Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1α and mPA-PLA1β

We have identified a novel phospholipase A(1), named mPA-PLA(1)beta, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA(1)alpha. The sequence of mPA-PLA(1)beta 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)alpha. mPA-PLA(1)beta contains a short lid and deleted beta9 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)alpha and phosphatidylserine-specific PLA(1). Both mPA-PLA(1)beta and mPA-PLA(1)alpha recombinant proteins exhibited PA-specific PLA(1) activity and were vanadate-sensitive. When mPA-PLA(1)beta-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA(1)alpha and beta-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)alpha protein was recovered from the cell supernatant. By contrast, mPA-PLA(1)beta was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA(1)beta has higher affinity to heparin than mPA-PLA(1)alpha. We also found that the membrane-associated mPA-PLA(1)s were insoluble in solubilization by 1% Triton X-100 and were detected in Triton X-100-insoluble buoyant fractions of sucrose gradients. The present study raises the possibility that production of LPA by mPA-PLA(1)alpha and -beta occurs on detergent-resistant membrane domains of the cells where they compete with lipid phosphate phosphatase for PA.