ABCMdb

PMID: 17947449

Kagawa T, Watanabe N, Mochizuki K, Numari A, Ikeno Y, Itoh J, Tanaka H, Arias IM, Mine T
Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells.
Am J Physiol Gastrointest Liver Physiol. 2008 Jan;294(1):G58-67. Epub 2007 Oct 18., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Arg1050Cys ABCB11 p.Lys461Glu ABCB11 p.Arg1057* ABCB11 p.Ala570Thr 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 (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Login to comment 14 ABCB11 p.Arg1057* Bsep protein expression levels correlated closely with transport activity, except for R1057X. Login to comment 15 ABCB11 p.Asp482Gly The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). Login to comment 16 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Arg1057* BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. Login to comment 18 ABCB11 p.Arg1057* The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. Login to comment 19 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Arg1050Cys ABCB11 p.Lys461Glu ABCB11 p.Arg1057* ABCB11 p.Ala570Thr In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C Ͼ D482G Ͼ E297G Ͼ K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. Login to comment 29 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Of these, D482G and E297G mutations are most frequent (35). Login to comment 30 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Studies of TC transport activity and intracellular trafficking by D482G and E297G mutants have reported inconstant results. Login to comment 32 ABCB11 p.Asp482Gly D482G mutant Bsep was localized in the apical membrane of HepG2 (28) and Madin-Darby canine kidney (MDCK) cells (41), and in the cytoplasm of MDCK II cells (8). Login to comment 87 ABCB11 p.Asp482Gly Briefly, MDCK II cells grown on 24-mm Transwell membrane inserts were transfected with WT or D482G mutant Bsep. Login to comment 115 ABCB11 p.Asp482Gly RESULTS TC transport activity, protein expression, and mRNA expression of the D482G mutant. Login to comment 116 ABCB11 p.Asp482Gly The D482G mutant is the most frequently observed mutation in PFIC2. Login to comment 118 ABCB11 p.Asp482Gly In a polarized MDCK II monolayer, the D482G mutation revealed 32.3 Ϯ 5.8% (mean Ϯ SD) TC transport activity of that observed in WT (Fig. 2A). Login to comment 121 ABCB11 p.Asp482Gly The D482G mutant produced the same two bands, suggesting normal maturation of Bsep protein. Login to comment 124 ABCB11 p.Asp482Gly These results reveal that the observed decrease in TC transport activity of the D482G mutation is attributable to decrease in Bsep protein expression. Login to comment 126 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly The mRNA expression of D482G was slightly higher than that of WT; however, the difference was not statistically significant (Fig. 2C), which suggests that the D482G mutant Bsep protein may be degraded faster than is the WT. Login to comment 127 ABCB11 p.Asp482Gly Next, we tested the subcellular distribution of the D482G mutant (Fig. 3). Login to comment 128 ABCB11 p.Asp482Gly MDCK II cells were transfected with WT or D482G Bsep, and the fluorescent signals were observed by confocal laser scanning microscopy. Login to comment 129 ABCB11 p.Asp482Gly The D482G mutant was predominantly distributed along apical membranes similar to that of WT (Fig. 3A). Login to comment 131 ABCB11 p.Asp482Gly As expected, WT and D482G Bsep were not detected in the basolateral membrane (Fig. 3B). Login to comment 133 ABCB11 p.Asp482Gly D482G mutant protein was present in the apical membrane at 30.6 Ϯ 14.4% of the level of WT. Login to comment 139 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Arg1050Cys ABCB11 p.Lys461Glu ABCB11 p.Ala570Thr ABCB11 p.Arg1057Cys The positions of 8 PFIC2 mutations (E297G, K461E, D482G, G982R, R1057C, R1153C, 3767-3768insC, and R1268Q) and 2 BRIC2 mutations (A570T and R1050C) are indicated by ଝ and ଙ, respectively. Login to comment 142 ABCB11 p.Asp482Gly G60 RAPID DEGRADATION OF PFIC2, BRIC2 MUTANT AJP-Gastrointest Liver Physiol • VOL 294 • JANUARY 2008 • www.ajpgi.org Biochemical half-life of the D482G mutant. Login to comment 143 ABCB11 p.Asp482Gly To explore the mechanism(s) for attenuation of protein expression of the D482G mutant, the biochemical half-life was determined in MDCK II cells by analyzing Bsep protein expression after inhibiting further protein synthesis with cycloheximide treatment (Fig. 4). Login to comment 145 ABCB11 p.Asp482Gly Band C of the WT was still present in 12 h, whereas, in D482G, bands disappeared in 4 h. Login to comment 146 ABCB11 p.Asp482Gly The calculated half-life of band C in the D482G was 1.35 h, which was significantly shorter than that