Identification of functional domains of chicken interleukin 2.
Interleukin 2 (IL-2) is an essential cytokine that plays a pivotal role in the replication, maturation and differentiation of lymphocytes. In this study, the functional domains of chicken IL-2 (chIL-2) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. Nine neutralizing mAbs to chIL-2 were produced using the recombinant chIL-2 monomer expressed in prokaryotic cells as an immunogen and used to finely map the functional domains of the chIL-2 protein. The mimotopes of nine anti-chIL-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 chIL-2 molecule, that is, N(26)K(27)I(28)H(29)L(30)E(31)L(32)P(35)Q(43)Q(44)T(45)L(46)Q(47)C(48)Y(49)L(50) (domain I), E(68)E(69)F(70)K(79)K(82)S(83)L(84)T(85)G(86)L(87) (domain II) and N(88)H(89)G(91)K(104)F(105)P(106)D(107)E(111)L(112)Y(118)L(119) (domain III). The neutralizing mAbs to chIL-2 inhibited the in vitro lymphocyte proliferation stimulated by three peptide domains of chIL-2. The predicted tertiary structure of chIL-2 reveals that domain I was positioned in the long A-B loop and the N terminal of Helix B, domain II was mostly situated in Helix C, and domain III was distributed in the C-D loop and Helix D. These data demonstrate the functional domains of chIL-2 and provide a clue for elucidating the interaction between chIL-2 and its receptor.
Gu
Jianyou
J
Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China.
Ruan
Xizhen
X
Huang
Zhenyu
Z
Chen
Jigang
J
Zhou
Jiyong
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D001483:Base Sequence; D002645:Chickens / Q000235:genetics* / Q000276:immunology*; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D017931:DNA Primers; D007376:Interleukin-2; D019151:Peptide Library; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins