ABCMdb

PMID: 14672610

Plass JR, Mol O, Heegsma J, Geuken M, de Bruin J, Elling G, Muller M, Faber KN, Jansen PL
A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump.
J Hepatol. 2004 Jan;40(1):24-30., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCB11 p.Asp482Gly In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function. Login to comment 4 ABCB11 p.Asp482Gly Methods: Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. Login to comment 6 ABCB11 p.Asp482Gly Results: The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Login to comment 7 ABCB11 p.Asp482Gly Protein expression and canalicular sorting were strongly affected by the D482G mutation. Login to comment 10 ABCB11 p.Asp482Gly Conclusions: These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump. Login to comment 24 ABCB11 p.Asp482Gly Abbreviations: ABC, ATP-binding cassette; ATPase, adenosine triphosphatase; BCV, bile canalicular-like vacuole; BSEP, human bile salt export pump; mBsep, mouse bile salt export pump; D482G, aspartate- to-glycine mutation at position 482 in BSEP/Bsep; EGFP, enhanced green fluorescent protein; NBD, nucleotide binding domain; PCR, polymerase chain reaction; PFIC, progressive familial intrahepatic cholestasis; PNGase F, peptide N-glycosidase F; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TCA, taurocholate. Login to comment 40 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly We investigated the effects of the PFIC-2 related missense mutation D482G, an aspartate to glycine change at position 482 [10], occurring in the first nucleotide-binding domain of BSEP, on the adenosine triphosphatase (ATPase) activity, transport activity, sorting, and expression of the protein. Login to comment 42 ABCB11 p.Asp482Gly In fact, the D482G mutation causes a temperature-sensitive reduction of BSEP protein expression, probably caused by reduced protein stability. Login to comment 48 ABCB11 p.Asp482Gly HepG2 cells were stably transfected with pEGFP-C1-mBsep[WT] or pEGFP-C1-mBsep[D482G] using the calcium phosphate precipitation method [12] and geneticin-resistant clones were selected. Login to comment 55 ABCB11 p.Asp482Gly Site-directed mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) to introduce the D482G mutation in pFASTBAC1-mBsep and pEGFP-C1-mBsep (primers used: mD482Gsense/antisense). Login to comment 87 ABCB11 p.Asp482Gly The D482G mutation is present in the first nucleotide-binding domain of BSEP, which is highly conserved between species (Fig. 1). Login to comment 90 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly The mBsep[D482G] protein is still functional as a bile salt transporter Wild type and D482G mutant mBsep were expressed at comparable levels in Sf21 insect cells (Fig. 2A, insert). Login to comment 94 ABCB11 p.Asp482Gly Similar results were obtained with membrane fractions containing mBsep[D482G] (black bars). Login to comment 96 ABCB11 p.Asp482Gly This shows that the ATPase activity of both WT and D482G mBsep are stimulated by the substrates of the transporter. Login to comment 97 ABCB11 p.Asp482Gly The bile salt transport activity of the wild type and mutant mBsep is shown in Fig. 2B. Both mBsep[WT] - and mBsep[D482G] -vesicles show similar TCA uptake rates (32.7 ^ 1.5 versus 30.5 ^ 3.3 pmol TCA&#b7;min21 &#b7;mg21 protein). Login to comment 99 ABCB11 p.Asp482Gly The EGFP-mBsep[D482G] is inefficiently targeted to the canalicular membrane in HepG2 cells To study the protein stability and subcellular sorting of wild type and mutant mBsep, we generated stable HepG2 cell lines that express these proteins, N-terminally tagged to the EGFP. Login to comment 111 ABCB11 p.Asp482Gly The EGFP-mBsep[D482G] mutant protein was detected in the BCV`s but, in addition, a considerable