ABCMdb

PMID: 17855769

Lam P, Pearson CL, Soroka CJ, Xu S, Mennone A, Boyer JL
Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases.
Am J Physiol Cell Physiol. 2007 Nov;293(5):C1709-16. Epub 2007 Sep 13., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Login to comment 10 ABCB11 p.Glu297Gly Bile salt transport was retained in all but the E297G mutant. Login to comment 11 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT Ͼ N591S Ͼ R1050C ϳ A570T ϳ E297G ϾϾ D482G. Login to comment 14 ABCB11 p.Asp482Gly Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic. Login to comment 32 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly These studies suggest that two PFIC2 mutations, D482G and E297G, lead to reduced total protein expression presumably due to folding, processing, and/or trafficking defects (8, 19, 22, 30). Login to comment 39 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly 0363-6143/07 $8.00 Copyright (c) 2007 the American Physiological Societyhttp://www.ajpcell.org C1709 trast to these conclusions, we find that all five green fluorescent protein (GFP)-tagged mutant proteins, including D482G and E297G, traffic to the cell surface as detected by confocal microscopy and by biotinylation of plasma membrane Bsep when expressed in MDCK and HEK293 cells, respectively. Login to comment 41 ABCB11 p.Glu297Gly Since ATPase and bile salt transport activity when expressed in Sf9 cells are also normal (with the exception of the E297G mutant) the severity of the clinical phenotype correlates most closely with mutation-induced defects in cell surface expression of the mature protein. Login to comment 45 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr All point mutations (D482G, E297G, A570T, R1050C, N591S) were introduced with the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Login to comment 64 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr PFIC2 (D482G, E297G), BRIC2 (A570T, R1050C), and ICP (N591S) mutations that are investigated in this study are indicated in italics. Login to comment 67 ABCB11 p.Val444Ala V444A (F) has been identified as a polymorphic site (14). Login to comment 84 ABCB11 p.Asp482Gly Stably transfected D482G HEK293 cells were treated with and without 2 ␮M MG-132 for 16 h. Login to comment 94 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr Sf9 cells infected with recombinant virus (mock, WT, D482G, E297G, A570T, R1050C, and N591S) were harvested, and cell membranes were prepared as described previously (23). Login to comment 107 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr The subcellular distributions of D482G, E297G, A570T, R1050C, and N591S were examined first because intracellular accumulation of misfolded proteins has been shown for many diseases. Login to comment 118 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr The level of cell surface expression of Bsep protein was quantitated by densitometry and determined to be in the following order: WT (100%) Ͼ N591S (75.6 Ϯ 15.6%) Ͼ E297G (38.5 Ϯ 12.6%) ϳ R1050C (35.6 Ϯ 14.5%) ϳ A570T (29.5 Ϯ 8.8%) ϾϾ D482G (5.7 Ϯ 2.3%). Login to comment 120 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr The total expression (cell surface and intracellular proteins) also showed changes similar to those in cell surface expression: WT (100%) Ͼ N591S (73.5 Ϯ 4.3%) Ͼ E297G (33.7 Ϯ 20.4%) ϳ R1050C (26.7 Ϯ 16.0%) ϳ A570T (11.9 Ϯ 5.2%) ϾϾ D482G (2.5 Ϯ 2.0%) (Fig. 4B). Login to comment 132 ABCB11 p.Asp482Gly We colocalized the D482G mutant with endogenous ubiquitin and with transfected rat ubiquitin tagged with hemagglutinin (Fig. 5C). Login to comment 135 ABCB11 p.Asp482Gly To further confirm that Bsep is ubiquitinated, we immunoprecipitated D482G molecules from stably transfected cells and blotted the immunoprecipitates with an anti-ubiquitin antibody (Fig. 5D). Login to comment 137 ABCB11 p.Asp482Gly MG-132 treatment increased both the D482G protein and its ubiquitinated form. Login to comment 138 ABCB11 p.Asp482Gly These results indicate that the proteasome is involved in the turnover of D482G protein. Login