Publications

• Identification of the expressed form of human cytosolic phospholipase A2beta (cPLA2beta): cPLA2beta3 is a novel variant localized to mitochondria and early endosomes.

  Ghosh M., Loper R., Gelb M.H., Leslie C.C.

  In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the ... >> More

  In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA(2)beta. BEAS-2B cells contain three different RNA splice variants of cPLA(2)beta (beta1, beta2, and beta3). cPLA(2)beta1 is identical to the previously cloned cPLA(2)beta, predicted to encode a 114-kDa protein. However, cPLA(2)beta2 and cPLA(2)beta3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA(2)beta in BEAS-2B cells is the translated product of cPLA(2)beta3. cPLA(2)beta3 exhibits calcium-dependent PLA(2) activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA(2)alpha, cPLA(2)beta3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA(2)beta3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites. << Less

  J. Biol. Chem. 281:16615-16624(2006) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Cloning and recombinant expression of a novel human low molecular weight Ca(2+)-dependent phospholipase A2.

  Chen J., Engle S.J., Seilhamer J.J., Tischfield J.A.

  Extensive biochemical studies of phospholipase A2s (PLA2s) over the last two decades indicate that there are likely to be several distinct PLA2 genes in mammals. Here we report the cloning of a 1-kilobase pair cDNA encoding a novel human low molecular weight PLA2. The cDNA appears to encode a 118- ... >> More

  Extensive biochemical studies of phospholipase A2s (PLA2s) over the last two decades indicate that there are likely to be several distinct PLA2 genes in mammals. Here we report the cloning of a 1-kilobase pair cDNA encoding a novel human low molecular weight PLA2. The cDNA appears to encode a 118-amino acid mature peptide (M(r) = 13,592) preceded by a 20-residue prepeptide. The deduced amino acid sequence encodes a protein that lacks one of the seven disulfide bridges found in similar PLA2s and, therefore, represents a class of enzymes distinct from the mammalian group I and group II enzymes. An RNA blot hybridized with the cDNA exhibited a putative 1.2-kilobase pair transcript in heart and, less abundantly, in lung, as well as multiple putative transcripts in placenta. When the cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium. Conditioned medium optimally hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+. In assays done with individual substrates, L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine, or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol. << Less

  J. Biol. Chem. 269:2365-2368(1994) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.

  Murakami M., Masuda S., Ueda-Semmyo K., Yoda E., Kuwata H., Takanezawa Y., Aoki J., Arai H., Sumimoto H., Ishikawa Y., Ishii T., Nakatani Y., Kudo I.

  Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into H ... >> More

  Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis. << Less

  J. Biol. Chem. 280:14028-14041(2005) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Role of calcium-independent phospholipase A2 in complement-mediated glomerular epithelial cell injury.

  Cohen D., Papillon J., Aoudjit L., Li H., Cybulsky A.V., Takano T.

  In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to morphological changes in GEC and proteinuria, in association with phospholipase A(2) (PLA(2)) activation. The present study addresses the role of calcium-independent PLA(2) (iPLA(2)) i ... >> More

  In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to morphological changes in GEC and proteinuria, in association with phospholipase A(2) (PLA(2)) activation. The present study addresses the role of calcium-independent PLA(2) (iPLA(2)) in GEC injury. iPLA(2)beta short and iPLA(2)gamma were expressed in cultured rat GEC and normal rat glomeruli. To determine whether iPLA(2) is involved in complement-mediated arachidonic acid (AA) release, GEC were stably transfected with iPLA(2)gamma or iPLA(2)beta cDNAs (GEC-iPLA(2)gamma; GEC-iPLA(2)beta). Compared with control cells (GEC-Neo), GEC-iPLA(2)gamma and GEC-iPLA(2)beta demonstrated greater expression of iPLA(2) proteins and activities. Complement-mediated release of [(3)H]AA was augmented significantly in GEC-iPLA(2)gamma compared with GEC-Neo, and the augmented [(3)H]AA release was inhibited by the iPLA(2)-directed inhibitor bromoenol lactone (BEL). For comparison, overexpression of iPLA(2)gamma also amplified [(3)H]AA release after incubation of GEC with H(2)O(2), or chemical anoxia followed by reexposure to glucose (in vitro ischemia-reperfusion injury). In parallel with release of [(3)H]AA, complement-mediated production of prostaglandin E(2) was amplified in GEC-iPLA(2)gamma. Complement-mediated cytotoxicity was attenuated significantly in GEC-iPLA(2)gamma compared with GEC-Neo, and the cytoprotective effect of iPLA(2)gamma was reversed by BEL, and in part by indomethacin. Overexpression of iPLA(2)beta did not amplify complement-dependent [(3)H]AA release, but nonetheless attenuated complement-mediated cytotoxicity. Thus iPLA(2)gamma may be involved in complement-mediated release of AA. Expression of iPLA(2)gamma or iPLA(2)beta induces cytoprotection against complement-dependent GEC injury. Modulation of iPLA(2) activity may prove to be a novel approach to reducing GEC injury. << Less

