title: Grouper interleukin-12, linked by an ancient disulfide-bond architecture, exhibits cytokine and chemokine activities.
abstract: Interleukin-12 (IL-12) is a pleiotropic cytokine which bridges innate and adaptive immunity in defense against pathogens. IL-12 proved to be an effective and successful adjuvant to enhance both the innate and adaptive immune responses and could be applicable for a rationale vaccine formulation in fish against pathogen infection. We have cloned the p35 and p40 cDNAs of IL-12 from orange-spotted grouper (Epinephelus coioides). Grouper IL-12 most resembles with sea bass orthologues; moderate to low identity with other teleost and mammalian counterparts. The structural model of grouper IL-12 heterodimer revealed NC(141)F three amino acid patch of grouper p35, which is present in teleost p35 but absent in mammalian and avian p35, and is spatially nearby the conserved cysteine residue located at A-helix of p35 to form a disulfide bond when the 14aa peptide located at loop 1 of grouper p35 was aligned with human corresponding exon 4, instead of exon 5. The results indicated that the loss of this 3aa patch during evolution was compensated by the duplication of exon 4 in mammalian p35 to gain another cysteine residue to form a disulfide bond, evidenced by chicken p35 which does not contain NCF corresponding 3-aa patch nor exon 4 duplication. Accordingly, the inter-chain disulfide bond of IL-12 heterodimer is conserved from teleost to mammalian IL-12. A single chain grouper IL-12 (scgIL-12) construct linked by (G4S)3 was successfully expressed in baculovirus-insect cell system; its identity has been confirmed by LC/MS/MS. In addition, the biological activity of recombinant scgIL-12 (rscgIL-12) are demonstrated for its stimulation of PBL proliferation, chemotactic migration, induction of TNF-α gene expression and a plausible adjuvant effect of prolonged protection against parasite infection in fish. We illustrated the first time in lower vertebrate that grouper IL-12 possesses both cytokine and chemokine activities.
authors.affiliation: Institute of Zoology, National Taiwan University, Taipei City 106, Taiwan.
authors.forename: Jui-Ling
authors.initials: JL
authors.lastname: Tsai
authors.affiliation: Institute of Zoology, National Taiwan University, Taipei City 106, Taiwan.
authors.forename: T A Jose
authors.initials: TA
authors.lastname: Priya
authors.affiliation: Department of Bioinformatics, Chung Hua University, Hsin-Chu City 300, Taiwan.
authors.forename: Kuang-Yu
authors.initials: KY
authors.lastname: Hu
authors.affiliation: Laboratory of Sensory Biology, Institute of Cellular and Organismic Biology, Academic Sinica, Yi-Lan County 262, Taiwan.
authors.forename: Hong-Young
authors.initials: HY
authors.lastname: Yan
authors.affiliation: Institute of Biological Chemistry, Academic Sinica, Taipei City 115, Taiwan. Electronic address: santai@gate.sinica.edu.tw.
authors.forename: San-Tai
authors.initials: ST
authors.lastname: Shen
authors.affiliation: Institute of Zoology, National Taiwan University, Taipei City 106, Taiwan; Department of Life Science, National Taiwan University, Taipei City 106, Taiwan. Electronic address: song@ntu.edu.tw.
authors.forename: Yen-Ling
authors.initials: YL
authors.lastname: Song
mesh_terms: D056704:Adaptive Immunity / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D002633:Chemotaxis / Q000276:immunology; D003001:Cloning, Molecular; D007113:Immunity, Innate / Q000235:genetics* / Q000276:immunology; D018664:Interleukin-12 / Q000235:genetics* / Q000276:immunology; D053765:Interleukin-12 Subunit p35 / Q000235:genetics / Q000276:immunology; D053762:Interleukin-12 Subunit p40 / Q000235:genetics / Q000276:immunology; D007963:Leukocytes, Mononuclear; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010473:Perciformes / Q000235:genetics* / Q000276:immunology; D010802:Phylogeny; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
chemical_list: D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D012313:RNA
keywords: Chemotactic migration; Grouper (Epinephelus coioides); IL-12 structure; PBL proliferation; TNF-α induction