Identification Of Functional Domains Of Poultry Interleukin 2, And Interaction Of Chicken Interleukin 2 And Its Receptors

Interleukin 2 (IL-2) is an essential cytokine that plays a pivotal role in the growth and differentiation of lymphocytes. In this study, the functional domains of chicken IL-2 (cIL-2) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. Nine neutralizing mAbs to cIL-2 were produced using the recombinant cIL-2 monomer expressed in prokaryotic cells as an immunogen and used to finely map the functional domains of the cIL-2 protein. The mimotopes of nine anti-cIL-2 mAbs, including KIELPSL, EHLDXNDSLYL, NHLXGXY, WHLPPSL, EFKASXL, TENPFPE, SGLYL, AHGYWEL and HHGYWEL, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute three conformational functional domains in the cIL-2 molecule, that is, N26K27I28H29L30E31L32P35Q43Q44T45L46Q47C48Y49L50 (domainⅠ), E68E69F70K79K82S83L84T85G86L87 (domainⅡ) and N88H89G91K104F105p106D107E111 L112Y118L119 (domainⅢ). The neutralizing mAbs to cIL-2 inhibited the in vitro lymphocyte proliferation stimulated by three peptide domains of cIL-2. The predicted tertiary structure of cIL-2 reveals that domainⅠwas positioned in the long A-B loop and the N terminal of Helix B, domainⅢwas mostly situated in Helix C, and domainⅢwas distributed in the C-D loop and Helix D.The functional domains of duck IL-2 (dIL-2) were finely mapped with four neutralizing mAbs to dIL-2. The mimotopes of four anti-dIL-2 mAbs, including LVXGSMPS, KPHKHHXHHSHM, HVPNERYPLR and WXXXKAKP, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute one functional domain in the dIL-2 molecule, that is, Y32T33P34N35D36T37K38E39C40S41W42Q43T44 (domainⅠ).The predicted tertiary structure of dIL-2 reveals four-helix bundle structure without two beta sheets and domain I was positioned in the long A-B loop and the N terminal of Helix B.The functional domains of goose IL-2 (gIL-2) were finely mapped with five neutralizing mAbs to gIL-2. The mimotopes of five anti-gIL-2 mAbs, including HHDPWDXLP, ESLSRXXMXXLXP, SHHLPTSXL, HPDPWDAPLSS and HEPWQLXL, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute one functional domain in the gIL-2 molecule, that is,T11I14K15D16E18K19L20G21T22S23M24K25L29E30L31Y32T33P34E36S41W42Q43T44L45 Q46 (domain I). The predicted tertiary structure of gIL-2 reveals four-helix bundle structure with two beta sheets and domain I was positioned in the Helix A, A-B loop and the N terminal of Helix B.Compare the functional domains of poultry IL-2 (cIL-2, dIL-2 and gIL-2), one common key domain was situated in the long A-B loop. The N terminal of poultry IL-2s were important for their bioactivities.The functional domains of chicken CD25 (cCD25) were mapped with two neutralizing mAbs to cCD25, which were identified by inhibition of IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that cCD25 molecules are expressed on the surface of splenic mononuclear cells. The mimotopes of two anti-cCD25 mAbs, including PVDRPRD and SLGKXTPVDEPXY, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute one functional domain in the cCD25 molecule, that is, S119V120G121K152W153T154P155V156D157R158P159C160T161 (domainⅠ) The predicted tertiary structure of cCD25 shows that functional domain I is positioned at the interdomain, paritial Sheet I and the subsequent connected-peptide with membrane.The functional domains of chicken CD132 (cCD132) were mapped with one neutralizing mAb to cCD132, which were identified by inhibition of IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that cCD132 molecules are expressed on the surface of splenic mononuclear cells. The functional domain of cCD132 that binds to chicken interleukin 2, Q84E94L95Q96N97L98, was found through phage display and peptide-competitive ELISA, and its critical residue Q96 was further identified. A tertiary structure model shows that the functional domain is positioned at the elbow-like junction of domain 1 and domain 2.Using phage display and competitive ELISA, a key binding epitope of cIL-2 to cIL-2R was identified and situated in the cIL-227-41 positioned in the long A-B loop. This fact implies that the interaction between cIL-2Ra and y subunits could be present. The binding epitopes of cIL-2Ra and y to cIL-2 were also identified by phage display and competitive ELISA.α99-112 of cCD25 was primary to bind cIL-227-41.γ119-137 of cCD132 was primary to bind cIL-227-41 and cIL-279-96.γ82-101 was the second site binding to cIL-279-96. The key binding epitopes of cIL-2R to cIL-2, namelyα99-112 andγ119-137, formed the common interaction interface to bind the cIL-227-41. The interaction between cIL-2 and cIL-2R (a and y) was further confirmed by the inhibition of lymphocyte proliferation in vitro. These results provide experimental evidences and structure models for elucidating the interaction between cIL-2 and cIL-2R.