JBC Advanced Peptides, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M200330200 on April 1, 2002

J. Biol. Chem., Vol. 277, Issue 23, 20942-20948, June 7, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/23/20942    most recent
M200330200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Tienhoven, M.
Right arrow Articles by Glynn, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Tienhoven, M.
Right arrow Articles by Glynn, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Human Neuropathy Target Esterase Catalyzes Hydrolysis of Membrane Lipids*

Marianne van TienhovenDagger, Jane AtkinsDagger§, Yong Li, and Paul Glynn

From the MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom

Received for publication, January 11, 2002, and in revised form, March 27, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A2 (iPLA2) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA2 inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (Vmax ~0.01 µmol/min/mg and Km ~0.4 mM for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of 14C-labeled products from two differentially labeled 14C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (Vmax ~20 µmol/min/mg and Km ~0.05 mM for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (Vmax ~1 µmol/min/mg and Km ~0.4 mM for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuropathy target esterase (NTE)1 is an integral membrane protein in neurons and some non-neural cell types; it was originally identified as the primary site of action for those organophosphates which, in humans and other vertebrates, cause a syndrome characterized by axonal degeneration (1). The NTE homologue in Drosophila, the swiss cheese protein, is essential for fly brain development (2). In the mouse, NTE is present in neurons from their earliest appearance in the nervous system and so is well placed to play a similar role in mammalian neural development (3). NTE also has a homologue in yeast (4), suggesting that it has functions beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.

In keeping with its reactivity with organophosphates, NTE belongs to the serine hydrolase class of enzymes. Since its discovery more than 30 years ago (5), NTE has been detected in vitro by assays with non-physiological ester substrates, most commonly with phenyl valerate (6). Clues to the cellular functions of NTE might be provided by identifying its natural substrate. Data on this point are sparse, but using artificial substrates, NTE activity in brain homogenates has been shown to catalyze hydrolysis of ester rather than peptide or amide bonds (7, 8). In addition, inhibition of the phenyl valerate hydrolase activity of NTE by a homologous series of alkyl saligenin cyclic phosphonates (9) and alkyl-thiotrifluoromethyl ketones (10) suggests that the active site of the enzyme has a preference for esters of carboxylic acids with an alkyl chain length of at least 9-10 carbon atoms (1). This hints that the natural substrate of NTE may be an ester of a fatty acid, such as a phospholipid or an acylglycerol.

Human NTE is a polypeptide of 1327 residues and has two major domains (4) as follows: a C-terminal catalytic domain containing the active site serine residue (Ser-966) that reacts with organophosphates and phenyl valerate; and an N-terminal putative regulatory domain that contains sequences similar to cyclic AMP-binding proteins. We have expressed in Escherichia coli a recombinant protein (corresponding to NTE residues 727-1216) called NEST which composes the esterase domain of NTE (11). NEST is very hydrophobic, and the purified protein must be incorporated into phospholipid liposomes to acquire a conformation with full phenyl valerate hydrolase activity (12). In this respect, NEST appears similar to enzymes such as cytoplasmic phospholipase A2 (13) and triacylglycerol lipase (14) which show the phenomenon of interfacial activation, i.e. they exhibit full catalytic activity only when at a lipid-water interface. Here, we use NEST to investigate the possibility that NTE has lipase- or phospholipase-A-like activity with membrane-associated lipids as its potential substrates.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 1-Palmitoyl, 2-oleoyl-[oleoyl-1-14C]phosphatidylcholine (52 mCi/mmol) and dioleoyl-[dioloeoyl-1-14C]phosphatidylcholine (104 mCi/mmol) were from PerkinElmer Life Sciences. Methyl arachidonyl fluorophosphonate (MAFP), 2-arachidonoylglycerol, anandamide, and N-oleoylethanolamine were from CN Biosciences; bromoenol lactone and arachidonoyl trifluoromethyl ketone (-TFMK) were from Affiniti. All other lipid substrates, putative lipase inhibitors, and bee venom secretory phospholipase A2 were purchased from Sigma. Lipid substrates were prepared by evaporating chloroform solutions under a stream of nitrogen gas and then resuspending the residue in PEN buffer (50 mM sodium phosphate, 0.5 mM EDTA, 300 mM NaCl, pH 7.8) by sonication (MSE Sanyo Soniprep 150 probe sonicator) at maximum power for 10 min to give stock suspensions at a concentration of 5 mM. Free fatty acid assay kit (half-micro test) was from Roche Diagnostics. The sources of all other materials have been described previously (11).

Expression and Isolation of NEST-- NEST (human NTE residues 727-1216 with N-terminal T7 and C-terminal His6 tags) was expressed in E. coli BL21 pLysS, extracted, and isolated by nickel chelate and gel filtration chromatography in solutions containing CHAPS as described previously (11). NEST, with its catalytic serine residue mutated to alanine (11), was expressed and isolated in identical fashion. Before assaying its phenyl valerate hydrolase activity, purified NEST was diluted with 0.3% CHAPS to a protein concentration of 0.1 mg/ml, mixed with dioleoylphosphatidylcholine (DOPC; 10 mg/ml in 10% (w/v) CHAPS) in a ratio of 1:4 (w/w), and then dialyzed overnight at room temperature against PEN buffer.

Enzyme Assays-- Phenyl valerate hydrolase activity was determined by the method of Johnson (6) as described previously (11). Purified NEST-DOPC complexes were diluted in 50 mM Tris-HCl, 1 mM EDTA to a protein concentration of 20-80 ng/ml and then incubated for 20 min at 37 °C with inhibitors before addition of phenyl valerate in Triton X-100 (final concentrations of 1.4 and 0.24 mM, respectively) and incubation for a further 20 min.

NEST lipase activity was assayed by determining the liberation of free fatty acid from various lipid substrates. For lysophospholipids and monoacylglycerols, incubations contained 2.5 mM substrate, NEST (0.1-3.0 µg of protein), and 2.4 mM CHAPS in a final volume of 0.05 or 0.1 ml of PEN buffer.

For diacylphospholipids and all other putative lipid substrates 2.0 mM substrate was incubated with 25 µg of NEST, 3.2 mM CHAPS in a final volume of 0.5 ml of PEN buffer. Incubations were at 37 °C and were terminated at the times indicated in the figures (including zero time) by addition of MAFP to a final concentration of 20 µM. Free fatty acid was determined using a kit from Roche Diagnostics based on the coupled enzyme assay of Shimizu et al. (15).

Zero time values (i.e. MAFP added before NEST) were subtracted from values determined at all subsequent time points. Data shown are the means of at least two separate experiments.

For more detailed evaluation of phospholipase A2 activity, 14C-labeled phospholipids (~0.4 × 106 dpm) were incubated at 37 °C with NEST (8-20 µg) in 0.05 ml of PEN buffer containing 2-4 mM CHAPS. Reactions were stopped by the addition of 0.1 ml of chloroform/methanol/acetic acid (2:1:0.02), and after vortexing and centrifugation, the organic phase solvent was evaporated, and the residue was redissolved in ethanol containing oleic acid (20 mg/ml). Aliquots were subjected to thin layer chromatography on Silica G plates run in hexane/ether/acetic acid (80:20:1). In this system, fatty acids are well resolved from phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) which remain very close to the origin. Spots containing either phospholipid or fatty acid were scraped into scintillation vials, and radioactivity was determined by scintillation counting (16). To resolve [14C]LPC, samples (mixed with unlabeled 1-oleoyl-LPC) were run on Silica G plates in chloroform/methanol/formic acid/water (60:30:8:2) (17). In this system, LPC has an RF ~0.5, whereas PC and fatty acids co-migrate near the solvent front.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NEST and Calcium-independent Phospholipase A2, Similarities in Active Site Sequence and Inhibitor Sensitivities of Phenyl Valerate Hydrolase Activity-- A BLAST search identified significant sequence similarity (28% identity) between an ~160-residue region of NEST and calcium-independent phospholipase A2 (iPLA2; Fig. 1). Among key conserved residues in both proteins were those corresponding to the active site serine (Ser-966) and two aspartates (Asp-960 and Asp-1086) shown previously to be essential for the phenyl valerate hydrolase activity of NEST (11). Outside this region there was no homology between NTE and iPLA2 (Fig. 1).


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 1.   Sequence similarity in the active site regions of iPLA2 and NTE. a, iPLA2, NTE, and NEST (the recombinant esterase domain of NTE) are represented as horizontal bars with the region of homology shaded. The location of the putative N-terminal transmembrane helix of the NTE is indicated by a vertical bar. b, the region of homology is shown with identical residues indicated white-on-black, similar residues in bold, and residues essential for the catalytic activity of NEST marked with an arrowhead.

We investigated whether compounds reported to be potent inhibitors of iPLA2 would also inhibit phenyl valerate hydrolase activity of NEST-DOPC preparations. Bromoenol lactone inhibits iPLA2 with an IC50 value (after a 5-min preincubation) of 60 nM (18). Under standard conditions for assay of NEST-DOPC phenyl valerate hydrolase activity (see "Experimental Procedures"), we determined an IC50 value of 94 ± 20 nM (n = 5) for bromoenol lactone. MAFP inhibits iPLA2 with an IC50 in the submicromolar range (19) and NEST-DOPC phenyl valerate hydrolase activity with an IC50 of 2.0 ± 0.9 nM (n = 3). Finally, micromolar concentrations of two trifluoromethyl ketones (TFMK) inhibit iPLA2 activity, with palmitoyl-TFMK 4 times more potent than arachidonoyl-TFMK (18). These two compounds also inhibited NEST-DOPC phenyl valerate hydrolase activity but, in contrast to iPLA2, arachidonoyl-TFMK (IC50 = 2.5 µM; n = 2) was 10 times more potent than palmitoyl-TFMK (IC50 = 28 µM; n = 2).

