Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog

Jonah E Zarrow; Jianhua Tian; Brendan Dutter; Kwangho Kim; Amanda C Doran; Gary A Sulikowski; Sean S Davies

doi:10.1016/j.jlr.2021.100156

Selective measurement of NAPE-PLD activity via a PLA_1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog

J Lipid Res. 2022 Jan;63(1):100156. doi: 10.1016/j.jlr.2021.100156. Epub 2021 Nov 26.

Authors

Jonah E Zarrow¹, Jianhua Tian², Brendan Dutter², Kwangho Kim³, Amanda C Doran⁴, Gary A Sulikowski⁵, Sean S Davies⁶

Affiliations

¹ Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN, USA; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA.
² Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA.
³ Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA; Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
⁴ Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
⁵ Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA; Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
⁶ Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA. Electronic address: sean.davies@vanderbilt.edu.

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.

Keywords: N-acylethanolamide; N-acylphosphatidylethanolamide; NAPE-PLD; atherosclerosis; biothionol; diabetes; fluorescence; nociception; phospholipase A1; tetrahydrolipstatin.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Phosphatidylethanolamines*

Substances

N-acylphosphatidylethanolamine
Phosphatidylethanolamines

Abstract

Publication types

MeSH terms

Substances

Grants and funding