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Originally published In Press as doi:10.1074/jbc.M602925200 on July 12, 2006

J. Biol. Chem., Vol. 281, Issue 36, 26081-26088, September 8, 2006
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Cyclic Phosphatidic Acid Is Produced by Autotaxin in Blood*

Satomi Tsuda{ddagger}, Shinichi Okudaira§, Keiko Moriya-Ito{ddagger}, Chie Shimamoto{ddagger}, Masayuki Tanaka§, Junken Aoki§, Hiroyuki Arai§, Kimiko Murakami-Murofushi{ddagger}, and Tetsuyuki Kobayashi{ddagger}||1

From the {ddagger}Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo-Ku, Tokyo 112-8610, Japan, §Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-Ku, Tokyo 113-0033, Japan, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, and ||Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

Received for publication, March 28, 2006 , and in revised form, July 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid (LPA), was previously identified in human serum. Although cPA possesses distinct physiological activities not elicited by LPA, its biochemical origins have scarcely been studied. In the present study, we assayed cPA formation from lysophosphatidylcholine in fetal bovine serum and found significant activity of transphosphatidylation that generated cPA. The cPA-producing enzyme was purified from fetal bovine serum using five chromatographic steps yielding a 100-kDa protein with cPA biosynthetic activity. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of its tryptic peptides revealed that the enzyme shared identical fragments with human autotaxin, a serum lysophospholipase D that produces LPA. Western blot analysis demonstrated that the 100-kDa protein was specifically recognized by an anti-human autotaxin antibody. Moreover, recombinant rat autotaxin was found to generate cPA in addition to LPA. No significant cPA- or LPA-producing activity was detected in autotaxin-depleted serum from bovine or human prepared by immunoprecipitation with an anti-autotaxin monoclonal antibody. These results indicate that the generation of cPA and LPA in serum is mainly attributed to autotaxin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid (LPA)2 is a bioactive lipid mediator with diverse biological activities, including induction of cancer cell invasion, regulation of actin stress fiber formation, and stimulation of cell proliferation (1–3). The signaling effects of LPA are mediated by at least five G protein-coupled receptors as LPA1–5 (4, 5).

Cyclic phosphatidic acid (cPA), which has a cyclic phosphate at the sn-2 and -3 positions of glycerol, is a unique analog of LPA. It was originally isolated from myxoamoebae of a true slime mold, Physarum polycephalum, and designated PHYLPA (6, 7). cPA was also detected as a physiological constituent of human serum (8). In contrast to LPA, cPA shows an antiproliferative activity in fibroblasts (9, 10) and an inhibitory activity toward cancer cell invasion and metastasis (11–13). It affects cellular functions, including regulation of actin stress fiber formation (14), and has neurotrophic effects on cultured embryonic hippocampal neurons (15). These findings suggest that cPA may act as a distinct physiological modulator of various cell functions. cPA activates LPA receptors (LPA1,2,3), although it is less potent than LPA (16, 17). It is also shown that cPA may directly inhibit Cdc25 phosphatase, suppressing the cell cycle progression at the G1 phase (18). However, its metabolic pathways remain to be elucidated.

Phospholipase D (PLD) catalyzes the transphosphatidylation of alcohols with phospholipids (19); however, the efficiency of transphosphatidylation relative to hydrolysis differs with various PLD enzymes and assay conditions. We found the almost exclusive formation of cPA from lysophosphatidylcholine (LPC) by PLD isolated from Actinomadura sp. No. 362, which under certain conditions preferentially transphosphatidylates rather than hydrolyzes LPC (18, 20). It is thus possible that cPA is produced through transphosphatidylation reaction mediated by a PLD-like enzyme present in blood.

