A Novel Phosphatidic Acid-selective Phospholipase A1That Produces Lysophosphatidic Acid
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m201659200
ISSN1083-351X
AutoresHirofumi Sonoda, Junken Aoki, Tatsufumi Hiramatsu, Mayuko Ishida, Koji Bandoh, Yuki Nagai, Ryo Taguchi, Keizo Inoue, Hiroyuki Arai,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoLysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A1 (PLA1) that produces 2-acyl-LPA. The PLA1 was identified in the GenBankTM data base as a close homologue of phosphatidylserine (PS)-specific PLA1(PS-PLA1). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA3, a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA1 and suggest that it has a role in LPA production. Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A1 (PLA1) that produces 2-acyl-LPA. The PLA1 was identified in the GenBankTM data base as a close homologue of phosphatidylserine (PS)-specific PLA1(PS-PLA1). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA3, a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA1 and suggest that it has a role in LPA production. lysophosphatidic acid phosphatidic acid phospholipase A1 membrane-associated PA-selective PLA1 phosphatidylserine PS-specific PLA1 phospholipase A2 type IIA secretory PLA2 phospholipase D phosphatidylcholine phosphatidylethanolamine lysophosphatidylserine lysophosphatidylcholine lysophosphatidylethanolamine lysophosphatidylinositol endothelial differentiation gene expressed sequence tags bovine serum albumin open reading frame glyceraldehyde-3-phosphate dehydrogenase standard saline citrate concentration of intracellular calcium ion electrospray ionization mass spectrometry wild type reverse transcription 4-morpholineethanesulfonic acid mass spectrometry phorbol 12-myristate 13-acetate cyclic PA novel PLA1 Lysophosphatidic acid (1- or 2-acyl-lysophosphatidic acid; LPA)1 is a lipid mediator with multiple biological functions (1Tokumura A. Prog. Lipid Res. 1995; 34: 151-184Crossref PubMed Scopus (165) Google Scholar, 2Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 3Moolenaar W.H. Exp. Cell Res. 1999; 253: 230-238Crossref PubMed Scopus (371) Google Scholar). These include induction of platelet aggregation, smooth muscle contraction, and stimulation of cell proliferation. LPA also promotes specific responses of the cytoskeleton such as generation of actin stress fibers in fibroblasts or inhibition of neurite outgrowth in neuronal cells. LPA evokes its multiple effects through G-protein-coupled receptors that are specific to LPA (see below), with consequent activation of phospholipase C (PLC) and phospholipase D (PLD), Ca2+ mobilization, inhibition of adenylyl cyclase, activation of mitogen-activated protein kinase, and transcription of serum-response-element transcriptional reporter genes, such as c-fos. Recent studies (4Chun J. Contos J.J. Munroe D. Cell Biochem. Biophys. 1999; 30: 213-242Crossref PubMed Scopus (138) Google Scholar, 5Contos J.J. Ishii I. Chun J. Mol. Pharmacol. 2000; 58: 1188-1196Crossref PubMed Scopus (366) Google Scholar) have identified a new family of receptor genes for LPA. Members of this family include three G-protein-coupled receptors belonging to the endothelial differentiation gene (EDG) family, EDG2/LPA1 (6Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar), EDG4/LPA2 (7An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar), and EDG7/LPA3 (8Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murotushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). These proteins may explain various cellular responses to LPA (6Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar, 7An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 8Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murotushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar).In contrast to the signal transduction mediated by LPA receptors, the molecular mechanisms for LPA production are poorly understood. LPA is produced both in biological fluids such as serum (9Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar) and in various cells such as platelets (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (177) Google Scholar, 11Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (573) Google