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Cloning and Functional Characterization of Human Sodium-dependent Organic Anion Transporter (SLC10A6) *

Cloning and Functional Characterization of Human Sodium-dependent Organic Anion Transporter... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 27, pp. 19728 –19741, July 6, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cloning and Functional Characterization of Human Sodium-dependent Organic Anion Transporter (SLC10A6) Received for publication, March 28, 2007 Published, JBC Papers in Press, May 9, 2007, DOI 10.1074/jbc.M702663200 ‡1 ‡ ‡ § ¶ ‡ Joachim Geyer , Barbara Do¨ ring , Kerstin Meerkamp , Bernhard Ugele , Nadiya Bakhiya , Carla F. Fernandes , ‡ ¶ ‡ Jose´ R. Godoy , Hansruedi Glatt , and Ernst Petzinger From the Institute of Pharmacology and Toxicology, Justus-Liebig-University of Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany, University Hospital, Ludwig-Maximilians-University of Munich, 80337 Munich, Germany, and the Department of Nutritional Toxicology, German Institute of Human Nutrition Potsdam-Rehbru¨cke, 14558 Nuthetal, Germany We have cloned human sodium-dependent organic anion sion cloning from rat liver (2) and is exclusively expressed at the transporter (SOAT) cDNA, which consists of 1502 bp and sinusoidal membrane of hepatocytes (3, 4). Three years later, its encodes a 377-amino acid protein. SOAT shows 42% sequence intestinal counterpart was cloned from a hamster intestinal identity to the ileal apical sodium-dependent bile acid trans- cDNA library and was named apical sodium-dependent bile porter ASBT and 33% sequence identity to the hepatic Na / acid transporter (Asbt; Slc10a2) (5). In contrast to the basolat- taurocholate-cotransporting polypeptide NTCP. Immuno- eral localization of Ntcp, Asbt is highly expressed at the apical precipitation of a SOAT-FLAG-tagged protein revealed a brush border membrane of enterocytes of the terminal ileum glycosylated form at 46 kDa that decreased to 42 kDa after (6). Although sequence identity between NTCP and ASBT is PNGase F treatment. SOAT exhibits a seven-transmembrane quite low (at 35%), both carriers transport conjugated bile acids domain topology with an outside-to-inside orientation of the with high affinity (7–11). N-terminal and C-terminal ends. SOAT mRNA is most highly Due to their transport characteristics and expression pattern, expressed in testis. Relatively high SOAT expression was also NTCP and ASBT are important factors for the maintenance of detected in placenta and pancreas. We established a stable the enterohepatic circulation of bile acids mediating the first SOAT-HEK293 cell line that showed sodium-dependent step in the cellular uptake of bile acids through the membrane transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, barriers in the liver (NTCP) and intestine (ASBT). Since the bile and pregnenolone sulfate with apparent K values of 28.7, 12.0, acid reflux from the intestine is a major negative regulator of and 11.3 M, respectively. Although bile acids, such as tauro- the de novo bile acid synthesis from cholesterol in the liver, cholic acid, cholic acid, and chenodeoxycholic acid, were not ASBT is a promising drug target for cholesterol-lowering ther- substrates of SOAT, the sulfoconjugated bile acid taurolitho- apy (12). In fact, several compounds were able to significantly cholic acid-3-sulfate was transported by SOAT-HEK293 cells in lower plasma cholesterol levels and prevent atherosclerosis in a sodium-dependent manner and showed competitive inhibi- animal studies, and currently they are being tested in clinical tion of SOAT transport with an apparent K value of 0.24 M. trials (13). Several nonsteroidal organosulfates also strongly inhibited Recently, four new members of the SLC10 family were dis- SOAT, including 1-(-sulfooxyethyl)pyrene, bromosulfoph- covered and referred to as SLC10A3, SLC10A4, SLC10A5, and thalein, 2- and 4-sulfooxymethylpyrene, and -naphthylsulfate. sodium-dependent organic anion transporter (SOAT; Among these inhibitors, 2- and 4-sulfooxymethylpyrene were SLC10A6) (14). SLC10A3 (P3) was cloned from placenta and competitive inhibitors of SOAT, with apparent K values of 4.3 teratocarcinoma cDNA libraries in 1988, before NTCP and and 5.5 M, respectively, and they were also transported by ASBT had been discovered, and showed broad tissue expres- SOAT-HEK293 cells. sion (15). The second orphan transporter SLC10A4 seems to be predominantly expressed in the central nervous system and shares a common ancestor gene with NTCP. In contrast, SLC10 (solute carrier family 10) is well established as the SLC10A5 shows high expression in the liver, kidney, and intes- “sodium bile acid cotransporter family” (1). The first member of tine, which is very similar to the expression pattern of ASBT this transporter family, the Na /taurocholate-cotransporting (14). Until now, however, these orphan transporters have not polypeptide (Ntcp; Slc10a1), was identified in 1990 by expres- been subjected to intensive experimental expression analysis, and there is no published data indicating that they have any * This study was supported by the German Research Foundation (Deutsche function as solute carriers. Finally, in 2004, we cloned rat Soat, Forschungsgemeinschaft, Bonn, Germany) Grant GE1921/1-1 (to J. G. and which showed the highest phylogenetic relationship to ASBT K. M.) and Graduate Research Program 455 “Molecular Veterinary Medi- cine” (to B. D. and J. R. G.). The costs of publication of this article were but did not transport taurocholate (16). In this paper, we report defrayed in part by the payment of page charges. This article must there- on the cloning, membrane topology, and expression of human fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- SOAT and also provide its functional characterization in stably tion 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the Gen- transfected human embryonic kidney (HEK293) cells. Besides TM Bank /EBI Data Bank with accession number(s) AJ583502 and EF437223. sulfoconjugated steroid hormones, SOAT also transports tau- To whom correspondence should be addressed. Tel.: 49-641-9938404; Fax: 49-641-9938419; E-mail: joachim.m.geyer@vetmed.uni-giessen.de. rolithocholic acid-3-sulfate and sulfoconjugated pyrenes. 19728 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 This is an Open Access article under the CC BY license. Cloning and Characterization of Human SOAT EXPERIMENTAL PROCEDURES obtain a SOAT-pBluescript plasmid, the sticky ended SOAT cDNA fragment was directionally ligated downstream from a Materials and Chemicals—All of the chemicals, unless oth- T3 promoter into the pBluescript vector (Stratagene), which erwise stated, were from Sigma. Glycolithocholic acid was from was predigested with the respective restriction enzymes (SacII Calbiochem. Phenylsulfate, hydroquinone sulfate, -naphthyl- and XbaI). Three different clones were sequenced on both sulfate, 1-(-sulfooxyethyl)pyrene (1-SEP), 2-sulfooxymeth- strands, and the cDNA sequence was deposited in the Gen- ylpyrene (2-SMP), 4-sulfooxymethylpyrene (4-SMP), and TM TM Bank data base under GenBank accession number 5-sulfooxymethylfurfural were prepared from the correspond- AJ583502. In order to confirm transcription of the full-length ing hydroxyl compounds and sulfuric acid in dimethylform- SOAT mRNA sequence also on organs with high SOAT expres- amide using dicyclohexylcarbodiimide as the condensing sion (i.e. testis, placenta, and pancreas) (see below), RT-PCR agent, as described in detail elsewhere (17, 18). was also performed on human testis, placenta, and pancreas Radiochemicals—[ H]Dehydroepiandrosterone sulfate 3 3 3 cDNAs (BD Clontech) as described above, and PCR fragments ([ H]DHEAS, 60 Ci/mmol), [ H]estrone-3-sulfate ([ H]E S, 3 3 were verified by DNA sequencing. To confirm transport activ- 57 Ci/mmol), [ H]digoxin (24 Ci/mmol), and [ H]tauro- ity of human SOAT, the SOAT-pBluescript plasmid was used cholic acid (3.5 Ci/mmol) were purchased from PerkinElmer 3 3 14 14 for transport experiments with [ H]DHEAS and [ H]E Sin Life Sciences. [ C]Cholic acid (55 mCi/mmol), [ C]che- 1 Xenopus laevis oocytes as described in detail previously (16). nodeoxycholic acid (51 mCi/mmol), [ H]lithocholic acid (50 3 3 Identification of SOAT cDNA Ends by Rapid Amplification of Ci/mmol), [ H]pregnenolone-3-sulfate ([ H]PREGS, 20 cDNA Ends (RACE)-PCR—In order to obtain the full-length Ci/mmol), and [ H]deoxycholic acid (20 Ci/mmol) were SOAT mRNA transcript, we employed the GeneRacer method obtained from American Radiolabeled Chemicals. [ H]Es- based on RNA ligase-mediated and oligonucleotide-capping trone (76 Ci/mmol), [ H]estradiol-17-D-glucuronide (44 RACE according to the manufacturer’s protocol (Invitrogen). Ci/mmol), [ H]dehydroepiandrosterone (54 Ci/mmol), and Reverse transcription of 1 g of testis RNA (BD Clontech) was [ H]ouabain (23 Ci/mmol) were obtained from PerkinElmer 3 3 performed with the GeneRacer Oligo dT Primer 5-gct gtc aac Life Sciences. [ H]Taurolithocholic acid-3-sulfate ([ H]TLCS, gat acg cta cgt aac ggc atg aca gtg t -3 in a volume of 20 l 24.1 Ci/mmol) was generously donated by Werner Kramer using SuperScript III Reverse Transcriptase (Invitrogen). Initial (Sanofi-Aventis, Frankfurt am Main, Germany). 3- and 5-RACE reactions were performed using the gene-spe- Cloning of Human SOAT cDNA—Using BLAST searches of cific primers 5-ggc agc tcc tcc tct gaa ctg ttg-3 for 5-RACE the human genome with the cDNA sequences of the six coding TM amplification and 5-aat tac cct tgt gtg cct gac cat tc-3 for exons of rat Soat (Slc10a6) (GenBank accession number 3-RACE amplification. For each 50-l reaction, 1 l of cDNA, AJ583503), we obtained matches with six genomic sequence 0.5 l of AmpliTaq Gold DNA polymerase (Applied Biosys- fragments on human chromosome 4q21. These sequences were tems), and 6 l of MgCl (25 mM) were used, and amplification used for an RT-PCR-based strategy to obtain the full open read- 2 was performed under the following thermocycling conditions: ing frame cDNA sequence of human SOAT. The following oli- 1 cycle of 95 °C for 5 min; five cycles of 94 °C for 30 s and 72 °C gonucleotide primers were designed, including SacII/XbaI for 1 min; 5 cycles of 94 °C for 30 s and 70 °C for 1 min; 25 cycles restriction sites for PCR amplification: forward primer, 5-atg of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 1 min; and a final acc gcg gat gag agc caa ttg ttc cag cag ctc-3; reverse primer, extension of 72 °C for 10 min. To increase the yield and speci- 5-cgt cta gac tat tcg cat gaa gtg atg tgg cca act g-3. Although it ficity of the RACE products, additional nested PCR was per- has a relatively low expression in this organ (see below), human formed using the nested primers 5-gct gag ctg ctg gaa caa ttg SOAT was initially cloned from the adrenal gland. RT-PCR was gct c-3 for the nested 5-RACE reaction and 5-cct gtg gcc ttt performed from 1 g of human adrenal gland poly(A) RNA ggt gtc tat gtg-3 for the nested 3-RACE reaction under the (BD Clontech) using the Expand High Fidelity PCR System following conditions: one cycle of 95 °C for 5 min; 10 cycles of (Roche Applied Science) according to the following thermocy- 94 °C for 30 s, 70 °C for 30 s minus 0.5 °C each cycle, and 72 °C cling conditions: one cycle of 94 °C for 2 min; 10 cycles of 95 °C for 1 min; 30 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for for 15 s, 56 °C for 15 s minus 0.5 °C each cycle, and 72 °C for 1 1 min; and a final extension of 72 °C for 10 min. 3- and min; 30 cycles of 95 °C for 15 s, 51 °C for 15 s, and 72 °C for 1 min 5-RACE fragments were gel-purified and cloned into the plus 5 s each cycle; and final extension of 72 °C for 10 min. After pCR4-TOPO vector (Invitrogen). Three individual clones were the amplification reaction, samples were held at 4 °C until anal- sequenced on both strands for each PCR fragment. ysis. An aliquot of the PCR product was electrophoresed on an Establishment of the SOAT-HEK293 Cell Line—The recom- agarose gel. The amplicon of 1152 bp was excised form the gel binant human cell line T-REx SOAT-HEK293 was made using and digested for 90 min at 37 °C with SacII and XbaI. In order to the Flp-In expression system and the commercially available Flp-In T-REx 293 host cell line according to the manufacturer’s The abbreviations used are: 1-SEP, 1-(-sulfooxyethyl)pyrene; DMEM, Dul- instructions (Invitrogen). Flp-In T-REx 293 cells contain a sin- becco’s modified Eagle’s medium; PBS, phosphate-buffered saline; E S, gle, stably integrated Flp recombinase target (FRT) site at a estrone-3-sulfate; DHEAS, dehydroepiandrosterone sulfate; PREGS, preg- transcriptionally active genomic locus, which is maintained by nenolone sulfate; 2-SMP, 2-sulfooxymethylpyrene; 4-SMP, 4-sulfooxym- ethylpyrene; 1-SMP, 1-sulfooxymethylpyrene; TLCS, taurolithocholic acid- selection for zeocin resistance and ensures high level gene 3-sulfate; RT, reverse transcription; FRT, Flp recombinase target; PBS, expression from a target-integrated Flp-In expression vector. phosphate-buffered saline; HPLC, high pressure liquid chromatography; Briefly, SOAT cDNA spanning the whole open reading frame PNGase F, peptide:N-glycosidase F; DAPI, 4,6-diamidino-2-phenylindole; TMD, transmembrane domain; RACE, rapid amplification of cDNA ends. was subcloned from SOAT-pBluescript vector into the Flp-In JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19729 Cloning and Characterization of Human SOAT pcDNA5/FRT/TO expression vector carrying the FRT site and and washing five times with ice-cold PBS. Cell monolayers were the hygromycin resistance gene (Invitrogen). In the generated lysed in 1 N NaOH with 0.1% SDS, and the cell-associated radio- vector, further referred to as SOAT-pcDNA5, SOAT cDNA is activity was determined in a liquid scintillation counter. The under the control of the cytomegalovirus promoter and the protein content was determined according to Lowry using ali- tetracycline operator sequences (tetO ). In addition to the FRT quots of the lysed cells with bovine serum albumin as a standard site, Flp-In T-REx 293 cells stably express the tetracycline (19). repressor, which is maintained by selection for blasticidin Uptake Studies with 2-SMP and 4-SMP—Cells were seeded resistance. In the absence of tetracycline, tetracycline repressor in 24-well plates (2  10 cells in 1 ml of medium/well) 2 days effectively binds to the tetO sequence and blocks SOAT tran- before the experiment started. SOAT-HEK293 cells were incu- scription from the cytomegalovirus promoter. In order to bated in Ringer’s solution (130 mM NaCl, 4 mM KCl, 1 mM establish the SOAT-HEK293 cell line, the SOAT-pcDNA5 con- CaCl ,1mM MgSO ,20mM HEPES, 1 mM NaH PO , and 18 2 4 2 4 struct was cotransfected with the Flp recombinase expression mM glucose, pH 7.4) or an equimolar solution in which sodium vector pOG44 into Flp-In T-REx 293 host cells by Fugene 6 was replaced by choline in the presence of 10 M 2-SMP or transfection reagent according to the manufacturer’s protocol 4-SMP for 15 min at 37 °C. After aspiration of the transport (Roche Applied Science). Upon cotransfection, the SOAT cod- solution and three washes with ice-cold Ringer’s solution, cells ing sequence was integrated into the genome of the Flp-In were lysed with 0.25 ml of 1 N NaOH. After neutralization with HEK293 cells via Flp recombinase-mediated homologous 0.25 ml of 1 N HCl and protein precipitation with 1 ml of ace- recombination at the FRT site. Stable clones containing the tone, aliquots (usually 10 l) of the supernatant were injected SOAT open reading frame sequence under control of the cyto- into HPLC using a Shimadzu SIL-M10 Avp autosampler. Sam- megalovirus/tetO hybrid promoter were selected by culturing ples were separated using a Shimadzu SLC-10 Avp delivery sys- in selective medium containing 150 g/ml hygromycin and 50 tem equipped with a Phenomenex Gemini C18 column (250 g/ml blasticidin. After 10–14 days, single clones were isolated 3 mm; 5 m). The eluent was methanol containing 20% water from the remaining cell pool using cloning cylinders and tested and 0.05% triethylamine (v/v). The flow rate was 0.2 ml/min. for sodium-dependent [ H]DHEAS transport. The best trans- 2-SMP and 4-SMP were quantified from the fluorescence signal porting cell clone (further referred to as SOAT-HEK293) was ( 334 nm,  392 nm) using a Shimadzu SPD-M10 Avp ex em selected and used for further experiments. SOAT-HEK293 cells detector. were maintained in DMEM/F-12 medium (Invitrogen) supple- Expression of SOAT-FLAG-tagged and ASBT-FLAG-tagged mented with 10% fetal calf serum (Sigma), L-glutamine (4 mM), Proteins—A FLAG-tagged SOAT protein was generated by penicillin (100 units/ml), and streptomycin (100 g/ml) (fur- insertion of the FLAG-peptide (DYKDDDDK) to the C-termi- ther referred to as standard medium) at 37 °C, 5% CO , and 95% nal end of SOAT by QuikChange site-directed mutagenesis humidity. (Stratagene) of the SOAT-pcDNA5 construct. The following Transport Studies in SOAT-HEK293 Cells—For transport oligonucleotide sense and antisense primers were used: 5-cat studies, 12-well plates were coated with poly-D-lysine for better cac ttc atg cga aga tta caa gga tga cga cga taa gta ggc ggc cgc tcg attachment of the cells. 