STRUCTURAL STUDIES OF THE APOLIPOPROTEIN E SUBDOMAINS

Chi-Yuan Chou 1,2, Ming-Shi Shiao 1, and Gu-Gang Chang 2
1 Department of Medical Research and Education, Taipei Veterans General Hospital, Taiwan,
2 Faculty of Life Sciences, Institute of Biochemistry, and Structural Biology Program, National Yang-Ming University, Taipei, Taiwan

Introduction

Human apolipoprotein E (apoE) is a 34 kDa protein containing 299 amino acid residues that mediates the binding of various lipoprotein receptors, which are crucial for lipid transportation [1-4]. There are three major isoforms of human apoE (apoE2, apoE3, and apoE4). The most common isoform of apoE is apoE3, which contains cysteine and arginine at positions 112 and 158, respectively. Both positions contain cysteine residues in apoE2 and arginine residues in apoE4. Strong correlation of e4 allele with dyslipidemia and coronary heart disease has been demonstrated. The apoE4 isoform also contributes to the pathogenesis of Alzheimer’s disease [2,5-13]. ApoE associates with ß-amyloid peptides (Aß) to form novel monofibrils [11]. The pathogenesis of ApoE4 aggregation is an intriguing question.

Structural Features of the Apolipoprotein E

Recent evidence has indicated that apoE is folded into two distinct structural domains [14]. Crystal structure is available for the N-terminal domain, which indicates an amphipathic, extended, and anti-parallel four-helix bundle [15] The model structure of the C-terminal domain of apoE from residue 191 to 299 contains three predicted helices, which include residues 203-223, 225-266, and 268-289, respectively [15-17]. The end of second helix may play a key role in the lipid binding property of apoE [18-19]. The third helix induces the aggregation of C-terminus. However, when the following five residues (Phe-257, Trp-264, Val-269, Leu-279, and Val-287) were changed to polar, charged residues, it becomes monomer in solution [20]. Choy et al. [21] have also shown the intermolecular coiled-coil helical formation in the C-terminal domain. The C-terminal region of apoE may also interact with Aß in forming neurofibrillary tangles [10]. The N-terminal and C-terminal truncated apoE4 proteins occurred in Alzheimer’s disease brains. These truncated proteins induce intracellular neurofibrillary tangles-like inclusions in cultured neurons [10]. Further research into the role of C- and N-terminal domains in the formation of filaments and aggregates may add to our better understanding of the molecular pathogenesis of Alzheimer disease.

Effect of N/C Truncations on the Structure of Apolipoprotein E

To elucidate the structural difference of apoE3 and apoE4, we have designed several deletion mutants in which each one helix was successively and additively deleted from the N- or C-terminus. We have over-expressed and purified the full-length and N- or C-terminal-truncated human apoE3 and apoE4 proteins to apparent homogeneity. Their secondary structure was studied by circular dichroism, which estimates the secondary structure distribution (percentage of helical, ß-pleated sheet, ß-turn, and random coiled structure contents). The conformational change of the mutant proteins was examined by the binding of apoE proteins with the hydrophobic dye ANS, which was studied by fluorescence spectroscopy. The quaternary structural alteration and aggregation state of the mutant proteins were examined by analytical ultracentrifugation, which monitors the sedimentation of macromolecules in the centrifugal field that allows their hydrodynamic and thermodynamic properties characterized in solution, without interaction with any matrix or surface [17].
The sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation revealed apoE372-299 as a major species with sedimentation coefficient of 5.9 in solution. ApoE472-299 showed larger and more complicated species distribution. Both apoE3 and E4 N-terminal domain (1-191) showed monomer as the major component together with some tetramer. The oligomerization and aggregation of apoE protein increased while the C-terminal domain (192-271) was incorporated. The structural influence of the C-terminal domain to apoE protein is to assist the self-association with no significant isoform preference. Circular dichroism and fluorescence studies demonstrated that apoE472-299 possessed more a-helical structure with more hydrophobic residue exposure. The structural variance of N-terminal truncated apoE3 and apoE4 protein provides useful information to explain the more readily aggregating feature of apoE4 isoform and the implication of apoE4 in Alzheimer’s disease.

Biomedical Implications of the N/C Subdomains in Apolipoprotein E

To examine the effect of various N/C truncations on the functional roles of the apoE proteins, we injected these proteins into apoE(-) mice and checked their ability to lower the plasma cholesterol in vivo. Our functional assay showed that the full-length apoE3 and apoE4, apoE341-299, apoE441-299, and apoE472-299 lowered the plasma cholesterol to a similar extent. However, the efficacy of apoE372-299 was 30% lower than that of full-length or apoE472-299. It correlated well that helix 2 in the N-terminus is involved in the low density lipoprotein receptor binding, especially for apoE3. Our findings of structural variance of N-terminal truncated apoE3 and apoE4 provide useful information to explain the more readily aggregating feature of E4 isoform and the implication of E4 in Alzheimer’s disease. Our studies also suggest a direction on the rational drug design for inhibiting the aggregation of apoE4, which may delay the formation of neurofibrillary tangles or extracellular amyloid plaques.

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Please address correspondence to:
Professor Ming-Shi Shiao
Department of Medical Research and Education
Taipei Veterans General Hospital
201 Shih-Pai Road
Section 2
Taipei 112, Taiwan
E-mail: msshiao@vghtpe.gov.tw

Or
Professor Gu-Gang Chang
Faculty of Life Sciences
National Yang-Ming University
155 Li-Nong Street
Section 2
Taipei 112, Taiwan
E-mail: ggchang@ym.edu.tw

 

 

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