In the present study, we assayed cPA formation from LPC in fetal bovine serum (FBS), and we found significant transphosphatidylation and ensuing cPA generation. We purified and characterized this enzyme from FBS. Several lines of evidence support that the main cPA-generating activity in serum can be attributed to autotaxin (ATX), which was previously shown to be identical to serum lysophospholipase D (lysoPLD), producing LPA (21, 22). Immunoprecipitation with an anti-ATX antibody demonstrated that ATX was largely responsible for the lysoPLD activity involved in LPA and cPA generation in mammalian serum.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (2-NBD-PC) was obtained from Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc. (Alabaster, AL). Egg yolk LPC was purchased from Wako Chemical Ltd. (Tokyo, Japan). PLD from Actinomadura sp. No. 362 and Rhizopus delemer lipase were purchased from Seikagaku Corporation (Tokyo, Japan). FBS was obtained from Sigma. 1-[14C]16:0-LPC (2.1 GBq/mmol) and 14:0-LPC were commercial products of PerkinElmer Life Sciences and Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc. (Alabaster, AL), respectively.

Synthesis of 1-NBD-LPC—1-NBD-labeled lysophosphatidylcholine (1-NBD-LPC) was prepared from 2-NBD-PC as follows. 2-NBD-PC was deacylated by R. delemer lipase to prepare 2-NBD-LPC as described previously (23). The 2-NBD-LPC was then dissolved in 0.1 M Tris-HCl buffer (pH 9.0) and incubated at 37 °C for 3 h to generate 1-NBD-LPC through acyl migration (24). The lipid was extracted and purified by a Shimadzu (LC-9A) high-performance liquid chromatography system on a TSK ODS-80 column (4.6 x 150 mm, Tosoh Corporation, Tokyo, Japan) using acetonitrile:water (40:60) solvent containing 20 mM choline chloride at a flow rate of 1.0 ml/min. The peak with the retention time of 17.9 min was collected as a 1-NBD-LPC fraction, which was separated from the peak of 2-NBD-LPC at 15.4 min.

LysoPLD Assay with FBS—In the standard assay, samples of 5–10 µl were incubated for 1 h at 37°C with 20 nmol of substrate containing a 1:99 ratio of 1-NBD-labeled LPC to unlabeled LPC in the presence of 0.1 M Tris-HCl (pH 8.5), 1 M NaCl, 5 mM CaCl2, 5 mM MgCl2, and 0.1% bovine serum albumin (total volume 0.1 ml) mixed with a 0.4 volume of diethyl ether. The reaction was terminated by adding a 0.35 volume of 0.1 M citric acid and 5.4 volumes of chloroform:methanol (2:1). After vigorous stirring, the mixtures were centrifuged at 1,400 x g for 5 min. The upper phase was re-extracted once more with chloroform:methanol (2:1), and the lower phases were combined and dried under a N2 gas stream. The lipids were resuspended in a small amount of chloroform:methanol (2:1) and spotted onto Silica Gel 60 thin layer chromatography plates (E. Merck, Darmstadt, Germany). The plates were developed with a solvent system consisting of chloroform:methanol:acetic acid:1% sodium disulfide aqueous solution (100:40:12:5), and the fluorescence intensities were measured by a FLA-2000 fluorescence plate scanner (Fuji Photo Film Co., Ltd., Tokyo, Japan) with excitation at 480 nm and emission at 520 nm. cPA standard was prepared from LPC using Actinomadura PLD under the conditions described previously (25).