1.25  10 cells/well were plated and agt cta g-3 sense and 5-cta gac tcg agc ggc cgc cta ctt atc gtc gtc grown under standard medium for 72 h. SOAT expression was atc ctt gta atc ttc gca tga agt gat g-3 antisense. Correct clones induced by preincubation with tetracycline (1 g/ml). SOAT- were selected by DNA sequencing. Furthermore, an ASBT- nonexpressing control cells (Flp-In HEK293 cells) were not FLAG fusion protein was generated for comparative analysis. pretreated with tetracycline. Before starting the transport Briefly, the full open reading frame of human ASBT was ampli- experiments, cells were washed three times with phosphate- fied by RT-PCR from 1 g of small intestine poly(A) RNA (BD buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 1.5 mM Clontech). Gene-specific oligonucleotide primers were used KH PO , 7.3 mM Na HPO , pH 7.4, 37 °C) and preincubated containing SacII/XbaI restriction sites (5-cca gcc gcg gac cca 2 4 2 4 with sodium transport buffer containing 142.9 mM NaCl, 4.7 gca atg aat g-3 forward and 5-gtc ctc tag atg tct act ttt cgt cag mM KCl, 1.2 mM MgSO , 1.2 mM KH PO , 1.8 mM CaCl , and 20 gtt g-3 reverse), and PCR amplification was performed using 4 2 4 2 mM HEPES, adjusted to pH 7.4. When transport assays were the Expand High Fidelity PCR System (Roche Applied Sci- performed in sodium-free transport buffer, sodium chloride ence) according to the following touchdown schedule: 1 was substituted with equimolar concentrations of choline chlo- cycle of 94 °C for 2 min; 10 cycles of 94 °C for 15 s, 65 °C for ride. To determine the ion selectivity of SOAT transport, 30 s minus 0.5 °C each cycle, and 72 °C for 1 min; 30 cycles of sodium chloride in the transport buffer was also substituted 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min plus 10 s with equimolar concentrations of lithium chloride, potassium each cycle. The PCR product of the expected size was gel- chloride, N-methyl-D-glucamine, sodium gluconate, and potas- purified and digested for 90 min at 37 °C with SacII and XbaI. sium gluconate. Uptake experiments were initiated by replac- The sticky ended cDNA fragment was directionally ligated ing the preincubation buffer by 500 l of transport buffer con- downstream from a T3 promoter into the pBluescript vector taining the radiolabeled test compound and were performed at (Stratagene), which was predigested with SacII and XbaI. 37 °C. For inhibition studies, SOAT-HEK293 cells were prein- Sequence verification was done according to the reference TM cubated with transport buffer containing the inhibitor com- sequence with GenBank accession number NM_000452. pound for 30 s. Then transport measurements were started by Transport activity of ASBT was confirmed by transport adding the radiolabeled substrate at 37 °C. Transport and inhi- experiments in X. laevis oocytes with [ H]taurocholic acid as bition assays were terminated by removing the transport buffer the test compound (see above). For transfection of Flp-In 19730 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT HEK293 cells, the ASBT open reading frame sequence was exposed to Eastman Kodak Co. BioMax MR film (Sigma) at subcloned into the Flp-In pcDNA5/FRT/TO expression vec- 80 °C. tor, and the FLAG epitope was inserted at the C-terminal Antibody Preparation—The SOAT-(2–17) antibody was end of ASBT by QuikChange site-directed mutagenesis as raised in rabbits against amino acid residues 2–17 of the described above for SOAT. The following oligonucleotide deduced SOAT sequence (RANCSSSSACPANSSE). The syn- primers were used: 5-caa cct gac gaa aag gat tac aag gat gac thetic peptide was coupled via the carboxyl-terminal glutamic gac gat aag tag aca tct cga gtc-3 sense and 5-gac tcg aga tgt acid residue to keyhole limpet hemocyanin and used to immu- cta ctt atc gtc gtc atc ctt gta atc ctt ttc gtc agg ttg-3 antisense. nize two rabbits (Eurogentec). Antigenicity of the rabbit serum The SOAT-FLAG-pcDNA5 and ASBT-FLAG-pcDNA5 con- was confirmed by enzyme-linked immunosorbent assay analy- structs were verified by DNA sequencing, and positive sis using the synthetic peptide as the antigen. A second SOAT- clones were used for further experiments. (349–364) antibody was raised against amino acid residues Immunoprecipitation and Deglycosylation of the FLAG- 349–364 at the C terminus of SOAT. However, this antibody tagged Proteins—Flp-In T-REx 293 cells were seeded in showed no immunoreactivity against the synthetic peptide in 6-well plates coated with poly-D-lysine at a density of 1.0  enzyme-linked immunosorbent assay experiments and was not 10 cells/well in antibiotic-free DMEM/F-12 medium sup- applicable (Eurogentec). plemented with 10% fetal calf serum and 4 mML-glutamine. Immunofluorescence Microscopy of SOAT-HEK293 Cells— On the following day, the cells were transfected with 4 gof SOAT-HEK293 cells were grown on poly-D-lysine-coated SOAT-FLAG-pcDNA5 and ASBT-FLAG-pcDNA5 vector glass coverslips to 80% confluence in standard medium. DNA or with 4 g of pcDNA5 alone (control) by Lipofectamine SOAT expression was induced by tetracycline treatment (1 2000 reagent according to the manufacturer’s protocol g/ml) for at least 24 h. Noninduced control cells were (Invitrogen). After 4 h, the medium was changed to standard equally processed but were not incubated with tetracycline. medium, and expression of the FLAG-tagged proteins was On the next day, cells were washed three times with PBS and induced by tetracycline treatment (1 g/ml). On the next day, then incubated with the SOAT-(2–17) rabbit antibody (1:10) the cells were washed with PBS and starved in methionine-free for1hat room temperature without paraformaldehyde and cysteine-free DMEM medium (Sigma) supplemented with treatment. After rinsing and three times washing with PBS, 4mML-glutamine and 1 g/ml tetracycline for 1 h. Subse- the cells were incubated for1hat room temperature with the quently, 70 Ci of L-[ S] in vitro cell labeling mix (Amersham mouse fluorescein isothiocyanate-conjugated anti-rabbit Biosciences) was added, and the cells were incubated for an IgG antibody at 1:200 dilution (Sigma). After a final washing additional6hat37 °C,5%CO , and 95% humidity. The cells procedure, the cells were covered with a DAPI/methanol were washed with ice-cold PBS and incubated in 500 l of ice- solution containing 1 g/ml DAPI and incubated for 5 min at cold radioimmune precipitation buffer containing 150 mM 8 °C. The cells were washed with methanol/acetone (1:1), NaCl, 50 mM TrisHCl (pH 8.0), 1% Nonidet P-40, 0.5% (w/v) air-dried, and mounted on slides with Mowiol mounting sodium deoxycholic acid, 0.1% (w/v) SDS, and protease inhibi- medium. Fluorescence imaging was performed on a Leica tor mixture (Sigma) for 5 min under shaking. Cell lysates were DM6000B fluorescence microscope. Captured files were transferred to a microcentrifuge tube and incubated for an analyzed with the Leica FW4000 fluorescence work station additional 30 min under rotation at 4 °C. To remove any cell software. debris, the samples were centrifuged for 15 min at 4 °C, and the Immunofluorescence Microscopy of SOAT-FLAG-transfected supernatant was transferred to a fresh tube. Immunoprecipita- HEK293 Cells—For transient transfection, Flp-In HEK293 cells tion was performed by incubation with 5 g of the monoclonal were seeded in 24-well plates at a density of 2.5  10 cells/well mouse anti-FLAG antibody (Sigma) under rotation for1hat on poly-D-lysine-coated glass coverslips and grown to 80% 4 °C. Subsequently, 100 l of protein A-Sepharose (Sigma; 25% confluence in antibiotic-free DMEM/F-12 medium supple- suspension in radioimmune precipitation buffer) was added, mented with 10% fetal calf serum and 4 mML-glutamine. Cells and samples were incubated under rotation. After 1 h, Sepha- were transfected with 1 g of SOAT-FLAG-pcDNA5 vector rose beads were precipitated by centrifugation and washed DNA by Lipofectamine 2000 reagent (Invitrogen). The parental three times with ice-cold radioimmune precipitation buffer. pcDNA5 vector lacking any insert was used as control. After For deglycosylation with PNGase F, the protein A-Sepharose 4 h, the medium was changed to standard medium, and the beads were resuspended in 1 glycoprotein denaturation transfected cells were induced by tetracycline (1 g/ml) for at buffer and boiled for 10 min, and the eluted proteins were incu- least 24 h. After fixation with 2% paraformaldehyde in PBS for bated overnight at 37 °C with 1000 units of PNGase F in 1 G7 15 min at 4 °C, the cells were washed twice with PBS and incu- reaction buffer supplemented with 1% Nonidet P-40 (New Eng- bated with 20 mM glycine in PBS for 5 min. Subsequently, the land Biolabs). Nondeglycosylated samples were equally pro- cells were permeabilized for 5 min in PBT buffer (PBS contain- cessed but not incubated with PNGase F. All samples were ing 0.2% Triton X-100 and 20 mM glycine). Nonpermeabilized mixed with the same amount of 2 Laemmli buffer containing cells were not treated with PBT and were used for outside 10% -mercaptoethanol and boiled for 10 min. Finally, degly- epitope localization. The cells were placed in blocking solution cosylated and nondeglycosylated samples were separated by PBSG (1% bovine serum albumin and 4% normal goat serum in 12% SDS-PAGE. The gel was fixed in 30% methanol, 10% acetic PBS) for 30 min at room temperature and incubated with the acid (v/v) for 30 min and soaked in Amplify Fluorographic rea- rabbit anti-FLAG antibody (Sigma) at a 1:40,000 dilution in gent (Amersham Biosciences) for 30 min. Dried gels were PBSG overnight at 4 °C. The cells were washed three times with JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19731 Cloning and Characterization of Human SOAT TABLE 1 PBS and incubated with the goat Cy3-conjugated anti-rabbit Exon-intron organization of the human SLC10A6 gene on IgG antibody (Jackson Immunoresearch) at 1:800 in PBSG for chromosomal locus 4q21.3 1 h at room temperature. After triple washing with PBS, nuclei Nucleotide sequences of exon/intron junctions are indicated according to full- TM were stained by incubation with a DAPI/methanol solution length SOAT cDNA sequence (GenBank accession number EF437223) and H. TM sapiens chromosome 4, reference assembly (GenBank accession number containing 0.2 g/ml DAPI for 5 min at room temperature. The NC_000004.10). Intron sequences are represented in lowercase letters, and exon cells were washed with methanol, air-dried, and mounted on sequences are in uppercase letters. Exon Exon size 5-Splice donor 3-Splice acceptor Intron size slides with Mowiol mounting medium. Fluorescence imaging was performed as described above. bp kb 1 525 TCTCAG/gtaagt tttcag/CATCAG 15.3 Real Time Quantitative PCR—Relative SOAT expression 2 119 ACATAG/gtctgt ctttag/GAATTA 1.4 analysis was performed with ABI PRISM 7300 technology using 389 CTCAAG/gtgagt cttcag/ATTGGG 3.6 4 176 GCAAAG/gtatag tggtag/GTGCAG 2.4 human multiple tissue cDNA panels (BD Clontech) and cDNA 5 158 TTGCAG/gtgggt acctag/CATATC 1.5 synthesized from human adrenal gland and human mammary 6 435 TCAATG gland RNAs (BD Clontech). PCR amplification was achieved with TaqMan Gene Expression Assays Hs01399354_m1 for RESULTS human SOAT (SLC10A6) covering exon boundary 5–6 and Cloning of Human SOAT—Based on the cDNA sequence of Hs99999903_m1 for human -actin (Applied Biosystems). rat Soat, we used an RT-PCR-based approach to clone the full Expression data of -actin in each tissue were used as endoge- TM 1134-bp open reading frame of human SOAT (GenBank nous control. For each tissue, quadruplicate determinations accession number AJ583502). The full-length SOAT transcript were performed in a 96-well optical plate for both targets was obtained by RACE-PCR and revealed a 1502-bp cDNA, (SOAT and -actin) using 2.5 l of cDNA, 1.25 l of TaqMan including 5- and 3-untranslated regions of 148 and 220 bp, Gene Expression Assay, 12.5 TM l of TaqMan Universal PCR respectively (GenBank accession number EF437223). The Master Mix (Applied Biosystems), and 8.75 l of water in each human SLC10A6 gene is located on chromosome 4 and is 25-l reaction. The plates were heated for 10 min at 95 °C, and coded by six exons mapped in region 4q21.3. As summarized in subsequently 45 cycles of 15 s at 95 °C and 60 s at 60 °C were Table 1, the lengths of the SOAT exons account for 525, 119, 89, applied. Relative SOAT expression (C ) was calculated by T 176, 158, and 435 bp. All intron/exon boundaries were compat- subtracting the signal threshold cycle (C )of -actin from the ible with the canonical donor and acceptor consensus motifs. C value of SOAT. Subsequently, for each tissue, C values Each intron started with GT at the 5-splice donor site and T T were calculated by subtracting brain C (set as calibrator) ended with AG at the 3-splice acceptor site. The cloned SOAT from the C of each individual tissue and transformed by the cDNA sequence exactly matched the genomic sequence of TM Homo sapiens chromosome 4, reference assembly (GenBank 2 equation to show x-fold higher SOAT expression in the accession number NC_000004). The length of the human respective tissue. SLC10A6 gene accounts for 25.8 kb. Bioinformatics—The BLAST program available on the The SOAT protein consists of 377 amino acid residues with a World Wide Web was used to identify SOAT-encoding calculated molecular mass of 41.2 kDa. Fig. 1 shows the sequences in the human genome. Multiple sequence align- deduced amino acid sequence of human SOAT in alignment ments were conducted using the EBI ClustalW algorithm, avail- with human NTCP and ASBT. SOAT has a higher amino acid able on the World Wide Web, and alignment was visualized by sequence identity/similarity to ASBT (42%/70%), compared BOXSHADE, version 3.21. Amino acid identity values were with NTCP (33%/63%). At the C-terminal and N-terminal determined after pairwise optimal GLOBAL alignment with domains, the aligned proteins are clearly divergent, but a highly the BioEdit program, version 7.0.5.2 (20). For similarity calcu- homologous core region exists, representing the transmem- lations, the DAYHOFF similarity matrix was used. Membrane brane part of the proteins (residues 32–307 in the SOAT topology and putative membrane-spanning domains were sequence). As is the case for ASBT and NTCP, potential outer determined by the following programs: TMHMM (21), PRED- 4 14 157 facing N-glycosylation sites (residues Asn , Asn , and Asn ) TMR2 (22), MEMSAT (23), TMAP (24), TopPred II (25), and potential inner facing serine and threonine phosphoryla- TMpred (26), and HMMTOP (27). The NetNGlyc 1.0 program 60 126 259 310 335 336 tion sites (residues Ser , Ser , Ser , Thr , Ser , Thr , was used to predict N-linked glycosylation sites, and NetPhos 337 338 354 374 Ser , Ser , Thr , and Thr ) are present in the SOAT 2.0 was used to predict potential phosphorylation sites in the protein sequence. Further bioinformatic analyses revealed a SOAT protein (28). positively charged cluster in the SOAT C terminus at positions Statistical Analysis—Statistical significance for uptake 312–339 (net charge 9) and a tandem repeat “LTIP” at measurements with radiolabeled substrates was calculated residues 158–161 and 172–175. using Student’s t test. Statistical analysis of more than two SOAT Tissue Expression—The expression of SOAT in dif- groups was performed by one-way analysis of variance, fol- ferent human tissues was investigated by real time quantita- lowed by post hoc testing (Dunnett). Kinetic data from exper- tive PCR (Fig. 2A). Very low SOAT expression was found in iments measuring the uptake of radiolabeled substrates were brain, colon, kidney, liver, ovary, prostate, small intestine, fit to the Michaelis-Menten equation by nonlinear regres- spleen, and thymus. Expression levels were low also in the sion analysis. Dixon plot analysis was used for K adrenal gland, from which the SOAT was initially cloned. In calculations. contrast, SOAT was highly expressed in human testis, where 19732 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT dependence. Fig. 3 shows the time profile for [ H]DHEAS uptake by SOAT-expressing HEK293 cells and noninduced control cells. In the absence of tetracycline, no sig- nificant transport of [ H]DHEAS was observed. In contrast, SOAT- expressing HEK293 cells showed 3-fold (30 s) to 9-fold (10 min) higher [ H]DHEAS transport over the control cells. Using identical amounts of [ H]DHEAS (200 nM), transport studies were also per- formed in the absence of Na by equimolar substitution with cho- line. In these experiments, sodium replacement completely abolished SOAT-mediated DHEAS trans- port. The initial uptake velocity of [ H]DHEAS was also analyzed in SOAT-expressing HEK293 cells and revealed linear uptake over FIGURE 1. Amino acid sequence alignment of NTCP (SLC10A1), ASBT (SLC10A2), and SOAT (SLC10A6). The deduced amino acid sequences were aligned using the EBI ClustalW algorithm, and alignment was visu- 75 s at concentrations ranging alized by BOXSHADE. The portion of the sequence that must agree for shading was set to 0.8. Amino acid from 0.5 to 100 M DHEAS (Fig. identity is displayed with black shading, and amino acid similarities are highlighted in gray. Gaps () are intro- 4). Finally, the concentration de- duced to maximize alignment. 