MOLECULAR CLONING AND CHARACTERIZATION OF ARACHIDONATE 5- LIPOXYGENASE FROM BIG BELLY SEAHORSE Hippocampus abdominalis

Arachidonate lipoxygeneses are a heterogeneous family of lipid peroxidizing enzymes with ability to convert the oxygenating polyunsaturated fatty acids to corresponding hydroperoxy molecules. Arachidonate 5-lipoxygenase (ALOX5) facilitates biosynthesis of leukotrienes, mediators of inflammation derived from arachidonic acid (AA). In this study, ALOX5 of big-belly seahorse (Hippocampus abdominalis; HaALOX5) were identified and characterized at sequence, transcriptional and molecular levels. Coding sequence of HaALOX5 was 2025 bp that encode respective protein sequence with 674 amino acids in length. In silico analysis reveal that homology modelling of HaALOXY5 3D structure was resemble to the human ALOX5 monomer. Signal peptide was not detected on the amino acid sequence. The recombinant HaALOX5 catalyzed the conversion of AA to 5-Hydroxyeicosatetraenoic acid (5-HETE). Final product of 5-HETE was detected by RP-HPLC. Phylogenetic tree showed a neighboring evolutionary relationship for ALOX5 vertebrate counterparts, with close assembly to the fish homologs. ALOX5 transcripts was detected in all tissues with highest in intestine and lowest in ovary. In blood and intestine, the mRNA expression of ALOX5 showed similar pattern upon S. iniae challenge. In blood, upon Poly (I:C) challenge, expression was significantly elevated after 72 h post injection and upon LPS, similarly increased at 3 h. Our results enriched understanding the modulation of HaALOX-5 mRNA expression under conditions of pathogenic stresses.