Purification and ATPase Activity of Human ABCA1

Kei Takahashi,Yasuhisa Kimura,Noriyuki Kioka,Michinori Matsuo,Kazumitsu Ueda

doi:10.1074/jbc.m513783200

Abstract

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.

Highlights

Loaded onto apolipoprotein A-I (apoA-I) to form pre-␤ high density lipoprotein (HDL)
Several models have been proposed for the mechanism of ABCA1mediated pre-␤ HDL formation: (a) a two-step process model proposed by Fielding et al [2] and Wang et al [3]: ATP-binding cassette protein A1 (ABCA1) first mediates PL efflux to apoA-I, and this apolipoprotein-PL complex accepts free cholesterol (FC) in an ABCA1-independent manner
Because the ABCA1 K939M mutant is defective in its interaction with apoA-I [3, 5], it is possible that ATP binding and/or ATP hydrolysis cause conformational changes of ABCA1, which are required for the interaction with apoA-I, and ABCA1 functions as a regulator in HDL formation [12] as SUR does in the ATP-sensitive potassium channel complex [13]

Summary

Introduction

Loaded onto apoA-I to form pre-␤ HDL. It is clear that ABCA1 is involved in phosphatidylcholine (PC)-rich HDL generation in plasma, because plasma PL concentration of Abca1Ϫ/Ϫ mice was decreased by more than 75%, mostly due to a reduction of PC in HDL [1]; the molecular mechanism behind ABCA1-mediated pre-␤HDL formation is still poorly understood. To explore the mechanism of ABCA1-mediated pre-␤ HDL formation, we purified human ABCA1 as a detergent-soluble form and examined ATPase activity. These results suggest that purified ABCA1 retains conformation similar to ABCA1 endogenously expressed in human fibroblast membranes.

Results

Conclusion