Isolevuglandin-modified phosphatidylethanolamine is metabolized by NAPE-hydrolyzing phospholipase D

Lilu Guo; Stephen D. Gragg; Zhongyi Chen; Yongqin Zhang; Venkataraman Amarnath; Sean S. Davies

doi:10.1194/jlr.M042556

Isolevuglandin-modified phosphatidylethanolamine is metabolized by NAPE-hydrolyzing phospholipase D

^*Division of Clinical Pharmacology, and
^†Departments of Pathology and
^§Pharmacology, Vanderbilt University, Nashville, TN

Abstract

Lipid aldehydes including isolevuglandins (IsoLGs) and 4-hydroxynonenal modify phosphatidylethanolamine (PE) to form proinflammatory and cytotoxic adducts. Therefore, cells may have evolved mechanisms to degrade and prevent accumulation of these potentially harmful compounds. To test if cells could degrade isolevuglandin-modified phosphatidylethanolamine (IsoLG-PE), we generated IsoLG-PE in human embryonic kidney 293 (HEK293) cells and human umbilical cord endothelial cells and measured its stability over time. We found that IsoLG-PE levels decreased more than 75% after 6 h, suggesting that IsoLG-PE was indeed degraded. Because N-acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) has been described as a key enzyme in the hydrolysis of N-acyl phosphatidylethanoamine (NAPE) and both NAPE and IsoLG-PE have large aliphatic headgroups, we considered the possibility that this enzyme might also hydrolyze IsoLG-PE. We found that knockdown of NAPE-PLD expression using small interfering RNA (siRNA) significantly increased the persistence of IsoLG-PE in HEK293 cells. IsoLG-PE competed with NAPE for hydrolysis by recombinant mouse NAPE-PLD, with the catalytic efficiency (V_max/K_m) for hydrolysis of IsoLG-PE being 30% of that for hydrolysis of NAPE. LC-MS/MS analysis confirmed that recombinant NAPE-PLD hydrolyzed IsoLG-PE to IsoLG-ethanolamine. These results demonstrate that NAPE-PLD contributes to the degradation of IsoLG-PE and suggest that a major physiological role of NAPE-PLD may be to degrade aldehyde-modified PE, thereby preventing the accumulation of these harmful compounds.

Footnotes

↵1 To whom correspondence should be addressed. e-mail: sean.davies{at}vanderbilt.edu
Abbreviations:

al-PE

aldehyde-modified phosphatidylethanolamine

DPPE

1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine

Etn

ethanolamine

HEK293

human embryonic kidney 293

HUVEC

human umbilical cord endothelial cell

IsoLG

isolevuglandin

IsoLG-PE

isolevuglandin-modified phosphatidylethanolamine

MRM

multiple reaction monitoring

NAE

N-acyl ethanolamine

NAPE

N-acyl phosphatidylethanolamine

NAPE-PLD

N-acyl phosphatidylethanolamine hydrolyzing phospholipase D

NBD

(7-nitro-2-1,3-benzoxadiazol-4-yl)amino

PA

phosphatidic acid

PC

phosphatidylcholine

PE

phosphatidylethanolamine

PLD

phospholipase D

small interfering RNA

siRNA
This work was supported by funds from the Vanderbilt Department of Pharmacology (S.S.D.) and National Institutes of Health Grants OD-003137-01 (S.S.D.), P30 ES-000267 (Vanderbilt Center in Molecular Toxicology), and UL1 RR-024975 (Vanderbilt Institute for Clinical and Translation Research).