Purification of cPA Biosynthetic Enzyme from FBS—Fetal bovine serum (200 ml) was diluted with 4 volumes of 20 mM BisTris (pH 6.5) and fractionated by precipitation using 40–60% saturation of ammonium sulfate. After dialysis, proteins were fractionated by ion exchange chromatography using a CM Sephadex C-50 column (30 ml; Amersham Biosciences) in 20 mM BisTris (pH 6.5) containing 10% glycerol with a linear NaCl gradient from 0 to 1 M. The active fractions were loaded onto a Phenyl-Sepharose CL-4B column (8 ml; Amersham Biosciences) in 20 mM Tris-HCl (pH 7.5) containing 10% glycerol in a linear gradient of ammonium sulfate concentration from 1 to 0 M. Fractions containing the activity were loaded onto a Heparin-Sepharose CL-6B column (4 ml; Amersham Biosciences) in 20 mM Tris-HCl (pH 7.5) containing 10% glycerol and eluted with a linear gradient of NaCl concentration from 0 to 0.5 M. The active fractions were loaded onto a concanavalin A-Sepharose 4B column (2 ml, Sigma) in 20 mM Tris-HCl (pH 7.5) containing 10% glycerol, 0.5 M NaCl, and 0.1% CHAPS and were eluted with 0.3 M methyl-{alpha}-D-mannopyranoside at room temperature. The active fractions were loaded onto a POROS HQ anion exchange column (4.6 x 100 mm; Applied Biosystems, Tokyo, Japan) in 20 mM Tris-HCl (pH 8.5) containing 10% glycerol and 0.1% CHAPS and eluted with a linear gradient of NaCl concentration from 0 to 1 M. Proteins present in the chromatographic fractions were separated by 7.5% SDS-PAGE.

Identification of Purified Enzyme—The fractions containing cPA-generating activity after the final chromatographic step were combined and subjected to SDS-PAGE. After staining the gel with Coomassie Brilliant Blue, the 100-kDa band, predominant in the active fractions, was cut and treated with trypsin (Promega, Tokyo, Japan). The tryptic digest was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Ultraflex, Brucker Daltonics, Bremen, Germany) using BioTools software, version 3.0.

Western Blot Analysis—Proteins separated by SDS-PAGE on 7.5% gels were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), blocked using 0.05% (v/v) Triton X-100 and 5% (v/v) powdered milk in Tris-buffered saline (25 mM Tris-HCl (pH 7.4) containing 137 mM NaCl and 2.7 mM KCl). Rat monoclonal antibodies against human ATX (26) were used as primary antibodies, and an anti-rat IgG horseradish peroxidase-conjugated antibody (American Qualex, San Clemente, CA) was used as a secondary antibody, followed by signal development using the ECL kit from Amersham Biosciences.

Anti-ATX Monoclonal Antibody and Immunoprecipitation— Recombinant mouse ATX protein with a His tag at the N terminus was expressed and purified using a baculovirus system and nickel column chromatography (HisTrap HP, Amersham Biosciences), respectively. The wild-type ATX protein (50 µg) was used to immunize rats (WKY/Izm strain), and the antibody-secreting hybridoma cells were selected by screening with an enzyme-linked immunosorbent assay and immunoprecipitation. Of eleven anti-ATX-secreting hybridoma cell lines established, 5E5 was found to have activities to immunoprecipitate the ATX activity in serum from fetal bovine and human. The immunoglobulin subclass of 5E5 was determined as IgG1. To deplete ATX from the serum, 5E5-coupled Sepharose 4B was mixed with the serum, and the resulting supernatants were used as ATX-depleted serum.

Determination of LPA and LysoPLD Activity in ATX-depleted Serum—LPA concentrations in serum were determined by the colorimetric method as described previously (27). LysoPLD activity was determined by monitoring liberated choline from 14:0-LPC as described previously (21).

Plasmids and Recombinant Enzyme—Generation of baculovirus with the cDNA encoding the rat gene for ATX (corresponding to rat ATX-T) and the purification of recombinant ATX protein in baculovirus are described in Ref. 21.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
cPA-generating Activity Is Present in FBS—Our earlier work established the presence of albumin-bound cPA in human serum (8), suggesting the presence of cPA-producing enzyme(s) in the serum. We assayed the cPA biosynthetic enzyme activity in FBS using the 1-NBD-LPC substrate and found that a lipid product co-migrating with the cPA standard was slightly generated, concomitant with subtle production of LPA (Fig. 1A, lane 3). Through detailed examination of assay conditions optimal for the cPA production, the presence of diethyl ether and 1 M NaCl appeared to be effective (Fig. 1A, lane 4). The amount of cPA increased with incubation time and reached a maximum at 3 h (Fig. 1B). Under the two-phase reaction conditions used in the presence of diethyl ether and 1 M NaCl, the accumulation of LPA was much lower than that of cPA. A considerable amount of fluorescence was present in spots of free NBD-fatty acid near the solvent front, indicating that the deacylation by serum lysophospholipase A1 was also strong.


