Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A 1 (PLA 1 ) that produces 2-acyl-LPA. The PLA 1 was identified in the GenBank TM data base as a close homologue of phosphatidylserine (PS)-specific PLA 1 (PS-PLA 1 ). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA 3 , a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA 1 and suggest that it has a role in LPA production.

Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A 2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [ 3 H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserinephospholipase A 1 , is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.Membrane phospholipids of activated platelets serve as precursors for second messengers, such as eicosanoids, and also as footholds for blood coagulation. Platelets contain at least three types of phospholipase A; cytosolic phospholipase A 2 (cPLA 2 ) 1 (1, 2), secretory 14-kDa type II phospholipase A 2 (sPLA 2 ) (3), and another serine-phospholipid-selective phospholipase which has not yet been fully characterized (4 -6). cPLA 2 is involved in the production of eicosanoid by cleaving arachidonic acid at the sn-2 position of phospholipids in various types of cells. sPLA 2 is found in inflammatory sites (7,8) and is considered to be involved in inflammatory response progression and may participate in eicosanoid formation in certain cells, such as vascular endothelial cells, mast cells, neutrophils, hepatocytes, and others (9 -13). Thus sPLA 2 and cPLA 2 are well characterized, and their cDNA and genomic structures are also known (2, 14, 15), whereas no information about the structure of the last enzyme is available.Like sPLA 2 this new member of the phospholipase A family is secreted from activated rat platelets (4); partially purified preparations specifically act on phosphatidylserine (PS) (6) and lysophosphatidylserine (lyso-PS) (4, 5). This activity is inhibited both by diisopropyl fluorophosphate (DFP) and dithiothreitol to release a fatty acid of PS ...

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In this study we isolated and sequenced cDNA clones encoding human PS-PLA 1 , which showed 80% homology with rat PS-PLA 1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA 1 ), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA 1 ⌬C) that lacks two-thirds of the C-terminal domain of PS-PLA 1 . Unlike PS-PLA 1 , PS-PLA 1 ⌬C hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA 1 ⌬C exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA 1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA 1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).

Summary Saccharomyces cerevisiae