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• W2013019095 abstract "The ileal Na+/bile acid cotransporter plays a critical role in the reabsorption of bile acids from the small intestine. In the course of cloning and characterizing the human ileal Na+/bile acid cotransporter cDNA, a dysfunctional isoform was identified in a patient diagnosed with Crohn's disease. Expression studies using hamster-human ileal Na+/bile acid cotransporter cDNA chimeras narrowed the location of the defect to the carboxyl-terminal 94 amino acids. Comparison of the sequence of the dysfunctional isoform to that of a wild-type human ileal Na+/bile acid cotransporter genomic clone revealed a single C to T transition resulting in a proline to serine substitution at amino acid position 290. The inheritance of this mutation in the proband's family was confirmed by single-stranded conformation polymorphism analysis and DNA sequencing. In transfected COS-1 cells, the single amino acid change abolished taurocholate transport activity but did not alter the transporter's synthesis or subcellular distribution. This dysfunctional mutation represents the first known molecular defect in a human sodium-dependent bile acid transporter. The ileal Na+/bile acid cotransporter plays a critical role in the reabsorption of bile acids from the small intestine. In the course of cloning and characterizing the human ileal Na+/bile acid cotransporter cDNA, a dysfunctional isoform was identified in a patient diagnosed with Crohn's disease. Expression studies using hamster-human ileal Na+/bile acid cotransporter cDNA chimeras narrowed the location of the defect to the carboxyl-terminal 94 amino acids. Comparison of the sequence of the dysfunctional isoform to that of a wild-type human ileal Na+/bile acid cotransporter genomic clone revealed a single C to T transition resulting in a proline to serine substitution at amino acid position 290. The inheritance of this mutation in the proband's family was confirmed by single-stranded conformation polymorphism analysis and DNA sequencing. In transfected COS-1 cells, the single amino acid change abolished taurocholate transport activity but did not alter the transporter's synthesis or subcellular distribution. This dysfunctional mutation represents the first known molecular defect in a human sodium-dependent bile acid transporter. The enterohepatic circulation of bile acids is maintained at the cellular level by a series of membrane transporters and binding proteins(1Hofmann A.F. Sleisenger M.H. Fordtran J.S. Gastrointestinal Disease: Pathophysiology/Diagnosis/Management. Saunders, Philadelphia, PA1993: 127-150Google Scholar). In the small intestine, the first step in this process is mediated by a sodium-dependent transport system located at the apical brush-border membrane of the ileocyte. After uptake, the bile acids are directed across the ileocyte to the basolateral membrane (2Wilson F.A. Schultz T. Stanley S. Handbook of Physiology: The Gastrointestinal System Vol. IV. Waverly Press, Baltimore, MD1991: 389-404Google Scholar) and secreted into the portal circulation by a sodium-independent anion-exchange mechanism(3Weinberg S. Burckhardt G. Wilson F.A. J. Clin. Invest. 1986; 78: 44-50Crossref PubMed Scopus (68) Google Scholar). A number of the transport proteins and membrane carriers that participate in the enterohepatic circulation of bile acids have recently been identified(4Dawson P.A. Oelkers P. Curr. Opin. Lipidol. 1995; 6: 109-114Crossref PubMed Scopus (68) Google Scholar). By expression and hybridization techniques, the hamster (5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar) and rat (6Shneider B.L. Dawson P.A. Christie D.-M. Hardikar W. Wong M.H. Suchy F.J. J. Clin. Invest. 