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

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.

Keywords: N-acyl ethanolamine; N-acyl phosphatidylethanolamine; cytotoxicity; inflammation; isoketal; oxidative stress.

Fig. 1.

HEK293 cells degrade IsoLG-PE. Confluent HEK293 cells were incubated with IsoLG (1 μM) for 1 h to form IsoLG-PE, excess IsoLG was removed by washing with DMEM, and IsoLG-PE levels were measured at 0, 1, or 6 h postwash by LC-MS after conversion to glycerophosphoethanolamine (GPE) derivative. A: Levels of IsoLG-PE in HEK293 cells decrease with time. Bars represent mean ± SEM, n = 3, one-way ANOVA, P < 0.0001. B: Representative MRM-MS trace of IsoLG-PE (as GPE derivative) at each time point.

Fig. 2.

HUVECs degrade IsoLG-PE. Cells were incubated with IsoLG (1 μM) for 1 h at 37°C to form IsoLG-PE, excess IsoLG was removed by washing, and IsoLG-PE levels were measured at 0, 6, or 24 h postwash by LC-MS. Bars represent mean ± SEM, n = 2, one-way ANOVA, P = 0.0259.

Fig. 3.

Knockdown of NAPE-PLD in HEK293 cells inhibits degradation of IsoLG-PE. HEK293 cells were transfected either with control siRNA (ctrl siRNA) or with siRNA specific for NAPE-PLD (PLD siRNA) and the effect of NAPE-PLD knockdown on IsoLG-PE levels after incubation with IsoLG (1 μM) was determined. A: Normalized immunoblot band density for NAPE-PLD relative to actin. Bars represent mean ± SEM, n = 3, *P = 0.272 two-tailed t-test. B: Fold expression of NAPE-PLD mRNA relative to control siRNA-treated cells, measured by quantitative RT-PCR. Bars represent mean ± SEM, n = 3, *P = 0.477 two-tailed t-test. C: Effect of NAPE-PLD knockdown on IsoLG-PE levels. Bars represent mean ± SEM, n = 3; *P = 0.0292, 0 h versus 1 h postwash for control (ctrl) siRNA, two-tailed t-test; P = 0.7109 (ns), 0 h versus 1 h postwash for PLD siRNA, two-tailed t-test.

Fig. 4.

Expression and purification of recombinant mouse NAPE-PLD from E. coli. DNA encoding mouse NAPE-PLD was inserted into the pQE-80 His vector and the resulting plasmid transformed into E. coli. Lysates (Lys) were then incubated with Ni-NTA agarose beads, washed two times (W1 and W2), and then eluted four times with 1 ml of elution buffer (E1, E2, E3, and E4, respectively.) An aliquot of each was run on SDS-PAGE and visualized with Coomassie blue stain. The major protein in the E3 and E4 fractions was a protein at 46 kDa, matching the expected size for NAPE-PLD.

Fig. 5.

NAPE-PLD hydrolyzes IsoLG-PE to IsoLG-Etn. IsoLG-PE was incubated with NAPE-PLD (A) or without NAPE-PLD (B) and the extent of IsoLG-Etn formation determined by MS using MRM at m/z 378.3 → 152.1. C: MRM analysis at m/z 382.3 → 156.1 for synthetic d4-IsoLG-Etn in this same chromatographic system.

Fig. 6.

Kinetic studies of NAPE-PLD hydrolysis of IsoLG-PE and NAPE. Fluorescently labeled NBD-NAPE and NBD-IsoLG-PE were used to measure the rate of hydrolysis of each substrate to NBD-phosphatidate (NBD-PA), analyzed by fluorescence-coupled HPLC (460 nm excitation/530 nm emission). A: IsoLG-PE inhibits hydrolysis of NBD-NAPE by recombinant NAPE-PLD. IsoLG-PE (0–100 μM) was incubated with NAPE-PLD and 10 mM NBD-NAPE and the resulting rate of hydrolysis normalized to the rate without IsoLG-PE. B: Lineweaver-Burke plot of the concentration dependence for the rate of hydrolysis of NBD-NAPE by NAPE-PLD. Calculated K_m = 3.79 μM, calculated V_max = 49 nmol min⁻¹ mg⁻¹. C: Lineweaver-Burke plot of the concentration dependence for the rate of hydrolysis of NBD-IsoLG-PE by NAPE-PLD.

Fig. 7.

Metabolism of IsoLG-Etn by HEK293 cells. A: Relative levels of IsoLG-Etn over time in IsoLG pretreated HEK293 cells. Confluent HEK293 cells were incubated with IsoLG (1 μM) for 1 h to form IsoLG-PE, excess IsoLG was removed by washing with DMEM, and IsoLG-Etn levels were measured at 0, 1, or 6 h postwash by LC-MS. Bars represent mean ± SEM, n = 3, one-way ANOVA, P = 0.1618. B: Relative levels of IsoLG-Etn over time after incubation with HEK293 cell lysate. Lysates were prepared from HEK293 cells and synthetic IsoLG-Etn added for 0 to 6 h. After extraction, the levels of IsoLG-Etn remaining were determined by LC-MS. Bars represent mean ± SEM, n = 2, one-way ANOVA, P = 0.0077.

Fig. 8.

Schematic of IsoLG-PE metabolism. Lipid peroxidation of arachidonic acid generates a variety of lipid aldehydes including IsoLG. IsoLG reacts rapidly with PE to form IsoLG-PE, which is proinflammatory and cytotoxic. NAPE-PLD hydrolyzes IsoLG-PE to the less toxic metabolite IsoLG-Etn and then IsoLG-Etn is further metabolized by unknown mechanisms.