A TRAF and TNF receptor-associated protein (TTRAP) in mollusk with endonuclease activity

Abstract

Tumor necrosis factor (TNF) signaling pathway plays crucial roles in the regulation of various immune responses. In the present study, a TNF signaling pathway related regulatory factor, TRAF and TNF receptor-associated protein (TTRAP), was firstly identified from the mollusk Zhikong scallop Chlamys farreri (designated as CfTTRAP). The full-length cDNA of CfTTRAP was of 2326bp, containing an open reading frame (ORF) of 1008 bp encoding a polypeptide of 335 amino acids with the predicted molecular weight of 38.4 kDa. There was an Exo_endo_phos domain in CfTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. The recombinant protein of CfTTRAP exhibited prominent endonuclease activity to digest the genome DNA from C. farreri in the presence of Mg(2+), but it could not digest genome DNA of Escherichia coli and Bacillus subtilis, indicating CfTTRAP was a new member of Mg(2+)/Mn(2+)-dependent phosphodiesterase enzymes (MDP) superfamily. The mRNA transcripts of CfTTRAP were detected in all tested tissues of scallop, including muscle, mantle, gonad, gill, kidney and hemocytes. The expression level of CfTTRAP mRNA in hemocytes varied greatly after the stimulation of LPS, PGN or β-glucan. LPS induced significant down-regulation (P<0.05) of CfTTRAP mRNA expression, while PGN or β-glucan up-regulated the expression significantly (P<0.01), indicating that this regulatory factor was involved in modulating immune responses towards different stimulus. The present results provided new evidences for the potential roles of such molecule in C. farreri, and further confirmed the existence of TTRAP modulated TNF signaling pathway in mollusk.