Cloning and Characterization of a Lysosomal Phospholipase A2, 1-O-Acylceramide Synthase

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 (653) 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 (91) 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 (77) 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. The reagents and sources were as follows: hemagglutinin (HA) and c-Myc peptides and mouse anti-HA monoclonal antibody (clone 12CA5) from Roche Molecular Biochemicals; anti-c-Myc monoclonal antibody (clone 9E10, mouse ascites fluid), anti-FLAG monoclonal antibody M2, horseradish peroxidase-conjugated goat anti-mouse IgG antibody, diaminobenzidine, PE from bovine brain, dicetyl phosphate, and CAPS from Sigma; PVDF membrane (Westran) from Schleicher & Schüll; protein G-agarose beads from Invitrogen; and dioleoylphosphatidylcholine from Avanti. [3H]NAS was prepared in our laboratory (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Silica gel HPTLC plates (10 × 20 cm) were purchased from Merck. ACS was purified from bovine brain as previously described (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The protein content was determined by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as a standard. SDS-PAGE was performed following the method of Laemmli and Favre (6Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3030) Google Scholar). For determination of the amino-terminal amino acid sequence of ACS, the purified protein was separated using a 10% acrylamide gel and transferred to a PVDF membrane using transfer buffer (10 mmCAPS (pH 11) in 10% methanol) at a constant voltage of 50 V for 60 min at room temperature. The protein on the membrane was briefly stained with 0.1% Coomassie Brilliant Blue R-250 in 40% methanol and 1% acetic acid and destained with 50% methanol. The membrane was extensively rinsed with deionized water, and the protein band was excised. The excised PVDF sample was air-dried and analyzed in the Howard Hughes Medical Institute Biopolymer/W. M. Keck Foundation Biotechnology Research Laboratory at Yale University. For determination of partial amino acid sequences of tryptic fragments of ACS, the protein band in the gel was stained with 0.1% Coomassie Brilliant Blue R-250 in ethanol/acetic acid/water (9:2:9), extensively destained with ethanol/acetic acid/water (25:8:65), and excised. The gel slice was washed twice with 50% acetonitrile. Tryptic digestion of the band and sequence analysis were carried out at the Harvard Microchemistry Facility by microcapillary reverse-phase high performance liquid chromatography mass on a mass were from mouse and bovine with was performed to the with the kidney was purchased from Research The with at for 60 for and for min with and kidney cDNAs as The for were and for mouse LLPL, and for bovine LLPL, and and for human products were into the by of and The open reading frames of the LLPLs were excised and sites from the described were into and sites of or by T. S. J. 1998; PubMed Scopus Google to generate carboxyl-terminally tagged LLPL proteins. The of serine to alanine in the lipase motif sequence of mouse LLPL was by the method using and R.M. PubMed Scopus Google Scholar). COS-7 cells were in modified with 10% bovine For COS-7 cells were in the cells were transfected with purified using in of of modified bovine serum was of at and the cells were washed with of with of in for 20 min at and transferred into an The following were carried out at The cells were by at × for The of the cells was into of and (pH by was carried out for × at in a The was for at × The was as a and in protein analysis and enzyme The assay are described in the In of dioleoylphosphatidylcholine PE and dicetyl were as the acyl group in were and under a of 50 (pH was to the at a of of The were into the buffer for min in an using a The of were with of NAS or of [3H]NAS of bovine serum and sample at in a of of (pH The was by of of for min at × the was transferred into and under a of The was to an HPTLC and in a of acid The of was confirmed using in our In the assay using the was with in and for min at An of the was by a to a and by the to the of each of ceramide were to a In the assay using was under a with and of the obtained from carboxyl-terminally mouse LLPL-transfected cells was with anti-c-Myc monoclonal antibody in the or of of c-Myc or peptide in 50 (pH in a of for at The was with 60 of protein G-agarose beads in (pH and for at using a The was at for at The beads were washed with of (pH The beads were with of assay (pH bovine serum and for at room using a The was on at the of the and at for at The was with of of and for min at × The was analyzed as described 20 of the obtained from the carboxyl-terminally LLPL-transfected cells was with or without of in (pH in a of for at the of the the was by the method of and A. Biochem. PubMed Scopus Google Scholar). The was in of buffer of for were separated using a 10% acrylamide gel and transferred to a PVDF membrane using transfer buffer and in methanol) at a constant voltage of V for at The membrane was with of in and 20 (pH The membrane was with anti-c-Myc monoclonal antibody in for at room in a The membrane was briefly rinsed twice with washed for min with and with horseradish peroxidase-conjugated goat anti-mouse in for at room in a The was with and The was performed using the method of J. A. S. J. Cell Biol. PubMed Scopus Google with cells in a were with of modified with 10% serum and for with and A. the cells were washed twice with of with of and (pH and transferred into a The following were carried out at of buffer was to the and the cells were by at × for min and with of buffer A. The cell was with 20 of a The was with