Molecular characterization and functional analysis of TRAF6 in the spotted sea bass (Lateolabrax maculatus)

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial adapter protein in the toll-like receptor signaling pathway that triggers downstream molecules involved in innate immunity. Although TRAF6 has been well studied in mammals, the molecular information and function of TRAF6 in fish is still limited. Here, we identified and analyzed a TRAF6 homolog (LmTRAF6) from the spotted sea bass (Lateolabrax maculatus). Similar to its counterparts in mammals and other fish species, LmTRAF6 shares the domain topology containing one N-terminal RING, two TRAF-type zinc fingers, a coiled-coil region and a C-terminal MATH domain. Despite a sequence similarity of 60% with mammalian TRAF6s, LmTRAF6 shares higher similarities with teleost homologs (~68%-93%). The coding region of LmTRAF6 gene contains seven exons and six introns, which is consistent to the genetic organization in grouper and rock bream, but not in zebrafish, common carp and tetrapods (the sixth intron was lost resulting in a combined exon). Quantitative real-time polymerase chain reaction analysis revealed that LmTRAF6 transcripts were ubiquitously expressed in all tested tissues and upregulated after Vibrio. harveyi and S. agalactiae infection. LmTRAF6 could assist HEK293T cells to survive by inhibiting apoptosis under both V. harveyi and S. agalactiae stimulation. Intracellular localization showed that LmTRAF6 was localized mainly in the cytoplasm. Overexpression of wild-type (WT) LmTRAF6 and the truncated form of △MATH increased the ability of NF-κB in HEK293T cells, whereas truncations, including the △RING and △coiled-coil domain, did not significantly activate NF-κB, indicating that the RING finger and coiled-coil domain play crucial roles in downstream signal transduction. In addition, overexpression of LmTRAF6-WT significantly increased the activation of NF-κB in HEK293T cells under V. harveyi and S. agalactiae stimulation. These results suggest that LmTRAF6 activates NF-κB and plays a potential role in the immune defense system against bacterial infection.