Figure 1
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  		FIGURE 1.
cPA formation from LPC by FBS. A, thin layer chromatography of lipid products by FBS. 1-NBD-LPC was incubated at 37 °C with FBS for 1 h in the absence of diethyl ether and 1 M NaCl (lane 3). cPA production from 1-NBD-LPC was also examined by incubation with FBS for 0 h (lane 2) or 1 h (lane 4) in the presence of diethyl ether and 1 M NaCl. The positions of 1-NBD-cPA and 1-NBD-LPA on a plate were determined using products from Actinomadura PLD (lane 1). B, time course of cPA (filled circles) and LPA (open circles) production by FBS. Each point represents the mean of three determinations ± S.D.

 
Significant levels of cPA synthetic activity were also detected in rat and human serum (data not shown) confirming our previous report (18). Furthermore, cPA production in human plasma was almost comparable with that in human serum under the two-phase reaction conditions (data not shown).

Purification of the cPA-producing Enzyme from FBS—The cPA biosynthetic enzyme was purified from FBS using five chromatographic steps (Table 1). We obtained an ~2,500-fold purified preparation of cPA synthetic enzyme activity at the final purification step. The peaks activity generating LPA overlapped with that of the cPA-forming activity in the final purification step (Fig. 2A). There were no other fractions that possessed cPA- and/or LPA-producing activities from LPC through the purification steps.


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  		TABLE 1
Purification of cPA-producing enzyme from FBS using five chromatographic steps

 
The active fractions from the POROS HQ column were subjected to SDS-PAGE and silver-stained (Fig. 2B). A prominent 100-kDa protein band co-purified with cPA biosynthetic activity. Tryptic peptides from this band were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on the basis of peptide mass matching. A data base search of mass spectra revealed that the purified cPA biosynthetic enzyme shared several peptide fragments with human ATX, TARGWECTK (corresponding to the sequence from 97 to 105 of human ATX, AAECPAGFVRPPLIIFSVDGFR (154–175); YGPFGPEMTNPLREIDKIVGQLMDGLK (318–344); LHYANNRRIEDIHLLVERR (433–451); KPSGKCFFQGDHGFDNK (464–480), and LDELNKR (579–585)). Furthermore, immunoblot analysis of the active fractions from the POROS HQ column demonstrated that the 100-kDa protein was immunoreactive with a monoclonal antibody specific to human ATX (Fig. 2C).

To determine whether ATX (which was previously known as nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2)) itself produces cPA as well as LPA, recombinant rat ATX/NPP2 was expressed in Sf9 cells and tested in the cPA production assay. The recombinant ATX/NPP2, under the assay conditions used, generated cPA and LPA at comparable levels (Fig. 3), indicating that ATX/NPP2 is the enzyme responsible for the cPA synthetic activity in FBS.

Immunoprecipitaion of Serum with Anti-ATX Antibody— Immunoprecipitation of FBS with anti-ATX antibody was performed to examine whether other lysoPLD enzymes besides ATX are partly responsible for cPA production in serum. A monoclonal antibody against mouse ATX with an activity to immunoprecipitate ATX was established. The monoclonal antibody 5E5 was found to cross-react with bovine and human ATX. Using 5E5-coupled Sepharose 4B beads, ATX-depleted FBS was prepared. The production of LPA was hardly detectable upon incubation of the ATX-depleted FBS (Fig. 4A), indicating that the contribution of ATX to LPA production is extremely high. It was suggested that the bulk of LPA produced in the serum results from a sequential cleavage of phospholipids to lysophospholipids by phospholipases A1 and A2 and then to LPA by lysoPLD (28–31). Our present data strongly support the idea that the two-step process containing lysoPLD is a main pathway for LPA biosynthesis in serum. Furthermore, ATX appears to be almost the sole enzyme responsible for lysoPLD activity in mammalian serum.