3 3 pendence of [ H]DHEAS, [ H]E S, the SOAT mRNA levels detected were 678 times higher than and [ H]PREGS transport was examined. As shown in Fig. 5, in brain (the tissue with lowest SOAT expression). Relatively SOAT-specific uptake of these compounds showed saturation high SOAT expression was also observed in human placenta kinetics and followed the Michaelis-Menten equation. Kinetic and pancreas, and moderate expression was detected in parameters were determined by nonlinear regression analysis heart, lung, and mammary gland. Because it has been and yielded K values of 28.7  3.9, 12.0  2.3, and 11.3  3.0 reported that an exon-2-skipped, alternatively spliced form M and V values of 1899  81, 585  34, and 2168  134 max of ASBT is expressed in certain rat tissues (29), we per- pmol/mg of protein/min for DHEAS, E S, and PREGS, formed additional RT-PCR experiments covering the whole respectively. open reading frame of SOAT from human testis, placenta, Ion Dependence of SOAT-mediated Transport—To further and pancreas RNAs. As shown in Fig. 2B, unique PCR ampli- investigate the sodium dependence of SOAT transport, we per- cons were detected that migrated at the expected size of 1152 formed transport experiments with equimolar substitutions of bp on the agarose gel without occurrence of shorter SOAT sodium chloride in the transport buffer by other cations and/or transcripts. anions. As shown in Table 3, lithium chloride and potassium Transport Properties of Human SOAT—Functional char- chloride maintained 36 and 23% of the transport function in acterization of human SOAT was performed in stably trans- relation to sodium chloride, respectively. In contrast, choline fected SOAT-HEK293 cells using the Flp-In T-REx expres- and N-methyl-D-glucamine could not maintain [ H]DHEAS sion system, where SOAT expression is under control of the transport by SOAT. Replacement of chloride by gluconate in tetracycline-regulated cytomegalovirus/tetO hybrid pro- the sodium-containing and potassium-containing transport moter. SOAT expression was induced in SOAT-HEK293 buffers revealed similar transport activities. This indicates that cells by pretreatment with tetracycline. Transport experi- SOAT transport is independent of chloride but highly depends ments were performed with several radiolabeled steroid on the presence of sodium in the transport buffer. compounds. SOAT-specific transport activity was not Inhibition Studies with SOAT-HEK293 Cells—Inhibition detected with nonconjugated steroids (estrone and dehydro- experiments were performed in SOAT-HEK293 cells to inves- epiandrosterone), glucuronidated steroids (estradiol-17-D- tigate the substrate selectivity of SOAT (Fig. 6). Cis-inhibitory glucuronide and estrone-3-D-glucuronide), bile acids effects of the indicated compounds on SOAT-mediated uptake (taurocholic acid, cholic acid, chenodeoxycholic acid, of 2.5 M [ H]DHEAS were examined at 25 M concentrations. deoxycholic acid, and lithocholic acid), or heart glycosides Among the group of bile acids, the trihydroxylated bile acids (ouabain and digoxin) (Table 2). However, SOAT-specific taurocholic acid, glycocholic acid, and cholic acid were no transport in SOAT-HEK293 cells was observed for DHEAS, inhibitors for SOAT-mediated [ H]DHEAS transport. In con- estrone-3-sulfate (E S), and pregnenolone sulfate (PREGS). trast, dihydroxylated bile acids inhibited SOAT transport Further characterization of this transport addressed time markedly, whereby inhibitory potency was higher for the dependence, sodium dependence, and concentration 3,7-dihydroxylated bile acids than for the 3,12-dihy- JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19733 Cloning and Characterization of Human SOAT droxylated bile acids. The chenodeoxycholic acid was the most nodeoxycholic acid, this bile acid is not a substrate of SOAT effective and reduced SOAT-mediated transport to 15%. How- (Table 2). SOAT inhibition was also observed by the 3-mono- ever, as demonstrated in transport experiments with [ C]che- hydroxylated bile acids lithocholic acid, glycolithocholic acid, and taurolithocholic acids. Again, [ C]lithocholic acid was not transported by SOAT-HEK293 cells in direct transport exper- iments (Table 2). Finally, sulfoconjugated 3-monohydroxy- lated bile acids were tested. These sulfated bile acids are struc- turally similar to the sulfated steroid hormones bearing an anionic sulfate moiety and a lipophilic steroid nucleus. At a 10-fold molar excess of unlabeled compounds, uptake of 2.5 M [ H]DHEAS was reduced to less than 10% by taurolithocholic acid-3-sulfate (TLCS), glycolithocholic acid-3-sulfate, and lith- ocholic acid-3-sulfate. TLCS, which was the most potent SOAT inhibitor among the group of sulfoconjugated bile acids, was also used for competitive inhibition experiments. Here, uptakes of 0.5 and 2.5 M [ H]DHEAS by SOAT were inhibited by increasing concentrations of TLCS (Fig. 7C). An apparent K value of 0.24 M was determined from Dixon plot transforma- tion; this is 2 orders of magnitude lower than the apparent K value for DHEAS (i.e. 28.7 M). Furthermore, cis-inhibitory effects of the SOAT transport were examined with a set of xenobiotic organosulfates. At 25 M concentrations, SOAT-mediated DHEAS transport was reduced to 4% by 1-SEP, to 18% by bromosulfophthalein, to 25% by 2-SMP and 4-SMP, and to 43% by -naphthylsulfate. In the case of 2-SMP and 4-SMP, additional inhibition experi- ments were performed using 100 and 500 nM concentrations of the SOAT substrate E S (Fig. 7, A and B). K values, determined 1 i from Dixon plots, were 4.3 and 5.5 M for 2-SMP and 4-SMP, respectively. In contrast, other sulfoconjugated organic mole- cules had little or no inhibitory activity for SOAT transport at 25 M concentrations. These included ethylsulfate, phenylsul- FIGURE 2. Expression of SOAT in various human tissues. SOAT tissue fate, phenylethylsulfate, 2-propylsulfate, 5-sulfooxymethylfur- expression was analyzed by quantitative real time PCR analysis (A) and con- fural, hydroquinone sulfate, 4-methylumbelliferylsulfate, and ventional PCR (B) using human multiple cDNA panels and cDNAs synthesized from human adrenal gland and mammary gland RNAs. Gene-specific primers indoxylsulfate (Fig. 6). Furthermore, a series of differently sub- and probes were used as outlined under “Experimental Procedures.” A, rela- T stituted naphthyl derivatives were tested to discriminate tive SOAT expression was calculated by the 2 method and represents SOAT expression that is x times higher in the respective tissue than in brain whether the sulfate moiety can be replaced by other groups for (set as calibrator). The values represent means  S.E. of quadruplicate meas- SOAT inhibition. However, in contrast to -naphthylsulfate, urements. B, RT-PCR experiments covering the whole open reading frame of SOAT. -naphthylisothiocyanate, -naphthylphosphate, and -naph- TABLE 2 3 14 Uptake of various H-labeled and C-labeled compounds by SOAT-HEK293 cells SOAT-HEK293 cells were seeded in 12-well plates and grown to confluence under standard conditions. Tetracycline (1 g/ml medium) was added to induce SOAT expression. Flp-In HEK293 cells were used as control. Cells were incubated with the indicated radiolabeled compound. After 10 min, the medium was removed, and each cell monolayer was washed and processed to determine the protein content and cell-associated radioactivity. The values represent means  S.E. of two independent experiments, each with triplicate determinations. Compound SOAT-HEK293 Control Ratio (SOAT/control) pmol/mg protein/10 min pmol/mg protein/10 min DHEAS (0.2 M) 13.1  0.7 1.1  0.04 12.2 E S (0.2 M) 10.2  0.5 0.8  0.1 12.1 PREGS (0.2 M) 146.1  6.5 19.5  3.7 7.5 Estrone (1 M) 21.5  0.5 21.7  0.7 1.0 Dehydroepiandrosterone (1 M) 31.0  1.3 33.4  0.7 0.9 Estradiol-17-D-glucuronide (1 M) 4.8  0.6 4.4  0.5 1.1 Estrone-3-D-glucuronide (1 M) 17.3  2.7 15.9  2.5 1.1 Taurocholic acid (1 M) 1.7  0.3 1.8  0.2 1.0 Cholic acid (2.5 M) 20.5  4.4 21.0  2.4 1.0 Chenodeoxycholic acid (2.5 M) 387.1  37.9 414.6  49.7 0.9 Deoxycholic acid (2.5 M) 124.9  4.9 124.3  2.4 1.0 Lithocholic acid (2.5 M) 281.4  20.8 311.0  11.0 0.9 Ouabain (1 M) 1.5  0.06 1.5  0.1 1.0 Digoxin (1 M) 6.2  0.3 6.2  0.1 1.0 Uptake values by SOAT-HEK293 cells were significantly different from control cells with p 0.001 (Student’s t test). 19734 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT FIGURE 3. Time course and sodium dependence of the transport of 200 nM [ H]DHEAS into SOAT-HEK293 cells. For SOAT expression, cells were pre- treated with 1 g/ml tetracycline (filled squares); noninduced control cells were not pretreated with tetracycline (open squares). Cells were incubated with 200 nM [ H]DHEAS at 37 °C for different time intervals in sodium chloride medium (continuous line) or in sodium-free choline chloride medium (broken line). At the indicated time points, cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent means  S.E. of two independent experiments, each with triplicate determinations. FIGURE 5. Concentration dependence of SOAT-mediated uptake of 3 3 3 FIGURE 4. Initial uptake velocity of [ H]DHEAS into SOAT-HEK293 cells. [ H]DHEAS (A), [ H]E S(B), and [ H]PREGS (C). SOAT-HEK293 cells were For SOAT expression, cells were pretreated with 1 g/ml tetracycline, and pretreated with 1 g/ml tetracycline. Flp-In HEK293 cells were used as con- uptakes of the indicated concentrations of DHEAS were measured over 15, trol. Cells were incubated with the indicated concentrations of [ H]DHEAS (A), 3 3 30, 45, 60, and 75 s. Cells were washed with ice-cold PBS, lysed, and subjected [ H]E S(B), and [ H]PREGS (C) for 1 min at 37 °C. The medium was removed, to scintillation counting. The values represent means of duplicate determina- and each cell monolayer was washed and processed to determine the protein tions of representative experiments. content and cell-associated radioactivity. SOAT-specific uptake was calcu- lated by subtracting nonspecific uptake of the Flp-In HEK293 control cells (open squares) from uptake into SOAT-HEK293 cells (filled squares) and is thylamine had no inhibitory effect on the DHEAS transport in shown by broken lines. The values represent means  S.E. of duplicate exper- iments, each with triplicate determinations. Michaelis-Menten kinetic param- SOAT-HEK293 cells (Fig. 6). eters were calculated from SOAT-specific uptakes by nonlinear regression Transport of [ H]TLCS, 2-SMP, and 4-SMP in SOAT- analysis and revealed K of 28.7  3.9 M and V of 1899  81 pmol/mg of m max HEK293 Cells—Since TLCS, 2-SMP, and 4-SMP competitively protein/min for DHEAS, K of 12.0 2.3 M and V of 585 34 pmol/mg of m max protein/min for E S, and K of 11.3 3.0M and V of 2168 134 pmol/mg 1 m max inhibited SOAT transport, we further investigated whether of protein/min for PREGS. they are also substrates of SOAT. No radiolabeled compounds were available for 2-SMP and 4-SMP, so we determined the intracellular accumulation of 2-SMP and 4-SMP in the SOAT- observed if the experiments were performed in Na -containing HEK293 cells with an HPLC-based method using fluorescence transport buffer and was completely abolished if sodium was detection. As shown in Fig. 7, D–G, 2-SMP, 4-SMP, and TLCS substituted by equimolar concentrations of choline, thus indi- were transported in SOAT-expressing HEK293 cells with a cating that 2-SMP, 4-SMP, and TLCS were transported by ratio of about 2 over control cells. This transport was only SOAT in a sodium-dependent manner (Fig. 7, D and E). Fur- JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19735 Cloning and Characterization of Human SOAT TABLE 3 thermore, 4-SMP transport was blocked by a 5-fold molar Influence of equimolar substitutions of NaCl on H DHEAS uptake excess of E S in the transport medium (Fig. 7F). In contrast, by SOAT-HEK293 cells transport of 50 nM TLCS was inhibited by not less than 50 M Uptake of 200 nM H DHEAS was measured at 37 °C in SOAT-HEK293 cells after DHEAS (Fig. 7G), which is consistent with the lower K value of preincubation with tetracycline (1 g/ml). 142 mM NaCl was used in the transport i buffer for the 100% control experiment and was substituted with equimolar con- TLCS compared with 4-SMP. centrations of sodium gluconate, potassium gluconate, lithium chloride, potassium Membrane Topology of Human SOAT—Analysis of the chloride, N-methyl-D-glucamine, and choline chloride. After 5 min, the transport medium was removed, and the cell monolayer was washed and subjected to radio- membrane topology of human SOAT was performed with activity and protein measurements. The values represent means  S.D. of two different topology prediction programs. TMHMM, PRED- independent experiments, each with triplicate determinations. TMR2, MEMSAT, TMAP, TopPred II (GES-scale), and Percentage DHEAS uptake of control TMpred proposed a membrane topology of SOAT with eight pmol/mg protein/5 min % transmembrane domains and an extracellular location of the Sodium chloride (control) 10.4  0.5 100 N-terminal and C-terminal domains (Fig. 8A). In contrast, Lithium chloride 3.8  0.3 36 HMMTOP analysis preferred a model with nine transmem- Potassium chloride 2.4  0.3 23 Choline chloride 0.4  0.04 3.8 brane domains, and TopPred II (KD-scale) calculated a sev- N-Methyl-D-glucamine 0.3  0.07 2.7 en-TMD topology. In all predictions, the N terminus of Sodium gluconate 9.9  0.8 95 Potassium gluconate 1.6  0.1 15 SOAT has an extracellular orientation and contains 30 Uptake values after equimolar substitution of NaCl were significantly different amino acid residues. This orientation is predicted due to a from control experiments with p 0.01 (one-way analysis of variance with cluster of positively charged amino acid residues just down- Dunnett post hoc analysis). stream from TMD 1 (net charge of the N terminus 4, net charge of the first intracellular loop 3). The C terminus is inside in the seven-TMD and nine-TMD models but has an extracellular orientation in the eight-TMD topology (Fig. 8A). Similar dis- crepancies from in silico topology predictions were obtained for NTCP and ASBT. For these SLC10 carriers, experimental data clearly favored a seven-TMD topology. To determine whether a seven- TMD topology can be applied also for SOAT, we directly compared the hydrophobicity profiles of SOAT, ASBT, and NTCP in an overlay of the individual hydro- phobicity plots. As shown in Fig. 8B, hydrophobicity values of SOAT and ASBT are nearly iden- tical, indicating that both carriers show similar membrane topology. However, both proteins differ from the NTCP hydrophobicity pattern, particularly concerning amino acid residues 70 –170, which represent transmembrane helices 2– 4. Localization of the N-terminal and C-terminal Domains of FIGURE 6. Inhibitory potency of bile acids, nonsteroidal organosulfates, and naphthyl derivatives on SOAT—Membrane expression SOAT transport. Sodium-dependent uptake of 2.5 M [ H]DHEAS was measured in the presence of 25 M and the C/N terminus orientation inhibitory compounds. Cells were seeded in 12-well plates and grown to confluence. For transport experi- ments, SOAT expression was induced by preincubation with tetracycline (1 g/ml). Inhibition experiments of human SOAT were analyzed in were started by preincubation with the respective inhibitor at 37 °C for 30 s. Subsequently, [ H]DHEAS was vitro in SOAT-HEK293 cells and added, and the incubation was continued for 5 min at 37 °C. Inhibition studies were terminated by removing HEK293 cells expressing the the transport buffer and washing with ice-cold PBS. Cells not incubated with any inhibitor served as positive control (set to 100%). The values represent the percentage of DHEAS transport activity in the presence of the SOAT-FLAG fusion protein in indicated inhibitor relative to the positive control and are expressed as means  S.E. of triplicate determina- which the FLAG motif (DYKD- tions of representative experiments. The values were significantly different from positive controls; *, p 0.01 (one-way analysis of variance with Dunnett post hoc analysis). DDDK) was attached to the C-ter- 19736 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT FIGURE 7. 