Identification of a Mutation in the Ileal Sodium-dependent Bile Acid Transporter Gene That Abolishes Transport Activity

Melissa H Wong,Peter Oelkers,Paul A Dawson

doi:10.1074/jbc.270.45.27228

Melissa H Wong, Peter Oelkers + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.45.27228

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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.

Highlights

The enterohepatic circulation of bile acids is maintained at the cellular level by a series of membrane transporters and binding proteins [1]
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
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

Summary

Introduction

The enterohepatic circulation of bile acids is maintained at the cellular level by a series of membrane transporters and binding proteins [1]. 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. 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.

Results

Conclusion