N-acylphosphatidylethanolamine-hydrolyzing phospholipase D is an important determinant of uterine anandamide levels during implantation

Implantation requires reciprocal interaction between blastocysts and a receptive uterus. In mice, one important player in this dialogue involves endocannabinoid signaling via cannabinoid receptor CB1. Anandamide is an endogenous cannabinoid ligand, and its levels are spatiotemporally regulated in the uterus during early pregnancy, showing lower levels in the receptive uterus and at the implantation site. However, the mechanism by which differential uterine anandamide gradients are established under different pregnancy status is not clearly understood. Using multiple approaches, we show here that uterine anandamide levels conducive to implantation are primarily regulated by spatiotemporal expression of Nape-Pld, the gene encoding N-acylphosphatidylethanolamine-hydrolyzing phospholipase D that generates anandamide. The expression is well correlated with its activity and anandamide levels. This study is clinically relevant, since elevated anandamide levels in peripheral circulation are associated with spontaneous pregnancy failure in women.     

Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho(N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of N-acylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and -independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1-alkenyl-2-hydroxy-glycero-3-phospho(N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg(2+)-dependent "lysophospholipase D". Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho-N-acylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.     

Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages.

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.     

Inhibition of phospholipase D alpha by N-acylethanolamines

N-Acylethanolamines (NAEs) are endogenous lipids in plants produced from the phospholipid precursor, N-acylphosphatidylethanolamine, by phospholipase D (PLD). Here, we show that seven types of plant NAEs differing in acyl chain length and degree of unsaturation were potent inhibitors of the well-characterized, plant-specific isoform of PLD-PLD alpha. It is notable that PLD alpha, unlike other PLD isoforms, has been shown not to catalyze the formation of NAEs from N-acylphosphatidylethanolamine. In general, inhibition of PLD alpha activity by NAEs increased with decreasing acyl chain length and decreasing degree of unsaturation, such that N-lauroylethanolamine and N-myristoylethanolamine were most potent with IC(50)s at submicromolar concentrations for the recombinant castor bean (Ricinus communis) PLD alpha expressed in Escherichia coli and for partially purified cabbage (Brassica oleracea) PLD alpha. NAEs did not inhibit PLD from Streptomyces chromofuscus, and exhibited only moderate, mixed effects for two other recombinant plant PLD isoforms. Consistent with the inhibitory biochemical effects on PLD alpha in vitro, N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) and Commelina communis at low micromolar concentrations. Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLD alpha physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.     

Involvement of N-acylethanolamine-hydrolyzing acid amidase in the degradation of anandamide and other N-acylethanolamines in macrophages

Bioactive N-acylethanolamines including the endocannabinoid anandamide are known to be hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). In addition, we recently cloned an isozyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)", which is active only at acidic pH [Tsuboi, Sun, Okamoto, Araki, Tonai, Ueda, J. Biol. Chem. 285 (2005) 11082-11092]. However, physiological roles of NAAA remained unclear. Here, we examined a possible contribution of NAAA to the degradation of various N-acylethanolamines in macrophage cells. NAAA mRNA as well as FAAH mRNA was detected in several macrophage-like cells, including RAW264.7, and mouse peritoneal macrophages. The homogenates of RAW264.7 cells showed both the NAAA and FAAH activities which were confirmed with the aid of their respective specific inhibitors, N-cyclohexanecarbonylpentadecylamine (CCP) and URB597. As analyzed with intact cells, RAW264.7 cells and peritoneal macrophages degraded anandamide, N-palmitoylethanolamine, N-oleoylethanolamine, and N-stearoylethanolamine. Pretreatment of the cells with CCP or URB597 partially inhibited the degradation, and a combination of the two compounds caused more profound inhibition. In contrast, the anandamide hydrolysis in mouse brain appeared to be principally attributable to FAAH despite the expression of NAAA in the brain. These results suggested that NAAA and FAAH cooperatively degraded various N-acylethanolamines in macrophages.     

Molecular characterization of a phospholipase D generating anandamide and its congeners

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.     

Regional distribution and age-dependent expression of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D in rat brain

The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.     

Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold

Recently, the zinc metallo-hydrolase family of the beta-lactamase fold has grown quite rapidly, accompanied by the accumulation of sequence and structure data. The variety of the biological functions of the family is higher than expected. In addition, the members often have mosaic structures with additional domains. The family includes class B beta-lactamase, glyoxalase II, arylsulfatase, flavoprotein, cyclase/dehydrase, an mRNA 3'-processing protein, a DNA cross-link repair enzyme, a DNA uptake-related protein, an alkylphosphonate uptake-related protein, CMP-N-acetylneuraminate hydroxylase, the romA gene product, alkylsulfatase, and insecticide hydrolases. In this minireview, the functional and structural varieties of the growing protein family are described.     

