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...

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.

Here, we demonstrate for the first time that the hollow-fibre bioreactor is an excellent tool for the production of Drosophila-expressed recombinant proteins. Using the example of the soluble extracellular portion of the human IL-5 (interleukin 5) receptor alpha expression in S2 (Schneider's Drosophila melanogaster cell line 2) cells, we found that it is possible to produce multi-milligram amounts of functional recombinant protein continuously for several months on a laboratory scale with minimal maintenance requirements. The insect cells grow to high density and express concentrated functional recombinant protein in a small volume, simplifying and economizing downstream purification.