2-SMP, 4-SMP, and TLCS as competitive inhibitors and substrates of SOAT. SOAT-HEK293 cells were pretreated with tetracycline (1 g/ml) to induce SOAT expression. For studying inhibition kinetics, SOAT-HEK293 cells were incubated in the presence (A and B) of 100 and 500 nM [ H]E S and increasing concentrations of 2-SMP (A) and 4-SMP (B) for 2 min at 37 °C and of 0.5 M and 2.5 M [ H]DHEAS and increasing concentrations of TLCS for 5 min at 37 °C (C). Cells were washed and processed to determine protein content and cell-associated radioactivity. Values are means  S.E. of triplicate determinations of representative experiments. K values were calculated from Dixon plots to 4.3 M for 2-SMP, 5.5 M for 4-SMP, and 0.24 M for TLCS. For studying SOAT- mediated uptake, SOAT-HEK293 cells (SOAT) and Flp-In HEK293 (control) were incubated with 10 M 2-SMP and 10 M 4-SMP for 15 min at 37 °C (D and F, filled bars) as well as with 50 nM [ H]TLCS for 5 min at 37 °C (E and G, filled bars). Cells were washed and processed to determine protein content and substrate uptake using HPLC analysis with fluorescence detection for 2-SMP and 4-SMP and using liquid scintillation counting for [ H]TLCS. Uptake of 10 M 2-SMP and 50 nM [ H]TLCS was also analyzed in Na -free exposure medium (D and E, open bars). In addition, uptake of 10 M 4-SMP was measured in the presence of 50 M E S (F), and uptake of [ H]TLCS was analyzed in the presence of 5 and 50 M DHEAS as competing substrates (G). Values are means  S.E. of three independent experiments and were significantly different from control; *, p 0.05 (Student’s t test). minal end of SOAT. To confirm the extracellular orientation microscopy under permeabilized and nonpermeabilized of the N terminus, we generated a SOAT antibody (SOAT- conditions. FLAG-directed fluorescence staining was only (2–17)) directed against the N-terminal 2–17 amino acids. observed if the cells were permeabilized by Triton X-100, Using this antibody, SOAT expression was analyzed in and it was undetectable in the nonpermeabilized cells (Fig. SOAT-HEK293 cells that were either induced or nonin- 8D). This clearly indicates a cytosolic orientation of the duced by tetracycline treatment and were kept under native SOAT C terminus and excludes an eight-TMD topology. (nonpermeabilized) conditions (Fig. 8C). Fluorescence sig- Immunoprecipitation and Deglycosylation of the SOAT- nals were only observed in the SOAT-expressing HEK293 FLAG Protein—SOAT-FLAG-pcDNA5-transfected HEK293 cells, and no cell-associated fluorescence was detected in the cells were also used for radiolabeling and immunoprecipita- noninduced control cells. Since SOAT-HEK293 cells were tion experiments of the SOAT-FLAG protein with a mono- not fixed and not permeabilized for these experiments clonal anti-FLAG antibody (Fig. 9). After separation of the before incubation with the SOAT-(2–17) antibody, the N precipitated cell extract, specific bands were detected at 46 terminus of SOAT must be located in the extracellular com- and 42 kDa. The SOAT-FLAG protein consists of 377  8 partment. In order to discriminate also the inside/outside amino acids with a predicted molecular mass of 41 kDa orientation of the C terminus, we generated a second SOAT (SOAT)  1 kDa (FLAG epitope). The higher apparent antibody, which was directed against amino acids 349 –364 molecular mass of 46 kDa after immunoprecipitation was of the C terminus (SOAT-(349 –364)). This antibody failed due to posttranslational modifications in the HEK293 cells. to show in vitro immunoreactivity against the SOAT-(349 – Since SOAT encodes three potential N-linked glycosylation 364) peptide, so we decided to attach the FLAG epitope tag sites, N-glycosylation of the SOAT-FLAG protein was exam- to the SOAT C terminus, which was then detected by using a ined by PNGase F digestion of the immunoprecipitated cell commercial anti-FLAG antibody. A SOAT-FLAG-pcDNA5 extract. As shown in Fig. 9, the apparent molecular mass of construct was generated by site-directed mutagenesis and the SOAT-FLAG protein was decreased from 46 to 42 kDa transfected into HEK293 cells to evaluate the accessibility of after PNGase F incubation. This change in apparent molec- the C-terminal FLAG epitope by immunofluorescence ular mass is consistent with the addition of at least one JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19737 Cloning and Characterization of Human SOAT FIGURE 8. Membrane expression and C/N terminus orientation of SOAT. A, proposed membrane topology of human SOAT based on the analyses of different topology prediction programs for eukaryotic proteins. The cylinders indicate the predicted transmembrane domains, and loops are depicted as lines. Topology models of human SOAT with seven, eight, and nine TMDs are shown. B, hydrophobicity profiles of human SOAT in comparison with human ASBT and NTCP. Increasing numerical values displayed on the y axis correspond to increasing hydrophobicity. The residue-specific hydropho- bicity index was calculated over a window of 11 residues by the GES-scale method of the TopPred II program. The plot shows an alignment of SOAT, NTCP, and ASBT amino acid sequences. The x axis refers to the amino acid residue number in the respective protein sequences. C and D orientation of the N-terminal and C-terminal ends of SOAT were determined by immunofluorescence microscopy of stably transfected SOAT-HEK293 cells (C) and Flp-In HEK293 cells transfected with the SOAT-FLAG-pcDNA5 construct (D). SOAT-HEK293 cells were grown on glass coverslips, and SOAT expression was induced by pretreatment with tetracycline (tet). Control cells were untreated with tetracycline (tet). Cells were not fixed or permeabilized. SOAT expression was analyzed with the SOAT-(2–17) antibody and an fluorescein isothiocyanate-conjugated secondary antibody (green fluorescence). Nuclei were stained with DAPI (blue fluorescence). The orientation of the C terminus was assessed in Flp-In HEK293 cells transfected with the SOAT-FLAG-pcDNA5 construct under permeabilized and nonpermeabilized conditions. The C-terminal FLAG epitope was detected by an anti-FLAG primary antibody and a Cy3-conjugated secondary antibody (orange fluorescence). Experimental data clearly exclude the eight-TMD model for human SOAT. Scale bar,25 m. N-linked carbohydrate chain to any of the potential N-linked all physiological dihydroxylated and trihydroxylated bile acids 4 14 157 glycosylation sites of SOAT (Asn , Asn , and Asn ). Based with a preference for the taurine and glycine amidated conju- on these data, molecular masses of 45 and 41 kDa can be gates versus the unconjugated forms (9–11, 31). SOAT is the estimated for the untagged glycosylated and nonglycosylated third member of the SLC10 transporter family that we have SOAT proteins, respectively. For comparison, also the functionally characterized in this paper. Transport studies in ASBT-FLAG protein was examined and also revealed a SOAT-HEK293 cells revealed no transport activity for tauro- decrease of its apparent molecular mass from 40 to 37 kDa cholic acid and cholic acid, thus indicating that SOAT, after PNGase F treatment, as has been previously reported although belonging to the bile acid transporter family SLC10, is (6, 7, 30). not a typical bile acid transporter, such as NTCP and ASBT. The latter also do not share identical substrate patterns. In con- DISCUSSION trast to ASBT, to which SOAT has closest homology, the sub- Transport Function of NTCP, ASBT, and SOAT—In the past strate specificity of NTCP is not strictly limited to bile acids and decade, the transport function of NTCP and ASBT were exten- also includes sulfoconjugated steroid hormones, such as E S (9, sively studied in several cell systems and revealed transport of 10). Additionally, it has been reported by Craddock and co- 19738 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT interference with SOAT-mediated transport. Concerning the negatively charged sulfate moiety, replacement of this group on a naphthyl core molecule by phosphate (-naphthylphos- phate), amine (-naphthylamine), and isothiocyanate (-naph- thylisothiocyanate) completely abolished all inhibitory potency of the respective -naphthyl derivative, thus indicating that the interaction of the sulfate moiety with the SOAT binding site is essential. Toxicological Aspects—The high affinity SOAT inhibitors 2-SMP and 4-SMP were also transported by SOAT in a sodium- dependent manner, thus showing that substrate recognition by SOAT also covers xenobiotics. 2-SMP and 4-SMP are isomers FIGURE 9. Immunoprecipitation and deglycosylation of SOAT-FLAG and ASBT-FLAG fusion proteins. HEK293 cells were transfected with the of 1-SMP that have longer half-life in water than 1-SMP ( 1 SOAT-FLAG-pcDNA5 and ASBT-FLAG-pcDNA5 constructs or vector alone day versus 2.8 min) (18). Thus, transport studies were conve- (control), and cells were induced by tetracycline treatment (1 g/ml). Cells niently performed with these long lived isomers. These com- were starved in medium without methionine and cysteine, followed by 35 35 [ S]labeling with L-[ S] in vitro cell labeling mix. Cell lysates were used for pounds are of particular toxicological importance, since cova- immunoprecipitation with an anti-FLAG antibody. Aliquots of the immuno- lent binding to DNA, causing mutations and neoplasia, was precipitated proteins were incubated with PNGase F (lanes 2, 4, and 6)or water (lanes 1, 3, and 5). The samples were separated by electrophoresis on a observed for 1-SMP (35, 36). This metabolite is formed from 12% SDS-polyacrylamide gel, and the dried gel was exposed to standard x-ray 1-methylpyrene, present at high levels in cigarette smoke, via film for visualization. 1-hydroxymethylpyrene by sulfoconjugation. The highest level of DNA adducts by 1-SMP was observed in kidney, where the workers (9) that the sulfoconjugated bile acid chenodeoxy- organic anion transporters OAT1 and OAT3 are expressed, cholate-3-sulfate is not a substrate of ASBT but is weakly trans- yielding a kidney-directed organotropism of 1-SMP. As shown ported by NTCP. Also, SOAT showed transport of a here, sulfoconjugated pyrenes, such as 2-SMP and 4-SMP, are sulfoconjugated bile acid, TLCS. Since the K value of TLCS is 2 also substrates of SOAT. Because of its predominant expression orders of magnitude lower than the K value of DHEAS and in testis, we conclude that testes are also exposed to electro- since uptake of TLCS into SOAT-HEK293 cells was inhibited philic adduct-forming pyrene sulfates by uptake via SOAT. by not less than a 1000-fold molar excess of DHEAS, SOAT This uptake might even be related to the well known risk of seems to be a high affinity transporter for sulfoconjugated bile testicular cancer in tobacco smokers (37). acids. Sulfoconjugation of bile acids is increased under choles- Phylogenetic Relationship of SOAT, ASBT, and NTCP—The tatic conditions and also takes place in the fetus (32, 33). Since evolutionary origin of the SLC10 transporter family was SOAT is highly expressed in human placenta, this carrier might recently shown by a phylogenetic analysis of the SLC10A1– be involved in the fetal-to-maternal transfer of sulfoconjugated SLC10A6 genes from several mammalian and nonmammalian bile acids for their elimination through the mother (34). In species (14). This analysis revealed two major clades of genes. addition to the sulfoconjugated bile acids, other bile acids were Clade I comprises SLC10A1 (NTCP), SLC10A2 (ASBT), potent inhibitors, albeit no substrates of SOAT transport, fol- SLC10A4, and SLC10A6 (SOAT) genes; clade II contains lowing the order: 3-monohydroxylated bile acids 3,7- SLC10A3 and SLC10A5 genes. Within clade I, SOAT is the dihydroxylated bile acids 3,12-dihydroxylated bile acids. sister group to ASBT, and SLC10A4 is the sister group to On the other hand, SOAT substrates include the sulfoconju- NTCP. This phylogenetic relationship explains the high gated steroid hormones DHEAS, E S, and PREGS with appar- sequence homology between SOAT and ASBT as well as the ent K values of 28.7, 12.0, and 11.3 M, respectively. These lower sequence homology between ASBT and NTCP. Func- SOAT substrates (TLCS, DHEAS, E S, and PREGS) share a tional transport properties of SLC10 carriers can overlap but hydrophilic, negatively charged, sulfate moiety that is linked to might also be very divergent. A likely explanation would be that a hydrophobic, hydrocarbon steroid nucleus and that seems to the common ancestor gene for SOAT, ASBT, and NTCP be basically required for substrate recognition by SOAT. The exerted transport of bile acids (either sulfoconjugated or non- specific recognition of each substrate by its sulfate group is sulfoconjugated) plus sulfoconjugated steroids but separated corroborated by the observation that neither glucuronidated them during later subdivision into ASBT (only nonsulfoconju- steroids, nonsulfoconjugated steroids, nor nonsulfoconjugated gated bile acids), SOAT (only sulfoconjugated bile acids and bile acids are transported by SOAT-HEK293 cells. sulfoconjugated steroids), and NTCP (bile acids, sulfoconju- Interaction of SOAT with Nonsteroidal Organosulfates— gated bile acids, and sulfoconjugated steroids). At present, the Some relatively large sulfoconjugated molecules also potently varying local organ expression of these three SLC10 transport- inhibited SOAT transport. These included 1-SEP, bromosul- ers combined with their individual substrate pattern reflects fophthalein, 2-SMP, 4-SMP, and -naphthylsulfate. On the and causes broad physiological plasticity. other hand, the uptake of [ H]DHEAS by SOAT was not Sodium Dependence of SOAT—The driving force for the affected by the sulfoconjugates of very small molecules, such as NTCP-mediated and ASBT-mediated transport of bile acids is ethylsulfate, 2-propylsulfate, phenylsulfate, phenylethylsulfate, provided by the inwardly directed Na gradient, which is main- and hydroquinone sulfate. Thus, it appears that a sulfated two- tained by the activity of the Na /K -ATPase in the plasma hydrocarbon ring structure is required at a minimum for any membrane as well as the negative intracellular potential. As JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19739 Cloning and Characterization of Human SOAT demonstrated for rat Ntcp and human ASBT, these transport- the transport characteristics of SOAT, indicating that SOAT is ers perform an electrogenic transport cycle and move two Na involved in the DHEAS transport into placenta trophoblasts. ions for each bile acid molecule (9, 38–40). A comparable After the first trimester of pregnancy, human placenta is also transport mechanism is also suggested for SOAT, because the the main biosynthetic source of progesterone, which is an transport of TLCS, DHEAS, E S, PREGS, 2-SMP, and 4-SMP by essential hormone to sustain pregnancy (54). The progesterone SOAT is also strictly sodium-dependent. Nonetheless, the cat- precursor pregnenolone is synthesized in the trophoblasts from ion selectivity of SOAT is not absolutely identical with that of cholesterol but also derives from PREGS, which is synthesized ASBT. Whereas Li maintained about 40% of the SOAT trans- at high levels in the adrenal gland and is delivered via the mater- port function compared with Na ,Li is not accepted as a nal and fetal blood circulation (55). We suppose that SOAT, stimulating co-substrate of ASBT. On the other hand, equimo- which mediates cellular PREGS uptake, is involved in this proc- lar substitutions of Na by choline abolished the transport ess and therefore could also contribute to placenta progester- function of both carriers (5, 9). one synthesis. Membrane Expression and Topology of NTCP, ASBT, and In conclusion, we have functionally characterized the novel SOAT—Hydrophobicity analyses of NTCP and ASBT pro- sodium-dependent organic anion transporter SOAT of posed 7–9 TMDs, but experimental data strongly support a humans. This carrier is expected to have physiological meaning seven-TMD topology with an exoplasmic N terminus and a for hormone response of testis and placenta on sulfoconjugated cytoplasmic C terminus (4, 8, 30, 41–43). For human SOAT, steroid hormones and also for their toxicologic exposure to only one topology prediction program (TopPred II/KD-scale) sulfoconjugated pyrene carcinogens as well as for placental supported this membrane topology, whereas most other calcu- transport of sulfoconjugated bile acids. lations yielded eight transmembrane domains with an exoplas- mic orientation of the N-terminal and C-terminal ends. In this Acknowledgments—We thank Klaus Schuh and Monika Rex-Haffner for technical help. paper, an N /C trans-orientation was experimentally dem- exo cyt onstrated, which is in accordance with the membrane topology of NTCP and ASBT but clearly eliminates a model with eight REFERENCES TMDs. Our experimental setup was not able to discriminate 1. 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Engst, W., Landsiedel, R., Hermersdorfer, H., Doehmer, J., and Glatt, H. 52. Ugele, B., and Simon, S. (1999) J. Steroid Biochem. Mol. Biol. 71, 203–211 (1999) Carcinogenesis 20, 1777–1785 53. Ugele, B., St. Pierre, M. V., Pihusch, M., Bahn, A., and Hantschmann, P. 37. Srivastava, A., and Kreiger, N. (2004) Cancer Epidemiol. Biomarkers Prev. (2003) Am. J. Physiol. 284, E390–E398 13, 49–54 54. Miller, W. L. (1998) Clin. Perinatol. 25, 799–817 38. Weinman, S. A. (1997) Yale J. Biol. Med. 70, 331–340 55. de Peretti, E., and Mappus, E. (1983) J. Clin. Endocrinol. Metab. 57, 39. Weinman, S. A., Carruth, M. W., and Dawson, P. A. (1998) J. Biol. Chem. 550–556 JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19741 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Cloning and Functional Characterization of Human Sodium-dependent Organic Anion Transporter (SLC10A6) *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2007 Elsevier Inc.
