ABCG2 p.Asn418Gln
| Predicted by SNAP2: | A: D (85%), C: D (91%), D: D (85%), E: D (91%), F: D (95%), G: D (91%), H: D (80%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), P: D (91%), Q: D (91%), R: D (91%), S: D (85%), T: D (85%), V: D (91%), W: D (95%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, E: N, 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] N-Linked glycosylation of the human ABC transporte... Biochemistry. 2005 Apr 12;44(14):5420-9. Diop NK, Hrycyna CA
N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane.
Biochemistry. 2005 Apr 12;44(14):5420-9., 2005-04-12 [PMID:15807535]
Abstract [show]
The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa plasma membrane protein that can confer multidrug resistance to cells in culture when overexpressed. Both transiently and stably expressed ABCG2 are glycosylated, and treatment with peptide N-glycosidase F reduces the apparent molecular mass on SDS-PAGE gels to approximately 60 kDa. Sequence analysis revealed three potential N-linked glycosylation sites in human ABCG2 at amino acids 418, 557, and 596. Site-directed mutagenesis experiments, in which each Asn was changed to Gln independently, revealed that only asparagine 596 is N-linked glycosylated. These data provide the first direct identification of the modified residue in ABCG2 and evidence for the localization of loop 5 to the extracellular space, previously only predicted from hydropathy analysis. Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 (N596Q) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives. Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein. The ABCG2 (N596Q) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2. Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated ( approximately 2-fold) ATPase activities of ABCG2 (N596Q) were affected compared to glycosylated ABCG2. Although subtle defects in transporter trafficking and function may exist, these data taken together suggest that N-glycosylation at arginine 596 is not essential for the expression, trafficking to the plasma membrane, or the overall function of ABCG2.
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No. Sentence Comment
32 In this study, we identified Asn 596 as the single N-linked glycosylated amino acid in ABCG2 using chemical treatments with tunicamycin or PNGase F and site-directed mutagenesis, constructing and evaluating three N f Q variants (N418Q, N557Q, and N596Q).
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ABCG2 p.Asn418Gln 15807535:32:229
status: VERIFIED52 The following genetic variants were obtained: Asn 418 (N418Q), Asn 557 (N557Q), and Asn 596 (N596Q).
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ABCG2 p.Asn418Gln 15807535:52:55
status: VERIFIED59 A 70-80% confluent monolayer of HeLa cells was infected with the vTF 7-3 vaccinia virus (10 pfu/cell) and cotransfected with an appropriate amount of a pTM1 expression plasmid containing ABCG2 or the following ABCG2 variants: N418Q, N557Q, and N596Q.
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ABCG2 p.Asn418Gln 15807535:59:226
status: VERIFIED153 Site-directed mutagenesis was performed on the human ABCG2 cDNA to generate three mutant variants of the half-transporter where the Asn residues were replaced by Gln residues: ABCG2 (N418Q), ABCG2 (N557Q), and ABCG2 (N596Q).
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ABCG2 p.Asn418Gln 15807535:153:183
status: VERIFIED156 ABCG2 (N418Q) and ABCG2 (N557Q) migrated as a range of bands between the 50 and 75 kDa markers and are indistinguishable from the glycosylated ABCG2 control (Figure 4A, lanes 1, 3, and 5).
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ABCG2 p.Asn418Gln 15807535:156:7
status: VERIFIED161 Upon treatment with PNGase F, both ABCG2 variants N418Q (Figure 4A, lane 4) and N557Q (Figure 4A, lane 6) also migrate to the same lower molecular mass position (~60 kDa) as the PNGase F treated ABCG2 glycosylated protein and the untreated N596Q protein (Figure 4A, lanes 2, 4, 6, and 7).
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ABCG2 p.Asn418Gln 15807535:161:50
status: VERIFIED181 The negative control membranes, containing the empty vector pTM1, show a basal activity of approximately 6.5 ( 1.6 nmol π min-1 mg-1 and FIGURE 4: Immunoblot detection of ABCG2 and the N418Q, N557Q, and N596Q ABCG2 variants.
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ABCG2 p.Asn418Gln 15807535:181:191
status: VERIFIED