©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Mutation in the Ileal Sodium-dependent Bile Acid Transporter Gene That Abolishes Transport Activity (*)

(Received for publication, June 14, 1995)

Melissa H. Wong(§)(¶) Peter Oelkers(§)(**) Paul A. Dawson (§§)

From the Department of Internal Medicine, Division of Gastroenterology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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.


INTRODUCTION

The enterohepatic circulation of bile acids is maintained at the cellular level by a series of membrane transporters and binding proteins(1) . 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 (2) and secreted into the portal circulation by a sodium-independent anion-exchange mechanism(3) . A number of the transport proteins and membrane carriers that participate in the enterohepatic circulation of bile acids have recently been identified(4) . By expression and hybridization techniques, the hamster (5) and rat (6) ileal Na/bile acid cotransporter (ISBT) (^1)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(5, 6) . 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. (^2)In this paper, we describe the identification and characterization of a naturally occurring dysfunctional mutation in the HISBT gene.


EXPERIMENTAL PROCEDURES

General Methods

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 N(2) and stored at -70 °C. Total cellular RNA was isolated by the guanidinium isothiocyanate/CsCl centrifugation procedure(7) . 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(8) . [^3H]Taurocholic acid (2.0-2.6 Ci/mmol) was purchased from DuPont NEN. TranS-label (a mixture of [S]Met and [S]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(5) .

Synthesis of a Human ISBT Probe and gt10 cDNA Library Screening

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(9) . 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(7) .

Construction and Screening of a gt10 cDNA Library

A gt10 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 gt10 DNA. After in vitro packaging using a Gigapack II Gold Cloning kit (Stratagene; La Jolla, CA), the phage (1.5 times 10^6 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 times SSC, 50 mM sodium phosphate, pH 6.8, 2 times Denhardt's solution, 100 µg/ml denatured salmon sperm DNA, and 1 times 10^6 cpm/ml of radiolabeled probe. After hybridization, filters were washed in 0.2 times SSC, 0.1% (w/v) SDS at 65 °C for 30 min. Positive clones were plaque purified; plate lysate DNA was isolated (10) and subcloned into pBluescript KS II for restriction mapping and DNA sequencing.

Construction of Hamster/Human Chimeras and Wild-type HISBT

The dysfunctional HISBT cDNA, indicated as HISBT(m), was subcloned into EcoRI-digested pCMV5 (11) 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 H13 SacI-EcoRI (nucleotides 967-1253; amino acids 284-348). The constructs were verified by dideoxy nucleotide sequencing.


Figure 3: Schematic representation of chimeras constructed between the human mutant and hamster ileal Nabile acid cotransporters. Two sets of hamster/human mutant ISBT cDNA chimeras were constructed. A, in the first set, a conserved HincII site at codon 153 was used to create the human/hamster (HA) and hamster/human (AH) ISBT chimeras. B, in the second set of chimeras, a conserved BanI site at codon 254 in the third extracellular loop of the protein was used to construct the human/human/hamster (HHA) and human/hamster/human (HAH) chimeras. The barrels represent putative transmembrane domains; the lines represent the cytoplasmic and extracellular regions; the Y symbol indicates N-linked glycosylation sites. The locations of restriction sites used to construct the chimeras are labeled and indicated by the boldface arrows. Human sequence is represented in white and the hamster sequence in black.



Antibody Preparation and Immunoblotting

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(12) . 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 (13) . The IgG was then purified from rabbit serum by protein A-Sepharose chromatography(14) .

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(15) . 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% beta-mercaptoethanol), boiled for 5 min, and resolved by SDS-PAGE on 10% acrylamide gels. Immunoblotting was performed as described previously (14) 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).

Expression and Deglycosylation of ISBT and HISBT(m)

On day 0, 5 times 10^5 COS cells/60-mm dish were plated in medium A. On day 1, duplicate dishes of cells were transfected with 2 µg of beta-galactosidase (beta-gal)(5) , hamster ISBT(5) , or HISBT(m) expression plasmid by the DEAE-dextran method(5) . 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 TranS-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 times g. Cell pellets were lysed in buffer B (25 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM CaCl(2), 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 times 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 CaCl(2) (TBS-C); RepliGen, Cambridge, MA) for 12 h at 4 °C. Immune complexes were recovered by centrifugation at 10,000 times 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% beta-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 times 10^3 units of endoglycosidase Hf (New England Biolabs; Beverly, MA) in 50 mM sodium citrate, pH 5.5, 0.5% SDS, and 1% beta-mercaptoethanol. For peptide:N-glycosidase F digestion, aliquots of the eluted protein were incubated for 1 h at 37 °C with 1 times 10^3 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% beta-mercaptoethanol. The samples were then brought to 3% SDS, 5% glycerol, 30 mM Tris-HCl, pH 6.8, 10 mM EDTA, and 2.5% beta-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.

