CDNA cloning and expression of a novel group IB phospholipase A2 in the intestine of the red sea bream, Pagrus (Chrysophrys) major

Abstract

A cDNA encoding a novel phospholipase A2 (PLA2), which was named IN PLA2, was cloned from the intestine of the red sea bream. The amino acid sequence of IN PLA2 showed 49–75% homology with those of red sea bream group IB sPLA2, hepatopancreas DE-1 and DE-2 PLA2, and gill G-3 PLA2. IN PLA2 consists of a prepropeptide of 24 amino acid residues, followed by a mature protein. IN PLA2 contains 14 cysteines, and includes Cys11, the calcium binding loop and the pancreatic loop that are commonly conserved in group IB sPLA2 enzymes. In addition, IN PLA2 is a cationic protein with a pI of 8.52. Therefore, IN PLA2 was identified as a novel group IB sPLA2 isoform in red sea bream. IN PLA2 mRNA was found by northern blot analysis to be expressed mainly in the pyloric caeca and the intestine, and was detected in the goblet cells of the intestine by in situ hybridization. The expression level of IN PLA2 mRNA was elevated by intravenous injection of lipopolysaccharide—the outer-membrane component of Gram-negative bacteria. These results suggest that IN PLA2 is secreted from the goblet cells of the intestine in response to stimulus such as bacterial infection, and that it contributes to antimicrobial defense in addition to the digestion of dietary phospholipids in the gastrointestinal tract.