ABCMdb

PMID: 10767346

Dixon PH, Weerasekera N, Linton KJ, Donaldson O, Chambers J, Egginton E, Weaver J, Nelson-Piercy C, de Swiet M, Warnes G, Elias E, Higgins CF, Johnston DG, McCarthy MI, Williamson C
Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.
Hum Mol Genet. 2000 May 1;9(8):1209-17., 2000-05-01 [PubMed]

Sentences

No. Mutations Sentence Comment

44 ABCB4 p.Ala546Asp In addition to the normal cytosine at this position, an adenine was also detected; this results in the substitution of the wild-type alanine with a mutant aspartic acid (A546D). Login to comment 64 ABCB4 p.Ala544Asp Mature A544D-P-gp1 at the cell surface is functional There are no established systems for quantification of PC translocation in mammalian systems making it currently not possible to study the function of the MDR3 protein. Login to comment 69 ABCB4 p.Ala546Asp We therefore introduced the equivalent A546D mutation into the first NBD of P-gp1 and studied the functional consequences of the change in transiently transfected mammalian cells. Login to comment 71 ABCB1 p.Ala544Asp The mutated fragment was subcloned into the plasmid pMDR1-wt to generate pMDR1-A544D, and used to transiently transfect human epithelial kidney (HEK293T) cells. Login to comment 75 ABCB1 p.Ala544Asp In contrast, cells transiently transfected with pMDR1-A544D formed two distinct populations. Login to comment 76 ABCB1 p.Ala544Asp The first population behaved similarly to the control cells with no UIC2-PE fluorescence and high R123 fluorescence (Fig. 3b, lower right quadrant), consistent with failure to express A544D-P-gp1 at the cell surface (and thus accumulate R123). Login to comment 90 ABCB4 p.Ala544Asp R123 fluorescence (Fig. 3b, upper left quadrant) consistent with cell surface expression of A544D-P-gp1 and extrusion of R123. Login to comment 91 ABCB4 p.Ala544Asp Further confirmation that the extrusion of R123 in the A544D-P-gp1-expressing cells is due to the function of the mutant P-gp was obtained by incubating the cells with the P-gp1-inhibitor cyclosporin A. Login to comment 93 ABCB4 p.Ala544Asp These data are typical of cells expressing functional P-gp1 (28) and indicate that the A544D-P-gp1 mutant is functional if it reaches the cell membrane. Login to comment 94 ABCB4 p.Ala546Asp ABCB4 p.Ala544Asp Evidence that A544D-P-gp1 is a trafficking mutant The finding that substitution of an aspartic acid for an alanine in a highly conserved region of NBD1 did not alter the ability of the mature protein to transport R123 was unexpected and suggested that the equivalent A546D-MDR3 protein mutant would also be functional. Login to comment 103 ABCB1 p.Ala544Asp Cells transfected with pMDR1-A544D also formed two populations (Fig. 4); the first population (M3) had a mean UIC2-PE fluorescence of 10 [barely above the mean (= 3) of the negative control cells], and the second population (M4) had a mean of 50. Login to comment 106 ABCB1 p.Ala544Asp Western blot analysis of A544D-P-gp1 Transiently transfected cells normally express two forms of P-gp1, discernible by polyacrylamide gel electrophoresis (PAGE) and western analysis: immature, non-or core-glyco- Figure 3. Login to comment 107 ABCB1 p.Ala544Asp FACS analysis of HEK293T cells transiently transfected with pCIneo-βgal (a), or pMDR1-A544D (b and c). Login to comment 112 ABCB1 p.Ala544Asp This allowed the intracellular, cell surface and total P-gp of wt-P-gp1 and A544D to be calculated (calculated values are shown in italics). Login to comment 113 ABCB1 p.Ala544Asp Mean fluorescence (relative arbitrary fluorescence units) A544D-P-gp1 Wt-P-gp1 HEK293T Intact cells 40 171 3 F&P then labelled with UIC2-PE 197 497 13 Labelled with UIC2-PE then F&P 32 144 3 Intracellular P-gp 155 343 - Cell surface P-gp 37 168 - Total P-gp 192 511 - sylated P-gp1 and mature glycosylated P-gp1. Login to comment 116 ABCB1 p.Ala544Asp If the A544D mutation impairs the trafficking of the protein from the ER to the cell membrane then it might be expected to alter the ratio of immature to mature protein found in the cell. Login to comment 117 ABCB1 p.Ala544Asp PAGE and western analysis (Fig. 5) showed that wild-type P-gp1 was expressed at higher levels than A544D-P-gp1 and was predominantly present as the 170 kDa mature, glycosylated form. Login to comment 118 ABCB1 p.Ala544Asp In contrast, the A544D-P-gp1 was predominantly found in the 140 kDa immature form. Login to comment 120 ABCB1 p.Ala544Asp