ABCMdb

PMID: 18698235

Kobayashi K, Ito K, Takada T, Sugiyama Y, Suzuki H
Functional analysis of nonsynonymous single nucleotide polymorphism type ATP-binding cassette transmembrane transporter subfamily C member 3.
Pharmacogenet Genomics. 2008 Sep;18(9):823-33., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC3 p.Arg1381Ser Results After transient transfection of cDNA into HeLa cells, it was found that R1381S ABCC3 exhibits intracellular accumulation of immature protein, the localization of which was mostly merged with a marker for the endoplasmic reticulum. Login to comment 5 ABCC3 p.Ser346Phe ABCC3 p.Ser607Asn Two kinds of SNPs type ABCC3 (S346F and S607N) lost their transport activity for [3 H]estradiol-17b-D-glucuronide in membrane vesicles from Sf9 cells infected with the recombinant baculoviruses, although the band length and the amount of protein expression remained normal. Login to comment 6 ABCC3 p.Pro920Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Gly11Asp ABCC3 p.Val765Leu In contrast, the cellular localization, protein expression and function of other eight kinds of SNPs type ABCC3 (G11D, R99Q, V765L, P920S, R923Q, R1286G, R1348C, and Q1365R ABCC3) remained normal. Login to comment 7 ABCC3 p.Ser346Phe ABCC3 p.Arg1381Ser ABCC3 p.Ser607Asn Conclusion The results of this study suggest that the possession of R1381S, S346F, and S607N types of ABCC3 sequences may be a possible risk factor for the acquisition of hepatotoxicity, due to their poor ability to transport toxic compounds across the sinusoidal membrane. Login to comment 40 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu Using site-directed mutagenesis, SNP-type ABCC3 (G11D, R99Q, S346F, S607N, V765L, P920S, R923Q, R1286G, R1348C, Q1365R, and R1381S ABCC3) was constructed on a pBluescript SK ( - ) vector. Login to comment 80 ABCC3 p.Arg1348Cys For example, R1348C was found with high frequency in African-American (5.17%) and sub-Saharan-African (7.5%) populations, but not found in other populations, even if similar sample sizes were screened . Login to comment 81 ABCC3 p.Arg1381Ser In contrast, R1381S was only found in the Mexican population, but not in other populations (Table 1). Login to comment 86 ABCC3 p.Arg1381Ser Except for R1381S ABCC3, other all SNP-type ABCC3 were localized on the plasma membrane of HeLa cells, as observed for the wild-type ABCC3. Login to comment 87 ABCC3 p.Arg1381Ser R1381S ABCC3 was mostly located intracellularly (Fig. 2a). Login to comment 88 ABCC3 p.Arg1381Ser Although the localization of R1381S ABCC3 on the plasma membrane was observed in some HeLa cells, this cell population accounted for only approximately 20% of the transfected cells, which was in marked contrast to the wild-type ABCC3. Login to comment 89 ABCC3 p.Arg1381Ser Then, we examined the localization of R1381S ABCC3 by double-label experiments with anti-ABCC3 antiserum and anti-calnexin antibody, which allows staining of the endoplasmic reticulum (ER) (Fig. 2b). Login to comment 91 ABCC3 p.Arg1381Ser In contrast, the localization of R1381S ABCC3 mostly merged with that of calnexin. Login to comment 92 ABCC3 p.Arg1381Ser It was found that a fraction of the R1381S molecules were located on intracellular organelles other than ER in HeLa cells (Fig. 2b). Login to comment 93 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu Protein expression of ATP-binding cassette transmembrane transporter subfamily C member 3 in HeLa cells The protein expression and modification of ABCC3 in HeLa cells were determined by Western blot analysis Fig. 1 G11D 0.18% R99Q 0.54% S346F 0.91% S607N 0.99% V765L 1.09% P920S 3.88% R923Q 0.55% R1286G 0.18% R1348C 1.65% R1381S 0.22% Q1365R 0.33% Outside Inside NBD2 NBD1 Predicted secondary structure of ATP-binding cassette transmembrane transporter subfamily C member 3 showing the position of the eleven nonsynonymous variants. Login to comment 103 ABCC3 p.Arg1381Ser Similar band patterns were observed for other SNP-type ABCC3 except for R1381S ABCC3. Login to comment 104 ABCC3 p.Arg1381Ser For R1381S ABCC3, the shortest 160-kDa band was the major product (Fig. 3a). Login to comment 107 ABCC3 p.Arg1381Ser As shown in Fig. 3b, a strong 160-kDa band and faint higher molecular weight bands were observed in R1381S without any treatment. Login to comment 108 ABCC3 p.Arg1381Ser In addition, treatment of R1381S ABCC3 