Gastroenterology

Gastroenterology

Volume 123, Issue 5, November 2002, Pages 1659-1666
Gastroenterology

Basic–Liver, Pancreas, and Biliary Tract
Functional expression of the canalicular bile salt export pump of human liver*,**

https://doi.org/10.1053/gast.2002.36587Get rights and content

Abstract

Background & Aims: Hepatic bile salt secretion is an essential function of vertebrate liver. Rat and mouse bile salt export pump (Bsep) are adenosine triphosphate (ATP)-dependent bile salt transporters. Mutations in human BSEP were identified as the cause of progressive familial intrahepatic cholestasis type 2. BSEP protein is highly identical with its rat and mouse orthologs and has not yet been functionally characterized; the effect of BSEP mutations on its function has also not been studied. Therefore, the aim of this study was to functionally characterize human BSEP. Methods: Complementary DNA for BSEP was isolated from human liver and expressed with the baculovirus system in Sf9 cells. ATP-dependent bile salt transport assays were performed with Sf9 cell vesicles expressing BSEP and a rapid filtration assay. Results: Cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif within its coding region. BSEP expressed in Sf9 cells transports different bile salts in an ATP-dependent manner with Michaelis constant values as follows: taurocholate, 7.9 ± 2.1 μmol/L; glycocholate, 11.1 ± 3.3 μmol/L; taurochenodeoxycholate, 4.8 ± 1.7 μmol/L; tauroursodeoxycholate, 11.9 ± 1.8 μmol/L. The rank order of the intrinsic clearance of bile salts was taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate. Conclusions: This study characterizes human BSEP as an ATP-dependent bile salt export pump with transport properties similar to its rat and mouse orthologs. Expression of BSEP in Sf9 cells will enable functional characterization of the consequences of mutations in the human BSEP gene.

GASTROENTEROLOGY 2002;123:1659-1666

Section snippets

Materials

[3H]Taurocholic acid (2 Ci/mmol) and [3H]cholic acid (27.5 Ci/mmol) were obtained from NEN Life Science Products (Boston, MA). [3H]-labeled glycocholic, taurochenodeoxycholic, and tauroursodeoxycholic acids of high specific activity (14–30 Ci/mmol) were prepared as described previously.14, 15, 16 All other chemicals and reagents were of analytic grade and were readily available from commercial sources.

Isolation of human BSEP

Cloning of human BSEP was started with the amplification of a 450–base pair complementary DNA

Results

Isolation of BSEP from human liver required repeated screening of a human liver cDNA library with successive isolation of several complementary cDNA fragments, which then could be ligated together to yield the full-length human BSEP cDNA. Our experience supports previous experiences that full-length clones of BSEP are absent from human cDNA libraries.12 The reason for the failure to generate full-length BSEP cDNA clones could be explained by the presence of cryptic E. coli promoter sequence

Discussion

The present study shows the isolation and functional characterization of the BSEP of human liver. In contrast to its rat and mouse orthologs, the successful cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif (Figure 1). In addition, its functional expression in Sf9 cells was dependent on the back-mutation of some, most probably artificially introduced, amino acid replacements to the published amino acid sequences (Table 1). Thus, the only “abnormal” amino acid

Acknowledgements

The authors thank Monika Gersbach for excellent technical assistance.

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    *

    Address requests for reprints to: Peter J. Meier-Abt, M.D., Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland. e-mail: [email protected]; fax: (41) 1-255-4411.

    **

    Supported by the Swiss National Science Foundation (grant 31-64140.00).

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