From the Departments of a Physiology and Biophysics, and e Pharmaceutical Science, the f Charles B. Stout Neuroscience Mass Spectrometry Laboratory, the Departments of g Neurology and h Biochemistry, the University of Tennessee, Memphis, Tennessee 38163, i Matreya Inc., Pleasant Gap, Pennsylvania 16823, j LXR Biotechnology Inc., Richmond, California 94804, and the b Institute of Enzymology, Biological Research Center of the Hungarian Academy of Sciences, Karolina Street 29, Budapest 1113, Hungary
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ABSTRACT |
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Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1H and 13C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 µM was only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065-C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.
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INTRODUCTION |
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The autacoids, 1-oleoyl-2-lyso-sn-glycero-3-phosphate (lysophosphatidic acid, LPA),1 sphingosine 1-phosphate (SPP), and sphingosylphosphorylcholine (SPC) have been the subject of intense investigation fueled by the diversity of biological responses that they elicit in a variety of cell types (for reviews see Refs. 1 and 2). Perhaps the three most interesting cellular responses elicited by these autacoids include the following: mitogenesis/antimitogenesis (3-6), effect on cell shape and motility (7-11), and anti-apoptotic action (12, 13). The somewhat overlapping spectrum of cellular responses elicited by SPP and LPA has led to the hypothesis that these mediators might activate the same receptors (14, 15). This concept is supported by other investigators, who found that SPC not only activated similar cellular responses and signal transduction pathways as LPA but that these two lipids also showed heterologous desensitization, suggesting that they activated the same receptor (16, 17). On the other hand, several reports have found no evidence for heterologous desensitization between sphingolipid mediators and glycerolipid mediators, leading to a controversy in the field (18).
We report that part of this controversy appears to be due to a glycerolipid that has been present in a commercially available SPC preparation used by many investigators. The impurity was identified as 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP), an analog of LPA. Alkenyl-GP is generated in the manufacturing process of SPC from ethanolamine-containing lysoplasmalogen (alkenyl-GPE), which is present in the sphingomyelin (SM) fraction, the precursor used to manufacture SPC. We provide a method of purification and the full structural elucidation of this novel LPA analog using NMR, FT, IR, and mass spectrometry (MS) and also enzymatic and chemical analytical methods. We also describe a high yield, semi-synthetic method for the production of alkenyl-GP. Alkenyl-GP, like its acyl counterpart, activates the extracellular signal-regulated protein kinases (ERKs) 1 and 2 and acts as a strong mitogen in Swiss 3T3 cells. Alkenyl-GP has recently been described as an injury-induced component of the anterior chamber fluid of the eye (19), suggesting that this novel lipid is a naturally occurring member of the acidic phospholipid autacoid (APA) family.
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MATERIALS AND METHODS |
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Lipids-- SPC was obtained either from Sigma or Matreya Inc. (Pleasant Gap, PA). SM and polar brain phospholipid extracts were purchased from Sigma, Matreya, and Avanti Polar Lipids (Alabaster, AL). Oleoyl-LPA was from Avanti Polar Lipids. RO56 (1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine, ethanolamine-containing lysoplasmalogen, alkenyl-GPE) was obtained from Matreya Inc. or was prepared as described below. Phosphorous was determined according to Ames and Dubin (20).
Acidic Methanolysis of SM and Brain Lipid Extracts-- Acidic methanolysis was carried out according to Gaver and Sweeley (21). This procedure is the same one that Sigma uses to manufacture SPC. Briefly, the reaction mixture was prepared by adding 8.6% (v/v) concentrated hydrochloric acid and 9.4% water to methanol (MeOH). Either SM or brain polar lipids dissolved in the reaction mixture at a 2.5 mg/ml final concentration was hydrolyzed at 65 °C in pressure-sealed vials with stirring for up to 18 h.
