Abstract 834: Residues Leu261, Trp264 and Phe265 Account for Apolipoprotein E Induced Dyslipidemia and Affect the Biogenesis of Apolipoprotein E Containing High Density Lipoprotein
Apolipoprotein E (ApoE) is one of the most important proteins of the lipoprotein transport system. At physiological levels ApoE plays a critical role in lipid homeostasis and in atheroprotection. At high concentrations ApoE induces hypertriglyceridemia in humans and experimental animals. Previous studies showed that the residues responsible for hypertriglyc-eridemia are within the 261–269 region. We have studied in vivo three sets of ApoE4 mutations within this region using adenovirus mediated gene transfer in apoE−/− mice. We have found that a single (ApoE4[F265A], mutA) and a double (ApoE4[L261A, W264A], mutB) aminoacid substitution induced dyslipidemia characterized by high cholesterol and triglyceride levels. In both mutants triglycerides were distributed in the VLDL/IDL region. In mutA 75% of the cholesterol was distributed in the VLDL whereas in mutB 35% of cholesterol was distributed in VLDL and the remainder in the HDL region. Adenovirus mediated gene transfer of an ApoE mutant carrying a triple aminoacid substitution (ApoE4[L261A, W264A, F265A], mutC) did not induce hypertriglyceridemia and resulted in great increase in HDL cholesterol levels. Analysis of the lipid composition of the FPLC fractions indicated that 80% of the cholesterol was in the HDL region and consisted mainly (80%) of esterified cholesterol. Consistently with the FPLC analysis, SDS-PAGE of plasma fractions obtained by gradient density ultracentrifugation of plasma of apoE−/− mice expressing mutC showed that this ApoE4 mutant was distributed mainly in the HDL region. Electron microscopy analysis of the HDL fractions showed that overexpression of mutC resulted in the formation of spherical particles. In contrast, the wild type (wt) ApoE4, when expressed at similar levels with mutC, caused severe dyslipidemia, enrichment of the HDL and VLDL fractions with free cholesterol and formation of discoidal HDL. The findings establish that aminoacids L261, W264 and F265 account for the hypertriglyceridemia that is induced by wt ApoE. Substitutions of these aminoacids by Ala improve the biological function of ApoE by preventing ApoE induced hypetriglyceridemia and promoting the formation of spherical ApoE containing HDL.