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 adenoiruses 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 HDL(2/3) particles. 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) 100143, 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.