ABCMdb

PMID: 15791618

Hayashi H, Takada T, Suzuki H, Akita H, Sugiyama Y
Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11.
Hepatology. 2005 Apr;41(4):916-24., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly However, the mechanisms for the deficiency in the function of two mutations (E297G and D482G), which are frequently found in European patients, have not yet been identified. Login to comment 3 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Introduction of E297G and D482G mutations into the human BSEP gene by site-directed mutagenesis resulted in a significant reduction in the BSEP expression level, which was associated with impaired membrane trafficking. Login to comment 4 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Most of the D482G BSEP and some of the E297G BSEP underwent only core glycosylation and appeared to be predominantly located in the endoplasmic reticulum. Login to comment 7 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In conclusion, E297G and D482G mutations result in impaired membrane trafficking, whereas the transport functions of these mutants remain largely unchanged. Login to comment 9 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly T he efficient biliary excretion of monovalent bile acids is mediated by the bile salt export pump (BSEP/ABCB11), an ATP-binding cassette transmembrane transporter located on the bile canalicular membrane.1 The function of BSEP/ABCB11 has been clarified by examining the adenosine triphosphate (ATP)- dependent transport of monovalent bile acids (such as taurocholic acid) in isolated bile canalicular membrane vesicles and/or membrane vesicles isolated from cells transfected with the complementary DNA (cDNA) for BSEP.2,3 Many studies have also been performed in patients and it has been shown that the hereditary defect in the expression of BSEP results in the acquisition of progressive familial intrahepatic cholestasis type 2 (PFIC2).4,5 Genomic analysis of PFIC2 patients has revealed that many kinds of missense, premature termination, frame shift, and splicing junction mutations are associated with the BSEP gene.4 Among these, E297G and D482G, two missense mutations in the second intracellular loop and in the first ATP-binding domain, respectively, are frequently observed in PFIC2 patients. Login to comment 21 ABCB11 p.Glu297Gly 916 was shown that the introduction of some mutations in the consensus region (such as E297G) into the rat and mouse Bsep genes resulted in altered cellular distribution of the Bsep. Login to comment 23 ABCB11 p.Asp482Gly In addition, the mechanism remains to be clarified for other important mutations, including D482G, which is very common in European PFIC2 patients. Login to comment 28 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly We focused particularly on E297G and D482G mutations, which are frequently found in European PFIC2 patients.6 Materials and Methods Materials. Login to comment 44 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly For the determination of BSEP messenger RNA (mRNA) levels, MDCK II cells were seeded 24 hours before infection at a density of 1.3 ϫ 106 cells per 10-cm dish and were infected with recombinant adenoviruses containing wild-type, E297G, and D482G BSEP cDNA at a multiplicity of infection (MOI) of 50. Login to comment 67 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly To determine the localization of BSEP, MDCK II cells were seeded on glass coverslips 24 hours before infection at a density of 3 ϫ 105 cells/well in 12-well plates and were infected with recombinant adenoviruses at 25 MOI (wild-type and E297G BSEP) or 250 MOI (D482G BSEP). Login to comment 74 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly To examine the cellular localization and transport function of the two mutants (E297G and D482G), we constructed recombinant adenoviruses containing wild-type and mutated BSEP cDNA. Login to comment 87 ABCB11 p.Asp482Gly Although the wild-type BSEP was detected as approximately 170 kDa, D482G BSEP was detected as approximately 150 kDa. Login to comment 88 ABCB11 p.Glu297Gly In addition, E297G BSEP was detected as approximately 170 kDa and approximately 150 kDa bands. Login to comment 90 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly These results suggest that D482G BSEP and some of the E297G BSEP molecules are present as immature endoplasmic reticulum (ER)-resident forms. Login to comment 93 ABCB11 p.Asp482Gly Although wild-type BSEP is predominantly localized in the apical membrane at 25 MOI (Fig. 3A), there was very little expression of D482G BSEP in most cells at 25 MOI because of the low expression level, as