ABCG2 p.Asn596Ala
| Predicted by SNAP2: | A: D (85%), C: D (91%), D: D (91%), E: D (91%), F: D (95%), G: D (85%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), P: D (91%), Q: D (85%), R: D (91%), S: D (85%), T: D (80%), V: D (91%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: N, T: D, V: D, W: D, Y: D, |
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[hide] Characterization of polarized expression of point-... Pharm Res. 2005 Mar;22(3):458-64. Takada T, Suzuki H, Sugiyama Y
Characterization of polarized expression of point- or deletion-mutated human BCRP/ABCG2 in LLC-PK1 cells.
Pharm Res. 2005 Mar;22(3):458-64., [PMID:15835752]
Abstract [show]
PURPOSE: In polarized cells, such as hepatocytes and intestinal epithelial cells, transporters are localized on the apical or basolateral membranes and play important roles in the vectorial transport of their substrates. In the current study, we have aimed to clarify the mechanism for the cellular sorting of human breast cancer resistance protein (BCRP/ABCG2), which is expressed on the apical membrane of many tissues and functions as an efflux transporter. METHODS: After the expression vector, including wild type or mutants of human BCRP cDNA, was transfected into LLC-PK1 cells, immunohistochemical staining and Western blot analyses were performed to characterize the cellular localization and the status of BCRP, respectively. RESULTS: The transfected cDNA product of wild-type BCRP was expressed on the apical membrane in LLC-PK1 cells. Glycosylation consensus sequences-disrupted mutants showed the apical localization as the wild type, whereas the apical-selective expression disappeared when disulfide bonds could not be formed. Furthermore, examination of the localization of deletion mutants of human BCRP emphasized the importance of some peptide sequences. The region between the N-terminal and ATP-binding cassette and proximal C-terminal region, both of which are well conserved in various animal species, were found to be significant for proper localization. CONCLUSIONS: These results suggest that, although the presence of N-glycan does not affect the localization of BCRP, disulfide bonds and some peptide sequences in both the N- and C-terminals are necessary for the apical expression of BCRP.
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No. Sentence Comment
36 Using the site-directed mutagenesis technique, several mutants of BCRP (N418A, N596A, N418A&N596A BCRP and C592A, C603A&C608A, C592A&C603A&C608A BCRP) were constructed on pcDNA3.1(+) vector.
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ABCG2 p.Asn596Ala 15835752:36:79
status: VERIFIED57 Western blot analysis revealed that the modification was on the latter asparagine (Fig. 3B); the N418A mutant was N-glycosylated, whereas the N596A and N418A&N596A mutants did not exhibit any alteration in molecular weight following PNGase F treatment.
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ABCG2 p.Asn596Ala 15835752:57:142
status: VERIFIED[hide] Absence of N-linked glycosylation does not affect ... Cancer Chemother Pharmacol. 2005 Oct;56(4):344-50. Epub 2005 May 5. Mohrmann K, van Eijndhoven MA, Schinkel AH, Schellens JH
Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2).
Cancer Chemother Pharmacol. 2005 Oct;56(4):344-50. Epub 2005 May 5., [PMID:15875186]
Abstract [show]
Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette (ABC) multidrug transporter that confers resistance to various anticancer drugs like topotecan and mitoxantrone. To obtain more insight in its cellular functioning, we investigated phosphorylation and N-linked glycosylation of BCRP. In the epithelial Madin-Darby canine kidney (MDCK) cell line, we did not detect phosphorylation of BCRP, in contrast to MRP2, which was phosphorylated. In the ovarian carcinoma cell line T8 also no phosphorylated BCRP was detected. As BCRP in both lines effectively transports drugs, it appears that phosphorylation of BCRP (if it occurs at all) is not needed for drug transport. We further mutated the asparagine residues 418, 557 and 596 in three putative N-linked glycosylation motifs of BCRP to alanines. Mutant proteins were expressed in CHO9 and MDCKII cells by transient transfection and characterized by Western blot and immunofluorescence analysis. We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated. The mutation of asparagine 596 (or 418) had little effect on the subcellular localization of BCRP, indicating that N-linked glycosylation is not essential for routing to the plasma membrane. However, BCRP-N557A and the triple mutant were mainly localized intracellularly, probably in the endoplasmic reticulum, suggesting that this mutation disrupted proper routing of BCRP.
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No. Sentence Comment
6 We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated.
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ABCG2 p.Asn596Ala 15875186:6:24
status: VERIFIED45 BCRP-N418A, BCRP-N557A and BCRP-N596A were made using pcDNA3/BCRP wild type (GenBank accession number AY017168; generously provided by Susan Bates) as template.
