The residues 35proline and 41cysteine of chicken IL-2 are critical for binding to chicken CD25

Abstract

The interleukin 2 (IL-2) immunoregulatory cytokine produces its biological effects by binding sequentially to its cell receptor subunits, alpha (CD25), beta (CD122), and common gamma chain (CD132). In this study, we identified the critical amino acid residues of chicken IL-2 (chIL-2) for binding to chicken CD25 (chCD25) by an Ag-capture ELISA, screening of a phage display peptide library, peptide-competitive ELISA and an in vitro T-cell proliferation assay. Specific ligand elution of phage bound to chCD25 and chIL-2 suggested that the P 35T 36C 41T 42Q 43L 46Q 47C 48Y 49L 50G 51 motif within chIL-2 molecule interacts with the S 99F 100C 101G 102M 103P 104Q 105T 106V 107P 108S 111L 112 motif of chCD25 molecule (chCD25 99–112), whereas the peptide competition ELISA assay showed the residues 27KIHLELYTPTETQEC 41 within chIL-2 (chIL-2 27–41) bound to the chCD25 protein. Lymphocyte proliferation and inhibition assays further confirmed that the binding of chIL-2 27–41 to the chCD25 molecule was inhibited by the chCD25 99–112 peptide. Site-specific mutation of the 35P and 41C residues in chIL-2 27–41 resulted in the lack of its ability to induce lymphocyte proliferation and binding to the chCD25 molecule. These findings demonstrate that chIL-2 27–41 and chCD25 99–112 are the binding domains between the chIL-2 and chCD25 molecules, and that the residues P 35 and C 41 within chIL-2 27–41 are the critical sites for its bioactivity and interaction with chCD25.