Inhibition of the phenyl valerate hydrolase activity of NEST-DOPC preparations by fatty acid TFMKs led us to ask whether fatty acids themselves would be inhibitory. Preincubating NEST-DOPC with 50 µM oleic acid abolished phenyl valerate hydrolase activity, whereas palmitic and arachidonic acids at this concentration had marginal (25% inhibition) or no effect, respectively (Fig. 2a). Similarly, palmitate and arachidonate at concentrations of 30 µM did not inhibit iPLA2, although this study did not report the effect of oleic acid (18).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2.   Inhibition of the phenyl valerate hydrolase activity of NEST after preincubation with free fatty acids and lysophospholipids. NEST-DOPC was preincubated (20 min; 37 °C) with the following: a, fatty acids dissolved in ethanol (final concentration, 1%); b, 1-palmitoyl-LPC (1-P-lysoPC), 1-O-hexadecyl-LPC (1-O-H-lysoPC) and 1-O-hexadecyl, 2-arachidonoyl-PC (1-O-H, 2-A-PC) dissolved in 1.6 mM CHAPS; after which phenyl valerate substrate was added to a final concentration of 1.4 mM and the assay done as under "Experimental Procedures."

As free fatty acid and lysophospholipid are the products of PLA2 activity, we examined possible effects of lysophospholipids on NEST-DOPC phenyl valerate hydrolase activity by preincubating with the enzyme before addition of substrate: 1-palmitoyl-LPC was inhibitory with an IC50 value of ~15 µM, and an alkyl ether analogue, 1-O-hexadecyl-LPC, was even more potent with an IC50 < 7.5 µM (Fig. 2b). By contrast, 1-O-hexadecyl, 2-arachidonoyl-PC had only a marginal effect (Fig. 2b).

To investigate the nature of the inhibition of phenyl valerate hydrolase activity of NEST-DOPC by oleic acid and lysophospholipids, we directly added these inhibitors to assays in the presence of varying concentrations of phenyl valerate substrate (Fig. 3). Oleic acid was much less potent as an inhibitor when added direct to the assay (Fig. 3a) than when preincubated with the enzyme before addition of substrate (cf. Fig. 2a). For example, in the presence of the standard concentration of phenyl valerate (1.4 mM), 50 µM oleic acid caused only ~15% inhibition, increasing to ~45% inhibition in the presence of 0.35 mM phenyl valerate (Fig. 3a). By contrast, LPC was almost as potent when added direct to the assay as when preincubated and at 25 µM caused 50% inhibition in the presence of 1.4 mM phenyl valerate (Fig. 3b). Lineweaver-Burk (1/v versus 1/s) transformations (not shown) of the data of the type in Fig. 3 were readily fit to straight lines allowing determination of kinetic values for phenyl valerate substrate (Vmax = 4.82 ± 0.52 mmol/min/mg; Km = 0.76 ± 0.09 mM; n = 5). However, in the presence of either oleic acid or LPC, Lineweaver-Burk plots intersected the y axis at a lower value than in the absence of these inhibitors, suggesting an increase in Vmax. It is notable that preincubation of NEST-DOPC with 10 µM fatty acid caused a small (20%) increase in phenyl valerate hydrolase activity (Fig. 2a). Thus, the interactions between NEST-DOPC, CHAPS, phenyl valerate, and either oleic acid or LPC are complex and cannot be described kinetically in terms of pure competitive or non-competitive inhibition.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   Inhibition of the phenyl valerate hydrolase activity of NEST by direct addition to assay medium of oleic acid (a) or lysophospholipid (b). Assays in 2 ml of PEN buffer contained 2.4 mM CHAPS, phenyl valerate (0.35, 0.47, 0.7, and 1.4 mM), and oleic acid (a) or 1-palmitoyl-LPC (b) at the concentrations (µM) indicated in the figure. Reactions were started by addition of NEST-DOPC (75 ng protein) and stopped after 20 min at 37 °C.

NEST Slowly Liberates Free Fatty Acid from Phospholipids-- NEST is extracted and isolated from bacterial lysates in solutions containing CHAPS, and under these conditions, its phenyl valerate hydrolase activity is lost, partly because this activity requires association with phospholipid and partly because concentrations of CHAPS greater than its critical micellar concentration (4-8 mM) are inhibitory (12). Restoration of esterase activity to purified NEST requires its incorporation into phospholipid (DOPC) liposomes concomitant with removal of CHAPS by overnight dialysis at room temperature. Determination of free oleic acid concentration in the dialyzed samples yielded a value of 0.12 ± 0.02 mM (n = 7), compared with an original DOPC concentration of ~0.5 mM. Free oleic acid was not detected in dialyzed mixtures of DOPC and detergent with S966A mutant NEST, indicating that generation of the fatty acid required catalytic activity.

