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• W2016277586 abstract "Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A2 and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A2 activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized withN-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS. Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A2 and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A2 activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized withN-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS. N-acetylsphingosine Madin-Darby canine kidney phosphatidylethanolamine phosphatidylcholine 1-O-acylceramide synthase lecithin:cholesterol acyltransferase LCAT-like lysophospholipase hemagglutinin 3-(cyclohexylamino)propanesulfonic acid polyvinylidene difluoride high performance thin-layer chromatography expressed sequence tag For the last decade, ceramide has been thought to play an important role in cell signal transduction involving cell growth, proliferation, differentiation, stress responses, and apoptosis (1Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). The ceramide levels within cells are regulated by several well defined metabolic pathways. We recently studied the metabolism ofN-acetylsphingosine (NAS)1 in Madin-Darby canine kidney (MDCK) cells (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In that study, NAS was actively metabolized and was not an inert compound, as had been previously suggested (3Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar). NAS was converted to other sphingolipids, including sphingosine, C2-sphingomyelin, C2-glucosylceramide, long-chain ceramide, long-chain sphingomyelin, and long-chain glucosylceramide. An unexpected product was also detected. This metabolite was a highly nonpolar compound and identified as 1-O-acyl-NAS.This discovery led to the discovery of a new enzyme activity, one that catalyzes the esterification of the hydroxyl group at C-1 in the ceramide molecule under acidic conditions. The enzyme does not require divalent cations for its activity. Glycerophospholipids (in particular, phosphatidylethanolamine (PE) and phosphatidylcholine (PC)) were identified as acyl group donors in the reaction. The acyl group at thesn-2-position in the phospholipid is transferred to an acceptor molecule, e.g. ceramide or water. If the acceptor is ceramide, 1-O-acylceramide is formed. However, if the acceptor is water, free fatty acid is released. It was also observed that a short-chain rather than a long-chain ceramide is preferred as an acceptor. These observations raised the possibility that this new enzyme regulates a novel pathway of ceramide metabolism.The new enzyme, named 1-O-acylceramide synthase (ACS), was purified from bovine brain and further characterized (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). ACS is a water-soluble glycoprotein with a molecular mass of 45 kDa and a single polypeptide chain, which specifically binds to concanavalin A-conjugated agarose. The enzyme has a pH optimum at 4.5 and has both phospholipase A2 and transacylase activities. The enzyme activity is calcium-independent. Therefore, ACS may be classified as a calcium-independent phospholipase A2.In this study, we report that a BLAST search revealed that the peptides obtained from ACS purified from bovine brain share amino acid sequences with a recently reported human gene termed lecithin:cholesterol acyltransferase (LCAT)-like lysophospholipase (LLPL) (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar). To understand the biological function of ACS, the ACS gene was sequenced on the basis of its similarity to LLPL, and the gene products were characterized.DISCUSSIONThis cloning study revealed that the previously reported LLPL gene encodes ACS. The gene product expressed in LLPL-transfected cells has both transacylase and phospholipase A2 activities under acidic conditions, but lacks significant LCAT and lysophospholipase activities under either acidic or neutral conditions.Previously, Taniyama et al. (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar) reported that human LLPL expressed in COS-7 cells is a secreted protein that has lysophospholipase activity under neutral conditions. In this study, we failed to observe any significant increase in lysophospholipase activity in the cultured medium of COS-7 cells transfected with recombinant LLPL DNA. This was true even when lysophospholipase activity was assayed at concentrations exceeding the critical micellar concentration of lyso-PC. A very slight but significant increase in lysophospholipase activity in the soluble fraction of the LLPL-transfected cells was observed when assayed at lyso-PC concentrations greater than the critical micellar concentration. The specific activity of lysophospholipase was significantly lower than that of ACS. Purified ACS from bovine brain has weak enzyme activity as a phospholipase A1 (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, the lysophospholipase activity observed in recombinant LLPL probably reflects the minor phospholipase A1 activity of ACS. We conclude that the LLPL gene product mainly functions as ACS or as a phospholipase A2, but not as a lysophospholipase.Human recombinant LLPL is inactivated with diisopropyl fluorophosphate, indicating that the active site contains a serine residue (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar). In our study, the replacement of serine with alanine within the lipase motif (AXSXG) of recombinant LLPL resulted in a loss