Phosphatidyl Group Research Articles

A polyamino acid-based phosphatidyl polymer library for in vivo mRNA delivery with spleen targeting ability.

A qualified delivery system is crucial for the successful application of messenger RNA (mRNA) technology. While lipid nanoparticles (LNPs) are currently the predominant platform for mRNA delivery, they encounter challenges such as high inflammation and difficulties in targeting non-liver tissues. Polymers offer a promising delivery solution, albeit with limitations including low transfection efficiency and potential high toxicity. Herein, we present a poly(L-glutamic acid)-based phosphatidyl polymeric carrier (PLG-PPs) for mRNA delivery that combines the dual advantages of phospholipids and polymers. The PLGs grafted with epoxy groups were firstly modified with different amines and then with alkylated dioxaphospholane oxides, which provided a library of PLG polymers grafted with various phosphatidyl groups. In vitro studies proved that PLG-PPs/mRNA polyplexes exhibited a significant increase in mRNA expression, peaking 14 716 times compared to their non-phosphatidyl parent polymer. Impressively, the subset PA8-PL3 not only facilitated efficient mRNA transfection but also selectively delivered mRNA to the spleen instead of the liver (resulting in 69.73% protein expression in the spleen) once intravenously administered. This type of phosphatidyl PLG polymer library provides a novel approach to the construction of mRNA delivery systems especially for spleen-targeted mRNA therapeutic delivery.

A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain

BackgroundSeveral types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins.ResultsThe engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved.ConclusionsThis study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable.

Factor effects and mechanisms of the adsorption of Hg(II), Cd(II) and Ni(II) on charged liposomes

Negatively charged liposomes prepared by solvent evaporation method were characterized by transmission electron microscopy, Raman spectroscopy and differential scanning calorimetry. The adsorption of Ni(II), Cd(II) and Hg(II) on the liposomes was systematically investigated. Special attention was paid to the effect of pH, ionic strength and the particle size of liposomes on the adsorption behavior. The increase of pH (from 6.5 to 8.1) enhanced the adsorption capacity (Qe) of three metals due to its effect on metal ions speciation and the conformation of polar phosphatidyl groups (active sites) on the liposome surfaces. The presence of ions slightly weakened the Qe of Ni(II) (p>0.05) and Cd(II) (p<0.05); while for Hg(II) adsorption at higher ionic strength (0.5mol/L), obvious stratification and precipitation was observed. Particle size of liposomes also influenced the adsorption process. Specifically, smaller unilamellar liposome vesicles after extrusion were more beneficial to the adsoprtion of Hg(II) (p<0.01); while larger multilamellar vesicles carried more negative charges benefit the adsoprtion of Ni(II) (p<0.01) and Cd(II) (p<0.05) rather than smaller unilamellar vesicles. Additionally, the sudden rises of the adsorption amount were observed for Cd(II) and Hg(II) and to a less extent for Ni(II), probably because of the changes of the structures or properties of liposomes, i.e., the fusion of vesicles, in the presence of high concentration of heavy metals.

Saturated phosphatidylcholine as matrix former for oral extended release dosage forms

The aim of this study was to evaluate the suitability of saturated phosphatidylcholine (Phospholipon® 90H) as extended release excipient in matrix tablets for three model drugs with different aqueous solubility (theophylline, caffeine and diprophylline). The tablets could be prepared by direct compression because of the favorable phospholipid powder flow properties (Carr's index: 12.64 and angle of repose: 28.85) and good compactibility. Tablets of low porosity were formed already at low pressure of 40MPa and with drug loadings up to 70% due to high plasticity of the phospholipid. Extended drug release was achieved with the drugs of different solubility and at various drug loadings. For example, the caffeine release time (t80%) from 8mm tablets ranged from 1.5h to 18h at 70% and 10% drug loading, respectively. The drug release was governed by diffusion and could therefore be modelled by Fick's law of diffusion. Drug release profiles were thus a function of drug solubility, drug loading and tablet dimension. Matrix tablets of caffeine (20% drug loading) showed robust dissolution with regard to agitation (50–100rpm) and ionic strength of the release media (100–600 mOsmol/kg). Caffeine release was pH-dependent with a faster drug release at acidic pH, which was attributed to a protonization of the phosphatidyl group of the matrix-former and thus a higher hydrophilicity.

