Tumor necrosis factor alpha-induced protein 8-like mediates immune responses of Procambarus clarkii through protein-protein interactions.

Procambarus clarkii is an economically important cultivated freshwater crayfish species in China. Intensive aquaculture and challenging environmental conditions cause diseases that kill crayfish and impact productivity. Clarification of immune mechanisms could assist the breeding of disease-resistant crayfish and improve survival rate. Here, a tumor necrosis factor alpha-induced protein 8-like gene (TNFAIP8L) encoding a 187 amino acid protein in P. clarkii was characterized, and phylogenetic analysis revealed high homology with genes in other crustaceans. Recombinant TNFAIP8L protein was successfully expressed and purified, and pull-down, mass spectrometry, molecular docking, and western blotting identified hemocyanin B and heat shock protein 60 (HSP60) as TNFAIP8L-interacting proteins. Enzyme-linked immunosorbent assay experiments revealed a higher affinity for hemocyanin B than for HSP60. Expression patterns of TNFAIP8L in different tissues and under immune challenge were determined by real-time PCR. TNFAIP8L was expressed in all tissues examined with highest levels in hemocytes, gills, and intestines. Following immune challenge, TNFAIP8L was down-regulated in hemocytes and gills. RNA interference and overexpression of TNFAIP8L induced the expression of immune-related genes Toll, Serpin, B-cell lymphoma (Bcl), Lectin, Defensin, Crustin, and anti-lipopolysaccharide factor (ALF) in hemocytes and gills. Together, the results suggest that TNFAIP8L mediates immune responses in P. clarkii.