Epitope Randomization Redefines the Functional Role of Glutamic Acid 110 in Interleukin-5 Receptor Activation

Wu, Sheng Jiun; Tambyraja, Rabindra R; Zhang, Wentao; Zahn, Stefan; Godillot, Alexis P.; Chaiken, Irwin M.

doi:10.1074/jbc.275.10.7351

Cited by 14 publications

(16 citation statements)

References 33 publications

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“…We surmise that targeting site I will be important to derive a specific antagonist for IL5R␣, whereas blocking site II offers the opportunity to interfere with IL5 biological activity induced by multisubunit receptor recruitment involving ␤c (see below). This can be seen in the IL5 ligand side, as we previously found that the roles of the receptor-binding epitope on IL5 can be separated into receptor recognition residues such as Arg 91 and receptor activation residues such as Glu 110 (35,36). In this regard, considering their charge complementarity, it would be reasonable to speculate that Arg 91 and Glu 110 of IL5 are more likely to be candidates for binding partners of sites I and II on IL5R␣, respectively.…”

Section: Af17121 Utilizes the Receptor Epitope-mentioning

confidence: 93%

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: Af17121 Utilizes the Receptor Epitope-mentioning

confidence: 93%

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Ishino

Urbina

Bhattacharya

et al. 2005

Journal of Biological Chemistry

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“…Site-directed mutagenesis, as well as other studies have shown the importance of charged residues in the CD turn (in particular Glu 89 and Arg 91 ) for receptor ␣ chain binding. [12][13][14][15][16] Moreover, phagebased CD turn randomization 17,18 has led to identification of a functionally active IL-5 mimetic containing the CD turn sequence 88 SLRGG 92 (Ref. 17).…”

Section: Introductionmentioning

confidence: 99%

“…We gratefully acknowledge the Fannie E. Ripple Foundation for technical support and Dr. Anna Tretiakova for , Glu 18 , and Arg 6 are shown as ball-and-stick models and labeled by residue type and number. The other residues are shown by stick models, except that two cysteine residues are shown as CPK models.…”

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confidence: 99%

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor α

et al. 2004

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The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.

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“…This verified the importance of the positive charge in the CD strand for IL5R recruitment and subsequent receptor activation. We also found that tryptophan replacement of Glu 110 did not affect high-affinity binding of IL5 to IL5R but caused a substantial reduction in biological activity, suggesting that negative charge at the position 110 might be important for c subunit recruitment [55], at least indirectly. Recalling that the stoichiometry of IL5: IL5R binding is 1: 1 [49] and monomeric IL5 construct with [ 88 SLRGG 92 , E110W] mutation (Fig.…”

Section: Ligand Epitopes That Drive Il5-il5r Complexmentioning

confidence: 91%

Structure-Based Rationale for Interleukin 5 Receptor Antagonism

Ishino

Harrington²,

Gopi³

et al. 2008

CPD

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Human interleukin 5 (IL5) is the major hematopoietin that stimulates the proliferation, migration and activation of eosinophils and is implicated in the pathogenesis of inflammatory and other myeloproliferative diseases. IL5 functions through the signaling of a common receptor subunit beta (beta c), in a receptor activation process that requires initial recruitment of an IL5 specific receptor subunit alpha (IL5Ralpha), for cytokine presentation to beta c. Important advances have been made to understand molecular mechanisms of cytokine recognition and receptor antagonism. Mutational studies indicate that a pair of charge complementary regions play an essential role in specific interaction between IL5Ralpha and IL5. Moreover, peptide studies with the IL5 system have identified a cyclic peptide inhibitor, AF17121, which binds specifically to IL5Ralpha by mimicking the cytokine. A key receptor-recognition pharmacophore has been identified in this peptide inhibitor, and sites of inhibitor recognition can be proposed in the homology-deduced structural model of IL5Ralpha. These results provide an experimental platform to derive enhanced-potency peptidomimetic inhibitors. Such inhibitors have potential use as tools to evaluate the role of eosinophilia in disease and as potential leads to antagonists to treat hyper-eosinophilic diseases such as eosinophilic esophagitis, asthma and chronic myeloproliferative leukemias.

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Epitope Randomization Redefines the Functional Role of Glutamic Acid 110 in Interleukin-5 Receptor Activation

Cited by 14 publications

References 33 publications

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor α

Structure-Based Rationale for Interleukin 5 Receptor Antagonism

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