Chromofuscus Phospholipase D Research Articles

Direct determination of phospholipase D activity by infrared spectroscopy

To determine phospholipase D (PLD) activity, an infrared spectroscopy assay was developed, based on the phosphate vibrational mode of phospholipids such as dimyristoylphophatidylcholine (DMPC), lysophosphatidylglycerol (lysoPG), dipalmitoylphosphatidylethanolamine (DPPE), and lysophosphatidylserine (lysoPS). The phosphate bands served to monitor the hydrolysis rates of phospholipids with PLD. The measurements could be performed within less than 20min with 10μl of buffer containing 2 to 40mM DMPC and 10 to 200ng of Streptomyces chromofuscus PLD (corresponding to 350–7000pmol of DMPC hydrolyzed per minute). The limit of sensitivity was approximately 10ng of PLD at 100mM Tris–HCl (pH 8.0) with 10mM Ca2+ and 2.5mgml−1 Triton X-100. Reproducible specific activity of PLD (35±5nmol of hydrolyzed DMPCmin−1μg−1 PLD) measured by the infrared assay remained stable over 50 to 200ng of PLD and over 5 to 40mM DMPC. The feasibility of this assay to determine the hydrolysis rate of other phospholipids such as lysoPG, DPPE, and lysoPS was confirmed. The IC50 of cobalt (800±200μM), a known S. chromofuscus PLD inhibitor, was measured by means of the infrared assay, demonstrating that this assay can be used to screen PLD activity and/or the specificity of its inhibitors.

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.

Expression and characterization of a heterodimer of Streptomyces chromofuscus phospholipase D

Streptomyces chromofuscus phospholipase D (PLD) is secreted by the bacterium and proteolytically cleaved to a more active form (PLD 37/18) where the two parts of the molecule are still tightly associated. Based on previous sequencing results of authentic PLD 37/18, we have constructed a vector consisting of separate ORFs for the N-terminal and C-terminal portions of S. chromofuscus PLD and overexpressed active heterodimeric PLD. Neither fragment cloned separately folded properly. The identity of each peptide was confirmed by peptide-mass fingerprinting with MALDI-TOF mass spectrometry. The recombinant complex had a specific activity about six times higher than that of the recombinant intact PLD enzyme and was no longer activated by phosphatidic acid (PA). Phosphotransferase activity, binding affinity to phospholipid vesicles, loss of product activation, pH profile and pH-related Ca 2+ activation and inhibition were comparable to authentic PLD 37/18 purified from S. chromofuscus growth medium. PLD 37 alone could also be isolated; the enzyme was active but not as stable as PLD 37/18. These experimental results strongly support the hypothesis that the C-terminal peptide is necessary for correct folding and insertion of catalytic metal ions. However, they suggest the ligands involved in Fe 3+ coordination must be altered upon cleavage of the protein. Asp389, in the C-terminal fragment, whose replacement impairs Fe 3+ binding to the protein, must be replaced by another ligand, since the N-terminal fragment, once folded, is active. In the process of cloning the two peptides, the complete signal sequence for this protein was also determined. The signal peptide of S. chromofuscus PLD enzyme contained a twin arginine motif suggesting that S. chromofuscus PLD, like Bacillus subtilis phoD, is most likely secreted by the TAT translocation pathway under the transcriptional control of the pho regulon.

Transphosphatidylation activity of Streptomyces chromofuscus phospholipase D in biomimetic membranes.

The phospholipase D (PLD) from Streptomyces chromofuscus belongs to the superfamily of PLDs. All the enzymes included in this superfamily are able to catalyze both hydrolysis and transphosphatidylation activities. However, S. chromofuscus PLD is calcium dependent and is often described as an enzyme with weak transphosphatidylation activity. S. chromofuscus PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid. Previous studies have shown that phosphatidic acid-calcium complexes are activators for the hydrolysis activity of this bacterial PLD. In this work, we investigated the influence of diacylglycerols (naturally occurring alcohols) as candidates for the transphosphatidylation reaction. Our results indicate that the transphosphatidylation reaction may occur using diacylglycerols as a substrate and that the phosphatidylalcohol produced can be directly hydrolyzed by PLD. We also focused on the surface pressure dependency of PLD-catalyzed hydrolysis of phospholipids. These experiments provided new information about PLD activity at a water-lipid interface. Our findings showed that classical phospholipid hydrolysis is influenced by surface pressure. In contrast, phosphatidylalcohol hydrolysis was found to be independent of surface pressure. This latter result was thought to be related to headgroup hydrophobicity. This work also highlights the physiological significance of phosphatidylalcohol production for bacterial infection of eukaryotic cells.

Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis.

A kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca(2+) in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca(2+), and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate-limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca(2+), not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca(2+) relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx(4)D-motif PLDs as well. For S. chromofuscus PLD, Ca(2+) is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca(2+)-PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C-terminal residue in S. chromofuscus PLD can modulate the extent of product activation.

Using O-( n-alkyl)- N-( N, N′-dimethylethyl)phosphoramidates to investigate the role of Ca 2+ and interfacial binding in a bacterial phospholipase D

O-( n-alkyl)- N-( N, N′-dimethylethyl)phosphoramidates ( n=6, 8, and 10; C n PNC) were synthesized and characterized as inhibitors of phospholipase D (PLD) activity toward phosphatidylcholine presented as monomers, micelles, and bilayers. Detailed studies with recombinant Streptomyces chromofuscus PLD, a Ca 2+-activated enzyme that does not show large changes in catalytic activity toward the same substrate as a monomer or micelle, showed that the longer the inhibitor chain length, the more potent C n PNC is as a competitive inhibitor toward all the substrates. However, the physical state of the inhibitor did affect the maximum inhibition attainable. For a fixed concentration of diC 4PC (monomer substrate), C n PNC inhibition reached a maximum around the CMC of the inhibitor; the inhibition was reduced at higher inhibitor concentrations, in part caused by the lower solubility of the aggregated inhibitor. With diC 4PC as the substrate and using concentrations of C 10PNC that were below its CMC, the K i for C 10PNC was 0.030±0.003 mM, ∼13-fold less than the K m for substrate. Aggregated substrates showed significant inhibition of PLD by C n PNC, although as the substrate chain length increased, inhibition by a given C n PNC was diminished. With POPC vesicles, the apparent K i for C 10PNC was 0.030 of the apparent K m. The availability of these inhibitors allowed us to show that PC analogues can bind to the active site of S. chromofuscus PLD in the absence of Ca 2+. Once bound at the active site, the inhibitor does not significantly affect the divalent ion-dependent partitioning of the enzyme to PC surfaces. Of the two other PLD enzymes examined, cabbage PLD, but not Streptomyces sp. PMF, was able to catalyze the cleavage of the PN bond. Differential susceptibility of PLDs to these phosphoramidates may eventually be useful in studying PLD isozymes in cells.

Cloning, overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D.

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca(2+)-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca(2+)-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca(2+)-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10(-5) and 10(-4). The Cys mutants still bound Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.

Involvement of Phospholipase D in Sphingosine 1-Phosphate-induced Activation of Phosphatidylinositol 3-Kinase and Akt in Chinese Hamster Ovary Cells Overexpressing EDG3

Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.

Transphosphatidylation capacity of phospholipase D from cabbage (Brassica oleracea L. var. capitata L.) leaves and Streptomyces chromofuscus

The phospholipase D (PLD) was purified from leaves of cabbage (Brassica oleracea L. var. capitata L.). The molecular weight of the enzyme was estimated as 87 and 73.5-kDa on SDS-PAGE and gel filtration using Superdex 200 HR 10/30 column. The transphosphatidylation capacity of cabbage PLD was shown to be higher than that of Streptomyces chromofuscus PLD. Although cabbage PLD showed a single band, Streptomyces chromofuscus PLD gave several bands on nondenaturing and denaturing PAGE. These results indicated that the cabbage PLD was a monomer protein, while the Streptomyces chromofuscus PLD was hetero oligomer, having several types with different molecular weights. Furthermore, the cabbage PLD included the duplicated HxKxxxxD catalytic motifs in the molecule, but the Streptomyces chromofuscus PLD did not. Previously, YAMANE et al. reported that the transphosphatidylation capacity of Streptomyces sp. PLD was higher than that of Streptomyces chromofuscus. These results indicated that the high degree of the transphosphatidylation capacity of cabbage PLD was due to the existence of HxKxxxxD catalytic motifs.