buffer and for min at × The was at × 20 The obtained from the was in of buffer and with of buffer A. of of and (pH was on of and (pH on the of the × The was on the and for min at band was observed the and the band that was and was observed the and of of was from the using a and a activity was as described activity was determined as described by J. A. S. J. Cell Biol. PubMed Scopus Google Scholar). activity was determined by the method of and A. 10: Scopus Google H. J. S. 10: PubMed Scopus Google Scholar). ACS was purified from bovine brain, and the tryptic fragments were sequenced by mass BLAST search revealed that the partial amino acid sequences of ACS are highly to the amino acid sequence from 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). Therefore, the gene product expressed from the LLPL gene was to be identical to ACS. BLAST search of the was performed to that with the human LLPL sequence and and with the and of the human LLPL coding and and with the and of human LLPL a were on to the coding This sequence has been to the under to a LLPL expression is higher in kidney (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). Therefore, kidney was for further products of mouse and bovine kidney cDNAs with to single that were in The products were into and sequenced in both The sequence obtained from mouse is identical to the gene reported in the The amino acid sequences of human, mouse, and bovine LLPLs were The human and mouse LLPL cDNAs acid with an acidic the other the bovine LLPL encodes a acid polypeptide with a for signal peptide sequence that human and mouse LLPLs a signal sequence cleavage and alanine The cleavage for bovine LLPL is and alanine mature LLPL contains amino to a molecular mass of previously alanine of human LLPL is the amino-terminal amino acid of 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). of the purified bovine protein yielded a sequence of indicating that the signal peptide cleavage of bovine LLPL may alanine and In each LLPL sequence contains a lipase motif of which is also in (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 C. PubMed Scopus Google and N-linked glycosylation in bovine and in human and The mouse LLPL gene was in the expression of The open reading of LLPL was cloned into the sites of pcDNA3 to generate carboxyl-terminally tagged proteins with or c-Myc was transfected into COS-7 cells with ACS activity in the of the cell was observed using of dicetyl phosphate, and NAS under acidic conditions. The prepared from mouse LLPL-transfected cells of and of fatty acid as a function of a enzyme activity was observed in each from cells transfected with carboxyl-terminally and The from each mouse LLPL-transfected cell had transacylase activity with that from non-transfected cells The did not LLPL activity or These that LLPL has the same transacylase and phospholipase A2 activities as ACS. were to that LLPL has ACS activity. The of mouse LLPL-transfected COS-7 cells was with anti-c-Myc monoclonal antibody and protein G-agarose In this the protein G-agarose beads were with the was with anti-c-Myc antibody, of and of fatty acid were observed with protein G-agarose beads However, the was with anti-c-Myc antibody in the of an of c-Myc no significant products were with protein G-agarose beads the other the was with anti-c-Myc antibody in the of an of of and of fatty acid were observed with protein G-agarose beads These that the protein expressed in cells transfected with mouse LLPL specifically binds to anti-c-Myc monoclonal antibody and transacylase and phospholipase A2 activities at pH This confirmed that LLPL expressed in cells transfected with LLPL has ACS activity. Therefore, ACS is a gene product of the LLPL to (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 the amino acid sequence of human LLPL is to human human LLPL did not activity under neutral assay conditions. We determined mouse LLPL has activity. from the a phospholipase A2 activity was observed in the from LLPL-transfected cells under acidic However, no significant esterification of was observed in the same under either acidic or neutral (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 also reported that human LLPL has lysophospholipase activity. We lysophospholipase activity in the of mouse LLPL-transfected cells. the did not significant in lysophospholipase activity under either acidic or neutral In this study, we the same assay of and under neutral as reported by (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 no lysophospholipase activity was in the with LLPL-transfected in with the of (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). The was than the of In lipase activity is as the phospholipid the Therefore, was to LLPL or the in our has lysophospholipase activity. significant in lysophospholipase activity was observed in the of the LLPL-transfected cells under either acidic or neutral conditions. We a significant in lysophospholipase activity in the of the LLPL-transfected cells under acidic not However, the activity of ACS in the same was higher than that of a LLPL has a lipase in the sequence of LLPL was by the method using R.M. PubMed Scopus Google Scholar). The of the LLPL-transfected cells did not significant enzyme activity analysis that the protein expression of LLPL in COS-7 cells was to that of LLPL These the that the serine residue in the lipase motif is for enzyme activity. There is a the molecular mass reported for human LLPL expressed in COS-7 cells and that observed for purified bovine ACS To this COS-7 cells were transfected with mouse, human, and bovine LLPL and the expressed LLPLs were The of the transfected cells had levels of ACS activity The protein expression