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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.
AuthorsJui-Ling Tsai, T A Jose Priya, Kuang-Yu Hu, Hong-Young Yan, San-Tai Shen, Yen-Ling Song
JournalFish & shellfish immunology (Fish Shellfish Immunol) Vol. 36 Issue 1 Pg. 27-37 (Jan 2014) ISSN: 1095-9947 [Electronic] England
PMID24176819 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2013 Elsevier Ltd. All rights reserved.
Chemical References
  • Interleukin-12 Subunit p35
  • Interleukin-12 Subunit p40
  • Recombinant Proteins
  • Tumor Necrosis Factor-alpha
  • Interleukin-12
  • RNA
Topics
  • Adaptive Immunity (genetics, immunology)
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Proliferation (drug effects)
  • Chemotaxis (immunology)
  • Cloning, Molecular
  • Immunity, Innate (genetics, immunology)
  • Interleukin-12 (genetics, immunology)
  • Interleukin-12 Subunit p35 (genetics, immunology)
  • Interleukin-12 Subunit p40 (genetics, immunology)
  • Leukocytes, Mononuclear
  • Models, Molecular
  • Molecular Sequence Data
  • Perciformes (genetics, immunology)
  • Phylogeny
  • RNA (chemistry, genetics)
  • Recombinant Proteins (immunology)
  • Reverse Transcriptase Polymerase Chain Reaction (veterinary)
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Tumor Necrosis Factor-alpha (immunology)

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