of the phospholipase A2 and transacylase activities. Therefore, the serine residue in the lipase motif must be essential for enzyme activity. As reported for human LLPL (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar, 14Jonas A. Biochim. Biophys. Acta. 2000; 1529: 245-256Crossref PubMed Scopus (304) Google Scholar), the catalytic triad of serine 181, aspartic acid 345, and histidine 377 of LCAT is also conserved in mouse and bovine LLPLs (Fig. 1). This observation is consistent with the view that the serine residue in the motif is an active site and that the enzyme forms an acyl-enzyme intermediate via the hydroxyl group of the serine (Fig.10).The deduced amino acid sequences of mouse, human, and bovine LLPLs indicate that each entire sequence has a signal sequence cleavage site and N-linked glycosylation sites (Fig. 1). Based on the presence of the cleavage site, the processed LLPL would be predicted to consist of 379 amino acid residues, corresponding to a molecular mass of 43 kDa. This value is in agreement with the observed results following treatment of each recombinant LLPL with endoglycosidase F1. Based on amino-terminal sequence analysis of bovine brain LLPL, bovine LLPL is presumed to consist of 378 amino acid residues. These data support the interpretation that the precursor protein of LLPL is post-translationally modified by both signal peptide cleavage and N-linked glycosylation.The observations that ACS has an acidic pH optimum andN-linked oligomannose and co-localizes with β-hexosaminidase in MDCK cells strongly indicate that ACS is a lysosomal enzyme. Deglycosylation of recombinant LLPLs had no effect on ACS activity. Thus, the oligomannose in ACS is probably involved in sorting ACS to lysosome.The family of phospholipase A2 enzymes has expanded greatly in recent years (15Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1201) Google Scholar). With this expansion, the criteria for recognition of an enzyme as a phospholipase A2 and its assignment to one of the 11 currently recognized groups have become more stringent. Currently, there are no lysosomal phospholipases A2 that meet such criteria. In 1997, a calcium-independent phospholipase A2 activity that was inhibited by serine hydrolase inhibitors was described (16Kim T.S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M.K. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This enzyme was subsequently identified as a 1-cysteine peroxiredoxin (17Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar); and thus, its characterization as a phospholipase A2 has been challenged.We conclude that the product of the gene encoding LLPL is not a lysophospholipase, but ACS. Moreover, ACS is identified as a lysosomal enzyme and has the dual enzyme activities of a calcium-independent transacylase and phospholipase A2. A reaction mechanism for ACS is proposed (Fig. 10). In this model, the enzyme reacts with phospholipid and forms an acyl-enzyme intermediate at serine 165 of the mature protein based on the putative cleavage site. The acyl group of the intermediate is then transferred to a hydroxyl group of water or lipophilic alcohol such as ceramide.We believe that ACS is a lysosomal phospholipase A2, but its biological function is unknown. Several possible functions may be entertained, but remain to be tested. ACS may play a primary role in regulating the levels of ceramide within cells. Many investigators have postulated that ceramide may mediate cell differentiation and death responses. A means for regulating ceramide content, particularly through lysosome-mediated pathways, may be important in such a system. In this regard, ACS may function to terminate the biological activities of ceramide generated through signaling pathways perhaps by sequestering ceramide within lysosomes or by facilitating its movement across membrane leaflets. Alternatively, ACS produces a novel metabolite, 1-O-acylceramide. We have previously observed that phospholipids containing arachidonate can serve as the acyl donor for the transacylase reaction (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It will be interesting to determine whether 1-O-arachidonylceramide is a source for biologically active arachidonic acid in cells. Finally, it is also conceivable that water is the usual acceptor for ACS and that its primary function is to serve as a lysosomal phospholipase A2 with PE and PC as preferred substrates and their respective lysolipids and free fatty acids as products.Based on the characterization reported in this study, the nomenclature used to refer to this lysosomal phospholipase is inaccurate. LCAT-like lysophospholipase does not describe the primary activity of this protein and should probably be discarded. Similarly, 1-O-acylceramide synthase may not reflect the primary lipid pathway catalyzed by the phospholipase because its in vivoactivity has yet to be established. We propose therefore that the name lysosomal phospholipase A2 be used in reference to this enzyme. For the last decade, ceramide has been thought to play an important role in cell signal transduction involving cell growth, proliferation, differentiation, stress responses, and apoptosis (1Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). The ceramide levels within cells are regulated by several well defined metabolic pathways. We recently studied the metabolism ofN-acetylsphingosine (NAS)1 in Madin-Darby canine kidney (MDCK) cells (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In that study, NAS was actively metabolized and was