Purification and ATPase Activity of Human ABCA1
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m513783200
ISSN1083-351X
AutoresKei Takahashi, Yasuhisa Kimura, Noriyuki Kioka, Michinori Matsuo, Kazumitsu Ueda,
Tópico(s)Systemic Lupus Erythematosus Research
ResumoATP-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-β high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-β HDL. To explore the mechanism of ABCA1-mediated pre-β 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. β-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. 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-β high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-β HDL. To explore the mechanism of ABCA1-mediated pre-β 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. β-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. ATP-binding cassette protein A1 (ABCA1) 2The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2-dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-β-d-maltoside; NTA, nitrilotriacetic acid. plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It has been reported that apolipoprotein A-I (apoA-I) binds to ABCA1 and cellular free cholesterol (FC) and phospholipids (PL) are loaded onto apoA-I to form pre-β HDL. It is clear that ABCA1 is involved in phosphatidylcholine (PC)-rich HDL generation in plasma, because plasma PL concentration of Abca1–/– mice was decreased by more than 75%, mostly due to a reduction of PC in HDL (1Francone O.L. Subbaiah P.V. van Tol A. Royer L. Haghpassand M. Biochemistry. 2003; 42: 8569-8578Crossref PubMed Scopus (37) Google Scholar); however, the molecular mechanism behind ABCA1-mediated pre-βHDL formation is still poorly understood. Several models have been proposed for the mechanism of ABCA1-mediated pre-β HDL formation: (a) a two-step process model proposed by Fielding et al. (2Fielding P.E. Nagao K. Hakamata H. Chimini G. Fielding C.J. Biochemistry. 2000; 39: 14113-14120Crossref PubMed Scopus (183) Google Scholar) and Wang et al. (3Wang N. Silver D.L. Thiele C. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar): ABCA1 first mediates PL efflux to apoA-I, and this apolipoprotein-PL complex accepts FC in an ABCA1-independent manner. This model is based on two types of experiments: (i) vanadate, glibenclamide, and cyclodextrin show differential inhibitory effects upon PL and FC efflux to apoA-I, and (ii) medium containing apoA-I conditioned on smooth muscle cells leads to FC efflux from vascular endothelial cells that do not express ABCA1. (b) A concurrent process model: FC and PL efflux by ABCA1 to apoA-I are tightly coupled to each other (4Smith J.D. Le Goff W. Settle M. Brubaker G. Waelde C. Horwitz A. Oda M.N. J. Lipid Res. 2004; 45: 635-644Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). (c) PS flipping model: ABCA1 mediates the translocation of PS to the outer leaflet, and extracellular exposure of PS promotes apoA-I binding to the cell surface and subsequent translocation of PC and cholesterol to apoA-I (5Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (467) Google Scholar). ABC proteins involved in xenobiotic efflux, such as MDR1 and MRP1, harness the energy liberated from ATP to drive the conformation changes that move xenobiotics across the membrane, and mutations in the ATP binding domain abolish the transport activity of these proteins (6Kimura Y. Matsuo M. Takahashi K. Saeki T. Kioka N. Amachi T. Ueda K. Curr. Drug Metab. 2004; 5: 1-10Crossref PubMed Scopus (44) Google Scholar, 7Haimeur A. Conseil G. Deeley R.G. Cole S.P. Curr. Drug Metab. 2004; 5: 21-53Crossref PubMed Scopus (453) Google Scholar). Like these xenobiotic transporters, ABCA1 K939M mutant, in which lysine, indispensable for the hydrolysis of ATP by various ABC proteins (8Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 9Lerner-Marmarosh N. Gimi K. Urbatsch I.L. Gros P. Senior A.E. J. Biol. Chem. 1999; 274: 34711-34718Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 10Sun H. Smallwood P.M. Nathans J. Nat. Genet. 