Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog |
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Authors: | Jonah E. Zarrow Jianhua Tian Brendan Dutter Kwangho Kim Amanda C. Doran Gary A. Sulikowski Sean S. Davies |
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Affiliation: | 1. Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA;2. Department of Pharmacology, Vanderbilt University, Nashville, TN, USA;3. Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA;4. Department of Chemistry, Vanderbilt University, Nashville, TN, USA;5. Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA |
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Abstract: | N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase enzyme that converts NAPEs to bioactive N-acyl-ethanolamides. Altered NAPE-PLD activity may contribute to pathogenesis of obesity, diabetes, atherosclerosis, and neurological diseases. Selective measurement of NAPE-PLD activity is challenging, however, because of alternative phospholipase pathways for NAPE hydrolysis. Previous methods to measure NAPE-PLD activity involved addition of exogenous NAPE followed by TLC or LC/MS/MS, which are time and resource intensive. Recently, NAPE-PLD activity in cells has been assayed using the fluorogenic NAPE analogs PED-A1 and PED6, but these substrates also detect the activity of serine hydrolase-type lipases PLA1 and PLA2. To create a fluorescence assay that selectively measured cellular NAPE-PLD activity, we synthesized an analog of PED-A1 (flame-NAPE) where the sn-1 ester bond was replaced with an N-methyl amide to create resistance to PLA1 hydrolysis. Recombinant NAPE-PLD produced fluorescence when incubated with either PED-A1 or flame-NAPE, whereas PLA1 only produced fluorescence when incubated with PED-A1. Furthermore, fluorescence in HepG2 cells using PED-A1 could be partially blocked by either biothionol (a selective NAPE-PLD inhibitor) or tetrahydrolipstatin (an inhibitor of a broad spectrum of serine hydrolase-type lipases). In contrast, fluorescence assayed in HepG2 cells using flame-NAPE could only be blocked by biothionol. In multiple cell types, the phospholipase activity detected using flame-NAPE was significantly more sensitive to biothionol inhibition than that detected using PED-A1. Thus, using flame-NAPE to measure phospholipase activity provides a rapid and selective method to measure NAPE-PLD activity in cells and tissues. |
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Keywords: | NAPE-PLD phospholipase A1 fluorescence biothionol tetrahydrolipstatin diabetes atherosclerosis nociception ABHD4" },{" #name" :" keyword" ," $" :{" id" :" kwrd0065" }," $$" :[{" #name" :" text" ," _" :" alpha/beta-hydrolase-4 AEA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0075" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" N" },{" #name" :" __text__" ," _" :" -arachidonyl-ethanolamide Bith" },{" #name" :" keyword" ," $" :{" id" :" kwrd0085" }," $$" :[{" #name" :" text" ," _" :" bithionol BMDM" },{" #name" :" keyword" ," $" :{" id" :" kwrd0095" }," $$" :[{" #name" :" text" ," _" :" bone marrow-derived macrophage GP-NAE" },{" #name" :" keyword" ," $" :{" id" :" kwrd0105" }," $$" :[{" #name" :" text" ," _" :" glycerophospho-NAE HEK293" },{" #name" :" keyword" ," $" :{" id" :" kwrd0115" }," $$" :[{" #name" :" text" ," _" :" human embryonic kidney 293 cell line NAE" },{" #name" :" keyword" ," $" :{" id" :" kwrd0125" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" N" },{" #name" :" __text__" ," _" :" -acyl-ethanolamide NAPE" },{" #name" :" keyword" ," $" :{" id" :" kwrd0135" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" N" },{" #name" :" __text__" ," _" :" -acyl-phosphatidylethanolamine OEA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0145" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" N" },{" #name" :" __text__" ," _" :" -oleoyl-ethanolamide PEA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0155" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" N" },{" #name" :" __text__" ," _" :" -palmitoyl-ethanolamide PLC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0165" }," $$" :[{" #name" :" text" ," _" :" phospholipase C PLD" },{" #name" :" keyword" ," $" :{" id" :" kwrd0175" }," $$" :[{" #name" :" text" ," _" :" phospholipase D THL" },{" #name" :" keyword" ," $" :{" id" :" kwrd0185" }," $$" :[{" #name" :" text" ," _" :" tetrahydrolipstatin |
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