Oxysterols are substrates for cholesterol sulfotransferase
2007; Elsevier BV; Volume: 48; Issue: 6 Linguagem: Inglês
10.1194/jlr.m700018-jlr200
ISSN1539-7262
AutoresHirotoshi Fuda, Norman B. Javitt, Kuniko Mitamura, Shigeo Ikegawa, Charles A. Strott,
Tópico(s)Steroid Chemistry and Biochemistry
ResumoOxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7α/7β-hydroxycholesterol and 5α,6α/5β,6β-epoxycholesterol as well as the 7α-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds. Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7α/7β-hydroxycholesterol and 5α,6α/5β,6β-epoxycholesterol as well as the 7α-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds. 2-hydroxypropyl-β-cyclodextrin Cell Counting Kit-8 electrospray ionization 7-ketocholesterol 7-ketocholesterol sulfate 3′-phosphoadenosine 5′-phosphosulfate In contrast to the many studies that have established the deleterious effects of oxysterols on biologic processes (1.Brown A.J. Mander E.L. Gelissen I.C. Kritharides L. Dean R.T. Jessup W. Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes.J. Lipid Res. 2000; 41: 226-237Abstract Full Text Full Text PDF PubMed Google Scholar, 2.Lemaire-Ewing S. Prunet C. Montange T. Vejux A. Berthier A. Bessede G. Corcos L. Gambert P. Neel D. Lizard G. Comparison of the cytotoxic, pro-oxidant and pro-inflammatory characteristics of different oxysterols.Cell Biol. Toxicol. 2005; 21: 97-114Crossref PubMed Scopus (168) Google Scholar, 3.Leonarduzzi G. Vizio B. Sottero B. Verde V. Gamba P. Mascia C. Chiarpotto E. Poli G. Biasi F. Early involvement of ROS overproduction in apoptosis induced by 7-ketocholesterol.Antioxid. Redox Signal. 2006; 8: 375-380Crossref PubMed Scopus (63) Google Scholar, 4.Smith L.L. Johnson B.H. Biological activities of oxysterols.Free Radic. Biol. Med. 1989; 7: 285-332Crossref PubMed Scopus (333) Google Scholar), there have been few studies focusing on metabolic pathways for their disposal. Of particular interest are the high levels of oxysterols in atheromas because of their association with instability and rupture, a prelude to myocardial infarction (5.Carpenter K.L. Taylor S.E. van der Veen C. Williamson B.K. Ballantine J.A. Mitchinson M.J. Lipids and oxidised lipids in human atherosclerotic lesions at different stages of development.Biochim. Biophys. Acta. 1995; 1256: 141-150Crossref PubMed Scopus (178) Google Scholar, 6.Garcia-Cruset S. Carpenter K.L. Guardiola F. Mitchinson M.J. Oxysterols in cap and core of human advanced atherosclerotic lesions.Free Radic. Res. 1999; 30: 341-350Crossref PubMed Scopus (51) Google Scholar). A major oxysterol found in atheromas as well as other tissues is 7-ketocholesterol (7-KC), which is known from cell culture studies to induce cell injury at concentrations present in vivo (7.Leonarduzzi G. Gamba P. Sottero B. Kadl A. Robbesyn F. Calogero R.A. Biasi F. Chiarpotto E. Leitinger N. Sevanian A. et al.Oxysterol-induced up-regulation of MCP-1 expression in macrophage cells.Free Radic. Biol. Med. 2005; 39: 1152-1161Crossref PubMed Scopus (72) Google Scholar, 8.Lizard G. Monier S. Cordelet C. Gesquiere L. Deckert V. Gueldry S. Largrost L. Gambert P. Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1190-1200Crossref PubMed Scopus (186) Google Scholar); for this reason, there exists a particular focus on metabolic pathways that can lead to a reduction in its toxicity. For example, it has been shown that 7-KC is a substrate for 27-hydroxylation, thus forming a more water-soluble triol that decreases the intracellular concentration of 7-KC in macrophages (9.Babiker A. Andersson O. Lund E. Xiu R.J. Deeb S. Reshef A. Leitersdorf E. Diczfalusy U. Bjorkhem I. Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.J. Biol. Chem. 1997; 272: 26253-26261Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 10.Brown A.J. Watts G.F. Burnett J.R. Dean R.T. Jessup W. Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture.J. Biol. Chem. 2000; 275: 27627-27633Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11.Jessup W. Brown A.J. Novel routes for metabolism of 7-ketocholesterol.Rejuvenation Res. 2005; 8: 9-12Crossref PubMed Scopus (33) Google Scholar). We recently reported that in addition to accelerating transport, 27-hydroxylation also prevents the loss of cell viability and in coculture nullifies the toxicity of 7-KC (12.Lee J.W. Fuda H. Javitt N.B. Strott C.A. Rodriguez I.R. Expression and localization of sterol 27-hydroxylase (CYP27A1) in monkey retina.Exp. Eye Res. 2006; 83: 465-469Crossref PubMed Scopus (49) Google Scholar), findings consonant with the differential effects of oxysterols when used in combination (13.O'Sullivan A.J. O'Callaghan Y.C. O'Brien N.M. Different effects of mixtures of cholesterol oxidation products on bovine aortic endothelial cells and human monocytic U937 cells.Int. J. Toxicol. 2005; 24: 173-179Crossref PubMed Scopus (18) Google Scholar). Another potential metabolic pathway for the metabolism of oxysterols became apparent when it was found that one member of the SULT2 family of cytosolic sulfotransferases, SULT2B1b, has a particular affinity for cholesterol (14.Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from dehydroepiandrosterone sulfotransferase, and differential tissue expression.Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar). Although it is well recognized that sulfonation of steroid hormones affects their biologic activity (15.Hähnel R. Twaddle E. Ratajczak T. The specificity of the estrogen receptor of human uterus.J. Steroid Biochem. 1973; 4: 21-31Crossref PubMed Scopus (125) Google Scholar) and can influence their disposal (16.Bongiovanni A.M. Cohn R.M. Clinical aspects of steroid conjugation.in: Bernstein S. Solomon S. Chemical and Biological Aspects of Steroid Conjugation. Springer-Verlag, New York1970: 409-453Crossref Google Scholar), the concept that an analogous pathway exists for C27 sterols has received limited attention. Cytosolic sulfotransferases make up a superfamily of enzymes of which the SULT2 family sulfonates steroids/sterols (17.Nagata K. Yamazoe Y. Pharmacogenetics of sulfotransferase.Annu. Rev. Pharmacol. Toxicol. 2000; 40: 159-176Crossref PubMed Scopus (177) Google Scholar). The SULT2 family is further differentiated into two subfamilies: SULT2A1, the prototypical steroid sulfotransferase commonly referred to as dehydroepiandrosterone sulfotransferase, and SULT2B1. The SULT2B1 gene, because of an alternative exon 1 and differential splicing, encodes two isoforms, SULT2B1a and SULT2B1b (18.Her C. Wood T.C. Eichler E.E. Mohrenweiser H.W. Ramagli L.S. Siciliano M.J. Weinshilboum R.M. Human hydroxysteroid sulfotransferase SULT2B1: two enzymes encoded by a single chromosome 19 gene.Genomics. 1998; 53: 284-295Crossref PubMed Scopus (135) Google Scholar). Whereas human SULT2A1 and SULT2B1a avidly sulfonate the steroid pregnenolone, they do not use cholesterol effectively as a substrate; on the other hand, the SULT2B1b isozyme sulfonates cholesterol with the highest efficiency and, therefore, represents the physiologic cholesterol sulfotransferase (19.Fuda H. Lee Y.C. Shimizu C. Javitt N.B. Strott C.A. Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase.J. Biol. Chem. 2002; 277: 36161-36166Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In this report, we present evidence that MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC than 293T cells, which do not express this isozyme. Interestingly, however, using transfection of 293T cells, we were able to establish that the level of SULT2B1b expression correlated with a reduction in the toxicity level of 7-KC. In additional studies, we extended the range of C27 sterols that are substrates for SULT2B1b to include the 5α,6α/5β,6β-epoxy, 7α/7β-hydroxy, and 7α-hydroperoxide derivatives of cholesterol, thus expanding the potential role of this novel pathway in modulating in vivo the injurious effects of both oxysterols and hydroperoxides. Finally, we also present evidence for the first time that the sulfoconjugate of 7-KC does indeed occur in vivo, as demonstrated for human atheromatous tissue. Cholesterol, 7-KC, 7α-/7β-hydroxycholesterol, and 5α,6α-/5β,6β-epoxycholesterol were purchased from Steraloids (Newport, RI). Methanol, acetonitrile, and ammonium acetate for liquid chromatography-mass spectrometry were of HPLC grade and obtained from Nacalai Tesque (Kyoto, Japan). Distilled water of HPLC grade was from Wako Pure Chemical Industries (Osaka, Japan). Cholesterol 7α-hydroperoxide was prepared from cholesterol using hematoporphyrin and visible light according to the procedure described in detail previously (20.Rodriguez-Estrada M.T. Costa A. Pedillo M. Caboni M.F. Lercker G. Comparison of cholesterol oxidation product preparation methods for subsequent gas chromatographic analysis.J. AOAC Int. 2004; 87: 474-480Crossref PubMed Google Scholar). The isotopes [35S]3′-phosphoadenosine 5′-phosphosulfate (PAPS; 1.1 Ci/mmol) and [3H]7-KC (40 Ci/mmol) were purchased from Perkin-Elmer Life Sciences (Boston, MA) and American Radiolabeled Chemicals (St. Louis, MO), respectively. Iodine crystals, 2-hydroxypropyl-β-cyclodextrin (BCD), PAPS, and polyethylenimine were obtained from Sigma-Aldrich (St. Louis, MO). Organic solvents were purchased from either J. T. Baker or Mallinckrodt (Phillipsburg, NJ). Silica gel TLC plates were obtained from Analtech (Newark, DE), and Immobilon-P was from Millipore (Bedford, MA). TOPO TA Cloning Kit, pcDNA3.1(+), DMEM, FBS, and Antibiotic-Antimycotic were from Invitrogen (Carlsbad, CA). PfuUltra Hotstart DNA Polymerase was purchased from Stratagene (La Jolla, CA). Oligonucleotides were purchased from Operon Biotechnologies (Huntsville, AL). Cell Counting Kit-8 (CCK-8) was from Dojindo Molecular Technologies (Gaithersburg, MD). Goat anti-rabbit IgG conjugated to horseradish peroxidase and the LumiGLO Chemiluminescent Substrate System were obtained from KPL (Gaithersburg, MD). Proteinase inhibitor cocktail was purchased from Roche (Indianapolis, IN), and the BCA protein assay kit was from Pierce (Rockford, IL). BioMax XAR film was obtained from Kodak (Rochester, NY). Melting point was measured with a micro hot-stage apparatus and is uncorrected. The 1H-NMR spectrum was recorded with a JEOL EX-270 spectrometer (JEOL, Tokyo, Japan) operated at 270.05 MHz. Chemical shifts are given as the δ value with tetramethylsilane as an internal standard (s, singlet; d, doublet; m, multiplet). LC-MS analysis was performed using a Finnigan LTQ linear ion-trap mass spectrometer (Thermo Electron, San Jose, CA) equipped with an electrospray ionization (ESI) source and coupled to a Paradigm MS4 pump (Michrom Bioresources, Auburn, CA) and an autosampler (HTC PAL; CTC Analytics, Zwingen, Switzerland). The ionization conditions for verifying the structure of 7-ketocholesterol sulfate (7-KCS) were as follows: ion source voltage, −4 kV; capillary temperature, 270°C; capillary voltage, −33 V; sheath gas (nitrogen gas) flow rate, 50 arbitrary units; auxiliary gas (nitrogen gas) flow rate, 5 arbitrary units; tube lens offset voltage, −135 V. For tandem MS analysis, helium gas was used as the collision gas and the normalized collision energy was set at 25%. The LC separations were conducted on