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.
α-Tocopherol (vitamin E) transfer protein (α-TTP) regulates the secretion of α-tocopherol from liver cells. Missense mutations of some arginine residues at the surface of α-TTP cause severe vitamin E deficiency in humans, but the role of these residues is unclear. Here, we found that wild-type α-TTP bound phosphatidylinositol phosphates (PIPs), whereas the arginine mutants did not. In addition, PIPs in the target membrane promoted the intermembrane transfer of α-tocopherol by α-TTP. The crystal structure of the α-TTP-PIPs complex revealed that the disease-related arginine residues interacted with phosphate groups of the PIPs and that the PIPs binding caused the lid of the α-tocopherol-binding pocket to open. Thus, PIPs have a role in promoting the release of a ligand from a lipid-transfer protein.
We have identified a novel phospholipase A 1 , named mPA-PLA 1 , which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA 1 ␣. The sequence of mPA-PLA 1  encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA 1 , mPA-PLA 1 ␣. mPA-PLA 1  contains a short lid and deleted 9 loop, which are characteristics of PLA 1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA 1 ␣ and phosphatidylserine-specific PLA 1 . Both mPA-PLA 1  and mPA-PLA 1 ␣ recombinant proteins exhibited PA-specific PLA 1 activity and were vanadate-sensitive. When mPA-PLA 1 -expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA 1 ␣ and -expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA 1 ␣ protein was recovered from the cell supernatant. By contrast, mPA-PLA 1  was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA 1  has higher affinity to heparin than mPA-PLA 1 ␣. We also found that the membrane-associated mPA-PLA 1 s were insoluble in solubilization by 1%
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