cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A2 (PLA2), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA2 protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA2 protein, suggesting the presence of multiple forms in the starfish PLA2. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA2. In phylogenetic tree, the starfish PLA2 should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA2, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA2 from porcine pancreas

Bacterial expression and characterization of starfish phospholipase A2

Phospholipase A2 (PLA2) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA2 from porcine pancreas. To investigate enzymatic properties of the starfish PLA2 in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA2 cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA2 protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-β-(–)-thiogalactopyranoside. The recombinant PLA2 produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris–HCl buffer (pH 8.0). Renatured PLA2 was purified by subsequent column chromatographies on DEAE–cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA2 was replaced by an Ala in the recombinant PLA2, the recombinant enzyme showed essentially the same properties as did the native PLA2 with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca2+ requirement