Figure 2
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  		FIGURE 2.
Purification of cPA-producing enzyme and identification. A, elution profile of cPA biosynthetic activity from FBS on anion exchange chromatography (POROS HQ). The cPA (filled circles)- and LPA (open circles)- producing activities in each fraction were determined under standard assay conditions. B, active fractions from the POROS HQ column were subjected to SDS-PAGE, and proteins were detected by silver staining. Arrows indicate the protein bands with cPA-producing activities. C, Western blot analysis. The ATX bands were detected using anti-human ATX as a primary antibody.

 


Figure 3
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  		FIGURE 3.
Conversion of 1-NBD-LPC into cPA and LPA by recombinant rat ATX. 1-NBD-LPC was incubated with Actinomadura PLD (lane 1), recombinant rat ATX expressed in Sf9 cells (lane 2), cPA-producing enzyme partially purified from FBS (lane 3), FBS (lane 4), or without enzyme (lane 5) under standard assay conditions. Results were typical of three separate experiments.

 


Figure 4
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  		FIGURE 4.
LPA- and cPA-producing activities in ATX-depleted serum prepared by immunoprecipitation. A, ATX-depleted FBS was prepared by mixing Sepharose 4B coupled with anti-ATX monoclonal antibody with FBS to obtain the supernatants. Either ATX-depleted FBS (filled circles) or control IgG-depleted FBS (open circles) was incubated at 37 °C for the indicated times, and the concentrations of LPA produced were determined colorimetrically. B, 1-NBD-LPC was incubated with either ATX-depleted or control IgG-depleted serum from fetal bovine and human under standard assay conditions. C, LysoPLD activities of ATX-depleted (filled bars) or control IgG-depleted (open bars) serum from fetal bovine and human were determined by monitoring liberated choline from 14:0-LPC.

 
The biosynthetic activity of cPA from LPC also disappeared almost completely in ATX-depleted serum prepared from fetal bovine or human, as shown in Fig. 4B. In addition, lysoPLD activity was greatly diminished in the ATX-depleted serum from fetal bovine or human (Fig. 4C). In this experiment, the lysoPLD activity was determined by choline release from LPC. Accordingly, the activity is considered to reflect the total activity of cPA and LPA formation. These observations indicate that ATX is largely responsible for the lysoPLD activity involved in not only LPA but also cPA biosynthesis in mammalian serum.


Figure 5
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  		FIGURE 5.
Effects of diethyl ether, NaCl, and metal ions on production of cPA and LPA. 1-NBD-LPC was incubated with a partially purified preparation from FBS (A) or recombinant rat ATX expressed in Sf9 cells (B) under standard assay conditions in the presence (+) or absence (–) of diethyl ether and/or 1 M NaCl. Lipids were extracted and analyzed by thin layer chromatography. Amounts of fluorescent cPA (filled bars) and LPA (open bars) were determined by a fluoroimage analyzer and expressed as a percentage relative to the activities of cPA production in the presence of diethyl ether and 1 M NaCl. Each point represents the mean of three determinations ± S.D. The differences between the means were compared by Student's t test. a, significantly different from diethyl ether (–) and NaCl (–) (p < 0.05). b, significantly different from diethyl ether (–) and NaCl (–) or diethyl ether (+) and NaCl (–) (p < 0.05). c, significantly different from any other combination (p < 0.05). Either cPA biosynthetic enzyme partially purified from FBS or recombinant rat ATX was incubated with 1-NBD-LPC under standard assay conditions in the presence or absence (–) of metal ions (5 mM)(C). The cPA-producing activities were expressed as a percentage relative to the activities without metal ions. Each point represents the mean of three determinations ± S.D. The differences between the means were compared by Student's t test (*, p < 0.05).

