Identification of a Novel Growth Factor-like Lipid, 1-O-cis-Alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (Alkenyl-GP) That Is Present in Commercial Sphingolipid Preparations
1998; Elsevier BV; Volume: 273; Issue: 22 Linguagem: Inglês
10.1074/jbc.273.22.13461
ISSN1083-351X
AutoresKároly Liliom, David J. Fischer, TamaÖs ViraÖg, Guoping Sun, Duane D. Miller, Jih-Lie Tseng, Dominic M. Desiderio, Michael Seidel, James R. Erickson, Gábor Tigyi,
Tópico(s)Cellular transport and secretion
ResumoLysophosphatidic 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 and13C 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 μmwas 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. 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 and13C 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 μmwas 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. The autacoids, 1-oleoyl-2-lyso-sn-glycero-3-phosphate (lysophosphatidic acid, LPA), 1The 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. 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. 1Tokumura A. Prog. Lipid Res. 1995; 34: 151-184Crossref PubMed Scopus (166) Google Scholar and 2Spiegel S. Merril A.H.J. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (650) Google Scholar). Perhaps the three most interesting cellular responses elicited by these autacoids include the following: mitogenesis/antimitogenesis (3van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (681) Google Scholar, 4Tigyi G. Dyer D. Miledi R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1908-1912Crossref PubMed Scopus (146) Google Scholar, 5Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (815) Google Scholar, 6Desai N.N. Spiegel S. Biochem. Biophys. Res. Commun. 1991; 181: 361-366Crossref PubMed Scopus (105) Google Scholar), effect on cell shape and motility (7Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar, 8Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3831) Google Scholar, 9Imamura F. Horai T. Mukai M. Shinkai K. Sawada M. Akedo H. Biochem. Biophys. Res. Commun. 1993; 193: 497-503Crossref PubMed Scopus (176) Google Scholar, 10Sadahira Y. Ruan F. Hakomori S. Igarashi Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9686-9690Crossref PubMed Scopus (237) Google Scholar, 11Seufferlein T. Rozengurt E. J. Biol. Chem. 1995; 270: 24343-24351Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), and anti-apoptotic action (12Umansky S.R. Shapiro J.P. Cuenco G.M. Foehr W.M. Bathurst I.C. Tomei L.D. Cell Death Differ. 1997; 4: 608-616Crossref PubMed Scopus (40) Google Scholar, 13Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Cosos O.A. Gutkind J.S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1350) Google Scholar). 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 (14Durieux M.E. Carlisle S.J. Salafranca M.N. Lynch K.R. Am. J. Physiol. 1993; 264: C1360-C1364Crossref PubMed Google Scholar,15Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Abstract Full Text PDF PubMed Scopus (108) Google Scholar). 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 (16Xu Y. Fang X.J. Casey G. Mills G.B. Biochem. J. 1995; 309: 933-940Crossref PubMed Scopus (252) Google Scholar, 17Xu Y. Casey G. Mills G.B. J. Cell. Physiol. 1995; 163: 441-450Crossref PubMed Scopus (78) Google Scholar). 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 (18Jalink K. Hengeveld T. Mulder S. Postma F.R. Simon M.F. Chap H. van der Marel G.A. van Boom J.H. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1995; 307: 609-616Crossref PubMed Scopus (112) Google Scholar). 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 (19Liliom K. Guan Z. Tseng H. Desiderio D.M. Tigyi G. Watsky M. Am. J. Physiol. 1998; 274: C1065-C1074Crossref PubMed Google Scholar), suggesting that this novel lipid is a naturally occurring member of the acidic phospholipid autacoid (APA) family. 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 (20Ames B.N. Dubin R.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar). Acidic methanolysis was carried out according to Gaver and Sweeley (21Gaver R.C. Sweeley C.C. J. Am. Oil Chem. Soc. 1965; 42: 294-298Crossref PubMed Scopus (474) Google Scholar). 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. The lower phase (60 g) of the Folch (22Folch J. Lees M. Sloane-Stanley G.A. J. Biol. Chem. 1957; 226: 497-502Abstract Full Text PDF PubMed Google Scholar) extract from bovine brain was dissolved in 500 ml of 0.5 mmethanolic 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 nHCl, 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. 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 (23Wright R.S. J. Chromatogr. 1971; 59: 220-221Crossref PubMed Scopus (141) Google Scholar) 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. (24Buenemann M. Brandts B.K. Pott L. Liliom K. Tseng J.-L. Desiderio D.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5524-5537Google Scholar), 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. 1H, 13C, and31P NMR spectra were recorded with a Bruker model 300 MHz ARX (Brueker Instruments Inc., Billerica, MA) spectrometer.1H chemical shifts (δ in ppm) were calibrated against CD3OD at δ = 3.30 ppm and 13C chemical shifts against CD3OD at δ = 49.00 ppm. 31P chemical shifts were calibrated against 0.0485 m triphenylphosphate in CDCl3 at δ = 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. Alkenyl-GP was prepared by a two-step procedure, using PLD followed by PLA2digestion 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 0.2m acetate diethyl ether buffer, pH 5.6, according to Kates (25Kates M. Burdon R.H. van Knippenberg P.H. Techniques in Lipidology. 