Preparation of RO56 (Alkenyl-GPE) from Brain
Phospholipids--
The lower phase (60 g) of the Folch (22) extract
from bovine brain was dissolved in 500 ml of 0.5 M
methanolic sodium hydroxide that contained a small amount (~2%) of
H2O, in which the sodium hydroxide was initially dissolved.
After a 4-h incubation at room temperature, the pH of the reaction
mixture was adjusted to 7.0-7.5 by the addition of 6 N
HCl, and the cloudy mixture was filtered. The solvents were evaporated,
and the solid was extracted (3 ×) with a mixture of
CHCl3:MeOH:H2O (400:200:150, v/v); the bottom layer was retained. The lipids present in the bottom layer were concentrated to a small volume (~10 ml) by evaporation and were applied to a column of 180 g of silica gel 60 (Merck). Lipids were
eluted under 12 p.s.i. N2 pressure in three steps,
starting with 2 liters of CHCl3:MeOH (90:10, v/v), followed
by 4 liters of CHCl3:MeOH (87:13, v/v), and finally by 3 liters of CHCl3:MeOH:H2O (87:13:1, v/v). After
evaporation of the solvents from the last elution step, the residue was
dissolved in 10 ml of CHCl3, to which 100 ml of acetone was
added. SM remained in solution, whereas the precipitate after an
overnight incubation at 20 °C from this mixture was isolated by
filtration and was designated RO56. This procedure yielded 1.5 g
of the RO56 compound, which was later identified as alkenyl-GPE.
Separation Techniques-- Thin layer chromatography (TLC) was performed on K6 60-Å silica gel plates (Whatman Inc., Fairfield, NJ, 20 × 20 cm, thickness 250 µm), and lipids were visualized by primuline staining (23) before eluting the lipids for bioassay. Spots marked with a pencil were scraped off the plate into glass vials, and the lipids were extracted (3 ×) from the silica gel with a large excess (10 × v/w) of methanol. The methanol-soluble material was filtered through a 0.2-µm Teflon syringe filter and was dried under a stream of N2.
TLC separations used four different solvent systems as follows: solvent 1 consisted of BuOH:HAc:H2O (3:1:1); solvent 2 of CHCl3:acetone:MeOH:HAc:H2O (10:4:3:2:1); solvent 3 of CHCl3:MeOH:H2O, 28% NH4OH (20:30:4:1), and solvent 4 of CHCl3:MeOH:H2O (60:30:5, all v/v). In some experiments, for the detection of primary amine and phosphate moieties, lipids were stained with ninhydrin (Sigma) or molybdenum blue (Sigma) spray reagents, respectively. The HPLC purification of the SPC stereoisomers was performed according to Buenemann et al. (24), with a 5-µm silica-packed Microsorb column (250 × 4.6 mm, Rainin Instruments, Woburn, MA). Solvent delivery and injection were done by a Waters HPLC system controlled by the Millennium 2010 version 2.1 software package (Waters Instruments, Milford, MA). Elution of the lipids was monitored by a Varex Evaporative Light-Scattering Detector (Burtonsville, MD) through a metering valve; the effluent was split (1:4) between the detector and a fraction collector, respectively. HPLC-grade solvents were from J. T. Baker Inc.Instrumental Analysis--
1H, 13C, and
31P NMR spectra were recorded with a Bruker model 300 MHz
ARX (Brueker Instruments Inc., Billerica, MA) spectrometer. 1H chemical shifts ( in ppm) were calibrated against
CD3OD at
= 3.30 ppm and 13C chemical shifts
against CD3OD at
= 49.00 ppm. 31P chemical
shifts were calibrated against 0.0485 M triphenylphosphate in CDCl3 at
= 0 ppm. Interpretation of NMR spectra was
accomplished with the assistance of ACD/HNMR and CNMR v2.03 software
package (Advanced Chemistry Development Inc., Toronto, Canada).