amount of protein was retained in the cytoplasm (Figs. 3D-F). Login to comment 113 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly The D482G mutation results in unstable and immature mBsep protein Next, we analyzed the correlation between the mRNA and protein level of cells stably expressing EGFP-mBsep[WT] and EGFP-mBsep[D482G] . Login to comment 115 ABCB11 p.Asp482Gly However, the EGFP-mBsep[D482G] signal at this position is hardly detectable (Fig. 4, lane 1). Login to comment 119 ABCB11 p.Asp482Gly PNGase F-treatment of EGFP-mBsep[D482G] resulted in the disappearance of the minor 190 kDa band. Login to comment 121 ABCB11 p.Asp482Gly This shows that the 160 kDa EGFP-mBsep[D482G] protein band is full-length, but improperly glycosylated protein. Login to comment 122 ABCB11 p.Asp482Gly Moreover, the specific amount of EGFP-mBsep[D482G] protein appeared lower than the EGFP-mBsep[WT] protein. Login to comment 123 ABCB11 p.Asp482Gly Therefore, we determined the specific mRNA levels of EGFP-mBsep[WT] and EGFP-mBsep[D482G] in the corresponding cell lines. Login to comment 124 ABCB11 p.Asp482Gly In contrast to the difference in protein level, the relative amount of EGFP-mBsep[D482G] was 4.5-fold higher compared to the EGFP-mBsep[WT] mRNA level (Fig. 5A). Login to comment 125 ABCB11 p.Asp482Gly These data show that high mRNA levels for EGFP-mBsep[D482G] do not give rise to similarly high protein levels, relative to the results obtained for EGFP-mBsep[WT] . Login to comment 129 ABCB11 p.Asp482Gly Therefore, we cultured both the EGFP-mBsep[WT] and EGFP-mBsep[D482G] HepG2 cells at 308C and determined the specific mRNA and protein levels. Login to comment 130 ABCB11 p.Asp482Gly Surprisingly, we found that the relative mRNA levels for both EGFP-mBsep[WT] and EGFP-mBsep[D482G] increased approximately 12-fold in cells grown at 308C compared to cells grown at 378C (Fig. 5A). Login to comment 131 ABCB11 p.Asp482Gly However, the 4.5-fold difference between EGFP-mBsep[D482G] and EGFP-mBsep[WT] remained despite the overall higher mRNA levels. Login to comment 132 ABCB11 p.Asp482Gly In cells grown at 308C, the protein level of both EGFP-mBsep[WT] and EGFP-mBsep[D482G] increased significantly when compared to cells at 378C (Fig. 5B). Login to comment 133 ABCB11 p.Asp482Gly However, the EGFP-mBsep[D482G] showed a much stronger increase in protein level than the EGFP-mBsep[WT] . Login to comment 134 ABCB11 p.Asp482Gly Moreover, at 308C the EGFP-mBsep[D482G] appeared predominantly in fully glycosylated form. Login to comment 135 ABCB11 p.Asp482Gly Under these conditions, the EGFP-mBsep[D482G] protein level was approximately 5-fold higher than EGFP-mBsep[WT] as determined by densitometry. Login to comment 137 ABCB11 p.Asp482Gly Confocal laser scanning microscopy revealed that at 308C, the EGFP-mBsep[D482G] protein was efficiently targeted to the BCV`s (Figs. 3G-I). Login to comment 139 ABCB11 p.Asp482Gly The D482G mutation does not block the ATPase and transport activity of mBsep. Login to comment 140 ABCB11 p.Asp482Gly (A) Membrane vesicles containing comparable levels of wild type or D482G mutant mBsep (insert) are subjected to an ATPase assay in the absence or presence of 30 mM TCA and 200 mM orthovanadate as described in Section 2. Login to comment 148 ABCB11 p.Asp482Gly Discussion In this study, we analysed the effect of the PFIC-2 mutation, D482G, on Bsep function and intracellular expression. Login to comment 151 ABCB11 p.Asp482Gly The D482G-mutant Bsep protein appears to be temperature-sensitive. Login to comment 152 ABCB11 p.Asp482Gly At 308C, high levels of normally glycosylated, and correctly sorted Bsep[D482G] were detected. Login to comment 156 ABCB11 p.Asp482Gly Efficient sorting of the D482G mutant