A missense mutation in ABCB4 gene involved in progressive familial intrahepatic cholestasis type 3 leads to a folding defect that can be rescued by low temperature

Abstract

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease characterized by early onset of cholestasis that leads to cirrhosis and liver failure before adulthood. PFIC3 may be improved by chronic administration of ursodeoxycholic acid, although in many cases liver transplantation is the only therapy. The disease is caused by mutations of the adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4) [multidrug resistance 3 (MDR3)] gene encoding a specific hepatocellular canalicular transporter involved in biliary phosphatidylcholine secretion. Several mutations have been reported; however, the effect of individual mutations has not been investigated. ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), the multidrug transporter responsible for drug resistance of cancer cells. We have studied the effect of mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4 and ABCB1. Plasmids encoding the wild-type human ABCB4 or rat ABCB1-green fluorescing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells. Expression studies showed that ABCB4 was localized at the bile canalicular membrane in HepG2 cells and at the apical surface in MDCK cells, whereas the I541F mutant was intracellular. In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticulum and cis-Golgi, and remained partially endoH-sensitive. After shifting cells to 27°C, ABCB1-I541F was expressed at the apical cell surface in a mature and active form. Similarly, ABCB4 was significantly trafficked to the membrane of bile canaliculi in HepG2 cells. Conclusion: Mutation I541F causes mislocalization of both ABCB4 and ABCB1. Intracellular retention of ABCB4-I541F can explain the disease in PFIC3 patients bearing this mutation. The observation that plasma membrane expression and activity can be rescued by low temperature opens perspectives to develop novel therapies for the treatment of PFIC3. (HEPATOLOGY 2009;49:1218-1227

Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction

“…The role of the missense mutations in the pathogenesis of ABCB4-related disease remains speculative because only in a few studies 19,23,24 their pathogenicity has been correlated with direct or indirect evidence of anomalous floppase activity.…”

Effects of Cellular, Chemical, and Pharmacological Chaperones on the Rescue of a Trafficking-defective Mutant of the ATP-binding Cassette Transporter Proteins ABCB1/ABCB4

“…Cell Culture, Transfection, and Drug Treatments-MadinDarby canine kidney (MDCK) II cells and hepatocellular carcinoma, human HepG 2 cells were grown at 37°C in Dulbecco's modified medium as reported (10). The generation of MDCK cells stably expressing GFP-ABCB1, GFP-ABCB1-I541F, ABCB4 and ABCBA-I541F has been previously described (10).…”

“…The generation of MDCK cells stably expressing GFP-ABCB1, GFP-ABCB1-I541F, ABCB4 and ABCBA-I541F has been previously described (10). Before the experiments, cells were treated overnight with 10 mM sodium butyrate to increase the cytomegalovirus promoter transcriptional activity.…”

“…Cells expressing ABCB4 were fixed with methanol/acetone (4:1, v/v) at Ϫ20°C. Incubations with primary and secondary antibodies were performed as described (10). Confocal imaging was acquired with a Leica TCS SP2 laser scanning spectral system attached to a DMR inverted microscope with a 63/1.4 immersion objective.…”

“…The mutation is located in the first nucleotide-binding domain, which is highly homologous between ABCB1 and ABCB4. This mutation had similar effect when reproduced in either ABCB1 or ABCB4, leading to intracellular trafficking defect and ER/Golgi retention (10). Nevertheless, intracellular traffic was restored when cells were grown at 27°C, and the rescued ABCB1-I541F mutant was functional.…”