2000; 26: 242-246Crossref PubMed Scopus (166) Google Scholar, 11Takada Y. Yamada K. Taguchi Y. Kino K. Matsuo M. Tucker S.J. Komano T. Amachi T. Ueda K. Biochim. Biophys. Acta. 1998; 1373: 131-136Crossref PubMed Scopus (39) Google Scholar), was substituted by methionine, was impaired in apoA-I-dependent PL and cholesterol efflux (3Wang N. Silver D.L. Thiele C. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 5Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (467) Google Scholar). As the translocation (flip-flop) of PLs rarely spontaneously occurs in lipid bilayers, and this process is highly energy-dependent, ABCA1 is suggested to flip-flop PLs depending on ATP hydrolysis. However, because the ABCA1 K939M mutant is defective in its interaction with apoA-I (3Wang N. Silver D.L. Thiele C. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 5Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (467) Google Scholar), it is also 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 (12Szakacs G. Langmann T. Ozvegy C. Orso E. Schmitz G. Varadi A. Sarkadi B. Biochem. Biophys. Res. Commun. 2001; 288: 1258-1264Crossref PubMed Scopus (48) Google Scholar) as SUR does in the ATP-sensitive potassium channel complex (13Ueda K. Komine J. Matsuo M. Seino S. Amachi T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1268-1272Crossref PubMed Scopus (135) Google Scholar). Human ABCA7, which has the highest homology (66.1%) to ABCA1, mediates the apoA-I-dependent efflux of phospholipids and cholesterol as ABCA1 (14Abe-Dohmae S. Ikeda Y. Matsuo M. Hayashi M. Okuhira K. Ueda K. Yokoyama S. J. Biol. Chem. 2004; 279: 604-611Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar); however, human ABCA7 mediates cholesterol release with much less efficiency than ABCA1 (15Hayashi M. Abe-Dohmae S. Okazaki M. Ueda K. Yokoyama S. J. Lipid Res. 2005; 46: 1703-1711Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and it has been reported that phospholipids but not cholesterol are loaded onto apoA-I by mouse ABCA7 (16Wang N. Lan D. Gerbod-Giannone M. Linsel-Nitschke P. Jehle A.W. Chen W. Martinez L.O. Tall A.R. J. Biol. Chem. 2003; 278: 42906-42912Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). These results may not be explained by the two-step process model and suggest that ABCA1 and ABCA7 have different substrate specificities for transport and that cholesterol is one of the transport substrates for ABCA1. To explore the mechanism of ABCA1-mediated pre-β HDL formation, we purified human ABCA1 as a detergent-soluble form and examined ATPase activity. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic PC, and the ATPase activity was inhibited by glibenclamide but not by vanadate. ABCA1 ATPase was reduced by the addition of cholesterol. Materials—Bovine serum albumin, leupeptin, aprotinin, trypsin, glibenclamide, stigmasterol, campesterol, and Na2ATP were purchased from Sigma. Baculovirus transfer vector (pVL1392) and baculovirus (Baculogold™) were obtained from Invitrogen. Anti-penta-His antibody was obtained from Qiagen. l-α-Lecitin (20%) from soybean and sphingomyelin from egg yolk were obtained from Sigma. Synthesized phospholipids, PC, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol were purchased from Avanti polar lipids. β-Sitosterol and brassicasterol were purchased from Tama Biochemical Co., Ltd. Other chemicals were purchased from Wako Pure Chemical Industries Ltd. KM3073 monoclonal antibody was generated against the first extracellular domain (35– 635 amino acid) of human ABCA1 in rats as described previously (17Munehira Y. Ohnishi T. Kawamoto S. Furuya A. Shitara K. Imamura M. Yokota T. Takeda S. Amachi T. Matsuo M. Kioka N. Ueda K. J. Biol. Chem. 2004; 279: 15091-15095Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Anti-ABCA1 NBF2 rabbit polyclonal antibody, which specifically interacts with the second nucleotide binding fold (NBF2) (data not shown), was generated against the purified NBF2 of human ABCA1 (1908–2159 amino acids). Anti-ABCA1 C terminus rabbit antibody was a kindly gift from Dr. Shinji Yokoyama, Nagoya City University Graduate School of Medical Sciences. Construction of Transfer Vector—The 3′ end of human ABCA1 cDNA (18Tanaka A.R. Ikeda Y. Abe-Dohmae S. Arakawa R. Sadanami K. Kidera A. Nakagawa S. Nagase T. Aoki R. Kioka N. Amachi T. Yokoyama S. Ueda K. Biochem. Biophys. Res. Commun. 