Deletion of Amino Acids Glu146→Arg160 in Human Apolipoprotein A-I (ApoA-ISeattle) Alters Lecithin:Cholesterol Acyltransferase Activity and Recruitment of Cell Phospholipid

Abstract

Human apolipoprotein A-I (apoA-I) has an important role in the efflux of cholesterol from peripheral cells, the first step in reverse cholesterol transport. Deletion of amino acids Glu146→Arg160 in apoA-I (apoA-I_Seattle) removes a large section of a lipid binding helix and is associated in vivo with an atherogenic lipoprotein profile characterized by a deficiency in high-density lipoproteins (HDL). In the present study, we asked whether apoA-I_Seattle had normal ability to recruit lipids from cells and to form nascent high-density lipoprotein (HDL) particles and whether the altered secondary structure affected lecithin:cholesterol acyltransferase (LCAT) activity. Wild-type apoA-I and apoA-I_Seattle expressed in transfected Chinese hamster ovary cells formed nascent HDL particles with similar density distribution and protein-to-lipid ratio. Phospholipid subclass distribution of apoA-I_Seattle nascent HDL demonstrated a significant increase in sphingomyelin and phosphatidylethanolamine compared to wild type. ApoA-I_Seattle nascent HDL had a unique size distribution compared to wild-type nascent HDL; large (9−20 nm) particles predominated while there were virtually no small (7.5 nm) particles. LCAT reactivity was impaired by apoA-I_Seattle nascent HDL where cholesterol esterification was only half that of wild-type complexes. The apoA-I_Seattle conformation on nascent HDL was studied with a panel of monoclonal antibodies (Mabs) specific for apoA-I. Mabs that recognize the putative LCAT activation site, residues 95−122, had normal reactivity. As expected, the Mabs that recognized residues 141−164 were unreactive because of the 146−160 deletion; in addition, there was low reactivity with a Mab that recognizes residues 220−242. The data suggest that apoA-I residues 146−160 and/or 220−242 partake in normal LCAT activation and that cooperative interactions between helices may be important for maximal cholesterol esterification.