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.     

Molecular and biochemical properties and physiological roles of plant phospholipase D.

Recent advances have thrust the study of plant phospholipase D (PLD) into the molecular era. This review will highlight some of the recent progress made in elucidating the molecular and biochemical nature of plant PLDs as well as their roles in plant physiology.     

Regulation of phospholipase D

Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N-terminus and have demonstrated that a conserved sequence at the C-terminus is required for catalysis. The N- and C-terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non-phosphorylating mechanism. Small G proteins of the Rho and ADP-ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C-terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.     

Regulation of phospholipase D

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H₂O₂ treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.     

Cross-talk between receptor-regulated phospholipase D and phospholipase C in brain

Because receptors, G proteins, and phospholipases all exist within a membrane lipid environment, it is not unreasonable to assume that an enzyme capable of changing the lipid environment can affect the coupling relationship among these signal transducing components. Our previous study showed that a muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D via a G protein in brain. We demonstrate here that phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase D are simultaneously activated within 15 s by muscarine in the presence of 1 microM GTP gamma S. More important, inhibition of phospholipase D by zinc attenuated carbamylcholine-induced activation of phospholipase C by 30%. Our additional evidence strongly indicates that the receptor-regulated phospholipase D plays an important modulatory role in agonist-stimulated phosphatidylinositol breakdown. This modulatory effect may be achieved by changing the membrane microenvironment in which phospholipase C and phosphoinositol lipids reside, consequently amplifying the inositol phospholipid signaling process. Our results lead us to postulate that the potential interaction between two different signaling pathways may provide a cell with intracellular coordination and enable the cell to achieve functional responses.     

Immobilized phospholipase D]

Conditions of phospholipase D adsorption on silica gels have been studied. The immobilized phospholipase D is shown to differ from the soluble form in thermostability, pH optima and activation conditions. A question is discussed as to the connection of the use of activators and the adsorption immobilization. It is assumed that phospholipase D belongs to enzymes, functioning only in the immobilized state.     

Kinetics of myocardial phospholipase D

Myocardial phospholipase D (PLD) is located in different subcellular membranes, including sarcolemma (SL) and sarcoplasmic reticulum (SR). In this study, the kinetics of PLD-dependent hydrolytic and transphosphatidylation activities were examined in SL and SR fractions isolated from rat heart by measuring the formation of phosphatidic acid and phosphatidylethanol, respectively. The results showed that, compared to SR PLD, SL PLD had a higher V_max, i.e. 373 vs. 70 nmol/mg protein/h for the hydrolytic activity and 415 vs. 60 nmol/mg protein/h for the transphosphatidylation activity. In comparison with the SR enzyme, SL PLD had a lower K_m value for the hydrolytic activity (0.46 vs. 0.65 mM), but a higher K_m for the transphosphatidylation activity (225 vs. 179 mM). These distinctive kinetic parameters suggest that SL PLD and SR PLD may be isoforms of the enzyme and/or have different membrane domain. Therefore, SL- and SR-localized PLD activities may be under independent control mechanism(s) and play distinct roles in normal conditions and pathological processes.     

Regulation of phospholipase D.

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H(2)O(2) treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.     

Disentangling confusions in inflorescence morphology: Patterns and diversity of reproductive shoot ramification in angiosperms

Terminology of inflorescence diversity has often been used in a confusing way in the literature, partly because it was based on uncritical and outdated definitions. In particular, the terms cyme, thyrse, and panicle have been misused. Although a more critical classification worked out by several authors is available, it is unfortunately not in general use because most of the relevant publications are written in German. In addition, some terms have not been used in the same way by morphologists and developmental geneticists. The present review attempts to remedy the situation with a simple outline of a classification based on: (i) different branching patterns; (ii) differential elongation of axes of different orders; and (iii) repetition of basic ramification patterns in different ways. Racemose and cymose branching are two extreme patterns; the former with limitation of axial orders to two, the second with limitation of lateral axes of each order to two. In a branching system, a sequence of racemose → cymose and, within the cyme, of dichasial → monochasial is common, but the reverse sequence generally does not occur. Systematic and evolutionary aspects of inflorescences are briefly discussed. Branching patterns are often stable in larger clades.Infiorescences of mutants studied in developmental genetic studies are mainly altered in flower or branch numbers or relative branch length, but not in branching patterns. This is also a contribution towards the goal of a unified terminology for the different fields of biology dealing with inflorescences.     

Recognition of phospholipids in Streptomyces phospholipase D

To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.     

Sensor of phospholipids in Streptomyces phospholipase D

Recently, we identified Ala426 and Lys438 of phospholipase D from Streptomyces septatus TH-2 (TH-2PLD) as important residues for activity, stability and selectivity in transphosphatidylation. These residues are located in a C-terminal flexible loop separate from two catalytic HxKxxxxD motifs. To study the role of these residues in substrate recognition, we evaluated the affinities of inactive mutants, in which these residues were substituted with Phe and His, toward several phospholipids by SPR analysis. By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD mutant. We demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids.     

