Purification and ATPase activity of human ABCA1.
ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.
Takahashi
Kei
K
Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Kimura
Yasuhisa
Y
Kioka
Noriyuki
N
Matsuo
Michinori
M
Ueda
Kazumitsu
K
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D005905:Glyburide / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007313:Insecta; D007425:Intracellular Membranes / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008075:Lipoproteins, HDL / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010743:Phospholipids / Q000378:metabolism; D010840:Phytosterols / Q000378:metabolism; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry; D012855:Sitosterols / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D003902:Detergents; D008055:Lipids; D008075:Lipoproteins, HDL; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D010743:Phospholipids; D010840:Phytosterols; D012855:Sitosterols; C483858:phosphatidylethanolamine; C021273:campesterol; C025473:gamma-sitosterol; D012685:Sepharose; D002784:Cholesterol; D014357:Trypsin; D000251:Adenosine Triphosphatases; D002794:Choline; D005905:Glyburide