Membrane localization of N-acylphosphatidylethanolamine in central neurons: Studies with exogenous phospholipases

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [ 3H]γ-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [ 3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A 2 (PLA 2) ( Apis mellifera) and PLC ( B. cereus) mobilized [ 3H]arachidonate and [ 3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that ∼ 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE.

PLD AUGMENTS CALCIUM INDUCED DEGRANULATION IN STREPTOLYSIN O PERMEABILIZED NEUTROPHILS. • 901

Neutrophil degranulation is characterized by the fusion of azurophil and specific granules with the plasma membrane. This process is preceded by a complex series of alterations in membrane phospholipid composition mediated, at least in part, by phospholipases C and D. The activation of phospholipase C activation initiates a rise in intracellular Ca2+, while phospholipase D (PLD) hydrolyzes membrane phosphatidylcholine (PC) to phosphatidic acid(PA). (This conversion of PC to PA converts a nonfusogenic phospholipid to a fusogenic one). Using a Streptolysin O permeabilized cell model, we examined the potential role of PLD metabolism on Ca2+-induced degranulation in human neutrophils. Neutrophils permeabilized with streptolysin O can be induced to release their granular contents in response to elevated extracellular [Ca2+]. We found that the addition of exogenous(Streptomyces chromofuscus) PLD caused a dose-dependent augmentation in the Ca2+-induced release of both specific (lactoferrin) as well as azurophil (β-glucuronidase) granular contents (Table 1). Secretion was dependent on both permeabilization as well as the preactivation of PLD with non-stimulatory [Ca2+], indicating that actual metabolism by PLD was important and that it was targeting an intracellular membrane. This work was funded by NICHD and NIAID grants HD28820 and AI20065

Phosphatidylcholine Hydrolysis and DNA Synthesis in Cultured Retinal Capillary Pericytes

In order to verify the role of activation of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) in the initiation of mitogenic process of retinal capillary pericytes, platelet-derived growth factor (PDGF), a known PC hydrolysis stimulator, and exogenous PLD have been used to stimulate pericytes. Exogenous PLD ( Streptomyces chromofuscus PLD) or PDGF BB homodimer (PDGF) was added to a medium of quiescent pericytes prelabeled with [ 32P]orthophosphate. In the presence of ethanol (300 mm), phosphatidic acid (PA) and its stable transphosphatidylated product, phosphatidylethanol (PEt), were determined. In parallel, [ 3H]thymidine incorporation was measured. Downregulation of PKC was achieved by long-term treatment with a phorbol ester. The addition of exogenous PLD or PDGF stimulated both [ 3H]thymidine incorporation and [ 32P]PEt formation in a similar kinetic fashion, suggesting that PC hydrolysis is involved in PDGF-mitogenic signaling pathway. PDGF-stimulated [ 3H]PA formation was significantly higher in the presence than in the absence of PA phosphohydrolase (PAP) inhibitor, indicating the activation of PLD/PAP pathway. In the presence of ethanol, a substantial level of PA at the steady state can be abolished by an inhibitor of diacylglycerol (DAG) kinase. This phenomenon indicates the existence of PC-phospholipase C (PLC)/DAG kinase pathway in PC hydrolysis. Insulin potentiated both PLD- and PDGF-induced DNA synthesis. Though similarities occur in the induction of DNA synthesis and PC hydrolysis by exogenous PLD and PDGF, the maximum extent of DNA synthesis of exogenous PLD was only approximately 43% of that induced by PDGF. Moreover, exogenous PLD-induced DNA synthesis was not blunted, while PDGF-elicited DNA synthesis was markedly reduced, by PKC downregulation. In addition, PDGF-induced PC hydrolysis was attenuated by a tyrosine kinase inhibitor, whereas exogenous PLD-induced PC hydrolysis was unchanged. Taken together, exogenous PLD may mimic PDGF action and partially account for the efficacy on DNA synthesis elicited by PDGF. The signal transduction initiated by exogenous PLD is able to bypass the PKC- and PTK-dependent activation of endogenous PLD. These findings provide evidence for the importance of PLD-mediated PC hydrolysis in pericyte DNA synthesis stimulated by PDGF.