2001; 283: 1019-1025Crossref PubMed Scopus (85) Google Scholar) was modified by elimination of the natural termination codon and insertion of a supplementary sequence containing a thrombin cleavage site, a biotinylation tag, and 10 consecutive histidine residues before the termination codon (5′-CTAGACTGGTTCCGCGTGGTTCCGGCTTGAATGATATATTCGAGGCCCAGAAGATAGAGTGGCATGAGGGAACTAGACTGGTTCCGCGTGGTTCCCACCATCACCATCACCATCACCATCACCATTGAG-3′). The modified ABCA1 cDNA (designated ABCA1-TATH) was inserted into the transfer vector pVL1392. To construct the NBF1 Walker A lysine mutant, ABCA1 K939M, DNA fragments containing a K939M missense mutation were generated by a two-step PCR method with two pairs of primers, 5′-GGGCCACAATGGAGCGGGGATGACGACCAC-3′ and 5′-CTGTCCCCCAGGACGTCCGCTTCATCCATG-3′ and 5′-GTGGTCGTCATCCCCGCTCCATTGTGGCCC-3′ and 5′-CTCAGTGGCTGTGATCATCAAGGGCATCG-3′. NBF2 Walker A lysine-1952 was substituted with methionine using a QuikChange site-directed mutagenesis kit (Stratagene) with a mutagenic primer, 5′-GGGGCTGGAATGTCATCAACTTTC-3′. The hMDR1 expression vector and recombinant virus were generated as described previously (6Kimura Y. Matsuo M. Takahashi K. Saeki T. Kioka N. Amachi T. Ueda K. Curr. Drug Metab. 2004; 5: 1-10Crossref PubMed Scopus (44) Google Scholar). Generation of Recombinant Baculovirus—Recombinant baculovirus containing ABCA1-TATH (BV-ABCA1-TATH) was generated by the co-transfection of Spodoptera frugiperda 9 (Sf9) cells with pVL1392-ABCA1-TATH and Baculogold™ DNA. BV-ABCA1-TATH was purified and amplified following the manufacturer's directions. The generated virus was kept at 4 °C in the dark. Expression of Human ABCA1 in Sf9 Cells—Sf9 cells were grown at 27 °C as a monolayer culture in Grace's insect medium (Invitrogen) with 10% fetal bovine serum or as a suspension culture in Grace's insect medium with 10% fetal bovine serum plus 0.1% purulonic F-68 (Invitrogen). Sf9 cells were infected with BV-hABCA1-TATH at a multiplicity of infection of 5. At 48 h after infection, cells were harvested and washed with ice-cold phosphate-buffered saline. Cells were stored at –80 °C. Preparation of Microsomal Membrane—All the steps in the preparation of the microsomal membrane fraction were performed at 0–4 °C. Sf9 cells were thawed and resuspended in 10×cell volume of sonication buffer containing 20 mm Tris-HCl (pH 7.5), 10% (v/v) glycerol, 1 mm EDTA, and protease inhibitor mixture (100 μg/ml (p-amidinophenyl)-methanesulfonyl fluoride, 10 μg/ml leupeptin, and 2 μg/ml aprotinin). The cell suspension was sonicated for 5 min (output 5, 10 rounds of sonication for 30 s + interval of 2 min) with a probe tip-type sonicator (Misonix Inc.) and centrifuged at 3,000 × g for 10 min to remove unbroken cells and nuclei. The supernatant was centrifuged at 40,000 × g for 60 min. The microsome pellet was resuspended in ice-cold buffer A containing 50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 30% (v/v) glycerol, 10 mm imidazole, 1 mm 2-mercaptoethanol, and protease inhibitor mix. The microsomal membrane suspension was passed through a 22-gauge syringe five times and stored at –80 °C. Micorosomal membrane protein (80–100 mg) was obtained from 10 g of wet cells. Glycosylation Analysis—The reaction of N-glycosidase F (NGF) was performed as described previously (18Tanaka A.R. Ikeda Y. Abe-Dohmae S. Arakawa R. Sadanami K. Kidera A. Nakagawa S. Nagase T. Aoki R. Kioka N. Amachi T. Yokoyama S. Ueda K. Biochem. Biophys. Res. Commun. 2001; 283: 1019-1025Crossref PubMed Scopus (85) Google Scholar). Lectin staining was performed as follows: After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane. The membrane was first blocked with block ACE (Dainihon Pharmaceutical) and incubated with 1 μg/ml of concanavalin A (Honen Corp.) in buffer containing 0.15 m NaCl, 0.01 m Tris-HCl (pH 7.5), and 0.05% Tween 20. The polyvinylidene difluoride membrane was then washed three times, and signals were detected with an ECL detection kit (Amersham Biosciences). Extraction of ABCA1 from Sf9 Membrane—The microsomal membrane was resuspended with buffer A containing 0.6 or 0.8% n-dodecyl-β-d-maltoside (DDM) (Dojindo) and