1995; 95: 745-754Crossref PubMed Google Scholar) ileal Na+/bile acid cotransporter (ISBT) 1The abbreviations used are: ISBTileal Na+/bile acid cotransporterHISBThuman wild-type ileal Na+/bile acid cotransporterPCRpolymerase chain reactionPBSphosphate-buffered salineHISBT(m)human mutant ileal Na+/bile acid cotransporter, HISBT(P290S)PAGEpolyacrylamide gel electrophoresisSSCPsingle stranded conformation polymorphismβ-galβ-galactosidaseNHS-LC-biotinsulfosuccinimidyl-6-(biotinamido) hexanoate. cDNAs were cloned and shown to encode 348-amino acid membrane glycoproteins. These studies have facilitated characterization of the structure, expression, and ontogeny of the ISBT(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar, 6Shneider B.L. Dawson P.A. Christie D.-M. Hardikar W. Wong M.H. Suchy F.J. J. Clin. Invest. 1995; 95: 745-754Crossref PubMed Google Scholar). To gain further insight into the role of the ISBT in bile acid metabolism, cholesterol homeostasis, and human disease, we recently isolated a human ISBT (HISBT) cDNA and mapped its chromosomal location. 2M. H. Wong, P. Oelkers, P. N. Rao, M. J. Pettenati, and P. A. Dawson, manuscript in preparation. In this paper, we describe the identification and characterization of a naturally occurring dysfunctional mutation in the HISBT gene. ileal Na+/bile acid cotransporter human wild-type ileal Na+/bile acid cotransporter polymerase chain reaction phosphate-buffered saline human mutant ileal Na+/bile acid cotransporter, HISBT(P290S) polyacrylamide gel electrophoresis single stranded conformation polymorphism β-galactosidase sulfosuccinimidyl-6-(biotinamido) hexanoate. Human ileal tissue (within 10 cm of the ileocecal valve) was obtained from a surgical specimen excised due to Crohn's disease. Tissues were frozen in liquid N2 and stored at -70°C. Total cellular RNA was isolated by the guanidinium isothiocyanate/CsCl centrifugation procedure(7Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Poly(A)+ RNA was isolated using oligo(dT)-cellulose spin columns from Pharmacia Biotech Inc. Genomic DNA was isolated from ileal tissue or peripheral white blood cells using the SDS-proteinase K procedure(8Strauss W.M. Janssen K. Current Protocols in Molecular Biology. Wiley & Sons, New York1994: 2.2.1-2.2.3Google Scholar). [3H]Taurocholic acid (2.0-2.6 Ci/mmol) was purchased from DuPont NEN. Tran35S-label (a mixture of [35S]Met and [35S]Cys) was obtained from ICN Biomedicals (Costa Mesa, CA). Unlabeled taurocholate was purchased from Sigma. COS-1 cells were from the American Type Culture Collection (Rockville, MD) and maintained in medium A that consisted of Dulbecco's modified Eagle's medium containing 4500 mg/liter D-glucose, 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.). For [3H]taurocholate uptake assays, COS cells were incubated in medium B, which consisted of a modified Hanks' balanced salt solution containing 137 mM NaCl(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar). The polymerase chain reaction was used to obtain a human ISBT DNA probe. First strand cDNA was synthesized from human ileal poly(A)+ RNA using a cDNA synthesis kit (Superscript kit; Life Technologies Inc.). For the PCR, oligonucleotide primers were synthesized corresponding to amino acid sequences conserved between the hamster ISBT and rat liver Na+/bile acid cotransporter(9Hagenbuch B. Stieger B. Foguet M. Lubbert H. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10629-10633Crossref PubMed Scopus (447) Google Scholar). The sense oligonucleotide primer 5′-CAGTTTGGAATCATGCCTCTC-3′ and antisense primer 5′-TGTTCTGCAACCCGGTTTCCA-3′ corresponded to amino acids 75-81 and 261-266 of the hamster ISBT. Amplification was performed using an annealing temperature of 45°C. A product of the expected size (576 base pairs) was isolated from a 0.8% (w/v) agarose gel and subcloned into a pT7Blue T vector (Novagen, Madison, WI). The inserts were sequenced by the dideoxy method(7Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A λgt10 cDNA library was constructed from human ileal poly(A)+ RNA using a cDNA synthesis kit (Superscript kit; Life Technologies Inc.). cDNA was synthesized using a combination of random hexamers and oligo(dT) primers and ligated to EcoRI-NotI adapters. cDNAs greater than 0.5 kilobases in length were isolated on a 1.0% (w/v) agarose gel and ligated to EcoRI-cleaved λgt10 DNA. After in vitro packaging using a Gigapack II Gold Cloning kit (Stratagene; La Jolla, CA), the phage (1.5 × 106 total plaques) were plated and transferred to replicate filters. The filters were hybridized for 16 h at 42°C in buffer containing 50% formamide, 5 × SSC, 50 mM sodium phosphate, pH 6.8, 2 × Denhardt's solution, 100 μg/ml denatured salmon sperm DNA, and 1 × 106 cpm/ml of radiolabeled probe. After hybridization, filters were washed in 0.2 × SSC, 0.1% (w/v) SDS at 65°C for 30 min. Positive clones were plaque purified; plate lysate DNA was isolated (10Xu S.