a semimicro column, TSKgel ODS-100V (5 μl, 150 × 2 mm inner diameter; Tosoh Co., Tokyo, Japan) by isocratic elution using acetonitrile-5 mM ammonium acetate buffer, pH 6.0 (3:1, v/v), as a mobile phase at a flow rate of 200 μl/min. To prepare the sodium salt of 7-KCS, a solution consisting of 50 mg of 7-KC in 1 ml of dry pyridine was added to freshly prepared sulfur trioxide-pyridine complex and stirred at room temperature overnight. After evaporation of the pyridine in vacuo at room temperature, the residue was redissolved in 5 ml of water and passed through a short pad of Cosmosil 140C18-OPN (Nacalai Tesque) on a sintered glass filter. After washing with water, the sterol sulfate was eluted with methanol and the eluate was chromatographed on silica gel using chloroform-methanol (7:1, v/v). The yield was 100%, and the colorless solid had a melting point of 125–129°C. For ESI-MS, m/z 479.4 [M-H]− (100%); for ESI-MS, m/z 96.8 [HSO4]−. For 1H-NMR (CDCl3) δ: 0.683 (3H, s, H-18), 0.863 (3H, d, J = 6.48 Hz, H-26 or H-27), 0.867 (3H, d, J = 6.75 Hz, H-26 or H-27), 0.923 (3H, d, J = 6.75 Hz, H-21), 1.197 (3H, s, H-19), 4.338 (1H, m, H-3α), 5.700 (1H, s, H-6). Human SULT2A1, SULT2B1a, and SULT2B1b were overexpressed in bacteria as glutathione S-transferase fusion proteins, cleaved, and affinity-purified as described previously (19.Fuda H. Lee Y.C. Shimizu C. Javitt N.B. Strott C.A. Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase.J. Biol. Chem. 2002; 277: 36161-36166Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). A 20 μl reaction volume contained 0.1 mM PAPS and purified enzyme preparation as described previously (14.Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from dehydroepiandrosterone sulfotransferase, and differential tissue expression.Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar). Briefly, a mixture consisting of SULT2A1 (4 μg), SULT2B1a (4 μg), and SULT2B1b (1 μg) in 0.1 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl2, 0.2 mM BCD, and [3H]7-KC in 4% ethanol was prepared. Reactions were carried out at 37°C for 5 min and stopped at 100°C for 5 min. After adding 10 μl of 5 mg/ml cholesterol sulfate as carrier, 5 μl aliquots were applied to TLC plates. Chromatography was carried out using the solvent system chloroform-methanol-acetone-acetic acid-water (8:2:4:2:1), after which the plates were dried and exposed to iodine vapor to visualize the location of 7-KCS. The iodine-adsorbed spots were excised and placed into counting vials containing 5 ml of scintillation cocktail, and the radioactivity was determined by liquid scintillation spectrometry. Human SULT2B1b was overexpressed in bacteria and affinity-purified as described above. A 20 μl reaction volume contained 0.1 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.2 mM BCD, oxysterol substrate, and 5 μM [35S]PAPS. The amount of SULT2B1b used for each reaction (μg/tube) was as follows: cholesterol (0.4 μg), 7-KC (1 μg), 7α-hydroxycholesterol (0.4 μg), 7β-hydroxycholesterol (1 μg), 5α,6α-epoxycholesterol (0.1 μg), and 5β,6β-epoxycholesterol (0.1 μg). Enzymatic reactions, chromatography, iodine-adsorbed staining, and liquid scintillation spectrometry counting were carried out as described above. PCR was used to isolate pcDNA-SULT2B1b. The PCR product was amplified using pGEX-6P-3-SULT2B1b (19.Fuda H. Lee Y.C. Shimizu C. Javitt N.B. Strott C.A. Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase.J. Biol. Chem. 2002; 277: 36161-36166Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) as a template and the SULT2B1b-specific primers 5′-TCTAGAATGGACGGGCCCGCCGAGCCCCAGATC-3′ (sense) and 5′-GCGGCCGCTTATGAGGGTCGTGGGTG-3′ (antisense). The underlined areas indicate XbaI and NotI sites, respectively. PCR conditions with PfuUltra Hotstart DNA Polymerase were as follows: denaturing at 95°C for 2 min, followed by 25 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The PCR product was subcloned into pCR2.1-TOPO using the TOPO TA Cloning Kit according to the manufacturer's protocol and sequenced. After digestion with XbaI and NotI, DNA was ligated to XbaI/NotI-digested pcDNA3-cMyc (kindly provided by Dr. Inohara Naohiro, Department of Pathology, University of Michigan Medical School). To isolate pcDNA-PAPS synthetase 1 (PAPSS1), after digestion of pGEX-6P-3-PAPSS1 with BamHI and NotI, DNA was ligated to BamHI/NotI-digested pcDNA3.1(+) (21.Fuda H. Shimizu C. Lee Y.C. Akita H. Strott C.A. Characterization and expression of human bifunctional 3′-phosphoadenosine 5′-phosphosulfate synthase isoforms.Biochem. J. 2002; 364: 497-504Crossref PubMed Google Scholar). 293T cells were generously provided by Dr. Pamela Schwartzberg at the National Human Genome Research Institute (National Institutes of Health, Bethesda, MD). MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown at 37°C in DMEM supplemented with 10% (v/v) FBS and Antibiotic-Antimycotic in 5% CO2. Media were changed every other day. 293T cells were seeded in 6 cm Falcon dishes (Franklin Lakes, NJ) at 16.5 × 105 cells/dish on the day before being transfected. Transfections were carried out using the Calphos Mammalian Transfection Kit according to the protocol of BD Biosciences Clontech (Mountain View, CA). pcDNA-SULT2B1b plus pcDNA-PAPSS1 or empty vector as a control was mixed with 2 M CaCl2 and HEPES-buffered saline and incubated for 20 min. After addition of the mixture, media were incubated for 8 h and changed to DMEM with 10% (v/v) FBS and Antibiotic-Antimycotic in 5% CO2. Transfected (48 h) 293T cells (10 × 104) were seeded using DMEM without phenol red on 24-well plates coated with 0.05% polyethylenimine and used to assay for sterol cytotoxicity, which was performed according to the manufacturer's protocol (Dojindo Molecular Technologies). Either 7-KC or 7-KCS dissolved in 45% (w/v) BCD was added to culture media, and the cells were incubated at 37°C in 5% CO2 for 24 h. After this time period, 20 μl of CCK-8 was added to the cultures and incubations were continued for an additional 1.5 h. Reactions were stopped by the addition of 50 μl of 1% (w/v) sodium dodecyl sulfate solution. Care was taken to avoid light exposure. Absorbance was measured at 450 nm using the μQuant plate reader (BioTek, Winooski, VT). Data were statistically analyzed by two-way ANOVA using GraphPad Prism (San Diego, CA). 293T cells were transfected with pcDNA-SULT2B1b and pcDNA-PAPSS1 or pcDNA3.1 as a control for 48 h as described previously and then reseeded in a 10 cm dish at 1 × 105 cells/dish. [3H]7-KCS (5 nM) in DMEM containing 30 μM BCD and 10% delipidated FBS (Monobind, Lake Forest, CA) and Antibiotic-Antimycotic was added to the cell culture and incubated for 48 h at 37°C in 5% CO2. The medium was extracted as described above and analyzed for 7-KCS formation. A model 1100 Hewlett-Packard instrument was used with a photoarray detector set at 210 and 225 nm. A C18 reverse-phase 250 × 4.6 mm chromatograph with a 4 μ silica column (catalog number OOG-4375-EO; Phenomenex, Torrance, CA) was used with a binary system of methanol-water beginning at 30% methanol and increasing to 100% methanol over a 30 min period and then continuing isocratically for an additional 30 min. The flow rate was constant at 0.8 ml/min. Samples were counted at constant efficiency using liquid scintillation spectrometry (Beckman). Transfected cells were washed two times with PBS and disrupted using a plastic cell scraper and cold RIPA lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100) with