with PNGase F gave a single 160-kDa band, whereas treatment with Endo H did not affect the band pattern. Login to comment 109 ABCC3 p.Arg1381Ser ABCC3 p.Arg1381Ser These results may be accounted for by assuming that the major 160-kDa band for R1381S ABCC3 may represent the nonglycosylated form and faint higher molecular weight bands of R1381S ABCC3 may represent the fully N-glycosylated form, thereby confirming resistance to Endo H treatment. Login to comment 110 ABCC3 p.Arg1381Ser ABCC3 p.Arg1381Ser Collectively, R1381S ABCC3 molecules are trapped mostly in the ER as the immature form, whereas only a minor population of R1381S ABCC3, which is resistant to Endo H digestion, may be located in cellular compartments other than ER. Login to comment 117 ABCC3 p.Arg1381Ser As observed for R1381S ABCC3, mutated rat Abcc2 provided substantial colocalization with calnexin in HEK293 cells. Login to comment 118 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu Transport activities of the ATP-binding cassette transmembrane transporter subfamily C member 3 variants The transport function of wild-type and SNP-type ABCC3 was studied by examining the ATP-dependent uptake of [3 H]E217bG into membrane vesicles isolated from Sf9 cells infected with recombinant baculoviruses Table 1 Allele frequency of ABCC3 SNPs examined in this study Ethnic population SNP sequence Asian (%) African-American (%) Pacific islander (%) Mexican (%) Caucasian (%) SubSaharan- African (%) Total (%) Reference G11D 0.83 (120) 0 (160) 0 (12) 0 (100) 0 (160) 0.18 (552) PharmGKB 0.6 (178)a Fukushima-Uesaka et al. 0.5 (206) Lang et al. R99Q 0 (120) 1.88 (160) 0 (12) 0 (100) 0 (160) 0.54 (552) PharmGKB S346F 0 (120) 0.62 (160) 0 (12) 0 (100) 2.5 (160) 0.91 (552) PharmGKB 0.5 (206) Lang et al. S607N 0 (296) 2.76 (290) 0 (10) 1 (100) 0 (210) 0.99 (906) PharmGKB 11.7 (120) dbSNP V765L 0 (120) 3.12 (160) 0 (12) 1 (100) 0 (160) 1.09 (552) PharmGKB P920S 0 (292) 10.3 (290) 0 (12) 0 (100) 2.4 (280) 3.88 (902) PharmGKB R923Q 0 (108) 0.62 (160) 0 (12) 0 (100) 1.25 (160) 0.55 (550) PharmGKB R1286G 0 (120) 0.62 (160) 0 (12) 0 (100) 0 (160) 0.18 (552) PharmGKB R1348C 0 (298) 5.17 (290) 0 (12) 0 (100) 0 (210) 1.65 (910) PharmGKB 7.5 (120) dbSNP Q1365R 0 (298) 1.05 (286) 0 (12) 0 (100) 0 (208) 0.33 (904) PharmGKB R1381S 0 (298) 0 (290) 0 (12) 2 (100) 0 (210) 0.22 (910) PharmGKB Allele frequencies (%) in individual ethnic population are shown. Login to comment 124 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu ABCC3 SNP analysis Kobayashi et al. 827 Fig. WT R1381S G11D R99Q S346F S607N V765L P920S R923Q R1286G WT ABCC3 Calnexin Calnexin merge R1381S merge ABCC3 R1348C Q1365R (a) (b) Subcellular localization of ATP-binding cassette transmembrane transporter subfamily C member 3 (ABCC3) in HeLa cells. Login to comment 138 ABCC3 p.Ser346Phe ABCC3 p.Ser607Asn As shown in Fig. 7, for all SNP-type variants except for S346F and S607N ABCC3, ATP-dependent uptake normalized by the protein expression was similar to the wild-type ABCC3. Login to comment 139 ABCC3 p.Ser346Phe ABCC3 p.Ser607Asn In contrast, S346F and S607N ABCC3-mediated transport was hardly detectable. Login to comment 140 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu Discussion On account of its inducible nature under cholestatic conditions, ABCC3/Abcc3 is considered to provide a compensatory efflux pathway for endogenous and exo- Table 2 Corresponding amino acid residues in other ABCC family proteins Protein Species G11D R99Q S346F S607N V765L P920S R923Q R1286G R1348C Q1365R R1381S ABCC3 Human G R S S V P R R R Q R Abcc3 Mouse G S T N V T K R V Q R Abcc3 Rat G S T N V P K S F Q R ABCC1 Human S I D S V R N V I K R ABCC2 Human - A L G T K R K I K R ABCC4 Human - - L L V Q R E W K R ABCC5 Human - - L L L - D E K K R Amino acid residues in other ABCC family proteins at the corresponding positions to 11 amino acid residues in human ABCC3 examined in this study. Login to comment 142 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg99Gln ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu Fig. 3 (a) (b) Treatment WT R1381S Endo HPNGaseEndo HPNGase 190 kDa 180 kDa 170 kDa 160 kDa 190 kDa 170 kDa 160 kDa WT G11D S346F R99Q S607N V765L P920S R923Q R1286G R1348C Q1365R R1381S pcDNA3.1(+) Western blot analysis of wild-type and single nucleotide polymorphism-type ATP-binding cassette transmembrane