{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T03:50:49Z","timestamp":1760241049758,"version":"build-2065373602"},"reference-count":44,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2019,11,15]],"date-time":"2019-11-15T00:00:00Z","timestamp":1573776000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100013061","name":"Jilin Scientific and Technological Development Program","doi-asserted-by":"publisher","award":["20180101244JC"],"award-info":[{"award-number":["20180101244JC"]}],"id":[{"id":"10.13039\/501100013061","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31272528","31772700"],"award-info":[{"award-number":["31272528","31772700"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004602","name":"Program for New Century Excellent Talents in University","doi-asserted-by":"publisher","award":["NCET-12-0232"],"award-info":[{"award-number":["NCET-12-0232"]}],"id":[{"id":"10.13039\/501100004602","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Interleukin-2 (IL-2) is a pleiotropic cytokine regulating the immune and nervous systems. Mammalian and bird IL-2s have different protein sequences, but perform similar functions. In the current study, two bands were detected by immunoblotting using an antibody against freshly purified chicken IL-2 (chIL-2). The molecular weight of the larger band was approximately twice as much of the chIL-2 monomer, although a chIL-2 complex or homodimer has never been reported. To explain this intriguing result, several dissociation reagents were used to examine the intermolecular forces between components of the proposed chIL-2 complex. It was found that intermolecular disulphide bond promotes homodimerization of chIL-2. Subsequently, mutation of Cys residues of chIL-2 revealed that mutation of all four Cys residues disrupted homodimerization, but a single, dual, or triple Cys mutation failed to disrupt homodimerization, suggesting that all four Cys residues on chIL-2 contribute to this dimerization. Functional analysis showed that both monomeric and dimeric chIL-2 consisting of either wild type or mutant chIL-2 were able to stimulate the expansion of CD4+ T cell in vivo or in vitro, and effectively bind to chIL-2 receptor. Overall, this study revealed that the recombinant chIL-2 purified from either Escherichia coli (E. coli) or Spodoptera frugiperda (Sf9) cells could homodimerize in vitro, with all four Cys residues on each chIL-2 protein contributing to this homodimerization, and dimerization and Cys mutation not impacting chIL-2 induced stimulation of chicken CD4+ T cells.<\/jats:p>","DOI":"10.3390\/ijms20225744","type":"journal-article","created":{"date-parts":[[2019,11,15]],"date-time":"2019-11-15T11:24:32Z","timestamp":1573817072000},"page":"5744","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Four Cysteine Residues Contribute to Homodimerization of Chicken Interleukin-2"],"prefix":"10.3390","volume":"20","author":[{"given":"Chen","family":"Deng","sequence":"first","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hailiang","family":"Tan","sequence":"additional","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongda","family":"Zhou","sequence":"additional","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mengyun","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"L\u00fc","sequence":"additional","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiacui","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"},{"name":"Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Institute of Zoonosis, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4896-2391","authenticated-orcid":false,"given":"Huanmin","family":"Zhang","sequence":"additional","affiliation":[{"name":"Avian Disease and Oncology Laboratory, Agriculture Research Service, United States Department of Agriculture, 4279 East Mount Hope Road, East Lansing, MI 48823, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Limei","family":"Han","sequence":"additional","affiliation":[{"name":"College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, 120 Dongling Road, Shenyang, Liaoning 110866, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5212-3609","authenticated-orcid":false,"given":"Yongxing","family":"Ai","sequence":"additional","affiliation":[{"name":"College of Animal Science, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"},{"name":"Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Institute of Zoonosis, Jilin University, 5333 XiAn Road, Changchun, Jilin 130062, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,11,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Moon, B., Kim, T., and Seoh, J. (2015). Functional modulation of regulatory T cells by IL-2. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0141864"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"6136","DOI":"10.4049\/jimmunol.172.10.6136","article-title":"IL-2 secretion by CD4+ T cells in vivo is rapid, transient, and influenced by TCR-specific competition","volume":"172","author":"Sojka","year":"2004","journal-title":"J. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.cyto.2004.06.010","article-title":"Overview of interleukin-2 function, production and clinical applications","volume":"28","author":"Gaffen","year":"2004","journal-title":"Cytokine"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2905","DOI":"10.3389\/fimmu.2018.02905","article-title":"Anti-cancer therapies employing IL-2 cytokine tumor targeting: Contribution of innate, adaptive and immunosuppressive cells in the anti-tumor efficacy","volume":"9","author":"Mortara","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1016\/j.coi.2011.08.003","article-title":"IL-2 family cytokines: New insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation","volume":"23","author":"Liao","year":"2011","journal-title":"Curr. Opin. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1142","DOI":"10.1038\/sj.embor.7401099","article-title":"Interleukin 2: From immunostimulation to immunoregulation and back again","volume":"8","author":"Bachmann","year":"2007","journal-title":"EMBO Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1002\/cyto.1132","article-title":"Different rates of CD4+ and CD8+ T-cell proliferation in interleukin-2\u2013treated human immunodeficiency virus-positive subjects","volume":"46","author":"Caggiari","year":"2001","journal-title":"Cytometry"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1046\/j.1365-2567.2001.01264.x","article-title":"The regulation of FasL expression during activation-induced cell death (AICD)","volume":"103","author":"Nguyen","year":"2001","journal-title":"Immunology"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"11082","DOI":"10.1073\/pnas.0403916101","article-title":"IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells","volume":"101","author":"Vereb","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2454","DOI":"10.1128\/JVI.78.5.2454-2459.2004","article-title":"Human CD4+CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens","volume":"78","author":"Aandahl","year":"2004","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1002\/hep.20454","article-title":"An immunomodulatory role for CD4+CD25+ regulatory T lymphocytes in hepatitis C virus infection","volume":"40","author":"Cabrera","year":"2004","journal-title":"Hepatology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"22913","DOI":"10.1074\/jbc.M116.729038","article-title":"Interleukin 2 activates brain microvascular endothelial cells resulting in destabilization of adherens junctions","volume":"291","author":"Wylezinski","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1159\/000014541","article-title":"Interleukin-2 and its effects in the central nervous system","volume":"7","author":"Jiang","year":"1998","journal-title":"Biol. Signals Recept."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1002\/jgm.677","article-title":"Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo","volume":"7","author":"Zhang","year":"2005","journal-title":"J. Gene Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1053\/j.seminoncol.2006.06.004","article-title":"Interleukin-2 therapy of metastatic renal cell carcinoma-predictors of response","volume":"33","author":"McDermott","year":"2006","journal-title":"Semin. Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1182\/blood-2003-12-4355","article-title":"IL-2\u2013induced CD4+ T-cell expansion in HIV-infected patients is associated with long-term decreases in T-cell proliferation","volume":"104","author":"Sereti","year":"2004","journal-title":"Blood"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1080\/03079450601113159","article-title":"Expression of chicken interleukin-2 by turkey herpesvirus increases the immune response against Marek\u2019s disease virus but fails to increase protection against virulent challenge","volume":"36","author":"Tarpey","year":"2007","journal-title":"Avian Pathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1111\/j.1365-3083.1986.tb01951.x","article-title":"Chicken T-cell growth factor: Use in the generation of a long-term cultured T-cell line and biochemical characterization","volume":"23","author":"Vainio","year":"1986","journal-title":"Scand. J. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1089\/107999002320271341","article-title":"Interleukin-2 directly induces activation and proliferation of chicken T cells in vivo","volume":"22","author":"Hilton","year":"2002","journal-title":"J. Interferon. Cytokine Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"533","DOI":"10.3389\/fmicb.2015.00533","article-title":"Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites","volume":"6","author":"Li","year":"2015","journal-title":"Front. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"8529","DOI":"10.1016\/j.vaccine.2007.10.006","article-title":"A recombinant turkey herpesvirus expressing chicken interleukin-2 increases the protection provided by in ovo vaccination with infectious bursal disease and infectious bronchitis virus","volume":"25","author":"Tarpey","year":"2007","journal-title":"Vaccine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/j.tvjl.2010.05.001","article-title":"The European ban on antibiotic growth promoters in animal feed: From challenges to opportunities","volume":"187","author":"Millet","year":"2011","journal-title":"Vet. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/S0165-2427(01)00414-7","article-title":"The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants","volume":"85","author":"Hilton","year":"2002","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"53","DOI":"10.2174\/221279680901151109161126","article-title":"An overview of protein-protein interaction","volume":"9","author":"Ali","year":"2015","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/j.tibs.2004.09.006","article-title":"The power of two: Protein dimerization in biology","volume":"29","author":"Marianayagam","year":"2004","journal-title":"Trends Biochem. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.bbrc.2007.12.123","article-title":"Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347","volume":"367","author":"Herscovitch","year":"2008","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3227","DOI":"10.1038\/ncomms4227","article-title":"A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes","volume":"5","author":"Kim","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"117","DOI":"10.3109\/08977199209046401","article-title":"Intermolecular cystine-bonding of murine interleukin 2 indicates that ligand dimerization is important for the formation of the high-affinity receptor complex","volume":"7","author":"Lother","year":"1992","journal-title":"Growth Factors"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3129","DOI":"10.1021\/bi00385a028","article-title":"Characterization of disulfide bonds in recombinant proteins: Reduced human interleukin 2 in inclusion bodies and its oxidative refolding","volume":"26","author":"Tsuji","year":"1987","journal-title":"Biochemistry"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1002\/mas.10025","article-title":"Protein disulfide bond determination by mass spectrometry","volume":"21","author":"Gorman","year":"2002","journal-title":"Mass Spectrom. Rev."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/j.cytogfr.2014.07.018","article-title":"The IL-2 cytokine family in cancer immunotherapy","volume":"25","author":"Sim","year":"2014","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Wrenshall, L.E., Clabaugh, S.E., Cool, D.R., Arumugam, P., Grunwald, W.C., Smith, D.R., Liu, G.C., and Miller, J.D. (2014). Identification of a cytotoxic form of dimeric interleukin-2 in murine tissues. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0102191"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"187.4","DOI":"10.4049\/jimmunol.194.Supp.187.4","article-title":"Dimeric IL-2 induces necrosis of interleukin-2 receptor positive cells","volume":"194","author":"Ganesan","year":"2015","journal-title":"J. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1096\/fasebj.9.11.7649410","article-title":"Self-association of interleukin 2 bound to its receptor","volume":"9","author":"Kaplan","year":"1995","journal-title":"FASEB J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/0169-328X(94)90086-8","article-title":"Linear dimeric interleukin-2 obtained by the use of a defective herpes simplex viral vector: Conformation-activity relationship","volume":"26","author":"Eizenberg","year":"1994","journal-title":"Mol. Brain Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"251","DOI":"10.3347\/kjp.2014.52.3.251","article-title":"A novel recombinant BCG vaccine encoding Eimeria tenella rhomboid and chicken IL-2 induces protective immunity against coccidiosis","volume":"52","author":"Wang","year":"2014","journal-title":"Korean J. Parasitol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Rueden, C.T., Schindelin, J., Hiner, M.C., DeZonia, B.E., Walter, A.E., Arena, E.T., and Eliceiri, K.W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinform., 18.","DOI":"10.1186\/s12859-017-1934-z"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"6233","DOI":"10.1039\/c2cp23749k","article-title":"Rotational dynamics of thiocyanate ions in highly concentrated aqueous solutions","volume":"14","author":"Kim","year":"2012","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1021\/je400903n","article-title":"Thermodynamic study of bile salts micellization","volume":"59","author":"Maestre","year":"2014","journal-title":"J. Chem. Eng. Data"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2328","DOI":"10.1021\/bi00357a005","article-title":"Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers","volume":"25","author":"Robinson","year":"1986","journal-title":"Biochemistry"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Patra, M., Mukhopadhyay, C., and Chakrabarti, A. (2015). Probing conformational stability and dynamics of erythroid and nonerythroid spectrin: Effects of urea and guanidine hydrochloride. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0116991"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.molimm.2013.12.003","article-title":"Dissociation and re-association studies on the interaction domains of Mannan-Binding Lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, provide evidence for heterodimer formation","volume":"59","author":"Hermann","year":"2014","journal-title":"Mol. Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e53340","DOI":"10.1371\/journal.pone.0053340","article-title":"Avirulent Marek\u2019s disease virus type 1 strain 814 vectored vaccine expressing avian influenza (AI) virus H5 haemagglutinin induced better protection than Turkey herpesvirus vectored AI vaccine","volume":"8","author":"Cui","year":"2013","journal-title":"PLoS ONE"},{"key":"ref_44","first-page":"2","article-title":"T cell enrichment by nonadherence to nylon","volume":"30","author":"Hathcock","year":"2001","journal-title":"Curr. Protoc. Immunol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/22\/5744\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:34:51Z","timestamp":1760189691000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/22\/5744"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,11,15]]},"references-count":44,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2019,11]]}},"alternative-id":["ijms20225744"],"URL":"https:\/\/doi.org\/10.3390\/ijms20225744","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2019,11,15]]}}}