proteinase inhibitor cocktail. After centrifugation for 20 min, the supernatant was collected and the protein concentration was determined using the BCA protein assay kit. Samples (40 μg) were subjected to electrophoresis on a NuPage 4–12% Bis-Tris gel (Invitrogen) using MOPS/SDS running buffer and transferred to a polyvinylidene fluoride membrane (Immobilon-P). Membranes were soaked in a solution of 5% dry milk in TBS containing 0.05% Tween 20 (T-TBS) for 30 min with gentle shaking. Membranes were then exposed to either SULT2B1b (1:2,000; HL4360 #9) or PAPSS1 (1:2,000; HL4004) overnight at 4°C and washed three times with T-TBS for 5 min. Goat anti-rabbit IgG conjugated to horseradish peroxidase (1:55,000) was added and incubated for 1 h at room temperature. After washing three times with T-TBS for 5 min, LumiGLO chemiluminescent substrate was used according to the manufacturer's protocol before exposing membranes to X-ray film for 1 min. 293T cells were transfected as described previously. After quantifying the cell number, cells were extracted with chloroform/1% Triton X-100 and the organic phase was collected. The chloroform was removed by heat block and the use of the Savant SpeedVac system (GMI, Ramsey, MN). The dried cholesterol content was measured using the Cholesterol/Cholesteryl Ester Quantitation Kit according to the manufacturer's instructions (BioVision, Mountain View, CA) and expressed as μg/106 cells. Fresh-frozen specimens of human atheroma, obtained from autopsies under an approved protocol, were generously supplied by Dr. Allen Burke at the CVPath Institute (Gaithersburg, MD). Approximately 500 mg sections were added to 9.5 ml of methanol, and using a microtip probe (Sonicator 3000; Misonix, Farmingdale, NY), the samples were sonicated (2.5 W) at intervals for a total of 3 min with temperature monitoring and cooling in ice water so that the temperature did not increase to >60°C. The specimens were then centrifuged, and the supernatant was decanted and taken to dryness. The residue was redissolved in 0.5 ml of methanol and centrifuged, and the supernatant was used for HPLC analysis. Reverse-phase HPLC was carried out using a Hewlett-Packard 1100 instrument and a 150 × 4.6 mm C18 column (Aqua 3 μ, 125 A; Phenomenex) that was maintained at 60°C. A gradient of methanol-water was used beginning at 45% methanol and increased to 100% methanol over a 40 min period with a constant flow rate of 0.8 ml/min. Under these conditions, it was determined that standards of 7-KCS and 7-KC had retention times of 26.5 and 34 min, respectively, using a multi-wavelength detector (210, 225, and 233 nm). Methanolic aliquots of the atheroma specimens were injected onto the column, and fractions were collected at 2 min intervals for 48 min. Fractions obtained from 26 to 28 min were pooled for each specimen and taken to dryness. The pooled and lyophilized HPLC samples were then dissolved in 50 μl of 75% acetonitrile, and 10 μl aliquots were used for LC-ESI-MS analysis. That SULT2B1b has a penchant for C27 sterols, in contrast to the SULT2A1 and SULT2B1a isozymes, is demonstrated in Fig. 1 . SULT2B1b is 1 order of magnitude more active in sulfonating 7-KC than the other two steroid sulfotransferases (Table 1). The ability of SULT2B1b to sulfonate C27 sterols in addition to cholesterol and 7-KC was extended to include the α- and β-isomers of 7-hydroxycholesterol and 5,6-epoxycholesterol (Table 2). Interestingly, although SULT2B1b sulfonated cholesterol with the highest efficiency, its efficiency in sulfonating 7α-hydroxycholesterol was nearly as good (Table 2). Additionally, SULT2B1b was able to sulfonate the 7α-hydroperoxide derivative of cholesterol, although not as effectively as