suggested by Western blot analysis (see Fig. 2A). Login to comment 94 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly An increase in the MOI to 250 resulted in significant expression of D482G BSEP, suggesting that D482G BSEP are expressed intracellularly (see Fig. 3A). Login to comment 95 ABCB11 p.Glu297Gly E297G BSEP was located in both the intracellular compartment and apical membrane at 25 MOI (see Fig. 3A). Login to comment 99 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Cell surface BSEP was detected in wild-type and E297G-expressing MDCK II cells as the mature form, whereas no D482G BSEP molecules were detectable on the cell surface (Fig. 3B). Login to comment 100 ABCB11 p.Asp482Gly This result is also consistent with the results of the Western blot analysis of the whole cell lysate in which the mature form of D482G BSEP was not detectable (see Fig. 3B). Login to comment 102 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly It has been reported that proteasomes play an important role in the degradation of incompletely folded and misfolded protein retained in the ER.17,18 Because the expression levels of E297G and D482G BSEP were lower than that of the wild-type BSEP-and because it is possible that these mutants are localized in the ER-proteasomes may be responsible for their degradation. Login to comment 103 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly To examine this hypothesis, cells were treated with 5 ␮mol/L MG132, a specific proteasome inhibitor, for 8 hours, and its effects on E297G and D482G BSEP were examined via Western blot anasysis. Login to comment 104 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Western blot analysis showed that MG132 treatment caused the accumulation of immature forms (150 kDa), particularly in E297G and D482G BSEP-expressing MDCK II cells (Fig. 4). Login to comment 106 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly It was found that MG132 treatment resulted in an increase in the number of wild-type, E297G, and D482G BSEP-expressing cells, and the degree of increase was particularly high for the latter two; in the absence of MG132, the number of E297G BSEP-expressing cells after adenovirus infection was only approximately 25% of those expressing wild-type BSEP, and only a minimal number of cells expressed D482G BSEP. Login to comment 107 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In contrast, in the presence of MG132, the number of cells expressing E297G and D482G BSEP was almost the same as that in wild-type BSEP-expressing cells after infection of adenoviruses at the same MOI (data not shown). Login to comment 108 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In the presence of MG132, E297G and D482G BSEP were predominantly expressed in the intracellular compartment, which is the same as that observed under control conditions (see Fig. 3A). Login to comment 115 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly (A) Results of Western blot analysis with crude membrane fraction prepared from MDCK II cells. MDCK II cells were infected with recombinant adrenoviruses at 250 MOI 48 hours before the experiments. Crude membrane fractions prepared from GFP (control), wild-type, E297G, and D482G BSEP-expressing MDCK II cells (80, 40, 80, and 80 ␮g protein, respectively) were analyzed. Login to comment 120 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Before the transport experiments, the expression level of BSEP was compared among the isolated membrane vesicles expressing wild-type, E297G, and D482G BSEP. Login to comment 121 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly D482G BSEP, as well as wild-type and E297G BSEP, were detected as approximately 170-kDa bands in isolated membrane vesicles (Fig. 5A). Login to comment 122 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The band density of the 170-kDa form of E297G and D482G in the isolated membrane vesicles was approximately 25% and 10% of wild-type BSEP, respectively (Fig. 5B). Login to comment 124 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The ATP-dependent uptake of taurocholate and glycocholate by wild-type, E297G and D482G BSEP-expressing isolated membrane vesicles was much higher than that by GFP-expressing vesicles (Fig. 6A). Login to comment 125 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly By normalizing the BSEP expression levels in the isolated membrane vesicles based on the results of Western blot analysis (see Fig. 5B), it was demonstrated that the transport of taurocholate and glycocholate mediated per unit mass of E297G and D482G BSEP molecules was not significantly different from that by wild-type