Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp)

Hayat El Maarouf, Frédéric Carrière, Mireille Rivière and Abdelkarim Abousalham,1

UPR 9025 du CNRS, Laboratoire de Lipolyse Enzymatique,31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLD{alpha} were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLD{alpha} yielded a protein (rPLD{alpha}a, 88 kDa) together with a shorter form (rPLD{alpha}b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD{alpha}a and rPLD{alpha}b showed that rPLD{alpha}b resulted from proteolytic cleavage at Gly8–Ile9. Immunoblotting showed that both rPLD{alpha}a and rPLD{alpha}b are recognized by a polyclonal antibody previously raised against native soybean PLD{alpha}. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD{alpha}, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD{alpha} is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLD{alpha}b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD{alpha}b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD{alpha}a was eluted after chelating calcium ions with EDTA. The purified rPLD{alpha} yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD{alpha} constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.

Keywords: baculovirus/calcium ions/C2 domain/insect cells/octyl-Sepharose/phospholipase D


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Phospholipase D (PLD) is a ubiquitous enzyme that hydrolyzes the terminal phospho diester bond of glycerophospholipids, leading to the formation of phosphatidic acid (PA) and a free polar head group such as choline in the case of phosphatidylcholine (PtdCho). Furthermore, this enzyme very efficiently catalyzes a transphosphatidylation reaction which, in the presence of a primary alcohol, leads to the formation of the corresponding phosphatidyl alcohol (Eibl and Kovatchev, 1981Go; Kobayashi and Kanfer, 1987Go). One of the PLD products, PA, has been found to serve as a second messenger in several physiological processes occurring in mammalian cells, including the oxidative burst in neutrophils, actin assembly, vesicle trafficking and secretion [see Exton (1999), Frohman et al. (1999) and Jones et al. (1999) for reviews]. In plants, PLD is known to be involved in many physiological processes, such as seed germination, the growth of seedlings, phosphatidylinositol metabolism in roots, senescence, fruit ripening and stress damage such as cold, water stress, wounding and pathogenic infection [see Abousalham et al. (1999) and Wang (1999) for reviews]. Recent evidence in barley (Ritchie and Gilroy, 1998Go) suggests that PLD activity in aleurone cells may be involved in signal transduction events controlling germination.

A eukaryotic PLD{alpha} cDNA was first cloned and sequenced from castor bean (Wang et al., 1994Go). The PLD{alpha} from maize (Ueki et al., 1995Go), rice (Ueki et al., 1995Go; Morioka et al., 1997Go), cabbage (Kim et al., 1999Go), cowpea (Vigna unguiculata) (El Maarouf et al., 1999Go), Pimpinella brachycarpa (Cha et al., 1997Go), Arabidopsis (Dyer et al., 1995Go; Pappan et al., 1997aGo; Qin et al., 1997Go) and yeast PLD (Rose et al., 1995Go) have since been cloned. In mammalian cells, two isozymes have been cloned and designated PLD1 and PLD2 (Hammond et al., 1995Go; Colley et al., 1997Go).

Three forms of PLD, known as PLD{alpha}, PLDß and PLD{gamma}, have been described in Arabidopsis thaliana. They are encoded by distinct genes and differ in their catalytic properties and regulatory processes (Pappan et al., 1997bGo, 1998Go; Qin et al., 1997Go). PLDß and PLD{gamma} require phosphoinositide as a cofactor and they are most active in vitro at micromolar calcium concentrations. PLD{alpha} is the main and most classical form: it is active in vitro at millimolar calcium concentrations and is phosphoinositide independent. The biochemical properties of this PLD{alpha} from several plant species have been studied and found to show considerable similarities. The enzyme was purified from cabbage leaves (Lambrecht and Ulbrich-Hohhmann, 1992Go; Abousalham et al., 1993Go), castor bean endosperm (Wang et al., 1993Go), rice (Ueki et al., 1995Go), soybean cells (Abousalham et al., 1995Go) and germinating sunflower seeds (Abousalham et al., 1997Go).

