Abstract
The deletion mutation Delta6 apolipoprotein A-I lacks residues 143-164 or repeat 6 in the mature apoA-I protein. In vitro studies show this mutation dramatically reduces the rate of lecithin:cholesterol acyltransferase (LCAT) catalyzed cholesterol esterification. The present study was initiated to investigate the effect of this mutation on in vivo high density lipoprotein (HDL) cholesterol esterification and metabolism. Transgenic mice expressing human Delta6 apoA-I (TgDelta6 +/+) were created and then crossed with apoA-I knockout mice (-/-) to generate mice expressing only human Delta6 apoA-I (TgDelta6 -/-). Human Delta6 apoA-I was associated with homogeneous sized alpha-HDL, when wild-type mouse apoA-I was present (in TgDelta6 +/+ and +/- mice). However, in the absence of endogenous mouse apoA-I, Delta6 apoA-I was found exclusively in cholesterol ester-poor HDL, and lipid-free HDL fractions. This observation coincides with the 6-fold lower cholesterol ester mass in TgDelta6 -/- mouse plasma compared with control. Structural studies show that despite the structural perturbation of a domain extending from repeat 5 to repeat 8 (137-178), Delta6 apoA-I binds to spherical unilamellar vesicles with only 2-fold less binding affinity. In summary, these data show a domain corresponding to apoA-I repeat 6 is responsible for providing an essential conformation for LCAT catalyzed generation of cholesterol esters. Deletion of apoA-I repeat 6 not only blocks normal levels of cholesterol esterification but also exerts a dominant inhibition on the ability of wild-type apoA-I to activate LCAT in vivo.
Highlights
The potent antiatherosclerotic properties associated with elevated plasma HDL1 levels in humans [1] and in animal models [2,3,4,5] are generally ascribed to apoA-I and its role in co
The antiatherogenic role of plasma high density lipoprotein (HDL) apoA-I in cholesterol removal has been recently questioned because of reports describing mutations within the apoA-I coding sequence, which lead to low concentrations of HDL apoA-I and for which little or no association to coronary heart disease has been demonstrated in affected individuals
These data suggest that plasma concentrations of ⌬6 apoA-I were dependent on the presence of plasma wild-type mouse apoA-I containing HDL
Summary
Creation of ApoA-I ⌬6 Transgenic Mice—The apoA-I ⌬6 mutation was prepared by polymerase chain reaction megaprimer mutagenesis using a 2.2-kilobase PstI DNA fragment of the human apoA-I gene as previously reported [40, 43]. In Vivo ApoA-I Turnover Studies—Purified mouse wild-type apoA-I (Biodesign Inc.) and ⌬6 apoA-I protein [40, 43] were radiolabeled with either 5 mCi of 131I or 125I (NEN Life Science Products), respectively, using Iodo-Beads (Pierce) according to standard procedures [54] Both radiolabeled proteins (ϳ7 ϫ 106 cpm) were incubated at 4 °C with the pooled fresh whole C57BL/6J mouse plasma for 1– 4 h. Radiolabled apoA-I was prepared for use in the SUV binding assay by reacting 0.5 mCi of 125I (NEN Life Science Products) with 500 g of either purified recombinant (wild-type or mutant ⌬6) apoA-I or purified plasma apoA-I in the presence of Iodo-Beads (Pierce) according to standard procedures [54]. B Apo A-I concentrations were measured by ELISA for human ⌬6 apoA-I. c Western blot analysis for mouse wild-type as described under “Experimental Procedures.” d–o One-way ANOVA; values with unlike superscripts are statistically different at p Ͻ 0.05.
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