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

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 P35T36C41T42Q43L46Q47C48Y49L50G51 motif within chIL-2 molecule interacts with the S99F100C101G102M103P104Q105T106V107P108S111L112 motif of chCD25 molecule (chCD2599–112), whereas the peptide competition ELISA assay showed the residues 27KIHLELYTPTETQEC41 within chIL-2 (chIL-227–41) bound to the chCD25 protein. Lymphocyte proliferation and inhibition assays further confirmed that the binding of chIL-227–41 to the chCD25 molecule was inhibited by the chCD2599–112 peptide. Site-specific mutation of the 35P and 41C residues in chIL-227–41 resulted in the lack of its ability to induce lymphocyte proliferation and binding to the chCD25 molecule. These findings demonstrate that chIL-227–41 and chCD2599–112 are the binding domains between the chIL-2 and chCD25 molecules, and that the residues P35 and C41 within chIL-227–41 are the critical sites for its bioactivity and interaction with chCD25.