A novel Fas ligand in mollusk abalone: Molecular characterization, immune responses and biological activity of the recombinant protein

Fas ligand is a member of the TNF superfamily that plays an important role by inducing apoptosis and homeostasis of immune responses. The gene encoding Fas ligand was isolated from a disk abalone (Haliotis discus discus) cDNA library, denoted as the Was ligand. It contains an 1832 bp transcript with a 945 bp open reading frame, encoding 315 amino acids. The Was ligand showed characteristic transmembrane and TNF family signature domains. The deduced amino acid comparison showed that the Was ligand exhibits 22.0, 16.1 and 14.5% identities to human Fas ligand, TNIF-alpha, and lymphotoxin (LT-alpha), respectively. Phylogenetic analysis indicates that the AbFas ligand belongs to the invertebrate TNF family and it is closely related to vertebrate Fas ligand counterparts. Quantitative real-time PCR analysis results showed that the Was ligand transcripts were constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and digestive gland in a tissue-specific manner. By immune stimulation, Was ligand mRNA was significantly (p < 0.05) up-regulated after infection with a mixture of bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes), viral haemorrhagic septicaemia virus (VHSV), and lipopolysaccharide (LPS) in abalone gills. The recombinant Was ligand was over-expressed in Escherichia coli (E coli) and purified using a pMAL protein fusion system. This recombinant AbFas ligand showed its biological activity by inducing both superoxide anion (O-2(-)) and H2O2 in human THP-1 cells in concentration-depend ant manner. Correlating the Was ligand transcriptional up-regulation against bacteria, virus and LPS with the biological activity of its recombinant protein, we could suggest that the abalone Fas ligand may control microbial infection by inducing O-2(-), H2O2 and other ROS. (C) 2009 Elsevier Ltd. All rights reserved.