Rational design of interleukin-21 antagonist through selective elimination of the gammaC binding epitope.
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.
Kang
Lishan
L
Novo Nordisk China R&D, Beijing 102206, China. LshK@novonordisk.com
Bondensgaard
Kent
K
Li
Tengkun
T
Hartmann
Rune
R
Hjorth
Siv A
SA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D015195:Drug Design; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics; D015847:Interleukin-4 / Q000737:chemistry / Q000235:genetics; D007378:Interleukins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D053655:Receptors, Interleukin-21 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D000098546:Interleukin-21
D000939:Epitopes; C508594:IL2 protein, human; C508598:IL4 protein, human; D007376:Interleukin-2; D007378:Interleukins; D046912:Multiprotein Complexes; D053655:Receptors, Interleukin-21; D011994:Recombinant Proteins; D015847:Interleukin-4; D000098546:Interleukin-21