title: Molecular characterization and functional analysis of TRAF6 in orange-spotted grouper (Epinephelus coioides).
abstract: Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer in both the TNFR superfamily and Toll-like receptor/interleukin 1R family. Although significant progress has been made in clarifying the role of TRAF6 in mammals, the function of TRAF6 in fish is still poorly understood. In this study, we cloned the orange-spotted grouper (Epinephelus coioides) TRAF6 (EcTRAF6) cDNA, with an open reading frame of 1713bp encoding 570 amino acids. Sequence analysis indicated that EcTRAF6 contains the four characteristic domains conserved in the TRAF family, including an N-terminal RING finger, two zinc fingers, a coiled-coil domain, and a C-terminal MATH domain. Homology alignment and phylogenetic analysis demonstrated that EcTRAF6 shares high sequence identity with TRAF6 of other fish species. The EcTRAF6 gene contains seven exons and six introns, which is similar to the organization in ayu, but not in the common carp, human, or mouse (six exons and five introns). EcTRAF6 transcripts were broadly expressed in all tissues tested, and increased after infection with Cryptocaryon irritans. Intracellular localization showed EcTRAF6 was distributed mainly in the cytoplasm. Over-expression of wild type (WT) EcTRAF6, truncated forms of EcTRAF6, including ΔZinc finger 2 and ΔMATH, and a mutant of C78A activated NF-κB strongly in HEK293T cells; whereas truncations, including ΔRING, ΔZinc finger 1 and Δcoiled-coiled, and a mutant of K132R induced the activity of NF-κB slightly compared to WT EcTRAF6, implying the latter has a more crucial role in downstream signal transduction. Together, these results suggested EcTRAF6 functions like that of mammals to activate NF-κB, and it might have an important role in host defense against parasitic infections.
authors.affiliation: Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, The School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 Guangdong Province, PR China.
authors.forename: Yan-Wei
authors.initials: YW
authors.lastname: Li
authors.affiliation: Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, The School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 Guangdong Province, PR China.
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, The School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 Guangdong Province, PR China.
authors.forename: Xi-Xi
authors.initials: XX
authors.lastname: Xiao
authors.affiliation: Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, 1 Xingyu Road, Liwan District, Guangzhou, 510380 Guangdong Province, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Zhao
authors.affiliation: School of Bioscience and Biotechnology, South China University of Technology, Guangzhou, 510006 Guangdong Province, PR China.
authors.forename: Xiao-Chun
authors.initials: XC
authors.lastname: Luo
authors.affiliation: College of Animal Science, South China Agricultural University, Guangzhou, 510642 Guangdong Province, PR China.
authors.forename: Xue-Ming
authors.initials: XM
authors.lastname: Dan
authors.affiliation: Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, The School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 Guangdong Province, PR China. Electronic address: lianxing@mail.sysu.edu.cn.
authors.forename: An-Xing
authors.initials: AX
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016798:Ciliophora / Q000276:immunology*; D016770:Ciliophora Infections / Q000276:immunology*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D005399:Fishes / Q000276:immunology*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D016328:NF-kappa B; D048029:TNF Receptor-Associated Factor 6
keywords: Cryptocaryon irritans; Epinephelus coioides; Over-expression; TRAF6