Functional characterization of ABCB4 mutations found in progressive familial intrahepatic cholestasis type 3

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.

Figure 1. Effect of ABCB4 mutants on transport activity.

Inside-out membrane vesicles were prepared after transfection of ABCB4 wild type or mutant-bearing plasmids into HEK-293T cells, and the ATPase activity induced by either paclitaxel (a) or phosphatidylcholine (b) was measured. The X-axis represents paclitaxel or phosphatidylcholine concentration. The Y-axis represents the amount of inorganic phosphate that was produced by the ATPase activity of MDR3. Data shown represent mean ± SD from five independent experiments and analyzed by one-way analysis of variance followed by Dunnett’s two-tailed test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. wild type (WT).

Figure 2. Effect of ABCB4 mutants on subcellular localization.

HEK-293T cells were transfected with ABCB4 wild type or mutant plasmids. Cells were fixed and permeabilized with methanol, and immunostained with anti-MDR3, and an endoplasmic reticulum maker BiP or a Golgi marker Gigantin antibodies. Scale bars, 10 μm.

Figure 3. Effect of ABCB4 mutants and cyclosporin A (CsA) on the surface expression of MDR3.

(a) HEK-293T cells transfected with ABCB4 wild type or mutant plasmids, and surface biotinylated. CsA was treated at 10 μM for 24 hours. Absence of the cytosolic protein aldolase A in the biotinylated fraction confirms cell surface protein-specific labeling in each experiment. (b) Quantification of surface expression of MDR3. Bar graphs represent band density of surface biotinylated MDR3 compared to the wild type ABCB4 and error bars indicate the SD for three independent experiments. ^*P < 0.05, Student’s t test.

Figure 4. Effect of MG132 or bafilomycin A₁ on MDR3 expression.

MDR3 expression was investigated after transfection with ABCB4 wild type or A364V mutant plasmids. Immunoblotting was performed after treatment with MG132 (a) or bafilomycin A₁ (b). Data shown represent mean ± SD from three independent experiments and analyzed by one-way analysis of variance followed by Dunnett’s two-tailed test. ^**P < 0.01 vs. expression of A364V without MG132 or bafilomycin A₁ treatment.

Figure 5. Effect of CsA on the transport activity of the mutant A364V.

Membrane vesicular ATPase assays were performed using paclitaxel (a) or phosphatidylcholine (b) after treatment with CsA. Data shown represent mean ± SD from three independent experiments, and the P values for comparison between the results obtained before and after CsA treatment, were calculated using Student’s t test. ^*P < 0.05, ^***P < 0.001 vs. transport activity of A364V without CsA treatment.