title: Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I.
abstract: ABCA1 mediates the efflux of cholesterol and phospholipids into apoA-I to form HDL, which is important in the prevention of atherosclerosis. To develop a novel method for the evaluation of HDL formation, we prepared an apoA-I-POLARIC by labeling the specific residue of an apoA-I variant with a hydrophobicity-sensitive fluorescence probe that detects the environmental change around apoA-I during HDL formation. apoA-I-POLARIC possesses the intact ABCA1-dependent HDL formation activity and shows 4.0-fold higher fluorescence intensity in HDL particles than in the lipid-free state. Incubation of apoA-I-POLARIC with ABCA1-expressing cells, but not ABCA1-non-expressing cells, caused a 1.7-fold increase in fluorescence intensity. Gel filtration analysis demonstrated that the increase in fluorescence intensity of apoA-I-POLARIC represents the amount of apoA-I incorporated into the discoidal HDL particles rather than the amount of secreted cholesterol. THP-1 macrophage-mediated HDL formation and inhibition of HDL formation by cyclosporine A could also be measured using apoA-I-POLARIC. Furthermore, HDL formation-independent lipid release induced by microparticle formation or cell death was not detected by apoA-I-POLARIC. These results demonstrate that HDL formation by ABCA1-expressing cells can be specifically detected by sensing hydrophobicity change in apoA-I, thus providing a novel method for assessing HDL formation and screening of the HDL formation modulator.
authors.affiliation: Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan.
authors.forename: Risa
authors.initials: R
authors.lastname: Omura
authors.affiliation: Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan.
authors.forename: Kohjiro
authors.initials: K
authors.lastname: Nagao
authors.affiliation: Kobe Pharmaceutical University, Kobe 658-8558, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kobayashi
authors.affiliation: Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Kazumitsu
authors.initials: K
authors.lastname: Ueda
authors.affiliation: Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008075:Lipoproteins, HDL / Q000737:chemistry* / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D064286:ATP Binding Cassette Transporter 1; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL
keywords: ATP binding cassette transporter A1; POLARIC; apolipoprotein A-I; cholesterol efflux; high density lipoprotein; hydrophobicity-sensitive fluorescence probe