  Am. J. Physiol. 294:F469-F479(2008) [PubMed] [EuropePMC]

• Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.

  Shinohara N., Uyama T., Jin X.H., Tsuboi K., Tonai T., Houchi H., Ueda N.

  A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined ... >> More

  A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities. << Less

  J. Lipid Res. 52:1927-1935(2011) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Purification and characterization of a catalytic domain of rat intestinal phospholipase B/lipase associated with brush border membranes.

  Tojo H., Ichida T., Okamoto M.

  A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at -35 degrees C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzy ... >> More

  A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at -35 degrees C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzyme exhibited broad substrate specificity including esterase, phospholipase A2, lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases inhibited purified enzyme. When the position of enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a approximately 150-kDa protein; autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH2-terminal amino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of approximately 38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F., Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and Tojo, H. (1988) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated phospholipase B/lipase. << Less

  J. Biol. Chem. 273:2214-2221(1998) [PubMed] [EuropePMC]

  This publication is cited by 23 other entries.

• Identification of a phospholipase B encoded by the LPL1 gene in Saccharomyces cerevisiae.

  Selvaraju K., Rajakumar S., Nachiappan V.

  Phospholipids also play a major role in maintaining the lipid droplet (LD) morphology. In our current study, deletion of LPL1 resulted in altered morphology of LDs and was confirmed by microscopic analysis. LPL1/YOR059c contains lipase specific motif GXSXG and acetate labeling in the LPL1 overexpr ... >> More

  Phospholipids also play a major role in maintaining the lipid droplet (LD) morphology. In our current study, deletion of LPL1 resulted in altered morphology of LDs and was confirmed by microscopic analysis. LPL1/YOR059c contains lipase specific motif GXSXG and acetate labeling in the LPL1 overexpressed strains depicted a decrease in glycerophospholipids and an increase in free fatty acids. The purified Lpl1p showed phospholipase activity with broader substrate specificity, acting on all glycerophospholipids primarily at sn-2 position and later at sn-1 position. Localization studies precisely revealed that Lpl1 is exclusively localized in the LD at the stationary phase. Site directed mutagenesis experiments clearly demonstrated that the lipase motif is vital for the phospholipase activity. In summary, our results demonstrate that yeast Lpl1 exerts phospholipase activity, plays a vital role in LD morphology, and its absence results in altered LD size. Based on the localization and enzyme activity we renamed YOR059c as LPL1 (LD phospholipase 1). << Less

  Biochim. Biophys. Acta 1842:1383-1392(2014) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity.

  Abe A., Shayman J.A.

  A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to th ... >> More

  A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2. << Less

  J. Biol. Chem. 273:8467-8474(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Cloning and characterization of novel mouse and human secretory phospholipase A2s.

  Ishizaki J., Suzuki N., Higashino K., Yokota Y., Ono T., Kawamoto K., Fujii N., Arita H., Hanasaki K.

  Mammalian secretory phospholipase A(2)s (sPLA(2)s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA(2) has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases ... >> More

  Mammalian secretory phospholipase A(2)s (sPLA(2)s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA(2) has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA(2) gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA(2) expressed in the spleen of group IIA sPLA(2)-deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA(2) genes. The human mature sPLA(2) protein consists of 125 amino acids (M(r) = 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA(2) with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA(2) should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA(2)s. When the cDNA was expressed in COS-7 cells, PLA(2) activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mM Ca(2+). In assays with individual substrates, L-alpha-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA(2) mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA(2)-deficient mice, suggesting its functional role in the progression of the inflammatory process. << Less

  J. Biol. Chem. 274:24973-24979(1999) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.