Redox status of serum apolipoprotein E and its impact on HDL cholesterol levels

Highlights

• An analytical method to determine the redox status of serum apoE is proposed.

• Reduced-form apoE displays a preference of VLDL fraction.

• The levels of reduced-form apoE show a significant correlation with HDL-C levels.

• Redox status of serum apoE is different among apoE phenotypes.

• Redox status of serum apoE may be a potent indicator to evaluate apoE-related diseases.

Abstract

Background

Apolipoprotein E (apoE) is closely involved in the pathogenesis of apoE-related diseases, such as Alzheimer's disease and cardiovascular disease. The redox modulation of cysteine-thiols in a protein is involved in various pathophysiological regulations; however, that of apoE has not been studied in detail. Herein, we devised an analytical method to determine the redox status of serum apoE and assessed its relation to serum cholesterol levels and apoE phenotype.

Methods

The present method was based on a band shift assay, using a photocleavable maleimide-conjugated polyethylene glycol.

Results

The basic characteristics of the present method were found to be satisfactory to determine the redox status of serum apoE quantitatively. Serum apoE was separated into its reduced-form (r-), non-reduced-form (nr-), apoE-AII complex, and homodimer using this method. R-apoE could be detected as a 40-kDa band, whereas nr-apoE remained as monomeric apoE. R-apoE displayed a preference for VLDL; however, the levels showed the correlation with HDL-cholesterol levels (p < 0.005). Redox status of serum apoE was significantly different among apoE phenotypes. The quantitative ratios of nr-apoE to total apoE in serum from subjects with apoE4/E3 were higher than in serum from subjects with apoE3/E3 (p < 0.0001) and apoE3/E2 (p < 0.001).

Conclusion

The redox status of serum apoE might be related to the synthesis of HDL. The information concerning the redox status of serum apoE provided by the present method may be a potent indicator to evaluate various apoE-related diseases.

Introduction

Apolipoprotein (apo) E is a 35-kDa plasma protein, comprising 299 amino acids, which is involved in lipid transport and metabolism as a component of lipoproteins [1], [2]. ApoE has three major isoforms, apoE2, apoE3, and apoE4, produced by three independent alleles at a single genetic locus [1].

Polymorphisms of apoE are associated with the pathogenesis of various diseases [3]. In particular, apoE4 is an identified risk factor of sporadic late-onset familial AD [4]. The incidence of AD is significantly higher in subjects with apoE4; however, 20–30% of AD patients have no apoE4 [4], [5]. Nevertheless, the precise role of apoE in the development of AD remains obscure.

One of the major characteristics distinguishing apoE2 and apoE3 from apoE4 is cysteine-arginine interchanges at residues 112 and/or 158. ApoE3, the most common of the three isoforms, contains a cysteine at residue 112 and an arginine at residue 158, whereas apoE4 has two arginines and apoE2 has two cysteines at both positions. The presence of cysteine at residues 112 and/or 158 allows apoE2 and apoE3 to form apoE homodimer and heterodimers (e.g., the apoE-AII complex) [6], [7]. Cysteine-arginine interchanges also reflect the differences in the distribution of apoE in lipoprotein particles. Weisgraber et al. [8] demonstrated that the preference of apoE3 for HDL and that of apoE4 for VLDL or IDL results from a difference in the charge at residue 112 of the apoE molecule and whether they can form the apoE-AII complex. These characteristics affect not only the structure of apoE, but also its pathophysiological functions. Indeed, the synthesis of HDL is enhanced significantly by the dimerization of apoE3 [9], [10]. Interestingly, Minagawa et al. [11] reported that the interaction between the cysteine-thiols of homocysteine (Hcy) and apoE3 interferes with apoE3 dimerization, and the consequent impaired ability of apoE3 to generate HDL to a level similar to that of apoE4. Hence, they concluded that hyperhomocysteinemia is a potent risk factor of AD for patients lacking apoE4.

A cysteine-thiol residue in a protein is susceptible to post-translational modifications such as hyperoxidation, nitrosylation, glutathiolation, and palmitoylation [12], [13]. Multiple lines of evidence suggest the possibility that cysteine-thiol residues and their modifications are closely involved in the modulation of various biological activities. For example, cysteine residues function as thiol-based redox regulatory switches in some signal pathways [14], [15], [16]. On the basis of these findings, we hypothesized that the redox status of apoE2 or apoE3, including the formation of disulfide-linked dimers, might affect their physiological functions, and some redox modulations might confer detrimental properties on apoE2 or apoE3, similar to apoE4, consequently influencing the pathology of various apoE-related diseases, such as AD and cardiovascular disease.

In this study, we attempted to devise the method that allows the comprehensive analysis of the apoE redox status based on a band shift assay using a photocleavable maleimide-conjugated polyethylene glycol (PEG-PC-Mal). We then characterized the redox status of serum apoE.