Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11
Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11
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DOI：
10.1002/hep.20627
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发表时间：
2005-04-01
期刊：
影响因子：
13.5
通讯作者：
Sugiyama, Y
中科院分区：
文献类型：
--
作者：
Hayashi, H;Takada, T;Sugiyama, Y
进行性家族性肝内胆汁淤积症2型(PFIC2)是由胆盐输出泵(BSEP/ABCB11)基因突变引起的。然而，欧洲患者中常见的两个突变(E297G和D482G)功能缺陷的机制尚未确定。在本研究中，我们分别检测了这两个突变体在人胚胎肾293和Madin-Darby犬肾II细胞中的转运活性和细胞定位。通过定点突变将E297G和D482G突变引入人BSEP基因，导致BSEP表达水平显著降低，这与膜转运受损有关。大多数D482G BSEP和部分E297G BSEP只进行了核心糖基化，似乎主要定位于内质网。MG132对蛋白酶体功能的抑制导致两个突变体的核心糖基化形式在细胞内积累。相反，牛磺胆酸盐和甘胆酸与从互补DNA转基因细胞分离的膜小泡的转运研究表明，这两个突变本身并没有显著影响BSEP的转运功能。总之，E297G和D482G突变导致膜转运功能受损，而这些突变的转运功能基本保持不变。
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. However, the mechanisms for the deficiency in the function of two mutations (E297G and D482G), which are frequently found in European patients, have not yet been identified. In the present study, we examined the transport activity and cellular localization of these two mutants in human embryonic kidney 293 and Madin-Darby canine kidney II cells, respectively. Introduction of E297G and D482G mutations into the human BSEP gene by site-directed mutagenesis resulted in a significant reduction in the BSEP expression level, which was associated with impaired membrane trafficking. Most of the D482G BSEP and some of the E297G BSEP underwent only core glycosylation and appeared to be predominantly located in the endoplasmic reticulum. The inhibition of proteasome function by MG132 resulted in the cellular accumulation of the core glycosylation form of the two mutants. In contrast, transport studies for taurocholate and glycocholate with membrane vesicles isolated from complementary DNA-transfected cells indicated that both mutations did not significantly affect the transport function of BSEP per se. In conclusion, E297G and D482G mutations result in impaired membrane trafficking whereas the transport functions of these mutants remain largely unchanged.