mBsep in HepG2 cells is temperature-sensitive. Login to comment 158 ABCB11 p.Asp482Gly At 378C, both EGFP-tagged wild type (A, C) and D482G mutant (D, F) mBsep sort to the BCV. Login to comment 159 ABCB11 p.Asp482Gly However, significant amounts of the D482G mutant mBsep are detected intracellular (D, F). Login to comment 162 ABCB11 p.Asp482Gly When cells were grown at 308C, the D482G mutant mBsep was solely observed in the BCV`s. Login to comment 167 ABCB11 p.Asp482Gly The D482G mutation leads to low levels and incompletely glycosylated mBsep protein. Login to comment 168 ABCB11 p.Asp482Gly Western blot analysis using a GFP antibody of approximately160 mg of total protein extracts from EGFP-mBsep[D482G] HepG2 cells (lane 1), and EGFP-mBsep[WT] cells (lanes 2 and 3). Login to comment 170 ABCB11 p.Asp482Gly Since the D482G mutation is present in the first NBD of BSEP, we first tested its effect on the ATPase activity. Login to comment 173 ABCB11 p.Asp482Gly The ATPase activity of the mBsep[D482G] -protein was also induced by TCA, be it to a lower level than the WT protein. Login to comment 175 ABCB11 p.Asp482Gly Taken together, these results show that the mBsep[D482G] is a functional bile salt transporter with biochemical characteristics comparable to the wild type protein. Login to comment 176 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly To study the effect of the D482G mutation on intracellular sorting and protein stability, we constructed stable HepG2 cell lines, expressing mBsep[WT] or mBsep[D482G] N-terminally tagged with EGFP. Login to comment 177 ABCB11 p.Asp482Gly Detectable amounts of EGFP-mBsep[D482G] protein were only observed in clones with significantly (5-fold) higher mRNA levels as compared to EGFP-mBsep[WT] . Login to comment 179 ABCB11 p.Asp482Gly This was confirmed by confocal laser scanning microscopy analyses, in which significant amounts of the EGFP-mBsep[D482G] protein were detected in a fine reticulum-like pattern in the cytoplasm. Login to comment 181 ABCB11 p.Asp482Gly Collectively, our findings suggest that the D482G mutation results in low BSEP protein levels and that the mutant protein is improperly glycosylated and targeted at physiological conditions. Login to comment 186 ABCB11 p.Asp482Gly Therefore, we cultured our stable cell lines at reduced (308C) temperatures and found that the mRNA level for both EGFP-mBsep[WT] and EGFP-mBsep[D482G] increased approximately 12-fold compared to the level in cells grown at 378C. Login to comment 192 ABCB11 p.Asp482Gly At 308C, both the mRNA and protein level of EGFP-mBsep[D482G] were approximately 5-fold higher than those for EGFP-mBsep[WT] mRNA level. Login to comment 196 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly They found that the D482G mutation reduced the taurocholate transport activity of rat Bsep by approximately 50% and GFP-tagged rBsep[D482G] was found at the apical membrane and in the cytoplasm of Madin-Darby canine kidney (MDCK) cells. Login to comment 197 ABCB11 p.Asp482Gly The correlation between GFP- rBsep[D482G] mRNA and protein levels was not determined in detail. Login to comment 198 ABCB11 p.Asp482Gly Our results show little or no effect of the D482G mutation on the BSEP transport function, but a very strong effect on protein stability, maturation and/or turnover. Login to comment 202 ABCB11 p.Asp482Gly The deleterious effect of the D482G mutation could Fig. 5. Login to comment 206 ABCB11 p.Asp482Gly (B) Total protein lysates from HepG2 cells expressing the wild type or D482G mutant mBsep were isolated. Login to comment 213 ABCB11 p.Asp482Gly In conclusion, our data show that PFIC-2 patients with the D482G mutation express a functional, but highly unstable bile salt export pump. Login to comment