We established conditions (2.5 mM phospholipid, 3.2 mM CHAPS) for assaying the lipase activity of NEST as described under "Experimental Procedures." After 1 h at 37 °C, ~0.8 µmol of oleic acid was liberated from DOPC per mg of NEST (Fig. 4a). This slow rate of hydrolysis was not substantially altered by several changes to the incubation conditions as follows: varying CHAPS concentration between 1.6 and 8 mM; incorporating DOPC into mixed lipid vesicles (various ratios of DOPC/dioleoylethanolamine/sphingomyelin/cholesterol); by adding calcium or magnesium ions; or by adding ATP, which has been reported to enhance iPLA2 activity (20) (data not shown).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   NEST liberates free fatty acid from phospholipids. NEST was incubated with 3.2 mM CHAPS and 2 mM substrate (dioleoyl phospholipids (a) and phosphatidylcholines (b)), and at the indicated times reaction was stopped by addition of MAFP, and free fatty acid was determined as under "Experimental Procedures." 1-P, 2-A-PC, 1-palmitoyl, 2-arachidonoyl-PC; 1-P, 2-O-PC, 1-palmitoyl, 2-oleoyl-PC; 1-O-H, 2-A-PC, 1-O-hexadecyl, 2-arachidonoyl-PC.

By comparing NEST-catalyzed hydrolysis of dioleoyl phospholipids, phosphatidylcholine (DOPC) was hydrolyzed more slowly than phosphatidylglycerol (DOPG) but faster than phosphatidic acid (DOPA) or phosphatidylethanolamine (DOPE) (Fig. 4a). Reaction rates, particularly for DOPG and DOPA, decreased with increasing incubation time even though less than 3% of the substrate was hydrolyzed after 1 h. Phosphatidylcholines (PC) with different acyl chains were compared as substrates for the putative PLA activity of NEST. The fastest initial rate (~0.12 µmol/min/mg) was observed with 1-palmitoyl, 2-arachidonoyl-PC and was 4-5 times faster than rates with DOPC, 1-palmitoyl, 2-oleoyl-PC, and dipalmitoyl-PC (Fig. 4b).

Whereas 1-O-hexadecadyl, 2-arachidonoyl-PC is a substrate for iPLA2 (20), it was not detectably hydrolyzed by NEST (Fig. 4b). This raised the possibility that NEST mediates selective cleavage of the sn-1 bond of phospholipids; to examine this issue we incubated NEST with differentially labeled [14C]phosphatidylcholines (Table I). Approximately twice as much 14C-labeled oleic acid was liberated from substrate labeled in both the sn-1 and sn-2 positions (1.75 ± 0.05 nmol) as from substrate labeled exclusively at the sn-2 position (0.83 ± 0.01 nmol). Furthermore, no 14C-labeled LPC was detected after reaction with the [(sn-1 + -2)-14C]PC substrate. By contrast, incubation of bee venom secretory PLA2 with the sn-2-labeled substrate resulted in essentially quantitative conversion to [14C]oleic acid and, with the (sn-1 + -2)-labeled substrate, quantitative conversion to [14C]oleic acid and [14C]LPC (Table I). These results strongly suggest that NEST mediates a very slow cleavage of the sn-2 bond of phospholipid followed by a rapid hydrolysis of the resulting lysophospholipid product, possibly before this product is released from the enzyme. Cleavage of the sn-2 bond of 1-O-hexadecyl, 2-arachidonoyl-PC by NEST would yield a non-hydrolyzable lysophospholipid product; the latter may dissociate only very slowly from the enzyme and hence inhibit binding of further phospholipid substrate, giving rise to the result observed in Fig. 4b.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Conversion of 14C-labeled phosphatidylcholines to labeled oleic acid and lysophosphatidylcholine by NEST and sPLA2
NEST (10 µg) or bee venom secretory PLA2; 1 µg) was incubated (37 °C; 1 h) in PEN buffer with 3.7 mM CHAPS and either 1.5 mM 1-palmitoyl, 2-oleoyl-[oleoyl-1-14C]PC ([sn-1 + 2-14C]PC) or 1.3 mM dioleoyl-[dioleoyl-1-14C]PC ([sn-1 + 2)-14C]PC). Reactions were stopped, and labeled oleic acid, and LPC were resolved and quantified as described under "Experimental Procedures."

NEST liberated [14C]oleic acid from sn-2-labeled PC at rates that were approximately linear over a period of 15 min (Fig. 5a). Thereafter, 15-min assays were used to determine the dependence of reaction rate on substrate concentration. Lineweaver-Burk transformations of these data (Fig. 5b) allowed determination of values for 1-palmitoyl, 2-oleoyl-PC of Vmax = 11.6 ± 1.1 nmol/min/mg and Km = 0.37 ± 0.12 mM (from three experiments).


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5.   Release of [14C]oleic acid from sn-2-labeled phospholipid, time course and dependence on substrate concentration. NEST (9.3 µg) was incubated (37 °C) in 0.05 ml of PEN buffer with 3.6 mM CHAPS and 1-palmitoyl, 2-oleoyl-[oleoyl-1-14C]PC (POPC) at either 0.1 or 2.1 mM (a) or concentrations from 0.36 to 2.16 mM (b). Reactions were stopped after 5, 15, and 45 min (a) or 15 min (b), and [14C]oleic acid was quantified as described under "Experimental Procedures."

