Publications

• Molecular characterization of a phospholipase D generating anandamide and its congeners.

  Okamoto Y., Morishita J., Tsuboi K., Tonai T., Ueda N.

  Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-p ... >> More

  Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class. << Less

  J. Biol. Chem. 279:5298-5305(2004) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines.

  Okamoto Y., Morishita J., Wang J., Schmid P.C., Krebsbach R.J., Schmid H.H., Ueda N.

  In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mo ... >> More

  In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298-5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50-90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells. << Less

  Biochem. J. 389:241-247(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Bile Acid Recognition by NAPE-PLD.

  Margheritis E., Castellani B., Magotti P., Peruzzi S., Romeo E., Natali F., Mostarda S., Gioiello A., Piomelli D., Garau G.

  The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide and other lipid signaling amides, including oleoylethanolamide and palmitoylethanolamide. These bioactive molecules play important roles i ... >> More

  The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide and other lipid signaling amides, including oleoylethanolamide and palmitoylethanolamide. These bioactive molecules play important roles in several physiological pathways including stress and pain response, appetite, and lifespan. Recently, we reported the crystal structure of human NAPE-PLD and discovered specific binding sites for the bile acid deoxycholic acid. In this study, we demonstrate that in the presence of this secondary bile acid, the stiffness of the protein measured by elastic neutron scattering increases, and NAPE-PLD is ∼7 times faster to catalyze the hydrolysis of the more unsaturated substrate N-arachidonyl-phosphatidylethanolamine, compared with N-palmitoyl-phosphatidylethanolamine. Chenodeoxycholic acid and glyco- or tauro-dihydroxy conjugates can also bind to NAPE-PLD and drive its activation. The only natural monohydroxy bile acid, lithocholic acid, shows an affinity of ∼20 μM and acts instead as a reversible inhibitor (IC₅₀ ≈ 68 μM). Overall, these findings provide important insights into the allosteric regulation of the enzyme mediated by bile acid cofactors and reveal that NAPE-PLD responds primarily to the number and position of their hydroxyl groups. << Less

  ACS Chem. Biol. 11:2908-2914(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structure of human N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: regulation of fatty acid ethanolamide biosynthesis by bile acids.

  Magotti P., Bauer I., Igarashi M., Babagoli M., Marotta R., Piomelli D., Garau G.

  The fatty acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions, such as innate immunity, energy balance, and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G pr ... >> More

  The fatty acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions, such as innate immunity, energy balance, and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G protein-coupled receptors (e.g., cannabinoid receptors) and nuclear receptors (e.g., PPAR-α). Here, we report the crystal structure of human NAPE-hydrolyzing phospholipase D (NAPE-PLD) at 2.65 Å resolution, a membrane enzyme that catalyzes FAE formation in mammals. NAPE-PLD forms homodimers partly separated by an internal ∼ 9-Å-wide channel and uniquely adapted to associate with phospholipids. A hydrophobic cavity provides an entryway for NAPE into the active site, where a binuclear Zn(2+) center orchestrates its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis. These elements offer multiple targets for the design of small-molecule NAPE-PLD modulators with potential applications in inflammation and metabolic disorders. << Less

  Structure 23:598-604(2015) [PubMed] [EuropePMC]

• Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-beta-lactamase family.

  Wang J., Okamoto Y., Morishita J., Tsuboi K., Miyatake A., Ueda N.

  In animal tissues, bioactive N-acylethanolamines including the endocannabinoid anandamide are formed from their corresponding N-acylphosphatidylethanolamines (NAPEs) by the catalysis of a specific phospholipase D (NAPE-PLD) that belongs to the metallo-beta-lactamase family. Despite its potential p ... >> More

  In animal tissues, bioactive N-acylethanolamines including the endocannabinoid anandamide are formed from their corresponding N-acylphosphatidylethanolamines (NAPEs) by the catalysis of a specific phospholipase D (NAPE-PLD) that belongs to the metallo-beta-lactamase family. Despite its potential physiological importance, NAPE-PLD has not yet been characterized with a purified enzyme preparation. In the present study we expressed a recombinant NAPE-PLD in Escherichia coli and highly purified it. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg2+ as well as Ca2+ and, hence, appeared to be constitutively active. The enzyme showed extremely high specificity for NAPEs among various glycerophospholipids but did not reveal obvious selectivity for different long chain or medium chain N-acyl species of NAPEs. These results suggested the ability of NAPE-PLD to degrade different NAPEs without damaging other membrane phospholipids. Metal analysis revealed the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies were addressed to several histidine and aspartic acid residues of NAPE-PLD that are highly conserved within the metallo-beta-lactamase family. Single mutations of Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 caused abolishment or remarkable reduction of the catalytic activity. Moreover, when six cysteine residues were individually mutated to serine, only C224S showed a considerably reduced activity. The activities of L207F and H380R found as single nucleotide polymorphisms were also low. Thus, NAPE-PLD appeared to function through a mechanism similar to those of the well characterized members of this family but play a unique role in the lipid metabolism of animal tissues. << Less

  J. Biol. Chem. 281:12325-12335(2006) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Presence and potential signaling function of N-acylethanolamines and their phospholipid precursors in the yeast Saccharomyces cerevisiae.

  Merkel O., Schmid P.C., Paltauf F., Schmid H.H.O.

  N-acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are trace constituents of vertebrate cells and tissues and much is known about their metabolism and possible function in animals. Here we report for the first time the identification and quantification of NAEs and NAPEs in seve ... >> More

  N-acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are trace constituents of vertebrate cells and tissues and much is known about their metabolism and possible function in animals. Here we report for the first time the identification and quantification of NAEs and NAPEs in several strains of the yeast Saccharomyces cerevisiae. Gas chromatography-mass spectrometry of appropriate derivatives revealed 16:0, 16:1, 18:0 and 18:1 N-acyl groups in both NAE and NAPE whose levels, in wild-type cells, were 50 to 90 and 85 to 750 pmol/micromol lipid P, respectively (depending on the phase of growth). NAPE levels were reduced by 45 to 60% in a strain lacking three type B phospholipases, suggesting their involvement in NAPE synthesis by their known transacylation activity. A yeast strain lacking the YPL103c gene, which codes for a protein with 50.3% homology to human NAPE-specific phospholipase D, exhibited a 60% reduction in NAE, compared to wild-type controls. The exposure of various yeast strains to peroxidative stress, by incubation in media containing 0.6 mM H(2)O(2), resulted in substantial increases in NAE. Because yeast cells lack polyunsaturated fatty acids, they offer a useful system for the study of NAE generation and its potential signaling and cytoprotective effects in the absence of polyunsaturated ("endocannabinoid") congeners. << Less

  Biochim. Biophys. Acta 1734:215-219(2005) [PubMed] [EuropePMC]