ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

• DOI: 10.1194/jlr.m033209
• Publication Date: 2012-11-20T06:45:16Z
• Authors: Hiroshi Hirayama; Yasuhisa Kimura; Noriyuki Kioka; Michinori Matsuo; Kazumitsu Ueda
• Keywords: ABCG1

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, biochemical properties ABCG1 have not been reported, and mechanism how involved in HDL remains unclear. We established a procedure to express purify human using suspension-adapted cell FreeStyle293-F. ABCG1, fused at C terminus with green fluorescent Flag-peptide, was solubilized n-dodecyl-β-D-maltoside purified via single round Flag-M2 antibody affinity chromatography. The reconstituted liposome various lipid compositions, ATPase activity analyzed. egg lecithin showed (150 nmol/min/mg), which inhibited by beryllium fluoride. phosphatidylserine liposome, stimulated cholesterol choline phospholipids (especially sphingomyelin), increased addition sphingomyelin. These results suggest that an active transporter possesses different binding sites sphingomyelin, may be synergistically coupled. Cholesterol component cellular membranes source steroid hormones. excess toxic cells becomes risk factor atherosclerosis. Therefore, level strictly regulated synthesis, intake, storage as esterified form, efflux. generation only pathway remove from peripheral tissues thus protect atherosclerosis (1Murphy A.J. Westerterp M. Yvan-Charvet L. Tall A.R. Anti-atherogenic mechanisms high density lipoprotein: effects on myeloid cells.Biochim. Biophys. Acta. 2012; 1821: 513-521Crossref PubMed Scopus (64) Google Scholar, 2Nagao K. Tomioka Ueda Function regulation ABCA1–membrane meso-domain organization reorganization.FEBS J. 2011; 278: 3190-3203Crossref (46) Scholar). Two ATP (ABC) transporters are generation. ABCA1 transports free phosphatidylcholine lipid-free apolipoprotein A-1 generates lipid-poor preHDL. predicted further transfer (3Gelissen I.C. Harris Rye K.A. Quinn C. Brown Kockx Cartland S. Packianathan Kritharides Jessup W. synergize mediate export apoA-I.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 534-540Crossref (340) 4Vaughan A.M. Oram J.F. or ABCG4 act sequentially generate cholesterol-rich HDL.J. Lipid Res. 47: 2433-2443Abstract Full Text PDF (231) Indeed, mice lacking accumulate lipids macrophages hepatocytes when fed high-fat high-cholesterol diet (5Baldan A. Pei Lee R. Tarr P. Tangirala R.K. Weinstein M.M. Frank Li A.C. Tontonoz Edwards P.A. Impaired development hyperlipidemic Ldlr−/− ApoE−/− transplanted Abcg1−/− bone marrow.Arterioscler. 2301-2307Crossref (151) Moreover, knockout both dramatic foam acceleration (6Yvan-Charvet Ranalletta Wang N. Han Terasaka Welch Combined deficiency promotes accumulation accelerates mice.J. Clin. Invest. 2007; 117: 3900-3908Crossref (445) data plays critical role homeostasis. reported mediates efflux phospholipids, especially sphingomyelin (SM) (7Kobayashi Takanezawa Y. Hirata T. Shimizu Misasa Kioka Arai H. Matsuo Efflux cholesterol, ABCG1.J. 1791-1802Abstract (161) Scholar), dependent SM (8Sano O. Kobayashi Nagao Kumagai Hanada Sphingomyelin-dependence mediated 48: 2377-2384Abstract (60) it unclear whether selectively SM. Furthermore, analysis although Cserepes et al. (9Cserepes Szentpetery Z. Seres Ozvegy-Laczka Langmann Schmitz G. Glavinas Klein I. Homolya Varadi al.Functional expression characterization proteins: indications heterodimerization.Biochem. Commun. 2004; 320: 860-867Crossref (85) Scholar) some crude insect cells. To address these issues, we then analyzed its activity. Our coupled L-α-lecithin yolk, Na2ATP, (PS), FLAG M2-agarose peptide (Sigma-Aldrich); FreeStyle 293-F cells, 293 medium, OPTI-MEM1 (Invitrogen); (PC) (Avanti Polar Lipids); phosphatidylethanolamine (PE) (Wako Pure Chemical Industries Ltd); anti-ABCG1 rabbit polyclonal (H65) (Santa Cruz Biotechnology); protease inhibitor (Roche Applied Science) were obtained. Suspension-adapted HEK293 (FreeStyle293-F) cultivated medium containing 25 μg/ml gentamicin avoid contamination microorganisms. Cells maintained 100 ml Erlenmeyer flasks shaken 130 rpm atmosphere 8% CO2; they passaged every 2 days. between 5 × 105 106 cells/ml. Large-scale culture performed 3-l spinner flask controller Cellmaster Model 1700 (Wakenyaku). temperature, dissolved oxygen, agitation speed 37°C, 3.0 ppm, rpm, respectively. One day before transfection, seeded 0.5 cells/ml, resulting 1.0 cells/ml log-phase growth transfection. cDNA tandemly repeated (eight times) glycine-threonine-serine sequence, (GFP), tag, 10 histidine residues pcDNA3.1. A stock solution (1 mg/ml) polyethyleneimine "MAX" (Polysciences) prepared H2O, sterilized filtration (0.2 μm), stored 4°C. For transfection large-scale culture, (7.5 mg) DNA (3 separately 150 mixed incubated 15 min being added culture. harvested 48 h after subcultured rpm. After incubation 24 h, transfected described above. another 0.02% BSA 20 content determined fluorescence enzyme assay (10Amundson D.M. Zhou Fluorometric method enzymatic determination cholesterol.J. Biochem. Methods. 