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 27, pp. 19728 –19741, July 6, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cloning and Functional Characterization of Human Sodium-dependent Organic Anion Transporter (SLC10A6) Received for publication, March 28, 2007 Published, JBC Papers in Press, May 9, 2007, DOI 10.1074/jbc.M702663200 ‡1 ‡ ‡ § ¶ ‡ Joachim Geyer , Barbara Do¨ ring , Kerstin Meerkamp , Bernhard Ugele , Nadiya Bakhiya , Carla F. Fernandes , ‡ ¶ ‡ Jose´ R. Godoy , Hansruedi Glatt , and Ernst Petzinger From the Institute of Pharmacology and Toxicology, Justus-Liebig-University of Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany, University Hospital, Ludwig-Maximilians-University of Munich, 80337 Munich, Germany, and the Department of Nutritional Toxicology, German Institute of Human Nutrition Potsdam-Rehbru¨cke, 14558 Nuthetal, Germany We have cloned human sodium-dependent organic anion sion cloning from rat liver (2) and is exclusively expressed at the transporter (SOAT) cDNA, which consists of 1502 bp and sinusoidal membrane of hepatocytes (3, 4). Three years later, its encodes a 377-amino acid protein. SOAT shows 42% sequence intestinal counterpart was cloned from a hamster intestinal identity to the ileal apical sodium-dependent bile acid trans- cDNA library and was named apical sodium-dependent bile porter ASBT and 33% sequence identity to the hepatic Na / acid transporter (Asbt; Slc10a2) (5). In contrast to the basolat- taurocholate-cotransporting polypeptide NTCP. Immuno- eral localization of Ntcp, Asbt is highly expressed at the apical precipitation of a SOAT-FLAG-tagged protein revealed a brush border membrane of enterocytes of the terminal ileum glycosylated form at 46 kDa that decreased to 42 kDa after (6). Although sequence identity between NTCP and ASBT is PNGase F treatment. SOAT exhibits a seven-transmembrane quite low (at 35%), both carriers transport conjugated bile acids domain topology with an outside-to-inside orientation of the with high affinity (7–11). N-terminal and C-terminal ends. SOAT mRNA is most highly Due to their transport characteristics and expression pattern, expressed in testis. Relatively high SOAT expression was also NTCP and ASBT are important factors for the maintenance of detected in placenta and pancreas. We established a stable the enterohepatic circulation of bile acids mediating the first SOAT-HEK293 cell line that showed sodium-dependent step in the cellular uptake of bile acids through the membrane transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, barriers in the liver (NTCP) and intestine (ASBT). Since the bile and pregnenolone sulfate with apparent K values of 28.7, 12.0, acid reflux from the intestine is a major negative regulator of and 11.3 M, respectively. Although bile acids, such as tauro- the de novo bile acid synthesis from cholesterol in the liver, cholic acid, cholic acid, and chenodeoxycholic acid, were not ASBT is a promising drug target for cholesterol-lowering ther- substrates of SOAT, the sulfoconjugated bile acid taurolitho- apy (12). In fact, several compounds were able to significantly cholic acid-3-sulfate was transported by SOAT-HEK293 cells in lower plasma cholesterol levels and prevent atherosclerosis in a sodium-dependent manner and showed competitive inhibi- animal studies, and currently they are being tested in clinical tion of SOAT transport with an apparent K value of 0.24 M. trials (13). Several nonsteroidal organosulfates also strongly inhibited Recently, four new members of the SLC10 family were dis- SOAT, including 1-(-sulfooxyethyl)pyrene, bromosulfoph- covered and referred to as SLC10A3, SLC10A4, SLC10A5, and thalein, 2- and 4-sulfooxymethylpyrene, and -naphthylsulfate. sodium-dependent organic anion transporter (SOAT; Among these inhibitors, 2- and 4-sulfooxymethylpyrene were SLC10A6) (14). SLC10A3 (P3) was cloned from placenta and competitive inhibitors of SOAT, with apparent K values of 4.3 teratocarcinoma cDNA libraries in 1988, before NTCP and and 5.5 M, respectively, and they were also transported by ASBT had been discovered, and showed broad tissue expres- SOAT-HEK293 cells. sion (15). The second orphan transporter SLC10A4 seems to be predominantly expressed in the central nervous system and shares a common ancestor gene with NTCP. In contrast, SLC10 (solute carrier family 10) is well established as the SLC10A5 shows high expression in the liver, kidney, and intes- “sodium bile acid cotransporter family” (1). The first member of tine, which is very similar to the expression pattern of ASBT this transporter family, the Na /taurocholate-cotransporting (14). Until now, however, these orphan transporters have not polypeptide (Ntcp; Slc10a1), was identified in 1990 by expres- been subjected to intensive experimental expression analysis, and there is no published data indicating that they have any * This study was supported by the German Research Foundation (Deutsche function as solute carriers. Finally, in 2004, we cloned rat Soat, Forschungsgemeinschaft, Bonn, Germany) Grant GE1921/1-1 (to J. G. and which showed the highest phylogenetic relationship to ASBT K. M.) and Graduate Research Program 455 “Molecular Veterinary Medi- cine” (to B. D. and J. R. G.). The costs of publication of this article were but did not transport taurocholate (16). In this paper, we report defrayed in part by the payment of page charges. This article must there- on the cloning, membrane topology, and expression of human fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- SOAT and also provide its functional characterization in stably tion 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the Gen- transfected human embryonic kidney (HEK293) cells. Besides TM Bank /EBI Data Bank with accession number(s) AJ583502 and EF437223. sulfoconjugated steroid hormones, SOAT also transports tau- To whom correspondence should be addressed. Tel.: 49-641-9938404; Fax: 49-641-9938419; E-mail: joachim.m.geyer@vetmed.uni-giessen.de. rolithocholic acid-3-sulfate and sulfoconjugated pyrenes. 19728 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 This is an Open Access article under the CC BY license. Cloning and Characterization of Human SOAT EXPERIMENTAL PROCEDURES obtain a SOAT-pBluescript plasmid, the sticky ended SOAT cDNA fragment was directionally ligated downstream from a Materials and Chemicals—All of the chemicals, unless oth- T3 promoter into the pBluescript vector (Stratagene), which erwise stated, were from Sigma. Glycolithocholic acid was from was predigested with the respective restriction enzymes (SacII Calbiochem. Phenylsulfate, hydroquinone sulfate, -naphthyl- and XbaI). Three different clones were sequenced on both sulfate, 1-(-sulfooxyethyl)pyrene (1-SEP), 2-sulfooxymeth- strands, and the cDNA sequence was deposited in the Gen- ylpyrene (2-SMP), 4-sulfooxymethylpyrene (4-SMP), and TM TM Bank data base under GenBank accession number 5-sulfooxymethylfurfural were prepared from the correspond- AJ583502. In order to confirm transcription of the full-length ing hydroxyl compounds and sulfuric acid in dimethylform- SOAT mRNA sequence also on organs with high SOAT expres- amide using dicyclohexylcarbodiimide as the condensing sion (i.e. testis, placenta, and pancreas) (see below), RT-PCR agent, as described in detail elsewhere (17, 18). was also performed on human testis, placenta, and pancreas Radiochemicals—[ H]Dehydroepiandrosterone sulfate 3 3 3 cDNAs (BD Clontech) as described above, and PCR fragments ([ H]DHEAS, 60 Ci/mmol), [ H]estrone-3-sulfate ([ H]E S, 3 3 were verified by DNA sequencing. To confirm transport activ- 57 Ci/mmol), [ H]digoxin (24 Ci/mmol), and [ H]tauro- ity of human SOAT, the SOAT-pBluescript plasmid was used cholic acid (3.5 Ci/mmol) were purchased from PerkinElmer 3 3 14 14 for transport experiments with [ H]DHEAS and [ H]E Sin Life Sciences. [ C]Cholic acid (55 mCi/mmol), [ C]che- 1 Xenopus laevis oocytes as described in detail previously (16). nodeoxycholic acid (51 mCi/mmol), [ H]lithocholic acid (50 3 3 Identification of SOAT cDNA Ends by Rapid Amplification of Ci/mmol), [ H]pregnenolone-3-sulfate ([ H]PREGS, 20 cDNA Ends (RACE)-PCR—In order to obtain the full-length Ci/mmol), and [ H]deoxycholic acid (20 Ci/mmol) were SOAT mRNA transcript, we employed the GeneRacer method obtained from American Radiolabeled Chemicals. [ H]Es- based on RNA ligase-mediated and oligonucleotide-capping trone (76 Ci/mmol), [ H]estradiol-17-D-glucuronide (44 RACE according to the manufacturer’s protocol (Invitrogen). Ci/mmol), [ H]dehydroepiandrosterone (54 Ci/mmol), and Reverse transcription of 1 g of testis RNA (BD Clontech) was [ H]ouabain (23 Ci/mmol) were obtained from PerkinElmer 3 3 performed with the GeneRacer Oligo dT Primer 5-gct gtc aac Life Sciences. [ H]Taurolithocholic acid-3-sulfate ([ H]TLCS, gat acg cta cgt aac ggc atg aca gtg t -3 in a volume of 20 l 24.1 Ci/mmol) was generously donated by Werner Kramer using SuperScript III Reverse Transcriptase (Invitrogen). Initial (Sanofi-Aventis, Frankfurt am Main, Germany). 3- and 5-RACE reactions were performed using the gene-spe- Cloning of Human SOAT cDNA—Using BLAST searches of cific primers 5-ggc agc tcc tcc tct gaa ctg ttg-3 for 5-RACE the human genome with the cDNA sequences of the six coding TM amplification and 5-aat tac cct tgt gtg cct gac cat tc-3 for exons of rat Soat (Slc10a6) (GenBank accession number 3-RACE amplification. For each 50-l reaction, 1 l of cDNA, AJ583503), we obtained matches with six genomic sequence 0.5 l of AmpliTaq Gold DNA polymerase (Applied Biosys- fragments on human chromosome 4q21. These sequences were tems), and 6 l of MgCl (25 mM) were used, and amplification used for an RT-PCR-based strategy to obtain the full open read- 2 was performed under the following thermocycling conditions: ing frame cDNA sequence of human SOAT. The following oli- 1 cycle of 95 °C for 5 min; five cycles of 94 °C for 30 s and 72 °C gonucleotide primers were designed, including SacII/XbaI for 1 min; 5 cycles of 94 °C for 30 s and 70 °C for 1 min; 25 cycles restriction sites for PCR amplification: forward primer, 5-atg of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 1 min; and a final acc gcg gat gag agc caa ttg ttc cag cag ctc-3; reverse primer, extension of 72 °C for 10 min. To increase the yield and speci- 5-cgt cta gac tat tcg cat gaa gtg atg tgg cca act g-3. Although it ficity of the RACE products, additional nested PCR was per- has a relatively low expression in this organ (see below), human formed using the nested primers 5-gct gag ctg ctg gaa caa ttg SOAT was initially cloned from the adrenal gland. RT-PCR was gct c-3 for the nested 5-RACE reaction and 5-cct gtg gcc ttt performed from 1 g of human adrenal gland poly(A) RNA ggt gtc tat gtg-3 for the nested 3-RACE reaction under the (BD Clontech) using the Expand High Fidelity PCR System following conditions: one cycle of 95 °C for 5 min; 10 cycles of (Roche Applied Science) according to the following thermocy- 94 °C for 30 s, 70 °C for 30 s minus 0.5 °C each cycle, and 72 °C cling conditions: one cycle of 94 °C for 2 min; 10 cycles of 95 °C for 1 min; 30 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for for 15 s, 56 °C for 15 s minus 0.5 °C each cycle, and 72 °C for 1 1 min; and a final extension of 72 °C for 10 min. 3- and min; 30 cycles of 95 °C for 15 s, 51 °C for 15 s, and 72 °C for 1 min 5-RACE fragments were gel-purified and cloned into the plus 5 s each cycle; and final extension of 72 °C for 10 min. After pCR4-TOPO vector (Invitrogen). Three individual clones were the amplification reaction, samples were held at 4 °C until anal- sequenced on both strands for each PCR fragment. ysis. An aliquot of the PCR product was electrophoresed on an Establishment of the SOAT-HEK293 Cell Line—The recom- agarose gel. The amplicon of 1152 bp was excised form the gel binant human cell line T-REx SOAT-HEK293 was made using and digested for 90 min at 37 °C with SacII and XbaI. In order to the Flp-In expression system and the commercially available Flp-In T-REx 293 host cell line according to the manufacturer’s The abbreviations used are: 1-SEP, 1-(-sulfooxyethyl)pyrene; DMEM, Dul- instructions (Invitrogen). Flp-In T-REx 293 cells contain a sin- becco’s modified Eagle’s medium; PBS, phosphate-buffered saline; E S, gle, stably integrated Flp recombinase target (FRT) site at a estrone-3-sulfate; DHEAS, dehydroepiandrosterone sulfate; PREGS, preg- transcriptionally active genomic locus, which is maintained by nenolone sulfate; 2-SMP, 2-sulfooxymethylpyrene; 4-SMP, 4-sulfooxym- ethylpyrene; 1-SMP, 1-sulfooxymethylpyrene; TLCS, taurolithocholic acid- selection for zeocin resistance and ensures high level gene 3-sulfate; RT, reverse transcription; FRT, Flp recombinase target; PBS, expression from a target-integrated Flp-In expression vector. phosphate-buffered saline; HPLC, high pressure liquid chromatography; Briefly, SOAT cDNA spanning the whole open reading frame PNGase F, peptide:N-glycosidase F; DAPI, 4,6-diamidino-2-phenylindole; TMD, transmembrane domain; RACE, rapid amplification of cDNA ends. was subcloned from SOAT-pBluescript vector into the Flp-In JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19729 Cloning and Characterization of Human SOAT pcDNA5/FRT/TO expression vector carrying the FRT site and and washing five times with ice-cold PBS. Cell monolayers were the hygromycin resistance gene (Invitrogen). In the generated lysed in 1 N NaOH with 0.1% SDS, and the cell-associated radio- vector, further referred to as SOAT-pcDNA5, SOAT cDNA is activity was determined in a liquid scintillation counter. The under the control of the cytomegalovirus promoter and the protein content was determined according to Lowry using ali- tetracycline operator sequences (tetO ). In addition to the FRT quots of the lysed cells with bovine serum albumin as a standard site, Flp-In T-REx 293 cells stably express the tetracycline (19). repressor, which is maintained by selection for blasticidin Uptake Studies with 2-SMP and 4-SMP—Cells were seeded resistance. In the absence of tetracycline, tetracycline repressor in 24-well plates (2  10 cells in 1 ml of medium/well) 2 days effectively binds to the tetO sequence and blocks SOAT tran- before the experiment started. SOAT-HEK293 cells were incu- scription from the cytomegalovirus promoter. In order to bated in Ringer’s solution (130 mM NaCl, 4 mM KCl, 1 mM establish the SOAT-HEK293 cell line, the SOAT-pcDNA5 con- CaCl ,1mM MgSO ,20mM HEPES, 1 mM NaH PO , and 18 2 4 2 4 struct was cotransfected with the Flp recombinase expression mM glucose, pH 7.4) or an equimolar solution in which sodium vector pOG44 into Flp-In T-REx 293 host cells by Fugene 6 was replaced by choline in the presence of 10 M 2-SMP or transfection reagent according to the manufacturer’s protocol 4-SMP for 15 min at 37 °C. After aspiration of the transport (Roche Applied Science). Upon cotransfection, the SOAT cod- solution and three washes with ice-cold Ringer’s solution, cells ing sequence was integrated into the genome of the Flp-In were lysed with 0.25 ml of 1 N NaOH. After neutralization with HEK293 cells via Flp recombinase-mediated homologous 0.25 ml of 1 N HCl and protein precipitation with 1 ml of ace- recombination at the FRT site. Stable clones containing the tone, aliquots (usually 10 l) of the supernatant were injected SOAT open reading frame sequence under control of the cyto- into HPLC using a Shimadzu SIL-M10 Avp autosampler. Sam- megalovirus/tetO hybrid promoter were selected by culturing ples were separated using a Shimadzu SLC-10 Avp delivery sys- in selective medium containing 150 g/ml hygromycin and 50 tem equipped with a Phenomenex Gemini C18 column (250 g/ml blasticidin. After 10–14 days, single clones were isolated 3 mm; 5 m). The eluent was methanol containing 20% water from the remaining cell pool using cloning cylinders and tested and 0.05% triethylamine (v/v). The flow rate was 0.2 ml/min. for sodium-dependent [ H]DHEAS transport. The best trans- 2-SMP and 4-SMP were quantified from the fluorescence signal porting cell clone (further referred to as SOAT-HEK293) was ( 334 nm,  392 nm) using a Shimadzu SPD-M10 Avp ex em selected and used for further experiments. SOAT-HEK293 cells detector. were maintained in DMEM/F-12 medium (Invitrogen) supple- Expression of SOAT-FLAG-tagged and ASBT-FLAG-tagged mented with 10% fetal calf serum (Sigma), L-glutamine (4 mM), Proteins—A FLAG-tagged SOAT protein was generated by penicillin (100 units/ml), and streptomycin (100 g/ml) (fur- insertion of the FLAG-peptide (DYKDDDDK) to the C-termi- ther referred to as standard medium) at 37 °C, 5% CO , and 95% nal end of SOAT by QuikChange site-directed mutagenesis humidity. (Stratagene) of the SOAT-pcDNA5 construct. The following Transport Studies in SOAT-HEK293 Cells—For transport oligonucleotide sense and antisense primers were used: 5-cat studies, 12-well plates were coated with poly-D-lysine for better cac ttc atg cga aga tta caa gga tga cga cga taa gta ggc ggc cgc tcg attachment of the cells. 