to comment 140 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly To investigate whether chemical chaperones could stabilize the D482G mutant, we found that reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate could enhance the expression of the mature D482G protein (band C) and surface expression in stably transfected HEK293 cells (Fig. 6). Login to comment 141 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly Recently, Hayashi and Sugiyama (7) also showed that sodium 4-phenylbutyrate enhances the expression of cell surface human D482G protein in MDCK cells. These results confirm that 4-phenylbutyrate has similar effect in stabilizing D482G mutant with human or rat gene. Login to comment 146 ABCB11 p.Asn591Ser Membrane vesicles containing WT Bsep (17.1 Ϯ 2.5 nmol Pi ⅐min-1 ⅐mg protein-1 ) and mutant N591S Bsep (18.4 Ϯ 1.53 nmol Pi ⅐min-1 ⅐mg protein-1 ) showed slightly higher basal ATPase activity than membrane vesicles containing the mock-transfected control (12.3 Ϯ 0.67 nmol Pi ⅐min-1 ⅐mg protein-1 ) (Fig. 7B). Login to comment 150 ABCB11 p.Asp482Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr The membrane vesicles expressing D482G, A570T, R1050C, or N591S showed similar levels of taurocholate uptake compared with WT Bsep. Login to comment 151 ABCB11 p.Glu297Gly The E297G mutation reduced the taurocholate uptake of Bsep to a level similar to membrane vesicles from mock-treated cells. Login to comment 152 ABCB11 p.Glu297Gly Although the mutations have significant effects on Bsep protein expression, the catalytic functions of the protein are maintained in all mutants except the E297G-expressing membrane vesicles. Login to comment 155 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr Cells were transiently transfected with pEGFPN1 (control), rat Bsep-GFP [wild type (WT)], D482G-GFP, E297G-GFP, A570T-GFP, R1050C-GFP, and N591S-GFP constructs. Login to comment 157 ABCB11 p.Asn591Ser In contrast, 4 of the mutant GFP-tagged Bsep proteins are variably localized to both intracellular and apical membranes, whereas localization of N591S-GFP is similar to WT. Login to comment 164 ABCB11 p.Asp482Gly ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr A PFIC2 mutation (D482G) is highly unstable (ϳ5% of WT), while the BRIC2 variants A570T and R1050C are expressed at the plasma membrane at intermediate levels (ϳ25% of WT). Login to comment 165 ABCB11 p.Asn591Ser In contrast, the surface expression of the ICP variant N591S is close to the level of the WT protein (ϳ70%). Login to comment 169 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr Cells were transfected with rat Bsep-GFP (WT), D482G-GFP, E297G-GFP, A570T-GFP, R1050C-GFP, and N591S-GFP constructs and were examined for GFP by confocal laser microscopy. Login to comment 171 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr D482G, E297G, A570T, and R1050C mutants partially colocalized with recycling endosome marker Rab11. Login to comment 172 ABCB11 p.Asn591Ser N591S was mainly expressed on the membrane surface, a pattern similar to the WT protein. Login to comment 176 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr D4, D482G; E2, E297G; A5, A570T; R10, R1050C; N5, N591S. Login to comment 183 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr P Ͻ 0.05, D482G, E297G, A570T, R1050C, and N591S compared with WT control. Login to comment 186 ABCB11 p.Asp482Gly This study shows that the D482G mutant is ubiquitinated and that proteasome inhibition increases the level of the mutant protein, thereby providing a means to regulate Bsep stability. Login to comment 191 ABCB11 p.Val444Ala A third patient with severe ICP was found to have combined homozygous alterations of a BSEP polymorphism (V444A) and a MDR3 missense mutation, possibly explaining the early onset and severity of the ICP (10). Login to comment 192 ABCB11 p.Asp482Gly Our results confirm previous results from this lab (30) and others (8, 19, 22) that have described a low expression of the PFIC2 mutant D482G in human, rat, and mouse Bsep following transfection into different mammalian