ABCB1 p.Thr811Ala
| Predicted by SNAP2: | A: D (53%), C: D (53%), D: D (91%), E: D (75%), F: D (85%), G: D (85%), H: D (80%), I: D (53%), K: D (80%), L: D (66%), M: D (66%), N: D (85%), P: N (53%), Q: D (85%), R: D (85%), S: N (66%), V: N (82%), W: D (85%), Y: D (85%), |
| 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, N: D, P: D, Q: D, R: D, S: D, V: N, W: D, Y: D, |
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[hide] The glycosylation and orientation in the membrane ... Biochemistry. 1999 Apr 20;38(16):5124-9. Loo TW, Clarke DM
The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates.
Biochemistry. 1999 Apr 20;38(16):5124-9., 1999-04-20 [PMID:10213617]
Abstract [show]
Multiple topologies have been detected for the COOH-terminal half of the human multidrug resistance P-glycoprotein (P-gp). In one topology, the predicted third cytoplasmic loop (CL3) is on the cytoplasmic side (P-gp-CL3-cyt) of the membrane. In an alternate topology, CL3 is on the extracellular side of the membrane (P-gp-CL3-ext). It is not known if both forms of P-gp are active because it is difficult to distinguish either topology in the full-length molecule. When the halves of P-gp are expressed as separate polypeptides, the two topologies of the C-Half are readily distinguished on SDS-PAGE, because only the C-Half (CL3-ext) is glycosylated. To test whether both topologies can fold into an active enzyme, we assayed for interaction between the N- and C-Halves of P-gp since functional P-gp requires interaction between both halves. In a mutant P-gp (E875C) that gave about equal amounts of both topologies, only the C-Half (CL3-cyt) could be recovered by nickel chromatography after coexpression with the histidine-tagged N-Half P-gp. The isolated N-Half and E875C C-Half (CL3-cyt) polypeptides, when expressed together, exhibited verapamil- and vinblastine-stimulated ATPase activities that were similar to the wild-type enzyme. We also found that biosynthesis of mutant E875C C-Half in the presence of the N-Half P-gp resulted in enhanced expression of C-Half (CL3-cyt). By contrast, interaction of C-Half (CL3-ext) with N-Half P-gp was not detected. These results show that the topology of the C-Half portion of P-gp greatly influences its interactions with the amino-terminal half of the molecule.
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No. Sentence Comment
101 Whole cell extracts of cells expressing the A52-tagged wild-type C-Half, mutant E875C C-Half or mutant T811A C-Half P-gps were treated with (+Endo H) or without (-Endo H) endoglycosidase H and subjected to SDS-PAGE and immunoblot analysis with monoclonal antibody A52.
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ABCB1 p.Thr811Ala 10213617:101:103
status: NEW