ABCMdb

PMID: 15875186

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., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCG2 p.Asn596Ala 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. Login to comment 8 ABCG2 p.Asn557Ala 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. Login to comment 45 ABCG2 p.Asn596Ala ABCG2 p.Asn418Ala ABCG2 p.Asn557Ala 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. Login to comment 46 ABCG2 p.Asn596Ala ABCG2 p.Asn418Ala ABCG2 p.Asn418Ala ABCG2 p.Asn418Ala ABCG2 p.Asn418Ala ABCG2 p.Asn557Ala ABCG2 p.Asn557Ala ABCG2 p.Asn557Ala 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. Login to comment 105 ABCG2 p.Asn418Ala Expression of BCRP-N418A resulted in detection of two BCRP bands at the same position as in wild-type BCRP transfectants, indicating that asparagine 418 is not significantly glycosylated. Login to comment 106 ABCG2 p.Asn557Ala ABCG2 p.Asn557Ala In BCRP-N557A transfectants, only the lower band was detected. As this band was PNGase F sensitive, BCRP-N557A was still glycosylated. Login to comment 110 ABCG2 p.Asn596Ala A protein containing all three mutations [triple (T) mutant] showed the same result as the BCRP-N596A mutant. Login to comment 130 ABCG2 p.Asn557Ala ABCG2 p.Asn557Ala These results suggest that both BCRP-N557A and the triple mutant were not properly targeted to the plasma membrane as a consequence of the N557A mutation. Login to comment 131 ABCG2 p.Asn596Ala 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. Login to comment 155 ABCG2 p.Asn596Ala Fig. 3 BCRP-N596A is not glycosylated. Login to comment 156 ABCG2 p.Asn596Ala ABCG2 p.Asn418Ala ABCG2 p.Asn557Ala 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. Login to comment 159 ABCG2 p.Asn557Ala N non-glycosylated C core-glycosylated G fully glycosylated In our experiments, BCRP-N557A was apparently core-glycosylated and localized in the ER. Login to comment 166 ABCG2 p.Asn557Arg However, expression of the mutant BCRP-N557R gave a low expression and the Western blot suggests that only one band was detected instead of the two bands in the other BCRP expressing cells in the same experiment. Login to comment 167 ABCG2 p.Asn557Arg This suggests that the BCRP-N557R mutant also has a (partial) problem with its folding in the ER. Login to comment 169 ABCG2 p.Asn557Ala We cannot exclude that in our study also a small fraction of BCRP-N557A reaches the plasma membrane. Login to comment 170 ABCG2 p.Asn557Asp ABCG2 p.Asn557Glu ABCG2 p.Asn557Arg ABCG2 p.Asn557His The described difference in resistance of the BCRP-N557H, BCRP-N557D, BCRP-N557E and BCRP-N557R mutants could be partially due to a small difference in folding of BCRP next to a difference in affinity. Login to comment 186 ABCG2 p.Asn596Ala ABCG2 p.Asn418Ala Annu Rev Cell Dev Biol 16:113-143 Fig. 4 Wild-type BCRP, BCRP-N418A and BCRP-N596A are localized at the plasma membrane. Login to comment 187 ABCG2 p.Asn557Ala BCRP-N557A and a triple mutant is localized intracellularly. Login to comment 188 ABCG2 p.Asn596Ala ABCG2 p.Asn418Ala ABCG2 p.Asn557Ala 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. Login to comment