cell lines. Login to comment 193 ABCB11 p.Glu297Gly With the exception of the E297G variant, all of the studied variants Fig. 5. Login to comment 196 ABCB11 p.Asp482Gly Twenty-five micrograms of protein was used for each reaction, except that 100 ␮g of D482G protein was used for digestion to detect expression (**). Login to comment 200 ABCB11 p.Asp482Gly C: Bsep-D482G-GFP (a) colocalizes with transfected hemagglutinin (HA)-tagged ubiquitin (b) with HA antibody. Login to comment 202 ABCB11 p.Asp482Gly Bar ϭ 5 ␮m. D: Western blots of total lysates or immunoprecipitates (IP) of D482G stably transfected HEK293 cells treated with and without MG-132 using anti-ubiquitin (ub) antibody or Bsep antibody (Bsep). Login to comment 204 ABCB11 p.Asp482Gly Temperature and chemical chaperones rescue mature D482G mutant protein. Login to comment 205 ABCB11 p.Asp482Gly A: stably transfected WT (top) and D482G (bottom) in HEK293 cells were incubated at 37°C or shifted to 27°C overnight. Login to comment 206 ABCB11 p.Asp482Gly There was a marked increase in plasma membrane expression of the D482G-GFP mutant compared with slight increase in plasma membrane expression of WT-GFP. Login to comment 207 ABCB11 p.Asp482Gly Western immunoblotting shows the increase in expression of mature and immature protein of D482G (bottom) compared with little change in expression of WT (top) after temperature rescue. Login to comment 208 ABCB11 p.Asp482Gly ABCB11 p.Asp482Gly B: chemical chaperones sodium butyrate (NaB) and sodium 4-phenylbutyrate (NaPB) and DMSO control (Ctl) were used to treat WT (top) and D482G (bottom) stably transfected HEK293 cells for 24 h. NaB and NaPB enhanced the expression of D482G at the plasma membrane and increased the expression levels of mature and immature protein (bottom). Login to comment 210 ABCB11 p.Glu297Gly In contrast, in membrane vesicles from HEK293 cells that overexpress the human E297G variant, taurocholate uptake activity is retained with the same transport efficiency as WT Bsep (8). Login to comment 211 ABCB11 p.Glu297Gly In addition, in patients the E297G mutation has been associated with different clinical phenotypes as well as in healthy relatives (9, 19, 28). Login to comment 212 ABCB11 p.Glu297Gly On the basis of this study and others, it appears that the biochemical data in the human HEK293 cells indicate that E297G mutation results in some protein expression and has residual bile acid transport activity. Login to comment 213 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly However, the absence of bile acid transport activity in Sf9 membrane vesicles expressing the E297G variant suggests that the mechanism for the E297G-associated defect is likely to be more complex. Login to comment 220 ABCB11 p.Asp482Gly Reduced temperature and 4-phenylbutyrate have been demonstrated in this study and by others (7) to increase mature and cell surface protein expression for the D482G mutant. Login to comment 228 ABCB11 p.Glu297Gly Bsep function [ATPase, taurocholate (TCA) transport] is retained in Bsep mutants except E297G mutant in Sf9 cells. Login to comment 229 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr A: immunoblot of the isolated Sf9 cell membrane proteins with anti-Bsep polyclonal antibody: GFP control expressed in HEK293 cells, WT expressed in HEK293 cells, rat liver homogenate (L), pFastBac1-Gus control (C), wild type (WT), D482G (D4), E297G (E2), A570T (A5), R1050C (R10), N591S (N5). Login to comment 231 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Arg1050Cys ABCB11 p.Ala570Thr B: TCA (200 ␮M) stimulates and orthovanadate inhibits ATPase activity in membrane vesicles containing comparable levels of WT or D482G, E297G, A570T, R1050C, and N591S mutant Bsep. Login to comment 234 ABCB11 p.Glu297Gly C: except for the E297G mutant, Bsep WT and mutant-expressing Sf9 cells demonstrate marked ATP stimulated [3 H] TCA in the same membrane vesicles as in Fig. 6C. Login to comment 237 ABCB11 p.Glu297Gly **P Ͻ 0.05 for E297G compared with WT control. 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