TY - JOUR
T1 - Engineering of a functional interleukin-5 monomer
T2 - A paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma
AU - Dickason, R. R.
AU - English, J. D.
AU - Huston, David P.
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1996
Y1 - 1996
N2 - 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 interdigitation 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 a-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.
AB - 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 interdigitation 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 a-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.
KW - Allergy and asthma
KW - Helical bundle proteins
KW - Interleukin-5
KW - Redesigning cytokines
KW - Signal transduction
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U2 - 10.1007/BF00204980
DO - 10.1007/BF00204980
M3 - Article
C2 - 8892059
AN - SCOPUS:0029793706
VL - 74
SP - 535
EP - 546
JO - Journal of Molecular Medicine
JF - Journal of Molecular Medicine
SN - 0946-2716
IS - 9
ER -