ABCMdb

PMID: 10213617

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 [PubMed]

Sentences

No. Mutations Sentence Comment

83 ABCB1 p.Glu875Cys During structure-function analysis studies (unpublished observations), we found a single mutant, E875C, that gave increased amounts of glycosylated C-Half polypeptide. Login to comment 85 ABCB1 p.Glu875Cys As shown in Figure 2 (lane 1), expression of the COOH-half of mutant E875C in HEK 293 cells resulted in the appearance of about equivalent amounts of the glycosylated (C-Half-CHO) and unglycosylated (C-Half) forms of the protein. Login to comment 95 ABCB1 p.Glu875Cys Mutation E875C is shown as a shaded circle in TM 10. Login to comment 101 ABCB1 p.Glu875Cys ABCB1 p.Thr811Ala 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. Login to comment 105 ABCB1 p.Glu875Cys Since mutant E875C gave relatively equal amounts of C-Half molecules with both topologies, it was used for further analysis. Login to comment 112 ABCB1 p.Glu875Cys To monitor the expression of the C-Half polypeptide, an A52 epitope tag was attached to mutant E875C C-Half molecule, while the N-Half was not tagged. Login to comment 113 ABCB1 p.Glu875Cys The untagged N-Half molecule of P-gp was coexpressed with the A52-tagged C-Half of mutant E875C in the presence or absence of drug substrate. Login to comment 114 ABCB1 p.Glu875Cys Figure 3 shows the relative expression levels of glycosylated (C-Half-CHO) or unglycosylated (C-Half) forms of mutant E875C C-Half when expressed in the presence or absence of untagged N-Half molecule and drug substrate (cyclosporin A). Login to comment 115 ABCB1 p.Glu875Cys When E875C C-Half was expressed alone, the CL3(cyt) and CL3(ext) topologies were present in about equivalent amounts (Figure 3, lane 2). Login to comment 116 ABCB1 p.Glu875Cys Similar results were obtained when the C-Half of mutant E875C was expressed alone in the presence of drug substrate (lane 3) or when it was coexpressed with the N-Half in the absence of substrate (lane 5). Login to comment 122 ABCB1 p.Glu875Cys In this assay, a histidine-tagged N-Half molecule was coexpressed with an A52 epitope-tagged E875C C-Half. Login to comment 125 ABCB1 p.Glu875Cys Histidine-tagged N-Half P-gp (N-Half-His) was coexpressed with the A52-tagged C-Half of mutant E875C (C-Half-A52) in HEK 293 cells, solubilized with n-dodecyl- -D-maltoside, and the solubilized material was subjected to nickel chromatography (18). Login to comment 133 ABCB1 p.Glu875Cys HEK 293 cells were transfected with A52-tagged mutant E875C C-Half cDNA with or without (+ or -) N-Half P-gp cDNA. Login to comment 137 ABCB1 p.Glu875Cys HEK 293 cells were cotransfected with histidine-tagged N-Half and A52-tagged mutant E875C C-Half cDNAs, and solubilized with n-dodecyl- -D-maltoside and the extract subjected to nickel-chelate chromatography using 10 mM imidazole in the washing steps. Login to comment 144 ABCB1 p.Glu875Cys The full-length mutant E875C P-gp also exhibited drug-stimulated ATPase activities that were about 80-90% of the wild-type enzyme (Figure 5). Login to comment 147 ABCB1 p.Glu875Cys Coexpression of wild-type N-Half-His with mutant E875C C-Half-A52 in the presence of cyclosporin A resulted in the formation of an active complex that had 85-95% of the activity of the wild-type enzyme (Figure 5). Login to comment 160 ABCB1 p.Glu875Cys When the C-Half of mutant E875C was synthesized to the presence of the N-Half polypeptide and cyclosporin A, only the amount of unglycosylated C-Half was increased. Login to comment 171 ABCB1 p.Glu875Cys It is surprising that a single mutation, E875C, promotes an alternative topology in P-gp. Login to comment 173 ABCB1 p.Glu875Cys When expressed in the presence of drug substrates, the mutant E875C showed vinblastineand verapamil-stimulated ATPase activity that was similar to that of wild-type enzyme (Figure 5). Login to comment 174 ABCB1 p.Glu875Cys In the absence of drug substrate, maturation of mutant E875C was inefficient, such that the majority of the product was a core-glycosylated 150 kDa protein (data not shown). Login to comment 177 ABCB1 p.Glu875Cys The FIGURE 5: Drug-stimulated ATPase activities of wild-type and mutant E875C P-gps. Login to comment 179 ABCB1 p.Glu875Cys Equivalent amounts of histidine-tagged full-length wild-type or mutant E875C P-gps isolated by nickel-chelate chromatography, were added to lipid and assayed for ATPase in the absence or presence verapamil (1 mM) or vinblastine (0.05 mM). Login to comment 180 ABCB1 p.Glu875Cys Histidine-tagged N-Half (N-Half-H) that was expressed alone or coexpressed with A52 epitope-tagged wild-type or mutant E875C C-Half were isolated by nickel-chelate chromatography. Login to comment 189 ABCB1 p.Glu875Cys In P-gp, it appears that the E875C mutation influences topological folding of the C-terminal domain such that increased amounts of CL-3(ext) are the major product. Login to comment 192 ABCB1 p.Glu875Cys It is therefore possible that the E875C mutation promotes the formation of P-gp with this topology. Login to comment