Isolation of a Human ISBT Genomic Clone

A human placental genomic DNA library in EMBL3 (Catalog no. HL1067j; Clontech; Palo Alto, CA) was screened using standard methods (7) with P-labeled probes derived from the coding region of the human ISBT cDNA. After screening a total of 2 times 10^5 bacteriophage, one positive clone was identified and plaque purified. Plate lysate DNA was isolated (10) and subcloned into pBluescript II KS for restriction enzyme mapping and DNA sequencing.

Sequence Determinations

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.

Single-stranded Conformational Analysis (SSCP)

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 MgCl(2), 50 µM of each dNTP, 1.75 µM of each oligonucleotide, and 1.7 µM [alpha-P]dCTP (3000 Ci/mmol) in a volume of 20 µl(16) . 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 times 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.

Cell Surface Biotinylation

To assess the surface expression of the wild-type and mutant HISBT proteins, cell surface biotinylation was performed(17) . Approximately 72 h after transfection with human wild-type ISBT, human mutant ISBT, or beta-gal expression plasmid, COS cells were trypsinized, washed with PBS, and resuspended at a density of 1.2 times 10^6 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 times 10^5 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 times 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 times 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.


RESULTS

Cloning and Expression of the Human ISBT cDNA

A human gt10 cDNA library was constructed using ileal tissue resected from a patient diagnosed with Crohn's disease. After screening 1.5 times 10^6 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(5) , 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.


Figure 1: Taurocholate uptake in hamster and human ileal Nabile acid cotransporter transfected COS cells. On day 0, 5 times 10^5 COS cells/60-mm dish were plated in medium A. On day 1, duplicate dishes of cells were transfected with 2 µg of pCMV-beta-galactosidase (betagal), pCMX-hamster ileal Na/bile acid cotransporter (ISBT), or pCMV5-human mutant ileal Na/bile acid cotransporter (HISBT(m)). On day 4, the cells were incubated in Dulbecco's modified Eagle's medium containing 4500 mg/liter D-glucose, 0.2% fatty acid-free bovine serum albumin, 100 units/ml penicillin, 100 µg/ml streptomycin, and supplemented with 5 µM [^3H]taurocholate (2.0 Ci/mmol). After 15 min at 37 °C, the medium was removed, and cell monolayers were washed and processed to determine cell-associated protein and radioactivity.



HISBT Synthesis and Glycosylation in Transfected COS Cells

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(5) , 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 2: Expression and endoglycosidase-sensitivity of the hamster and human ileal Nabile acid cotransporters in transfected COS cells. Cells were transfected with the indicated plasmids, labeled for 1 h with TranS-label, and chased for 1 h in the absence of radioactivity. Cell detergent lysates were subjected to immunoprecipitation with anti-ISBT peptide antibody. Immunoprecipitates were resuspended and incubated for 1 h in the presence of endoglycosidase Hf (Endo H, lanes 3 and 8) or PNgase (lanes 5 and 10). Control reactions were incubated in parallel for 1 h in the absence of endoglycosidase H (lanes 2 and 7) or peptide:N-glycosidase (lanes 4 and 9). The immunoprecipitates and glycosidase reactions were analyzed by SDS-PAGE on 10% acrylamide gels and fluorography. The arrows indicate the migration of the glycosylated and deglycosylated ISBTs.




Figure 5: Immunoblotting of ileal Nabile 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).




Figure 4: Taurocholate uptake activity of ileal Nabile acid cotransporter cDNA construct-transfected COS cells. On day 0, 1.50 times 10^6 COS cells/100-mm dish were plated in medium A. On day 1, cells were transfected with 5 µg of pCMV-beta-galactosidase (betagal), pCMX-hamster ileal Na/bile acid cotransporter (ISBT), pCMV5-Human mutant ileal Na/bile acid cotransporter (HISBT(m)), and the indicated chimera plasmids by the DEAE-dextran method. On day 2, each group of transfected cells was trypsinized, pooled, and replated in 24-well culture plates at 7 times 10^4 cells/well. On day 4, the cells were incubated in medium B containing 5 µM [^3H]taurocholate (0.4 Ci/mmol) for 10 min at 37 °C. The medium was removed, and cell monolayers were washed and processed to determine cell-associated protein and radioactivity. Each value represents the mean of duplicate wells. The results for panel A and panel B were obtained from separate transfection experiments performed on different days.