-Y. Gene Anal. Tech. 1986; 3: 90-91Crossref Scopus (14) Google Scholar) and subcloned into pBluescript KS II for restriction mapping and DNA sequencing. The dysfunctional HISBT cDNA, indicated as HISBT(m), was subcloned into EcoRI-digested pCMV5 (11Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar) for expression studies. Conserved sites for the restriction enzymes, HincII and BanI, were used to generate the HISBT(m) and hamster ISBT chimeras that are diagrammed (see Fig. 3). The pHA construct encoded HISBT(m) amino acids 1-153 and hamster ISBT amino acids 154-348 in the expression plasmid pCMV5. The pAH construct encoded hamster ISBT amino acids 1-153 and HISBT(m) amino acids 154-348 in the expression plasmid pcDNA I/Amp (Invitrogen; San Diego, CA). The pHHA construct encoded HISBT(m) amino acids 1-254 and hamster ISBT amino acids 255-348 in pCMV5. The pHAH construct encoded HISBT(m) amino acids 1-153, hamster ISBT amino acids 154-254, and HISBT(m) amino acids 255-348 in pCMV5. To construct the wild-type HISBT, the following fragments were ligated into pCMV5: HISBT(m) EcoRI-SacI (nucleotides 1-966; amino acids 1-283) and human ISBT cDNA clone λH13 SacI-EcoRI (nucleotides 967-1253; amino acids 284-348). The constructs were verified by dideoxy nucleotide sequencing. A synthetic peptide (Research Genetics, Huntsville, AL) corresponding to amino acids 335-348 of the hamster ISBT was coupled to tuberculin-purified protein derivative (Statens Seruminstitut, Copenhagen, Denmark) using glutaraldehyde(12Lachmann P.J. Strangeways L. Vyakarnam A. Evans G. CIBA Found. Symp. 1986; 119: 25-57PubMed Google Scholar). Three New Zealand White rabbits were immunized with 200 μg of coupled peptide in Freund's complete adjuvant. Rabbit serum was assayed for anti-ISBT antibody by immunoblotting using rat or hamster ileal brush-border membrane preparations (13Kessler M. Acuto O. Storelli C. Murer H. Muller M. Semenza G. Biochim. Biophys. Acta. 1978; 506: 136-154Crossref PubMed Scopus (1017) Google Scholar). The IgG was then purified from rabbit serum by protein A-Sepharose chromatography(14Dawson P.A. Metherall J.E. Ridgway N.D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1991; 266: 9128-9134Abstract Full Text PDF PubMed Google Scholar). For immunoblotting studies, cells were harvested in phosphate-buffered saline (PBS) and lysed in buffer A (15% SDS, 8 M urea, 10% sucrose, 62.5 mM Tris-HCl, pH 6.8, 10 mM EDTA, and 5 mM dithiothreitol) by repeated aspiration through a 25-gauge needle(15Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar). The samples were diluted 10-fold with Laemmli sample buffer (3% SDS, 5% glycerol, 30 mM Tris-HCl, pH 6.8, 10 mM EDTA, and 2.5%β-mercaptoethanol), boiled for 5 min, and resolved by SDS-PAGE on 10% acrylamide gels. Immunoblotting was performed as described previously (14Dawson P.A. Metherall J.E. Ridgway N.D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1991; 266: 9128-9134Abstract Full Text PDF PubMed Google Scholar) using rabbit anti-ISBT peptide antibody. The rabbit antibody was visualized using a horseradish peroxidase-conjugated goat anti-rabbit antibody and an enhanced chemiluminescent detection system (ECL; Amersham Corp). On day 0, 5 × 105 COS cells/60-mm dish were plated in medium A. On day 1, duplicate dishes of cells were transfected with 2 μg of β-galactosidase (β-gal)(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar), hamster ISBT(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar), or HISBT(m) expression plasmid by the DEAE-dextran method(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar). On day 4, cells were washed with PBS and incubated with methionine- and cysteine-free