Scholar) and ovarian cancer cells (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 13Eder A. Sasagawa T. Mao M. Aoki J. Mills G. Clin. Cancer Res. 2000; 6: 2482-2491PubMed Google Scholar). In these latter studies, it was speculated that LPA is produced by phospholipase A2 (PLA2) from phosphatidic acid (PA) that is generated as a result of PLD activation (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 13Eder A. Sasagawa T. Mao M. Aoki J. Mills G. Clin. Cancer Res. 2000; 6: 2482-2491PubMed Google Scholar). Tokumuraet al. (14Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar) demonstrated that LPA is also produced in plasma from lysophosphatidylcholine (LPC) by the action of lysophospholipase D, which may account for the accumulation of LPA in aged plasma.LPA, with various fatty acid species, has been detected in several biological systems. For example, human serum contains LPA with both saturated (16:0 and 18:0) and unsaturated (16:1, 18:1, 18:2, and 20:4) fatty acids (15Baker D.L. Desiderio D.M. Miller D.D. Tolley B. Tigyi G.J. Anal. Biochem. 2001; 292: 287-295Crossref PubMed Scopus (197) Google Scholar). A similar LPA species was detected in human platelets (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (177) Google Scholar). The activity of LPA has been shown to be modulated by the length, degree of unsaturation, and linkage to the glycerol backbone of the fatty acyl chain (16Sugiura T. Tokumura A. Gregory L. Nouchi T. Weintraub S.T. Hanahan D.J. Arch. Biochem. Biophys. 1994; 311: 358-368Crossref PubMed Scopus (84) Google Scholar, 17Gueguen G. Gaige B. Grevy J.M. Rogalle P. Bellan J. Wilson M. Klaebe A. Pont F. Simon M.F. Chap H. Biochemistry. 1999; 38: 8440-8450Crossref PubMed Scopus (75) Google Scholar, 18van Corven E.J. van Rijswijk A. Jalink K. van der Bend R.L. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1992; 281: 163-169Crossref PubMed Scopus (236) Google Scholar, 19Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Am. J. Physiol. 1994; 267: C204-C210Crossref PubMed Google Scholar, 20Perkins L.M. Ramirez F.E. Kumar C.C. Thomson F.J. Clark M.A. Nucleic Acids Res. 1994; 22: 450-452Crossref PubMed Scopus (14) Google Scholar, 21Tokumura A. Fukuzawa K. Tsukatani H. Lipids. 1978; 13: 572-574Crossref PubMed Scopus (140) Google Scholar). Of particular interest is the detection of 2-linoleoyl-LPA in ascites from ovarian cancer patients, which may account for the increased ability of the ascites to activate the growth of ovarian cancer cell lines (22Xu Y. Gaudette D.C. Boynton J.D. Frankel A. Fang X.J. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Holub B.J. Mills G.B. Clin. Cancer Res. 1995; 1: 1223-1232PubMed Google Scholar). We recently identified a novel LPA receptor, EDG7/LPA3, which shows a relatively high affinity for 2-acyl-LPA with unsaturated fatty acid (8Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murotushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 23Bandoh K. Aoki J. Taiva A. Tsujimoto M. Arai H. Inoue K. FEBS Lett. 2000; 478: 159-165Crossref PubMed Scopus (221) Google Scholar). It is generally accepted that the sn-1 position of glycerophospholipids is occupied by saturated fatty acids, whereas the sn-2 position is occupied by unsaturated fatty acids. This suggests that phospholipase A1 (PLA1) as well as PLA2 are involved in LPA production.PLA1 enzymes hydrolyze the sn-1 fatty acids from phospholipids. Although PLA1 activities are detected in many tissues and cell lines, a limited number of PLA1s have been purified and cloned. We have purified and cloned a cDNA for phosphatidylserine-specific PLA1 (PS-PLA1), a member of the lipase family, from the culture medium of activated rat platelets. PS-PLA1 specifically hydrolyzes PS (24Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and produces 2-acyl-lysophosphatidylserine (LPS), which is a lipid mediator for mast cells (25Hosono H. Aoki J. Nagai Y. Bandoh K. Ishida M. Taguchi R. Arai H. Inoue K. J. Biol. Chem. 2001; 276: 29664-29670Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), T cells (26Bellini F. Bruni A. FEBS Lett. 1993; 316: 1-4Crossref PubMed Scopus (62) Google Scholar), and neural cells (27Lourenssen S. Blennerhassett M.G. Neurosci. Lett. 1998; 248: 77-80Crossref PubMed