Functional expression of the canalicular bile salt export pump of human liver
Functional expression of the canalicular bile salt export pump of human liver
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DOI：
10.1053/gast.2002.36587
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发表时间：
2002-11-01
期刊：
影响因子：
29.4
通讯作者：
Meier, PJ
中科院分区：
文献类型：
--
作者：
Noé, J;Stieger, B;Meier, PJ
背景与目的：肝胆汁盐分泌是脊椎动物肝脏的基本功能。大鼠和小鼠胆盐输出泵（Bsep）是三磷酸腺苷（ATP）依赖的胆盐转运体。人类BSEP突变被确定为进行性家族性肝内胆汁淤积症2型的原因。BSEP蛋白与其大鼠和小鼠直系同源物高度相同，尚未对其功能进行表征; BSEP突变对其功能的影响也尚未研究。因此，本研究的目的是对人BSEP进行功能表征。方法：从人肝组织中提取BSEP的互补DNA，用杆状病毒系统在Sf 9细胞中表达。用表达BSEP的Sf 9细胞囊泡和快速过滤试验进行ATP依赖性胆盐转运试验。结果：克隆人BSEP需要在其编码区内的细菌隐蔽启动子基序失活。在Sf 9细胞中表达的BSEP以ATP依赖性方式转运不同的胆汁盐，米氏常数如下：牛磺胆酸盐，7.9 +/- 2.1 mumol/L;甘氨胆酸盐，11.1 +/- 3.3 mumol/L;牛磺鹅脱氧胆酸盐，4.8 +/- 1.7 mumol/L;牛磺鹅脱氧胆酸盐，11.9 +/- 1.8 mumol/L。胆盐的固有清除率顺序为牛磺鹅去氧胆酸盐>牛磺胆酸盐>牛磺熊去氧胆酸盐>甘氨胆酸盐。结论：本研究将人BSEP表征为ATP依赖性胆盐输出泵，其转运特性与其大鼠和小鼠直系同源物相似。BSEP在Sf 9细胞中的表达将能够对人BSEP基因突变的后果进行功能表征。
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 mumol/L; glycocholate, 11.1 +/- 3.3 mumol/L; taurochenodeoxycholate, 4.8 +/- 1.7 mumol/L; taurochenodeoxycholate, 11.9 +/- 1.8 mumol/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.