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. 2014 Jul;55(7):1310-23.
doi: 10.1194/jlr.M048348. Epub 2014 Apr 28.

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL

Panagiotis Fotakis  1 , Alexander Vezeridis  2 , Ioannis Dafnis  3 , Angeliki Chroni  3 , Dimitris Kardassis  4 , Vassilis I Zannis  2
Affiliations

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL

Panagiotis Fotakis et al. J Lipid Res. 2014 Jul.

Abstract

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.

Keywords: apolipoprotein E3; dominant type III hyperlipoproteinemia; gene transfer; high density lipoprotein; hypertriglyceridemia; lecithin:cholesterol acyl transferase; lipoprotein lipase; recombinant adenovirus.

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Figures

Fig. 1.
Fig. 1.
A–C: Plasma cholesterol, triglycerides, and apoE plasma protein levels of apoE−/− mice at different days postinfection with recombinant adenoviruses expressing the WT apoE3, the full-length apoE3[K146N/R147W], and the truncated apoE3[K146N/R147W]202 mutants and control truncated apoE4-202 and apoE4[R142C]202 forms as indicated. Plasma cholesterol (A), plasma triglycerides (B), plasma apoE (C). The plasma CE/TE ratios at different days postinfection are shown in supplementary Table II. The doses of the adenoviruses used were 5 × 108 pfu for the full-length forms and 2 × 109 pfu for the truncated forms. The apoE mRNA levels of the different apoE forms used are shown in supplementary Table IB. Parameters reported are mean ± SD based on analysis of four to six mice per experiment. *The changes in cholesterol and apoE levels between WT apoE3 and either apoE3[K146N/R147W] or apoE3[K146N/R147W]202 were statistically significant (P < 0.05). The changes in triglyceride levels between apoE3[K146N/R147W] and either WT apoE3 or apoE3[K146N/R147W]202 were statistically significant (P < 0.05). ^The changes in cholesterol and apoE levels between apoE3[K146N/R147W] and apoE3[K146N/R147W]202 were statistically significant (P < 0.05).
Fig. 2.
Fig. 2.
A–K: FPLC profiles of plasma cholesterol and triglycerides 4 days postinfection of apoE−/− mice expressing the WT apoE3, the full-length apoE3[K146N/R147W], and the truncated apoE3[K146N/R147W]202 mutants as indicated. FPLC cholesterol profile (A) and FPLC triglyceride profile (B). Analysis of the plasma of apoE−/− mice infected with adenoviruses expressing the WT apoE3 (C), the full-length apoE3[K146N/R147W] mutant (D), and the truncated apoE3[K146N/R147W]202 mutant (E) by density gradient ultracentrifugation and SDS-PAGE (Coomassie brilliant blue staining) of the resulting fractions. EM analysis of HDL fractions 6 and 7 obtained from apoE−/− mice expressing the WT apoE3 (F), the full-length apoE3[K146N/R147W] mutant (G), and the truncated apoE3[K146N/R147W]202 mutant (H). The photomicrographs were taken at 75,000× magnification and enlarged three times. EM analysis of fractions 4 and 5 of the WT and the mutant apoE forms are shown in supplementary Fig. IA, B, E. Two-dimensional gel electrophoresis of plasma of apoE−/− mice infected with adenoviruses expressing the WT apoE3 (I), the full-length apoE3[K146N/R147W] mutant (J), and the truncated apoE3[K146N/R147W]202 mutant (K). The relationship between the two-dimensional patterns of the WT apoE3, the apoE3[K146N/R147W]202 mutant, and the apoE3[K146N/R147W] and apoE3[K146N/R147W]202 mutants were established by mixing experiments shown in supplementary Fig. IF, G.
Fig. 3.
Fig. 3.
A–I: Plasma cholesterol, triglycerides, apoE levels, density gradient ultracentrifugation profiles, and EM analysis of plasma of apoA-I−/−×apoE−/− mice infected with recombinant adenoviruses expressing the WT apoE3, the full-length apoE3[K146N/R147W] mutant, and the truncated apoE3[K146N/R147W]202 mutant as indicated. Plasma cholesterol (A), plasma triglycerides (B), plasma apoE (C), density gradient ultracentrifugation and SDS-PAGE (Coomassie brilliant blue staining) analysis of plasma obtained from WT apoE3 (D), apoE3[K146N/R147W] (E), and apoE3[K146N/R147W]202 (F). EM analysis of HDL fractions 6 and 7 obtained by density gradient ultracentrifugation of plasma of WT apoE3 (G), apoE3[K146N/R147W] (H), and apoE3[K146N/R147W]202 (I). Parameters reported are mean ± SD based on analysis of four to six mice per experiment. *The changes in cholesterol and apoE levels between WT apoE3 and either apoE3[K146N/R147W] or apoE3[K146N/R147W]202 were statistically significant (P < 0.05). The changes in triglyceride levels between apoE3[K146N/R147W] and either WT apoE3 or apoE3[K146N/R147W]202 were statistically significant (P < 0.05).
Fig. 4.
Fig. 4.
A–E: Plasma cholesterol, triglycerides, apoE levels, and FPLC profiles of apoE−/− mice infected with recombinant adenoviruses expressing the full-length apoE3[K146N/R147W] alone or in combination with LCAT or LPL as indicated. Plasma cholesterol (A), plasma triglycerides (B), plasma apoE (C), FPLC cholesterol profiles (D), and FPLC triglyceride profile (E) of plasma of apoE−/− mice infected with the indicated adenoviruses. The doses of the adenoviruses used were 7 × 108 pfu for the apoE3[K146N/R147W] mutant and 5 × 108 pfu for LCAT and LPL. Parameters reported are mean ± SD based on analysis of four to six mice per experiment. *The changes in cholesterol levels between apoE3[K146N/R147W] and either apoE3[K146N/R147W]+LCAT or apoE3[K146N/R147W]+LPL were statistically significant (P < 0.05). The changes in triglyceride levels between apoE3[K146N/R147W] and either apoE3[K146N/R147W]+LCAT or apoE3[K146N/R147W]+LPL were statistically significant (P < 0.05). The changes in apoE levels between apoE3[K146N/R147W]+LPL and apoE3[K146N/R147W] for day 2 and between apoE3[K146N/R147W]+LPL and either apoE3[K146N/R147W] or apoE3[K146N/R147W]+LCAT for day 4 were statistically significant (P < 0.05). ^The changes in cholesterol levels between apoE3[K146N/R147W]+LPL and apoE3[K146N/R147W]+LCAT were statistically significant (P < 0.05).
Fig. 5.
Fig. 5.
A–D: Analysis of the plasma of apoE−/− mice infected with adenoviruses expressing the full-length apoE3[K146N/R147W] in combination with LPL (A) or LCAT (B) as indicated by density gradient ultracentrifugation and SDS-PAGE (Coomassie brilliant blue staining) of the resulting fractions. EM analysis of HDL fractions 6 and 7 obtained from apoE−/− mice expressing the full-length apoE3[K146N/R147W] in combination with LPL (C) or LCAT (D). The photomicrographs were taken at 75,000× magnification and enlarged three times. EM analysis of fractions 4 and 5 of the apoE3[K146N/R147W] in combination with LPL or LCAT are shown in supplementary Fig. 1C, D.
Fig. 6.
Fig. 6.
ABCA1-mediated cholesterol efflux from J774 mouse macrophages treated with cpt-cAMP using WT apoE3 or the full-length and truncated apoE3[K146N/R147W] mutants. Experiments were performed as described in the Materials and Methods. The data represent the average of two independent experiments in triplicate.
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