Epitope randomization redefines the functional role of glutamic acid 110 in interleukin-5 receptor activation

Sequence randomization through functional phage display of single chain hum an interleukin (IL)-5 was used to investigate the limits of replaceability of the Glu(110) residues that form a part of the receptor-binding epitope. Mutational analysis revealed unexpected affinity for IL-5 receptor a chain with variants containing E110W or E110Y. Escherichia coli-expressed Glu(110 ) variants containing E110W in the otherwise sequence-intact N-terminal hal f, including a variant with an E110A replacement in the sequence-disabled C -terminal half, were shown by their CD spectra to be folded into secondary structures similar to that of single chain human IL-5 (scIL-5), Biosensor k inetics analysis revealed that (E110W/A5)scIL-5 and (E110W/A6)scIL-5 had re ceptor a chain binding affinities similar to that of (wt/A5)scIL-5. However , (E110W/A6)scIL-5 had a significantly reduced bioactivity in TF-1 cell pro liferation compared with both (wt/A5)scIL-5 and (E110W/A5)scIL-5, and this activity reduction was disproportionately greater than the much smaller eff ect of Glu(110) mutation on receptor binding affinity. The marked and dispr oportionate decrease in TF-1 proliferation observed with (E110W/A6)scIL-5 s uggests a role for Glu(110) in the biological activity mediated by the sign al transducing receptor beta c subunit of the IL-5 receptor. This is also c onsistent with the lack of stimulation of JAK2 phosphorylation by the (E110 W/ A6)scIL-5 mutant in recombinant 293T cells, as compared with the concent ration-dependent stimulation seen for scIL-5. The results reveal the dispen sability of charge in the Glu(110) locus of IL-5 for receptor a chain bindi ng and, in contrast, its heretofore underappreciated importance for recepto r activation.