Identification by phage display of the linear continuous MRPr1 epitope in the multidrug resistance-associated protein (MRP1).
In order to study the structure of the multidrug resistance-associated protein (MRP1), which is one of the most important members of the ATP-binding cassette (ABC) protein family acting as drug-efflux systems, we have developed an epitope mapping-based strategy. By means of the mAb MRPr1, we have immunoselected clones from two distinct random peptide libraries displayed on phages and have identified several peptide sequences mimicking the internal conformation of this 190 kDa multidrug transporter protein. Phage clones able to block the immunolabeling of the MRPr1 antibody to MRP1-overexpressing multidrug resistance (MDR) H69/AR cells were isolated and, after sequencing the corresponding inserts, their amino acid sequence was compared to that of MRP1. This analysis led to the identification of the consensus sequence L.SLNWED, corresponding to the MRP1 segment LWSLNKED (residues 241-248). This MRP1 sequence is partially overlapping with the MRPr1 epitope GSDLWSLNKE (residues 238-247) previously mapped using peptide scanning techniques. These results demonstrate the high reliability of phage display technology to study not only the topography of complex integral membrane proteins such as MRP1, but also to help identify critical residues participating in the formation of the epitope structure.
Flego
Michela
M
Laboratory of Immunology, Istituto Superiore di Sanità, Viale R. Elena 299, I-00161, Rome, Italy.
Mennella
Vito
V
Moretti
Franca
F
Poloni
Francesca
F
Dupuis
Maria Luisa
ML
Ascione
Alessandro
A
Barca
Stefano
S
Felici
Franco
F
Cianfriglia
Maurizio
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D018604:Epitope Mapping; D005434:Flow Cytometry; D007120:Immunochemistry; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D019151:Peptide Library; D010449:Peptide Mapping
D004277:DNA, Single-Stranded; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019151:Peptide Library; C421109:multidrug resistance-associated protein 1