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.

Expression of Human-Hamster ISBT Chimeras in Transfected COS Cells

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. 2indicated 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 (beta-gal) cells. These bands may represent the apparent aggregation products described previously for the rat ISBT(6) . 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.

Sequence Analysis of an HISBT Genomic Clone and HISBT cDNA Clones

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 (HG8) 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 HG8 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 (H13, H17, and H22) encoded proline, and one clone (H19) encoded serine at codon 290 (data not shown).


Figure 6: Nucleotide sequence of dysfunctional ileal Nabile acid cotransporter. Sequence of the dysfunctional HISBT(m) cDNA (right) is compared with the wild-type sequence (left) obtained from genomic clone HG8. A single C to T nucleotide substitution changes the proline at amino acid position 290 to a serine. The nucleotide sequence has been submitted to the GenBank/EMBL Data Bank with accession number U10417.




Figure 7: Proposed membrane topology of the human ileal Nabile 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(34) . The orientation of the first transmembrane domain was assigned by analysis of the flanking positively charged amino acids as described by von Heijne(35) . 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.



Sequencing and SSCP Analysis of the HISBT(P290S) Mutation

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 of genomic DNA from the father (I.1) and brother (II.3) revealed single-stranded DNA conformers representing the two wild-type strands, indicating that they are homozygous for a proline at position 290. An additional single-stranded DNA conformer representing the mutant allele was readily detected in the proband (II.1), the proband's brother (II.2), and the proband's mother (I.2).


Figure 8: Pedigree and SSCP analysis of the HISBT(P290S) family. Individuals with the mutation are denoted by half-shaded symbols; the proband is indicated by the arrow. Genomic DNA from individual family members was prepared from lymphocytes and used for SSCP analysis as described under ``Experimental Procedures.'' The migration of the wild-type (wt) and mutant conformers is indicated.



Taurocholate Uptake Activity of the Wild-type HISBT and HISBT(P290S) in Transfected COS Cells

HISBT expression plasmids encoding a proline or a serine at position 290 were transfected into COS cells to confirm that the serine substitution at position 290 was responsible for lack of taurocholate uptake activity. Human ISBT protein expression and [^3H]taurocholate uptake activity were then assayed. By immunoblotting, both the wild type and mutant HISBT-transfected COS cells expressed similar amounts of protein (Fig. 9, inset); however, a dramatic difference in [^3H]taurocholate uptake activity was observed. The [^3H]taurocholate uptake activity of the HISBT(P290S)-transfected COS cells was indistinguishable from background, while the taurocholate uptake activity of the wild-type HISBT-transfected cells was saturable with a V(max) of approximately 96 pmol min mg of cell protein (Fig. 9) and a K(m) of 17 µM.


Figure 9: [^3H]Taurocholate uptake activity in HISBT and HISBT(P290S)-transfected COS cells. COS cells were plated and transfected with 5 µg of pCMV-beta-galactosidase (betagal; up triangle), pCMV5-Human mutant ileal Na/bile acid cotransporter (HISBT(P290S); circle), or pCMV5-Human wild-type ileal Na/bile acid cotransporter (HISBT; box), as described in the legend to Fig. 4. On day 4, the cells were incubated in medium B supplemented with the indicated concentration of [^3H]taurocholate (0.4 Ci/mmol) for 10 min at 37 °C. Cell monolayers were washed and processed to determine cell-associated protein and radioactivity. Each value represents the mean of duplicate wells. Eadie-Hofstee analysis of the wild-type HISBT data revealed a K for taurocholate uptake of 17 µm and a V(max) of 96 pmol min mg of cell protein. Inset, 20 µg of cell extract from a parallel aliquot of transfected COS cells was analyzed by immunoblotting as described under ``Experimental Procedures.''