Lysophospholipids Are the Most Avidly Hydrolyzed Lipid Substrate for NEST-- Fatty acid was generated rapidly when NEST was incubated with LPC. With 1-palmitoyl-LPC, initial rates of ~20 µmol/min/mg were about twice those observed with 1-oleoyl-LPC (Fig. 6a) and were ~200 times faster than those with 1-palmitoyl, 2-arachidonoyl-PC (cf. Fig. 4b). As with the diacylphospholipid substrates, rates declined after 2-5 min, and this was not due to substrate depletion.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 6.   NEST potently catalyzes lysophospholipid hydrolysis and does not require additional phospholipid. a, NEST was incubated with 1-palmitoyl-LPC or 1-oleoyl-LPC, and reactions were stopped at the indicated times with MAFP and free fatty acid determined as under "Experimental Procedures." b, NEST that had been incorporated into DOPC liposomes by overnight dialysis (see "Experimental Procedures") or NEST alone (both at 0.1 µg of protein/assay) was incubated with 1-palmitoyl-LPC, and free fatty acid was liberated determined as in a.

Among the lipid substrates tested with NEST, lysophospholipids gave by far the fastest initial rates. The affinity of the active site of NEST for lysophospholipids may underlie the potent inhibition by these compounds and their ether analogues of phenyl valerate hydrolase activity (cf. Figs. 2b and 3b). However, unlike phenyl valerate hydrolase activity, NEST lipase activity toward lysophospholipid (Fig. 6b) and other lipid substrates did not require incorporation into DOPC vesicles to achieve interfacial activation, suggesting that in these cases the substrate itself fulfills this requirement.

We established conditions under which linear rates of hydrolysis of 1-palmitoyl-LPC could be observed (Fig. 7a). Lineweaver-Burk transformation of data obtained under these conditions (Fig. 7b) allowed the determination of values for Vmax = 20.8 ± 2.5 µmol/min/mg and Km = 0.054 ± 0.006 mM (from four experiments).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7.   NEST-catalyzed hydrolysis of lysophospholipid: linear rate conditions (a) and dependence on substrate concentration (b). NEST (0.3 µg) was incubated (37 °C) in 0.5 ml of PEN buffer with 2 mM CHAPS and 1-palmitoyl-LPC at the concentrations (mM) indicated in the figure. Reactions were stopped at the times indicated, and free fatty acid formed was determined as described under "Experimental Procedures." Linear reaction rates derived from data in a were used for the 1/v versus 1/s transformation in b.

Other Naturally Occurring Lipid Substrates for NEST-- NEST hydrolyzed monoacylglycerols, with a marked preference for the sn-1 isomer. After a 5-min incubation, 8-10 times more fatty acid was liberated from 1-palmitoylglycerol than from the 2-isomer (Fig. 8a). Although the first 2-5 min of NEST-catalyzed hydrolysis of 1- and 2-oleoylglycerol and 2-arachidonylglycerol were at least as rapid as for 2-palmitoylglycerol, the rate diminished dramatically after this time (Fig. 8b). Thus, with certain members of each class of lipid substrate (phospholipids, lysophospholipids, and acylglycerols), NEST-catalyzed release of fatty acid shows an initial burst of variable duration and then slows markedly. A similar, poorly understood stalling in reaction rate has been reported for several phospholipases (21-23). Nevertheless, we were able to establish conditions under which approximately linear rates of hydrolysis of 1-palmitoylglycerol could be measured (Fig. 9a). Lineweaver-Burk transformations of these rates (Fig. 9b) allowed determination of values for Vmax = 1.37 ± 0.47 µmol/min/mg and Km = 0.37 ± 0.09 (from three experiments).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 8.   NEST-catalyzed hydrolysis of monoacylglycerols. NEST was incubated with 2.4 mM CHAPS and 2.5 mM substrate (a, 1-, and 2-palmitoylglycerols (1-PG, 2-PG); b, 2-palmitoyl-, 1-oleoyl-, 2-oleoyl-, 2-arachidonoylglycerols (1-oleoylglycerol (1-OG), 2-oleoylglycerol (2-OG), 2-arachidonoylglycerol (2-AG))) and at the indicated times the reaction was stopped with MAFP and free fatty acid determined as under "Experimental Procedures." Note different scales in a and b.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 9.   NEST-catalyzed hydrolysis of 1-palmitoylglycerol: linear rate conditions (a) and dependence on substrate concentration (b). NEST (4 µg) was incubated (37 °C) in 0.5 ml of PEN buffer with 2.4 mM CHAPS and 1-palmitoylglycerol at the concentrations (mM) indicated in the figure. At the times shown the reaction was stopped and free fatty acid determined as under "Experimental Procedures." Portions of the time course (a) with linear reaction rates were used to derive data for the 1/v versus 1/s plot (b).

In contrast to the relatively brisk hydrolysis of monoacylglycerols, NEST did not catalyze detectable fatty acid release from di- or triacylglycerols nor from a variety of acyl amides including sphingomyelin, anandamide, and N-oleoylethanolamine (data not shown). Free fatty acid was slowly liberated when a suspension of cholesteryl oleate was incubated with NEST, at rates similar to those with DOPC (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown here that NEST, the recombinant esterase domain of NTE, liberates fatty acid from phospholipids, monoacylglycerols, and lysophospholipids. Thus, more than 30 years after the discovery of NTE, we have identified naturally occurring, membrane-associated lipids as its potential endogenous substrates. This demonstration that NTE is a putative lipase provides new clues to its possible cellular functions.

