title: Enhanced apoA-I-dependent cholesterol efflux by ABCA1 from sphingomyelin-deficient Chinese hamster ovary cells.
abstract: ATP binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It is proposed that ABCA1 reorganizes the plasma membrane and generates more loosely packed domains that facilitate apoA-I-dependent cholesterol efflux. In this study, we examined the effects of the cellular sphingomyelin level on HDL formation by ABCA1 by using a Chinese hamster ovary-K1 mutant cell line, LY-A, which has a missense mutation in the ceramide transfer protein CERT. When LY-A cells were cultured in Nutridoma-BO medium and sphingomyelin content was reduced, apoA-I-dependent cholesterol efflux by ABCA1 from LY-A cells increased 1.65-fold compared with that from LY-A/CERT cells stably transfected with human CERT cDNA. Exogenously added sphingomyelin significantly reduced the apoA-I-dependent efflux of cholesterol from LY-A cells, confirming that the decrease in sphingomyelin content in the plasma membrane stimulates cholesterol efflux by ABCA1. The amount of cholesterol available to cold methyl-beta-cyclodextrin (MbetaCD) extraction from LY-A cells was increased by 40% by the expression of ABCA1 and was 1.6-fold higher than that from LY-A/CERT cells. This step in ABCA1 function, making cholesterol available to cold MbetaCD, was independent of apoA-I. These results suggest that the function of ABCA1 could be divided into two steps: (i) a flopping step to move phosphatidylcholine and cholesterol from the inner to outer leaflet of the plasma membrane, where cholesterol becomes available to cold MbetaCD extraction, and (ii) a loading step to load phosphatidylcholine and cholesterol onto apoA-I to generate HDL.
authors.affiliation: Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan.
authors.forename: Kohjiro
authors.initials: K
authors.lastname: Nagao
authors.forename: Kei
authors.initials: K
authors.lastname: Takahashi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hanada
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kioka
authors.forename: Michinori
authors.initials: M
authors.lastname: Matsuo
authors.forename: Kazumitsu
authors.initials: K
authors.lastname: Ueda
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D001693:Biological Transport, Active / Q000235:genetics; D016466:CHO Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism; D009154:Mutation; D013109:Sphingomyelins / Q000172:deficiency* / Q000494:pharmacology
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D013109:Sphingomyelins; D002784:Cholesterol