| Budget Amount *help |
¥3,610,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥210,000)
Fiscal Year 2007: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
(1) TWO N-LINKED GLYCANS ARE REQUIRED TO MAINTAIN TRANSPORT ACTIVITY OF THE BILE SALT EXPORT PUMP (ABCB11) IN MDCK II CELLS
The aim of this study was to elucidate the role of N-linked glycosylation in protein stability, intracellular trafficking and bile acid transport activity of the bile salt export pump (Bsep, ABCB 11). Rat Bsep was fused with a yellow fluorescent protein at the C-terminus and mutants in which Asn residues of putative glycosylation sites (Asn^<109>, Asn^<16>, Asn1^<22> and Asn^<125>) were sequentially replaced with GIn were constructed by site-directed mutagenesis: single mutant N109Q; double mutant N109Q + N116Q; triple mutant N109Q + N116Q + N122Q, and quadruple mutant N109Q + N116Q + N122Q + N125Q. Analysis by immunoblot and cleavage with glycosidases of cell lysates from transfected MDCK II cells demonstrated that each site carried a carbohydrate and no other sites were present on Bsep. The taurocholate transport activity determined in polarized MDCK II cells was
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diminished by the removal of glycans. This decrease was caused by the rapid decay of the mutant Bsep protein; the biochemical half-lives were 3.76 h, 3.65 h, 3.24 h, 1.35 h and 0.52 h in the wild-type, single, double, triple and quadruple mutant, respectively. The wild type, single and double mutants were distributed exclusively along the apical membranes, whereas triple and quadruple mutants remained intracellular. MG132 but not bafilomycin Al extended the half-life, suggesting the role of the proteasome in the degradation. There was a significant difference in the rate of degradation between single and double, and triple and quadruple mutants. To elucidate whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of N-glycan deficient Bsep mutants with various combinations and found that Bsep with one glycan was extremely unstable compared with Bsep harboring two and more glycans. In conclusion, at least two of four N-linked glycans were required for proper folding, protein stability, and subsequent intracellular trafficking and function in the apical membrane of Bsep.
(2) Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells
Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis(ERIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene, however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in, rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461Em G982R, R1153C, R1268Q, 3767-3768insC, and E1057X). Bsep protein expression levels correlated closely with transport activty, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35h vs. 3.49h in the form). BRIC2 mutants and three PFIC mutants (D482G, E297G, R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the C-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in propotion to rapid dagradation of Bsep protein in the mutants which ewre aligned in the following order, A570T and R1050C>D482G>E297G>K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2. Less
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