Although the active site of NEST has primary sequence similarity to iPLA2, it hydrolyzes diacylphospholipids much more slowly. Using sn-2-labeled [14C]PC we found a Vmax ~0.01 µmol/min/mg NEST, whereas initial rates of 1-5 µmol/min/mg have been reported for iPLA2 itself (20, 24). When assayed with relatively low detergent concentrations, both iPLA2 and calcium-dependent cytosolic PLA2 can display lysophospholipase activity of about the same magnitude (a few µmol/min/mg) as their maximal PLA2 activity (21, 28, 29). NEST shows no significant sequence similarity to the cytosolic 25-kDa lysophospholipid-specific lysophospholipases (25, 26), and yet it catalyzes hydrolysis of 1-palmitoyl-LPC (Vmax ~20 µmol/min/mg) much faster than initial rates (1-2 µmol/min/mg) reported for recombinant lysophospholipase (27).

Under the experimental conditions in this report, lysophospholipids were by far the most avidly hydrolyzed substrate for NEST. It has been stressed repeatedly that the rates and selectivities of bond cleavage observed in lipase assays in vitro are profoundly affected by the physicochemical nature of the substrate (14, 16, 20, 23). Nevertheless, it seems reasonable to consider the possibility that lysophospholipids may be a physiological substrate for NTE.

The presence of a homologue of NTE in yeast (4) suggests that this enzyme mediates a cellular process conserved throughout much of eukaryotic evolution. NTE itself is firmly associated with intracellular membranes, and from a combination of esterase assays on brain subcellular fractions (30), immunohistochemistry on brain sections (31), and distribution of green fluorescent protein-tagged NTE in transfected COS-7 cells,2 we have tentatively concluded that NTE may be localized predominantly in the endoplasmic reticulum and Golgi complex. What might be the biological significance of NTE-mediated lysophospholipase activity in the endoplasmic reticulum/Golgi of various cells from yeast to neurons?

There is a growing recognition that, along with other factors, the relative concentrations of diacyl- and lysophospholipids contribute to the degree of curvature of biological membranes and that this is an important determinant in the process of membrane fission, tubulation, and fusion (32-34). Addition to cultured mammalian cells of various PLA2 inhibitors, including bromoenol lactone and arachidonoyl-TFMK, causes reversible fragmentation of the Golgi complex (35, 36). It has been suggested that a PLA2 activity may be required for maintenance of Golgi complex architecture (35); this would result in continuous production of lysophospholipid within the Golgi. Indeed, in Golgi membranes (of rat kidney and liver), lysophospholipids compose 3-4% of the total phospholipid (37). It has been demonstrated that CtBP/BARS-catalyzed acylation of lysophosphatidic acid to form phosphatidic acid induces fission of Golgi membranes (33). Another mechanism for acutely reducing the steady-state lysophospholipid level might involve regulated lysophospholipase activity. If ligand binding to the N-terminal domain of the NTE, which has sequence similarity to cyclic AMP-binding proteins (4), modulates the lipase activity of NTE toward its natural substrate, then this might allow inducible alteration of Golgi architecture and contribute to processes such as regulated protein secretion.

    FOOTNOTES

* This work was supported by the MRC, a studentship from the MRC (to J. A.), and a scholarship from the Leonardo da Vinci fund (to M. v. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Both authors contributed equally to this work.

§ Present address: Adprotech, Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, UK.

To whom correspondence should be addressed. Tel.: 44 116 252 5598; Fax: 44 116 252 5616; E-mail: pg8@le.ac.uk.