transporter subfamily C member 3 (ABCC3) proteins expressed in HeLa cells. Login to comment 150 ABCC3 p.Ser346Phe ABCC3 p.Arg1381Ser ABCC3 p.Ser607Asn It was found that R1381S ABCC3 accumulates in the ER as an immature form with a molecular weight of 160 kDa (Figs 2 and 3), whereas S346F and S607N ABCC3 lost their transport function (Fig. 7). Login to comment 151 ABCC3 p.Arg1381Ser It is possible that R1381S could not fold properly and, consequently was trapped in the ER with calnexin, a molecular chaperon in the ER. Login to comment 152 ABCC3 p.Arg1381Ser Altered transport activity was expected for such misfolded protein; however, R1381S ABCC3 exhibited apparently normal transport activity for E217bG when expressed in insect Sf9 cells. Login to comment 153 ABCC3 p.Arg1381Ser To account for the unaltered transport activity of R1381S ABCC3 expressed in Sf9 cells, two hypotheses may be postulated. Login to comment 154 ABCC3 p.Arg1381Ser ABCC3 p.Arg1381Ser One possibility is that R1381S ABCC3 may be properly folded in Sf9 cells and the other is that the R1381S ABCC3 is still functional even if this molecule is not properly folded. Login to comment 156 ABCB11 p.Glu297Gly A similar phenomenon that the protein accumulated in ER seems potentially functional has been reported for ABCB11 mutations, such as E297G Fig. 4 rat Abcc2-WT rat Abcc2 Calnexin rat Abcc2 Calnexin merge merge rat Abcc2-R1388S Subcellular localization of mutated rat Abcc2 in HEK293 cells. Login to comment 160 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Gly11Asp ABCC3 p.Val765Leu ABCC2 p.Ser607Asn Fig. 5 WT 170 kDa Band density 1.00 1.01 0.77 1.21 1.16 0.95 1.00 1.01 0.91 1.29 1.10 G11D S346F S607N V765L P920S R923Q R1286G R1348C Q1365R R1381S GFP Western blot analysis of wild-type or single nucleotide polymorphisms-type ATP-binding cassette transmembrane transporter subfamily C member 3 (ABCC3) proteins in membrane vesicles isolated from Sf9 cells. Login to comment 169 ABCB11 p.Asp482Gly 830 and D482G, both of which are frequently found in progressive familial intrahepatic cholestasis type 2 patients [39,40]. Login to comment 171 ABCC3 p.Arg1381Ser The cellular localization of R1381S ABCC3 should also be discussed in more detail. Login to comment 172 ABCC3 p.Arg1381Ser Although R1381S ABCC3 exhibited altered localization in HeLa cells, this should be regarded with caution, because sorting machinery is sometimes cell type-specific. Login to comment 173 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr For example, E297G ABCB11 is mislocalized to ER when heterologously expressed in MDCK cells [39,40], although the patient with compound heterozygosity for E297G and R432T ABCB11 showed normal canalicular localization, and the primary cause of dysfunction of ABCB11 was attributed to the reduced transport function [41]. Login to comment 178 ABCC3 p.Ser346Phe ABCC3 p.Ser607Asn The transport function of S346F and S607N ABCC3 was shown to be reduced using E217bG as a substrate, although these two type ABCC3 SNPs were observed in abundance on the membrane surface of HeLa cells as wild-type ABCC3 (Fig. 2a). Login to comment 188 ABCC3 p.Arg1297His Lee et al. [32] focused on SNP R1297H, one of the nonsynonymous ABCC3 SNPs frequently observed in Caucasians with an allele frequency of 8%, although this SNP did not affect ABCC3 protein localization and function when expressed in MDCK cells [32]. Login to comment 190 ABCC3 p.Ser346Phe ABCC3 p.Pro920Ser ABCC3 p.Arg1381Ser ABCC3 p.Arg923Gln ABCC3 p.Gln1365Arg ABCC3 p.Arg1348Cys ABCC3 p.Arg1286Gly ABCC3 p.Ser607Asn ABCC3 p.Gly11Asp ABCC3 p.Val765Leu These are Q741Stop and Fig. 7 10 8 ATP-dependentuptakenormalizedbyrelative ABCC3expressionlevel(pmol/mgprotein/2min) 6 * * ** ** ** ** ** ** ** 4 2 WT G11D S346F S607N V765L P920S R923Q R1286G R1348C Q1365R R1381S GFP 0 Uptake of [3 H]estradiol-17b-D-glucuronide (E217bG) by ATP-binding cassette transmembrane transporter subfamily C member 3 (ABCC3) variants. Login to comment 200 ABCC3 p.Ser346Phe ABCC3 p.Ser607Asn In conclusion, we have analyzed 11 nonsynonymous SNPs found in the SNP database of ABCC3 and found that three of these are expected to be nonfunctional for clear reasons; the impaired function of S346F and S607N is because of impaired transport function, whereas that of R1381R is because of impaired intracellular trafficking. Login to comment