The residues (35)proline and (41)cysteine 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 P(35)T(36)C(41)T(42)Q(43)L(46)Q(47)C(48)Y(49)L(50)G(51) motif within chIL-2 molecule interacts with the S(99)F(100)C(101)G(102)M(103)P(104)Q(105)T(106)V(107)P(108)S(111)L(112) motif of chCD25 molecule (chCD25(99-112)), whereas the peptide competition ELISA assay showed the residues (27)KIHLELYTPTETQEC(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 (35)P and (41)C 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.
Gu
Jianyou
J
Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou 310029, PR China.
Xu
Jieping
J
Huang
Zhenyu
Z
Liao
Min
M
Zhou
Jiyong
J
D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D003545:Cysteine; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008213:Lymphocyte Activation; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019151:Peptide Library; D011392:Proline; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D013154:Spleen / Q000473:pathology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
D030161:Avian Proteins; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D010446:Peptide Fragments; D019151:Peptide Library; D011392:Proline; D003545:Cysteine