A zebrafish (Danio rerio) bloodthirsty member 20 with E3 ubiquitin ligase activity involved in immune response against bacterial infection.

The tripartite motif (TRIM)-containing proteins exhibit various activities and play important roles in the immune system through regulating signaling pathways. Bloodthirsty gene is a multigene subset of TRIM genes. In this study we identified and characterized a new member of the bloodthirsty subset of TRIM genes, btr20, in zebrafish (Danio rerio). The gene is located on chromosome 19 and forms a cluster with btr18, btr21, btr22 and an E3 ubiquitin ligase TRIM39-like gene. Deduced btr20 represents a RBCC-B30.2 TRIM protein containing 544 amino acids. The mRNA expression level of btr20 was highest in intestine and gill, followed by in spleen and kidney. Challenge experiment with Aeromonas hydrophila strain NJ-1 showed that the levels of btr20 and NF-κB mRNA were remarkably upregulated in the four tissues mentioned above. btr20 was localized in the cytoplasm and formed aggregate in human embryonic kidney cell line 293T. In vitro self-ubiquitylation experiment demonstrated that btr20 has E3 ubiquitin ligase activity that can be self-ubiquitylated with most E2 enzymes, especially UbcH6. The results suggested that btr20 may involve in the anti-microbial activity in the immune system as an E3 ubiquitin ligase.