1999; 38: 43-52Crossref (215) media no control subtracted. resuspended buffer 50 mM Tris-HCl (pH 7.5), 10% (v/v) glycerol, NaCl, 1 EDTA, 2-mercaptoethanol, inhibitors (complete EDTA-free; Roche Science). suspension sonicated (output 7, rounds sonication s interval 30 s) probe sonicator (Misonix Inc.) centrifuged 1,500 g unbroken nuclei. supernatant treated M NaCl peripherally anchored proteins 45,000 min. pellet ice-cold membrane passed through homogenizer −80°C. Crude (50 Tris [pH 7.4], inhibitor) detergents Insoluble materials removed centrifugation (15,000 min), loaded onto Superose 6 column (10/300; Amersham Biosciences) pre-equilibrated SEC 7.5], dithiothreitol, 0.03% C12E8). GFP detected in-line detector (Ex 480 nm/Em 510 nm). All purification steps 0–4°C. 0.8% (DDM) (Anatrace) kept ice occasional gentle mixing. insoluble (45,000 g, min). beads (Sigma-Aldrich) 12 washed four times 10× bed volume DDM three 0.05% DDM. Proteins eluted 1× 3× peptides. eluate concentrated ultrafiltration (100 kDa cut-off PES membrane; Sartorius Stedim Biotech) 0.1–0.3 mg/ml protein. Lipids chloroform dried evaporation reaction 0.1 EGTA) mg/ml. flash-frozen liquid N2 slowly thawed room temperature. This step five make large multilamellar vesicles. vesicles extruded 200-nm polycarbonate filter form unilamellar μl aliquot vesicles, 75 buffer, 0.1% destabilize liposomes. Purified μg) detergent-destabilized liposomes (11Geertsma E.R. Nik Mahmood N.A. Schuurman-Wolters G.K. Poolman B. Membrane reconstitution ABC assays translocator function.Nat. Protoc. 2008; 3: 256-266Crossref (179) mixture under detergent, 50% slurry Bio-Beads stepwise fashion: min, 40 reconstitution, concentrations measuring fluorescence. previously (12Kimura Kato Modulation drug-stimulated MDR1/P-glycoprotein cholesterol.Biochem. 401: 597-605Crossref (97) minor modifications. Reconstituted Tris/HCl EGTA, 3 MgCl2) 37°C stopped adding EDTA. analyze effect methyl-β-cyclodextrin (MβCD)-conjugated activity, Michaelis–Menten equation computer-fitted experimental data: v=Vcmax[S]/(Km+[S])+V0where Vcmax enhanced V0 basal [S] concentration MβCD-conjugated cholesterol,. Fitting carried out KaleidaGraph software, values V0, Vcmax, Km extracted. Vmax included value. ethanol MβCD 80°C, stirred until initially precipitated completely dissolved. MβCD) Proteoliposome cholesterol-MβCD complex temperature Experiments triplicate, presented means ± SD. statistical significance mean unpaired t-test. P value less than 0.05 considered statistically significant. Human Flag-peptide (ABCG1-GFP) stability screening. ABCG1-GFP expressed comparable (Fig. 1A). Similar wild-type localized mainly plasma 1B, C). found HDL-dependent also 1D). can used host expressing ABCG1. Selection optimal detergent purifying maintain during procedures (13Herget Kreissig Kolbe Scholz Tampe Abele Purification antigen transport TAP: prerequisite stoichiometry hydrolysis.J. Chem. 2009; 284: 33740-33749Abstract (42) 14Galian Manon F. Dezi Torres Ebel Levy D. Jault J.M. Optimized heterodimeric highly stable amenable 2-D crystallization.PLoS ONE. 6: e19677Crossref (28) examine detergents, molecular size monodispersity exclusion chromatography (FSEC) (15Kawate Gouaux E. Fluorescence-detection size-exclusion precrystallization screening integral proteins.Structure. 14: 673-681Abstract (502) separated gel 2). most examined around 280 symmetrical peak shape. weight consistent estimated one dimer (200 kDa) micelles, suggesting examined. smaller peaks cleaved fusion endogenous (supplementary . Among Fos-choline-14 highest efficacy solubilizing ABCG1-GFP. ABCG1(KM)-GFP, lysine residue hydrolysis replaced methionine, bioreactor. 