in WT (3.49 h, P Ͻ 0.05, Fig. 4B). Login to comment 147 ABCB11 p.Asp482Gly Likewise the half-life of band B in the D482G was shorter than that in WT (0.41 h vs. 1.16 h, P Ͻ 0.05, Fig. 4B). Login to comment 148 ABCB11 p.Asp482Gly These results suggest that the D482G protein is degraded faster than is the WT. Login to comment 152 ABCB11 p.Asp482Gly Bands C of WT at 12 h and D482G at 4 h were denser in the presence of MG132. Login to comment 153 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly The half-life of each band was significantly extended by MG132 in WT and D482G; from 3.49 to 14.1 h in WT band C, from 1.35 to 13.2 h in D482G band C, from 1.16 to 2.95 h in WT band B, and from 0.41 to 1.73 h in D482G band B (Fig. 5C). Login to comment 155 ABCB11 p.Asp482Gly These results suggest that the WT and D482G mutant Bsep proteins are degraded in proteasomes rather than in lysosomes. Login to comment 164 ABCB11 p.Glu297Gly The E297G mutation revealed ϳ10.2 Ϯ 6.6% TC transport activity of WT (Fig. 6A). Login to comment 165 ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Lys461Glu ABCB11 p.Arg1057* Other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X) did not show significant TC transport activity. Login to comment 166 ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr In contrast, two BRIC2 mutants exhibited considerable transport activity (A570T: 52.2 Ϯ 13.0%, R1050C: 58.7 Ϯ 9.9% of the WT). Login to comment 168 ABCB11 p.Glu297Gly ABCB11 p.Arg1057* The E297G mutant was expressed at 16.1 Ϯ 6.8% of that of WT, whereas expression of the other mutants, except for R1057X, was at trace level (Fig. 6, B and C). Login to comment 169 ABCB11 p.Arg1057* R1057X was expressed as much as was the WT at ϳ120 kDa, which is consistent with the molecular size anticipated after truncated mutant protein. Login to comment 170 ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr A570T and R1050C Fig. 2. Login to comment 171 ABCB11 p.Asp482Gly Taurocholate (TC) transport activity and expression of Bsep protein and mRNA of the D482G mutant expressed in MDCK II cells. Login to comment 172 ABCB11 p.Asp482Gly A: MDCK II cells grown on Transwell membrane inserts were cotransfected with Ntcp and beta-gal or Ntcp and either wild-type (WT) or the D482G mutant Bsep. Login to comment 174 ABCB11 p.Asp482Gly Each value represents mean Ϯ SD of 6 independent experiments performed in duplicate. B: MDCK II cells were transiently transfected with WT or D482G mutant Bsep and lysed. Five micrograms of cell lysates were separated by SDS-PAGE and subjected to immunoblot analysis using antibody to GFP. Login to comment 177 ABCB11 p.Asp482Gly C: MDCK II cells were transiently transfected with an empty vector or WT or D482G mutant Bsep, and total RNA was extracted 24 h after transfection. cDNA was obtained by reverse transcription with SuperScript III and random hexamers, and quantitative real-time PCR was performed using TaqMan technology on ABI 7700 sequence detection system. Login to comment 181 ABCB11 p.Ala570Thr mRNA expression levels examined in MDCK II cells expressing these mutants except for A570T were slightly higher than those with WT, but the difference was not statistically significant (Fig. 6D). Login to comment 182 ABCB11 p.Arg1057* TC transport activity was significantly correlated with Bsep protein expression levels when R1057X was excluded from analysis (Fig. 6E, P Ͻ 0.001). Login to comment 184 ABCB11 p.Glu297Gly ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Arg1050Cys ABCB11 p.Lys461Glu ABCB11 p.Arg1057* ABCB11 p.Ala570Thr Subcellular distribution study revealed that E297G, R1057X, A570T, and R1050C mutants were predominantly located along the apical membrane, whereas the other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, and 3767-3768insC) remained intracellular (Fig. 7). Login to comment 185 ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr In summary, BRIC2 mutants (A570T and R1050C) were expressed substantially, trafficked correctly, and maintained half of transport activity. Login to comment 186 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Two PFIC2 mutants (D482G and E297G) were expressed at 10-30% of WT, trafficked correctly, and exhibited 10-30% activity. Login to comment 187 ABCB11 p.Arg1057* The R1057X mutant was well expressed and trafficked correctly, but it lacked activity. Login to comment 188 ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Lys461Glu Other PFIC2 mutant proteins (K461E, G982R, R1153C, R1268Q, and 3767-3768insC) were unstable and lost all transport activity. Login to comment 197 ABCB11 p.Asp482Gly From the observation that a representative mutant, D482G, had a shorter biochemical half-life than the