BSEP (Figs. 5B and 6B). Login to comment 127 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Wild-type, E297G, and D482G BSEP-mediated initial ATP-dependent uptake rates were saturable with apparent Km values of 4.61 Ϯ 0.91, 5.41 Ϯ 0.27, and 14.3 Ϯ 2.0 ␮mol/L, respectively (Fig. 6C). Login to comment 128 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The maximum taurocholate transport velocity (Vmax) for wild-type, E297G, and D482G BSEP were 2310 Ϯ 220, 485 Ϯ 15, and 761 Ϯ 60 pmol/min/mg isolated membrane vesicle protein, respectively. Login to comment 132 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly MDCK II cells were infected with the recombinant adenoviruses at 25 MOI (wild-type and E297G BSEP) or 250 MOI (D482G BSEP) 48 hours before the experiments. Login to comment 140 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly mass of E297G and D482G BSEP molecules was calculated to be 94% and 329%, respectively, of wild-type BSEP. Login to comment 141 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Discussion In the present study, we analyzed the consequence of two frequently found PFIC2 mutations (E297G and D482G) in vitro to investigate the pathogenesis of PFIC2. Login to comment 143 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Although quantitative PCR showed no difference in mRNA levels between the wild-type and two mutated BSEP (see Fig. 1), Western blot analysis indicated reduced expression of D482G and E297G BSEP (see Fig. 2A). Login to comment 144 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In addition, the molecular weight of most of the D482G BSEP and some of the E297G molecules was approximately 150 kDa (see Fig. 2A). Login to comment 147 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly This suggestion is also consistent with the immunofluorescence observations which indicated that D482G and E297G BSEP molecules are located intracellularly (see Fig. 3A). Login to comment 148 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The results of the surface biotinylation study also suggested that the D482G BSEP and core glycosylated form of E297G BSEP are located intracellularly (see Fig. 3B). Login to comment 149 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In addition, the results of the MG132 treatment experiment suggested that E297G and D482G BSEP molecules are degraded by the proteasome pathway (see Fig. 4). Login to comment 151 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Although the immunohistochemical studies indicate a loss of BSEP on the canalicular membrane in PFIC2 patients with the E297G mutation, we found that some E297G BSEP with a molecular mass of approximately 170 kDa were expressed on the apical membrane (see Fig. 3B). Login to comment 152 ABCB11 p.Glu297Gly At the present moment, we do not know the reason why some E297G BSEP molecules are trafficked in a normal manner. Login to comment 154 ABCB11 p.Glu297Gly Alternatively, it is also possible that the reduction in the expression level on the apical membrane of E297G BSEP (approximately 25% of wild-type BSEP; see Fig. 3B) may be related to the pathogenesis of PFIC2. Login to comment 156 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Western blot analysis of the isolated membrane vesicles indicated the presence of approximately 170-kDa molecules for wild-type, E297G, and D482G BSEP. Login to comment 157 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Moreover, it was found that the amount of 150-kDa molecules for E297G and D482G BSEP in the isolated membrane vesicles was lower than that in the crude membrane fraction. Login to comment 163 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Effects of proteasome inhibitors on the localization of BSEP in MDCK II cells. MDCK II cells were infected with recombinant adenoviruses at 250 MOI 48 hours before the experiments. Crude membrane fractions prepared from GFP (control), wild-type, E297G, and D482G BSEP-expressing MDCK II cells (80, 40, 80, and 80 ␮g protein, respectively), treated with or without 5 ␮mol/L MG132 for 8 hours before the preparation of crude membrane, were subjected to Western blot analysis. Login to comment 165 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Functional analysis using these membrane vesicles indicated that the transport of taurocholate and glycocholate in E297G and D482G BSEP-expressing isolated membrane vesicles was significantly higher than that in control isolated membrane vesicles (see Fig. 6A). Login to comment 166 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Based on the hypothesis that the transport activity per unit mass of BSEP molecules can