Crystal structure of a phospholipase D family member

The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 A resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.     

The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components.

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.     

Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases.

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endonuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino-terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single-strand DNA and an artificial substrate, bis(4-nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 A resolution.     

Vaccenic acid suppresses intestinal inflammation by increasing anandamide and related N-acylethanolamines in the JCR:LA-cp rat

Vaccenic acid (VA), the predominant ruminant-derived trans fat in the food chain, ameliorates hyperlipidemia, yet mechanisms remain elusive. We investigated whether VA could influence tissue endocannabinoids (ECs) by altering the availability of their biosynthetic precursor, arachidonic acid (AA), in membrane phospholipids (PLs). JCR:LA-cp rats were assigned to a control diet with or without VA (1% w/w), cis-9, trans-11 conjugated linoleic acid (CLA) (1% w/w) or VA+CLA (1% + 0.5% w/w) for 8 weeks. VA reduced the EC, 2-arachidonoylglycerol (2-AG), in the liver and visceral adipose tissue (VAT) relative to control diet (P < 0.001), but did not change AA in tissue PLs. There was no additive effect of combining VA+CLA on 2-AG relative to VA alone (P > 0.05). Interestingly, VA increased jejunal concentrations of anandamide and those of the noncannabinoid signaling molecules, oleoylethanolamide and palmitoylethanolamide, relative to control diet (P < 0.05). This was consistent with a lower jejunal protein abundance (but not activity) of their degrading enzyme, fatty acid amide hydrolase, as well as the mRNA expression of TNFα and interleukin 1β (P < 0.05). The ability of VA to reduce 2-AG in the liver and VAT provides a potential mechanistic explanation to alleviate ectopic lipid accumulation. The opposing regulation of ECs and other noncannabinoid lipid signaling molecules by VA suggests an activation of benefit via the EC system in the intestine.     

The immunoglobulin fold family: sequence analysis and 3D structure comparisons.

Fifty-two 3D structures of Ig-like domains covering the immunoglobulin fold family (IgFF) were compared and classified according to the conservation of their secondary structures. Members of the IgFF are distantly related proteins or evolutionarily unrelated proteins with a similar fold, the Ig fold. In this paper, a multiple structural alignment of the conserved common core is described and the correlation between corresponding sequences is discussed. While the members of the IgFF exhibit wide heterogeneity in terms of tissue and species distribution or functional implications, the 3D structures of these domains are far more conserved than their sequences. We define topologically equivalent residues in the Ig-like domains, describe the hydrophobic common cores and discuss the presence of additional strands. The disulfide bridges, not necessary for the stability of the Ig fold, may have an effect on the compactness of the domains. Based upon sequence and structure analysis, we propose the introduction of two new subtypes (C3 and C4) to the previous classifications, in addition to a new global structural classification. The very low mean sequence identity between subgroups of the IgFF suggests the occurrence of both divergent and convergent evolutionary processes, explaining the wide diversity of the superfamily. Finally, this review suggest that hydrophobic residues constituting the common hydrophobic cores are important clues to explain how highly divergent sequences can adopt a similar fold.     

Molecular characterization of maize acetylcholinesterase: a novel enzyme family in the plant kingdom

Acetylcholinesterase (AChE) has been increasingly recognized in plants by indirect evidence of its activity. Here, we report purification and cloning of AChE from maize (Zea mays), thus providing to our knowledge the first direct evidence of the AChE molecule in plants. AChE was identified as a mixture of disulfide- and noncovalently linked 88-kD homodimers consisting of 42- to 44-kD polypeptides. The AChE hydrolyzed acetylthiocholine and propyonylthiocholine, but not S-butyrylthiocholine, and the AChE-specific inhibitor neostigmine bromide competitively inhibited its activity, implying that maize AChE functions in a similar manner as the animal enzyme. However, kinetic analyses indicated that maize AChE showed a lower affinity to substrates and inhibitors than animal AChE. The full-length cDNA of maize AChE gene is 1,471 nucleotides, which encode a protein having 394 residues, including a signal peptide. The deduced amino acid sequence exhibited no apparent similarity with that of the animal enzyme, although the catalytic triad was the same as in the animal AChE. In silico screening indicated that maize AChE homologs are widely distributed in plants but not in animals. These findings lead us to propose that the AChE family, as found here, comprises a novel family of the enzymes that is specifically distributed in the plant kingdom.     

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

Beta-ketophosphonates as beta-lactamase inhibitors: Intramolecular cooperativity between the hydrophobic subsites of a class D beta-lactamase

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme. Reduction of the keto group to form the corresponding beta-hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme's active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA-1 active site. Overall, the results suggest that quite effective inhibitors of class C and some class D beta-lactamases could be designed, based on the beta-ketophosphonate platform.     

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

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.