Although they occur in many plants, bacteria and animals, no information is available so far about the three-dimensional structure of plant PLD. This gap has been mainly due to the lack of availability of large amounts of purified protein. The cDNA cloning of PLD from several species opened up the possibility of establishing high expression systems for PLD. Only two plant PLD{alpha} cDNAs, from Ricinus communis and cabbage, were found to be expressed in Escherichia coli JM109 (Wang et al., 1994Go; Kim et al., 1999Go). These rPLD{alpha} showed a comparable low level of activity to that of the purified enzyme. Furthermore, the rPLD{alpha} were able to catalyze the hydrolytic and transphosphatidylation reactions. No rPLD{alpha} could be purified, however, because of the low levels of expression obtained with E.coli.

In this study, we overcame these limitations as far as the level of expression is concerned by using the baculovirus/insect cell expression system, which has been used successfully to produce a large number of eukaryotic proteins, including several lipases. Here we report the expression of rPLD{alpha} from cowpea in insect cells, along with the corresponding one-step purification procedure.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Materials

PtdCho from egg yolk (type XI-E), octyl-Sepharose CL-4B, choline oxidase (from Arthrobacter globiformis), horseradish peroxidase (type IV) and protease inhibitors were obtained from Sigma Chemical. Silica gel 60 F254 thin-layer chromatography (TLC) plates were purchased from Merck. All the other reagents used were of the best grade commercially available.

DNA source and manipulations

A cDNA encoding the PLD{alpha} from cowpea (Vigna unguiculata) was obtained as described previously (El Maarouf et al., 1999Go). The PLD{alpha} cDNA (2911 bp, with a 5' non-coding region of 224 bp) was originally inserted into the 5'EcoRI and 3'NotI restriction sites of the {lambda} Ziplox plasmid (Invitrogen). A new EcoRI site was inserted by PCR upstream of the PLD{alpha} start codon in order to remove the 5' non-coding region. A 2700 bp EcoRI/BamHI DNA fragment containing the entire PLD{alpha} coding region was further subcloned into the EcoRI/BamHI sites of the pVL1392 baculovirus transfer vector (Invitrogen). All plasmids were produced and amplified in E.coli after electroporation of ElectroMAX DH10B cells (Life Technologies). Plasmid DNAs were isolated from E.coli cultures using the alkaline lysis procedure (Sambrook et al., 1989Go) and purified using the Wizard DNA purification system (Promega). Digestion with restriction enzymes and ligation with T4 DNA ligase were performed as recommended by the enzyme supplier (New England Biolabs). The PCR reactions were carried out using pfu DNA polymerase (Stratagene). All DNA constructs were checked by performing DNA sequencing (ESGS, France).

Production of rPLD{alpha} using the baculovirus expression system

Recombinant baculovirus production and rPLD{alpha} expression were performed as described previously (Bezzine et al., 1999Go). The pVL1392 baculovirus transfer vector containing the PLD{alpha} cDNA was used for the co-transfection of Sf9 insect cells (Spodoptera frugiperda) with the linearized genomic DNA from Autographa californica baculovirus (AcMNPV from the BaculoGold transfection kit, Pharmingen). The Sf9 cells were grown in monolayers at 27°C in tissue culture flasks, using TNM-FH medium (Sigma Chemical) supplemented with 10% fetal calf serum (Bio-Whittaker) and 1% of an antibiotic–antimycotic solution (Gibco BRL-Life Technologies) containing 10 000 units/ml penicillin G, 10 000 µg/ml streptomycin and 25 µg/ml amphotericin B (Fungizone). The insect cell co-transfection procedure was performed using 1 µg of linear AcMNPV DNA and 3 µg of the recombinant pVL1392 mixed with cationic liposomes, added to a 60 mm Petri dish containing 2x106 Sf9 cells grown in TNM-FH medium. The culture supernatants were collected after the lysis of all the cells (approximately 7 days post-infection) and were used as the primary stocks of the recombinant baculovirus expressing rPLD{alpha}. An additional cell infection stage was performed to amplify the virus and generate the high-titer virus stocks (107–109 pfu/ml or plaque-forming units per ml of culture) required for the recombinant protein production. The production of the rPLD{alpha} was carried out using another cell line from Trichoplusiani (High Five), grown in a monolayer up to 5x107 cells per 175 cm2 culture flask, in 25 ml EX-CELL 400 medium (Valbiotech) supplemented with 0.5% of the previously mentioned antibiotic–antimycotic solution. An inoculum of the recombinant baculovirus encoding rPLD{alpha} was added to the cell culture at a multiplicity of infection of around 10 pfu/cell. Six hours post-infection, the culture supernatant was removed by aspiration and replaced by 25 ml of fresh EX-CELL 400 medium. The baculovirus-infected insect cell cultures were harvested at various times after the infection in order to determine the time course of the expression of rPLD{alpha} and to determine whether any proteolytic degradation had occurred.