not an inert compound, as had been previously suggested (3Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar). NAS was converted to other sphingolipids, including sphingosine, C2-sphingomyelin, C2-glucosylceramide, long-chain ceramide, long-chain sphingomyelin, and long-chain glucosylceramide. An unexpected product was also detected. This metabolite was a highly nonpolar compound and identified as 1-O-acyl-NAS. This discovery led to the discovery of a new enzyme activity, one that catalyzes the esterification of the hydroxyl group at C-1 in the ceramide molecule under acidic conditions. The enzyme does not require divalent cations for its activity. Glycerophospholipids (in particular, phosphatidylethanolamine (PE) and phosphatidylcholine (PC)) were identified as acyl group donors in the reaction. The acyl group at thesn-2-position in the phospholipid is transferred to an acceptor molecule, e.g. ceramide or water. If the acceptor is ceramide, 1-O-acylceramide is formed. However, if the acceptor is water, free fatty acid is released. It was also observed that a short-chain rather than a long-chain ceramide is preferred as an acceptor. These observations raised the possibility that this new enzyme regulates a novel pathway of ceramide metabolism. The new enzyme, named 1-O-acylceramide synthase (ACS), was purified from bovine brain and further characterized (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). ACS is a water-soluble glycoprotein with a molecular mass of 45 kDa and a single polypeptide chain, which specifically binds to concanavalin A-conjugated agarose. The enzyme has a pH optimum at 4.5 and has both phospholipase A2 and transacylase activities. The enzyme activity is calcium-independent. Therefore, ACS may be classified as a calcium-independent phospholipase A2. In this study, we report that a BLAST search revealed that the peptides obtained from ACS purified from bovine brain share amino acid sequences with a recently reported human gene termed lecithin:cholesterol acyltransferase (LCAT)-like lysophospholipase (LLPL) (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar). To understand the biological function of ACS, the ACS gene was sequenced on the basis of its similarity to LLPL, and the gene products were characterized. DISCUSSIONThis cloning study revealed that the previously reported LLPL gene encodes ACS. The gene product expressed in LLPL-transfected cells has both transacylase and phospholipase A2 activities under acidic conditions, but lacks significant LCAT and lysophospholipase activities under either acidic or neutral conditions.Previously, Taniyama et al. (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar) reported that human LLPL expressed in COS-7 cells is a secreted protein that has lysophospholipase activity under neutral conditions. In this study, we failed to observe any significant increase in lysophospholipase activity in the cultured medium of COS-7 cells transfected with recombinant LLPL DNA. This was true even when lysophospholipase activity was assayed at concentrations exceeding the critical micellar concentration of lyso-PC. A very slight but significant increase in lysophospholipase activity in the soluble fraction of the LLPL-transfected cells was observed when assayed at lyso-PC concentrations greater than the critical micellar concentration. The specific activity of lysophospholipase was significantly lower than that of ACS. Purified ACS from bovine brain has weak enzyme activity as a phospholipase A1 (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, the lysophospholipase activity observed in recombinant LLPL probably reflects the minor phospholipase A1 activity of ACS. We conclude that the LLPL gene product mainly functions as ACS or as a phospholipase A2, but not as a lysophospholipase.Human recombinant LLPL is inactivated with diisopropyl fluorophosphate, indicating that the active site contains a serine residue (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar). In our study, the replacement of serine with alanine within the lipase motif (AXSXG) of recombinant LLPL resulted in a loss of the phospholipase A2 and transacylase activities. Therefore, the serine residue in the lipase motif must be essential for enzyme activity. As reported for human LLPL (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar, 14Jonas A. Biochim. Biophys. Acta. 2000; 1529: 245-256Crossref PubMed Scopus (304) Google Scholar), the catalytic triad of serine 181, aspartic acid 345, and histidine 377 of LCAT is also conserved in mouse and bovine LLPLs (Fig. 1). This observation is consistent with the view that the serine residue in the motif is an active site and that the enzyme forms an acyl-enzyme intermediate via the hydroxyl group of the serine (Fig.10).The deduced amino acid sequences of mouse, human, and bovine LLPLs indicate that each entire sequence has a signal sequence cleavage site and N-linked glycosylation sites (Fig. 1). Based on the presence of the cleavage site, the processed LLPL would be predicted to consist of 379 amino acid residues, corresponding to a molecular mass of 43 kDa. This value is in agreement with the observed results following treatment of each recombinant LLPL with endoglycosidase F1. Based on amino-terminal sequence analysis of bovine brain LLPL, bovine LLPL is presumed to consist of 378 amino acid residues. These data support the interpretation that the precursor protein of LLPL is post-translationally modified by both signal peptide cleavage and N-linked glycosylation.The