cholesterol, 7α-hydroxycholesterol, and 7-KC (Table 3). By contrast, the rate of dehydroepiandrosterone sulfate formation by SULT2B1b was <10% of that occurring with the prototypical steroid sulfotransferase, SULT2A1 (data not shown).TABLE 1.SULT2 enzyme 7-KC kinetic valuesEnzymeKmVmaxkcatkcat/KmMnmol/min/mgs−1M−1 s−1SULT2B1b7.7 × 10−629.12.0 × 10−22.6 × 103SULT2B1a2.4 × 10−56.74.2 × 10−31.8 × 102SULT2A11.7 × 10−57.03.9 × 10−32.3 × 1027-KC, 7-ketocholesterol. Human SULT2 enzymes were overexpressed as fusion proteins, cleaved, and affinity-purified. SULT2A1 (4 μg), SULT2B1a (4 μg), and SULT2B1b (1 μg) were placed in 0.1 mM Tris-HCl (pH 7.5) containing 0.1 mM 3′-phosphoadenosine 5′-phosphosulfate (PAPS), 5 mM MgCl2, 0.2 mM 2-hydroxypropyl-β-cyclodextrin (BCD), and [3H]7-KC. Reactions were carried out at 37°C for 5 min and stopped at 100°C for 5 min, and reactants were analyzed as described in Experimental Procedures. Open table in a new tab TABLE 2.SULT2B1b substrate kinetic valuesSubstrateKmVmaxkcatkcat/KmMnmol/min/mgs−1M−1 s−1Cholesterol1.1 × 10−625.21.7 × 10−21.6 × 1047α-Hydroxycholesterol2.3 × 10−650.33.5 × 10−21.5 × 1045α,6α-Epoxycholesterol2.5 × 10−619.71.4 × 10−25.4 × 1037-KC5.5 × 10−638.22.6 × 10−24.8 × 1037β-Hydroxycholesterol5.9 × 10−636.22.5 × 10−24.2 × 1035β,6β-Epoxycholesterol3.9 × 10−622.61.6 × 10−24.0 × 103The reaction mixture (20 μl) consisted of 0.1 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.2 mM BCD, substrate, and 5 μM [35S]PAPS. The optimal amount of human SULT2B1b, which was overexpressed as a fusion protein, cleaved, and affinity-purified, was determined for each substrate as follows: cholesterol (0.4 μg), 7-KC (1 μg), 7α-hydroxycholesterol (0.4 μg), 7β-hydroxycholesterol (1 μg), 5α,6α-epoxycholesterol (0.1 μg), and 5β,6β-epoxycholesterol (0.1 μg). Reactions were carried out at 37°C for 5 min and stopped at 100°C for 5 min, and the sulfonated products were analyzed as described in Experimental Procedures. Open table in a new tab TABLE 3.SULT2B1b sulfonation of C27 oxysterolsSterol SubstrateEnzyme Activitynmol/min/mgCholesterol7.57-KC7.17α-Hydroxycholesterol21.07β-Hydroxycholesterol1.97α-Hydroperoxide4.8Reactions were carried out for 20 min at 37°C in a solution of 0.08 M Tris (pH 7.1) containing 87 M [35S]PAPS, 4 mM MgCl2, and 1.3 mM BCD. Substrate and protein concentrations were 20 M and 10 g/ml, respectively. [35S]sterol sulfate formation was analyzed after extraction with methylene blue (14.Javitt N.B. Lee Y.C. Shimizu C. Fuda H. Strott C.A. Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from dehydroepiandrosterone sulfotransferase, and differential tissue expression.Endocrinology. 2001; 142: 2978-2984Crossref PubMed Scopus (75) Google Scholar). Open table in a new tab 7-KC, 7-ketocholesterol. Human SULT2 enzymes were overexpressed as fusion proteins, cleaved, and affinity-purified. SULT2A1 (4 μg), SULT2B1a (4 μg), and SULT2B1b (1 μg) were placed in 0.1 mM Tris-HCl (pH 7.5) containing 0.1 mM 3′-phosphoadenosine 5′-phosphosulfate (PAPS), 5 mM MgCl2, 0.2 mM 2-hydroxypropyl-β-cyclodextrin (BCD), and [3H]7-KC. Reactions were carried out at 37°C for 5 min and stopped at 100°C for 5 min, and reactants were analyzed as described in Experimental Procedures. The reaction mixture (20 μl) consisted of 0.1 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.2 mM BCD, substrate, and 5 μM [35S]PAPS. The optimal amount of human SULT2B1b, which was overexpressed as a fusion protein, cleaved, and affinity-purified, was determined for each substrate as follows: cholesterol (0.4 μg), 7-KC (1 μg), 7α-hydroxycholesterol (0.4 μg), 7β-hydroxycholesterol (1 μg), 5α,6α-epoxycholesterol (0.1 μg), and 5β,6β-epoxycholesterol
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