 


Figure 6
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  		FIGURE 6.
LPA production from cPA by recombinant rat ATX. 1-NBD-cPA at a concentration of 200 µM was incubated with recombinant rat ATX under standard assay conditions (lanes 1 and 2) or without ATX (lane 3) in the presence of both 5 mM Ca2+ and 5 mM Mg2+ (lanes 1 and 3) or 5 mM Co2+ (lane 2).

 
Characterization of cPA-producing Activities of ATX—We characterized cPA biosynthetic activities using either the partially purified enzyme from FBS or the recombinant rat ATX protein. Because transphosphatidylation reactions are frequently performed in an aqueous/organic two-phase system (32) or in the presence of high concentrations of salt (33), the effects of these systems were examined. The cPA biosynthetic activity of the partially purified enzyme increased when 1 M NaCl was present and was significantly enhanced by the addition of both diethyl ether and NaCl (Fig. 5A). LPA production from LPC by the purified enzyme was also augmented in the presence of diethyl ether and/or NaCl. The cPA:LPA ratio was not altered under any condition. The facilitatory effect on cPA and LPA production by NaCl was also observed with the recombinant ATX, whereas the addition of diethyl ether did not affect the activity (Fig. 5B). These results raise the possibility of the existence of a serum factor that enhances the lysoPLD activity in a diethyl ether-dependent manner.

Divalent cations, especially Co2+ have been found to enhance the lysoPLD activity in rat serum (34). We found that Co2+ increased the lysoPLD activity of purified protein to produce LPA, whereas cPA formation was scarcely affected (Fig. 5C). The activation by other divalent cations, such as Ca2+ and Mg2+, was much smaller. Similar results were obtained with the recombinant protein (Fig. 5C).

When the recombinant ATX was incubated with 1-NBD-cPA in the presence of Ca2+ and Mg2+, a small amount of 1-NBD-LPA was produced (Fig. 6). The presence of Co2+ increased the hydrolytic activity of cPA to LPA, consistent with observations shown in Fig. 5C. The data raise the possibility that divalent cations determine the products of ATX (LPA or cPA).


Figure 7
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  		FIGURE 7.
Formation of cPA and LPA from 1-[14C]16:0-LPC by the purified enzyme under near physiological conditions. cPA-producing enzyme partially purified from FBS was incubated for 2 h at 37 °C with 0.2 (lane 3), 0.5 (lane 4), and 1.0 mM 1-[14C]16:0-LPC (lane 5) in the presence of 0.1 M Tris-HCl (pH 8.5), 140 mM NaCl, 2.5 mM CaCl2, 1.0 mM MgCl2, and 0.7 µM CoCl2. The concentrations of these metal ions are equivalent to those in human serum (42). 1-[14C]16:0-LPC (0.5 mM) was incubated with Actinomadura PLD (lane 1) or without enzyme (lane 2) under the same conditions. The apparent Km and Vmax values were determined by varying the substrate concentrations more widely.