Infrared spectra were recorded in CHCl3 on a Perkin-Elmer
model 2000 FTIR (50/60 Hz) spectrophotometer. Mass spectra were
obtained from an AutoSpec Q (E1BE2qQ) tandem
mass spectrometer (VG Fisions, Altrincham, UK) with fast atom
bombardment (FAB) ionization, using a glycerol matrix (1 µl). Spectra
were analyzed with the VG Opus level 1.7f software package, and CsI was
used for the mass calibration of the instrument.
Enzymatic Synthesis of Alkenyl-GP-- Alkenyl-GP was prepared by a two-step procedure, using PLD followed by PLA2 digestion of 1-O-cis-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (alkenyl-acyl-GPE) obtained from Serdary Research (London, Ontario, Canada). Six µmol of alkenyl-acyl-GPE were digested with 250 units of cabbage PLD (Type V, Sigma) that was suspended in 1 ml of 0.2 M acetate diethyl ether buffer, pH 5.6, according to Kates (25). After 4 h, the ether was removed with a stream of N2 gas, and the lipids were extracted by phase separation after adding 3 volumes of CHCl3:MeOH (3:2, v/v) to the aqueous reaction mixture.
Phospholipase A2 (from Naja naja, Sigma)-catalyzed removal of the fatty acid side chain from the sn-2 position was carried out in a non-aqueous medium, according to Kates (25). After a 4-h digestion at room temperature, the solvents were evaporated with a stream of N2, and the reaction products were analyzed by TLC, and the plate was developed in solvent 2.Enzymatic Dephosphorylation of Alkenyl-GP-- Alkenyl-GP (100 µg/reaction) was digested with Escherichia coli alkaline phosphatase (AP, EC 3.1.3.1, Worthington) with a specific activity of 37 units/mg, as described by Blank and Snyder (26). Control reactions received equal amounts of heat-inactivated enzyme. After 2 h, the reaction was terminated, and the lipids were extracted (37).
Hydrolysis of the Vinyl-Ether Bond by HCl Vapor-- SPC (1 mg, Sigma) and alkenyl-GP (50 µg) were spotted onto a TLC plate, which was placed into a chromatography tank that contained concentrated HCl at the bottom. The HCl vapor was allowed to react with the vinyl-ether bond for 15 min. Subsequently, the lipids were scraped off, extracted with large excess of methanol, dried under a stream of N2 at 40 °C, and dissolved in 200 µl of methanol. As a control, the same lipids were processed the same way without the exposure to HCl vapor.
Electrophysiological Recording from Xenopus Oocytes-- Oocytes were obtained from xylazine-anesthetized adult Xenopus laevis frogs (Carolina Scientific, Burlington, NC) under aseptic conditions and were prepared for experiments as described previously (33). Lipids for bioassay were always complexed with fatty acid-free BSA (Sigma, for complexing procedure see below), diluted in frog Na+-Ringer's solution (120 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.0), and were applied through superfusion to the oocyte, at a flow-rate of 5 ml/min. Membrane currents were recorded with a NIC-310 digital oscilloscope (Nicolet, Madison, WI).
Cell Proliferation Assay-- DNA synthesis induced by the different lipids was determined using [3H]thymidine (Amersham Pharmacia Biotech, 185 GBq/mmol) incorporation into confluent cultures of Swiss 3T3 fibroblasts, as described by Desai and Spiegel (6). Lipids, dissolved in methanol, were complexed with fatty acid-free BSA (Sigma) in Ca2+-free Hanks' basal salt solution at a 1:1 molar ratio and were applied to the cells at a 10 µM final concentration. After an 18-h incubation in the presence of the lipids, 1 µCi of [3H]thymidine was added to each well; cells were harvested 6 h later to quantify the amount of acid-insoluble radioactivity (27).
Assay for ERK1/ERK2--
The combined activity of the ERK1 and
ERK2 was determined by a method described elsewhere (28, 29).