medium A for 45 min. The cells were then incubated in 1 ml of methionine and cysteine-free medium A containing 200 μCi/ml Tran35S-label for 1 h. After the pulse, the cells were washed once with PBS and chased for 1 h in medium A supplemented with 100 μM unlabeled methionine and 100 μM unlabeled cysteine. The cells were then scraped in PBS and recovered by centrifugation at 400 × g. Cell pellets were lysed in buffer B (25 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM CaCl2, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin) by repeated aspiration through a 25-gauge needle. The samples were centrifuged at 10,000 × g for 2 min at 4°C, and the cell supernatant was immunoprecipitated by incubation with 9 μg of anti-ISBT peptide antibody plus 50 μl of protein A-agarose (50% suspension in 25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1 mM CaCl2 (TBS-C); RepliGen, Cambridge, MA) for 12 h at 4°C. Immune complexes were recovered by centrifugation at 10,000 × g for 30 s. Pellets were washed twice with buffer B and once with TBS-C. The protein A-agarose beads were resuspended in 0.5% SDS and 1%β-mercaptoethanol and boiled for 10 min to elute the immunoprecipitated protein. For endoglycosidase H digestion, aliquots of eluted protein were incubated for 1 h at 37°C with 1 × 103 units of endoglycosidase Hf (New England Biolabs; Beverly, MA) in 50 mM sodium citrate, pH 5.5, 0.5% SDS, and 1%β-mercaptoethanol. For peptide:N-glycosidase F digestion, aliquots of the eluted protein were incubated for 1 h at 37°C with 1 × 103 units of peptide:N-glycosidase F (New England Biolabs) in 50 mM sodium phosphate, pH 7.5, 1% Nonidet P-40, 0.5% SDS, and 1%β-mercaptoethanol. The samples were then brought to 3% SDS, 5% glycerol, 30 mM Tris-HCl, pH 6.8, 10 mM EDTA, and 2.5%β-mercaptoethanol, boiled for 5 min, and resolved by SDS-PAGE on 10% acrylamide gels. After electrophoresis, gels were soaked for 60 min in Entensify (DuPont NEN), dried, and exposed to Amersham Hyperfilm. A human placental genomic DNA library in λEMBL3 (Catalog no. HL1067j; Clontech; Palo Alto, CA) was screened using standard methods (7Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with 32P-labeled probes derived from the coding region of the human ISBT cDNA. After screening a total of 2 × 105 bacteriophage, one positive clone was identified and plaque purified. Plate lysate DNA was isolated (10Xu S.-Y. Gene Anal. Tech. 1986; 3: 90-91Crossref Scopus (14) Google Scholar) and subcloned into pBluescript II KS for restriction enzyme mapping and DNA sequencing. A region of the HISBT gene encompassing the point mutation was amplified by PCR using flanking oligonucleotides and subcloned into a pT7 Blue T vector. The sense oligonucleotide primer 5′-ACACGCAGCTATGTTCCACCATCGT-3′ corresponded to HISBT nucleotides 915-939 (amino acids 266-274); the antisense primer 5′-TGAAATGGGATTGGCATGATTCCT-3′ corresponded to flanking intron sequence. The amplification was carried out with 100 ng of genomic DNA for 35 cycles using an annealing temperature of 65°C. The 150-nucleotide product was resolved on a 1.5% agarose gel and isolated for subcloning. For each subject, 8-13 individual clones harboring inserts were subjected to dideoxy nucleotide sequencing using pT7 Blue-specific primers. As a control for contamination, parallel reactions were performed in the absence of genomic DNA. After an initial denaturation step at 100°C for 5 min, PCR amplification was performed with 100 ng of genomic DNA for 30 cycles using an annealing temperature of 68°C. The reaction buffer contained 1.75 mM MgCl2, 50 μM of each dNTP, 1.75 μM of each oligonucleotide, and 1.7 μM [α-32P]dCTP (3000 Ci/mmol) in a volume of 20 μl(16Orita M. Suzuki Y. Hayashi K. Genomics. 