1.25  10 cells/well were plated and agt cta g-3 sense and 5-cta gac tcg agc ggc cgc cta ctt atc gtc gtc grown under standard medium for 72 h. SOAT expression was atc ctt gta atc ttc gca tga agt gat g-3 antisense. Correct clones induced by preincubation with tetracycline (1 g/ml). SOAT- were selected by DNA sequencing. Furthermore, an ASBT- nonexpressing control cells (Flp-In HEK293 cells) were not FLAG fusion protein was generated for comparative analysis. pretreated with tetracycline. Before starting the transport Briefly, the full open reading frame of human ASBT was ampli- experiments, cells were washed three times with phosphate- fied by RT-PCR from 1 g of small intestine poly(A) RNA (BD buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 1.5 mM Clontech). Gene-specific oligonucleotide primers were used KH PO , 7.3 mM Na HPO , pH 7.4, 37 °C) and preincubated containing SacII/XbaI restriction sites (5-cca gcc gcg gac cca 2 4 2 4 with sodium transport buffer containing 142.9 mM NaCl, 4.7 gca atg aat g-3 forward and 5-gtc ctc tag atg tct act ttt cgt cag mM KCl, 1.2 mM MgSO , 1.2 mM KH PO , 1.8 mM CaCl , and 20 gtt g-3 reverse), and PCR amplification was performed using 4 2 4 2 mM HEPES, adjusted to pH 7.4. When transport assays were the Expand High Fidelity PCR System (Roche Applied Sci- performed in sodium-free transport buffer, sodium chloride ence) according to the following touchdown schedule: 1 was substituted with equimolar concentrations of choline chlo- cycle of 94 °C for 2 min; 10 cycles of 94 °C for 15 s, 65 °C for ride. To determine the ion selectivity of SOAT transport, 30 s minus 0.5 °C each cycle, and 72 °C for 1 min; 30 cycles of sodium chloride in the transport buffer was also substituted 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min plus 10 s with equimolar concentrations of lithium chloride, potassium each cycle. The PCR product of the expected size was gel- chloride, N-methyl-D-glucamine, sodium gluconate, and potas- purified and digested for 90 min at 37 °C with SacII and XbaI. sium gluconate. Uptake experiments were initiated by replac- The sticky ended cDNA fragment was directionally ligated ing the preincubation buffer by 500 l of transport buffer con- downstream from a T3 promoter into the pBluescript vector taining the radiolabeled test compound and were performed at (Stratagene), which was predigested with SacII and XbaI. 37 °C. For inhibition studies, SOAT-HEK293 cells were prein- Sequence verification was done according to the reference TM cubated with transport buffer containing the inhibitor com- sequence with GenBank accession number NM_000452. pound for 30 s. Then transport measurements were started by Transport activity of ASBT was confirmed by transport adding the radiolabeled substrate at 37 °C. Transport and inhi- experiments in X. laevis oocytes with [ H]taurocholic acid as bition assays were terminated by removing the transport buffer the test compound (see above). For transfection of Flp-In 19730 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT HEK293 cells, the ASBT open reading frame sequence was exposed to Eastman Kodak Co. BioMax MR film (Sigma) at subcloned into the Flp-In pcDNA5/FRT/TO expression vec- 80 °C. tor, and the FLAG epitope was inserted at the C-terminal Antibody Preparation—The SOAT-(2–17) antibody was end of ASBT by QuikChange site-directed mutagenesis as raised in rabbits against amino acid residues 2–17 of the described above for SOAT. The following oligonucleotide deduced SOAT sequence (RANCSSSSACPANSSE). The syn- primers were used: 5-caa cct gac gaa aag gat tac aag gat gac thetic peptide was coupled via the carboxyl-terminal glutamic gac gat aag tag aca tct cga gtc-3 sense and 5-gac tcg aga tgt acid residue to keyhole limpet hemocyanin and used to immu- cta ctt atc gtc gtc atc ctt gta atc ctt ttc gtc agg ttg-3 antisense. nize two rabbits (Eurogentec). Antigenicity of the rabbit serum The SOAT-FLAG-pcDNA5 and ASBT-FLAG-pcDNA5 con- was confirmed by enzyme-linked immunosorbent assay analy- structs were verified by DNA sequencing, and positive sis using the synthetic peptide as the antigen. A second SOAT- clones were used for further experiments. (349–364) antibody was raised against amino acid residues Immunoprecipitation and Deglycosylation of the FLAG- 349–364 at the C terminus of SOAT. However, this antibody tagged Proteins—Flp-In T-REx 293 cells were seeded in showed no immunoreactivity against the synthetic peptide in 6-well plates coated with poly-D-lysine at a density of 1.0  enzyme-linked immunosorbent assay experiments and was not 10 cells/well in antibiotic-free DMEM/F-12 medium sup- applicable (Eurogentec). plemented with 10% fetal calf serum and 4 mML-glutamine. Immunofluorescence Microscopy of SOAT-HEK293 Cells— On the following day, the cells were transfected with 4 gof SOAT-HEK293 cells were grown on poly-D-lysine-coated SOAT-FLAG-pcDNA5 and ASBT-FLAG-pcDNA5 vector glass coverslips to 80% confluence in standard medium. DNA or with 4 g of pcDNA5 alone (control) by Lipofectamine SOAT expression was induced by tetracycline treatment (1 2000 reagent according to the manufacturer’s protocol g/ml) for at least 24 h. Noninduced control cells were (Invitrogen). After 4 h, the medium was changed to standard equally processed but were not incubated with tetracycline. medium, and expression of the FLAG-tagged proteins was On the next day, cells were washed three times with PBS and induced by tetracycline treatment (1 g/ml). On the next day, then incubated with the SOAT-(2–17) rabbit antibody (1:10) the cells were washed with PBS and starved in methionine-free for1hat room temperature without paraformaldehyde and cysteine-free DMEM medium (Sigma) supplemented with treatment. After rinsing and three times washing with PBS, 4mML-glutamine and 1 g/ml tetracycline for 1 h. Subse- the cells were incubated for1hat room temperature with the quently, 70 Ci of L-[ S] in vitro cell labeling mix (Amersham mouse fluorescein isothiocyanate-conjugated anti-rabbit Biosciences) was added, and the cells were incubated for an IgG antibody at 1:200 dilution (Sigma). After a final washing additional6hat37 °C,5%CO , and 95% humidity. The cells procedure, the cells were covered with a DAPI/methanol were washed with ice-cold PBS and incubated in 500 l of ice- solution containing 1 g/ml DAPI and incubated for 5 min at cold radioimmune precipitation buffer containing 150 mM 8 °C. The cells were washed with methanol/acetone (1:1), NaCl, 50 mM TrisHCl (pH 8.0), 1% Nonidet P-40, 0.5% (w/v) air-dried, and mounted on slides with Mowiol mounting sodium deoxycholic acid, 0.1% (w/v) SDS, and protease inhibi- medium. Fluorescence imaging was performed on a Leica tor mixture (Sigma) for 5 min under shaking. Cell lysates were DM6000B fluorescence microscope. Captured files were transferred to a microcentrifuge tube and incubated for an analyzed with the Leica FW4000 fluorescence work station additional 30 min under rotation at 4 °C. To remove any cell software. debris, the samples were centrifuged for 15 min at 4 °C, and the Immunofluorescence Microscopy of SOAT-FLAG-transfected supernatant was transferred to a fresh tube. Immunoprecipita- HEK293 Cells—For transient transfection, Flp-In HEK293 cells tion was performed by incubation with 5 g of the monoclonal were seeded in 24-well plates at a density of 2.5  10 cells/well mouse anti-FLAG antibody (Sigma) under rotation for1hat on poly-D-lysine-coated glass coverslips and grown to 80% 4 °C. Subsequently, 100 l of protein A-Sepharose (Sigma; 25% confluence in antibiotic-free DMEM/F-12 medium supple- suspension in radioimmune precipitation buffer) was added, mented with 10% fetal calf serum and 4 mML-glutamine. Cells and samples were incubated under rotation. After 1 h, Sepha- were transfected with 1 g of SOAT-FLAG-pcDNA5 vector rose beads were precipitated by centrifugation and washed DNA by Lipofectamine 2000 reagent (Invitrogen). The parental three times with ice-cold radioimmune precipitation buffer. pcDNA5 vector lacking any insert was used as control. After For deglycosylation with PNGase F, the protein A-Sepharose 4 h, the medium was changed to standard medium, and the beads were resuspended in 1 glycoprotein denaturation transfected cells were induced by tetracycline (1 g/ml) for at buffer and boiled for 10 min, and the eluted proteins were incu- least 24 h. After fixation with 2% paraformaldehyde in PBS for bated overnight at 37 °C with 1000 units of PNGase F in 1 G7 15 min at 4 °C, the cells were washed twice with PBS and incu- reaction buffer supplemented with 1% Nonidet P-40 (New Eng- bated with 20 mM glycine in PBS for 5 min. Subsequently, the land Biolabs). Nondeglycosylated samples were equally pro- cells were permeabilized for 5 min in PBT buffer (PBS contain- cessed but not incubated with PNGase F. All samples were ing 0.2% Triton X-100 and 20 mM glycine). Nonpermeabilized mixed with the same amount of 2 Laemmli buffer containing cells were not treated with PBT and were used for outside 10% -mercaptoethanol and boiled for 10 min. Finally, degly- epitope localization. The cells were placed in blocking solution cosylated and nondeglycosylated samples were separated by PBSG (1% bovine serum albumin and 4% normal goat serum in 12% SDS-PAGE. The gel was fixed in 30% methanol, 10% acetic PBS) for 30 min at room temperature and incubated with the acid (v/v) for 30 min and soaked in Amplify Fluorographic rea- rabbit anti-FLAG antibody (Sigma) at a 1:40,000 dilution in gent (Amersham Biosciences) for 30 min. Dried gels were PBSG overnight at 4 °C. The cells were washed three times with JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19731 Cloning and Characterization of Human SOAT TABLE 1 PBS and incubated with the goat Cy3-conjugated anti-rabbit Exon-intron organization of the human SLC10A6 gene on IgG antibody (Jackson Immunoresearch) at 1:800 in PBSG for chromosomal locus 4q21.3 1 h at room temperature. After triple washing with PBS, nuclei Nucleotide sequences of exon/intron junctions are indicated according to full- TM were stained by incubation with a DAPI/methanol solution length SOAT cDNA sequence (GenBank accession number EF437223) and H. TM sapiens chromosome 4, reference assembly (GenBank accession number containing 0.2 g/ml DAPI for 5 min at room temperature. The NC_000004.10). Intron sequences are represented in lowercase letters, and exon cells were washed with methanol, air-dried, and mounted on sequences are in uppercase letters. Exon Exon size 5-Splice donor 3-Splice acceptor Intron size slides with Mowiol mounting medium. Fluorescence imaging was performed as described above. bp kb 1 525 TCTCAG/gtaagt tttcag/CATCAG 15.3 Real Time Quantitative PCR—Relative SOAT expression 2 119 ACATAG/gtctgt ctttag/GAATTA 1.4 analysis was performed with ABI PRISM 7300 technology using 389 CTCAAG/gtgagt cttcag/ATTGGG 3.6 4 176 GCAAAG/gtatag tggtag/GTGCAG 2.4 human multiple tissue cDNA panels (BD Clontech) and cDNA 5 158 TTGCAG/gtgggt acctag/CATATC 1.5 synthesized from human adrenal gland and human mammary 6 435 TCAATG gland RNAs (BD Clontech). PCR amplification was achieved with TaqMan Gene Expression Assays Hs01399354_m1 for RESULTS human SOAT (SLC10A6) covering exon boundary 5–6 and Cloning of Human SOAT—Based on the cDNA sequence of Hs99999903_m1 for human -actin (Applied Biosystems). rat Soat, we used an RT-PCR-based approach to clone the full Expression data of -actin in each tissue were used as endoge- TM 1134-bp open reading frame of human SOAT (GenBank nous control. For each tissue, quadruplicate determinations accession number AJ583502). The full-length SOAT transcript were performed in a 96-well optical plate for both targets was obtained by RACE-PCR and revealed a 1502-bp cDNA, (SOAT and -actin) using 2.5 l of cDNA, 1.25 l of TaqMan including 5- and 3-untranslated regions of 148 and 220 bp, Gene Expression Assay, 12.5 TM l of TaqMan Universal PCR respectively (GenBank accession number EF437223). The Master Mix (Applied Biosystems), and 8.75 l of water in each human SLC10A6 gene is located on chromosome 4 and is 25-l reaction. The plates were heated for 10 min at 95 °C, and coded by six exons mapped in region 4q21.3. As summarized in subsequently 45 cycles of 15 s at 95 °C and 60 s at 60 °C were Table 1, the lengths of the SOAT exons account for 525, 119, 89, applied. Relative SOAT expression (C ) was calculated by T 176, 158, and 435 bp. All intron/exon boundaries were compat- subtracting the signal threshold cycle (C )of -actin from the ible with the canonical donor and acceptor consensus motifs. C value of SOAT. Subsequently, for each tissue, C values Each intron started with GT at the 5-splice donor site and T T were calculated by subtracting brain C (set as calibrator) ended with AG at the 3-splice acceptor site. The cloned SOAT from the C of each individual tissue and transformed by the cDNA sequence exactly matched the genomic sequence of TM Homo sapiens chromosome 4, reference assembly (GenBank 2 equation to show x-fold higher SOAT expression in the accession number NC_000004). The length of the human respective tissue. SLC10A6 gene accounts for 25.8 kb. Bioinformatics—The BLAST program available on the The SOAT protein consists of 377 amino acid residues with a World Wide Web was used to identify SOAT-encoding calculated molecular mass of 41.2 kDa. Fig. 1 shows the sequences in the human genome. Multiple sequence align- deduced amino acid sequence of human SOAT in alignment ments were conducted using the EBI ClustalW algorithm, avail- with human NTCP and ASBT. SOAT has a higher amino acid able on the World Wide Web, and alignment was visualized by sequence identity/similarity to ASBT (42%/70%), compared BOXSHADE, version 3.21. Amino acid identity values were with NTCP (33%/63%). At the C-terminal and N-terminal determined after pairwise optimal GLOBAL alignment with domains, the aligned proteins are clearly divergent, but a highly the BioEdit program, version 7.0.5.2 (20). For similarity calcu- homologous core region exists, representing the transmem- lations, the DAYHOFF similarity matrix was used. Membrane brane part of the proteins (residues 32–307 in the SOAT topology and putative membrane-spanning domains were sequence). As is the case for ASBT and NTCP, potential outer determined by the following programs: TMHMM (21), PRED- 4 14 157 facing N-glycosylation sites (residues Asn , Asn , and Asn ) TMR2 (22), MEMSAT (23), TMAP (24), TopPred II (25), and potential inner facing serine and threonine phosphoryla- TMpred (26), and HMMTOP (27). The NetNGlyc 1.0 program 60 126 259 310 335 336 tion sites (residues Ser , Ser , Ser , Thr , Ser , Thr , was used to predict N-linked glycosylation sites, and NetPhos 337 338 354 374 Ser , Ser , Thr , and Thr ) are present in the SOAT 2.0 was used to predict potential phosphorylation sites in the protein sequence. Further bioinformatic analyses revealed a SOAT protein (28). positively charged cluster in the SOAT C terminus at positions Statistical Analysis—Statistical significance for uptake 312–339 (net charge 9) and a tandem repeat “LTIP” at measurements with radiolabeled substrates was calculated residues 158–161 and 172–175. using Student’s t test. Statistical analysis of more than two SOAT Tissue Expression—The expression of SOAT in dif- groups was performed by one-way analysis of variance, fol- ferent human tissues was investigated by real time quantita- lowed by post hoc testing (Dunnett). Kinetic data from exper- tive PCR (Fig. 2A). Very low SOAT expression was found in iments measuring the uptake of radiolabeled substrates were brain, colon, kidney, liver, ovary, prostate, small intestine, fit to the Michaelis-Menten equation by nonlinear regres- spleen, and thymus. Expression levels were low also in the sion analysis. Dixon plot analysis was used for K adrenal gland, from which the SOAT was initially cloned. In calculations. contrast, SOAT was highly expressed in human testis, where 19732 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT dependence. Fig. 3 shows the time profile for [ H]DHEAS uptake by SOAT-expressing HEK293 cells and noninduced control cells. In the absence of tetracycline, no sig- nificant transport of [ H]DHEAS was observed. In contrast, SOAT- expressing HEK293 cells showed 3-fold (30 s) to 9-fold (10 min) higher [ H]DHEAS transport over the control cells. Using identical amounts of [ H]DHEAS (200 nM), transport studies were also per- formed in the absence of Na by equimolar substitution with cho- line. In these experiments, sodium replacement completely abolished SOAT-mediated DHEAS trans- port. The initial uptake velocity of [ H]DHEAS was also analyzed in SOAT-expressing HEK293 cells and revealed linear uptake over FIGURE 1. Amino acid sequence alignment of NTCP (SLC10A1), ASBT (SLC10A2), and SOAT (SLC10A6). The deduced amino acid sequences were aligned using the EBI ClustalW algorithm, and alignment was visu- 75 s at concentrations ranging alized by BOXSHADE. The portion of the sequence that must agree for shading was set to 0.8. Amino acid from 0.5 to 100 M DHEAS (Fig. identity is displayed with black shading, and amino acid similarities are highlighted in gray. Gaps () are intro- 4). Finally, the concentration de- duced to maximize alignment. 