Capillary electrophoresis as a method to determine underivatized urinary lipoarabinomannans, a biomarker of active tuberculosis caused by Mycobacterium tuberculosis.

Tuberculosis is a devastating contagious disease caused by Mycobacterium tuberculosis. This is the first report describing the development of novel capillary electrophoresis methods to detect lipoarabinomannans shed into the blood circulation by replicating bacteria. The novelty of the methods is the detection without derivatization. The lipoarabinomannan is detected owing to the ionization of the diverse functional groups of the structure, such as the multibranched mannan domain or the phosphatidyl group. Four alkaline solutions were used; normal polarity in three of them and reversed polarity in one. Urinary lipoarabinomannans by saccharide domains were identified with direct absorbance detection. The accuracy and the analytical sensitivity were then validated with cello-, manno- and xylooligosaccharides. Lipoarabinomannan detection was feasible within 20 min (RSD 2.1%). This method worked at the dynamic range of 0.1-10 μg/mL. With reversed polarity, indirect absorbance detection, and pH 9.0 electrolyte were used, the analytes migrated already within 5 min (RSD 0.01%). Inorganic nonabsorbing ions were used for this method optimization. This improvement resulted in the detection limit of 1 pg/mL in water and in the linear dynamic range of 1 pg/mL to 10 ng/mL. In conclusion, the described method has great potential as a point-of-care assay for clinical use.

PH-Driven Ordering Transitions in Liquid Crystal Induced by Conformational Changes of Cardiolipin.

We report an investigation of interfacial phenomena occurring at aqueous-liquid crystal (LC) interfaces that triggers an orientational ordering transition of the LC in the presence of cardiolipin (CL) by varying pH, salt concentration and valence. In particular, the effects of three different conformational isomeric forms of the CL are observed to cause the response of the LC ordering to vary significantly from one to another at those interfaces. An ordering transition of the LC was observed when the CL is mostly in undissociated (at pH 2) and/or in bicyclic (at pH 4) conformation in which LC shows changes in the optical appearance from bright to dark. By contrast, no change in the optical appearance of the LC was observed when the pH of the system increases to 8 or higher in which the CL mostly exists in the open conformation. Fluorescence microscopy measurements further suggest that pH-dependent conformational forms of the CL have different ability to self-assemble (thus different packing efficiency) at aqueous-LC interfaces leading to dissimilar orientational behavior of the LC. Specifically, we found that change in headgroup-headgroup repulsion of the central phosphatidyl groups of the CL plays a key role in tuning the lipid packing efficiency and thus responses to interfacial phenomena. Orientational ordering transition of the LC was also observed as a function of increasing the ionic strength (buffer capacity) and strongly influenced in the presence of mono and divalent cations. Langmuir-Blodgett (LB) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements provide further insight in modulation of the lipid packing efficiency and alkyl chain conformation of the CL at different pH and ionic conditions. Overall, the results presented in this paper establish that LCs offer a promising approach to differentiate different conformations (label free detection) of the CL through ordering transition of the LC at aqueous-LC interfaces.