PLD{alpha} assay

PLD{alpha} activity was assayed spectrophotometrically (Abousalham et al., 1997Go) by measuring the free choline released, upon PtdCho hydrolysis, using a continuous method, adapted for use with microplates (96 wells) from Takrama and Taylor (1991). Choline was continuously transformed into betaine by means of choline oxidase, which simultaneously yielded hydrogen peroxide. The H2O2 was used instantaneously by the added peroxidase (in the presence of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate) to give a colored product absorbing light at ~500 nm. The absorbance was measured at 490 nm, using a Dynatech MR 5000 microplate reader.

Egg PtdCho was dispersed in an equimolar mixture of SDS and Triton-X100. The mixed micellar solution was shaken for 30 s using a Vortex mixer (3000 r.p.m.), sonicated for 10 min using a Bioblock T460 sonicator and vortex mixed for 30s. The assay mixture (150 µl) contained 50 mM Tris–HCl, pH 8.0, 20 mM CaCl2, 1.7 mM 4-aminoantipyrine, 9 mM sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, 0.5 U choline oxidase and 0.5 U peroxidase. After a 10 min period of stabilization, the reaction was initiated by adding the enzyme and the substrate (egg PtdCho mixed micelles, 0.26 mM, final concentration) and absorbance measurements were carried out every 30 s for 5 min. Control assays without PLD{alpha} were tested simultaneously. The amount of free choline released was quantified based on a calibration curve obtained with pure choline.

One unit of PLD{alpha} activity was defined as the amount of enzyme which releases 1 µmol/min of choline under the experimental conditions specified above.

TLC assay for transphosphatidylation activity

The reaction mixture (0.5 ml final volume) was composed of 0.26 mM egg PtdCho dispersed in an equimolar mixture of SDS and Triton-X100, 50 mM Tris–HCl (pH 8), 20 mM CaCl2 and 2% ethanol (final concentration). After incubating this mixture with the appropriate amount of enzyme for 10 min at 37°C, the phospholipids were extracted with 0.8 ml of chloroform–methanol (2:1, v/v) after vigorous shaking. The lower chloroform phase was separated, evaporated and the lipid residue was dissolved in 50 µl of chloroform and then analyzed by TLC. Sample migration was performed on a silica gel 60 TLC plate using chloroform–methanol–acetic acid (65:15:1, v/v/v) as the developing solvent. The phospholipid spots were revealed by exposing the plate to iodine vapor.