3 3 pendence of [ H]DHEAS, [ H]E S, the SOAT mRNA levels detected were 678 times higher than and [ H]PREGS transport was examined. As shown in Fig. 5, in brain (the tissue with lowest SOAT expression). Relatively SOAT-specific uptake of these compounds showed saturation high SOAT expression was also observed in human placenta kinetics and followed the Michaelis-Menten equation. Kinetic and pancreas, and moderate expression was detected in parameters were determined by nonlinear regression analysis heart, lung, and mammary gland. Because it has been and yielded K values of 28.7  3.9, 12.0  2.3, and 11.3  3.0 reported that an exon-2-skipped, alternatively spliced form M and V values of 1899  81, 585  34, and 2168  134 max of ASBT is expressed in certain rat tissues (29), we per- pmol/mg of protein/min for DHEAS, E S, and PREGS, formed additional RT-PCR experiments covering the whole respectively. open reading frame of SOAT from human testis, placenta, Ion Dependence of SOAT-mediated Transport—To further and pancreas RNAs. As shown in Fig. 2B, unique PCR ampli- investigate the sodium dependence of SOAT transport, we per- cons were detected that migrated at the expected size of 1152 formed transport experiments with equimolar substitutions of bp on the agarose gel without occurrence of shorter SOAT sodium chloride in the transport buffer by other cations and/or transcripts. anions. As shown in Table 3, lithium chloride and potassium Transport Properties of Human SOAT—Functional char- chloride maintained 36 and 23% of the transport function in acterization of human SOAT was performed in stably trans- relation to sodium chloride, respectively. In contrast, choline fected SOAT-HEK293 cells using the Flp-In T-REx expres- and N-methyl-D-glucamine could not maintain [ H]DHEAS sion system, where SOAT expression is under control of the transport by SOAT. Replacement of chloride by gluconate in tetracycline-regulated cytomegalovirus/tetO hybrid pro- the sodium-containing and potassium-containing transport moter. SOAT expression was induced in SOAT-HEK293 buffers revealed similar transport activities. This indicates that cells by pretreatment with tetracycline. Transport experi- SOAT transport is independent of chloride but highly depends ments were performed with several radiolabeled steroid on the presence of sodium in the transport buffer. compounds. SOAT-specific transport activity was not Inhibition Studies with SOAT-HEK293 Cells—Inhibition detected with nonconjugated steroids (estrone and dehydro- experiments were performed in SOAT-HEK293 cells to inves- epiandrosterone), glucuronidated steroids (estradiol-17-D- tigate the substrate selectivity of SOAT (Fig. 6). Cis-inhibitory glucuronide and estrone-3-D-glucuronide), bile acids effects of the indicated compounds on SOAT-mediated uptake (taurocholic acid, cholic acid, chenodeoxycholic acid, of 2.5 M [ H]DHEAS were examined at 25 M concentrations. deoxycholic acid, and lithocholic acid), or heart glycosides Among the group of bile acids, the trihydroxylated bile acids (ouabain and digoxin) (Table 2). However, SOAT-specific taurocholic acid, glycocholic acid, and cholic acid were no transport in SOAT-HEK293 cells was observed for DHEAS, inhibitors for SOAT-mediated [ H]DHEAS transport. In con- estrone-3-sulfate (E S), and pregnenolone sulfate (PREGS). trast, dihydroxylated bile acids inhibited SOAT transport Further characterization of this transport addressed time markedly, whereby inhibitory potency was higher for the dependence, sodium dependence, and concentration 3,7-dihydroxylated bile acids than for the 3,12-dihy- JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19733 Cloning and Characterization of Human SOAT droxylated bile acids. The chenodeoxycholic acid was the most nodeoxycholic acid, this bile acid is not a substrate of SOAT effective and reduced SOAT-mediated transport to 15%. How- (Table 2). SOAT inhibition was also observed by the 3-mono- ever, as demonstrated in transport experiments with [ C]che- hydroxylated bile acids lithocholic acid, glycolithocholic acid, and taurolithocholic acids. Again, [ C]lithocholic acid was not transported by SOAT-HEK293 cells in direct transport exper- iments (Table 2). Finally, sulfoconjugated 3-monohydroxy- lated bile acids were tested. These sulfated bile acids are struc- turally similar to the sulfated steroid hormones bearing an anionic sulfate moiety and a lipophilic steroid nucleus. At a 10-fold molar excess of unlabeled compounds, uptake of 2.5 M [ H]DHEAS was reduced to less than 10% by taurolithocholic acid-3-sulfate (TLCS), glycolithocholic acid-3-sulfate, and lith- ocholic acid-3-sulfate. TLCS, which was the most potent SOAT inhibitor among the group of sulfoconjugated bile acids, was also used for competitive inhibition experiments. Here, uptakes of 0.5 and 2.5 M [ H]DHEAS by SOAT were inhibited by increasing concentrations of TLCS (Fig. 7C). An apparent K value of 0.24 M was determined from Dixon plot transforma- tion; this is 2 orders of magnitude lower than the apparent K value for DHEAS (i.e. 28.7 M). Furthermore, cis-inhibitory effects of the SOAT transport were examined with a set of xenobiotic organosulfates. At 25 M concentrations, SOAT-mediated DHEAS transport was reduced to 4% by 1-SEP, to 18% by bromosulfophthalein, to 25% by 2-SMP and 4-SMP, and to 43% by -naphthylsulfate. In the case of 2-SMP and 4-SMP, additional inhibition experi- ments were performed using 100 and 500 nM concentrations of the SOAT substrate E S (Fig. 7, A and B). K values, determined 1 i from Dixon plots, were 4.3 and 5.5 M for 2-SMP and 4-SMP, respectively. In contrast, other sulfoconjugated organic mole- cules had little or no inhibitory activity for SOAT transport at 25 M concentrations. These included ethylsulfate, phenylsul- FIGURE 2. Expression of SOAT in various human tissues. SOAT tissue fate, phenylethylsulfate, 2-propylsulfate, 5-sulfooxymethylfur- expression was analyzed by quantitative real time PCR analysis (A) and con- fural, hydroquinone sulfate, 4-methylumbelliferylsulfate, and ventional PCR (B) using human multiple cDNA panels and cDNAs synthesized from human adrenal gland and mammary gland RNAs. Gene-specific primers indoxylsulfate (Fig. 6). Furthermore, a series of differently sub- and probes were used as outlined under “Experimental Procedures.” A, rela- T stituted naphthyl derivatives were tested to discriminate tive SOAT expression was calculated by the 2 method and represents SOAT expression that is x times higher in the respective tissue than in brain whether the sulfate moiety can be replaced by other groups for (set as calibrator). The values represent means  S.E. of quadruplicate meas- SOAT inhibition. However, in contrast to -naphthylsulfate, urements. B, RT-PCR experiments covering the whole open reading frame of SOAT. -naphthylisothiocyanate, -naphthylphosphate, and -naph- TABLE 2 3 14 Uptake of various H-labeled and C-labeled compounds by SOAT-HEK293 cells SOAT-HEK293 cells were seeded in 12-well plates and grown to confluence under standard conditions. Tetracycline (1 g/ml medium) was added to induce SOAT expression. Flp-In HEK293 cells were used as control. Cells were incubated with the indicated radiolabeled compound. After 10 min, the medium was removed, and each cell monolayer was washed and processed to determine the protein content and cell-associated radioactivity. The values represent means  S.E. of two independent experiments, each with triplicate determinations. Compound SOAT-HEK293 Control Ratio (SOAT/control) pmol/mg protein/10 min pmol/mg protein/10 min DHEAS (0.2 M) 13.1  0.7 1.1  0.04 12.2 E S (0.2 M) 10.2  0.5 0.8  0.1 12.1 PREGS (0.2 M) 146.1  6.5 19.5  3.7 7.5 Estrone (1 M) 21.5  0.5 21.7  0.7 1.0 Dehydroepiandrosterone (1 M) 31.0  1.3 33.4  0.7 0.9 Estradiol-17-D-glucuronide (1 M) 4.8  0.6 4.4  0.5 1.1 Estrone-3-D-glucuronide (1 M) 17.3  2.7 15.9  2.5 1.1 Taurocholic acid (1 M) 1.7  0.3 1.8  0.2 1.0 Cholic acid (2.5 M) 20.5  4.4 21.0  2.4 1.0 Chenodeoxycholic acid (2.5 M) 387.1  37.9 414.6  49.7 0.9 Deoxycholic acid (2.5 M) 124.9  4.9 124.3  2.4 1.0 Lithocholic acid (2.5 M) 281.4  20.8 311.0  11.0 0.9 Ouabain (1 M) 1.5  0.06 1.5  0.1 1.0 Digoxin (1 M) 6.2  0.3 6.2  0.1 1.0 Uptake values by SOAT-HEK293 cells were significantly different from control cells with p 0.001 (Student’s t test). 19734 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT FIGURE 3. Time course and sodium dependence of the transport of 200 nM [ H]DHEAS into SOAT-HEK293 cells. For SOAT expression, cells were pre- treated with 1 g/ml tetracycline (filled squares); noninduced control cells were not pretreated with tetracycline (open squares). Cells were incubated with 200 nM [ H]DHEAS at 37 °C for different time intervals in sodium chloride medium (continuous line) or in sodium-free choline chloride medium (broken line). At the indicated time points, cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent means  S.E. of two independent experiments, each with triplicate determinations. FIGURE 5. Concentration dependence of SOAT-mediated uptake of 3 3 3 FIGURE 4. Initial uptake velocity of [ H]DHEAS into SOAT-HEK293 cells. [ H]DHEAS (A), [ H]E S(B), and [ H]PREGS (C). SOAT-HEK293 cells were For SOAT expression, cells were pretreated with 1 g/ml tetracycline, and pretreated with 1 g/ml tetracycline. Flp-In HEK293 cells were used as con- uptakes of the indicated concentrations of DHEAS were measured over 15, trol. Cells were incubated with the indicated concentrations of [ H]DHEAS (A), 3 3 30, 45, 60, and 75 s. Cells were washed with ice-cold PBS, lysed, and subjected [ H]E S(B), and [ H]PREGS (C) for 1 min at 37 °C. The medium was removed, to scintillation counting. The values represent means of duplicate determina- and each cell monolayer was washed and processed to determine the protein tions of representative experiments. content and cell-associated radioactivity. SOAT-specific uptake was calcu- lated by subtracting nonspecific uptake of the Flp-In HEK293 control cells (open squares) from uptake into SOAT-HEK293 cells (filled squares) and is thylamine had no inhibitory effect on the DHEAS transport in shown by broken lines. The values represent means  S.E. of duplicate exper- iments, each with triplicate determinations. Michaelis-Menten kinetic param- SOAT-HEK293 cells (Fig. 6). eters were calculated from SOAT-specific uptakes by nonlinear regression Transport of [ H]TLCS, 2-SMP, and 4-SMP in SOAT- analysis and revealed K of 28.7  3.9 M and V of 1899  81 pmol/mg of m max HEK293 Cells—Since TLCS, 2-SMP, and 4-SMP competitively protein/min for DHEAS, K of 12.0 2.3 M and V of 585 34 pmol/mg of m max protein/min for E S, and K of 11.3 3.0M and V of 2168 134 pmol/mg 1 m max inhibited SOAT transport, we further investigated whether of protein/min for PREGS. they are also substrates of SOAT. No radiolabeled compounds were available for 2-SMP and 4-SMP, so we determined the intracellular accumulation of 2-SMP and 4-SMP in the SOAT- observed if the experiments were performed in Na -containing HEK293 cells with an HPLC-based method using fluorescence transport buffer and was completely abolished if sodium was detection. As shown in Fig. 7, D–G, 2-SMP, 4-SMP, and TLCS substituted by equimolar concentrations of choline, thus indi- were transported in SOAT-expressing HEK293 cells with a cating that 2-SMP, 4-SMP, and TLCS were transported by ratio of about 2 over control cells. This transport was only SOAT in a sodium-dependent manner (Fig. 7, D and E). Fur- JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19735 Cloning and Characterization of Human SOAT TABLE 3 thermore, 4-SMP transport was blocked by a 5-fold molar Influence of equimolar substitutions of NaCl on H DHEAS uptake excess of E S in the transport medium (Fig. 7F). In contrast, by SOAT-HEK293 cells transport of 50 nM TLCS was inhibited by not less than 50 M Uptake of 200 nM H DHEAS was measured at 37 °C in SOAT-HEK293 cells after DHEAS (Fig. 7G), which is consistent with the lower K value of preincubation with tetracycline (1 g/ml). 142 mM NaCl was used in the transport i buffer for the 100% control experiment and was substituted with equimolar con- TLCS compared with 4-SMP. centrations of sodium gluconate, potassium gluconate, lithium chloride, potassium Membrane Topology of Human SOAT—Analysis of the chloride, N-methyl-D-glucamine, and choline chloride. After 5 min, the transport medium was removed, and the cell monolayer was washed and subjected to radio- membrane topology of human SOAT was performed with activity and protein measurements. The values represent means  S.D. of two different topology prediction programs. TMHMM, PRED- independent experiments, each with triplicate determinations. TMR2, MEMSAT, TMAP, TopPred II (GES-scale), and Percentage DHEAS uptake of control TMpred proposed a membrane topology of SOAT with eight pmol/mg protein/5 min % transmembrane domains and an extracellular location of the Sodium chloride (control) 10.4  0.5 100 N-terminal and C-terminal domains (Fig. 8A). In contrast, Lithium chloride 3.8  0.3 36 HMMTOP analysis preferred a model with nine transmem- Potassium chloride 2.4  0.3 23 Choline chloride 0.4  0.04 3.8 brane domains, and TopPred II (KD-scale) calculated a sev- N-Methyl-D-glucamine 0.3  0.07 2.7 en-TMD topology. In all predictions, the N terminus of Sodium gluconate 9.9  0.8 95 Potassium gluconate 1.6  0.1 15 SOAT has an extracellular orientation and contains 30 Uptake values after equimolar substitution of NaCl were significantly different amino acid residues. This orientation is predicted due to a from control experiments with p 0.01 (one-way analysis of variance with cluster of positively charged amino acid residues just down- Dunnett post hoc analysis). stream from TMD 1 (net charge of the N terminus 4, net charge of the first intracellular loop 3). The C terminus is inside in the seven-TMD and nine-TMD models but has an extracellular orientation in the eight-TMD topology (Fig. 8A). Similar dis- crepancies from in silico topology predictions were obtained for NTCP and ASBT. For these SLC10 carriers, experimental data clearly favored a seven-TMD topology. To determine whether a seven- TMD topology can be applied also for SOAT, we directly compared the hydrophobicity profiles of SOAT, ASBT, and NTCP in an overlay of the individual hydro- phobicity plots. As shown in Fig. 8B, hydrophobicity values of SOAT and ASBT are nearly iden- tical, indicating that both carriers show similar membrane topology. However, both proteins differ from the NTCP hydrophobicity pattern, particularly concerning amino acid residues 70 –170, which represent transmembrane helices 2– 4. Localization of the N-terminal and C-terminal Domains of FIGURE 6. Inhibitory potency of bile acids, nonsteroidal organosulfates, and naphthyl derivatives on SOAT—Membrane expression SOAT transport. Sodium-dependent uptake of 2.5 M [ H]DHEAS was measured in the presence of 25 M and the C/N terminus orientation inhibitory compounds. Cells were seeded in 12-well plates and grown to confluence. For transport experi- ments, SOAT expression was induced by preincubation with tetracycline (1 g/ml). Inhibition experiments of human SOAT were analyzed in were started by preincubation with the respective inhibitor at 37 °C for 30 s. Subsequently, [ H]DHEAS was vitro in SOAT-HEK293 cells and added, and the incubation was continued for 5 min at 37 °C. Inhibition studies were terminated by removing HEK293 cells expressing the the transport buffer and washing with ice-cold PBS. Cells not incubated with any inhibitor served as positive control (set to 100%). The values represent the percentage of DHEAS transport activity in the presence of the SOAT-FLAG fusion protein in indicated inhibitor relative to the positive control and are expressed as means  S.E. of triplicate determina- which the FLAG motif (DYKD- tions of representative experiments. The values were significantly different from positive controls; *, p 0.01 (one-way analysis of variance with Dunnett post hoc analysis). DDDK) was attached to the C-ter- 19736 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT FIGURE 7. 