Subconfluent cultures (75%) of Swiss 3T3 cells were serum-starved for
6 h and were treated with a 1 µM concentration of
each lipid for 10 min. The total protein concentration of the
supernatant was determined according to Bradford (30). Total cell
protein (200 µg) was used for each assay. Samples were treated with
ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA) antisera at a
1:100 dilution for 1 h before incubation with 20 µl of protein
G-agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. Beads
were washed twice with 1 ml of lysis buffer and once with kinase buffer
(30 mM Tris-HCl, pH 8.0, 20 mM
MgCl2, and 2 mM MnCl2). The kinase
reactions were started by adding 30 µl of kinase buffer that
contained 10 µM ATP, 2.5 µCi of
[-32P]ATP (Amersham Pharmacia Biotech, 370 GBq/mmol),
and 7 µg of myelin basic protein (MBP, Sigma) to each sample and were
incubated for 30 min at 30 °C. MBP was separated on a 14%
SDS-polyacrylamide gel (31), stained with Coomassie Blue, and dried.
MBP bands were excised, and the incorporated radioactivity was
determined by scintillation counting. Results represent the mean
(n = 3) of enzyme assays and are representative of
three experiments.
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RESULTS |
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The Novel Lipid Present in Commercial SPC Is Responsible for the
LPA-like Activity--
In Xenopus oocytes, APA mediators
elicit Ca2+-activated oscillatory Cl currents
through the activation of multiple APA receptor subtypes, which couple
to the inositol trisphosphate-Ca2+ signaling system (7,
32). To confirm its LPA-like effects, SPC was applied to oocytes that
were voltage-clamped at
60 mV, and membrane currents were recorded.
The SPC preparation obtained from Sigma, which is used by many of the
investigators who reported its LPA-like effects (6, 11, 16, 17,
33-36), elicited oscillatory currents with a threshold concentration
in the low micromolar range (Fig. 1).
Because this particular brand of SPC is only 85% pure, according to
the manufacturer, we also purchased SPC (nominal purity = 99%)
from Matreya Inc. Surprisingly, the Matreya brand of SPC was inactive
in the oocyte bioassay (Fig. 1). To eliminate batch-to-batch
variations, we tested multiple batches of SPC from Sigma (Fig. 1) and
Matreya. All Sigma SPC samples tested to date activated oscillatory
Cl
currents in the oocyte, whereas none of the Matreya
brand SPCs was active up to 100 µM (data not shown). Due
to the consistent differences between the two brands of SPC, HPLC was
applied to test the purity of the different SPCs and to determine
whether any difference in the L-threo- and
D-erythro stereoisomeric composition of the two
preparations could account for the difference in their biological
activity. Of the two SPC stereoisomers, we have previously shown that
only the D-erythro-SPC isomer (eSPC) activated
the sphingolipid receptors in atrial myocytes, whereas the
L-threo isomer was inactive (24). As shown in
Fig. 2, HPLC separation of the Sigma
brand SPC, using the solvent system originally developed for the
separation of SPC stereoisomers (24), showed several impurities that
eluted before D-erythro- (Rt = 32 min) and L-threo-SPC (Rt = 41 min). Bioassays of the 1-min fractions collected during the
elution showed that the compound(s) responsible for eliciting
oscillatory Cl
currents did not co-purify with either SPC
stereoisomer but did correspond to a small peak in the impurities that
eluted between 10 and 13 min.
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Elucidation of the Structure of RO56-- The ninhydrin and molybdenum blue staining of RO56 suggested that this lipid was a phospholipid with a free amino group, possibly with an ethanolamine moiety. Moreover, the poor yield of the acid methanolysis reaction was presumed to be due to RO56 being a plasmalogen-like substance. Because we had milligram amounts of RO56, we first focused on determining its structure.
A FTIR spectrum of RO56 was obtained in CHCl3: 732 cmHigh Yield Synthesis of the Active Compound Using Base Hydrolysis
of Alkenyl-GPE--
The vinyl-ether bond in alkenyl-GPE is unstable in
acid but is stable in base. Treatment of alkenyl-GPE with NaOH can
hydrolyze the ethanolamine moiety and generate alkenyl-GP
(2a and 2b, Fig.
