The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II - PubMed (original) (raw)

The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II

Lin Wang et al. J Clin Invest. 2002 Oct.

Abstract

PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II-associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep.

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Figures

Figure 1

PFIC II mutations reside in regions highly conserved throughout the vertebrate phylum. (a) Sequence alignment of mutation sites in BSEP/Bsep from human, rat, mouse, rabbit, and skate using the Clustal alignment software MegAlign (DNASTAR Inc., Madison, Wisconsin, USA). The PFIC II mutations in human BSEP and the corresponding residues in other Bseps are shown in bold. (b) The positions of G238V, E297G, C336S, D482G, G982R, R1153C, and R1268Q are indicated by stars in a predicted topology model of rat Bsep. The Walker A and Walker B regions are shown in boxes, and a number of potential glycosylation sites are indicated by the branch structures in the first extracellular loop of Bsep. The predicted 12 transmembrane-spanning segments in Bsep are shown in boxes labeled 1–12.

Figure 2

Expression of Bsep-GFP in MDCK cells and HEK 293 cells. (a) After transient transfection with Bsep-GFP, MDCK cells were processed for immunofluorescence using antibodies against rat Bsep, gp-135, and NaK-ATPase, and imaged to locate the GFP fusion protein and apical and basolateral membranes of MDCK cells. In each panel, the top part shows the en face image and the bottom part shows the Z-sectioning image. Bsep-GFP colocalized with gp-135 but was distinct from NaK-ATPase. Bar, 5 μm. (b) Bsep-GFP was expressed in either a stable MDCK cell line or HEK 293 cells by transient expression. Both en face and Z-sectioning images of the GFP fusion protein are presented. Bar, 5 μm. Total membrane and cytosol fractions were prepared from (c) MDCK cell lines either stably expressing Bsep-GFP or expressing GFP alone, or (d) from transfected and mock-treated HEK 293 cells. These fractions (100 μg protein per lane) and a rat liver plasma membrane preparation (LPM) (100 μg protein per lane) were separated on a 6.5% Laemmli gel, and Bsep-GFP was detected by Western blotting using an antibody against rat Bsep. The position of Bsep-GFP in the total membrane fraction of MDCK cells is indicated by the arrow. Rat Bsep and Bsep-GFP have apparent molecular weights of approximately 160 kDa and 190 kDa, respectively.

Figure 3

PFIC II mutations have a heterogeneous effect on the localization of Bsep-GFP in MDCK cells. MDCK cells were transiently transfected with wild-type (WT) Bsep-GFP (a) or Bsep PFIC II mutants (b–h) for 72 hours and imaged by confocal microscopy. In each panel, the top part shows the en face image and the bottom part shows the Z-sectioning image. Bar, 10 μm.

Figure 4

G238V is degraded by proteasomes in MDCK cells. MDCK cells were transiently transfected with wild-type Bsep-GFP (bottom row) or G238V (top row) for 48 hours. The transfected cells were then either treated with 5 μmol/ml MG-132 for an additional 2, 8, or 12 hours or left untreated (controls, indicated as 0 h). All images were acquired under identical conditions, in which gain was optimized for the treated cells in order to avoid saturation of the signal. Under these conditions, G238V was barely detectable in MDCK cells not treated with MG-132 (G238V, 0 h). Protein aggregates were seen as a number of signals at perinuclear positions in the cytoplasm. In each panel, the top part shows the en face image and the bottom part shows the Z-sectioning image.

Figure 5

PFIC II mutations exhibit heterogeneous effects on Bsep taurocholate transport activity. (a) Sf9 cell vesicles (100 μg) expressing Bsep or Bsep PFIC II mutant were analyzed by Western blotting as indicated. (b) ATP-dependent [3H]taurocholate transport was measured using membrane vesicles isolated from Sf9 cells expressing Bsep or PFIC II mutants. Vesicle uptake of [3H]taurocholate (2.5 μM) was determined in the presence and absence of ATP (5 mM). Data represent the mean ± SD of three determinations. The difference between taurocholate uptake measured in the presence and absence of ATP was defined as ATP-dependent taurocholate uptake.

References

1. Meier PJ, Stieger B. Bile salt transporters. Annu Rev Physiol. 2002;64:635–661. - PubMed
1. Boyer, J.L., and Nathanson, M.H. 1999. Bile formation. In Schiff’s diseases of the liver. 8th edition. E.R. Schiff, M.F. Sorrell, and W.C. Maddrey, editors. Lippincott-Raven Publishers. Philadelphia, Pennsylvania, USA. 119–146.
1. Gerloff T, et al. The sister P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem. 1998;273:10046–10050. - PubMed
1. Wang R, et al. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in non-progressive but persistent intrahepatic cholestasis. Proc Natl Acad Sci USA. 2001;98:2011–2016. - PMC - PubMed
1. Strautnieks SS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet. 1998;20:233–238. - PubMed

The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II - PubMed (original) (raw)