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Engineering of a functional interleukin-5 monomer: a paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma

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Abstract

Interleukin (IL) 5 specifically induces the differentiation of eosinophils which are central to the pathogenesis of allergies and asthma. Structurally, IL-5 is a unique member of the short-chain helical bundle subfamily of cytokines. In contrast to other subfamily members which fold unimolecularly into a single helical bundle, IL-5 forms a pair of helical bundles by the inter-digitation of two identical monomers covalently linked by a pair of intermolecular disulfide bonds. Although a native IL-5 monomer lacks bioactivity, we recently reported the engineering of an insertional mutant of IL-5 (designated mono5) which folds unimolecularly into a single helical bundle and has biological activity similar to that of native IL-5. Here we demonstrate no differences in signal transduction pathways utilized by mono5 and IL-5, as determined by western blot analysis of early tyrosine phosphorylation events, Jak2 activation, and mitogen-activated protein kinase activation. However, binding studies utilizing conformationally dependent neutralizing anti-IL-5 monoclonal antibodies localized a tertiary structural perturbation near the insert of mono5. This perturbation enabled localization of a limited region of the tertiary structure of IL-5 that engages the IL-5 receptor α-chain. Fluorescent labeling studies further revealed that the cysteines of mono5 contained free sulfhydryl groups, thereby demonstrating that the role of the disulfide bonds of IL-5 is the structural maintenance of other functional domains. The retention of conformational epitopes by mono5, but not IL-5, under reducing conditions and the equivalent thermostability of mono5 and IL-5 despite the absence of a disulfide bond in mono5 indicated that the conformation assumed by mono5 is very stable. In addition to providing the structural framework for designing novel IL-5 agonists and antagonists, the knowledge gained from the development of mono5 will enable other helical bundle proteins to be redesigned with therapeutic potential.

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Abbreviations

ELISA :

Enzyme-linked immunosorbent assay

IFN :

Interferon

IL :

Interleukin

mAb :

Monoclonal antibody

MAPK :

Mitogen-activated protein kinase

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, FBRN B567, 77030, Houston, TX, USA

    R. R. Dickason

  2. Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, FBRN B567, 77030, Houston, TX, USA

    J. D. English

  3. Department of Medicine and Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, FBRN B567, 77030, Houston, TX, USA

    D. P. Huston

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Dickason, R.R., English, J.D. & Huston, D.P. Engineering of a functional interleukin-5 monomer: a paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma. J Mol Med 74, 535–546 (1996). https://doi.org/10.1007/BF00204980

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