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ABCG2 p.Asn596Ala 15875186:45:32
status: VERIFIED46 BCRP-N418A/ N557A was made using overlap extension PCR with pGEM-T Easy/BCRP-N418A as template and BCRP-N418A/N557A/N596A was made with pGEM-T Easy/ BCRP-N418A/N557A as template again using overlap extension PCR.
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ABCG2 p.Asn596Ala 15875186:46:116
status: VERIFIED110 A protein containing all three mutations [triple (T) mutant] showed the same result as the BCRP-N596A mutant.
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ABCG2 p.Asn596Ala 15875186:110:96
status: VERIFIED131 The non-glycosylated BCRP-N596A mutant, however, showed a normal plasma membrane staining in both CHO9 and MDCKII cells, indicating that N-glycosylation of BCRP is not important for its plasma membrane targeting.
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ABCG2 p.Asn596Ala 15875186:131:26
status: VERIFIED155 Fig. 3 BCRP-N596A is not glycosylated.
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ABCG2 p.Asn596Ala 15875186:155:12
status: VERIFIED156 Empty vector pcDNA3 or pcDNA3 with BCRP or mutant BCRP, BCRP-N418A, BCRP-N557A and BCRP-N596A, was transiently transfected into CHO9 cells or MDCKII cells using FuGENE6.
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ABCG2 p.Asn596Ala 15875186:156:88
status: VERIFIED186 Annu Rev Cell Dev Biol 16:113-143 Fig. 4 Wild-type BCRP, BCRP-N418A and BCRP-N596A are localized at the plasma membrane.
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ABCG2 p.Asn596Ala 15875186:186:77
status: VERIFIED188 Empty vector pcDNA3 or pcDNA3 with BCRP or mutant BCRP, BCRP-N418A, BCRP-N557A and BCRP-N596A, was transiently transfected into CHO9 cells or MDCKII cells using FuGENE6.
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ABCG2 p.Asn596Ala 15875186:188:88
status: VERIFIED[hide] Tunicamycin potentiates cisplatin anticancer effic... Mol Cancer Ther. 2013 Dec;12(12):2874-84. doi: 10.1158/1535-7163.MCT-13-0201. Epub 2013 Oct 15. Hou H, Sun H, Lu P, Ge C, Zhang L, Li H, Zhao F, Tian H, Zhang L, Chen T, Yao M, Li J
Tunicamycin potentiates cisplatin anticancer efficacy through the DPAGT1/Akt/ABCG2 pathway in mouse Xenograft models of human hepatocellular carcinoma.
Mol Cancer Ther. 2013 Dec;12(12):2874-84. doi: 10.1158/1535-7163.MCT-13-0201. Epub 2013 Oct 15., [PMID:24130050]
Abstract [show]
Hepatocellular carcinoma is highly chemoresistant, and ATP-binding cassette subfamily G member 2 (ABCG2) is thought to play a critical role in this drug resistance. The present study aims to develop effective therapeutic strategies to decrease ABCG2 expression level and to surmount drug resistance in hepatocellular carcinoma chemotherapy. First, we verified a positive correlation between the ABCG2 protein level and the drug resistance of hepatocellular carcinoma cell lines. ABCG2 was preferentially expressed in highly chemoresistant hepatocellular carcinoma cancer stem cells (CSC) enriched with CD133. In addition, ABCG2 was N-linked glycosylated in hepatocellular carcinoma cells, and this modification was involved in sustaining its protein stability. The N-linked glycosylation (NLG) inhibitor tunicamycin dramatically reduced ABCG2 expression, altered its subcellular localization, and reversed its drug efflux effect in multiple hepatocellular carcinoma cell lines. Furthermore, tunicamycin reduced the expression levels of several CSC markers and suppressed the tumorigenicity of CD133(+) CSCs. Tunicamycin combined with cisplatin (CDDP) inhibited proliferating cell nuclear antigen (PCNA) expression and increased the cleavage of PARP; this effect was partially rescued by the overexpression of ABCG2 or Akt-myr. The combination therapy more effectively suppressed tumor growth in xenograft mice than did single-agent therapy with either drug. Finally, the CDDP treatment combined with UDP-GlcNAc-dolichol-phosphate N-acetylglucosamine-1 phosphate transferase (DPAGT1) knockdown recapitulated the effect observed when CDDP was used in combination with tunicamycin. In summary, our results suggest that tunicamycin may reverse the drug resistance and improve the efficacy of combination treatments for hepatocellular carcinomas by targeting the DPAGT1/Akt/ABCG2 pathway.
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No. Sentence Comment
190 A previous report showed that NLG is not essential for routing ABCG2 to the plasma membrane because the N596A mutation had little effect on ABCG2 subcellular localization (28).
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ABCG2 p.Asn596Ala 24130050:190:104
status: NEW