Purification of rPLD{alpha}

Recombinant baculovirus-infected High Five cell cultures were harvested after 3 or 4 days, in order to prevent the proteolysis of the recombinant protein from occurring owing to intracellular proteases being released into the medium during cell lysis. The purification of rPLD{alpha} was carried out as described by Abousalham et al. (1993), with some modifications. A 100 ml volume of the culture medium was centrifuged at 10 000 g for 10 min and the cell pellet was discarded. Protease inhibitors were added to the cell culture medium in order to prevent proteolysis. The culture supernatant was either lyophilized and stored at –20°C or dialyzed overnight against 30 mM PIPES buffer (pH 6.2), 50 mM CaCl2 and applied to an octyl-Sepharose CL-4B column (2.5x20 cm) equilibrated in 30 mM PIPES buffer (pH 6.2), 50 mM CaCl2. The flow-rate was 1 ml/min and the protein elution profile was recorded spectrophotometrically at 280 nm. The column was rinsed with at least 80 ml of the same buffer until the absorbance reached the baseline level. The elution of the proteins bound to the column was first performed with 10 mM PIPES buffer (pH 6.2), 30 mM CaCl2 and then with 10 mM PIPES buffer (pH 6.2), 0.1 mM EDTA to remove high calcium affinity proteins. The 10 ml fractions containing PLD{alpha} activity were pooled and concentrated 10–20-fold by ultrafiltration in an Amicon cell with a PM 30 membrane. The pure rPLD{alpha} was aliquoted and stored at –80°C in 30% glycerol.

Protein quantitation, electrophoresis and immunoblotting

Protein concentrations were determined routinely using the Bradford procedure (Bradford, 1976Go) with Bio-Rad Dye Reagent and bovine serum albumin as the standard. Samples were separated by 10% SDS–PAGE as described by Laemmli (1970) and transferred to nitrocellulose membranes. Non-specific protein binding sites were blocked by incubating the membranes for 1 h with 5% (w/v) dried skimmed milk in TBS. Membranes were then incubated for 2 h with 1 µg/ml of anti-soybean PLD{alpha} antibodies. Unbound primary antibodies were removed by performing six washes (for 5 min each) with TBS containing 0.05% (v/v) Tween 20. The membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical) (diluted 1:2000 with TBS–Tween 20) and developed with enhanced chemiluminescence Western blotting solutions (Amersham), in line with the manufacturer's recommendations.

N-Terminal amino acid sequencing

The N-terminal sequence of the purified rPLD{alpha} was determined using a gas-phase sequencer (Applied Biosystems, Model 470 A). The phenylthiohydantoin amino acid derivatives were identified by performing HPLC using a C18 column (Brownlee, 5 µm, 2.1x220 mm), eluted with a methanol gradient (10–46%) in 7 mM sodium acetate (pH 4.84) and quantified by an integration program on a Waters Model 840 data control station.

Mass spectrometric determination

Matrix-assisted laser desorption/ionization (MALDI) MS was performed on a reflectron time-of-flight mass spectrometer equipped with delayed extraction (Voyager DE-RP, PerSeptive Biosystems). The purified rPLD{alpha} was first dissolved in 1 µl of acetic acid and then diluted with 9 µl of water. The sample (0.7 µl) was mixed directly on the support with an equal volume of sinapinic matrix (saturated solution of sinapinic acid in 40% acetonitrile–60% water containing 0.1% trifluoroacetic acid).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Expression of rPLD{alpha} in insect cells

rPLD{alpha} was expressed in High Five insect cells using the baculovirus expression system. The rPLD{alpha} was found to be secreted by insect cells grown in monolayers. The first 34 amino acids encoded by the PLD{alpha} cDNA make up a secretory signal which is also recognized and processed in insect cells. Several other recombinant lipolytic enzymes have also been successfully expressed and secreted by insect cells using their own signal peptide, e.g. pancreatic lipases (Bezzine et al., 1999Go) and gastric lipase (Dupuis et al., 1997Go). The active fraction of the rPLD{alpha} was found to be mainly present in the serum-free medium of infected cells. Culture medium samples were collected every day post-infection by the baculovirus. After adding protease inhibitors, these samples were centrifuged and the supernatant was analyzed to determine the PLD{alpha} activity. The time course of rPLD{alpha} expression is shown in Figure 1Go. rPLD{alpha} activity was detected in the culture medium at 3 days post-infection. The activity accumulated in the medium and reached a maximum at 4 days post-infection and the corresponding amount of rPLD{alpha} was estimated to be ~40 mg/l, based on the known specific activity of PLD. From 6 to 8 days post-infection, a slight decrease in the activity was observed, indicating that some proteolytic degradation of the recombinant protein might have occurred.



