ABCB1 p.Glu875Cys
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PMID: 10213617
[PubMed]
Loo TW et al: "The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates."
No.
Sentence
Comment
83
During structure-function analysis studies (unpublished observations), we found a single mutant, E875C, that gave increased amounts of glycosylated C-Half polypeptide.
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ABCB1 p.Glu875Cys 10213617:83:97
status: NEW85 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.
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ABCB1 p.Glu875Cys 10213617:85:69
status: NEW95 Mutation E875C is shown as a shaded circle in TM 10.
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ABCB1 p.Glu875Cys 10213617:95:9
status: NEW101 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.Glu875Cys 10213617:101:80
status: NEW105 Since mutant E875C gave relatively equal amounts of C-Half molecules with both topologies, it was used for further analysis.
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ABCB1 p.Glu875Cys 10213617:105:13
status: NEW112 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.
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ABCB1 p.Glu875Cys 10213617:112:95
status: NEW113 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.
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ABCB1 p.Glu875Cys 10213617:113:90
status: NEW114 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).
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ABCB1 p.Glu875Cys 10213617:114:118
status: NEW115 When E875C C-Half was expressed alone, the CL3(cyt) and CL3(ext) topologies were present in about equivalent amounts (Figure 3, lane 2).
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ABCB1 p.Glu875Cys 10213617:115:5
status: NEW116 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).
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ABCB1 p.Glu875Cys 10213617:116:56
status: NEW122 In this assay, a histidine-tagged N-Half molecule was coexpressed with an A52 epitope-tagged E875C C-Half.
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ABCB1 p.Glu875Cys 10213617:122:93
status: NEW125 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).
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ABCB1 p.Glu875Cys 10213617:125:95
status: NEW133 HEK 293 cells were transfected with A52-tagged mutant E875C C-Half cDNA with or without (+ or -) N-Half P-gp cDNA.
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ABCB1 p.Glu875Cys 10213617:133:54
status: NEW137 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.
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ABCB1 p.Glu875Cys 10213617:137:84
status: NEW144 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).
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ABCB1 p.Glu875Cys 10213617:144:23
status: NEW147 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).
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ABCB1 p.Glu875Cys 10213617:147:49
status: NEW160 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.
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ABCB1 p.Glu875Cys 10213617:160:26
status: NEW171 It is surprising that a single mutation, E875C, promotes an alternative topology in P-gp.
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ABCB1 p.Glu875Cys 10213617:171:41
status: NEW173 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).
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ABCB1 p.Glu875Cys 10213617:173:62
status: NEW174 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).
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ABCB1 p.Glu875Cys 10213617:174:55
status: NEW177 The FIGURE 5: Drug-stimulated ATPase activities of wild-type and mutant E875C P-gps.
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ABCB1 p.Glu875Cys 10213617:177:72
status: NEW179 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).
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ABCB1 p.Glu875Cys 10213617:179:71
status: NEW180 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.
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ABCB1 p.Glu875Cys 10213617:180:119
status: NEW189 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.
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ABCB1 p.Glu875Cys 10213617:189:29
status: NEW192 It is therefore possible that the E875C mutation promotes the formation of P-gp with this topology.
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ABCB1 p.Glu875Cys 10213617:192:34
status: NEW
PMID: 10681495
[PubMed]
Loo TW et al: "The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis."
No.
Sentence
Comment
65
Twelve of the 125 P-gp mutants (TM4/TM12 constructs L227C/S993C, V231C/S993C, W232C/S993C, A233C/S993C, I235C/S993C, and L236C/S993C; TM5/TM12 constructs A295C/S993C and I299C/S993C; TM10/TM6 constructs V874C/P350C, E875C/ P350C, and M876C/P350C; and TM11/TM6 construct G939C/ P350C), however, had slower mobilities in SDS-PAGE after treatment with oxidant.
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ABCB1 p.Glu875Cys 10681495:65:216
status: NEW77 In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Glu875Cys 10681495:77:1221
status: NEW82 Cyclosporin A inhibited the cross-linking of mutants L227C(TM4)/S993C(TM12), W232C(TM4)/ S993C(TM12), I299C(TM5)/S993(TM12), E875C (TM10)/ P350C(TM6), and M876C(TM10)/P350C(TM6).
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ABCB1 p.Glu875Cys 10681495:82:125
status: NEW99 Mutants L227C/S993C, V231C/ S993C, W232C/S993C, A233C/S993C, I235C/S993C, L236C/ S993C, A295C/S993C, I299C/S993C, V874C/P350C, E875C/ P350C, M876C/P350C, and G939C/P350C were inhibited by 81, 88, 90, 89, 93, 81, 78, 87, 87, 77, 70, and 78%, respectively.
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ABCB1 p.Glu875Cys 10681495:99:127
status: NEW118 TABLE II Minimum temperature required for cross-linking Residues TM segments 4 °C 21 °C 37 °C L227C/S993C 4/12 -a - ϩ V231C/S993C 4/12 - ϩ ϩ W232C/S993C 4/12 - ϩ ϩ A233C/S993C 4/12 ϩb ϩ ϩ I235C/S993C 4/12 ϩ ϩ ϩ L236C/S993C 4/12 ϩ ϩ ϩ A295C/S993C 5/12 - ϩ ϩ I299C/S993C 5/12 ϩ ϩ ϩ V874C/P350C 10/6 - ϩ ϩ E875C/P350C 10/6 - - ϩ M876C/P350C 10/6 - ϩ ϩ G939C/P350C 11/6 - ϩ ϩ a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Glu875Cys 10681495:118:444
status: NEW
PMID: 21182301
[PubMed]
Loo TW et al: "The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants."
No.
Sentence
Comment
309
It was not surprising that the mutations affected rhodamine B-stimulated ATPase activity because it was previously shown that treatment of mutants W232C, N296C, and T945C (E875C was not tested) with a thiol-reactive analogue of rhodamine inhibited activity (15).
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ABCB1 p.Glu875Cys 21182301:309:172
status: NEW366 It was previously reported that residue Glu875 could contribute to folding of TM segments because the E875C mutation altered the topology of C-half Pgp (45).
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ABCB1 p.Glu875Cys 21182301:366:102
status: NEW