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 AbFas ligand. It contains an 1832bp transcript with a 945bp open reading frame, encoding 315 amino acids. The AbFas ligand showed characteristic transmembrane and TNF family signature domains. The deduced amino acid comparison showed that the AbFas ligand exhibits 22.0, 16.1 and 14.5% identities to human Fas ligand, TNF-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 AbFas ligand transcripts were constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and digestive gland in a tissue-specific manner. By immune stimulation, AbFas 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 AbFas 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 H(2)O(2) in human THP-1 cells in concentration-dependant manner. Correlating the AbFas 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-), H(2)O(2) and other ROS.
De Zoysa
Mahanama
M
Department of Marine Life Sciences, College of Ocean Science, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
Nikapitiya
Chamilani
C
Moon
Dong-Oh
DO
Whang
Ilson
I
Kim
Gi-Young
GY
Lee
Jehee
J
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000276:immunology; D001483:Base Sequence; D053222:Fas Ligand Protein / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D049851:Gastropoda / Q000145:classification / Q000235:genetics* / Q000276:immunology* / Q000382:microbiology; D005786:Gene Expression Regulation / Q000187:drug effects; D015723:Gene Library; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000276:immunology* / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D000276:Adjuvants, Immunologic; D053222:Fas Ligand Protein; D008070:Lipopolysaccharides; D011994:Recombinant Proteins