain interaction. This fragment also contained the key residue (E255) which was thought to be involved in ApoE4 domain interaction. The AUC data suggested that ApoE(222-271) existed in a monomer-trimer equilibrium in the aqueous solution. At 283 K, the major form of ApoE(222-271) was a trimer which contained an α-helix located at residues 231-262. At 310K, the dominated form of ApoE(222-271) was a monomer which contained three short α-helices located at residues 236-243, 245-250, and 252-258. At 293 K, the ratio of trimeric form to monomeric form of ApoE(222-271) in solution state is 1:3. According to these structural information, the oiligomization of ApoE(222-271) would induce conformational change (the formation of α-helix). The results of AUC analysis also suggested that ApoE(222-271) formed complex with DHPC micelle, and the complex contained one ApoE(222-271) molecule and one DHPC micelle which contained ~44 DHPC molecules. When ApoE(222-271) formed complex with DHPC micelle, it adopted an α-helical structure, spanning from residue 228 to 264. This result may suggest that the interaction of ApoE with lipids would result in the formation of α-helix. Under the condition which ApoE(222-271) existed in a monomeric form, ApoE4(1-191) was added into the solution for observing the domain interaction of ApoE. The NMR data suggested that the residues involved in the domain interaction were R240, Q248, Q249, I250, A254, and L261-F266. Previous study suggested that the COOH-terminal domain interacted with the NH2-terminal domain of ApoE4 through a single residue (E255). This discrepancy may be because of the different methods used for observing the domain interaction. However, these residues all located at the hydrophilic surface of amphipathic α-helix. The results of this study provided the structural information of ApoE COOH-terminal domain and the information of domain interaction of ApoE4. These may help us gain more insight into the molecular mechanism of ApoE domain interaction.