ABCB1 p.Asn99Ala
| Predicted by SNAP2: | A: N (53%), C: D (63%), D: D (66%), E: D (63%), F: N (57%), G: D (53%), H: N (61%), I: N (53%), K: D (63%), L: N (57%), M: N (61%), P: D (63%), Q: N (61%), R: D (66%), S: N (61%), T: N (66%), V: N (53%), W: D (71%), Y: N (53%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: D, G: N, H: N, I: N, K: N, L: N, M: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D, |
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[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]
Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.
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No. Sentence Comment
128 Mutants ⌬NBD2- P-gp, M68R/⌬NBD2-P-gp, and ⌬NBD2-P-gp lacking the three glycosylation sites (N91A, N94A, N99A) were expressed in HEK 293 cells, and whole cell SDS extracts were subjected to immunoblot analysis.
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ABCB1 p.Asn99Ala 18596043:128:125
status: NEW160 Whole cell extracts of HEK 293 cells expressing mutants ⌬NBD2-P-gp with no changes (None), the M68R mutation, or N91A, N94A and N99A changes to the glycosylation sites (Unglycos) were subjected to immunoblotanalysis.Thepositionsofmature,immature,andunglycosylated(Unglycos) forms of ⌬NBD2 P-gp are indicated.
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ABCB1 p.Asn99Ala 18596043:160:135
status: NEW[hide] Identification of residues in the drug-binding sit... J Biol Chem. 1997 Dec 19;272(51):31945-8. Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PMID:9405384]
Abstract [show]
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.
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No. Sentence Comment
65 B, whole cell extracts of HEK 293 cells expressing wild-type, Cys-less, glycosylation-deficient N91A/N94A/N99A and single Cys mutants that exhibited little or no verapamil-stimulated ATPase activity were subjected to immunoblot analysis as described under "Experimental Procedures."
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ABCB1 p.Asn99Ala 9405384:65:106
status: NEW90 This appeared to be a degradation product rather than a nonglycosylated product because it had a higher mobility than the glycosylation-deficient P-glycoprotein (N91A/N94A/N99A) (Fig. 2B, lanes 23 and 24).
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ABCB1 p.Asn99Ala 9405384:90:172
status: NEW[hide] Quality control by proteases in the endoplasmic re... J Biol Chem. 1998 Dec 4;273(49):32373-6. Loo TW, Clarke DM
Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein.
J Biol Chem. 1998 Dec 4;273(49):32373-6., 1998-12-04 [PMID:9829963]
Abstract [show]
Human P-glycoprotein is synthesized in HEK 293 cells as two major products: the 150-kDa core-glycosylated intermediate and the 170-kDa mature proteins. The 150- and 170-kDa proteins were not detected in mutants such as G341C. The major protein in this mutant was a 130-kDa proteolytic degradation product. This result suggested that the mutant protein was misfolded and sensitive to proteolytic digestion during or immediately after synthesis. We found that mutation of Arg113, located in the first extracellular loop of P-glycoprotein and near the consensus glycosylation sites, to Ala, Lys, Glu, Met, or Cys blocked formation of the 130-kDa product. Introduction of R113A into mutant G341C resulted in the synthesis of a mature (170 kDa) and functional transporter. Similarly, when R113A was introduced into misprocessed mutants, there was increased synthesis of the 150-kDa core-glycosylated intermediate. Maturation of the core-glycosylated intermediate into the mature enzyme, however, was not observed. These results suggest that polytopic proteins are accessible to proteases in the lumen of the endoplasmic reticulum during biosynthesis and that proteases are important contributors to the quality control mechanism involved in protein folding. It is also shown that unstable proteins can be made more stable by removal of hypersensitive proteolytic sites.
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No. Sentence Comment
33 A glycosylation-deficient mutant was made by mutating the three consensus glycosylation sites (N91A,N94A,N99A).
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ABCB1 p.Asn99Ala 9829963:33:105
status: NEW71 The 130-kDa product was also observed when the glycosylation-deficient (N91A,N94A,N99A) mutant was expressed in the presence of proteasome inhibitors (Fig. 1, lanes 5 and 6).
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ABCB1 p.Asn99Ala 9829963:71:82
status: NEW93 HEK 293 cells were transfected with Cys-less (C-less), mutant G341C (in Cys-less background), or glycosylation-deficient (N91A,N94A,N99A; -Glycos.)
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ABCB1 p.Asn99Ala 9829963:93:132
status: NEW[hide] Purification and characterization of N-glycosylati... Arch Biochem Biophys. 2001 Apr 1;388(1):171-7. Urbatsch IL, Wilke-Mounts S, Gimi K, Senior AE
Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.
Arch Biochem Biophys. 2001 Apr 1;388(1):171-7., [PMID:11361134]
Abstract [show]
P-glycoprotein confers multidrug resistance in mammalian cells and basic structure-function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al. (1999) J. Biol. Chem. 274, 34711-34718). The degree of glycosylation of these preparations was unknown, and was of relevance for crystallization studies. Therefore mutant proteins in which the N-glycosylation sites were eliminated (Asn --> Gln in mouse MDR3 Pgp, Asn --> Gln or Ala in human MDR1 Pgp) were expressed in P. pastoris and purified to homogeneity. Yields of mutant Pgp were the same as for parent wild-type proteins. Nucleotide-binding and catalytic (ATPase) characteristics were completely normal in the mutant proteins. Mass spectrometry indicated that mutant and wild-type proteins did not differ significantly in mass, demonstrating that the wild-type proteins contain no N-glycosylation.
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No. Sentence Comment
45 A BglII-BglII fragment from expression vector pMT21 containing mutations N91A, N94A, and N99A in the human MDR1 sequence (21) was transferred into pHIL-MDR1.4-His10(⌬BglII) (above) yielding pHIL- AAAMDR1.4-His10.
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ABCB1 p.Asn99Ala 11361134:45:89
status: NEW