WT, rapid degradation of Bsep protein may be responsible for impaired function. Login to comment 204 ABCB11 p.Asp482Gly The biochemical half-life of both forms of the D482G mutant was significantly shorter than that of the WT (mature form: 1.35 h, and core-glycosylated form: 0.41 h, Fig. 4B). Login to comment 205 ABCB11 p.Asp482Gly These results suggest that after translation D482G Bsep protein is unstable and rapidly degraded. Login to comment 206 ABCB11 p.Asp482Gly In contrast to a previous study (28), D482G was completely glycosylated (Fig. 2B). Login to comment 209 ABCB11 p.Asp482Gly Subcellular localization of the WT and D482G mutant Bsep. Login to comment 210 ABCB11 p.Asp482Gly A: MDCK II cells grown on Transwell membrane inserts were transfected with the WT or D482G mutant Bsep. Login to comment 214 ABCB11 p.Asp482Gly The scale bars are 10 ␮m. B: MDCK II cells grown on 24-mm Transwell membrane inserts were transfected with the WT or the D482G mutant Bsep. Login to comment 228 ABCB11 p.Asp482Gly Biochemical half-life of the WT and D482G mutant Bsep in MDCK II cells. Login to comment 229 ABCB11 p.Asp482Gly A: MDCK II cells were transiently transfected with the WT or D482G mutant Bsep. Login to comment 230 ABCB11 p.Asp482Gly After 24 h cells were further cultured in the presence of cycloheximide (20 ␮g/ml) and lysed at 0, 3, 6, and 12 h (WT) or at 0, 1, 2 and 4 h (D482G). Login to comment 237 ABCB11 p.Asp482Gly A and B: MDCK II cells were transiently transfected with the WT or D482G mutant Bsep. Login to comment 238 ABCB11 p.Asp482Gly After 24 h, cells were cultured with cycloheximide (20 ␮g/ml) in the absence or presence of either MG132 (10 ␮M) (A) or bafilomycin A1 (1 ␮M) (B) and lysed at 0, 3, 6, and 12 h (WT) or at 0, 1, 2, and 4 h (D482G). Login to comment 244 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly In the eight PFIC2 mutants studied, D482G and E297G were predominantly distributed in the apical membrane and exhibited TC transport activity (D482G: 32.3% and E297G: 10.2% of WT). Login to comment 245 ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Lys461Glu The mature form of Bsep protein (band C) of K461E, G982R, R1153C, R1268Q, and 3767-3768insC mutants was hardly detected (Fig. 6B) and, consequently, TC transport activity was abolished (Fig. 6A). Login to comment 247 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Our observation that the D482G and E297G mutants exhibited less impaired transport activity than other mutants is in agreement with the clinical phenotype. Login to comment 248 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The response to biliary diversion is better in PFIC2 patients carrying D482G and E297G mutations than in those with other mutations (27). Login to comment 250 ABCB11 p.Arg1057* We demonstrated that rapid degradation of Bsep protein is responsible for the defect of TC transport activity with exception of the R1057X mutation. Login to comment 262 ABCB11 p.Arg1057* Correlation between TC transport activity and Bsep protein expression of WT and mutants Bsep excluding R1057X was tested for significance by Spearman rank correlation. Login to comment 286 ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr TC transport activity was 50-60% in BRIC2 mutants (A570T and R1050C) and 0-30% in PFIC2 mutants. Login to comment 288 ABCB11 p.Glu297Gly Furthermore the E297G mutation, which is responsible for PFIC2, also occurs in BRIC2 patients (37). Login to comment 289 ABCB11 p.Arg1057* Therefore, although the BSEP genotype appears to play an important role in influencing clinical severity, other precipitating factors, including viral infection and pregnancy (37), may participate. In conclusion, our study demonstrates that rapid protein degradation is responsible for decreased TC transport activity in all PFIC2 and BRIC2 mutations except R1057X. Login to comment 290 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Arg1268Gln ABCB11 p.Arg1153Cys ABCB11 p.Gly982Arg ABCB11 p.Arg1050Cys ABCB11 p.Lys461Glu ABCB11 p.Arg1057* ABCB11 p.Ala570Thr From the view of maintenance of TC transport activity, the mutants could be aligned in the following order: A570T and R1050C Ͼ D482G Ͼ E297G Ͼ K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. Login to comment 291 ABCB11 p.Arg1057* R1057X is expressed stably and localized correctly but loses its activity (defective activity). Login to comment 295 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly When we were preparing the manuscript, a paper appeared in which 4-phenylbutyrate (4PBA) was demonstrated to extend the half-life of cell surface-resident WT, E297G, and D482G BSEP by 1.8-, 2.5-, and 3.3-fold, respectively, in MDCK II cells (7). Login to comment