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. 2011 Jul;52(7):1363-72.
doi: 10.1194/jlr.M012989. Epub 2011 Apr 19.

Alteration of negatively charged residues in the 89 to 99 domain of apoA-I affects lipid homeostasis and maturation of HDL

Andreas K Kateifides  1 , Irina N Gorshkova, Adelina Duka, Angeliki Chroni, Dimitris Kardassis, Vassilis I Zannis
Affiliations

Alteration of negatively charged residues in the 89 to 99 domain of apoA-I affects lipid homeostasis and maturation of HDL

Andreas K Kateifides et al. J Lipid Res. 2011 Jul.

Abstract

In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.

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Figures

Fig. 1.
Fig. 1.
1. FPLC profiles of total cholesterol (A) or triglycerides (B) of apoA-I−/− mice infected with adenoviruses expressing the WT apoA-I, apoA-I[D89A/E91A/E92A], and apoA-I[K94A/K96A] as indicated. Plasma samples were obtained four days postinfection.
Fig. 2.
Fig. 2.
2. Analyses of plasma of apoA-I−/− mice infected with adenoviruses expressing the WT apoA-I (A, D, G), the apoA-I[K94A/K96A] (B, E, H), and the apoA-I[D89A/E91A/E92A] (C, F, I) by density gradient ultracentrifugation, SDS-PAGE, two-dimensional gel electrophoresis, and EM. A–C: SDS-PAGE analysis of density gradient ultracentrifugation fractions. D–F: EM pictures of HDL fractions 6-7 obtained from apoA-I−/− mice expressing the WT and mutant forms of apoA-I following density gradient ultracentrifugation of plasma as indicated. The photomicrographs were taken at 75,000× magnification and enlarged three times. G–I: Analysis of plasma obtained from mice expressing the WT apoA-I or the mutant forms as indicated following two-dimensional gel electrophoresis and Western blotting.
Fig. 3.
Fig. 3.
3. Analyses of plasma of apoA-I−/− mice infected with adeno­viruses expressing the apoA-I[D89A/E91D/E92A] and hLPL. A: SDS-PAGE analysis of fractions obtained by density gradient ultracentrifugation of plasma of apoA-I−/− mice infected with 2 × 109 pfu of adenovirus expressing apoA-I[D89A/E91A/E92A] and 5 × 108 pfu of an adenovirus expressing the hLPL. B: EM picture of HDL fractions 6-7 obtained from apoA-I−/− mice expressing the apoA-I[D89A/E91A/E92A] mutant and human lipoprotein lipase, following density gradient ultracentrifugation as indicated in panel A. The sample was concentrated two times. The photomicrographs were taken at 75,000× magnification and enlarged three times. C: Analysis of plasma obtained from mice expressing the apoA-I[D89A/E91A/E92A] mutant and hLPL following two-dimensional gel electrophoresis and Western blotting.
Fig. 4.
Fig. 4.
4. Physicochemical properties of the WT apoA-I, apoA-I[D89A/E91A/E92A], and apoA-I[K94A/K96A]. A: Far-UV CD spectra of the apoA-I forms recorded at 25°C. Each sample contained 25 μg/ml protein in 10 mM PBS. Each spectrum is the average of four scans. B: Thermal unfolding of the apoA-I forms monitored by the ellipticity at 222 nm. Protein concentration in each sample was 40 μg/ml in 10 mM PBS. C: ANS fluorescence spectra obtained in the presence of the WT apoA-I, apoA-I[D89A/E91A/E92A], apoA-I[K94A/K96A] mutant, carbonic anhydrase, or in buffer alone, as indicated. The two latter spectra are superimposed. Final sample concentrations are 125 μM ANS and 25 μg/ml protein in 10 mM PBS. D: The time course of DMPC clearance by the apoA-I forms or in buffer alone. DMPC multilamellar vesicles (100 μg/ml lipids) were pre-incubated at 24°C, and the clearance was triggered by addition of protein to reach final concentration in cuvette 40 μg/ml. The turbidity was monitored by absorbance at 325 nm at the controlled temperature 24°C. *, WT apoA-I; •, apoA-I[D89A/E91A/E92A]; ▵, apoA-I[K94A/K96A], indicated by arrows.
Fig. 5.
Fig. 5.
5. Binding of the WT apoA-I and the apoA-I[D89A/E91A/E92A] mutant to triglyceride-rich emulsion particles. A: Portion of bound protein at different PC:protein ratios. B: Bound WT apoA-I or apoA-I[D89A/E91A/E92A] per one emulsion particle. *, WT apoA-I; •, apoA-I[D89A/E91A/E92A].
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