1989; 5: 874-879Crossref PubMed Scopus (3316) Google Scholar). The antisense primer 5′-GCGAGCTGGAAAATGCTGTAGATGA-3′ corresponded to HISBT nucleotides 1014-990 (amino acids 298-291); the sense primer 5′-CATGTGCTCTCTTTAACATCTTCTT-3′ corresponded to the flanking intron sequence. Following the PCR, each sample was diluted 1:10 with Stop solution (90% formamide, 25 mM EDTA, 0.02% bromphenol blue, 0.02% xylene cyanol) and boiled for 5 min prior to resolving on a 10% acrylamide, 10% glycerol, 2 × TBE (180 mM Tris base, 180 mM boric acid, and 4 mM EDTA) gel at room temperature under constant voltage (350 V) for 20 h. The gel was dried and exposed to film with an intensifying screen at -70°C for 4 h. A mock reaction omitting genomic DNA was run in parallel as a control for contamination. To assess the surface expression of the wild-type and mutant HISBT proteins, cell surface biotinylation was performed(17Lantz L.M. Holmes K.L. BioTechniques. 1995; 18: 58-60Google Scholar). Approximately 72 h after transfection with human wild-type ISBT, human mutant ISBT, or β-gal expression plasmid, COS cells were trypsinized, washed with PBS, and resuspended at a density of 1.2 × 106 cells/ml in PBS containing 1 mM sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin; Pierce). The cells were incubated for 30 min at 20°C, washed 3 times with PBS containing 50 mM glycine, and counted. Aliquots of 6 × 105 cells were lysed in buffer B by repeated aspiration through a 25-gauge needle. After a 20-min incubation on ice, samples were centrifuged at 10,000 × g for 15 min. The supernatants were incubated with 50 μl of protein A-agarose for 12 h at 4°C prior to immunoprecipitation to reduce nonspecific binding to the resin. After centrifuging the samples for 2 min at 10,000 × g, the supernatants were removed and incubated with 3 μg of rabbit anti-ISBT peptide antibody plus 60 μl of protein A-agarose for 3 h at 4°C. As a control for specificity, parallel reactions were incubated with 3 μg of anti-ISBT peptide antibody in the presence of 20 μg of peptide antigen. Immunoprecipitates were washed 4 times with buffer B, once with TBS-C, and resuspended in 125 μl of buffer A. An aliquot corresponding to the indicated number of cells was prepared for SDS-PAGE by diluting with three volumes of Laemmli sample buffer and boiling for 6 min. Proteins were separated by SDS-PAGE on a 10% acrylamide gel, transferred to nitrocellulose, and detected with horseradish peroxidase-conjugated streptavidin (Amersham Corp.). Biotinylated proteins were visualized using ECL. A human λgt10 cDNA library was constructed using ileal tissue resected from a patient diagnosed with Crohn's disease. After screening 1.5 × 106 plaques with a PCR-derived human ISBT cDNA probe, seven positive cDNA clones were identified. Restriction enzyme mapping revealed that six of the seven clones encoded partial cDNAs. The seventh clone, HISBT(m), encompassed the entire predicted coding sequence of the human ileal Na+/bile acid cotransporter and was sequenced on both strands. The HISBT(m) clone was 1490 base pairs in length and encoded a 118-nucleotide 5′-untranslated region, a 1047-nucleotide coding sequence, and a 325-nucleotide 3′-untranslated region. To compare the bile acid transport properties of the human ISBT cDNA to the previously isolated hamster ISBT(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar), the HISBT(m) clone was inserted into the expression vector pCMV5. After transfection of the hamster ISBT into COS cells, [3H]taurocholate uptake was stimulated almost 600-fold over the mock-transfected background (Fig. 1). Surprisingly, the [3H]taurocholate uptake activity of parallel dishes of HISBT(m)-transfected COS cells was only slightly higher than background. To confirm that the HISBT protein was synthesized in HISBT(m)-transfected COS cells, a pulse-labeling and immunoprecipitation experiment was performed. As shown in Fig. 2, equivalent amounts of hamster ISBT (43 kDa; lanes 1 and 2) and human ISBT (40 kDa; lanes 6 and 7) are synthesized in the ISBT- and HISBT(m)-transfected COS cells. Since both the hamster and human ISBT cDNAs encode 348-amino acid proteins with predicted molecular masses of 38 kDa, the differences in their apparent molecular masses may be due to posttranslational modifications. The hamster ISBT encodes three potential N-linked glycosylation sites(5Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar), whereas the human ISBT encodes only two. The N-linked glycosylation of the hamster