Abstract
Multidrug resistance 3 (MDR3), encoded by the ATP-binding cassette, subfamily B, member 4 gene (ABCB4), localizes to the canalicular membrane of hepatocytes and translocates phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare hepatic disease caused by genetic mutations of ABCB4. In this study, we characterized 8 ABCB4 mutations found in PFIC3 patients, using in vitro molecular assays. First, we examined the transport activity of each mutant by measuring its ATPase activity using paclitaxel or phosphatidylcholine. Then, the pathogenic mechanisms by which these mutations affect MDR3 were examined through immunoblotting, cell surface biotinylation, and immunofluorescence. As a result, three ABCB4 mutants showed significantly reduced transport activity. Among these mutants, one mutation A364V, located in intracellular domains, markedly decreased MDR3 expression on the plasma membrane, while the others did not affect the expression. The expression of MDR3 on the plasma membrane and transport activity of A364V was rescued by a pharmacological chaperone, cyclosporin A. Our study provides the molecular mechanisms of ABCB4 mutations and may contribute to the understanding of PFIC3 pathogenesis and the development of a mutation-specific targeted treatment for PFIC3.
Highlights
Of the ABCB4 promoter variants and found that two common ABCB4 promoter haplotypes led to significantly decreased promoter activity
Our study provides the molecular pathogenic mechanisms of ABCB4 mutations, and our findings may contribute to the development of a new drug for the treatment of Progressive familial intrahepatic cholestasis type 3 (PFIC3) or other multidrug resistance 3 (MDR3)-deficiency related diseases
Among the 29 ABCB4 mutations identified by Degiorgio et al.[7], we excluded nonsense mutations and mutations that were found in other MDR3-deficient phenotypes or those that affect amino acid residues, which are conserved in other ABC transporters, in particular, the MDR1 transporter, as these had already been investigated[20,21,22]
Summary
Of the ABCB4 promoter variants and found that two common ABCB4 promoter haplotypes led to significantly decreased promoter activity. To evaluate the effect of these mutations on protein function, they mapped each variant on the predicted tertiary structure of MDR3, and found that 10 out of 29 mutations were located on transmembrane domains (TMs). TM 7, which may be required for the translocation of phosphatidylcholine, was most commonly affected. These studies did not perform a functional characterization of mutations. We selected 8 missense mutations of ABCB4 that were first reported by Degiorgio et al.[7] and investigated the effect of each mutant on MDR3 transport activity or expression by using a range of in vitro assays such as a membrane vesicular adenosine triphosphatase (ATPase) assay, immunoblotting, surface biotinylation assay, and immunofluorescence. Our study provides the molecular pathogenic mechanisms of ABCB4 mutations, and our findings may contribute to the development of a new drug for the treatment of PFIC3 or other MDR3-deficiency related diseases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
