RESUMO
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.
Assuntos
Apolipoproteínas E/sangue , HDL-Colesterol/sangue , Apolipoproteína A-II/sangue , Apolipoproteína A-II/química , Apolipoproteína A-II/isolamento & purificação , Apolipoproteína E2/sangue , Apolipoproteína E2/química , Apolipoproteína E2/isolamento & purificação , Apolipoproteína E3/sangue , Apolipoproteína E3/química , Apolipoproteína E3/isolamento & purificação , Apolipoproteína E4/sangue , Apolipoproteína E4/química , Apolipoproteína E4/isolamento & purificação , Apolipoproteínas E/química , Apolipoproteínas E/isolamento & purificação , HDL-Colesterol/química , Cisteína/química , Diamida/química , Dimerização , Ditiotreitol/química , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Indicadores e Reagentes/química , Peso Molecular , Oxirredução , Processos Fotoquímicos , Polietilenoglicóis/química , Solubilidade , Reagentes de Sulfidrila/química , Raios UltravioletaRESUMO
Since the discovery of the association of apolipoprotein E (apoE) 4 with Alzheimer's disease 17 years ago, numerous in vitro experiments with the apoE isoforms (apoE2, apoE3, and apoE4) have been performed to try to understand the basis for this association. The majority of these studies used commercial sources for apoE, but some used recombinant protein. In either case, these studies were most often conducted without considering the ramifications of the structural and biophysical differences among the three isoforms or without adequate quality control of the preparations. Here, we present a protocol for producing recombinant apoE that we have used successfully in our laboratory for the last 20 years. We also review the considerations that are critical for obtaining reliable and interpretable results with the end product.