and human ISBTs synthesized in transfected COS cells was examined by endoglycosidase Hf and peptide:N-glycosidase F-digestion of immunoprecipitated cell extracts. After removal of N-linked glycosylation with endoglycosidase Hf or peptide:N-glycosidase F, the hamster and human ISBTs comigrated with an identical apparent molecular mass of approximately 35 kDa (Fig. 2; lanes 3, 5, 8, and 10). The migration of the deglycosylated and glycosylated forms of ISBT are indicated by the arrows in Fig. 2. This change in apparent molecular weight is consistent with the addition of two N-linked carbohydrate chains to the hamster ISBT and one N-linked chain to the human. In addition to the 35 and 40/43 kDa bands, a 29 kDa band was also observed in cell extracts from hamster or human ISBT-transfected COS cells. The origin of this product is unclear. The 29-kDa protein is not present in mock-transfected cells (see Fig. 5) and may be generated by proteolysis of the ISBT protein.Figure 5:Immunoblotting of ileal Na+/bile acid cotransporter cDNA construct-transfected COS cells. COS cells were transfected with the indicated plasmid as described in the legend to Fig. 4. 72 h after transfection, the cells were lysed in buffer A and processed for SDS-PAGE on 10% acrylamide gels and immunoblotting. Approximately 15 μg of cell protein was electrophoresed except for HISBT(m) and HAH where 6 μg was analyzed (lanes 3, 8, and 10).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The pulse labeling studies indicated that the lack of taurocholate uptake activity in the HISBT(m)-transfected cells was not due to a defect in HISBT protein synthesis. Other possible explainations for the absence of activity include a block in HISBT protein trafficking to the plasma membrane or a defect in the transport mechanism itself. To identify the location of the apparent defect in the HISBT(m) clone, chimeras between the hamster ISBT and HISBT(m) cDNAs were constructed by taking advantage of several conserved restriction enzyme sites (Fig. 3). The first set of chimeras utilized the HincII site at codon 153 that divided the ISBT cDNA roughly in half. After transfection into COS cells, the chimera encoding hamster ISBT amino acids 154-348 (pHA) exhibited [3H]taurocholate uptake activity similar to the wild-type hamster ISBT, while the chimera encoding human ISBT amino acids 154-348 (pAH) expressed approximately 20-fold less activity (Fig. 4A). This analysis indicated that the defect in HISBT(m) was located in the carboxyl-terminal half of the protein. To further localize the defect between amino acids 154 and 348, a second set of chimeras, pHHA and pHAH, were generated using the conserved BanI site at codon 254 (Fig. 3). The [3H]taurocholate uptake activity in the hamster ISBT and pHHA-transfected COS cells were similar, whereas the taurocholate uptake activity of the pHAH-transfected cells was significantly reduced (Fig. 4B). These results localized the HISBT(m) defect to the region between amino acids 255 and 348. While the pulse-labeling study in Fig. 2 indicated that similar amounts of the hamster ISBT and HISBT(m) protein were synthesized, that study did not provide a measure of the steady-state protein mass. To determine if differences in ISBT protein levels accounted for the reduced taurocholate uptake activity, immunoblotting was performed with the COS cell extracts from Fig. 4. In agreement with the pulse-labeling studies in Fig. 2, immunoblotting analysis of transfected COS cell extracts detected proteins of 43 and 40 kDa for the hamster (Fig. 5, lanes 2 and 7) and human ISBT proteins (Fig. 5, lanes 3 and 8), respectively. In contrast to the dramatic differences in taurocholate uptake activity (Fig. 4), similar amounts of ISBT protein were detected in the hamster and HISBT(m)-transfected COS cells (Fig. 5). Additional higher molecular weight bands were also detected in the hamster and human ISBT-transfected cells but not in the mock-transfected (β-gal) cells. These bands may represent the apparent aggregation products described previously for the rat ISBT(6Shneider B.L. Dawson P.A. Christie D.