Scopus (42) Google Scholar). We recently showed that PS-PLA1 stimulates histamine release from rat peritoneal mast cells by hydrolyzing PS exposed on the surface of some cell types such as apoptotic cells and activated platelets (25Hosono H. Aoki J. Nagai Y. Bandoh K. Ishida M. Taguchi R. Arai H. Inoue K. J. Biol. Chem. 2001; 276: 29664-29670Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Accordingly we searched GenBankTM for sequences similar to PS-PLA1 and found one PS-PLA1homologue. Here we demonstrate that the PS-PLA1 homologue is a membrane-associated PA-selective PLA1(mPA-PLA1) that can produce a bioactive lysophospholipid, 2-acyl-LPA, by hydrolyzing PA generated by PLD.DISCUSSIONThe metabolic pathways for LPA synthesis are currently poorly understood, and at least three pathways have been postulated. In the first pathway, LPA is converted from PA by PLA1 or PLA2, which has been observed to occur in erythrocytes and ovarian cancer cells (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 13Eder A. Sasagawa T. Mao M. Aoki J. Mills G. Clin. Cancer Res. 2000; 6: 2482-2491PubMed Google Scholar, 33Fourcade O. Simon M.F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (491) Google Scholar). In the second pathway, which may occur in platelets, diacylglycerol produced by PLC, could be deacylated by diacylglycerol lipase, with the resulting monoacylglycerol being further phosphorylated into LPA (34Mauco G. Chap H. Simon M.F. Douste B.L. Biochimie (Paris). 1978; 60: 653-661Crossref PubMed Scopus (125) Google Scholar, 35Gaits F. Fourcade O., Le, B.F. Gueguen G. Gaige B. Gassama D.A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. FEBS Lett. 1997; 410: 54-58Crossref PubMed Scopus (147) Google Scholar). The third pathway involves lysophospholipase D acting on LPC in plasma and may explain the large accumulation of LPA in aged plasma (14Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar). A similar reaction may occur on the cell surface, in which LPC was converted to LPA by bacterial PLD (36van Dijk M.C. Postma F. Hilkmann H. Jalink K. van Blitterswijk W.J. Moolenaar W.H. Curr. Biol. 1998; 8: 386-392Abstract Full Text Full Text PDF PubMed Google Scholar). Enzymes involved in these processes of LPA synthesis have not been characterized fully. However, several PLA2 isoforms identified and characterized biochemically have been implicated in LPA production. For example, studies using inhibitors of PLA2isoforms have suggested that sPLA2-IB, Ca2+-independent PLA2, and cytosolic PLA2 were partially involved in the LPA production of ovarian cancer cells (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 13Eder A. Sasagawa T. Mao M. Aoki J. Mills G. Clin. Cancer Res. 2000; 6: 2482-2491PubMed Google Scholar). It was also proposed that sPLA2-IIA was able to produce LPA by hydrolyzing PA exposed on the cell surface after phospholipid scrambling (37le Balle F. Simon M.F. Meijer S. Fourcade O. Chap H. Adv. Enzyme Regul. 1999; 39: 275-284Crossref PubMed Scopus (24) Google Scholar) or by hydrolyzing PA on membrane microvesicles shed from erythrocytes (33Fourcade O. Simon M.F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (491) Google Scholar).The present investigation led to several interesting observations, allowing us to propose a role of a novel PLA1 molecule, mPA-PLA1, in LPA production. Our results from this study are as follows. (i) A low level of LPA that could activate EDG7 was continuously produced and released into the medium in Sf9-mPA-PLA1 cells. (ii) The production of LPA in Sf9-mPA-PLA1 cells was significantly increased after PLD administration (Figs. 6 and 7). (iii) The expression of mPA-PLA1 did not promote accumulation of any lysophospholipids including LPC, LPE, LPI, and LPS in the cells (Fig.4). (iv) We also observed that cPA was equally detected in the media from Sf9-mPA-PLA1, Sf9-WT, and Sf9-mutPLA1 cells only after the PLD treatment (Fig.7). The bacterial PLD (from Actinomadura) used in this study converts lysophospholipids (LPC, LPE, LPS, and LPI) to cPA but not to LPA. 2T. Kobayashi, Ochanomizu University, personal communication. All these results clearly indicate that mPA-PLA1 produces LPA by hydrolyzing PA. We could not detect PLA1 activity of mPA-PLA1 toward exogenously