2nd Ed. Elsevier Science Publishers B.V., Amsterdam1986: 405-409Google Scholar). 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 thesn-2 position was carried out in a non-aqueous medium, according to Kates (25Kates M. Burdon R.H. van Knippenberg P.H. Techniques in Lipidology. 2nd Ed. Elsevier Science Publishers B.V., Amsterdam1986: 405-409Google Scholar). 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. 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 (26Blank M.L. Snyder F. Biochemistry. 1970; 9: 5034-5036Crossref PubMed Scopus (31) Google Scholar). Control reactions received equal amounts of heat-inactivated enzyme. After 2 h, the reaction was terminated, and the lipids were extracted (37Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 31: 911-917Crossref Scopus (42865) Google Scholar). 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 N2at 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. 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 (33Desai N.N. Carlson R.O. Mattie M.E. Olivera A. Buckley N.E. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1993; 121: 1385-1395Crossref PubMed Scopus (115) Google Scholar). 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). 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 (6Desai N.N. Spiegel S. Biochem. Biophys. Res. Commun. 1991; 181: 361-366Crossref PubMed Scopus (105) Google Scholar). 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 μmfinal 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 (27Tigyi G. Dyer D.L. Miledi R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1908-1912Crossref PubMed Google Scholar). The combined activity of the ERK1 and ERK2 was determined by a method described elsewhere (28Cook S.J. Beltman J. Cadwallader K.A. McMahon M. McCormick F. J. Biol. Chem. 1997; 272: 13309-13319Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29Cadwallader K. Beltman J. McCormick F. Cook S. Biochem. J. 1997; 321: 795-804Crossref PubMed Scopus (48) Google Scholar). 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 (30Bradford M.A. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216391) Google Scholar). 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 mmMgCl2, 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 [γ-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 (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar), 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. 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 (7Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar,32Tigyi G. Dyer D. Matute C. Miledi R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1521-1525Crossref PubMed Scopus (81) Google Scholar). 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 (6Desai N.N. Spiegel S. Biochem. Biophys. Res. Commun. 1991; 181: 361-366Crossref PubMed Scopus (105) Google Scholar, 11Seufferlein T. Rozengurt E. J. Biol. Chem. 1995; 270: 24343-24351Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Xu Y. Fang X.J. Casey G. Mills G.B. Biochem. J. 1995; 309: 933-940Crossref PubMed Scopus (252) Google Scholar, 17Xu Y. Casey G. Mills G.B. J. Cell. Physiol. 1995; 163: 441-450Crossref PubMed Scopus (78) Google Scholar,33Desai N.N. Carlson R.O. Mattie M.E. Olivera A. Buckley N.E. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1993; 121: 1385-1395Crossref PubMed Scopus (115) Google Scholar, 34Pushkareva M. Hannun Y.A. Biochim. Biophys. Acta. 1994; 1221: 54-60Crossref PubMed Scopus (14) Google Scholar, 35Berger A. Rosenthal D. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5885-5889Crossref PubMed Scopus (48) Google Scholar, 36Seufferlein T. Rozengurt E. J. Biol. Chem. 1995; 270: 24334-24342Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), 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- andd-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 thel-threo isomer was inactive (24Buenemann M. Brandts B.K. Pott L. Liliom K. Tseng J.-L. Desiderio D.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5524-5537Google Scholar). As shown in Fig. 2, HPLC separation of the Sigma brand SPC, using the solvent system originally developed for the separation of SPC stereoisomers (24Buenemann M. Brandts B.K. Pott L. Liliom K. Tseng J.-L. Desiderio D.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5524-5537Google Scholar), showed several impurities that eluted before d-erythro- (R t= 32 min) and l-threo-SPC (R t= 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.Figure 2HPLC purification of Sigma brand SPC.SPC (1 mg) was injected onto a 250 × 4.6-mm silica analytical column and was eluted using a gradient described by Buenemann et al. (24Buenemann M. Brandts B.K. Pott L. Liliom K. Tseng J.-L. Desiderio D.