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 ␣ (IL5R␣) 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 IL5R␣ interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide⅐receptor complex is comparable with that of the cytokine⅐receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5R␣, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its Asthma is an incurable disease characterized by eosinophil bronchial inflammation and tissue remodeling of the airway wall (1). Human interleukin (IL) 1 -5 is a key cytokine that plays a critical role in the differentiation, proliferation, migration, and activation of eosinophils and has been implicated in the pathogenesis of eosinophil-associated allergic inflammation such as asthma (2, 3). Injection of IL5 increases eosinophil numbers in the blood, bone marrow, spleen, and peritoneal cavities (4, 5). This increase can be prevented by the passive administration of anti-IL5 or anti-IL5 receptor antibodies (5, 6). One distinguishing characteristic of IL5 versus other cytokines is that IL5 functions selectively to activate eosinophils (7). These data argue that IL5 plays a central role in the development of allergen-induced eosinophils and suggest that the ability to block IL5 activity evokes the potential for alternative treatment for asthma.At the molecular level, IL5 leads to biological functions through recruitment of a cell-surface receptor composed of two polypeptide chains, ␣ and ␤ (8). The ␣ chain is IL5-specific and is called IL5 receptor ␣ (IL5R␣), whereas the ␤ chain is shared with IL3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) (9 -11) and is called the common ␤ chain (␤c). Despite a high degree of amino acid sequence similarity of the ␣ chains for IL5, IL3, and GM-CSF, their interaction with cognate cytokine is strictly specific, and no cross-reactivity is found among these cytokines. Previous observations argue that receptor subunit recruitment occurs stepwise, with initial formation of the IL5⅐IL5R␣ complex required for ␤c binding to induce cytoplasmic signal transduction (12). IL5R␣ alone binds IL5 with an equilibrium dissociation constant of 0.8 nM when expressed in COS cells, and this binding affinity is increased by only 1.5-fold when ␣ and ␤ chains a...

Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging

“…2C) and that this increased FRET was abolished by the addition of AF17121 (Fig. 2D), an IL-5R␣ antagonist peptide (37,43). AF17121 inhibition of the IL-5/IL-5R␣ interaction at the ligand-binding epitope in IL-5R␣ is consistent with the view that complex formation of IL-5 with the extracellular region of IL-5R␣ is necessary to enable closer proximity between ␣-␤c subunits in the heteromeric complex.…”

“…2B). IL-5 has been shown to bind to the extracellular region of IL-5R␣ (both soluble and full-length forms) in a dose-dependent manner (18,37,52,55). No evidence exists to support direct binding of IL-5 to the ␤c subunit on its own (13,56).…”

“…This visible increase in fluorescence of donor after photobleaching suggested that FRET was occurring between the tagged IL-5R␣ and ␤c subunits in the presence of IL-5. This response was inhibited by AF17121, a peptide antagonist of IL-5R␣ (37,43) (Fig. 2B, third row).…”

“…Protein Expression and Purification-The human IL-5 protein was expressed and purified as previously described (18,37). AF17121 peptide was synthesized and purified as before (33,42).…”

A Protein Engineering Approach Differentiates the Functional Importance of Carbohydrate Moieties of Interleukin-5 Receptor α

“…Asn 196 of IL5Rα and Asn 200 of IL12 are colored green, and other asparagine residues for N-glycosylation are colored yellow. The IL5-binding residues at site I and II epitopes, which have been found previously, 12,32 are shown as a CPK model. The loop regions are represented as coils and β strands as ribbons.…”