Abstract
Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE2-G > PGF2α-G > PGD2-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.
Highlights
Prostaglandin glycerol esters are rapidly hydrolyzed in biological systems
By comparing Prostaglandin glycerol esters (PG-Gs) hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis
Characterization of PGE2-G Hydrolysis in Cancer Cells— MDA-MB-231 breast cancer cells were initially tested for hydrolytic activity by quantitatively monitoring the formation of PGE2 from exogenously provided PGE2-G by LC-MS/MS
Summary
Prostaglandin glycerol esters are rapidly hydrolyzed in biological systems. Results: Complementary approaches demonstrated that lysophospholipase A2 hydrolyzes prostaglandin glycerol esters. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. Nomura et al [34, 35] coupled fluorophosphonate probe binding with mass spectrometric proteomics techniques, known as activity-based protein profiling with multidimension protein identification technology, to determine the relative activity levels of serine hydrolases across different cancer cell lines Utilizing these inventories and comparing the relative activities of individual serine hydrolases to PGE2-G hydrolase activities has allowed us to identify lysophospholipase A2 (LYPLA2) as a principal hydrolase responsible for PG-G metabolism in human cells. In contrast to other PG-G-hydrolyzing enzymes, we found that LYPLA2 exerted no action on 2-AG or AEA but did hydrolyze 1-AG
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