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Fig. 1. Time-dependent expression of rPLD{alpha} in baculovirus-infected High Five cells. A monolayer of High Five cells was infected at a density of 5x107 cells/175 cm2 culture flask and a multiplicity of infection of 10. At the indicated times post-infection, an aliquot of the culture medium was recovered and analyzed for PLD{alpha} activity, as described in Materials and methods. Values are means ± SD from three experiments.

 
In parallel with the activity measurements, the rPLD{alpha} secreted into the insect cell medium was analyzed by SDS–PAGE with Coomassie Brilliant Blue staining (Figure 2AGo) and also by immunoblotting (Figure 2BGo). We observed that the patterns from SDS–PAGE and immunoblotting follow the activity measurements. A protein with a molecular mass of ~90 kDa was detected in the cell supernatant 3 days post-infection (Figure 2AGo, line 3). A second protein with a lower molecular mass of ~88 kDa also appeared at 4 days post-infection (Figure 2AGo, lines 4–8) and then accumulated in the medium. To check further that the protein expressed was in fact rPLD{alpha}, Western blot analysis was performed, using an anti-native soybean PLD{alpha} antibody previously obtained in our laboratory. This antibody detected two protein bands corresponding in size to the proteins expressed as previously seen by protein staining (Figure 2BGo). Some smaller immunoreactive bands were also observed, which suggested that the PLD{alpha} expressed may have been partly degraded by various intracellular proteases released during the insect cell lysis. No protein bands were detected by immunoblotting the non-infected cells medium (data not shown). Membrane and cytosol from infected cells samples were also analyzed and no significant PLD{alpha} activity was detected in these fractions (data not shown).



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Fig. 2. (A) SDS–PAGE and (B) immunoblotting of rPLD{alpha} expressed in baculovirus-infected High Five cells. Each day after the initial infection of a monolayer of High Five cells, aliquots of the culture medium were recovered and analyzed by SDS–PAGE and immunoblot analysis. Lines 1–8 correspond to days 1–8 post-infection. The first line on the left corresponds to a control sunflower PLD{alpha}. Molecular mass markers are indicated by arrows on the right.

 
Purification of rPLD{alpha}

Most plant PLD{alpha} bound strongly to a hydrophobic gel in the presence of Ca2+ ions (Lambrecht and Ulbrich-Hohhmann, 1992Go; Abousalham et al., 1993Go). We used this property to purify the main form of rPLD{alpha} (rPLD{alpha}b) collected from the insect cell culture media. The purification was performed in a one-step procedure using an octyl-Sepharose CL-4B column. The activity of the rPLD{alpha}b was eluted by decreasing the concentration of Ca2+ ions in the buffer from 50 to 30 mM (Figure 3Go). Highly purified rPLD{alpha}b devoid of any detectable contaminants gives a single protein band at a position corresponding to a molecular mass of 88 kDa (Figure 3Go inset, lane 2). The specific activities and recovery yield of the enzyme after the purification step are summarized in Table IGo. A 17-fold purification was achieved with the supernatant of insect cell culture media and the overall recovery was 83%. From 100 ml of insect cell culture media, about 1 mg of pure rPLD{alpha}b was obtained with a specific activity of 25 U/mg (Table IGo).



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Fig. 3. Purification of rPLD{alpha}b expressed in baculovirus-infected High Five cells. Pure rPLD{alpha}b was obtained using a one-step purification procedure on an octyl-Sepharose CL-4B column (2.5x20 cm). Elution was performed stepwise in PIPES buffer (pH 6.2) containing successively 50, 30 and 0 mM of CaCl2 as described in Materials and methods. Protein elution was recorded by measuring the absorbance at 280 nm. Inset: SDS–PAGE (10% acrylamide) stained with Coomassie Brilliant Blue to reveal the proteins. Lane 1, crude medium of baculovirus-infected High Five cells at 4 days post-infection; lane 2, octyl-Sepharose pooled fractions containing active rPLD{alpha}b. Molecular mass markers are indicated by arrows on the left.

