Receptor epitope usage by an interleukin-5 mimetic peptide.
The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) 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 IL5Ralpha 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 IL5Ralpha, 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 IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) 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.
Ishino
Tetsuya
T
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Urbina
Cecilia
C
Bhattacharya
Madhushree
M
Panarello
Dominick
D
Chaiken
Irwin
I
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D016207:Cytokines / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007377:Interleukin-3 / Q000378:metabolism; D015848:Interleukin-5 / Q000737:chemistry* / Q000378:metabolism; D053666:Interleukin-5 Receptor alpha Subunit; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013879:Thioredoxins / Q000737:chemistry; D013997:Time Factors
D016207:Cytokines; D000939:Epitopes; D005353:Fibronectins; C485918:IL5RA protein, human; D007377:Interleukin-3; D015848:Interleukin-5; D053666:Interleukin-5 Receptor alpha Subunit; D010455:Peptides; D011108:Polymers; D018123:Receptors, Interleukin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D001120:Arginine; D000409:Alanine