(Received for publication, June 14, 1995)
From the
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(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) (
)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. (
)In this paper, we
describe the identification and characterization of a naturally
occurring dysfunctional mutation in the HISBT gene.
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.
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%
-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).
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, [H]taurocholate
uptake was stimulated almost 600-fold over the mock-transfected
background (Fig. 1). Surprisingly, the
[
H]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
10
COS cells/60-mm dish were
plated in medium A. On day 1, duplicate dishes of cells were
transfected with 2 µg of pCMV-
-galactosidase (
gal), 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
[
H]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.
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 Tran
S-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
10
COS cells/100-mm dish
were plated in medium A. On day 1, cells were transfected with 5 µg
of pCMV-
-galactosidase (
gal), 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
10
cells/well. On day 4, the cells were incubated
in medium B containing 5 µM
[
H]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.
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 (-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.
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.
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.
Figure 9:
[H]Taurocholate
uptake activity in HISBT and HISBT(P290S)-transfected COS cells. COS
cells were plated and transfected with 5 µg of
pCMV-
-galactosidase (
gal;
), pCMV5-Human
mutant ileal Na
/bile acid cotransporter (HISBT(P290S);
), or pCMV5-Human wild-type ileal Na
/bile acid
cotransporter (HISBT;
), as described in the legend to Fig. 4. On day 4, the cells were incubated in medium B
supplemented with the indicated concentration of
[
H]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
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.''
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
10
) 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
10
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).
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. ()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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U10417[GenBank].