observations that ACS has an acidic pH optimum andN-linked oligomannose and co-localizes with β-hexosaminidase in MDCK cells strongly indicate that ACS is a lysosomal enzyme. Deglycosylation of recombinant LLPLs had no effect on ACS activity. Thus, the oligomannose in ACS is probably involved in sorting ACS to lysosome.The family of phospholipase A2 enzymes has expanded greatly in recent years (15Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1201) Google Scholar). With this expansion, the criteria for recognition of an enzyme as a phospholipase A2 and its assignment to one of the 11 currently recognized groups have become more stringent. Currently, there are no lysosomal phospholipases A2 that meet such criteria. In 1997, a calcium-independent phospholipase A2 activity that was inhibited by serine hydrolase inhibitors was described (16Kim T.S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M.K. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This enzyme was subsequently identified as a 1-cysteine peroxiredoxin (17Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar); and thus, its characterization as a phospholipase A2 has been challenged.We conclude that the product of the gene encoding LLPL is not a lysophospholipase, but ACS. Moreover, ACS is identified as a lysosomal enzyme and has the dual enzyme activities of a calcium-independent transacylase and phospholipase A2. A reaction mechanism for ACS is proposed (Fig. 10). In this model, the enzyme reacts with phospholipid and forms an acyl-enzyme intermediate at serine 165 of the mature protein based on the putative cleavage site. The acyl group of the intermediate is then transferred to a hydroxyl group of water or lipophilic alcohol such as ceramide.We believe that ACS is a lysosomal phospholipase A2, but its biological function is unknown. Several possible functions may be entertained, but remain to be tested. ACS may play a primary role in regulating the levels of ceramide within cells. Many investigators have postulated that ceramide may mediate cell differentiation and death responses. A means for regulating ceramide content, particularly through lysosome-mediated pathways, may be important in such a system. In this regard, ACS may function to terminate the biological activities of ceramide generated through signaling pathways perhaps by sequestering ceramide within lysosomes or by facilitating its movement across membrane leaflets. Alternatively, ACS produces a novel metabolite, 1-O-acylceramide. We have previously observed that phospholipids containing arachidonate can serve as the acyl donor for the transacylase reaction (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It will be interesting to determine whether 1-O-arachidonylceramide is a source for biologically active arachidonic acid in cells. Finally, it is also conceivable that water is the usual acceptor for ACS and that its primary function is to serve as a lysosomal phospholipase A2 with PE and PC as preferred substrates and their respective lysolipids and free fatty acids as products.Based on the characterization reported in this study, the nomenclature used to refer to this lysosomal phospholipase is inaccurate. LCAT-like lysophospholipase does not describe the primary activity of this protein and should probably be discarded. Similarly, 1-O-acylceramide synthase may not reflect the primary lipid pathway catalyzed by the phospholipase because its in vivoactivity has yet to be established. We propose therefore that the name lysosomal phospholipase A2 be used in reference to this enzyme. This cloning study revealed that the previously reported LLPL gene encodes ACS. The gene product expressed in LLPL-transfected cells has both transacylase and phospholipase A2 activities under acidic conditions, but lacks significant LCAT and lysophospholipase activities under either acidic or neutral conditions. Previously, Taniyama et al. (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar) reported that human LLPL expressed in COS-7 cells is a secreted protein that has lysophospholipase activity under neutral conditions. In this study, we failed to observe any significant increase in lysophospholipase activity in the cultured medium of COS-7 cells transfected with recombinant LLPL DNA. This was true even when lysophospholipase activity was assayed at concentrations exceeding the critical micellar concentration of lyso-PC. A very slight but significant increase in lysophospholipase activity in the soluble fraction of the LLPL-transfected cells was observed when assayed at lyso-PC concentrations greater than the critical micellar concentration. The specific activity of lysophospholipase was significantly lower than that of ACS. Purified ACS from bovine brain has weak enzyme activity as a phospholipase A1 (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, the lysophospholipase activity observed in recombinant LLPL probably reflects the minor phospholipase A1 activity of ACS. We conclude that the LLPL gene product mainly functions as ACS or as a phospholipase A2, but not as a lysophospholipase. Human recombinant LLPL is inactivated with diisopropyl fluorophosphate, indicating that the active site contains a serine residue (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar). In our study, the replacement of serine with alanine within the lipase motif (AXSXG) of recombinant LLPL resulted in a loss of the phospholipase A2 and transacylase activities. Therefore, the serine residue in the lipase motif must be essential for enzyme activity. As reported for human LLPL (5Taniyama Y. Shibata S. Kita S. Horikoshi K. Fuse H. Shirafuji H. Sumino Y. Fujino M. Biochem. Biophys. Res. Commun. 