added PA liposome using a conventional assay for PLA1 or A2. It can be speculated that the availability of exogenous substrate to the enzyme is limited, as mPA-PLA1 is tightly associated with membrane phospholipids. mPA-PLA1 may hydrolyze such phospholipids, which surround the enzyme on the plasma membrane, after the phospholipids are converted to PA.PA is a very minor component of phospholipids in mammalian cells and also in Sf9 cells (38Marheineke K. Grunewald S. Christie W. Reilander H. FEBS Lett. 1998; 441: 49-52Crossref PubMed Scopus (120) Google Scholar). This is consistent with the result that the LPA level was very low under normal conditions (Figs. 5 and 6). It is thus reasonable to assume that the rate-limiting step for LPA production in this pathway is generation of PA. PA could be generated by PLD or sequentially by PLC and diacylglycerol kinase. We observed that exogenously added PLD strongly promoted the production of LPA (Figs. 6 and 7) and that PMA-stimulated production of LPA was suppressed by a PLD inhibitor, 1-butanol (Fig. 5). Thus, it is likely that PLD is involved in the production of LPA mediated by mPA-PLA1. In mammalian cells, the molecular identities of the two isozymes of PLD, PLD1 and PLD2, have been elucidated. Among these two isozymes, PLD1 is activated by PMA both in vivo and in vitrothrough an activation of protein kinase Cα (39Min D.S. Park S.K. Exton J.H. J. Biol. Chem. 1998; 273: 7044-7051Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although information about PLD isozyme(s) in Sf9 insect cells is limited (40Hoer A. Schoneberg T. Harteneck C. Cetindag C. Oberdisse E. Biochim. Biophys. Acta. 1998; 1393: 325-335Crossref PubMed Scopus (2) Google Scholar), the observation that PMA stimulated LPA formation in Sf9-mPA-PLA1 cells (Fig. 5) suggests an involvement of a PLD1-like molecule in the insect cells. Consistent with this, it is reported by Shen et al. (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar) that LPA is produced and secreted from ovarian cancer cells after they were treated with PMA.LPA produced by mPA-PLA1 in Sf9 cells was rich in oleic acid (18:1) and palmitoleic acid (16:1) (Fig. 7). Marheinekeet al. (38Marheineke K. Grunewald S. Christie W. Reilander H. FEBS Lett. 1998; 441: 49-52Crossref PubMed Scopus (120) Google Scholar) reported that the major fatty acids in the phospholipids from Sf9 cells were oleic acid, palmitoleic acid, and stearic acid (18:0), with a small amount of palmitic acid (16:0). This explains why LPA with linoleic acid (18:2) and arachidonic acid (20:4), which are the major fatty acids at the sn-2 position of phospholipids of mammalian cells, was not detected. We observed that mPA-PLA1 is abundantly expressed in human platelets that have been characterized well as LPA-producing cells (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (177) Google Scholar, 11Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (573) Google Scholar). In activated platelet, LPA with both saturated (16:0, 18:0) and unsaturated 16:1, 18:1, 18:2, and 20:4 has been detected. This suggests that both PLA1 and PLA2 isozymes are involved in the LPA production in the cells.Although it is possible that EDG7 is activated by an entity other than LPA, this seems unlikely for two reasons. First, the amount of LPA in the conditioned medium of Sf9-mPA-PLA1 cells treated with PLD is ∼5 μm based on the MS analysis (Fig. 7) and 4 μm based on the dose response of EDG7 activation (Fig.3C). Second, the amount of LPA in the conditioned medium of untreated Sf9-mPA-PLA1 cells based on the bioassay is ∼400 nm, a concentration that cannot be detected by MS analysis under the present conditions. These observations support the idea that LPA is the component that activated EDG7.What molecular structures determine the enzymatic activity of PLA1? Guinea pig pancreatic lipase-related protein 2 (GPLRP2), which is 63% identical to that of human pancreatic lipase, differs from classical pancreatic lipases in that it displays both lipase and PLA1 activity (41Gassama-Diagne A. Fauvel J. Chap H. Methods Enzymol. 1991; 197: 316-325Crossref PubMed Scopus (8) Google Scholar, 42Hjorth A. Carriereá F. Cudrey C. Woldike H. Boel E. Lawson D.M. Ferrato F. Cambillau C. Dodson G.G. Thim L. Biochemistry. 