4). A solution of alkenyl-GPE (15 mg, 33 µmol) in CHCl3, was dried with argon at room temperature,
and the resulting material was dissolved in 10 ml of MeOH saturated
with NaOH. The mixture was sealed in a pressure-resistant vial and was
heated to ~100 °C in an oil bath for 2 h with stirring. The
reaction mixture was allowed to cool to room temperature and was
neutralized with 1 N HCl in MeOH to pH ~7.5. The
precipitated NaCl was removed by filtration, and the filtrate was
concentrated at 30 °C on a rotary evaporator. The residual solid was
extracted (3 ×) with 5 ml of MeOH, and the combined extracts were
evaporated to ~0.5 ml with argon. The extract was assayed in the
oocyte bioassay and elicited oscillatory Cl currents (not
shown).
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Purification of the Active Compounds from the Alkenyl-GPE Hydrolysate-- The crude methanol extract of the hydrolysate, dissolved in 0.5 ml of MeOH, was applied to silica gel TLC plates, and the plate was developed for 1 h with solvent 3 (see "Materials and Methods"). Primuline-staining showed two major (Rf 0.31 and 0.42) and five minor bands (Rf values 0.61, 0.66, 0.85, 0.92, and 0.97). The bands were scraped off the plates, and the lipids were extracted with three washes of 3 ml of MeOH and concentrated to ~0.5 ml. The concentrated fractions were tested in the oocyte bioassay. Only two bands, migrating close to each other with Rf values of 0.31 and 0.42, showed activity (not shown). The overall yield of the active compounds was ~40%, which was substantially higher than the <1% yield of the acid methanolysis reaction. Retention factors for the two active compounds in the four different TLC solvent systems are shown in Table I.
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Structure Elucidation of the Active Compounds--
The two active
bands were separated a second time by TLC, which was ineffective in
completely separating the two because both compounds showed mutual
cross-contamination. Therefore, instrumental analysis was first
performed with a mixture of the two active compounds. FTIR spectrum of
the compounds in CHCl3: 1080 cm1 (P-O-C),
1216 cm
1 (=C-O, P=O), 1654 cm
1 (C=C), and
2550-3500 cm
1 (CH2, CH3, and
OH). 1H NMR in CD3OD:
0.89 (t,
J = 6.4 Hz, 3H), 1.15-1.50 (m, 24H), 1.91-2.19 (m,
4H), 3.70-3.95 (m, 5H), 4.19-4.34 (m, 1H), 5.20-5.41 (m, 1H), 5.99 (dt, J1 = 6.2 Hz, 1/2H, 2a), 6.03 (dt, J1 = 6.3 Hz, 1/2H, 2b).
13C NMR in CD3OD:
14.42, 23.71, 24.89 (24.92), 28.11 (28.14), 30.02, 30.31, 30.42, 30.59, 30.63, 30.78, 30.83, 30.95, 30.98, 32.92 (33.06), 60.63, 64.25 (d), 66.99 (d), 71.59 (d), 72.23 (d), 74.22, 75.10 (d), 107.51 (107.71), 130.83 (130.86), and
146.45 (146.54). 31P NMR in CD3OD appear at
26.00 and 26.32, respectively.
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Biological Activities of Alkenyl-GP-- In the oocyte bioassay alkenyl-GP had an apparent EC50 of 4.6 nM, whereas the apparent EC50 for LPA was 16.3 nM. (Fig. 6A). Heterologous desensitization analysis showed that LPA completely desensitized the alkenyl-GP response (Fig. 6B). In contrast, alkenyl-GP only partially desensitized the response to LPA (Fig. 6C) indicating that it was a receptor subtype-selective agonist.
