Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatment with LCAT

Abstract

In the present study we have used adenovirus-mediated gene transfer of apoA-I (apolipoprotein A-I) mutants in apoA-I-/- mice to investigate how structural mutations in apoA-I affect the biogenesis and the plasma levels of HDL (high-density lipoprotein). The natural mutants apoA-I(R151C)Paris, apoA-I(R160L)Oslo and the bioengineered mutant apoA-I(R149A) were secreted efficiently from cells in culture. Their capacity to activate LCAT (lecithin:cholesterol acyltransferase) in vitro was greatly reduced, and their ability to promote ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux was similar to that of WT (wild-type) apoA-I. Gene transfer of the three mutants in apoA-I-/- mice generated aberrant HDL phenotypes. The total plasma cholesterol of mice expressing the apoA-I(R160L)Oslo, apoA-I(R149A) and apoA-I(R151C)Paris mutants was reduced by 78, 59 and 61% and the apoA-I levels were reduced by 68, 64 and 55% respectively, as compared with mice expressing the WT apoA-I. The CE (cholesteryl ester)/TC (total cholesterol) ratio of HDL was decreased and the apoA-I was distributed in the HDL3 region. apoA-I(R160L)Oslo and apoA-I(R149A) promoted the formation of prebeta1 and alpha4-HDL subpopulations and gave a mixture of discoidal and spherical particles. apoA-I(R151C)Paris generated subpopulations of different sizes that migrate between prebeta and alpha-HDL and formed mostly spherical and a few discoidal particles. Simultaneous treatment of mice with adenovirus expressing any of the three mutants and human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It also led to the formation of spherical HDL particles consisting mostly of alpha-HDL subpopulations of larger size. The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a potential therapeutic intervention for HDL abnormalities that result from specific mutations in apoA-I.

Figure 1. apoA-I expression and in vitro functional assays

(A) SDS/PAGE analysis of 100 μl of culture medium of HTB13 cells infected with control adenovirus expressing GFP, adenovirus expressing WT or the mutants apoA-I(R151C)_Paris, apoA-I(R160L)_Oslo and apoA-I(R149A). apoA-I levels can be assessed qualitatively by comparison with the intensity of the band of a sample containing 1 μg of BSA. ‘M’ indicates protein markers. (B) Activation of LCAT by rHDL containing WT or mutant apoA-I forms. The apparent K_m and V_max values are given at the bottom of the Figure. The catalytic efficiency of the enzyme, expressed as the V_max (app)/K_m (app) ratio, is plotted and expressed as percentage of WT control. Values are the means±S.D. for three independent experiments performed in duplicate. (C) cAMP-dependent (ABCA1-mediated) cholesterol efflux from J774 murine macrophages in the presence of WT apoA-I, apoA-I(R151C)_Paris, apoA-I(R160L)_Oslo or apoA-I(R149A), as indicated, was determined as described in the Experimental section. Values are the means±S.D. for three independent experiments performed in duplicate.

Figure 2. FPLC profiles of TC in plasma of apoA-I^−/− mice infected with 1×10⁹ pfu of adenoviruses expressing the WT apoA-I, apoA-I(R151C)_Paris, apoA-I(R160L)_Oslo, apoA-I(R149A) or the control protein GFP

Plasma samples were obtained from mice infected with the recombinant adenoviruses, 4 days post-infection.

Figure 3. Analyses of plasma of apoA-I^−/− mice infected with adenoviruses expressing the WT apoA-I (A, E, J), the apoA-I(R160L)_Oslo (B, F, K), apoA-I(R149A) (C, G, L), the apoA-I(R151C)_Paris (D, H, M) or the control adenovirus expressing GFP (I) by density-gradient ultracentrifugation, SDS/PAGE and two-dimensional gel electrophoresis

(A–C) SDS/PAGE analysis of density-gradient ultracentrifugation fractions. The densities of the fractions are indicated on the top of the Figure. (E–I) EM pictures of HDL fraction 6 obtained from apoA-I^−/− mice expressing the WT apoA-I, the mutant forms or the control adenovirus expressing GFP, following density-gradient ultracentrifugation of plasma. The arrows indicate discoidal HDL particles. (J–M) Two-dimensional (2D) gel electrophoresis and Western-blot analysis of plasma obtained from mice infected with adenoviruses expressing the WT or the mutant forms of apoA-I, as indicated. The arrows indicate the different HDL subpopulations.

Figure 4. Analyses of plasma of apoA-I^−/− mice infected with a combination of adenoviruses expressing human LCAT (5×10⁸ pfu) and apoA-I(R160L)_Oslo (1×10⁹ pfu), apoA-I(R149A) (1×10⁹ pfu) or apoA-I(R151C)_Paris (1×10⁹ pfu) mutants

(A–C) FPLC profiles of TC and comparison with the corresponding profiles of plasma obtained from apoA-I^−/− mice expressing the WT apoA-I or each of the three apoA-I mutants alone, as indicated. The CE/TC ratio of HDL fractions 15–22 of the FPLC is indicated. (D–F) SDS/PAGE profiles of fractions obtained by density-gradient ultracentrifugation analysis of plasma of apoA-I^−/− mice expressing the mutant forms of apoA-I in combination with the human LCAT, as indicated. (G–I) EM pictures of HDL fraction 6, following density-gradient ultracentrifugation of plasma obtained from apoA-I^−/− mice expressing the indicated mutant forms in combination with human LCAT. (J–L) Two-dimensional gel electrophoresis and Western blot analysis of plasma obtained from mice infected with adenoviruses expressing the WT or the mutant forms of apoA-I, as indicated. The arrows indicate the different HDL subpopulations.