Multisite mutagenesis of interleukin 5 differentiates sites for receptor recognition and receptor activation

Abstract

Multisite mutagenesis of single-chain and monomeric forms of human interleukin 5 (IL-5) was performed to investigate mechanistic features of receptor activation and the possibility of differentiating sites of activation from those for receptor interaction. The normally dimeric human IL-5 contains two domains, each containing a four-helix bundle. IL-5 has previously been re-engineered into the monomeric, one-domain GM1 form by introducing an eight-residue linker between the third and fourth helices. In this study, we tested a combination of mutations in a single-chain IL-5 (scIL-5) construct, [89SLRGG92,W110/89AAAAA92,A110]scIL-5. This mutein was found to retain substantial IL-5 receptor α-chain binding but with selectively suppressed proliferation of the IL-5-dependent cell line TF-1.28. This result confirms recent findings that IL-5 receptor α-chain recognition can be supported by the 89SLRGG92 epitope and that, in contrast, Glu110 is important in receptor activation. On the basis of this result, two mutants of GM1 were constructed with the intent to retain receptor α-chain binding while modifying receptor activation epitopes. In the first, [88SLRGG92,W110]GM1, the wild-type CD-loop sequence 89EERRR92 was converted to the mimotope 89SLRGG92, and Glu 110 to Trp. In the second, [A13,A110]GM1, wild-type Glu 13, and Glu 110 were both mutated to Ala. GM1 and mutants were expressed in high yield in Escherichia coli, purified under denaturing conditions from inclusion bodies, and refolded. Monomers were screened for binding to shIL-5Rα-Fc using optical biosensor and ELISA and for bioactivity by proliferation of TF-1.28 cells. Both [88SLRGG92,W110]GM1 and [A13,A110]GM1 were found to interact with the shIL-5Rα-Fc, with affinities of 69-585 nM, 2-15-fold weaker than that of the original GM1. The mutants also were able to compete with IL-5 for binding to shIL-5Rα in an ELISA. In contrast, both mutants exhibited a disproportionately decreased capacity to stimulate TF-1.28 cell proliferation. [A13,A110]GM1 bioactivity was 160-fold lower than that of GM1, while that for the [88SLRGG92,W110]GM1 mutant was 2600-fold lower. The largely retained IL-5 receptor α-chain binding affinities versus relatively suppressed bioactivities of [A13,A110]GM1 and [88SLRGG92,W110]GM1 variants, in particular the latter, point to the existence of separable IL-5 epitopes for receptor binding and activation and establish the potential to design smaller IL-5 mimetic antagonists.