title: Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody.
abstract: The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.
authors.affiliation: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Gutay-Tóth
authors.affiliation: Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Hungary.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Fenyvesi
authors.affiliation: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Bársony
authors.affiliation: CycloLab Cyclodextrin Research & Development Laboratory, Ltd., Hungary.
authors.forename: Lajos
authors.initials: L
authors.lastname: Szente
authors.affiliation: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Goda
authors.affiliation: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Szabó
authors.affiliation: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary. Electronic address: bacso@med.unideb.hu.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Bacsó
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D001667:Binding, Competitive; D002784:Cholesterol / Q000378:metabolism*; D000939:Epitopes; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014162:Transfection; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000911:Antibodies, Monoclonal; D000939:Epitopes; D047392:beta-Cyclodextrins; D002784:Cholesterol
keywords: 15D3; Antibody competition test; Conformation; Cyclodextrin; Flow cytometric detergent resistance; P-glycoprotein; UIC2