of mouse, human, and bovine LLPLs was also as confirmed by with anti-HA monoclonal antibody mouse, human, and bovine LLPLs expressed in COS-7 cells molecular of and to amino acid both mouse and human LLPLs N-linked glycosylation LLPL has N-linked glycosylation Therefore, the in molecular were thought to be to in glycosylation in brain ACS binds to concanavalin beads and is specifically with from the beads (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The of each with an enzyme that and resulted in a in molecular mass for each were at a molecular mass of 42 kDa This is than that from the amino acid sequence for each These that the amino acid sequence for each of a protein of LLPL with a signal peptide In each mature protein of LLPL is a high glycoprotein with the same size protein In did not significant on enzyme activity not The N-linked in LLPL may be in the protein that ACS has an acidic pH optimum and N-linked (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (91) Google A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These that ACS is a a lysosomal that ACS activity is high in cells with other not Therefore, cells were to the of ACS using the method of fractionation J. A. S. J. Cell Biol. PubMed Scopus Google Scholar). and of the activity in the was in the and at × and of the ACS activity in the was in the and activity was not in the The was to fractionation and of the activity was in and indicating that the and of the ACS activity was in and The in the observed and ACS activities is to the of ACS by 40% of the transacylase activity in the was by In the obtained from the or not or yielded a at the in a of acid These that ACS activity in is by the of and the of ACS activity is to that of activity. the activity in was higher than that in indicating that of the were in These the that ACS is a lysosomal This 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 significant and lysophospholipase activities under either acidic or neutral conditions. (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 reported that human LLPL expressed in COS-7 cells is a protein that has lysophospholipase activity under neutral conditions. In this study, we to significant in lysophospholipase activity in the of COS-7 cells transfected with LLPL This was lysophospholipase activity was at the of significant in lysophospholipase activity in the of the LLPL-transfected cells was observed at than the The activity of lysophospholipase was than that of ACS. ACS from bovine brain has enzyme activity as a phospholipase (4Abe A. Shayman J.A. J. Biol. Chem. 1998; 273: 8467-8474Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Therefore, the lysophospholipase activity observed in LLPL the phospholipase activity of ACS. We that the LLPL gene product as ACS or as a phospholipase not as a LLPL is with indicating that the 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 of LLPL resulted in a of the phospholipase A2 and transacylase activities. Therefore, the serine residue in the lipase motif be for enzyme activity. 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 A. Biophys. 2000; PubMed Scopus Google the catalytic of serine acid and of is also in mouse and bovine LLPLs This is with the that the serine residue in the motif is an and that the enzyme an the hydroxyl group of the serine The amino acid sequences of mouse, human, and bovine LLPLs that each sequence has a signal sequence cleavage and N-linked glycosylation sites on the of the cleavage site, the LLPL be to of amino acid to a molecular mass of This is in with the observed following of each LLPL with on amino-terminal sequence analysis of bovine brain LLPL, bovine LLPL is to of amino acid These the that the protein of LLPL is post-translationally modified by both signal peptide cleavage and N-linked The observations that ACS has an acidic pH optimum and with in cells that ACS is a lysosomal Deglycosylation of LLPLs had no on ACS activity. the in ACS is in ACS to The of phospholipase A2 has in Biophys. 2000; PubMed Scopus Google Scholar). this the for of an enzyme as a phospholipase A2 and its to one of the are no lysosomal A2 that In a calcium-independent phospholipase A2 activity that was by serine was described C. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This enzyme was identified as a J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and its as a phospholipase A2 has been We that the product of the gene LLPL is not a ACS. ACS is identified as a lysosomal enzyme and has the enzyme activities of a calcium-independent transacylase and phospholipase A2. for ACS is In this the enzyme with phospholipid and an at serine of the mature protein on the cleavage site. The acyl group of the is transferred to a hydroxyl group of or as ceramide. We that ACS is a lysosomal phospholipase its biological function is may be to be ACS may play a role in the levels of ceramide within cells. that ceramide may cell and for ceramide may be important in a In this ACS may function to the biological activities of ceramide by ceramide within or by its membrane ACS a novel We previously observed that as the acyl for the transacylase (2Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). It be to is a for acid in cells. is also that is the acceptor for ACS and that its function is to as a lysosomal phospholipase A2 with PE and as preferred and respective and free fatty as on the reported in this study, the to to this lysosomal phospholipase is LCAT-like lysophospholipase does not the activity of this protein and be 1-O-acylceramide synthase may not the pathway by the phospholipase its in has to be We propose that the lysosomal phospholipase A2 be in to this We and for and