1993; 32: 4702-4707Crossref PubMed Scopus (157) Google Scholar). Based on the three-dimensional structures of GPLRP2 and human pancreatic lipase, as well as a modeling of hornet PLA1, two domain structures, the lid domain and the β9 loop, have been suggested to play an essential role in substrate selectivity toward triacylglycerides and phospholipids (43Carriereá F. Withers M.C. van Tilbeurgh H. Roussel A. Cambillau C. Verger R. Biochim. Biophys. Acta. 1998; 1376: 417-432Crossref PubMed Scopus (119) Google Scholar). The lid domain in lipases, which overlies the active site (44Winkler F.K. D'Arcy A. Hunziker W. Nature. 1990; 343: 771-774Crossref PubMed Scopus (1028) Google Scholar), has been suggested to be involved in substrate recognition (45Jennens M.L. Lowe M.E. J. Biol. Chem. 1994; 269: 25470-25474Abstract Full Text PDF PubMed Google Scholar). One striking feature of molecules belonging to the lipase family which show PLA1 activity is existence of “short” or “mini” lids. In most of the lipases the lids are composed of 22 or 23 amino acids. By contrast, GPLRP2, hornet PLA1, PS-PLA1, and mPA-PLA1 have short lids composed of 5, 7, 12, and 12 amino acids, respectively (Fig.1B). The other domain structure that is capable of determining the substrate specificity of PLA1/lipase is the β9 loop, which is also located in the vicinity of the active site of lipases. The loop is present in human pancreatic lipase and GPLRP2 (showing lipase activity), whereas it is absent in hornet PLA1, PS-PLA1, and mPA-PLA1. Thus, simultaneous deletions of the β9 loop and the lid domain may determine the molecular characteristics of PLA1 in the lipase family. These molecular features may allow us to identify other PLA1 isozymes in the future.mPA-PLA1 and PS-PLA1 form a subfamily within the lipase family (Fig. 1C). PS-PLA1 produces LPS from PS (24Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 46Nagai Y. Aoki J. Sato T. Amano K. Matsuda Y. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 11053-11059Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), a potential lysophospholipid mediator with an activity to stimulate mast cell degranulation (47Martin T.W. Lagunoff D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4997-5000Crossref PubMed Scopus (14) Google Scholar, 48Smith G.A. Hesketh T.R. Plumb R.W. Metcalfe J.C. FEBS Lett. 1979; 105: 58-62Crossref PubMed Scopus (54) Google Scholar) and neurite outgrowth (27Lourenssen S. Blennerhassett M.G. Neurosci. Lett. 1998; 248: 77-80Crossref PubMed Scopus (42) Google Scholar). Recently we showed (25Hosono H. Aoki J. Nagai Y. Bandoh K. Ishida M. Taguchi R. Arai H. Inoue K. J. Biol. Chem. 2001; 276: 29664-29670Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) that PS-PLA1 also functions as a synthetic enzyme of LPS. It is thus reasonable to assume, from both structural and functional points of view, that these two PLA1s have specialized common function(s) to produce lysophospholipid mediators. Lysophosphatidic acid (1- or 2-acyl-lysophosphatidic acid; LPA)1 is a lipid mediator with multiple biological functions (1Tokumura A. Prog. Lipid Res. 1995; 34: 151-184Crossref PubMed Scopus (165) Google Scholar, 2Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 3Moolenaar W.H. Exp. Cell Res. 1999; 253: 230-238Crossref PubMed Scopus (371) Google Scholar). These include induction of platelet aggregation, smooth muscle contraction, and stimulation of cell proliferation. LPA also promotes specific responses of the cytoskeleton such as generation of actin stress fibers in fibroblasts or inhibition of neurite outgrowth in neuronal cells. LPA evokes its multiple effects through G-protein-coupled receptors that are specific to LPA (see below), with consequent activation of phospholipase C (PLC) and phospholipase D (PLD), Ca2+ mobilization, inhibition of adenylyl cyclase, activation of mitogen-activated protein kinase, and transcription of serum-response-element transcriptional reporter genes, such as c-fos. Recent studies (4Chun J. Contos J.J. Munroe D. Cell Biochem. Biophys. 1999; 30: 213-242Crossref PubMed Scopus (138) Google Scholar, 5Contos J.J. Ishii I. Chun J. Mol. Pharmacol. 