Published, JBC Papers in Press, April 1, 2002, DOI 10.1074/jbc.M200330200

2 Y. Li and P. Glynn, unpublished data.

    ABBREVIATIONS

The abbreviations used are: NTE, neuropathy target esterase; DOPA, dioleoylphosphatidic acid; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; DOPG, dioleoylphosphatidylglycerol; iPLA2, calcium-independent phospholipase A2; LPC, lysophosphatidylcholine; MAFP, methyl arachidonoyl fluorophosphonate; NEST, NTE esterase domain; PC, phosphatidylcholine; TFMK, trifluoromethyl ketone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Glynn, P. (2000) Prog. Neurobiol. 61, 61-74[CrossRef][Medline] [Order article via Infotrieve]
2. Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., and Benzer, S. (1997) J. Neurosci. 17, 7425-7432[Abstract/Free Full Text]
3. Moser, M., Stempfl, T., Li, Y., Glynn, P., Buettner, R., and Kretzschmar, D. (2000) Mech. Dev. 90, 279-282[CrossRef][Medline] [Order article via Infotrieve]
4. Lush, M. J., Li, Y., Read, D. J., Willis, A. C., and Glynn, P. (1998) Biochem. J. 332, 1-4
5. Johnson, M. K. (1969) Biochem. J. 114, 711-714[Medline] [Order article via Infotrieve]
6. Johnson, M. K. (1977) Arch. Toxicol. 37, 113-115[CrossRef][Medline] [Order article via Infotrieve]
7. Johnson, M. K. (1975) Biochem. Pharmacol. 24, 797-805[CrossRef][Medline] [Order article via Infotrieve]
8. Johnson, M. K. (1982) Rev. Biochem. Toxicol. 4, 141-212
9. Wu, S.-Y., and Casida, J. E. (1992) Chem. Res. Toxicol. 5, 680-684[CrossRef][Medline] [Order article via Infotrieve]
10. Thomas, T. C., Szekas, A., Rojas, S., Hammock, B. D., Wilson, B. W., and MacNamee, M. G. (1990) Biochem. Pharmacol. 40, 2587-2596[CrossRef][Medline] [Order article via Infotrieve]
11. Atkins, J., and Glynn, P. (2000) J. Biol. Chem. 275, 24477-24483[Abstract/Free Full Text]
12. Atkins, J., Luthjens, L. H., Hom, M. L., and Glynn, P. (2002) Biochem. J. 361, 119-123[CrossRef][Medline] [Order article via Infotrieve]
13. Dessen, A. (2000) Biochim. Biophys. Acta 1488, 40-47[Medline] [Order article via Infotrieve]
14. Derewenda, Z. S., and Sharp, A. M. (1993) Trends Biochem. Sci. 18, 20-25[CrossRef][Medline] [Order article via Infotrieve]
15. Shimizu, S., Tani, Y., Yamada, H., Tabata, M., and Murachi, T. (1980) Anal. Biochem. 107, 193-198[CrossRef][Medline] [Order article via Infotrieve]
16. Reynolds, L. J., Washburn, W. N., Deems, R. A., and Dennis, E. A. (1991) Methods Enzymol. 197, 3-23[CrossRef][Medline] [Order article via Infotrieve]
17. Yokoyama, K., Fuzuki, S., and Setaka, M. (2000) J. Lipid Res. 41, 142-147[Abstract/Free Full Text]
18. Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) J. Biol. Chem. 270, 445-450[Abstract/Free Full Text]
19. Lio, Y.-C., Reynolds, L. J., Balsinde, J., and Dennis, E. A. (1996) Biochim. Biophys. Acta 1302, 55-60[Medline] [Order article via Infotrieve]
20. Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233[Abstract/Free Full Text]
21. Leslie, C. C. (1991) J. Biol. Chem. 266, 11366-11371[Abstract/Free Full Text]
22. Pete, M. J., and Exton, J. (1996) J. Biol. Chem. 271, 18114-18121[Abstract/Free Full Text]
23. Lin, Q., Higgs, H. N., and Glomset, J. A. (2000) Biochemistry 39, 9335-9344[CrossRef][Medline] [Order article via Infotrieve]
24. Tang, J., Kriz, R. W., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575[Abstract/Free Full Text]
25. Sugimoto, H., Hayashi, H., and Yamashita, S. (1996) J. Biol. Chem. 271, 7705-7711[Abstract/Free Full Text]
26. Wang, A., Deems, R. A., and Dennis, E. A. (1997) J. Biol. Chem. 272, 12723-12729[Abstract/Free Full Text]
27. Wang, A., Yang, H.-C., Friedman, P., Johnson, C. A., and Dennis, E. A. (1999) Biochim. Biophys. Acta 1437, 157-169[Medline] [Order article via Infotrieve]
28. Lio, Y.-C., and Dennis, E. A. (1998) Biochim. Biophys. Acta 1392, 320-332[Medline] [Order article via Infotrieve]
29. Loo, R. W., Conde-Frieboes, K, Reynolds, L. J., and Dennis, E. A. (1997) J. Biol. Chem. 272, 19214-19219[Abstract/Free Full Text]
30. Richardson, R. J., Davis, C. S., and Johnson, M. K. (1979) J. Neurochem. 32, 607-615[CrossRef][Medline] [Order article via Infotrieve]
31. Glynn, P., Holton, J. L., Nolan, C. C., Read, D. J., Brown, L., Hubbard, A., and Cavanagh, J. B. (1998) Neuroscience 83, 295-302[CrossRef][Medline] [Order article via Infotrieve]
32. Chemomordik, L., Kozlov, M. M., and Zimmerberg, J. (1995) J. Membr. Biol. 146, 1-14[Medline] [Order article via Infotrieve]
33. Weigert, R., Silletta, M. G., Spano, S., Turacchio, G., Cericola, G., Colanzi, A., Senatore, S., Mancini, R., Polishchuk, E. V., Salmona, M., Facchiano, F., Burger, K. N. J., Mironov, A., Luini, A., and Corda, D. (1999) Nature 402, 429-433[CrossRef][Medline] [Order article via Infotrieve]
34. Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A. V., Witke, W., Huttner, W. B., and Soling, H.-D. (1999) Nature 401, 133-141[CrossRef][Medline] [Order article via Infotrieve]