Phospholipase D fromStreptomyces antibioticus: Cloning, sequencing, expression, and relationship to other phospholipases

The extracellular phospholipase D (PLD) gene from Streptomyces antibioticus was cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form in E. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCl and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S. antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus and Streptomyces sp., and contained a conserved region with S. chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region XPLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.

Hydrolytic activity of phospholipase D from plants and Streptomyces spp. against phosphatidylcholine monolayers at the polarized oil/water interface

A previously proposed method of measuring phospholipase D (PLD) activity using the change in electrical capacitance of a phospholipid monolayer at the polarized oil/water interface has been applied, with some modifications, to a comparative study of the hydrolytic activity of four PLDs frequently used in transphosphatidylation. Distinctive differences in the activities of PLDs from Streptomyces spp. and PLDs from plants were revealed. The two PLDs from Streptomyces spp. hydrolyzed the monolayer completely, whereas peanut and cabbage PLDs hydrolyzed up to 75% and 60% of the monolayer respectively. Peanut PLD did not show an absolute requirement for Ca 2+, whereas the hydrolytic activity of S. chromofuscus PLD was appreciably enhanced at 50 mM Ca 2+. The rate of hydrolysis for PLDs from plants showed little dependence on interfacial potential, while the rate for PLDs from Streptomyces spp. was highest when the potential of the aqueous phase was −90 mV relative to that of the oil phase. The Frumkin two-parallel-plate condenser model for the adsorption of PLD provided a good description of the observed potential-dependent activity of PLD.

Fluorescence microscopic imaging of hydrolysis of phospholipid monolayers by phospholipase D at the air-water interface

The enzymatic hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) by phospholipase D (PLD) at the air-water interface was investigated with epifluorescence microscopy. In the PC-PA mixed monolayer doped with octadecyl rhodamine B, the gel phase of PA was observed as dark domains of 2–5 μm diameter at a surface pressure above 25 mN m −1. When PC was spread over a subphase containing Streptomyces chromofuscus PLD, the assemblies of the circular dark domains whose diameter was 2–5 μm appeared at 25 mN m −1. In the case of peanut PLD, few dark domains were observed and their diameter was less than 1 μm. The observed morphological difference in the assemblies of the hydrolysis product by peanut and Streptomyces PLDs indicates that the two PLDs act differently on the monolayer, as has been suggested in the hydrolysis of the monolayer by these PLDs at the oil-water interface. The difference in the hydrolytic action of Streptomyces and plant PLDs suggests that the segregation of the product, PA, from the reaction system as a solid phase is important in efficient hydrolysis.

Phospholipase D mimics platelet-derived growth factor as a competence factor in vascular smooth muscle cells.

Recent studies have suggested the importance of phosphatidylcholine (PC) metabolism in growth factor-stimulated cells. In these cells, PC is hydrolyzed not only by PC-specific phospholipase C but also by phospholipase D (PLD). In the present investigation, we show that the simple addition of PC-hydrolyzing PLD from Streptomyces chromofuscus to the culture medium of vascular smooth muscle cells elicits choline release into the medium accompanied by the formation of phosphatidic acid. In the presence of ethanol, this treatment elicits a formation of phosphatidylethanol (PEt) at the expense of phosphatidic acid. Furthermore, we show here that exogenous addition of S. chromofuscus PLD induces a marked DNA synthesis in quiescent vascular smooth muscle cells. This DNA synthesis induced by S. chromofuscus PLD is, like platelet-derived growth factor (PDGF)-elicited DNA synthesis, largely dependent on the presence of insulin. In addition, S. chromofuscus PLD-induced PEt formation and DNA synthesis were not affected by protein kinase C down-regulation, whereas PDGF-induced PEt formation and DNA synthesis were significantly inhibited. These observations strongly suggest that protein kinase-dependent activation of PLD is involved in mitogenic signal in PDGF-stimulated cells and that exogenously added PLD acts as a competence factor in the same way as PDGF.