protease inhibitor mix and kept on ice with occasional gentle mixing for 30 min. The insoluble fraction was removed by centrifugation (100,000 × g, 60 min) in a TLA 100.4 rotor (Beckman). Purification with Ni2+-NTA-Agarose—All purification steps were performed at 0– 4 °C. Extracted proteins were applied to Ni2+-NTA-agarose (Qiagen) pre-equilibrated with buffer A. The mixture was rotated for 18 h. The resin was then washed with 20× bed volume of buffer A containing 0.1% DDM and 20 mm imidazole. ABCA1 or MDR1 was eluted with 2× bed volume of buffer A containing 0.1% DDM and 200 mm imidazole. The eluate was concentrated by ultrafiltration using a microcon YM-100 (Millipore) to 0.1– 0.3 mg/ml protein. Protein concentrations were determined by the Bradford method using a protein assay kit (Bio-Rad) (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Bovine serum albumin was used as a standard. Purification with Anion Exchange Chromatography Sepharose, DE52— Proteins were mixed with pre-equilibrated DE52 Sepharose (Whatman) in DE52 binding buffer (50 mm Tris-HCl (pH 7.4), 30%(v/v) glycerol, 0.1% DDM, 1 mm 2-mercaptoethanol), and the mixture was rotated for 12 h. ABCA1 was eluted with DE52 binding buffer containing NaCl (50∼200 mm) for 60 min. The flow-through fraction and eluate were concentrated by ultrafiltration using a microcon YM-100 to 0.1– 0.2 mg/ml protein. Cell Culture and Membrane Preparation—WI-38 human fibroblast cells and HEK293 stably expressing human ABCA1, generated by hygromycin selection as described previously (17Munehira Y. Ohnishi T. Kawamoto S. Furuya A. Shitara K. Imamura M. Yokota T. Takeda S. Amachi T. Matsuo M. Kioka N. Ueda K. J. Biol. Chem. 2004; 279: 15091-15095Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), were grown at 37 °C in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. WI-38 cells were seeded into a 100-mm dish at 5 × 106 cells. To induce ABCA1 expression, cells were cultured in the medium containing 10 μm TO901317 and 5 μm 9-cis-retinoic acid for 24 h. Crude membranes were prepared by nitrogen cavitation method as described previously (18Tanaka A.R. Ikeda Y. Abe-Dohmae S. Arakawa R. Sadanami K. Kidera A. Nakagawa S. Nagase T. Aoki R. Kioka N. Amachi T. Yokoyama S. Ueda K. Biochem. Biophys. Res. Commun. 2001; 283: 1019-1025Crossref PubMed Scopus (85) Google Scholar). Reconstitution of Purified ABCA1 with Lipids—l-α-Lecitin (20%) from soybean, sphingomyelin, or synthesized phospholipids dissolved in chloroform were dried by evaporation and resuspended in reaction buffer, 40 mm Tris-HCl (pH 7.5), 0.1 mm EGTA to a final concentration of 5 mg/ml. When sterols were incorporated into the liposomes, sterols were mixed with phospholipids in chloroform at first, then dried by evaporation and resuspended in reaction buffer according to the standard procedure (20Rujanavech C. Silbert D.F. J. Biol. Chem. 1986; 261: 7215-7219Abstract Full Text PDF PubMed Google Scholar). The suspension was sonicated in a bath sonicator. To examine the effect of apoA-I, apoA-I (500 ng) was added to the suspension either before or after sonication. The lipid stock was stored at 4 °C under N2 gas and kept in a dark place. Purified protein (100 ng) was mixed with 500 ng of lipid, and the mixture was incubated at 23 °C for 20 min. The reconstitution buffer for MDR1 contained 2 mm dithiothreitol. Assay of ATPase Activity—Reactions were carried out in 16 μl of 40 mm Tris-HCl (pH 7.5), 0.1 mm EGTA, 10 mm Na2ATP (pH 7.0), and 10 mm MgCl2 at 37 °C for 30 min. Reactions were initiated by the addition of purified reconstituted ABCA1 (100 ng) and stopped by the addition of 14 μl of 12% SDS and vigorous mixing. ATPase activity was analyzed using two methods: (i) measuring released ADP by high performance liquid chromatography with titanium dioxide (21Kimura Y. Shibasaki S. Morisato K. Ishizuka N. Minakuchi H. Nakanishi K. Matsuo M. Amachi T. Ueda M. Ueda K. Anal. Biochem. 2004; 326: 262-266Crossref PubMed Scopus (40) Google Scholar) and (ii) measuring released Pi by the Pi-Mo method (22Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (418) Google Scholar, 23Drueckes P. Schinzel R. Palm D. Anal. Biochem. 