Molecular characteristics and function study of TNF receptor-associated factor 5 from grouper (Epinephelus coioides)

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a key adapter molecule that participates in numerous signaling pathways. The function of TRAF5 in fish is largely unknown. In the present study, a TRAF5 cDNA sequence (EcTRAF5) was identified in grouper (Epinephelus coioides). Similar to its mammalian counterpart, EcTRAF5 contained an N-terminal RING finger domain, a zinc finger domain, a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. The EcTRAF5 protein shared relatively low sequence identity with that of other species, but clustered with TRAF5 sequences from other fish. Real-time PCR analysis revealed that EcTRAF5 mRNA was broadly expressed in numerous tissues, with relatively high expression in skin, hindgut, and head kidney. Additionally, the expression of EcTRAF5 was up-regulated in gills and head kidney after infection with Cryptocaryon irritans. Intracellular localization analysis demonstrated that the full-length EcTRAF5 protein was uniformly distributed in the cytoplasm; while a deletion mutant of the coiled-coil domain of EcTRAF5 was observed uniformly distributed in the cytoplasm and the nucleus. After exogenous expression in HEK293T cells, TRAF5 significantly activated NF-κB. The deletion of the EcTRAF5 RING domain or of the zinc finger domain dramatically impaired its ability to activate NF-κB, implying that the RING domain and the zinc finger domain are required for EcTRAF5 signaling.