be calculated by considering the BSEP expression level in the isolated membrane vesicles, it was found that the transport of taurocholate and glycocholate mediated per unit mass of E297G and D482G BSEP molecules was not reduced compared with wild-type BSEP (see Figs. 5B and 6B). Login to comment 167 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly The Km values for taurocholate of wild-type, E297G, and D482G BSEP molecules were consistent with previous observations obtained using membrane vesicles isolated from human wild-type BSEP cDNA-infected Sf9 and Sf High Five cells.2,3 Based on these results, it appears that the transport function of two mutated BSEP molecules (E297G and D482G) per se remains normal (see Fig. 6). Login to comment 168 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly These results suggest that, if E297G and D482G are matured and consequently expressed on the membrane surface, these mutated transporters are able to transport BSEP substrates. Login to comment 169 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly Our results using mutated human BSEP were different fromthosereportedinapreviousstudy,inwhichthecauseof PFIC2wasexaminedusingmutatedratBsep,7 buttheywere consistentwiththosereportedrecently,inwhichtheeffectof D482G mutation was examined using mouse Bsep.8 In the study using rat Bsep gene, the pathogenic mechanism for the D482G mutation was not identified because D482G rat Bsep is localized in the apical membrane as well as the cytoplasm of MDCK cells, and this mutation did not significantly affect taurocholate transport examined using Fig. 5. Login to comment 174 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 5 ␮g (upper lane), 15 ␮g (middle lane) and 30 ␮g (lower lane) protein of isolated membrane vesicles prepared from GFP (control), wild-type, E297G and D482G BSEP-expressing HEK 293 cells were subjected to Western blot analysis. Login to comment 176 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The results for wild-type (F), E297G (E), and D482G (■) BSEP are shown. Login to comment 177 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly (C) Results of Western blot analysis using crude membrane fraction and isolated membrane vesicles prepared from GFP (control), wild-type, E297G, and D482G BSEP-expressing HEK 293 cells (50, 20, 75, and 75 ␮g protein, respectively). Login to comment 179 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly membranevesiclesisolatedfromSf9cells.7 Incontrast,inthe study with mouse Bsep gene, it was found that D482G mutation resulted in impaired canalicular trafficking in HepG2 cells, although this mutation did not affect the transport of taurocholate examined using membrane vesicles isolated from Sf21 cells.8 Concerning E297G mutation, it has been shownthatE297GratBsepiswidelydistributedthroughout the cytoplasm, and the studies using isolated membrane vesicles indicated that this mutation resulted in the loss of taurocholate transport activity.7 The differences among these results involving the previous mutated rat Bsep, mouse Bsep, and the present mutated human BSEP may be explained by considering the species difference in the Bsep/BSEP sequence, although the homology of the amino acid sequence around E297G and D482G is quite high as far as human BSEPandratandmouseBsepareconcerned.Forexample,it is still possible that the introduction of the D482G mutation to human BSEP, but not rat Bsep, results in a conformational change in human BSEP so that D482G BSEP is bound to some molecular chaperons. Login to comment 180 ABCB11 p.Asp482Gly The features of D482G BSEP resemble those of the CFTR ⌬F508 mutant in that the deletion of phenylalanine at 508 results in the accumulation of the mutated protein in the ER followed by proteasome degradation. Login to comment 182 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly One of the methods being explored as a potential treatment of CFTR ⌬F508 patients is to administer drugs (such as sodium 4-phenylbutyrate) which are capable of trafficking the mutated protein to the apical membrane by inhibiting the binding to the molecular chaperons in the ER.21-23 After clarifying the mechanism for the intracellular retention of E297G and D482G BSEP, it is possible to identify agents able to target these mutated BSEP molecules to the apical surface. Login to comment 184 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In conclusion, the results of the present in vitro study suggest that E297G and D482G, which are frequently observed PFIC2 mutations, cause deficient BSEP maturation, although the transport functions of these mutants per se remain almost normal. Login to comment