Facile Synthesis of Phosphatidyl Saccharides for Preparation of Anionic Nanoliposomes with Enhanced Stability

Physical stability during storage and against processing such as dehyration/rehydration are the cornerstone in designing delivery vehicles. In this work, mono-, di- and tri-saccharides were enzymatically conjugated to phosphatidyl group through a facile approach namely phospholipase D (PLD) mediated transphosphatidylation in a biphasic reaction system. The purified products were structurally identified and the connectivities of carbohydrate to phosphatidyl moiety precisely mapped by 1H, 31P, 13C NMR pulse sequences and LC-ESI-FTMS. The synthetic phosphatidyl saccharides were employed as the sole biomimetic component for preparation of nanoliposomes. It was found that the critical micelle concentration (CMC) of phosphatidyl saccharides increases as more bulky sugar moiety (mono- to tri-) is introduced. Phosphatidyl di-saccharide had the largest membrane curvature. In comparison to the zwitterionic phosphatidylcholine liposome, all phosphatidyl saccharides liposomes are anionic and demonstrated significantly enhanced stability during storage. According to the confocal laser scan microscopy (CLSM) and atom force microscopy (AFM) analyses, the nanoliposomes formed by the synthetic phosphatidyl saccharides also show excellent stability against dehydration/rehydration process in which most of the liposomal structures remained intact. The abundance hydroxyl groups in the saccharide moieties might provide sufficient H-bondings for stabilization. This work demonstrated the synthesized phosphatidyl saccharides are capable of functioning as enzymatically liable materials which can form stable nanoliposomes without addition of stabilizing excipients.

Oligonucleotides Conjugated to Natural Lipids: Synthesis of Phosphatidyl-Anchored Antisense Oligonucleotides

Antisense oligonucleotides are promising therapeutic agents against a variety of diseases. Effective delivery of these molecules is critical in view of their clinical application. Despite the richness of synthetic strategies addressed to the lipophilic modification of oligodeoxynucleotides (ODNs) for enhancing their pharmacokinetic behavior and trans-membrane delivery, the phosphatidyl group (1,2-di-O-acyl-sn-glycero-3-phosphoryl) has been never used as the lipophilic moiety of lipid-ODN conjugates. The present paper reports a general procedure for synthesizing 5'-phosphatidyl-ODNs. By this procedure, phosphatidyl conjugates of a VEGF antisense-ODN have been prepared, which differ in the fatty acid composition of their phosphatidyl moiety. These new lipid-ODN conjugates, which have been characterized on the basis of their physicochemical properties, showed an improved resistance to exonucleases and were able to lower the VEGF-mRNA expression in human SH-SY5Y neuroblastoma cells more effectively than the relevant free antisense-ODN did.

Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography

An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-( R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.

A Eukaryote-like Cardiolipin Synthase Is Present in Streptomyces coelicolor and in Most Actinobacteria

Cardiolipin (CL) is an anionic membrane lipid present in bacteria, plants, and animals, but absent from archaea. It is generally thought that bacteria use an enzyme belonging to the phospholipase D superfamily as cardiolipin synthase (Cls) catalyzing a reversible phosphatidyl group transfer from one phosphatidylglycerol (PG) molecule to another PG to form CL and glycerol. In contrast, in eukaryotes a Cls of the CDP-alcohol phosphatidyltransferase superfamily uses cytidine diphosphate-diacylglycerol (CDP-DAG) as the donor of the phosphatidyl group, which is transferred to a molecule of PG to form CL. Searching the genome of the actinomycete Streptomyces coelicolor A3(2) we identified a gene coding for a putative Cls of the CDP-alcohol phosphatidyltransferase superfamily (Sco1389). Here we show that expression of Sco1389 in a CL-deficient Rhizobium etli mutant restores CL formation. In an in vitro assay Sco1389 condenses CDP-DAG with PG to form CL and therefore catalyzes the same reaction as eukaryotic cardiolipin synthases. This is the first time that a CDP-alcohol phosphatidyltransferase from bacteria is shown to be responsible for CL formation. The broad occurrence of putative orthologues of Sco1389 among the actinobacteria suggests that CL synthesis involving a eukaryotic type Cls is common in actinobacteria.

Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease.

