Abstract
The cytokine interleukin (IL)-21 exerts pleiotropic effects acting through innate as well as adaptive immune responses. The activities of IL-21 are mediated through binding to its cognate receptor complex composed of the IL-21 receptor private chain (IL-21Ralpha) and the common gamma-chain (gammaC), the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15. The binding energy of the IL-21 ternary complex is predominantly provided by the high affinity interaction between IL-21 and IL-21Ralpha, whereas the interaction between IL-21 and gammaC, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling gammaC chain, while simultaneously maintaining a tight interaction with the private chain, would in theory represent candidates for IL-21 antagonists. We predicted the IL-21 residues, which compose the gammaC binding epitope using homology modeling and alignment with the related cytokines, IL-2 and IL-4. Next we systematically analyzed the predicted binding epitope by a mutagenesis study. Indeed two mutants, which have significantly impaired gammaC affinity with undiminished IL-21Ralpha affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists.
Highlights
Interleukin (IL),2-21 which is produced by activated CD4ϩ T cells, T-follicular helper cells, and natural killer T (NKT) cells, [1] has been demonstrated to exert pleiotropic effects on the proliferation, differentiation, and cytotoxicity of various classes of lymphoid cells
IL-21 signals through its receptor complex composed of the private chain IL-21R␣ and the common chain, ␥C, the latter of which is shared by five other cytokines: IL-2, IL-4, IL-7, IL-9, and IL-15 [1]
Preceding this structural knowledge of the IL-4 cytokine/cytokine receptor complex, IL-4 antagonists had been identified through systematic mutagenesis, seeking to abolish binding to the signaling receptor chain ␥C while preserving high affinity binding to the IL-4R␣ chain [8]
Summary
We have reported the three-dimensional structure of human IL-21 (hIL-21) resolved by heteronuclear NMR spectroscopy [4]. Residues predicted to constitute part of the ␥C binding interface, and possibly being implicated in the binding of this receptor chain, were identified by homology modeling based on the structures of IL-2 and IL-4 in complex with ␥C, and through knowledge of the NMR structure of IL-21. Through mutagenesis, these residues constituting the predicted ␥C binding epitope were explored with respect to their effect on the binding of ␥C and IL-21R␣. Through the combination of mutants demonstrated to have impaired ␥C affinity, while undiminished IL-21R␣ affinity, we have identified two hIL-21 double mutants as novel hIL-21 antagonists
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