Cell Surface Biotinylation of HISBT and HISBT(P290S) Expressed in Transfected COS Cells

The substitution of a proline for a serine at position 290 is responsible for the lack of taurocholate uptake activity in the mutant HISBT-transfected COS cells. Since proline residues are known to be important determinants of protein structure, this change may alter HISBT folding and prevent normal expression on the cell surface. The processing of N-linked carbohydrate in the Golgi apparatus, as measured by the loss of endoglycosidase H sensitivity, is often used to follow the transit of plasma membrane proteins out of the endoplasmic reticulum (18) . However, the functional hamster and human wild-type ISBTs as well as the dysfunctional HISBT(P290S) mutant remains sensitive to deglycosylation by endoglycosidase Hf (Fig. 2). Thus, this assay cannot be used to assess if the HISBT(P290S) protein has left the endoplasmic reticulum and trafficked through the Golgi apparatus.

To determine if the HISBT(P290S) mutant protein traffics to the plasma membrane, we performed cell surface biotinylation followed by immunoprecipitation with anti-ISBT antibody and streptavidin blotting. The wild-type HISBT was detected with horseradish peroxidase-conjugated streptavidin after surface labeling with NHS-LC-biotin and anti-ISBT precipitation; however, HISBT was not detected in the absence of biotinylation reagent or in the presence of competitor peptide antigen (data not shown). The cell surface expression of the wild-type HISBT and HISBT(P290S) were directly compared in transfected COS cells by surface labeling with NHS-LC-biotin. After quenching the biotinylation reagent, an aliquot of transfected COS cells was removed for immunoblotting, and the remaining cells were used for anti-ISBT immunoprecipitation and streptavidin blotting. By immunoblotting, similar amounts of HISBT and HISBT(P290S) protein were detected in cell extracts from the surface biotinylated COS cells (Fig. 10, lanes 2 and 3). Analysis of the cell surface expression by immunoprecipitation and streptavidin blotting also detected similar amounts of HISBT and HISBT(P290S) protein (Fig. 10, lanes 5 and 6). These results indicate that the proline to serine substitution in HISBT(P290S) does not interfere with trafficking to the cell surface but must interfere directly in the taurocholate transport process.


Figure 10: Immunoblotting and streptavidin detection of biotinylated HISBT and HISBT(P290S) in transfected COS cells. Detergent lysates were prepared from surface biotinylated transfected COS cells. Extract from an equivalent number of cells (1 times 10^4) was subjected to SDS-PAGE on a 10% acrylamide gel and immunoblotting with rabbit anti-ISBT antibody (1.8 µg/ml). The anti-ISBT antibody was detected using horseradish peroxidase-conjugated goat anti-rabbit antibody and visualized by ECL (lanes 1-3). The remaining cell extract was immunoprecipitated with rabbit anti-ISBT antibody. Immunoprecipitated cell extract corresponding to 7 times 10^5 cells was subjected to SDS-PAGE on a 10% acrylamide gel, transferred to nitrocellulose, and detected using horseradish peroxidase-conjugated streptavidin. The streptavidin conjugate was visualized by ECL (lanes 4-6).




DISCUSSION

We report the identification of a dysfunctional ileal Na/bile acid cotransporter isolated from a patient diagnosed with Crohn's disease. A single point mutation in the gene, substituting a serine for a proline at position 290, was responsible for the lack of taurocholate transport activity. Proline residue 290 is situated near the extracellular face of the transporter in the seventh putative transmembrane domain (Fig. 7) and is invariant in all of the liver and ileal Na/bile acid cotransporters identified to date(4-6, 9). The observation that proline residues are enriched in transporter protein transmembrane domains compared with other membrane proteins (20) suggests that prolines serve an important role in the transport process(21) . This has led to the hypothesis that prolines may be involved in the protein conformational changes that mediate solute transport across the membrane. More specifically, potential functions for transmembrane domain proline residues include 1) induction of conformational changes in transmembrane helices after cis-trans isomerization of proline-containing peptide bonds, 2) formation of the solute binding pocket due to bending of transmembrane helices, and 3) solute hydrogen bonding through the carbonyl groups in proline-containing peptide bonds (21, 22) . Interestingly, five of the seven predicted transmembrane segments of the HISBT protein contain proline residues that are conserved between the ileal and liver cotransporters(4) . The observation that HISBT(P290S) is expressed on the cell surface but fails to transport taurocholate supports the hypothesis that membrane prolines function in solute transport. Mechanistically, the mutation may act by blocking bile acid binding or by preventing a conformational change required for bile acid translocation. Additional structural and mechanistic studies will be required to determine the exact effect of the proline to serine substitution on bile acid transport.

