Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells.
Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or -2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography-MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.
Pettitt
T R
TR
CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, U.K.
McDermott
M
M
Saqib
K M
KM
Shimwell
N
N
Wakelam
M J
MJ
D000818:Animals; D002460:Cell Line; D004730:Endothelium, Vascular / Q000201:enzymology; D007700:Kinetics; D008322:Mammals; D013058:Mass Spectrometry; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D010739:Phospholipase D / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014162:Transfection
D010712:Phosphatidic Acids; D011994:Recombinant Proteins; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1