1999; 257: 50-56Crossref PubMed Scopus (32) Google Scholar, 14Jonas A. Biochim. Biophys. Acta. 2000; 1529: 245-256Crossref PubMed Scopus (304) Google Scholar), the catalytic triad of serine 181, aspartic acid 345, and histidine 377 of LCAT is also conserved in mouse and bovine LLPLs (Fig. 1). This observation is consistent with the view that the serine residue in the motif is an active site and that the enzyme forms an acyl-enzyme intermediate via the hydroxyl group of the serine (Fig.10). The deduced amino acid sequences of mouse, human, and bovine LLPLs indicate that each entire sequence has a signal sequence cleavage site and N-linked glycosylation sites (Fig. 1). Based on the presence of the cleavage site, the processed LLPL would be predicted to consist of 379 amino acid residues, corresponding to a molecular mass of 43 kDa. This value is in agreement with the observed results following treatment of each recombinant LLPL with endoglycosidase F1. Based on amino-terminal sequence analysis of bovine brain LLPL, bovine LLPL is presumed to consist of 378 amino acid residues. These data support the interpretation that the precursor protein of LLPL is post-translationally modified by both signal peptide cleavage and N-linked glycosylation. The observations that ACS has an acidic pH optimum andN-linked oligomannose and co-localizes with β-hexosaminidase in MDCK cells strongly indicate that ACS is a lysosomal enzyme. Deglycosylation of recombinant LLPLs had no effect on ACS activity. Thus, the oligomannose in ACS is probably involved in sorting ACS to lysosome. The family of phospholipase A2 enzymes has expanded greatly in recent years (15Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1201) Google Scholar). With this expansion, the criteria for recognition of an enzyme as a phospholipase A2 and its assignment to one of the 11 currently recognized groups have become more stringent. Currently, there are no lysosomal phospholipases A2 that meet such criteria. In 1997, a calcium-independent phospholipase A2 activity that was inhibited by serine hydrolase inhibitors was described (16Kim T.S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M.K. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This enzyme was subsequently identified as a 1-cysteine peroxiredoxin (17Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar); and thus, its characterization as a phospholipase A2 has been challenged. We conclude that the product of the gene encoding LLPL is not a lysophospholipase, but ACS. Moreover, ACS is identified as a lysosomal enzyme and has the dual enzyme activities of a calcium-independent transacylase and phospholipase A2. A reaction mechanism for ACS is proposed (Fig. 10). In this model, the enzyme reacts with phospholipid and forms an acyl-enzyme intermediate at serine 165 of the mature protein based on the putative cleavage site. The acyl group of the intermediate is then transferred to a hydroxyl group of water or lipophilic alcohol such as ceramide. We believe that ACS is a lysosomal phospholipase A2, but its biological function is unknown. Several possible functions may be entertained, but remain to be tested. ACS may play a primary role in regulating the levels of ceramide within cells. Many investigators have postulated that ceramide may mediate cell differentiation and death responses. A means for regulating ceramide content, particularly through lysosome-mediated pathways, may be important in such a system. In this regard, ACS may function to terminate the biological activities of ceramide generated through signaling pathways perhaps by sequestering ceramide within lysosomes or by facilitating its movement across membrane leaflets. Alternatively, ACS produces a novel metabolite, 1-O-acylceramide. We have previously observed that phospholipids containing arachidonate can serve as the acyl donor for the transacylase reaction (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It will be interesting to determine whether 1-O-arachidonylceramide is a source for biologically active arachidonic acid in cells. Finally, it is also conceivable that water is the usual acceptor for ACS and that its primary function is to serve as a lysosomal phospholipase A2 with PE and PC as preferred substrates and their respective lysolipids and free fatty acids as products. Based on the characterization reported in this study, the nomenclature used to refer to this lysosomal phospholipase is inaccurate. LCAT-like lysophospholipase does not describe the primary activity of this protein and should probably be discarded. Similarly, 1-O-acylceramide synthase may not reflect the primary lipid pathway catalyzed by the phospholipase because its in vivoactivity has yet to be established. We propose therefore that the name lysosomal phospholipase A2 be used in reference to this enzyme. We thank Drs. Masayuki Funaba, Yoshii Nishino, and Naohiro Inohara for valuable advice and encouragement." @default.
• W2016277586 created "2016-06-24" @default.
• W2016277586 creator A5019980109 @default.
• W2016277586 creator A5072799468 @default.
• W2016277586 creator A5083609672 @default.
• W2016277586 date "2002-03-01" @default.
• W2016277586 modified "2023-10-16" @default.
• W2016277586 title "Cloning and Characterization of a Lysosomal Phospholipase A2, 1-O-Acylceramide Synthase" @default.
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