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DISCUSSION |
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The present study has elucidated the structure of a novel LPA-like
autacoid, alkenyl-GP, which is present in the SPC from Sigma, a
preparation used by numerous investigators who report on the
overlapping biological responses between LPA and SPC. The manufacturer
carefully alerts the users that this particular product is only 85%
pure. Our experiments were prompted by our inability to reproduce
several published reports, using high purity SPC and SPP. For example,
in contrast to other investigators (14), we have not been able to
detect oscillatory Cl currents in oocytes elicited by
SPP, which was produced by PLD cleavage of SPC, nor have we been able
to detect these currents in response to pure SPC preparations up to 100 µM, except for the Sigma brand. Xu and co-workers (16,
17), using SPC from Sigma, showed heterologous desensitization between
LPA and SPC in ovarian and breast cancer cell lines and in Jurkat T
cells. These investigators, based on these cross-desensitization
studies, adapted the hypothesis originally put forward by Durieux and
colleagues (14) that glycerolipid and sphingolipid mediators were
acting on the same receptors that activate Ca2+
mobilization in different cell types. On the other hand, we have previously reported that in cardiac myocytes SPP and SPC elicited a
K+ current, with nanomolar EC50 concentrations,
through a receptor that was not activated by glycerolipids, including
LPA, phosphatidic acid, and lysophophatidylserine (24). Others (18, 39)
also could not verify cross-desensitization between SPP and LPA,
contradicting the hypothesis that LPA and sphingolipid mediators
activated the same putative receptor(s). Furthermore, recent reports on
the cloning of two LPA receptors found no evidence that these receptors were activated by SPP or SPC (40-42).
HPLC analysis of the Sigma brand SPC revealed that the LPA-like
biological activity responsible for eliciting oscillatory Cl currents in the Xenopus oocyte bioassay did
not co-purify with either one of the previously identified
D-erythro- or L-threo-SPC compounds, but rather eluted as a distinct peak (Rt = 11.5 min). The low (5-8%) abundance of this impurity initially did
not permit the isolation of sufficient quantities for instrumental chemical analysis other than FAB-MS. Nonetheless, FAB-MS revealed that
the active compound contained two major deprotonated molecule anions,
(M
H)
, at m/z 419 and 421, which were
different from the corresponding ions and fragments of SPC and SPP.
Moreover, the active impurity did not stain with ninhydrin, suggesting
that it did not contain a sphingoid base.
These observations prompted us to analyze the manufacturing process in which SPC is produced by Sigma. The acidic methanolysis procedure, in the presence of dilute HCl, yielded the active compound only if we used SM from Sigma; the hydrolysates prepared from SM from two other commercial sources did not generate the active compound. Importantly, when the acidic methanolysis reaction was performed without H2O, no detectable amounts of the active compound were found (data not shown). We speculate that a small amount of alkenyl-GP survives in the dilute aqueous-methanolic solution of HCl (~1 N) used in the manufacturing process, probably due to an equilibrium between the product and its precursor. Considering the 4.6 nM EC50 of alkenyl-GP, such a trace amount, present in the reaction mixture, could easily explain the biological activity. In stronger acid, alkenyl-GP completely decomposed, and therefore, no activity was observed. This scheme also indicates that there was no trace alkyl-GP in the starting material, as it would have survived due to its stability in strong acid.
Acidic methanolysis of the crude phospholipid fraction formed the active compound(s); therefore, we focused our attention on the possibility that other non-sphingolipid contaminants present in SM could give rise to the active compound(s). We analyzed the manufacturing process of SM and identified a ninhydrin-positive phospholipid that co-purifies with SM, from which it could only be removed by careful low pressure chromatography. This previously unidentified lipid, designated RO56 by Matreya researchers, migrated as a single spot in basic or acidic TLC solvents, and when exposed to acidic methanolysis, it became active in the oocyte bioassay. The very low yield of the acidic methanolysis reaction, the positive staining of the RO56 precursor with ninhydrin, together with other reports on the presence of plasmalogens in the base hydrolysates of the Folch extracts used in the manufacturing of SM, suggested the possibility that RO56 might be a plasmalogen.
