Topology of P-glycoprotein as determined by epitope mapping of MRK-16 monoclonal antibody.
There is growing evidence for the direct role of P-glycoprotein mediating multidrug resistance in tumor cells. P-glycoprotein is thought to function as an energy-dependent drug efflux pump. The monoclonal antibody MRK-16 binds to an external domain of P-glycoprotein and partially inhibits drug efflux in multidrug-resistant cells. As an approach toward elucidating the mechanism by which MRK-16 affects drug transport, we undertook the definition of the precise binding site of this antibody. In this study we have mapped the epitope of MRK-16 monoclonal antibody to a resolution of a single amino acid using a series of overlapping synthetic peptides. We demonstrate that MRK-16 recognizes only the class I isoform (MDR1) of human P-glycoprotein and that its epitope encompasses at least two (first and fourth) of the six predicted extracellular peptide loops. These results suggest that the epitope of MRK-16 is discontinuous and that the sequences involved which are separated by about 625 amino acids in the linear sequence must be spatially situated in close proximity in the native protein. Based on these results, we present a model for transmembrane alpha-helical packing of P-glycoprotein in the lipid bilayer. This may have implications for understanding the function of P-glycoprotein in drug transport.
Georges
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Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada.
Tsuruo
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T
Ling
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D004317:Doxorubicin; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D007951:Leukemia, Myeloid; D008051:Lipid Bilayers / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D004317:Doxorubicin