Point mutations have also been identified in the Na/glucose cotransporter SGLT1 that underlies intestinal glucose/galactose malabsorption(23, 24) . These mutations change an aspartate residue near the intracellular face of the first transmembrane domain to an asparagine or glycine. The molecular mechanism responsible for the lack of glucose transport activity has not yet been identified. Although the Na/glucose cotransporters appear to constitute a separate family that is structurally distinct from the Na/bile acid cotransporters(25) , analysis of these transport mutations should provide insight into the general mechanism of Na/solute cotransport.

The proband in this study was treated for ileal inflammation with minor granulomatous infiltration, consistent with the diagnosis of Crohn's disease. Crohn's disease is a chronic, idiopathic, inflammatory disorder characterized by transmural granulomatous lesions (26) usually involving the ileum and/or colon(27) . Although the proline to serine mutation was initially detected in the proband, the relationship of the HISBT mutation to the patient's disorder is not clear. The mRNA for the mutant allele is clearly expressed in this patient, as demonstrated by the cDNA cloning. However analysis of the patient's family indicated that the mother and brother also carry the mutation, though neither individual has evidence of Crohn's disease or inflammatory bowel disease. In addition, genotyping of a large cohort of patients with inflammatory bowel disease indicated that the mutation is not enriched in these patients compared with the general population. (^3)However, since the susceptibility to Crohn's disease is influenced by a wide range of genetic and environmental factors(27) , these studies have not ruled out a minor role of HISBT gene defects in this disease. The possibility that the mutation may increase the susceptibility to some forms of Crohn's disease or worsen its symptoms is currently being investigated.

Previous studies have described patients with chronic diarrhea, increased fecal bile acid loss, and symptomatic response to treatment with bile acid-binding resins such as cholestyramine(28, 29) . While the etiology of this ``bile salt wasting'' was not clear, a genetic defect in intestinal active bile acid transport was suggested as a possible cause(30) . In a more recent study, an apparent defect in ileal bile acid uptake was documented in two boys with lifelong diarrhea and steatorrhea who had an ultrastructurally normal ileum (31) . While suggestive, these studies have not demonstrated that a primary genetic defect in ileal Na/bile acid cotransport is the underlying cause of the patients' bile acid malabsorption, and this hypothesis remains controversial. A recent analysis of crude membrane vesicles prepared from ileal biopsy specimens from patients with abnormal in vivo ileal reabsorption of bile acids suggested that the sodium-dependent bile acid uptake was normal(32, 33) .

In this study, we have identified and characterized a naturally occurring dysfunctional mutation in the human ileal brush-border membrane Na/bile acid cotransporter. Further study of the effect of this mutation on the transport process may provide a unique insight into the molecular mechanism of Na/bile acid cotransport. In addition, analysis of the in vivo phenotype of this mutation will provide important information about the relationship between defects in the HISBT gene, bile acid malabsorption syndromes, and bile acid and cholesterol metabolism.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant DK47987 and American Heart Association (North Carolina affiliate) Grant-in-aid NC92GS15. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U10417[GenBank].

§
Contributed equally to this work.

Recipient of Predoctoral Fellowship DK08718 from the National Institutes of Health. Present address: Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110.

**
Supported by Cardiovascular Pathology National Service Training Award HL07115 from the National Institutes of Health.

§§
American Gastroenterology Association/Janssen Pharmaceutical Research Scholar. To whom correspondence should be addressed: Dept. of Internal Medicine, Div. of Gastroenterology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 910-716-4633; Fax: 910-716-6376.

(^1)
The abbreviations used are: ISBT, ileal Na/bile acid cotransporter; HISBT, human wild-type ileal Na/bile acid cotransporter; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; HISBT(m), human mutant ileal Na/bile acid cotransporter, HISBT(P290S); PAGE, polyacrylamide gel electrophoresis; SSCP, single stranded conformation polymorphism; beta-gal, beta-galactosidase; NHS-LC-biotin, sulfosuccinimidyl-6-(biotinamido) hexanoate.

(^2)
M. H. Wong, P. Oelkers, P. N. Rao, M. J. Pettenati, and P. A. Dawson, manuscript in preparation.

(^3)
P. Oelkers, H. Yang, J. I. Rotter, and P. A. Dawson, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Rebecca Daniel for assistance with the immunoprecipitation assays and Drs. Russell Howerton and Matthew Wood for assistance with patient samples. We also thank Dr. Helen Hobbs (University of Texas Southwestern Medical Center) for assistance and advice throughout these studies. We thank Ann Craddock for excellent technical assistance and Dr. Greg Shelness for critical reading of the manuscript.


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