lnterleukin-5 (IL-5) is a member of the short chain subfamily of the helical bundle family of cytokines. IL-5 signals through a heterodimeric receptor, IL-5R-alpha/IL-5R-beta, inducing pleotrophic effects on a number of leukocytes. Physiologically IL-5 aids in host defense against parasites and tumors while its dysregulation has been associated with asthma and allergic disease. Thus, IL-5 and its receptor are obvious targets for modulation of such inflammatory responses. The goal of this application is to understand the molecular interactions between IL-5 and its receptor subunits. Toward this goal, we have successfully modeled and expressed the first IL-5 monomer with biologic activity (designated mono5), thereby demonstrating that all the structural features necessary for IL-5 function are contained within a single helical bundle. In addition, a panel of anti-IL-5 neutralizing mAb, COS-7 expressed interspecies IL-5 chimeras, and molecular modeling have been used to localize five neutralizing epitopes and the species specificity residues of IL-5 within two domains.
The specific aims are proposed to: 1) test our hypothesis that mono5 serves as a paradigm for investigating whether homodimerization of native IL-5 is biologically advantageous or is a novel biologic mechanism for conservation of an essential functional motif within the helical bundle family of cytokines; and 2) define the precise structural requirements for engagement of IL-5 and its receptor.
Aim I will determine whether a homodimeric IL-5 configuration imparts a functional advantage over a monomeric helical bundle for receptor binding. Native IL-5 and mono5 will be studied for differences in receptor ligand binding kinetics. In addition, a loop 3 deletion mutant of the monomeric cytokine GM-CSF will be generated and analyzed for the predicted formation of an interdigitating homodimer with enhanced biological activity.
Aim II will test the hypothesis that residues proximate to the junction of loop 3 and helix D of IL-5 engage the IL-5R-alpha chain. Predicted residues will be assessed by defining the confirmational epitopes and bioactivity of IL-5 mutants generated by site-directed mutagenesis.
Aim III will test the hypothesis that residues proximate to loop 2 and the middle of helix A of IL-5 engage the IL-5R-beta chain, utilizing site-directed mutagenesis of COS-7 expressed IL-5. The structure/function analyses of IL-5 signaling should enable the development of IL-5 analogs capable of antagonizing IL-5-mediated inflammation.
