ABCMdb

PMID: 19185004

Delaunay JL, Durand-Schneider AM, Delautier D, Rada A, Gautherot J, Jacquemin E, Ait-Slimane T, Maurice M
A missense mutation in ABCB4 gene involved in progressive familial intrahepatic cholestasis type 3 leads to a folding defect that can be rescued by low temperature.
Hepatology. 2009 Apr;49(4):1218-27., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCB1 p.Ile541Phe ABCB1 p.Ile541Phe ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), themultidrugtransporterresponsiblefordrugresistanceofcancercells.Wehavestudiedtheeffectof mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4andABCB1.Plasmidsencodingthewild-typehumanABCB4orratABCB1-greenfluoresc- ing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells. Login to comment 4 ABCB4 p.Ile541Phe Expression studies showed that ABCB4 was localized at the bile canalicular membrane in HepG2 cells and at the apical surface in MDCK cells, whereas the I541F mutant was intracellular. Login to comment 5 ABCB4 p.Ile541Phe ABCB4 p.Ile541Phe In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticu- lumandcis-Golgi,andremainedpartiallyendoH-sensitive.Aftershiftingcellsto27°C,ABCB1-I541F wasexpressedattheapicalcellsurfaceinamatureandactiveform.Similarly,ABCB4wassignificantly trafficked to the membrane of bile canaliculi in HepG2 cells. Login to comment 6 ABCB4 p.Ile541Phe Conclusion: Mutation I541F causes mislocalizationofbothABCB4andABCB1.IntracellularretentionofABCB4-I541Fcanexplainthe disease in PFIC3 patients bearing this mutation. Login to comment 31 ABCB4 p.Ile541Phe We have studied the effect of mutation I541F, located in the first nucleotide-binding domain (NBD), which has been described in a homozygous patient with PFIC3.4 This female patient experienced chronic cholestasis since 1 year of age and developed cirrhosis. Login to comment 61 ABCB1 p.Ile541Phe ABCB1 p.Ile541Phe For construction of the ABCB4-I541F and ABCB1-I541F-GFP mutants, site-directed mutagenesis was performed using the QuikChange XL mutagenesis kit (Stratagene Europe, Amsterdam Zvidoost, The Netherlands). Login to comment 68 ABCB1 p.Ile541Phe Enrichment of the cell population with ABCB1-I541F-GFP-expressing cells was performed by fluorescence-activated cell sorting. Login to comment 100 ABCB4 p.Ile541Phe Results Expression of ABCB4 and ABCB4-I541F in HepG2 and MDCK Cells. Login to comment 102 ABCB4 p.Ile541Phe ABCB4 p.Ile541Phe To examine the cellular localization of ABCB4 and ABCB4-I541F, HepG2 cells were transiently transfected with the pcDNA3 plasmid encoding wild-type ABCB4 or ABCB4-I541F or with the empty plasmid. Login to comment 105 ABCB4 p.Ile541Phe Cells transfected with the plasmid encoding ABCB4 expressed the protein exclusively on the membranes of bile canaliculi, which correspond to the apical surface (Fig. 1A), consistent with the expected canalicular localization of ABCB4 in hepatocytes.15 By contrast, cells transfected with the plasmid encoding ABCB4-I541F did not express the protein at the membrane of bile canaliculi. Login to comment 109 ABCB4 p.Ile541Phe MDCK cells were transfected with the plasmids encoding ABCB4 and ABCB4-I541F or the empty plasmid. Login to comment 114 ABCB4 p.Ile541Phe By contrast, ABCB4-I541F was detected around the nuclei (Fig. 1D). Login to comment 116 ABCB4 p.Ile541Phe These expression studies show that ABCB4 is expressed at the apical membrane, whereas ABCB4-I541F leads to a major trafficking defect and accumulates within the cell cytoplasm, in both HepG2 and MDCK cells. Login to comment 117 ABCB4 p.Ile541Phe ABCB1-I541F-GFP Is Also Intracellular. Login to comment 118 ABCB4 p.Ile541Phe The I541F mutation is localized next to the ABC signature (LSSGGQ) in the first nucleotide-binding domain. Login to comment 123 ABCB4 p.Ile541Phe Expression of ABCB4 and ABCB4-I541F in HepG2 and MDCK cells. Login to comment 124 ABCB4 p.Ile541Phe ABCB4 p.Ile541Phe HepG2 cells transiently expressing ABCB4 (A) or ABCB4-I541F (B), and filter-grown MDCK cells stably expressing ABCB4 (C, E) or ABCB4-I541F (D, F) were fixed with methanol/acetone and processed for immunofluorescence using the P3II-26 monoclonal antibody and Alexa 488-conjugated anti-mouse immunoglobulin G. Login to comment 127 ABCB4 p.Ile541Phe ABCB4 p.Ile541Phe Bars, 10 ␮m. activity.20 In MDCK cells, ABCB1-GFP was exclusively expressed at the apical surface (Fig. 3A), as already reported.24 In contrast, ABCB1-I541F-GFP was intracellular, as observed for ABCB4-I541F. Login to comment 132 ABCB4 p.Ile541Phe ABCB1-I541F-GFP accumulated around the nuclei, in membrane compartments that appeared sometimes granular (Fig. 3B). Login to