35. de Figueiredo, P., Drecktrah, D., Katzenellenbogen, J. A., Strang, M., and Brown, W. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8642-8647[Abstract/Free Full Text]
36. Kuroiwa, N., Nakamura, M., Tagaya, M., and Takatsuki, A. (2001) Biochem. Biophys. Res. Commun. 281, 582-588[CrossRef][Medline] [Order article via Infotrieve]
37. Zambrano, F., Fleischer, S., and Fleischer, B. (1975) Biochim. Biophys. Acta 380, 357-369[Medline] [Order article via Infotrieve]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. H. Moon, C. M. Jenkins, D. J. Mancuso, J. Turk, and R. W. Gross
Smooth Muscle Cell Arachidonic Acid Release, Migration, and Proliferation Are Markedly Attenuated in Mice Null for Calcium-independent Phospholipase A2{beta}
J. Biol. Chem., December 5, 2008; 283(49): 33975 - 33987.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. C. Kienesberger, A. Lass, K. Preiss-Landl, H. Wolinski, S. D. Kohlwein, R. Zimmermann, and R. Zechner
Identification of an Insulin-regulated Lysophospholipase with Homology to Neuropathy Target Esterase
J. Biol. Chem., February 29, 2008; 283(9): 5908 - 5917.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. Bao, D. A. Jacobson, M. Wohltmann, A. Bohrer, W. Jin, L. H. Philipson, and J. Turk
Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2{beta} in pancreatic {beta}-cells and in iPLA2{beta}-null mice
Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E217 - E229.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
K. A. Poulsen, S. F. Pedersen, M. Kolko, and I. H. Lambert
Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants
Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1605 - C1615.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Bao, Y. Li, X. Lei, M. Wohltmann, W. Jin, A. Bohrer, C. F. Semenkovich, S. Ramanadham, I. Tabas, and J. Turk
Attenuated Free Cholesterol Loading-induced Apoptosis but Preserved Phospholipid Composition of Peritoneal Macrophages from Mice That Do Not Express Group VIA Phospholipase A2
J. Biol. Chem., September 14, 2007; 282(37): 27100 - 27114.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Kuwata, C. Fujimoto, E. Yoda, S. Shimbara, Y. Nakatani, S. Hara, M. Murakami, and I. Kudo
A Novel Role of Group VIB Calcium-independent Phospholipase A2 (iPLA2{gamma}) in the Inducible Expression of Group IIA Secretory PLA2 in Rat Fibroblastic Cells
J. Biol. Chem., July 13, 2007; 282(28): 20124 - 20132.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
P. A. Wilson, S. D. Gardner, N. M. Lambie, S. A. Commans, and D. J. Crowther
Characterization of the human patatin-like phospholipase family
J. Lipid Res., September 1, 2006; 47(9): 1940 - 1949.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Bao, H. Song, M. Wohltmann, S. Ramanadham, W. Jin, A. Bohrer, and J. Turk
Insulin Secretory Responses and Phospholipid Composition of Pancreatic Islets from Mice That Do Not Express Group VIA Phospholipase A2 and Effects of Metabolic Stress on Glucose Homeostasis
J. Biol. Chem., July 28, 2006; 281(30): 20958 - 20973.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Bao, A. Bohrer, S. Ramanadham, W. Jin, S. Zhang, and J. Turk
Effects of Stable Suppression of Group VIA Phospholipase A2 Expression on Phospholipid Content and Composition, Insulin Secretion, and Proliferation of INS-1 Insulinoma Cells
J. Biol. Chem., January 6, 2006; 281(1): 187 - 198.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. K. Nomura, D. Leung, K. P. Chiang, G. B. Quistad, B. F. Cravatt, and J. E. Casida
A brain detoxifying enzyme for organophosphorus nerve poisons
PNAS, April 26, 2005; 102(17): 6195 - 6200.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Murakami, S. Masuda, K. Ueda-Semmyo, E. Yoda, H. Kuwata, Y. Takanezawa, J. Aoki, H. Arai, H. Sumimoto, Y. Ishikawa, et al.
Group VIB Ca2+-independent Phospholipase A2{gamma} Promotes Cellular Membrane Hydrolysis and Prostaglandin Production in a Manner Distinct from Other Intracellular Phospholipases A2
J. Biol. Chem., April 8, 2005; 280(14): 14028 - 14041.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. M. Jenkins, D. J. Mancuso, W. Yan, H. F. Sims, B. Gibson, and R. W. Gross
Identification, Cloning, Expression, and Purification of Three Novel Human Calcium-independent Phospholipase A2 Family Members Possessing Triacylglycerol Lipase and Acylglycerol Transacylase Activities
J. Biol. Chem., November 19, 2004; 279(47): 48968 - 48975.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
O. Zaccheo, D. Dinsdale, P. A. Meacock, and P. Glynn
Neuropathy Target Esterase and Its Yeast Homologue Degrade Phosphatidylcholine to Glycerophosphocholine in Living Cells
J. Biol. Chem., June 4, 2004; 279(23): 24024 - 24033.
[Abstract] [Full Text] [PDF]