2000; 58: 1188-1196Crossref PubMed Scopus (366) Google Scholar) have identified a new family of receptor genes for LPA. Members of this family include three G-protein-coupled receptors belonging to the endothelial differentiation gene (EDG) family, EDG2/LPA1 (6Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar), EDG4/LPA2 (7An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar), and EDG7/LPA3 (8Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murotushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). These proteins may explain various cellular responses to LPA (6Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar, 7An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 8Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murotushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). In contrast to the signal transduction mediated by LPA receptors, the molecular mechanisms for LPA production are poorly understood. LPA is produced both in biological fluids such as serum (9Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar) and in various cells such as platelets (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (177) Google Scholar, 11Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (573) Google Scholar) and ovarian cancer cells (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 13Eder A. Sasagawa T. Mao M. Aoki J. Mills G. Clin. Cancer Res. 2000; 6: 2482-2491PubMed Google Scholar). In these latter studies, it was speculated that LPA is produced by phospholipase A2 (PLA2) from phosphatidic acid (PA) that is generated as a result of PLD activation (12Shen Z. Belinson J. Morton R.E., Xu, Y. Xu Y. Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 13Eder A. Sasagawa T. Mao M. Aoki J. Mills G. Clin. Cancer Res. 2000; 6: 2482-2491PubMed Google Scholar). Tokumuraet al. (14Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar) demonstrated that LPA is also produced in plasma from lysophosphatidylcholine (LPC) by the action of lysophospholipase D, which may account for the accumulation of LPA in aged plasma. LPA, with various fatty acid species, has been detected in several biological systems. For example, human serum contains LPA with both saturated (16:0 and 18:0) and unsaturated (16:1, 18:1, 18:2, and 20:4) fatty acids (15Baker D.L. Desiderio D.M. Miller D.D. Tolley B. Tigyi G.J. Anal. Biochem. 2001; 292: 287-295Crossref PubMed Scopus (197) Google Scholar). A similar LPA species was detected in human platelets (10Gerrard J.M. Robinson P. Biochim. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (177) Google Scholar). The activity of LPA has been shown to be modulated by the length, degree of unsaturation, and linkage to the glycerol backbone of the fatty acyl chain (16Sugiura T. Tokumura A. Gregory L. Nouchi T. Weintraub S.T. Hanahan D.J. Arch. Biochem. Biophys. 1994; 311: 358-368Crossref PubMed Scopus (84) Google Scholar, 17Gueguen G. Gaige B. Grevy J.M. Rogalle P. Bellan J. Wilson M. Klaebe A. Pont F. Simon M.F. Chap H. Biochemistry. 1999; 38: 8440-8450Crossref PubMed Scopus (75) Google Scholar, 18van Corven E.J. van Rijswijk A. Jalink K. van der Bend R.L. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1992; 281: 163-169Crossref PubMed Scopus (236) Google Scholar, 19Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Am. J. Physiol. 1994; 267: C204-C210Crossref PubMed Google Scholar, 20Perkins L.M. Ramirez F.E. Kumar C.C. Thomson F.J. Clark M.A. Nucleic Acids Res. 1994; 22: 450-452Crossref PubMed Scopus (14) Google Scholar, 21Tokumura A. Fukuzawa K. Tsukatani H. Lipids. 1978; 13: 572-574Crossref PubMed Scopus (140) Google Scholar). Of particular interest is the detection of 2-linoleoyl-LPA in ascites from ovarian cancer patients, which may account for the increased ability of the ascites to activate the growth of ovarian cancer cell lines (22Xu Y. Gaudette D.C. Boynton J.D. Frankel A. Fang X.J. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Holub B.J. Mills G.B. Clin. Cancer Res. 1995; 1: 1223-1232PubMed Google Scholar). We recently identified a novel LPA receptor, EDG7/LPA3, which shows a relatively high affinity for 2-acyl-LPA with unsaturated fatty acid (8Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murotushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Che
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