Cellular function of neuropathy target esterase in lysophosphatidylcholine action

doi:10.1016/j.taap.2008.07.015

Toxicology and Applied Pharmacology

Volume 232, Issue 3, 1 November 2008, Pages 376-383

https://doi.org/10.1016/j.taap.2008.07.015 Get rights and content

Abstract

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography–mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.

Introduction

Neuropathy target esterase (NTE) is a member of the serine hydrolase patatin-like phospholipase family (PNPLA) designated PNPLA6 (Wilson et al., 2006). Members of this family act as triglycerol lipases, retinylester lipases and phospholipases (PLA) (Kienesberger et al., 2008). NTE (a 1327-amino acid enzyme) and NEST (a recombinant enzyme comprised of human NTE residues 727–1216) hydrolyze lysophosphatidylcholine (LPC) much faster than phosphatidylcholine (PC) (van Tienhoven et al., 2002, Quistad et al., 2003) and may act as phospholipase B (PLB) enzymes (Glynn, 2006). NTE is expressed in brain, testis, muscle, and other tissues (Wilson et al., 2006). Human NTE-related esterase (PNPLA7), with 65% homology to NTE at the amino acid level (Winrow et al., 2003), hydrolyzes LPC and lysophosphatidic acid (LPA) but not PC or triglycerides and may play a role in energy metabolism (Kienesberger et al., 2008). Mouse NTE, Drosophila Swiss Cheese protein, and yeast YML059c are 97, 61 and 51% homologous to human NTE. The endogenous substrate of yeast NTE is proposed to be PC or LPC since YML059c degrades PC to glycerophosphocholine (GPC) in living cells (Zaccheo et al., 2004).

Metabolic reactions related to NTE and LPC cellular function are shown in Fig. 1. LPC levels of cellular membranes are tightly regulated by several LPC hydrolases (LPCHs) (Ross and Kish, 1994, Wang and Dennis, 1999). NTE hydrolyzes membrane-associated lipids, suggesting a cellular role in their metabolism (van Tienhoven et al., 2002). NTE, lysophospholipases (LysoPLAs) and other LPCHs hydrolyze LPC to GPC (Casida and Quistad, 2004), an important osmolyte in kidney cells (Gallazzini et al., 2006). LPC is generated upon activation of PLA₂ (Macdonald et al., 2004) and also by lecithin:cholesterol acyltransferase (LCAT) (Santamarina-Fojo et al., 2000). High LPC concentrations are cytotoxic and associated with demyelination (Hall, 1972, Weltzien, 1979). LPC is also an effector molecule for the G protein-coupled G2A receptor (Parks et al., 2006). LPC is present in human plasma at concentrations up to 230 μM (Kishimoto et al., 2002), although it is mostly bound to albumin (Switzer and Eder, 1965) and unavailable for acting as a signaling effector.

The toxicology of organophosphorus (OP) insecticides and chemical warfare agents involves acetylcholinesterase as the primary target and several other serine hydrolases as secondary targets (Casida and Quistad, 2004, Gupta, 2006). Of these, NTE has received the greatest attention because its inhibition and aging leads to OP-induced delayed neuropathy (OPIDN), with about 70,000 cases in the last 70 years (Ehrich and Jortner, 2001, Johnson and Glynn, 2001). The steps from NTE inhibition to development of neuropathy are unclear but involve degeneration of long peripheral axons (Moretto and Lotti, 2006). A role of NTE in maintaining the integrity of peripheral motor axons is indicated by a M1012V mutation associated with a hereditary disease characterized by progressive lower-extremity weakness and wasting of distal extremity muscles (Rainier et al., 2008). NTE is also critical in embryonic development (Winrow et al., 2003, Moser et al., 2004). An understanding of the cellular role of NTE is an important step in evaluating OP toxicology.

The goal of this study was to determine the cellular function of NTE relative to LPC and OP action primarily by using recombinant NTE (rNTE). The first step was to express full-length rNTE (not NEST or YML059c) in Neuro-2a and COS-7 cells. rNTE was then characterized as to inhibitor sensitivity and specificity relative to LPCHs in brain and blood. Next, the effect of expressed rNTE was examined on the sensitivity of the Neuro-2a and COS-7 cells to LPC. Finally the LPC pathway metabolites were compared in Neuro-2a cells without and with rNTE. The findings here (partly reviewed in Casida et al., 2008) show that NTE plays a key role in LPC homeostasis and action.

Section snippets

Chemicals and reagents

LPC-[¹⁴C]16:0 and PC-18:0/[¹⁴C]20:4 were from Amersham (Buckinghamshire, UK). 2-[³H-n-octyl]-4H-1,3,2-Benzodioxaphosphorin 2-oxide ([³H]OBDPO) was prepared in this laboratory (Yoshida et al., 1995). Chlorpyrifos oxon, paraoxon and glyphosate were from ChemService (Westchester, PA). Diisopropyl phosphonofluoridate and phenylmethanesulfonyl fluoride were from Sigma-Aldrich (St. Louis, MO). Compounds available from previous or analogous syntheses in this laboratory were: S- and R-octyl-BDPO,

rNTE expression in Neuro-2a and COS-7 cells

rNTE was expressed in Neuro-2a and COS-7 cells as a 182kDa GFP-fusion protein and identified by an NTE antibody (Fig. 2A) and a GFP antibody (Fig. 2B), while the control cells transfected with the empty vector expressed only the 27kDa GFP (Fig. 2B). The molecular mass of rNTE from COS-7 cells was verified by radiolabeling with [³H]OBDPO and SDS-PAGE giving a single radiolabeled protein band at the expected kDa (Fig. 2C). The transfection efficiencies were 25–35% and 50–60% for Neuro-2a and

rNTE as a model for endogenous NTE

NTE originated as an OPIDN target defined by an assay with hen brain membranes using a discriminatory substrate (sequentially phenylphenyl acetate, PV and LPC) and selective inhibitors (paraoxon-resistant and mipafox-sensitive) (Johnson, 1988). This definition is experimentally cumbersome because several enzymes hydrolyze these substrates in most tissues. The studies were greatly facilitated by using NTE as a recombinant enzyme (Lush et al., 1998) in a background lacking interfering activities.

Conflict of interest statement

The authors declare that there are no conflicts of interest in this study.

Acknowledgments

This work was supported by Grants 2 P01 ES09605 from the National Institute of Environmental Health Sciences (NIEHS) and the US EPA (to N.T.H.) and Grant R01 ES08762 from the NIEHS, NIH (to J.E.C). We thank Brenda Eskenazi of the University of California at Berkeley Center for Children's Environmental Health Research for support and guidance. The pNTE-GFP-N₁ plasmid was a gift from PaulGlynn (Medical Research Council Toxicology Unit, University of Leicester, United Kingdom) and the FP-biotin

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Cellular function of neuropathy target esterase in lysophosphatidylcholine action

Abstract

Introduction

Section snippets

Chemicals and reagents

rNTE expression in Neuro-2a and COS-7 cells

rNTE as a model for endogenous NTE

Conflict of interest statement

Acknowledgments

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Prog. Lipid Res.

Toxicol. Lett.

Biochem. Pharmacol.

J. Biol. Chem.

Clin. Biochem.

Toxicol. Lett.

J. Biol. Chem.

Blood

Blood

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Am. J. Hum. Genet.

Bioorg. Med. Chem. Lett.

J. Lipid Res.

J. Chromatogr. B

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Biochim. Biophys. Acta