1995; 230: 173-177Crossref PubMed Scopus (87) Google Scholar). The Pi released by the reaction with reconstituted ABCA1, predenatured by SDS, was subtracted as a negative control. Little or no ATP hydrolysis was observed in the reaction without protein. Expression of Human ABCA1 in Insect Cells—We constructed a plasmid, pVL1392-ABCA1-TATH, to express human ABCA1 fused with a histidine tag and biotinylation tag at the C terminus, and this was introduced into insect Sf9 cells. Individual plaques were examined for the expression, and Sf9 cells were infected by the purified virus for large scale production and purification. Human ABCA1 expressed in Sf9 cells, about 250 kDa, was found most abundantly in the microsome membrane fraction in 48 h (Fig. 1A), and ABCA1 in the nuclear fraction increased in 72 h. The amount of ABCA1 decreased in 120 h probably due to degradation. Therefore, Sf9 cells were harvested at 48–72 h after infection. The amount of ABCA1 in the microsome membrane fraction accounted for about 1% of total membrane proteins. Glycosylation of ABCA1 Expressed in the Sf9 Membrane—ABCA1 is glycosylated and has two large extracellular domains, which are suggested to be functionally important (24Fitzgerald M.L. Morris A.L. Rhee J.S. Andersson L.P. Mendez A.J. Freeman M.W. J. Biol. Chem. 2002; 277: 33178-33187Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 25Tanaka A.R. Abe-Dohmae S. Ohnishi T. Aoki R. Morinaga G. Okuhira K. Ikeda Y. Kano F. Matsuo M. Kioka N. Amachi T. Murata M. Yokoyama S. Ueda K. J. Biol. Chem. 2003; 278: 8815-8819Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The glycosylation of ABCA1 expressed in Sf9 membranes was examined. The mobility of ABCA1, expressed in Sf9 membranes, in SDS-PAGE became faster by NGF treatment (Fig. 1, B and C). ABCA1 reacted with concanavalin A, which reacts mainly with high mannose-type sugar chains, but ABCA1 treated with NGF did not (Fig. 1C). These results suggest that human ABCA1 expressed in Sf9 is modified with high mannose-type sugar chains. Solubility and Purification of ABCA1—As nonionic-detergent DDM was successfully used to solubilize recombinant ABC proteins (9Lerner-Marmarosh N. Gimi K. Urbatsch I.L. Gros P. Senior A.E. J. Biol. Chem. 1999; 274: 34711-34718Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 26Doerrler W.T. Raetz C.R. J. Biol. Chem. 2002; 277: 36697-36705Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 27Chen J. Sharma S. Quiocho F.A. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1525-1530Crossref PubMed Scopus (177) Google Scholar), human ABCA1 expressed in the Sf9 membrane was extracted with 0.6% and 0.8% DDM (Fig. 2A). ABCA1, extracted with 0.6% and 0.8% DDM (lanes 1 and 3), migrated a little slower than it remained in the insoluble fraction (lanes 2 and 4). Roughly 50% of ABCA1 was extracted with DDM, and efficiency was not significantly increased even with 1.0% DDM (data not shown). Extracted ABCA1 was purified using Ni2+-NTA-agarose resin (Fig. 2B). ABCA1 was recovered from the resin with 200 mm imidazole (lanes 10 and 11) in 80–90% purity judged from Coomassie Brilliant Blue staining. Purity was not significantly different between 0.6 and 0.8% DDM extraction. About 100 μg of purified ABCA1 was obtained from 250 mg of microsome membranes of Sf9 cells cultured in 1L suspension culture. Initially we planned to further purify ABCA1 with avidine column after in vitro biotinylation of the tag fused at the C terminus as reported previously (28Julien M. Kajiji S. Kaback R.H. Gros P. Biochemistry. 2000; 39: 75-85Crossref PubMed Scopus (16) Google Scholar); however, ABCA1 was not successfully recovered from the avidine column (data not shown). Therefore, we performed anion exchange chromatography using DE52 Sepharose (Fig. 2C). ABCA1, recovered from Ni2+-NTA-agarose resin, was concentrated and mixed with pre-equilibrated DE52 Sepharose. The majority of ABCA1 did not bind to DE52 Sepharose, and ABCA1 could be purified as a flow-through fraction of DE52 (Fig. 2C). ABCA1 was recovered in almost pure form from DE52 Sepharose judging from Coomassie Brilliant Blue (Fig. 2C) and silver staining (Fig. 2D). ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. 1B and 2E). Trypsin Sensitivity of ABCA1—To confirm that purified ABCA1 retained the correct conformation after the purification procedure, we examined the trypsin sensitivity of ABCA1. ABCA1, endogenously expressed in human fibroblast WI-38 cells, was partially digested by trypsin. ABCA1 was partially cleaved to produce four fragments, 170 and 150 kDa and subsequently 125 and 110 kDa, which were recognized by anti-ABCA1 extracellular domain 1 antibody (KM3073) (Fig. 3A) and two fragments, 170 and 120 kDa, which were recognized by anti-ABCA1 NBF2 antibody (Fig. 3B). These fragments may be assigned to fragments digested at site A, just after the sixth transmembrane α-helix, and at site B, just before the seventh transmembrane α-helix as shown in Fig. 3F. It is predicted that the limited digestion at site A produces 150- and 110-kDa fragments and their glycosylated forms, and the limited digestion at site B produces 155- and 110-kDa fragments and their glycosylated forms as a diagram (Fig. 3F). When these fragments were analyzed by SDS-PAGE under non-reducing conditions, they co-migrated with undigested ABCA1 of about 280 kDa (supplemental Fig. 2). These results suggested that N and C halves of ABCA1 were connected with disulfide bonds. When ABCA1 expressed in Sf9 cells was partially digested with trypsin, ABCA1 produced 140-kDa and subsequently 95-kDa fragments, which were recognized by KM3073 (Fig. 3C), and 155-kDa and subsequently 110-kDa fragments, which were recognized by anti-ABCA1 NBF2 antibody (Fig. 3D). All the produced fragments were smaller than the corresponding ones produced from ABCA1 of WI-38 cells probably due to glycosylation differences. When these fragments were analyzed by SDS-PAGE under non-reducing conditions, they co-migrated with undigested ABCA1 of about 250 kDa (supplemental Fig. 2). These results suggested that ABCA1 expressed in WI-38 cells and Sf9 cells contained similar typsin-sensitive sites (Fig. 3E). Trypsin limited-digestion of purified detergent-soluble ABCA1, producing N-terminal 140- and 95-kDa fragments and C-terminal 155and 110-kDa fragments (Fig. 3E). These fragments well corresponded to the fragments produced from ABCA1 endogenously expressed in WI-38 (Fig. 3, A and B), ABCA1 exogenously expressed in Sf9 cells (Fig. 3, C and D), and HEK293 cells (supplemental Fig. 1). These results suggest that purified ABCA1 retains conformation similar to ABCA1 endogenously expressed in human fibroblast membranes. ATPase Activity of ABCA1—The ATPase activity of purified ABCA1 reconstituted in soybean lipids was examined. ATP was hydrolyzed by proteoliposomes in a time-dependent manner at 37 °C, and released phosphate ions increased linearly during 30 min (Fig. 4). Hence, the following ATPase assays were performed at 37 °C for 30 min in this study. Next, we examined ATPase activity in the presence of various concentrations of MgATP. ATPase increased with increasing concentrations of ATP, and the apparent Km for ATP and Vmax were 112 μm and 455 nmol/min/mg of protein, respectively. The ATPase activity of purified reconstituted ABCA1 varied ranging from 400 to 900 nmol/min/mg of protein depending on preparations. Fig. 5 shows the representative data. To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. These lysines were reported to be indispensable for ATP hydrolysis of ABC proteins (29Gao M. Cui H.R. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 2000; 275: 13098-13108Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). These results suggest that the purified ABCA1 reconstituted into soybean lipids shows ATPase activity. Lipid Specificity in Stimulating ABCA1 ATPase—It has been reported that purified MDR1 shows ATPase activity only after reconstitution in liposomes and stimulation by transport substrates (30Doige C.A. Yu X. Sharom F.J. Biochim. Biophys. Acta. 1993; 1146: 65-72Crossref PubMed Scopus (174) Google Scholar). We compared the ATPase activity of ABCA1 and MDR1 purified with the same procedure (Fig. 6A). Purified human MDR1 (supplemental Fig. 3) reconstituted in soybean lipids showed minimu
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