Cloning, Expression And Functional Analysis Of TRAF3 In Humphead Snapper, Lutjanus Sanguineus

Humphead snapper(Lutjanus sanguineus) is one of the most important marine fish in the south coastal regions of China. In recent years, the outbreaks of serious infectious diseases have caused great economic lossses to the snapper aquaculture. However, the immune defense mechanisms of humphead snapper against pathogen invasion are largely unknown. Tumor necrosis factor receptor-associated factors are kinds of crucial signal transduction docking proteins that regulate various immune signal pathways. TRAF3, a distinct member of TRAF family, is recognized as signal transducer of diverse other receptor families, including receptors of the TNF families(TNFRs), NOD-like receptors(NLRs), Toll-like receptors(TLRs) family and interleukin-1 receptor(IL-1R) family.Notably, mounting evidence has revealed that TRAF3 is a highly versatile regulator that control diverse cellular processes, including proliferation, differentiation, apoptosis and immunity response. To investigate the role of TRAF3 in snapper immunity signal transduction, a novel TRAF3 gene(Ls-TRAF3) was cloned from humphead snapper, its expression and function characteristics were also investigated.1. In this study, the TRAF3 gene(Ls-TRAF3) was cloned from humphead snapper,Lutjanus sanguineus through RACE method. Biological informatics analysis showed that the full-length of Ls-TRAF3 cDNA was 3160 bp, containing a 5’ untranslated region(5’-UTR) of 253 bp, a 3’ untranslated region(3’-UTR) of 1119 bp and an open reading frame of 1788 bp, which encoded a polypeptide of 595 amino acids with an estimated molecular weight(MW) of 67.44 KDa and an estimated isoelectric point(pI) of 7.5. The deduced protein of Ls-TRAF3 contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain at the C-terminus, which were conserved from mammals to fish. Ls-TRAF3 encoded amino acid peptide which shared high homology with Maylandia zebra TRAF3(92%), Haplochromis burtoni TRAF3(91%) and Oncorhynchus mykiss TRAF3(79%). Phylogenetic analysis showed that Ls-TRAF3 had the closest distance with T. Rubripes.2. Ls-TRAF3 was ubiquitously expressed in all the tissues tested, with relative higher expression in spleen, head kidney and liver. Quantitative real-time PCR(qRT-PCR)analysis revealed that Ls-TRAF3 could be induced by bacteria or viral PAMP poly I:Cstimulation in head kidney leukocytes(HKLs). Here, we also demonstrated Ls-TRAF3 that,positively regulated IRF3 and Mx upon poly I:C stimuli, whereas prevented production of proinflammatory cytokines IL-6 after LPS injection. Those results revealed that Ls-TRAF3 was a highly versatile regulator that positively controls IFN-related genes production, but inhibited the secretion of inflammation cytokine IL-6. The yeast two hybrid system showed:Ls-TRAF3 could inteacted with Ls-CD40-IC. In addation, over-expression of wide type(WT) Ls-TRAF3 and truncated forms, including ?Zinc finger 1, ?Zinc finger 2 and ?coiled-coil suppressed NF-κB activity significantly, whereas the inhibitory effect of NF-κB was partially impaired when the RING finger or MATH domain deletion, suggesting the latter was more important for downstream signal transduction.In summary, Ls-TRAF3 plays an important role in humphead snapper immune response, which not only help to provide new theoretical basis for researching the functions of piscine TRAF3, but also provides a new strategy for fish immune signal transduction mechanisms.