Circular patterns of calcium oxalate monohydrate induced by defective Langmuir–Blodgett film on quartz substrates

The defective Langmuir–Blodgett (LB) film of dipalmitoylphosphatidylcholine (DPPC) on quartz injured by potassium oxalate (K 2C 2O 4) was used as a model system to induce growth of calcium oxalate crystals. Atomic force microscopy (AFM) indicated that circular defective domains with a diameter of 1–200 μm existed in the LB film. Scanning electron microscopy (SEM) showed circular patterns of aggregated calcium oxalate monohydrate (COM) crystallites were induced by these defective domains. It was ascribed to that the interaction between the negatively-charged oxalate ions and the phosphatidyl groups in DPPC headgroups makes the phospholipid molecules rearranged and exist in an out-of-order state in the LB film, especially at the boundaries of liquid-condensed (LC)/liquid-expanded (LE) phases, which provide much more nucleating sites for COM crystals.

Streptomyces Phospholipase D Mutants with Altered Substrate Specificity Capable of Phosphatidylinositol Synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 μM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.

Nitric Oxide Is Critical for Inducing Phosphatidic Acid Accumulation in Xylanase-elicited Tomato Cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.

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.

My Brief Encounter with the Phosphoinositides and IP3

. Before describing thisresearch, however, I should say how that choice came about. While in graduate school at theUniversity of Wisconsin, I had had the good fortune to study under Karl Paul Link, who waswidely renowned for his discovery of dicumarol and the synthesis of related blood anticoagu-lants such as warfarin, work that was recognized with two Lasker Awards (1). On the side,however, Link remained a carbohydrate chemist at heart, a hobby that had grown out of hisstudies on plant polysaccharides and uronic acids while a student and then a young facultymember. In fact, Stanford Moore had completed his doctoral dissertation with Link on amethod for characterizing aldo-monosaccharides as benzimidazole derivatives (2).I arrived at Madison in the fall of 1946, fresh from a stint in the United States Navy, andI found Link’s laboratory bursting at the seams with about 15 ex-GIs, all hard at work tryingto make up for lost time. During earlier investigations on the structure-function relationshipof coumarin anticoagulants, an attempt to synthesize the glucoside of dicumarol had beenfrustrated because the acetylated intermediate was degraded in alkali under conditions usedfor deacetylation (3). Because glycosides are acetals, which are typically acid-labile andalkali-stable, I found the anomaly intriguing and decided to study a variety of syntheticcompounds in an effort to understand the structural basis for alkali sensitivity (4). Thisresearch formed the core of my doctoral dissertation, and although I failed to recognize it at thetime, the chance exposure to carbohydrate chemistry was to have a lasting influence on thedirection my career would take.I continued my indoctrination in sugar chemistry during a postdoctoral year in Edinburgh,Scotland, with E. G. V. Percival in the new Department of Chemistry at Kings Buildingsheaded by Edwin Hirst. This was a time of economic depression in Britain, which was stillsuffering the aftermath of the war, and I discovered that I had left a well equipped laboratoryin Madison to engage an unexpectedly primitive research environment. Wisely I did not letthis change in fortunes discourage me. Instead I undertook a project dealing with the structureof maple sapwood starch and did the best that I could with the available facilities (5). Myefforts were well rewarded because, in the process, I became adept at the uses of analytical andpreparative filter paper and cellulose column chromatography, skills that were to be extremelyvaluable in my later research. The greatest challenge to my ingenuity, however, was toconstruct an electric stirring device from a small board-mounted motor, a couple of woodenpulleys, a piece of string, and a glass rod. The speed of the motor was regulated by adjustinglight bulbs that were wired in series with the power cord to draw off electricity, a crude buteffective method of control. I have always enjoyed working with my hands, so this mundaneproject even took on a certain appeal.Living in a new environment always has its fringe benefits. While in Edinburgh, I developeda special affection for the Scots and a better understanding for the lingering resentment that

Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells.

Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).

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.

Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain.

Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent-labelled substrate, 1-hexanoyl-2-{6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)-amino]hexanoyl}-sn-glycero-3-phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N-terminal half of the intact PLD containing one HKD motif, and the other was the C-terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C-terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C-terminal half of PLD, and that the N-terminal half did not contain such a nucleophile.