Engineering of a functional interleukin-5 monomer: a paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma.
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 alpha-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 conformation 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.
Dickason
R R
RR
Baylor College of Medicine, Department of Microbiology and Immunology, Houston, TX 77030, USA.
English
J D
JD
Huston
D P
DP
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000737:chemistry; D006967:Hypersensitivity / Q000276:immunology / Q000628:therapy; D007249:Inflammation / Q000276:immunology / Q000628:therapy; D015848:Interleukin-5 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D053613:Janus Kinase 1; D008958:Models, Molecular; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism; D013696:Temperature; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015848:Interleukin-5; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D053613:Janus Kinase 1