 
Because the NBD-labeled substrates are not natural compounds and supplementation of ether into the assay mixtures is non-physiological, we next examined the cPA formation by ATX under near physiological conditions from 1–16:0-LPC, which is one of the major molecular species of LPC in bovine and human serum. Various concentrations of 1-[14C]16:0-LPC were incubated with the purified enzyme in the presence of 140 mM NaCl and physiological concentrations of metal ions (Ca2+, Mg2+, and Co2+) without ether, and the reaction products were analyzed by thin layer chromatography. In addition to the predominant formation of LPA, significant levels of cPA were produced from 16:0-LPC by ATX (Fig. 7). These results demonstrate that cPA is generated by ATX under near physiological conditions from native LPC. The apparent Km of cPA-producing activity of purified enzyme for 1-[14C]16:0-LPC calculated from Lineweaver-Burk plots was 2.6 mM. The apparent Vmax of cPA- and LPA-producing activities were 0.32 and 1.2 nmol/h/ml, respectively, suggesting that the ratio of cPA to LPA produced by ATX at a high concentration of LPC is ~1:4 under the present conditions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The enzyme generating cPA was purified from FBS, and peptide mass matching analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that it shared several fragments with human ATX. In addition, we obtained several lines of evidence that ATX is capable of producing, not only LPA, but cPA as well. Immunoblot analysis confirmed that the 100-kDa protein present in active fractions is immunoreactive with anti-human ATX antibodies. Both cPA and LPA biosynthetic activities were detected in recombinant rat ATX expressed in Sf9 cells. These results indicate that ATX catalyzes both cPA biosynthesis as well as LPA production. ATX/NPP2 was originally identified as an autocrine motility-stimulating factor secreted by melanoma cells (35). ATX was found to possess an intrinsic lysoPLD activity that produces LPA (21, 22). It was also shown that ATX hydrolyzes sphingosylphosphorylcholine to produce sphingosine-1-phosphate (36), but the transphosphatidylation activity of ATX has not hitherto been reported. Our results indicate that ATX has the ability to synthesize cPA by its intramolecular transphosphatidylation action on LPC. There was the possibility that other lyso-PLD enzymes, besides ATX, are also responsible for cPA production in serum. However, it was demonstrated from our immunoprecipitation study that ATX was largely responsible for the lysoPLD activity involved in cPA generation in mammalian serum.

Mammalian genomes contain at least seven NPP-encoding genes, but only three of them, namely NPP1, ATX/NPP2, and NPP3, have been studied in some detail. These three NPPs release nucleotide 5'-monophosphates from a variety of nucleotides and nucleotide derivatives. Compared with NPP1 and NPP3, ATX/NPP2 shows rather poor activity against nucleotides. However, ATX/NPP2 acts as a lipid phosphodiesterase, lysophospholipase D, in contrast with NPP1 and NPP3. Cimpean et al. (37) suggest that the expression of lysophospholipase D activity depends on isoform-specific determinants in N-terminal and catalytic, as well as C-terminal domains of ATX/NPP2 and that sequences in all three domains together form a lysophospholipid-binding site. The catalytic mechanism of nucleotide pyrophosphatase/phosphodiesterase reaction of NPPs has been well studied. It is proposed that catalysis occurs by a two-step mechanism (38). The reaction mechanism of PLD might be similar to that of NPP. Hydrolysis and transphosphatidylation of LPC by PLD comprise a two-step "ping-pong" reaction mechanism with the formation of a phosphatidyl-enzyme intermediate. Friedman et al. (39) demonstrate, with Streptomyces chromofuscus PLD, that the free hydroxy group in the sn-2 position of LPC competes with ambient water molecules in the nucleophilic attack of the phosphatidyl intermediate to form intramolecular cyclization, producing cPA followed by hydrolysis to LPA. We have demonstrated previously that Actinomadura PLD can efficiently produce cPA but little LPA (18). cPA produced by Actinomadura PLD is considered to be dissociated from the enzyme without hydrolysis to LPA. A difference in the ring opening activities of these bacterial PLDs that generate LPA is reflected in the final concentrations of cPA and LPA after incubation. The present study demonstrated that ATX can produce not only LPA but cPA as well from LPC.

Although some of effects of cPA are homologous, many are opposite to those of LPA (18). The concentrations and/or localization of these lipid mediators should be stringently regulated in vivo. The addition of 1 M NaCl into the reaction mixture of recombinant ATX was found to enhance the formation of cPA rather than LPA as shown in Fig. 5B. It is known that lipolytic enzyme reactions are usually influenced by the physical state of hydrophobic substrates. Under the assay conditions employed, LPC may exist as micelles in an aqueous system, forming a monolayer at interfaces between the two phases in the presence of diethyl ether. A high concentration of salts could influence the physical state of hydrophobic molecules to increase the substrate availability for cPA formation. Thus, the physical state of the substrate (LPC) and the nature of the interface at which reaction takes place should play an important role on cPA and LPA formation by ATX in vivo.