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5524-5537Google Scholar). This method has been used to separated-erythro-SPC (R t ~32 min) from the biologically inactive stereoisomer,l-threo-SPC (R t ~41 min). The eluting material was monitored by an evaporative light-scattering detector through a metering valve that split the effluent in a 4:1 ratio between a fraction collector and the detector, respectively. Fractions (1 min) were collected and were tested in the oocyte bioassay at 100-fold dilution. The compound(s) that activated oscillatory Cl− currents eluted with the impurities with an apparent peak retention time of ~11.5 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both companies produce SPC by the acidic methanolysis of SM; this process can lead to the generation of several by-products besides SPC, depending on the conditions of the hydrolysis reaction and on the amount of water included in the reaction mixture (21Gaver R.C. Sweeley C.C. J. Am. Oil Chem. Soc. 1965; 42: 294-298Crossref PubMed Scopus (474) Google Scholar). Purified SM and brain-derived phospholipids were obtained from Sigma, Matreya, and Avanti Polar Lipids and were subjected to acid semi-aqueous methanolysis using the conditions originally described by Gaver and Sweely (21Gaver R.C. Sweeley C.C. J. Am. Oil Chem. Soc. 1965; 42: 294-298Crossref PubMed Scopus (474) Google Scholar) that are used to manufacture SPC by Sigma. After an 18-h aqueous acidic hydrolysis, the reaction mixtures were neutralized, and SPC was extracted according to Bligh and Dyer (37Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 31: 911-917Crossref Scopus (42865) Google Scholar). The extracted lipids were dissolved in methanol and were tested in the oocyte bioassay for the generation of compounds capable of eliciting oscillatory Cl− currents. None of the SM preparations from the different suppliers elicited oscillatory Cl− currents in the oocyte. However, only the hydrolysate from the Sigma brand SM elicited oscillatory Cl− currents (data not shown). Although the brain phospholipid extracts contained traces of biological activity, after 18 h of acidic hydrolysis all brain phospholipid hydrolysates were highly active and contained titers of the active compound(s) that were substantially (~10-fold) higher than that titer prior to hydrolysis. These observations suggested that a contaminating phospholipid precursor might be present in the Sigma SM, which, upon acid methanolysis, would lead to the generation of the active compound(s). To test this hypothesis, we turned our attention to a previously unidentified lipid fraction that co-elutes with SM in the manufacturing process. This lipid, which is removed by careful low pressure chromatography and cold acetone precipitation from the SM fraction in the manufacturing process by Matreya, could potentially contaminate the Sigma brand SM. This particular fraction, designated RO56 by Matreya, migrated as a single spot (R f 0.36) ahead of SM, when developed by TLC solvent 1, and stained positive with ninhydrin and molybdenum blue (Fig.3 A). Acid methanolysis of RO56, carried out the same way as in the manufacturing of SPC, led to the generation of an even faster migrating product (R f 0.42) that was no longer positive with ninhydrin but remained positive with molybdenum blue (Fig. 3 B). The bioassay of RO56 showed that the R f 0.36 compound was inactive, whereas the hydrolysate was highly potent in activating oscillatory Cl− currents (Fig. 3 C). A major problem of this reaction was the low yield (<1%) of the active compound; this prevented the collection of a sufficient quantity of the active material for IR and NMR analysis. However, the FAB−-MS spectra from the active lipid isolated from the hydrolysate of RO56 and of that lipid found in the active fraction isolated by HPLC from the Sigma SPC were both identical, which showed two deprotonated molecular anions, (M − H)−, atm/z 419 and 421, respectively. 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 cm−1 (cis-CH=CH-), 1076 cm−1(P-O-C), 1222 cm−1 (=CH-O, P=O), 1664 cm−1(C=C), 2550–3500 cm−1 (CH2, CH3, and NH3+). 1H NMR in CD3OD: δ 0.89 (t, J = 6.7 Hz, 3H), 1.20–1.45 (m, 30H), 1.90–2.20 (m, 4H), 3.13–3.16 (t,J = 4.9 Hz, 2H), 3.65–3.80 (m, 2H), 3.82–3.98 (m, 4H), 4.00–4.10 (m, 2H), 4.31 (dt, J 1 = 6.3 Hz;J 2 = 7.3 Hz, 1H), 5.25–5.41 (m, 1H), 5.99 (dt,J 1 = 6.2 Hz, J 2 = 1.4 Hz, 1H). 1H NMR of RO56 in CD3OD showed the presence of a vinyl-ether linkage, on which the two hydrogen atoms are in a cis-configuration (δ 5.99 ppm,J 1 = 6.2 Hz) since the trans-isomer would have a coupling constant greater than 10 Hz. The 1H NMR also showed the presence of an ethanolamine moiety (3.13–3.16 and 4.00–4.10 ppm in CD3OD). 13C NMR in CD3OD: δ 14.42, 23.73, 24.94, 28.12, 30.04, 30.33, 30.44, 30.65, 30.69, 30.79, 30.84, 30.97, 32.93, 33.07, 41.67 (d, J = 6.6 Hz), 62.91 (d,J = 5.2 Hz), 67.91 (d, J = 5.8 Hz), 70.95 (d, J = 7.6 Hz), 73.90, 108.01, 130.85, and 146.33. 13C NMR of RO56 in CD3OD confirmed the presence of the vinyl-ether linkage (δ 130.85 and 146.33 ppm). A single peak at 108.01 ppm indicated the existence of a second double bond in the hydrocarbon chain, which was non-conjugated with the vinyl-ether bond. The lack of a peak above 160 ppm confirms the absence of carbonyl groups in the molecule; that conclusion is consistent with the FTIR data. 31P NMR of RO56 in CD3OD appears at δ 18.17. FAB MS spectra (R = C8H17for each compound). Negative ion mode: (M − H)− atm/z 464 (Δ1, n = 4), 462 (Δ1,9, n = 4), and 436 (Δ1,n = 2). Positive ion mode: (M + H)+ atm/z 466 (Δ1, n = 4) and 464 (Δ1,9, n = 4), (M + Na)+ atm/z 486 (Δ1,9, n = 4) and 432 (Δ1, n = 0). Based on these analytic
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