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

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.

Highlights

To create a fluorogenic NAPE analog likely to be resistant to both PLA1 and PLA2 hydrolysis for our assay, we sought to generate a PED-A1 analog where the sn-1 ester bond of PED-A1 was substituted with either an ether bond or an N-methyl amide bond
The absorbance spectrums of flame-NAPE closely matched those of PED-A1 both prior to and after hydrolysis with Nape-pld (Fig. 2A), confirming the presence of the BODIPY group and the dinitrophenyl group for flame-NAPE
Our results demonstrate that the use of flame-NAPE provides a facile method to selectively assay NAPE-phospholipase D (PLD) activity in a variety of cell types

Summary

Introduction

At the 10 min time point, the average raw fluorescence measured in vehicle-treated HepG2 cells with flame-NAPE was 66% that of treated cells with PED-A1. To assess the effect of the N-methyl amide bond substitution on substrate hydrolysis by Nape-pld, varying concentrations of flame-NAPE or PED-A1 were incubated with recombinant mouse Nape-pld, and the resulting rate of fluorescence generation was measured.

Results

Conclusion