 

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Table I. Flow sheet of the rPLD{alpha}b purification procedure
 
In order to purify the rPLD{alpha}a, a transiently secreted PLD{alpha} form, the insect cell medium was collected on the third day post-infection and loaded onto an octyl-Sepharose CL-4B column as described in Materials and methods. Unlike the rPLD{alpha}b, the rPLD{alpha}a was eluted from the octyl-Sepharose column by chelating the calcium ions with EDTA (Figure 4Go). In the presence of calcium ions, this rPLD{alpha}a bound more strongly to the hydrophobic matrix than the rPLD{alpha}b. SDS–PAGE analysis of the purified rPLD{alpha}a gave a major band with the predicted molecular size of 90 kDa (Figure 4Go inset, lane 2). A 14-fold purification was achieved with the supernatant of insect cell culture media and the corresponding overall recovery was 64%. From 50 ml of insect cell culture media, ~0.6 mg of pure rPLD{alpha}a was obtained with a specific activity of 26.7 U/mg.



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Fig. 4. Purification of rPLD{alpha}a expressed in baculovirus-infected High Five cells. Pure rPLD{alpha} was obtained using a one-step purification procedure on an octyl-Sepharose CL-4B column (2.5x20 cm) as described in Figure 3Go. Inset: SDS–PAGE (10% acrylamide) stained with Coomassie Brilliant Blue to reveal the proteins. Lane 1, crude medium of baculovirus-infected High Five cells at 3 days post-infection; lane 2, octyl-Sepharose pooled fractions containing active rPLD{alpha}a. Molecular mass markers was indicated by arrows on the left.

 
Biochemical and structural characterization of rPLD{alpha}

The molecular masses obtained using mass spectrometric analysis were found to be 88 020 and 87 150 Da for rPLD{alpha}a and rPLD{alpha}b, respectively (Table IIGo), which is consistent with the apparent molecular mass values, ranging from 87 000 to 90 000, obtained with other purified plant PLD{alpha}. The N-terminal amino acid sequence of rPLD{alpha}a was QNIEETVGIGKGVTK, whereas it was IGKGVTK in the case of rPLD{alpha}b. rPLD{alpha}b may result from either a proteolytic degradation of rPLD{alpha}a with the cleavage site occurring between Gly8 and Ile9 (Table IIGo) or an alternative cleavage of the signal peptide. The sequential appearance of rPLD{alpha}a first and rPLD{alpha}b second, as shown from the electophoretic analysis in Figure 2Go, is in favor of the first hypothesis, however. Both rPLD{alpha}a and rPLD{alpha}b had pI values of ~4.7 (data not shown), which is in good agreement with the acidic values obtained previously with other purified plant PLD{alpha} (Lambrecht and Ulbrich-Hohhmann, 1992Go; Abousalham et al., 1993Go, 1995Go: Wang et al., 1993Go).


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Table II. Molecular characteristics and behavior of various plant PLD{alpha} on calcium-dependent octyl-Sepharose chromatography
 
To study further the PLD{alpha} activity of the rPLD{alpha}a and rPLD{alpha}b, TLC analysis was carried out to determine whether the concurrent production of PA occurred. In the presence of 2% ethanol, rPLD{alpha}b catalyzes the transphosphatidylation reaction, as shown by the formation of phosphatidylethanol (PEtOH) (Figure 5Go). Similar results were obtained using rPLD{alpha}a (data not shown).



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Fig. 5. Thin-layer chromatogram of the reaction products of egg PtdCho dispersed in an equimolar mixture of SDS and Triton-X100 incubated with rPLD{alpha}b as described in Materials and methods. Lane 1, reference PA; lane 2, reference PEtOH; lane 3, reaction products after 10 min incubation of PtdCho mixed micelles with rPLD{alpha}b; lane 4, control experiment without any rPLD{alpha}b. Arrows indicate the positions of PA and PEtOH and PtdCho.