NMR, FTIR, and MS were used to elucidate the structure of RO56. These techniques verified our hypothesis that RO56 was alkenyl-GPE. This conclusion was based on the identification of all the functional groups characteristic of the plasmalogen structure, particularly the cis-vinyl-ether bond. We also fulfilled the requirements for the proof of the chemical structure of plasmalogens put forward by Snyder and co-workers (43), using enzymatic and chemical modifications. Establishing that RO56 was alkenyl-GPE allowed us to develop a hypothetical scheme for the reactions that could lead to the generation of alkenyl-GP through the base hydrolysis of the ethanolamine moiety (Fig. 5).
To overcome the degradation of the vinyl-ether group under acidic conditions, and in order to synthesize a sufficient amount of alkenyl-GP for structural determination, we turned to basic hydrolysis conditions. We found that the yield of the basic reaction was at least 40 times greater than that of the acidic hydrolysis. However, TLC analysis of the basic hydrolysate of alkenyl-GPE revealed the generation of a second active lipid that migrated slightly behind (Rf 0.31; solvent 3) the active compound present in the acidic hydrolysate (Rf 0.42, solvent 3). The FAB-MS spectra of the two compounds, which showed identical molecule ions and fragmentation patterns, suggested that these compounds were regioisomers. 1H and 13C NMR spectra of the partially separated active lipids verified that these compounds were regioisomers of alkenyl-GP. By using the ACD computer program to model the NMR spectra of the two regioisomers, we found that the experimental data were consistent with the 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (2a) being more abundant (~80%) in the faster migrating band with the vinyl-ether hydrogen chemical shift at 5.99 ppm. In contrast, the compound in the slower migrating TLC band showed the abundance of the 1-O-cis-alk-1'-enyl-3-lyso-sn-glycero-2-phosphate (2b) regioisomer with the vinyl-ether hydrogen chemical shift at 6.03 ppm. The generation of regioisomers during base-catalyzed hydrolysis of phospholipids has been previously reported, and a scheme similar to the one proposed (Fig. 5) has been proposed by Hanahan et al. (44) for the base hydrolysis of platelet-activating factor. Because no significant differences were found in the biological activity of the two partially pure (~70-80%) regioisomers, we concluded that they both were biologically active, which underlines that phosphomonoesters at the sn-2 and sn-3 position both can activate APA receptors. Heterologous desensitization analysis in the oocyte indicated that alkenyl-GP activates only distinct subtypes of the APA receptors.
LPA is a potent mitogen in several cell types (45) but is antimitogenic in others (27, 46). The mitogenic response elicited by LPA has been linked to the activation of the Ras-ERK-signaling pathway (47, 48). SPC has also been shown to be mitogenic in Swiss 3T3 fibroblasts (6), and its mitogenic activity has also been linked to the activation of the ERKs (36). Alkenyl-GP, similar to LPA, activated the ERK1 and ERK2 protein kinases in Swiss 3T3 cells. Purified eSPC had only a marginal effect on the activation of ERK1 and ERK2. When added together with alkenyl-GP, eSPC showed an additive effect in activating ERKs. These results are in agreement with the report by Seufferlein and Rozengurt (36); however, those investigators used SPC from Sigma. By using peak integration for the impurity present in the Sigma SPC, we estimate that it constitutes ~5-8% of the total material. This amount of alkenyl-GP could account for the activation of ERKs reported by Seufferlein and Rozengurt (36).
LPA has generated considerable interest because of its mitogenic effect in a variety of cell types. We tested the purified, biologically active stereoisomer e-SPC, Sigma SPC, alkenyl-GP, and LPA for their effect on the proliferation of Swiss 3T3 cells, which is the cell line that has been used by other investigators who report on the LPA-like actions of SPC. Alkenyl-GP, similar to LPA, was highly mitogenic at micromolar concentrations in this cell line. Interestingly, eSPC alone was weakly mitogenic in our hands; however, when applied together with alkenyl-GP, it enhanced the mitogenic effect of alkenyl-GP. Sigma SPC, consistent with the presence of the contaminating alkenyl-GP impurity, was more potent than eSPC in causing mitogenesis.