ABCB1 p.Ala544Asp The ratio of cell surface to intracellular P-gp1 is also altered in the A544D mutant Additional evidence that A544D-P-gp1 is a trafficking mutant was provided by calculating the ratio of cell surface to intracellular P-gp1 by FACS analysis (Fig. 6 and Table 2). Login to comment 124 ABCB1 p.Ala544Asp These data, obtained for cells expressing wild-type P-gp1 and for cells expressing the A544D mutant, are summarized along with the background levels of fluorescence in Table 2. Login to comment 125 ABCB1 p.Ala544Asp Calculation of total P-gp1 level and the ratio of intracellular to cell surface P-gp clearly showed that cells transfected with pMDR1-A544D expressed a lower level of P-gp1 than cells transfected with pMDR1-wt (total fluorescence 192 and 511, respectively), and that much less of it got to the cell surface (only 19% of the total produced, compared with 33% of wild-type protein). Login to comment 127 ABCB4 p.Ala546Asp When considered together, these results demonstrate that not all of the mature protein is at the cell surface. These results show that the MDR3 mutation A546D causes abnormal protein glycosylation and trafficking when its equivalent is expressed in P-gp1. Login to comment 128 ABCB4 p.Ala546Asp DISCUSSION Our analysis of the sequence of the coding region of the MDR3 gene in eight patients with ICP and raised γ-GT has revealed the presence of a mutation in exon 14 (A546D) in one individual. Login to comment 135 ABCB4 p.Ala546Asp The occurrence of the A546D mutation, the substitution of a hydrophobic amino acid with a charged polar amino acid, in the highly conserved first NBD, suggested that this mutation would also result in loss-of-function of the MDR3 protein. Login to comment 136 ABCB4 p.Ala546Asp ABCB4 p.Ala544Asp In order to gain insight into the functional consequences of this mutation, we tested the effects of the equivalent mutation to A546D in P-gp1 (A544D). Login to comment 137 ABCB4 p.Ala544Asp Transient expression of A544D-P-gp1 in HEK293T cells, and subsequent FACS and western analysis suggested that this mutation reduces the abundance of the protein and impairs protein trafficking to the cell Figure 4. Login to comment 138 ABCB4 p.Ala544Asp UIC2-PE antibody labelling of intact HEK293T cells transiently transfected with pCIneo-βgal (grey intermittent line), pMDR1-wt (black) or pMDR1-A544D (grey). Login to comment 140 ABCB4 p.Ala544Asp The transfected populations were observed to consist of two separate populations (M1 and M2 for pMDR1-wt, M3 and M4 for pMDR1-A544D) with different amounts of protein at the cell surface. Login to comment 142 ABCB4 p.Ala544Asp ABCB4 p.Ala544Asp Western analysis of HEK293T cells transiently transfected with pCIneo- βgal (-VE), pMDR1-A544D (A544D) and pMDR1-wt (WT). Login to comment 147 ABCB4 p.Ala546Asp The A546D mutation in the MDR3 protein is likely to have a similar phenotype as there is considerable sequence identity between the proteins in this domain (87%), and because the NBDs of P-gp1 and the MDR3 protein have been shown to be functionally interchangeable (27). Login to comment 174 ABCB4 p.Ala544Asp FACS analysis of cells transiently transfected with pMDR1-A544D (a), pMDR1-wt (b) or pCIneo-βgal (c). Login to comment 199 ABCB4 p.Ala546Asp Study of the MDR3 protein A546D mutation in P-gp1 Plasmid pMDR-wt encodes wild-type P-gp1 with a hexa-histidine tag at the C-terminus and has been described previously (28). Login to comment 203 ABCB4 p.Ala546Asp Site-directed mutagenesis Alignment of the amino acid sequence of human P-gp1 with the MDR3 protein identified A544 as the P-gp1 equivalent of MDR3 protein A546. Site-directed mutagenesis was used to alter codon 544 of MDR1 to encode an aspartic acid, adopting the C→A tranversion of the second position of the codon analogous to the A546D mutant of the MDR3 protein. Login to comment 204 ABCB4 p.Ala544Asp The A544D mutation was introduced into NBD1 of P-gp1 by oligonucleotide-directed mutagenesis ('Altered sites` II; Promega) of pSBH. Login to comment 206 ABCB4 p.Ala544Asp The nucleotide sequence of the mutated DNA was verified by automated DNA sequencing as described above, prior to subcloning the mutated EcoRI-HindIII DNA fragment back into pMDR-wt to generate pMDR-A544D. Login to comment 208 ABCB1 p.Ala544Asp The mutated fragment was subcloned into pMDR1-wt to generate pMDR1-A544D for expression studies in mammalian cells. Login to comment