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.
Baylor College of Medicine, Department of Microbiology and Immunology, Houston, TX 77030, USA.
R R
RR
Dickason
J D
JD
English
D P
DP
Huston
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