-M. Hardikar W. Wong M.H. Suchy F.J. J. Clin. Invest. 1995; 95: 745-754Crossref PubMed Google Scholar). Immunoblotting was also performed on cell extracts from the ISBT chimera construct-transfected COS cells in Fig. 4. By immunoblotting, similar amounts of ISBT protein were detected in the pHA and pAH-transfected cells and the pHHA and pHAH-transfected cells. In addition, the different apparent molecular weights of the various chimeric ISBT proteins agreed with the predicted human or hamster glycosylation pattern (Fig. 5). These studies indicate that the lack of taurocholate activity in the HISBT(m) and ISBT chimera-transfected COS cells was not due to decreased ISBT protein accumulation. In the region between amino acids 255 and 348, the hamster ISBT and HISBT(m) differ at 23 positions. Examination of the sequence revealed that many of the changes were conservative and present in the putative cytoplasmic tail region. To determine which of these changes may be responsible for the lack of activity, a genomic clone (λHG8) was isolated that encoded the 3′ end of the HISBT gene. Amino acids 255-348 are encoded by two exons interrupted by a 2.8-kilobase intron at codon 307. Comparison of the genomic sequence of these exons to the HISBT(m) cDNA sequence revealed only a single C to T transition at nucleotide position 985 in the cDNA clone (Fig. 6). This transition resulted in a single amino acid change; the genomic λHG8 encoded a proline at codon 290 (CCG), whereas the HISBT(m) cDNA clone encoded a serine (TCG). Proline 290 lies near the extracellular face of the transporter in the seventh predicted transmembrane domain (Fig. 7). To determine if this point mutation represented a random cloning artifact, the remaining six HISBT cDNA clones were analyzed. Four of the clones encompassed this region and were sequenced. Three of the clones (λH13, λH17, and λH22) encoded proline, and one clone (λH19) encoded serine at codon 290 (data not shown).Figure 7:Proposed membrane topology of the human ileal Na+/bile acid and location of proline to serine substitution. The topology was predicted using Kyte-Doolittle hydropathy analysis over a sliding window of 11 amino acids(34Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16899) Google Scholar). The orientation of the first transmembrane domain was assigned by analysis of the flanking positively charged amino acids as described by von Heijne(35Sipos L. Von Heijne G. Eur. J. Biochem. 1993; 213: 1333-1340Crossref PubMed Scopus (252) Google Scholar). Transmembrane domains appear as boxes; glycosylation at Asn-10 is indicated by the branched symbol. The proline to serine substitution at position 290 (shaded box) is predicted to lie near the extracellular face of the transporter in the seventh transmembrane domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The identification of multiple independent cDNA clones encoding serine at codon 290 suggested that the library was derived from a patient who is heterozygous at this nucleotide position. To address this question, genomic DNA was isolated from ileal tissue and white blood cells obtained from the patient. The genomic DNA region flanking the point mutation was amplified by PCR and subcloned. 13 independent clones were sequenced; five clones encoded serine, and eight clones encoded proline at position 290. A similar analysis of lymphocyte genomic DNA from the proband's parents indicated that the mother was also heterozygous at this nucleotide position. To directly assay for the presence of the mutation in the proband's family, SSCP was used. The pedigree and results from the SSCP analysis are shown in Fig. 8. Analysis o" @default.
• W2013019095 created "2016-06-24" @default.
• W2013019095 creator A5042075065 @default.
• W2013019095 creator A5053795637 @default.
• W2013019095 creator A5055242592 @default.
• W2013019095 date "1995-11-01" @default.
• W2013019095 modified "2023-10-04" @default.
• W2013019095 title "Identification of a Mutation in the Ileal Sodium-dependent Bile Acid Transporter Gene That Abolishes Transport Activity" @default.
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