Receptor epitope usage by an interleukin-5 mimetic peptide

Abstract

The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor a (IL5Rα) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Rα interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide-receptor complex is comparable with that of the cytokine-receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Rα, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Rα by alanine substitution of Asp55, Asp56, Glu58, Lys186, Arg188, and Arg297 of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Rα, in particular Asp55, Arg188, and Arg297 in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.