Stability of phospholipase D in primary astrocytes

Induction of expression and proteolytic breakdown of phospholipase D (PLD) isoforms in primary astrocyte cultures have been investigated. Astrocytes express both PLD1 and 2 and are dependent on PLD activity for cell proliferation [K. K?tter, J. Klein, J. Neurochem. 73 (1999) 2517]. Competitive RT-PCR analysis demonstrated a higher level of PLD1 mRNA than PLD2 mRNA (8.9 vs. 0.9amol/microg RNA, respectively). Treatment of astroglial cultures with the phorbol ester, 4beta-phorbol-12beta,13alpha-dibutyrate (0.1 microM), for 24-48h selectively induced PLD1b but not PLD1a or 2 expression as shown by PCR and Western blot; the effect was sensitive to G? 6976. In cells transiently permeabilized with streptolysin-O, antisense oligonucleotides directed against PLD1 or 2 entered the cytoplasm as shown by immunofluorescence experiments but did not affect astroglial proliferation within 2-6 days. Treatment of the cultures with cycloheximide revealed that PLD1 and 2 proteins had biological half-lives of 2-3 days (PLD2) and 4-6 days (PLD1), respectively. It has been concluded that astroglial PLD1b is up-regulated by phorbol esters via protein kinase C activation. Down-regulation of PLD isoforms is prevented by extended biological half-lives of the PLD proteins.     

D5 dopamine receptor regulation of phospholipase D

D(1)-like receptors have been reported to decrease oxidative stress in vascular smooth muscle cells by decreasing phospholipase D (PLD) activity. However, the PLD isoform regulated by D(1)-like receptors (D(1) or D(5)) and whether abnormal regulation of PLD by D(1)-like receptors plays a role in the pathogenesis of hypertension are unknown. The hypothesis that the D(5) receptor is the D(1)-like receptor that inhibits PLD activity and serves to regulate blood pressure was tested using D(5) receptor mutant mice (D(5)(-/-)). We found that in the mouse kidney, PLD2, like the D(5) receptor, is mainly expressed in renal brush-border membranes, whereas PLD1 is mainly expressed in renal vessels with faint staining in brush-border membranes and collecting ducts. Total renal PLD activity is increased in D(5)(-/-) mice relative to congenic D(5) wild-type (D(5)(+/+)) mice. PLD2, but not PLD1, expression is greater in D(5)(-/-) than in D(5)(+/+) mice. The D(5) receptor agonist fenoldopam decreases PLD2, but not PLD1, expression and activity in human embryonic kidney-293 cells heterologously expressing the human D(5) receptor, effects that are blocked by the D(5) receptor antagonist SCH-23390. These studies show that the D(5) receptor regulates PLD2 activity and expression. The hypertension in the D(5)(-/-) mice is associated with increased PLD expression and activity. Impaired D(5) receptor regulation of PLD2 may play a role in the pathogenesis of hypertension.     

Modulation of phospholipase D activity in vitro

Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro. The present paper gives an overview on different factors which can modulate PLD activity and compares their influence on the enzymes from different sources. While no biological regulator can be recognized for extracellular bacterial PLDs, the most prominent specific activator of eukaryotic PLDs is phosphatidylinositol-4,5-bisphosphate (PIP₂). In a sophisticated interplay PIP₂ seems to cooperate with several regulatory proteins in mammalian PLDs, whereas in plant PLDs it mainly acts in concert with Ca²⁺ ions. Moreover, curvature, charges and heterogeneities of membrane surfaces are assessed as unspecific modulators. A possible physiological role of the transphosphatidylation reaction catalyzed by PLDs in competition with phospholipid hydrolysis is discussed.     

Phosphatidylcholine-specific phospholipase C-mediated induction of phospholipase D activity in Fas-expressing murine cells

We have previously reported that Fas cross-linking resulted in the activation of phosphatidylcholine-specific phospholipase C (PC-PLC) and the subsequent activation of protein kinase C (PKC) and phospholipase D (PLD) in A20 cells. In an attempt to correlate the existence of PC-PLC activity and activation of PLD by Fas activation among various Fas-expressing murine cell lines, we have investigated the effect of anti-Fas monoclonal antibody on PC-PLC and PLD activities in A20, P388D1 and YAC-1 cell lines. Upon treatment of anti-Fas monoclonal antibody to these three cell lines, the activation of PLD was only observed in A20 cells. When the effect of anti-Fas monoclonal antibody on PKC and PC-PLC activities in Fas-expressing clones were investigated, the activation of PKC and PC-PLC was detected only in A20 clones. Results presented here also show that exogenous addition of Bacillus cereus PC-PLC activates PC hydrolysis, PKC and PLD in all three murine cell lines. These findings suggest that the activation of PC-PLC is a necessary requirement for the activation of PLD by Fas cross-linking and cell lines devoid of functional PC-PLC activity could exhibit enhanced PLD activity by exogenous addition of PC-PLC.     

Cenozoic extinctions account for the low diversity of extant gymnosperms compared with angiosperms

We test the widely held notion that living gymnosperms are 'ancient' and 'living fossils' by comparing them with their sister group, the angiosperms. This perception derives partly from the lack of gross morphological differences between some Mesozoic gymnosperm fossils and their living relatives (e.g. Ginkgo, cycads and dawn redwood), suggesting that the rate of evolution of gymnosperms has been slow. We estimated the ages and diversification rates of gymnosperm lineages using Bayesian relaxed molecular clock dating calibrated with 21 fossils, based on the phylogenetic analysis of alignments of matK chloroplast DNA (cpDNA) and 26S nuclear ribosomal DNA (nrDNA) sequences, and compared these with published estimates for angiosperms. Gymnosperm crown groups of Cenozoic age are significantly younger than their angiosperm counterparts (median age: 32 Ma vs 50 Ma) and have long unbranched stems, indicating major extinctions in the Cenozoic, in contrast with angiosperms. Surviving gymnosperm genera have diversified more slowly than angiosperms during the Neogene as a result of their higher extinction rate. Compared with angiosperms, living gymnosperm groups are not ancient. The fossil record also indicates that gymnosperms suffered major extinctions when climate changed in the Oligocene and Miocene. Extant gymnosperm groups occupy diverse habitats and some probably survived after making adaptive shifts.     

Molecular diversity in Bacillus anthracis

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.