comment 133 ABCB1 p.Ile541Phe ABCB4 p.Ile541Phe These expression experiments show that mutation I541F causes a trafficking defect both in ABCB1 and ABCB4, and that ABCB1-GFP chimera provides an interesting model to further investigate the effect of I541F mutation. Login to comment 134 ABCB4 p.Ile541Phe ABCB1-I541F-GFP Is Retained in an Endoplasmic Reticulum/Golgi Compartment. Login to comment 135 ABCB4 p.Ile541Phe To precisely determine the sites of intracellular accumulation, different cellular compartments were identified with specific antibodies in MDCK/ABCB1-I541F-GFP cells grown to subconfluence. Login to comment 137 ABCB4 p.Ile541Phe In all of these colocalization experiments, ABCB1-I541F-GFP was observed exclusively in the cytoplasm, especially around the nuclei, whereas little ABCB1-GFP was intracellular (Fig. 4A). Login to comment 138 ABCB4 p.Ile541Phe ABCB1-I541F-GFP colocalized strongly with calnexin and to a lesser extent with protein disulfide isomerase, two endoplasmic reticulum (ER) markers (Fig. 4B). Login to comment 140 ABCB4 p.Ile541Phe In nontransfected cells, the distribution of calnexin was finely granular, whereas in ABCB1-I541F cells, bundles strongly labeled for calnexin colocalized with the fluorescent mutant (Fig. 4B). Login to comment 145 ABCB1 p.Ile541Phe The location of mutation I541F is indicated by an arrow. Login to comment 148 ABCB1 p.Ile541Phe Expression of ABCB1-GFP and ABCB1-I541F-GFP in MDCK cells. Login to comment 149 ABCB1 p.Ile541Phe (A) Confluent filter-grown MDCK cells stably expressing ABCB1-GFP (A, C) or ABCB1-I541F-GFP (B, D) were fixed with paraformaldehyde and examined for the fluorescence of GFP. Login to comment 152 ABCB1 p.Ile541Phe (B) Subconfluent MDCK cells stably expressing ABCB1-I541F-GFP (B, D) were fixed with paraformaldehyde and ethanol, and processed for immunofluorescence using the monoclonal C219 antibody and Cy3-conjugated anti-mouse immunoglobulin G. Login to comment 158 ABCB1 p.Ile541Phe ABCB1-I541F-GFP Is Not Processed Correctly. Login to comment 161 ABCB1 p.Ile541Phe To assess more precisely the processing defect caused by the mutation, the glycosylation status of ABCB1 and ABCB1-I541F was studied with endoglycosidases. Login to comment 166 ABCB1 p.Ile541Phe In contrast, ABCB1-I541F-GFP migrated as two bands of roughly equal intensity of 180 and 190 kDa (Fig. 5). Login to comment 168 ABCB1 p.Ile541Phe Thus, biochemical studies confirmed that the I541F-mutant does not mature as the wild-type protein during the biosynthetic process, and remains largely in a high-mannose endoH-sensitive form. Login to comment 172 ABCB1 p.Ile541Phe Colocalization of ABCB1 and ABCB1-I541F with markers of specific cellular compartments. Login to comment 173 ABCB1 p.Ile541Phe Subconfluent MDCK cells stably expressing ABCB1-GFP (A) or ABCB1-I541F-GFP (B) were fixed with 4% paraformaldehyde, permeabilized with 0.075% saponin, and processed for immunofluorescence using antibodies to calnexin, protein disulfide isomerase, giantin, Golgi matrix protein, early endosomal antigen, or lysosomal-associated membrane protein 2, and appropriate Cy3-conjugated secondary antibodies. Login to comment 176 ABCB1 p.Ile541Phe Note that the calnexin pattern (B, upper middle picture) is different in ABCB1-I541F-GFP-expressing cells (large asterisks) than in nontransfected cells (small asterisks), and colocalizes with the GFP signal. Login to comment 178 ABCB1 p.Ile541Phe Expression and glycosylation status of ABCB1-GFP and ABCB1-I541F-GFP in MDCK cells. Login to comment 179 ABCB1 p.Ile541Phe MDCK/ABCB1-GFP and MDCK/ ABCB1-I541F-GFP cells were lysed and immunoprecipitation was performed using a polyclonal anti-GFP antibody absorbed onto protein A-sepharose. Login to comment 183 ABCB1 p.Ile541Phe defects that can be overcome by lowering the temperature.25 MDCK-ABCB1-I541F-GFP cells were filter grown to confluence at 37°C, then the cells were switched or not to 27°C for 24 hours. Login to comment 184 ABCB1 p.Ile541Phe ABCB1 p.Ile541Phe Figure 6A shows that in control cells grown at 37°C, ABCB1-I541F-GFP was intracellular, whereas in cells grown at 27°C, ABCB1-I541F-GFP was expressed at the apical surface. Login to comment 187 ABCB1 p.Ile541Phe These results show that I541F is a temperature-sensitive mutation and that processing and trafficking of the mutant reverts toward that of wild-type on lowering temperature. Login to comment 188 ABCB1 p.Ile541Phe ABCB1-I541F-GFP Is Functional When Expressed at the Apical Membrane. Login to comment 194 ABCB1 p.Ile541Phe Only very weak activity was measured in MDCK/ ABCB1-I541F-GFP cells grown at 37°C; however, when