2-SMP, 4-SMP, and TLCS as competitive inhibitors and substrates of SOAT. SOAT-HEK293 cells were pretreated with tetracycline (1 g/ml) to induce SOAT expression. For studying inhibition kinetics, SOAT-HEK293 cells were incubated in the presence (A and B) of 100 and 500 nM [ H]E S and increasing concentrations of 2-SMP (A) and 4-SMP (B) for 2 min at 37 °C and of 0.5 M and 2.5 M [ H]DHEAS and increasing concentrations of TLCS for 5 min at 37 °C (C). Cells were washed and processed to determine protein content and cell-associated radioactivity. Values are means  S.E. of triplicate determinations of representative experiments. K values were calculated from Dixon plots to 4.3 M for 2-SMP, 5.5 M for 4-SMP, and 0.24 M for TLCS. For studying SOAT- mediated uptake, SOAT-HEK293 cells (SOAT) and Flp-In HEK293 (control) were incubated with 10 M 2-SMP and 10 M 4-SMP for 15 min at 37 °C (D and F, filled bars) as well as with 50 nM [ H]TLCS for 5 min at 37 °C (E and G, filled bars). Cells were washed and processed to determine protein content and substrate uptake using HPLC analysis with fluorescence detection for 2-SMP and 4-SMP and using liquid scintillation counting for [ H]TLCS. Uptake of 10 M 2-SMP and 50 nM [ H]TLCS was also analyzed in Na -free exposure medium (D and E, open bars). In addition, uptake of 10 M 4-SMP was measured in the presence of 50 M E S (F), and uptake of [ H]TLCS was analyzed in the presence of 5 and 50 M DHEAS as competing substrates (G). Values are means  S.E. of three independent experiments and were significantly different from control; *, p 0.05 (Student’s t test). minal end of SOAT. To confirm the extracellular orientation microscopy under permeabilized and nonpermeabilized of the N terminus, we generated a SOAT antibody (SOAT- conditions. FLAG-directed fluorescence staining was only (2–17)) directed against the N-terminal 2–17 amino acids. observed if the cells were permeabilized by Triton X-100, Using this antibody, SOAT expression was analyzed in and it was undetectable in the nonpermeabilized cells (Fig. SOAT-HEK293 cells that were either induced or nonin- 8D). This clearly indicates a cytosolic orientation of the duced by tetracycline treatment and were kept under native SOAT C terminus and excludes an eight-TMD topology. (nonpermeabilized) conditions (Fig. 8C). Fluorescence sig- Immunoprecipitation and Deglycosylation of the SOAT- nals were only observed in the SOAT-expressing HEK293 FLAG Protein—SOAT-FLAG-pcDNA5-transfected HEK293 cells, and no cell-associated fluorescence was detected in the cells were also used for radiolabeling and immunoprecipita- noninduced control cells. Since SOAT-HEK293 cells were tion experiments of the SOAT-FLAG protein with a mono- not fixed and not permeabilized for these experiments clonal anti-FLAG antibody (Fig. 9). After separation of the before incubation with the SOAT-(2–17) antibody, the N precipitated cell extract, specific bands were detected at 46 terminus of SOAT must be located in the extracellular com- and 42 kDa. The SOAT-FLAG protein consists of 377  8 partment. In order to discriminate also the inside/outside amino acids with a predicted molecular mass of 41 kDa orientation of the C terminus, we generated a second SOAT (SOAT)  1 kDa (FLAG epitope). The higher apparent antibody, which was directed against amino acids 349 –364 molecular mass of 46 kDa after immunoprecipitation was of the C terminus (SOAT-(349 –364)). This antibody failed due to posttranslational modifications in the HEK293 cells. to show in vitro immunoreactivity against the SOAT-(349 – Since SOAT encodes three potential N-linked glycosylation 364) peptide, so we decided to attach the FLAG epitope tag sites, N-glycosylation of the SOAT-FLAG protein was exam- to the SOAT C terminus, which was then detected by using a ined by PNGase F digestion of the immunoprecipitated cell commercial anti-FLAG antibody. A SOAT-FLAG-pcDNA5 extract. As shown in Fig. 9, the apparent molecular mass of construct was generated by site-directed mutagenesis and the SOAT-FLAG protein was decreased from 46 to 42 kDa transfected into HEK293 cells to evaluate the accessibility of after PNGase F incubation. This change in apparent molec- the C-terminal FLAG epitope by immunofluorescence ular mass is consistent with the addition of at least one JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19737 Cloning and Characterization of Human SOAT FIGURE 8. Membrane expression and C/N terminus orientation of SOAT. A, proposed membrane topology of human SOAT based on the analyses of different topology prediction programs for eukaryotic proteins. The cylinders indicate the predicted transmembrane domains, and loops are depicted as lines. Topology models of human SOAT with seven, eight, and nine TMDs are shown. B, hydrophobicity profiles of human SOAT in comparison with human ASBT and NTCP. Increasing numerical values displayed on the y axis correspond to increasing hydrophobicity. The residue-specific hydropho- bicity index was calculated over a window of 11 residues by the GES-scale method of the TopPred II program. The plot shows an alignment of SOAT, NTCP, and ASBT amino acid sequences. The x axis refers to the amino acid residue number in the respective protein sequences. C and D orientation of the N-terminal and C-terminal ends of SOAT were determined by immunofluorescence microscopy of stably transfected SOAT-HEK293 cells (C) and Flp-In HEK293 cells transfected with the SOAT-FLAG-pcDNA5 construct (D). SOAT-HEK293 cells were grown on glass coverslips, and SOAT expression was induced by pretreatment with tetracycline (tet). Control cells were untreated with tetracycline (tet). Cells were not fixed or permeabilized. SOAT expression was analyzed with the SOAT-(2–17) antibody and an fluorescein isothiocyanate-conjugated secondary antibody (green fluorescence). Nuclei were stained with DAPI (blue fluorescence). The orientation of the C terminus was assessed in Flp-In HEK293 cells transfected with the SOAT-FLAG-pcDNA5 construct under permeabilized and nonpermeabilized conditions. The C-terminal FLAG epitope was detected by an anti-FLAG primary antibody and a Cy3-conjugated secondary antibody (orange fluorescence). Experimental data clearly exclude the eight-TMD model for human SOAT. Scale bar,25 m. N-linked carbohydrate chain to any of the potential N-linked all physiological dihydroxylated and trihydroxylated bile acids 4 14 157 glycosylation sites of SOAT (Asn , Asn , and Asn ). Based with a preference for the taurine and glycine amidated conju- on these data, molecular masses of 45 and 41 kDa can be gates versus the unconjugated forms (9–11, 31). SOAT is the estimated for the untagged glycosylated and nonglycosylated third member of the SLC10 transporter family that we have SOAT proteins, respectively. For comparison, also the functionally characterized in this paper. Transport studies in ASBT-FLAG protein was examined and also revealed a SOAT-HEK293 cells revealed no transport activity for tauro- decrease of its apparent molecular mass from 40 to 37 kDa cholic acid and cholic acid, thus indicating that SOAT, after PNGase F treatment, as has been previously reported although belonging to the bile acid transporter family SLC10, is (6, 7, 30). not a typical bile acid transporter, such as NTCP and ASBT. The latter also do not share identical substrate patterns. In con- DISCUSSION trast to ASBT, to which SOAT has closest homology, the sub- Transport Function of NTCP, ASBT, and SOAT—In the past strate specificity of NTCP is not strictly limited to bile acids and decade, the transport function of NTCP and ASBT were exten- also includes sulfoconjugated steroid hormones, such as E S (9, sively studied in several cell systems and revealed transport of 10). Additionally, it has been reported by Craddock and co- 19738 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 27 •JULY 6, 2007 Cloning and Characterization of Human SOAT interference with SOAT-mediated transport. Concerning the negatively charged sulfate moiety, replacement of this group on a naphthyl core molecule by phosphate (-naphthylphos- phate), amine (-naphthylamine), and isothiocyanate (-naph- thylisothiocyanate) completely abolished all inhibitory potency of the respective -naphthyl derivative, thus indicating that the interaction of the sulfate moiety with the SOAT binding site is essential. Toxicological Aspects—The high affinity SOAT inhibitors 2-SMP and 4-SMP were also transported by SOAT in a sodium- dependent manner, thus showing that substrate recognition by SOAT also covers xenobiotics. 2-SMP and 4-SMP are isomers FIGURE 9. Immunoprecipitation and deglycosylation of SOAT-FLAG and ASBT-FLAG fusion proteins. HEK293 cells were transfected with the of 1-SMP that have longer half-life in water than 1-SMP ( 1 SOAT-FLAG-pcDNA5 and ASBT-FLAG-pcDNA5 constructs or vector alone day versus 2.8 min) (18). Thus, transport studies were conve- (control), and cells were induced by tetracycline treatment (1 g/ml). Cells niently performed with these long lived isomers. These com- were starved in medium without methionine and cysteine, followed by 35 35 [ S]labeling with L-[ S] in vitro cell labeling mix. Cell lysates were used for pounds are of particular toxicological importance, since cova- immunoprecipitation with an anti-FLAG antibody. Aliquots of the immuno- lent binding to DNA, causing mutations and neoplasia, was precipitated proteins were incubated with PNGase F (lanes 2, 4, and 6)or water (lanes 1, 3, and 5). The samples were separated by electrophoresis on a observed for 1-SMP (35, 36). This metabolite is formed from 12% SDS-polyacrylamide gel, and the dried gel was exposed to standard x-ray 1-methylpyrene, present at high levels in cigarette smoke, via film for visualization. 1-hydroxymethylpyrene by sulfoconjugation. The highest level of DNA adducts by 1-SMP was observed in kidney, where the workers (9) that the sulfoconjugated bile acid chenodeoxy- organic anion transporters OAT1 and OAT3 are expressed, cholate-3-sulfate is not a substrate of ASBT but is weakly trans- yielding a kidney-directed organotropism of 1-SMP. As shown ported by NTCP. Also, SOAT showed transport of a here, sulfoconjugated pyrenes, such as 2-SMP and 4-SMP, are sulfoconjugated bile acid, TLCS. Since the K value of TLCS is 2 also substrates of SOAT. Because of its predominant expression orders of magnitude lower than the K value of DHEAS and in testis, we conclude that testes are also exposed to electro- since uptake of TLCS into SOAT-HEK293 cells was inhibited philic adduct-forming pyrene sulfates by uptake via SOAT. by not less than a 1000-fold molar excess of DHEAS, SOAT This uptake might even be related to the well known risk of seems to be a high affinity transporter for sulfoconjugated bile testicular cancer in tobacco smokers (37). acids. Sulfoconjugation of bile acids is increased under choles- Phylogenetic Relationship of SOAT, ASBT, and NTCP—The tatic conditions and also takes place in the fetus (32, 33). Since evolutionary origin of the SLC10 transporter family was SOAT is highly expressed in human placenta, this carrier might recently shown by a phylogenetic analysis of the SLC10A1– be involved in the fetal-to-maternal transfer of sulfoconjugated SLC10A6 genes from several mammalian and nonmammalian bile acids for their elimination through the mother (34). In species (14). This analysis revealed two major clades of genes. addition to the sulfoconjugated bile acids, other bile acids were Clade I comprises SLC10A1 (NTCP), SLC10A2 (ASBT), potent inhibitors, albeit no substrates of SOAT transport, fol- SLC10A4, and SLC10A6 (SOAT) genes; clade II contains lowing the order: 3-monohydroxylated bile acids 3,7- SLC10A3 and SLC10A5 genes. Within clade I, SOAT is the dihydroxylated bile acids 3,12-dihydroxylated bile acids. sister group to ASBT, and SLC10A4 is the sister group to On the other hand, SOAT substrates include the sulfoconju- NTCP. This phylogenetic relationship explains the high gated steroid hormones DHEAS, E S, and PREGS with appar- sequence homology between SOAT and ASBT as well as the ent K values of 28.7, 12.0, and 11.3 M, respectively. These lower sequence homology between ASBT and NTCP. Func- SOAT substrates (TLCS, DHEAS, E S, and PREGS) share a tional transport properties of SLC10 carriers can overlap but hydrophilic, negatively charged, sulfate moiety that is linked to might also be very divergent. A likely explanation would be that a hydrophobic, hydrocarbon steroid nucleus and that seems to the common ancestor gene for SOAT, ASBT, and NTCP be basically required for substrate recognition by SOAT. The exerted transport of bile acids (either sulfoconjugated or non- specific recognition of each substrate by its sulfate group is sulfoconjugated) plus sulfoconjugated steroids but separated corroborated by the observation that neither glucuronidated them during later subdivision into ASBT (only nonsulfoconju- steroids, nonsulfoconjugated steroids, nor nonsulfoconjugated gated bile acids), SOAT (only sulfoconjugated bile acids and bile acids are transported by SOAT-HEK293 cells. sulfoconjugated steroids), and NTCP (bile acids, sulfoconju- Interaction of SOAT with Nonsteroidal Organosulfates— gated bile acids, and sulfoconjugated steroids). At present, the Some relatively large sulfoconjugated molecules also potently varying local organ expression of these three SLC10 transport- inhibited SOAT transport. These included 1-SEP, bromosul- ers combined with their individual substrate pattern reflects fophthalein, 2-SMP, 4-SMP, and -naphthylsulfate. On the and causes broad physiological plasticity. other hand, the uptake of [ H]DHEAS by SOAT was not Sodium Dependence of SOAT—The driving force for the affected by the sulfoconjugates of very small molecules, such as NTCP-mediated and ASBT-mediated transport of bile acids is ethylsulfate, 2-propylsulfate, phenylsulfate, phenylethylsulfate, provided by the inwardly directed Na gradient, which is main- and hydroquinone sulfate. Thus, it appears that a sulfated two- tained by the activity of the Na /K -ATPase in the plasma hydrocarbon ring structure is required at a minimum for any membrane as well as the negative intracellular potential. As JULY 6, 2007• VOLUME 282 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 19739 Cloning and Characterization of Human SOAT demonstrated for rat Ntcp and human ASBT, these transport- the transport characteristics of SOAT, indicating that SOAT is ers perform an electrogenic transport cycle and move two Na involved in the DHEAS transport into placenta trophoblasts. ions for each bile acid molecule (9, 38–40). A comparable After the first trimester of pregnancy, human placenta is also transport mechanism is also suggested for SOAT, because the the main biosynthetic source of progesterone, which is an transport of TLCS, DHEAS, E S, PREGS, 2-SMP, and 4-SMP by essential hormone to sustain pregnancy (54). The progesterone SOAT is also strictly sodium-dependent. Nonetheless, the cat- precursor pregnenolone is synthesized in the trophoblasts from ion selectivity of SOAT is not absolutely identical with that of cholesterol but also derives from PREGS, which is synthesized ASBT. Whereas Li maintained about 40% of the SOAT trans- at high levels in the adrenal gland and is delivered via the mater- port function compared with Na ,Li is not accepted as a nal and fetal blood circulation (55). We suppose that SOAT, stimulating co-substrate of ASBT. On the other hand, equimo- which mediates cellular PREGS uptake, is involved in this proc- lar substitutions of Na by choline abolished the transport ess and therefore could also contribute to placenta progester- function of both carriers (5, 9). one synthesis. Membrane Expression and Topology of NTCP, ASBT, and In conclusion, we have functionally characterized the novel SOAT—Hydrophobicity analyses of NTCP and ASBT pro- sodium-dependent organic anion transporter SOAT of posed 7–9 TMDs, but experimental data strongly support a humans. This carrier is expected to have physiological meaning seven-TMD topology with an exoplasmic N terminus and a for hormone response of testis and placenta on sulfoconjugated cytoplasmic C terminus (4, 8, 30, 41–43). For human SOAT, steroid hormones and also for their toxicologic exposure to only one topology prediction program (TopPred II/KD-scale) sulfoconjugated pyrene carcinogens as well as for placental supported this membrane topology, whereas most other calcu- transport of sulfoconjugated bile acids. lations yielded eight transmembrane domains with an exoplas- mic orientation of the N-terminal and C-terminal ends. In this Acknowledgments—We thank Klaus Schuh and Monika Rex-Haffner for technical help. paper, an N /C trans-orientation was experimentally dem- exo cyt onstrated, which is in accordance with the membrane topology of NTCP and ASBT but clearly eliminates a model with eight REFERENCES TMDs. Our experimental setup was not able to discriminate 1. 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Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Jul 6, 2007

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