Evolutionary and molecular characterization of fish specific decoy receptor 3 isoforms (TNFRSF6B.1 and 2) in rock bream (Oplegnathus fascatus)

TNFRSF6B, commonly referred to as decoy receptor 3, interacts with TNFSF6, TNFSF14, and TNFSF15, thereby imparting anti-apoptotic and anti-inflammatory properties. This study identifies two isoforms, TNFRSF6B.1 and TNFRSF6B.2, in rock bream (RB) and explores their evolutionary and molecular attributes. Phylogenetic analysis indicates that the teleost isoforms TNFRSF6B.1 and TNFRSF6B.2 have diverged from ancestral forms of TNFRSF6B and TNFRSF11B. Notably, the coelacanth TNFRSF6B is positioned phylogenetically closer to frogs, while TNFRSF6B.2 branches off from TNFRSF11B. Synteny analysis reveals that the TNFRSF6B isoforms in rainbow trout are situated within the conserved mammalian TNFRSF6B locus, whereas the teleost TNFRSF6B.1 and TNFRSF6B.2 genes are located within the conserved TNFRSF11B locus. However, the amino acid sequences of TNFRSF6B.1 and TNFRSF6B.2 exhibit greater similarity to TNFRSF6B than to TNFRSF11B, with homology percentages of 33.92 % and 36.31 % for TNFRSF6B, and 27.94 % and 29.98 % for TNFRSF11B, respectively. Furthermore, protein-protein interaction analyses demonstrate associations with typical ligands of TNFRSF6B, including TNFSF14, TNFSF6, TNFSF5, and TNFSF15. Tertiary structure modeling of receptor-ligand interactions has also identified conserved TNFSF15-binding residues in both TNFRSF6B.1 and TNFRSF6B.2. In healthy rock bream, TNFRSF6B.1 expression is predominantly observed in gill and body kidney, while TNFRSF6B.2 expression is highest in fin, followed by skin and gill. In vitro, experiment reveals that TNFRSF6B.1 expression is significantly upregulated by calcium ionophores at 4 and 8 h, as well as by LPS at 8 or 12 h. Conversely, TNFRSF6B.2 exhibits a more pronounced response to concanavalin A and phytohemagglutinin at 4 or 12 h compared to TNFRSF6B.1. Both isoforms are upregulated in response to recombinant RB-TNFSF15 protein (RB-rTNFSF15). In vivo, expression levels of these genes are not significantly altered by RB-rTNFSF15 in head kidney, spleen, and blood, but are significantly upregulated in liver. In gill, RB-TNFRSF6B.1 expression is upregulated, whereas RB-TNFRSF6B.2 expression is downregulated. These findings suggest that TNFRSF6B.1 and TNFRSF6B.2 play roles in inflammatory responses and mucosal immunity, particularly in relation to TNFSF15, akin to mechanisms observed in humans.