title: ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin.
abstract: ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.
authors.affiliation: Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hirayama
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Kimura
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: D000070998:ATP Binding Cassette Transporter, Subfamily G, Member 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology*; D003902:Detergents / Q000737:chemistry; D004357:Drug Synergism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C419353:ABCG1 protein, human; D000070998:ATP Binding Cassette Transporter, Subfamily G, Member 1; D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D010710:Phosphates; D013109:Sphingomyelins; D002784:Cholesterol; D000251:Adenosine Triphosphatases