cells were grown at 27°C for 24 hours before the assay, they accumulated significantly less fluorescent calcein (Fig. 6C). Login to comment 195 ABCB1 p.Ile541Phe These results show that mutation I541F does not impair activity, provided that the transporter can reach the plasma membrane. Login to comment 197 ABCB4 p.Ile541Phe To check whether the temperature rescue of ABCB1-I541F in MDCK cells might be relevant for PFIC3 therapy, we studied the effect of low temperature on ABCB4 expression in HepG2 cells. Login to comment 198 ABCB4 p.Ile541Phe Cells were transiently transfected with either ABCB4 or ABCB4-I541F cDNA. Login to comment 200 ABCB4 p.Ile541Phe Figure 7A shows that ABCB4-I541F was detected in significant amount at the membrane of bile canaliculi in cells grown at 27°C. Login to comment 204 ABCB4 p.Ile541Phe Low temperature rescues functional ABCB1-I541F at the apical membrane. Login to comment 205 ABCB4 p.Ile541Phe (A) MDCK/ABCB1-I541F-GFP cells were grown on filters until confluence at 37°C, then shifted or not to 27°C for 24 hours. Login to comment 211 ABCB4 p.Ile541Phe MDCK/ABCB1-I541F-GFP cells were grown at 37°C then shifted or not to 27°C 24 hours before the experiment. Login to comment 216 ABCB4 p.Ile541Phe ABCB4-I541F migrated as a single 140-kDa polypeptide in cells grown at 37°C. Login to comment 218 ABCB4 p.Ile541Phe These results show that, for ABCB1-I541F in MDCK cells, low temperature is effective on processing and bile canalicular delivery of the ABCB4 mutant in hepatic cells. Login to comment 221 ABCB4 p.Ile541Phe Homozygous I541F ABCB4 mutation was described in a patient with PFIC3 who presented with persistent cholestasis from the age of 1 year and developed severe portal hypertension.4 In the patient`s liver, immunohistochemistry did not show any canalicular staining for ABCB4.4 This suggested that in the liver in vivo, the mutant protein could not reach the canalicular membrane because of a targeting defect or protein degradation. Login to comment 222 ABCB4 p.Ile541Phe In both HepG2 and MDCK cells, we found that ABCB4-I541F accumulated intracellularly and was not detected at the canalicular membrane, as in the previously reported mutated patient. Login to comment 227 ABCB4 p.Ile541Phe The location of the I541F mutation, in the first NBD domain, next to the LSSGQ signature, predicted that ATP-binding or hydrolysis could be affected. Login to comment 229 ABCB4 p.Ala546Asp Dixon et al.26 have shown that the A546D mutation described in a patient with intrahepatic cholestasis of pregnancy affected the traffic of an ABCB1 mutant to the plasma membrane, but not its activity. Login to comment 230 ABCB4 p.Ala546Asp In the work of Dixon et al.,26 ABCB1 was used as a model to reproduce the A546D ABCB4 mutation. Login to comment 231 ABCB1 p.Ile541Phe Here, we also used ABCB1 and showed that the I541F mutation caused similar intracellular retention in both ABCB1 and ABCB4. Login to comment 235 ABCB4 p.Ile541Phe Many inherited diseases are known to arise because of point mutations within a gene that result in the production of proteins unable to assume a stable conformation within the cell.28,29 Mutations lead to non-native protein folding intermediates that are recognized by specialized chaperones and eventually targeted for destruction by the quality control machinery of the cell.30 The fact that trafficking of I541F-mutant can be rescued by lowering the temperature suggests that this mutation causes a folding defect. Login to comment 237 ABCB4 p.Ile541Phe In the case of I541F, both isoleucine and phenylalanine are hydrophobic amino acids, but isoleucine has a branched chain, whereas phenylalanine has a Fig. 7. Login to comment 238 ABCB4 p.Ile541Phe ABCB4-I541F reaches the bile canalicular membrane at low temperature. Login to comment 239 ABCB4 p.Ile541Phe HepG2 cells were transfected with either ABCB4 or ABCB4-I541F cDNA. Login to comment 247 ABCB4 p.Ile541Phe Our observation that folding of ABCB1-I541F at 27°C allows translocation of calcein indicates that, despite the mutation, the NBD domain is able to adopt a native transport-competent conformation. Login to comment 249 ABCB4 p.Ile541Phe Response to UDCA therapy is variable among PFIC3 patients.4 The patient harboring the I541F mutation was not improved by UDCA therapy and required liver transplantation. Login to comment 252 ABCB4 p.Ile541Phe The observation that the I541F-mutant is transport-competent if it successfully transits to the plasma membrane raises the possibility that strategies to influence protein folding inside cells might prove to have therapeutic value. Login to comment