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, [(89)SLRGG(92),W(110)/(89)AAAAA(92), A(110)]scIL-5. This mutein was found to retain substantial IL-5 receptor alpha-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 alpha-chain recognition can be supported by the (89)SLRGG(92) 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 alpha-chain binding while modifying receptor activation epitopes. In the first, [(88)SLRGG(92),W(110)]GM1, the wild-type CD-loop sequence (89)EERRR(92) was converted to the mimotope (89)SLRGG(92), and Glu110 to Trp. In the second, [A(13), A(110)]GM1, wild-type Glu13, and Glu110 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-5Ralpha-Fc using optical biosensor and ELISA and for bioactivity by proliferation of TF-1.28 cells. Both [(88)SLRGG(92),W(110)]GM1 and [A(13),A(110)]GM1 were found to interact with the shIL-5Ralpha-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-5Ralpha in an ELISA. In contrast, both mutants exhibited a disproportionately decreased capacity to stimulate TF-1. 28 cell proliferation. [A(13),A(110)]GM1 bioactivity was 160-fold lower than that of GM1, while that for the [(88)SLRGG(92),W(110)]GM1 mutant was 2600-fold lower. The largely retained IL-5 receptor alpha-chain binding affinities versus relatively suppressed bioactivities of [A(13),A(110)]GM1 and [(88)SLRGG(92),W(110)]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.

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

“…We surmise that targeting site I will be important to derive a specific antagonist for IL5R␣, whereas blocking site II offers the opportunity to interfere with IL5 biological activity induced by multisubunit receptor recruitment involving ␤c (see below). This can be seen in the IL5 ligand side, as we previously found that the roles of the receptor-binding epitope on IL5 can be separated into receptor recognition residues such as Arg 91 and receptor activation residues such as Glu 110 (35,36). In this regard, considering their charge complementarity, it would be reasonable to speculate that Arg 91 and Glu 110 of IL5 are more likely to be candidates for binding partners of sites I and II on IL5R␣, respectively.…”

Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

“…5A (14)). Although IL5 is a homodimeric protein, it has been shown that the stoichiometry of IL5⅐IL5R␣ interaction is 1:1 (10, 12), and a Langmuir 1:1 binding model has been used for global fitting analysis of IL5⅐IL5R␣ interaction (33). In this article, we first measured the association rates at flow rates of a range of 10 to 80 l/min to examine the mass transfer effect (Fig.…”

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor α

“…In the CD turn of this IL-5 mutant, the only charged residue is Arg 90 . Moreover, variants of IL-5 have been constructed which retain the SLRGG CD turn but replace other residues more important for biological activity, leading to the view 19 that the CD turn is important for driving receptor ␣ subunit recruitment but not receptor activation and that mimicking this region could lead to effective IL-5 antagonists.…”