Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids. (September 2016) - Jablonická, Veronika
Abstract: The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings ( Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla 2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus. Ps PLA2 contains a PLA2 signature domain (PA2c), including the Ca 2+ -binding loop (YGKYCGxxxxGC) and the catalytic site motif (DACCxxHDxC) with the conserved catalytic histidine and the calcium-coordinating aspartate residues. The aspartate of the His/Asp dyad playing an important role in animal sPLA2 catalysis is substituted by a serine residue. Furthermore, the Ps PLA2 sequence contains 12 conserved cysteine residues to form 6 structural disulfide bonds. The calculated molecular weight of the mature Ps PLA2 is 14.0 kDa. Based on the primary structure Ps PLA2 belongs to the XIB group of PLA2 s. Untagged recombinant Ps PLA2 obtained by expression in Escherichia coli, renaturation from inclusion bodies and purification by cation-exchange chromatography was characterized in vitro . The pH optimum for activity of Ps PLA2 was found to be pH 7, when using mixed micelles of 1, 2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) and Triton X-100. Ps PLA2 specifically cleaves fatty acids from the sn -2 position of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine and shows a pronounced preference for PC over phosphatidyl ethanolamine, -glycerol and -inositol. The active recombinant enzyme was tested in vitro against natural phospholipids isolated from poppy plants and preferably released the unsaturated fatty acids, linoleic acid and linolenic acid, from the naturally occurring mixture of substrate lipids. Graphical abstract: Highlights: Newly identified poppy phospholipase A2 fits into the XIB group of plant secretory PLA2 s. Ps PLA2 shows strong head-group preference for phosphatidylcholine. Ps PLA2 specifically cleaves fatty acids from the sn -2 position of phospholipids. Ps PLA2 preferably releases linoleic and linolenic acid from membrane phospholipids.