Distinct Central Amphipathic α-Helices in Apolipoprotein A-I Contribute to the in Vivo Maturation of High Density Lipoprotein by Either Activating Lecithin-Cholesterol Acyltransferase or Binding Lipids

Abstract Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Δ4-5A-I, Δ5-6A-I, and Δ6-7A-I, that have deletions removing regions coding for amino acids 100–143, 122–165, and 144–186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Δ5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Δ4-5A-I and Δ6-7A-I that formed more buoyant HDL2/3particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Δ5-6A-I and Δ6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Δ4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Δ6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Δ5-6A-I and greatly for Δ4-5A-I. We conclude from these findings that amino acids (aa) 100–143, specifically helix 4 (aa 100–121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144–186) centered around helix 6 (aa 144–165) is responsible for the activation of lecithin-cholesterol acyltransferase.