We found that the purified enzyme from FBS and the recombinant ATX each yield cPA that is hydrolyzed gradually to LPA. Co2+ has been shown to facilitate the hydrolysis of cPA to LPA by ATX. It was proposed that the catalytic site of ATX contains two essential divalent metals (38). Co2+ is presumed to activate a water molecule to hydrolyze the cPA-enzyme intermediate and release LPA. Co2+ may be one of the regulatory factors involved in serum cPA and LPA concentrations. A concentration of cPA in human serum is estimated to be ~0.1 µM (8), whereas it contains micromolar concentrations of LPA (28). Our present study suggested that the rate of cPA production could be 1/4 of that of LPA production under physiological conditions, indicating that the serum concentration difference between cPA and LPA cannot be solely attributed to the difference in production rate by ATX between them. It is known that LPA is further metabolized by being deacylated by lysophospholipases or dephosphorylated by lipid phosphate phosphatases in serum (40). The degradation of cPA, however, has not been characterized. Differences in the substrate specificity of these degradation enzymes may also account for the final concentrations of cPA and LPA in serum.

Information on biosynthetic pathways of LPA has recently been accumulated (28–31). It has been demonstrated that LPA is converted from phosphatidic acid in cells such as platelets and some cancer cells. By contrast, LPA is demonstrated to be mainly generated from lysophospholipids in serum and plasma. In the latter case, certain phospholipases, such as secretory phospholipase A2 (sPLA2-IIA), phosphatidylserine-specific phospholipase A1 (PS-PLA1), and lecithin-cholesterol acyl transferase (LCAT), are thought to be involved in the production of lysophospholipids, followed by lysoPLD action to form LPA (29). The lysoPLD activity producing LPA was first detected in rat plasma (41) and a responsible enzyme was identified as ATX (21, 22). However, the contribution of ATX to the overall production of LPA in serum has not been clarified. Our immunoprecipitation study using anti-ATX monoclonal antibody demonstrated that the lysoPLD activity in mammalian serum arises almost exclusively from ATX. In addition, the bulk of LPA produced in serum appears to result mainly from the two-step process containing lysoPLD.

The present study demonstrates that cPA production detected in FBS can be attributed mainly to the intramolecular transphosphatidylation reaction of ATX to lysophospholipids. cPA and LPA have similar structures, but a cyclic phosphate moiety is absolutely necessary for the biological activities of cPA that are distinct from those of LPA (18). Further analyses of regulatory mechanisms for the conversion of cPA to LPA will provide us with an understanding of how cPA coordinates with LPA and acts as a lipid modulator of various cellular functions.


   FOOTNOTES
 
* This work was supported in part by research grants from the ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. K.); the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, (to T. K.); National Institute of Biomedical Innovation, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency (to S. O., M. T., J. A. and H. A.); and the Terumo Life Science Foundation (to T. K.). Back

1 To whom correspondence should be addressed: Dept. of Biology, Faculty of Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan. Tel.: 81-3-5978-2604; Fax: 81-3-5978-2604; E-mail: tetkoba{at}cc.ocha.ac.jp.

2 The abbreviations used are: LPA, lysophosphatidic acid; ATX, autotaxin; PA, phosphatidic acid; PC, phosphatidylcholine; PLD, phospholipase D; cPA, cyclic PA (1-radyl-2,3-cyclic glycerophosphate); LPC, lysoPC; lysoPLD, lysophospholipaseD;FBS, fetal bovine serum;NBD,[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl; NPP, nucleotide pyrophosphatase/phosphodiesterase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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