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. 2013 Dec;54(12):3293-302.
doi: 10.1194/jlr.M043489. Epub 2013 Oct 12.

Significance of the hydrophobic residues 225-230 of apoA-I for the biogenesis of HDL

Panagiotis Fotakis  1 , Ioanna Tiniakou, Andreas K Kateifides, Christina Gkolfinopoulou, Angeliki Chroni, Efstratios Stratikos, Vassilis I Zannis, Dimitris Kardassis
Affiliations

Significance of the hydrophobic residues 225-230 of apoA-I for the biogenesis of HDL

Panagiotis Fotakis et al. J Lipid Res. 2013 Dec.

Abstract

We studied the significance of four hydrophobic residues within the 225-230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I⁻/⁻ mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I⁻/⁻ × apoE⁻/⁻ mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225-230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225-230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.

Keywords: apolipoprotein A-I mutations; dyslipidemia; high density lipoprotein biogenesis; pre-β- and α-HDL particles.

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Figures

Fig. 1.
Fig. 1.
Analysis of plasma of apoA-I−/− mice infected with adenoviruses expressing the WT apoA-I or apoA-I[225–230] by FPLC (A) and by density gradient ultracentrifugation and SDS-PAGE (B, C). EM analysis of HDL fractions 6 and 7 obtained from apoA-I−/− mice expressing the WT apoA-I (D) or apoA-I[225–230] mutant (E) following density gradient ultracentrifugation of plasma as indicated. The photomicrographs in this as well as in Figs. 2 and 3 were taken at 75,000× magnification and enlarged three times. The average diameter of the particles in this as well as in Figs. 2 and 3 was determined by measuring the diameter of 200 individual particles. Two-dimensional gel electrophoresis of plasma of apoA-I−/− mice infected with adenoviruses expressing WT apoA-I (F) or apoA-I[225–230] mutant (G).
Fig. 2.
Fig. 2.
Analysis of plasma of apoA-I−/− × apoE−/− mice infected with adenoviruses expressing the WT apoA-I or apoA-I[225–230] mutant by FPLC (A, B) and by density gradient ultracentrifugation and SDS-PAGE (C, D). EM analysis of HDL fractions 6 and 7 obtained from apoA-I−/− × apoE−/− mice expressing the WT apoA-I (E) or apoA-I[225–230] mutant (F) following density gradient ultracentrifugation of plasma as indicated. Two-dimensional gel electrophoresis of plasma of apoA-I−/− × apoE−/− mice infected with adenoviruses expressing WT apoA-I (G) or apoA-I[225–230] mutant (H).
Fig. 3.
Fig. 3.
Analyses of plasma of apoA-I−/− (A,C,E,G) or apoA-I−/− × apoE−/− mice (B, D, F, H) infected with adenoviruses expressing the apoA-I[225–230] mutant in combination with human LCAT. Plasma FPLC profiles of mice expressing the apoA-I[225–230] mutant alone or the apoA-I[225–230] mutant in combination with human LCAT in apoA-I−/− (A) or apoA-I−/− × apoE−/− background (B). SDS-PAGE of fractions obtained by density gradient ultracentrifugation from apoA-I−/− (C) or apoA-I−/− × apoE−/− mice (D) mice expressing the apoA-I[225–230] mutant and human LCAT. EM analysis of HDL fractions 6 and 7 obtained from apoA-I−/− (E) or apoA-I−/− × apoE−/− (F) mice expressing the apoA-I[225–230] mutant and human LCAT following density gradient ultracentrifugation of plasma as indicated. Two-dimensional gel electrophoresis of plasma of apoA-I−/− (G) or apoA-I−/− × apoE−/− (H) mice expressing the apoA-I[225–230] mutant and human LCAT .
Fig. 4.
Fig. 4.
ABCA1-mediated cholesterol efflux from J774 mouse macrophages treated with cpt-cAMP using WT apoA-I and the apoA-I[225–230] mutant as cholesterol acceptors (A). LCAT activation capacity of WT apoA-I and the apoA-I[225–230] mutant. Experiments were performed as described in Materials and Methods. The data represent the average from two independent experiments performed in triplicate (B).
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