Mapping of interleukin-5 functional domains and creation of a functional interleukin-5 monomer demonstrate that all structural features necessary for interleukin-5 biological activity are contained within a single helical bundle motif
The cytokine interleukin-5 (IL-5) specifically induces the maturation and activation of eosinophils, which are important in normal host defense and allergic disease. Crucial to the development of specific modulators of IL-5 is a comprehensive understanding of the interaction of IL-5 with its receptor complex, which consists of alpha and beta subunits. IL-5 is a unique member of the short chain subfamily of helical bundle cytokines. IL-5 is a glycosylated, disulfide-linked, interdigitating homodimer, which contains two canonical helical bundle motifs. This distinguishes IL-5 from the other subfamily members, which fold unimolecularly into a single helical bundle. I undertook a systematic approach to identify functional domains on IL-5 and solve the enigma surrounding its unique dimeric configuration. A library of neutralizing monoclonal antibodies (mAb) was generated and characterized. Two noncompetitive anti-IL-5 mAb were used to develop a highly sensitive sandwich ELISA capable of quantitating IL-5 in biological fluids. The anti-IL-5 mAb were also used to map functional domains on IL-5. These results predicted regions which engaged the IL-5 receptor. Each functional domain was defined twice on the IL-5 dimer. However, the functional significance of each domain pair could not be established. To determine whether a single helical bundle of IL-5 was independently functional or if IL-5 required both helical bundles for bioactivity, I created a novel monomer, which folded unimolecularly into a single IL-5 helical bundle motif. Generation of this cytokine, designated mono5, relied on the hypothesis that the shortened loop 3 of IL-5 physically restricts unimolecular completion of a single helical bundle. Structural and functional analyses demonstrated that mono5 was indeed a monomer that possessed full biological activity comparable to native IL-5 at saturating concentrations. Although slight tertiary structural differences were identified, the full functional capacity of mono5 demonstrated for the first time that, all structural features necessary for IL-5 biological activity are contained within a single helical bundle motif. The creation of biologically active mono5 revolutionizes the understanding of IL-5 functional structure. Furthermore, my loop 3 hypothesis is applicable to the entire family of helical bundle cytokines and may lead to the creation of other novel proteins.
Dickason, Richard Ray, "Mapping of interleukin-5 functional domains and creation of a functional interleukin-5 monomer demonstrate that all structural features necessary for interleukin-5 biological activity are contained within a single helical bundle motif" (1996). Texas Medical Center Dissertations (via ProQuest). AAI9619583.