Alkenyl-GP is a novel member of the APA family and is derived from a different group of lipids than LPA, which is the plasmalogens. Alkenyl-GP has been recently identified among a group of naturally occurring mitogenic lipids generated in response to injury (19). Plasmalogens, the precursors of alkenyl-GP, are abundant in certain tissues (49), particularly in the heart (50) and in the brain, where it constitutes over 32% of the myelin phospholipids (51). Enzymes capable of specifically cleaving the ethanolamine and choline substituents of the phosphate moiety of plasmalogens have also been identified in the brain (52) and other tissues (53). A lysoplasmalogenase enzyme (EC 3.3.2.2 and 3.3.2.5), specifically catalyzing the hydrolysis of the vinyl-ether bond of plasmalogens, has also been described in the brain (54). However, preliminary data indicate that alkenyl-GP is not cleaved by this enzyme2; the possibility that it could have a sufficiently long half-life in biological fluids is supported by its ~5 µM in vivo steady-state concentration in the anterior chamber fluid of the eye (19). A more detailed analysis of the synthesis and degradation of alkenyl-GP will be necessary in forthcoming studies.
Our data underline the utmost importance for the use of analytically pure reagents, and thus it appears necessary to re-evaluate the previous reports on the different actions of SPC because of the likely presence of alkenyl-GP, which could have led to mixed biological actions. For these experiments, chemical and regioisomer pure compounds must be used. At this stage of the research, we believe that the best method to resolve the controversy of whether APA receptors are activated by sphingolipids is to use the heterologous expression of the already cloned LPA receptor subtypes in a host cell that does not express endogenous receptors. Thus far, there is only one system that fulfills this criterion, the yeast expression system described by Erickson and colleagues (42). In this system SPP did not activate the Edg-2 receptor (42). However, this system has two potential shortfalls. It requires high micromolar concentrations of LPA to activate a reporter gene, which is much higher than the nanomolar concentration required for mammalian cells; and it only works for those LPA receptor types that can efficiently couple to the yeast mating pheromone pathway. A more detailed characterization of the receptor subtype-specific action of alkenyl-GP is provided elsewhere (55).
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ACKNOWLEDGEMENTS |
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We thank Dr. Ten-ching Lee (Oak Ridge Associated Universities) for suggestions and insightful comments; Dr. Alicja Bielawska (Duke University) for assistance collecting NMR spectra during the initial phases of the project; and Dr. Greg Wall (Sigma) for the support. We thank to Alina West and David Smith for the expert technical help.
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FOOTNOTES |
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* This work was supported in part by Grant IBN-9722969 from the National Science Foundation (to G. T.) and LXR Biotechnology Inc.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.
c Recipients of a postdoctoral fellowship from the Center for Neuroscience at the University of Tennessee, Memphis.
d Fellow of the American Heart Association Tennessee Affiliate and a recipient of National Institutes of Health Training Grant USPHS-HL07746.
k Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, College of Medicine, University of Tennessee, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-4793; Fax: 901-448-7126; E-mail: gtigyi{at}physio1.utmem.edu.
1 The abbreviations used are: LPA, lysophosphatidic acid; SPP, sphingosine 1-phosphate; SPC, sphingosylphosphorylcholine; alkenyl-GP, 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate; ERK, extracellular signal-regulated protein kinase(s); BuOH, butanol; HAc, glacial acetic acid; MeOH, methanol; MAP, mitogen-activated protein; SPC, sphingosylphosphorylcholine; FTIR, Fourier transform infrared spectrometry; MS, mass spectrometry; FAB-MS, fast atom bombardment-mass spectrometry; SM, sphingomyelin; alkenyl-GPE, ethanolamine-containing lysoplasmalogen, 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine; MBP, myelin basic protein; HPLC, high pressure liquid chromatography; AP, alkaline phosphatase; eSPC, D-erythro-SPC.
2 M. A. Jurkowitz-Alexander, personal communication.
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