Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou 310029, PR China.
Dev Comp Immunol. 2010 Aug;34(8):805-11. doi: 10.1016/j.dci.2010.02.013. Epub 2010 Mar 19.
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.
白细胞介素 2 (IL-2) 免疫调节细胞因子通过与细胞受体亚基 α（CD25）、β（CD122）和共同γ链（CD132）依次结合来发挥其生物学作用。在这项研究中，我们通过 Ag 捕获 ELISA、噬菌体展示肽文库筛选、肽竞争 ELISA 和体外 T 细胞增殖试验鉴定了鸡白细胞介素 2（chIL-2）与鸡 CD25（chCD25）结合的关键氨基酸残基。噬菌体与 chCD25 和 chIL-2 的特异性配体洗脱表明，chIL-2 分子内的 P(35)T(36)C(41)T(42)Q(43)L(46)Q(47)C(48)Y(49)L(50)G(51)基序与 chCD25 分子内的 S(99)F(100)C(101)G(102)M(103)P(104)Q(105)T(106)V(107)P(108)S(111)L(112)基序相互作用（chCD25(99-112)），而肽竞争 ELISA 试验表明 chIL-2 内的残基（27）KIHLELYTPTETQEC(41)（chIL-2(27-41)）与 chCD25 蛋白结合。淋巴细胞增殖和抑制试验进一步证实，chIL-2(27-41)与 chCD25 分子的结合被 chCD25(99-112)肽抑制。chIL-2(27-41)中（35）P 和（41）C 残基的定点突变导致其丧失诱导淋巴细胞增殖和与 chCD25 分子结合的能力。这些发现表明，chIL-2(27-41)和 chCD25(99-112)是 chIL-2 和 chCD25 分子之间的结合域，并且 chIL-2(27-41)内的残基 P(35)和 C(41)是其生物活性和与 chCD25 相互作用的关键位点。
