Abstract
P-glycoprotein (P-gp), an ATP-dependent efflux pump, is associated with the development of multidrug resistance in cancer cells. Antibody-mediated blockade of human P-gp activity has been shown to overcome drug resistance by re-sensitizing resistant cancer cells to anticancer drugs. Despite the potential clinical application of this finding, the epitopes of the three human P-gp-specific monoclonal antibodies MRK-16, UIC2 and 4E3, which bind to the extracellular loops (ECLs) have not yet been mapped. By generating human-mouse P-gp chimeras, we mapped the epitopes of these antibodies to ECLs 1 and 4. We then identified key amino acids in these regions by replacing mouse residues with homologous human P-gp residues to recover binding of antibodies to the mouse P-gp. We found that changing a total of ten residues, five each in ECL1 and ECL4, was sufficient to recover binding of both MRK-16 and 4E3 antibodies, suggesting a common epitope. However, recovery of the conformation-sensitive UIC2 epitope required replacement of thirteen residues in ECL1 and the same five residues replaced in the ECL4 for MRK-16 and 4E3 binding. These results demonstrate that discontinuous epitopes for MRK-16, UIC2 and 4E3 are located in the same regions of ECL1 and 4 of the multidrug transporter.
Highlights
Of 48 known human ABC (ATP-binding cassette) transporters, ABCB1, known as P-glycoprotein (P-gp) or multidrug resistance 1 (MDR1), is the most studied protein of this superfamily[1,2]
There are four domains in P-gp structure, transmembrane domain 1 (TMD1), nucleotide-binding domain 1 (NBD1), TMD2 and NBD2. As both human and mouse P-gp homologs arise from the folding of a single polypeptide chain that is transcribed and translated in the order (N-term) TMD1-NBD1-TMD2NBD2 (C-term), we used a four-letter code to refer to chimeras throughout the paper, where “H” corresponds to a domain based on the human amino acid sequence and “M” to a domain based on the mouse amino acid sequence
To determine whether the epitopes of any of the tested antibodies were located in the carboxy-terminal half of the molecule, we generated a chimeric P-gp in which the first half of the molecule was human (TMD1 and NBD1, HH) and the second half (TMD2 and NBD2, MM) was from mouse P-gp (HHMM chimera, Fig. 2C)
Summary
Of 48 known human ABC (ATP-binding cassette) transporters, ABCB1, known as P-glycoprotein (P-gp) or multidrug resistance 1 (MDR1), is the most studied protein of this superfamily[1,2]. There are at least three widely-used monoclonal antibodies (UIC217, MRK-1616 and 4E319) that can be used to inhibit human P-gp function (reviewed in Okochi et al.[20]). The MRK-16 antibody was generated by immunizing mice against adriamycin-resistant human myelogenous leukemia K-562 cells[16], whereas UIC2 was developed by immunizations of BALB/c mice with human P-gp expressing BALB/c 3T3-1000 cells[17]. Using cryo-EM, the binding of the Fab of UIC2 to mouse P-gp containing all six ECLs of the human transporter has recently been reported[21]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
