Cloning and expression of group IB phospholipase A2 isoforms in the red sea bream, Pagrus major

Two cDNA encoding red sea bream DE-1 and DE-2 phospholipases A2 (PLA2) were cloned from the hepatopancreas of red sea bream, Pagrus (Chrysophrys) major. The cDNA of DE-1 PLA2 encoded a mature protein of 125 amino acid residues with an apparent signal peptide of 20 residues and propeptide of 5 residues, and that of DE-2 PLA2, a mature protein of 126 amino acid residues with an apparent signal peptide of 17 residues and propeptide of 6 residues. Comparison of the predicted amino acid sequences for mature DE-1 and DE-2 PLA2 showed that both proteins contain 14 cysteines including Cys 11 and 77 and a pancreatic loop, which are commonly conserved in group IB PLA2; however, the identity in amino acid sequence between DE-1 and DE-2 PLA2 was low (47%). A previous report concerning the cDNA cloning of red sea bream gill G-3 PLA2 and the present results represent the first cloning and sequencing of three distinct isoforms of group IB PLA2 in a single fish species, red sea bream. Reverse transcription-polymerase chain reaction analysis showed that DE-1 PLA2 mRNA was expressed in the hepatopancreas, pyloric ceca, intestine, spleen, gonad, stomach, and kidney, whereas gill G-3 PLA2 mRNA was expressed only in the gills and gonad. The expression of DE-2 PLA2 mRNA was detected in all of the tissues analyzed. These results indicate that three distinct isoforms of group IB PLA2, DE-1 and DE-2 PLA2 in hepatopanceas and gill G-3 PLA2, are expressed in a tissue-specific manner in red sea bream.