Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

Ishino, Tetsuya; Pasut, Gianfranco; Scibek, Jeffery J.; Chaiken, Irwin M.

doi:10.1074/jbc.m309327200

Cited by 39 publications

(74 citation statements)

References 41 publications

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“…In particular, the mass transfer effect can be a major artifact when the association rate is fast or when a large amount of immobilization is required (30). Indeed, the binding of IL5 to sIL5R␣ was compromised by the mass transfer effect even when using as low a level of immobilization as possible (19). The presence of the mass transfer effect is easily detected, as the binding signal will be dependent on the level of immobilization.…”

Section: Design Production and Characterization Of Thioredoxinfusedmentioning

confidence: 96%

“…sIL5R␣ and its mutational variants (D55A, D56A, E58A, K186A, R188A, and R297A) were transiently expressed in Drosophila S2 cells and secreted into serum-free medium as described previously (19). Cell-free supernatant was collected after 2 days and stored at Ϫ20°C for the following binding analysis.…”

Section: S2 Cell Expression Of Il5r␣ and Its Mutational Variantsmentioning

confidence: 99%

“…Noncovalent Immobilization of IL5R␣ and Binding Assay of the Fusion Peptides-The conformation-sensitive anti-IL5R␣ monoclonal antibody ␣16 (26) was covalently immobilized on a CM5 sensor chip, and sIL5R␣ was captured by the antibody as described previously (19). Various concentrations of trx-AF17121 or trx-AF17121(AA) were passed over the antibody-captured sIL5R␣ surface.…”

Section: Spr Biosensor Binding Analysismentioning

confidence: 99%

“…Here, to validate the direct binding of AF17121 to IL5R␣ and the binding stoichiometry, we employed the sandwich SPRbased binding assay, which we have established for cytokinereceptor interaction (15,19). Fig.…”

Section: Design Production and Characterization Of Thioredoxinfusedmentioning

confidence: 99%

“…IL5R␣ comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region, and the membrane-proximal pair of D2 and D3 domains constitutes a cytokine recognition motif that generally can be recognized by a cytokine (18). Recently, we found that the IL5-binding residues are located in the D1 domain (Asp 55 , Asp 56 , and Glu 58 ) as well as in the D2 domain (Lys 186 and Arg 188 ) and the D3 domain (Arg 297 ) of IL5R␣ (19). The homology-deduced IL5R␣ structure indicates that the binding interface of IL5R␣ comprises a cluster of negatively charged residues from the D1 domain and a cluster of positively charged residues from the D2D3 tandem domain.…”

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confidence: 99%

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Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Ishino

Urbina

Bhattacharya

et al. 2005

Journal of Biological Chemistry

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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...

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Section: Design Production and Characterization Of Thioredoxinfusedmentioning

confidence: 96%

Section: S2 Cell Expression Of Il5r␣ and Its Mutational Variantsmentioning

confidence: 99%

Section: Spr Biosensor Binding Analysismentioning

confidence: 99%

Section: Design Production and Characterization Of Thioredoxinfusedmentioning

confidence: 99%

mentioning

confidence: 99%

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Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Ishino

Urbina

Bhattacharya

et al. 2005

Journal of Biological Chemistry

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Protein Recognition in Biology

McCourt

Nickels

Ishino

et al. 2007

Handbook of Biosensors and Biochips

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Protein recognition is pervasive in living systems. Interactions of proteins with each other and with other biomolecules occur in crowded and dynamic environments, ultimately leading to the multimolecular assemblies and networks that are recurrent components underpinning the molecular mechanisms of health and disease. Appreciating the nature and environment of protein interactions can help identify opportunities for molecular sensing in biological systems and guide the conditions needed for biomolecular sensing using biosensors and biochips.

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Recruitment pharmacophore for interleukin 5 receptor α antagonism

et al. 2006

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Interleukin-5 receptor alpha is a therapeutic target for hypereosinophilic diseases including allergic inflammations and asthma. The cyclic peptide AF17121 (Ac-VDE[CWRIIASHTWFC]AEE-CONH(2)) has been identified as a submicromolar inhibitor of interleukin 5 (IL5)-interleukin 5 receptor alpha (IL5Ralpha) interaction from a random peptide screen. However, this inhibitor has limitations as a drug lead because of its relatively large size. We used chemical synthesis of peptides with natural and non-natural amino acids along with kinetic binding and cell proliferation competition assays to expand definition of structural elements in the peptide that are important for receptor antagonism and to elucidate the underlying pharmacophore. We found that the specific steric array of hydrogen bonding groups in the Arg 6 guanido side chain is critical for receptor inhibition. We also investigated noncharged structural elements in AF17121. Screening a set of five hydrophobic residues showed that peptide function is strongly sensitive to variations in several of these residues, most prominently Ile 7 and Trp 13. We postulate that presentation of charged, hydrogen bonding and hydrophobic structural elements within the disulfide-constrained peptide drives IL5Ralpha recruitment by AF17121. We hypothesize from these results and previous receptor mutagenesis studies that Arg 6 recruitment of IL5Ralpha occurs through hydrogen bonding as well as charge-charge interactions with Asp 55 in site one of domain 1 of IL5Ralpha, and that this interaction is complemented by additional charged and hydrophobic interactions around the Asp 55 locus. Scaffolding a limited set of structural elements in the inhibitor pharmacophore may be useful for small molecule antagonist design inspired by the peptide.

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Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

Cited by 39 publications

References 41 publications

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Protein Recognition in Biology

Recruitment pharmacophore for interleukin 5 receptor α antagonism

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