3) Fig. II), Judging silver-stained SDS-PAGE gel, purity 80 90% 3A). assess oligomeric state, ABCG1(KM)-GFP FSEC 3B). main kDa, small shoulder observed 163 kDa. 47 likely moiety. elution profile similar wild-type, major fraction structure while degraded. pattern II). into kinetic parameters amount released ADP linearly within approximately 4A). following experiments. replacement Walker motif methionine largely impaired followed kinetics, maximum velocity calculated 0.95 0.12 6.9 nmol/min/mg, respectively 4B). Without did show 4C), bilayer environment function MDR1 (16Kimura Shibasaki Morisato Ishizuka Minakuchi Nakanishi Amachi Microanalysis high-performance titanium dioxide column.Anal. 326: 262-266Crossref (40) 17Doige C.A. Yu X. Sharom F.J. ATPase-active P-glycoprotein.Biochim. 1993; 1146: 65-72Crossref (173) Interestingly, even Typical release γ phosphate catalytically inactive analogs present (18Urbatsch I.L. Tyndall G.A. Tombline Senior A.E. P-glycoprotein catalytic mechanism: studies ADP-vanadate state.J. 2003; 23171-23179Abstract (76) mode measured presence ortho-vanadate fluoride 4C). Beryllium effectively whereas had inhibitory effect. pH dependency GTA increasing 4D). sharply pH. maximal 7.5, decreased SM, exported ABCG1-expressed Because activities generally substrates, PS PC, PE 5A). quite low (31.9 5.8 nmol/min/mg) compared higher (18:0), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (18:0-18:0), dose-dependent manner, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine half (in molar ratios) DSPC 9.5%, 8.3%, 14%, interacts head group about 1.7-fold PC. determine moiety affect 30% PC acyl chain structures 5B). equally 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1–18:1), (18:0–18:0), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0–18:1), lower 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0–14:0) liposome. should longer 16, but (saturated unsaturated) chains does It has several possess multiple substrate-binding (19Shapiro A.B. Ling V. Positively cooperative drug distinct specificities.Eur. 1997; 250: 130-137Crossref (437) 20Buxbaum Co-operative transported substrates resistance Mdr1.Eur. 265: 64-70Crossref (32) 21Loe D.W. Deeley R.G. Cole S.P. Verapamil stimulates glutathione 190-kDa multidrug (MRP1).J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed their affects each other, 2.5% 5% MβCD-conjugate 5C). 0.10 0.01 155.2 12.6 similar. When PC-containing 147.6 4.5 SM-containing shifted 0.06 mM, affected 153.3 1.9 nmol/min/mg. In this study, culture-adapted because cultured scale expected would best suited proteins. Flag-tag purification. first C-terminal GFP-fused membrane. functional localization half-size required function, concerned detergents. rather suggests once assembled endoplasmic reticulum. analysis, Fos-Choline-14 Fos-Choline-14, any indicating irreversibly inactivates without destroying form. Fos-choline-14, having tightly interact inactivate it. will interesting investigate site inactivation future studies. crucial (about ABCA1, MDR1, TAP (450, 1700, 1920 respectively) 22Takahashi Kimura ABCA1.J. 281: 10760-10768Abstract (87) 23Urbatsch Wilke-Mounts Gimi N-glycosylation mutant mouse P-glycoproteins Pichia pastoris cells.Arch. 2001; 388: 171-177Crossref (29) ABCA4 ABCG5/G8 (92 260 (24Tsybovsky Quazi Molday R.S. Palczewski Posttranslational modifications photoreceptor-specific ABCA4.Biochemistry. 50: 6855-6866Crossref (31) 25Johnson B.J. J.Y. Pickert Urbatsch Bile acids stimulate ABCG5/G8.Biochemistry. 2010; 49: 3403-3411Crossref (35) Thus, enough transporter. though analogs; selectivity ABCG5/8 (22Takahashi 26Wang Stalcup L.D. Harvey Weber Chloupkova Dumont M.E. Dean putative ABCG5 ABCG8.Biochemistry. 45: 9929-9939Crossref contrast, many transporters, including analogs. geometry slightly crystal maltose (27Oldham M.L. Chen Snapshots hydrolysis.Proc. Natl. Acad. Sci. USA. 108: 15152-15156Crossref (157) clear why others one. narrower range other (26Wang 28Urbatsch al-Shawi M.K. Characterization Chinese hamster P-glycoprotein.Biochemistry. 1994; 33: 7069-7076Crossref (225) 29Mao Q. Leslie E.M. MRP1 drug-selected H69AR 1461: 69-82Crossref (86) difference site. Substrate hydrolysis, substrate ATP-dependent specific reconstituting Cholesterol, 3-fold, ratios speculated due saturated if 16 hydroxyl exist interaction more two lowered affecting ATPase. binding. synergistic cholesterol. known spontaneously liquid-ordered microdomains (30Ramstedt Slotte J.P. Sphingolipids sterol-enriched ordered domains.Biochim. 1758: 1945-1956Crossref (156) efficiently microdomain speculate possible causes pseudo-synergistic coupling sites. Alternatively, changed fluidity liposomes, might recognition investigated further. summary, time cooperatively propose levels modification Download .pdf (.48 MB) Help pdf files n-octyl-β-D-glucoside