 
The present data show that rPLD{alpha}a and rPLD{alpha}b bind differently to an octyl-Sepharose column in the presence of Ca2+ ions. rPLD{alpha}b can be eluted by simply decreasing the calcium concentration from 50 to 30 mM, whereas rPLD{alpha}a is released from the column only in the complete absence of Ca2+ ions. This difference may be attributable to the removal of eight amino acids at the N-terminus of rPLD{alpha}a. One possibility is that this peptide might contain part of a region which is necessary for the binding of Ca2+ ions. In all plant PLD{alpha} cloned so far, sequence alignments have suggested the presence of a calcium/phospholipid binding C2 domain at their N-terminus (Ponting and Kerr, 1996Go; Qin et al., 1997Go). C2 domains often mediate the binding to phospholipids in a calcium-dependent manner and these C2 domains have been identified in a number of enzymes involved in signal transduction and/or phospholipid metabolism (Nalefski and Falke, 1996Go; Ponting and Parker, 1996Go). The three-dimensional structures of several C2 domains have been described, including those from synaptotagmin C2A (synC2A) (Sutton et al., 1995Go; Shao et al., 1996Go), phosphoinositide-specific phospholipase C{delta}1 (PLC{delta}1) (Essen et al., 1996Go, 1997Go) and 85 kDa cytosolic phospholipase A2 (Perisic et al., 1998Go; Xu et al., 1998Go). The C2 domain shows a ß-sandwich structure with two ß-sheets consisting of four antiparallel ß-strands each. Three connecting loops form the calcium binding regions (CBR1, CBR2 and CBR3). The C2 domains of synC2A and PLC{delta}1 represent two distinct topological folds, referred as topology I and topology II, respectively, differing slightly in their ß-strand connectivity (Nalefski and Falke, 1996Go). Several amino acid residues from CBR1, CBR2 and CBR3 are involved in the calcium binding sites, including five negatively charged residues that are highly conserved in C2 domains. Based on these data, the putative C2 domains of cowpea rPLD{alpha}a and rPLD{alpha}b were aligned with those of synC2A, PLC{delta}1, PLD{alpha}, PLDß and PLD{gamma} from Arabidopsis and PLD{alpha} from cabbage and castor bean (Figure 6Go). The most highly conserved segments in all the C2 domains analyzed here were located within strands ß3 and ß5 of SynC2A. These regions contain the sequences (P,L)Y(V,A) and NPx(F,W)x3Fx(F,V,I) (alternative residues at a single position are separated by commas and enclosed by parentheses and `x' may be occupied by an amino acid), which are largely hydrophobic and have been thought to maintain the structural integrity of the C2 domain (Sutton et al., 1995Go). Four acidic residues (E190, D200, D248, D250 for PLDß and E77, D87, D134, D136 for PLD{gamma}) out of the five observed in the calcium binding regions of the classical C2 domains are conserved in Arabidopsis PLDß and PLD{gamma}. Only two of these acidic residues are conserved in PLD{alpha} from Arabidopsis (E38, D97), castor bean (E7, D66), cabbage (E3, D62) and cowpea rPLD{alpha}a (E4, D63) (Figure 6Go). PLDß and PLD{gamma} were found to be most active in vitro at micromolar calcium concentrations whereas PLD{alpha} is active in vitro at millimolar calcium concentrations (Pappan et al., 1997aGo). The N-terminal amino acid sequence of cowpea rPLD{alpha}b starts with IGKGVTK and consequently lacks the first acidic amino acid residue, which might be involved in CBR1. This might induce a lower affinity of rPLD{alpha}b for calcium and, consequently, a lower affinity of the rPLD{alpha}b C2 domain for a hydrophobic interface. The full-length cowpea rPLD{alpha}a possesses the first acidic amino acid residue (E4), which might explain its higher calcium-dependent retention on an octyl-Sepharose column. The acidic residue E4 of rPLD{alpha}a is conserved in the N-terminal sequence of the native PLD{alpha} from cabbage, sunflower and soybean cells (Table IIGo). All these enzymes have been reported to show the same calcium-dependent retention behavior on an octyl-Sepharose column, from which they were eluted only by chelating the calcium ions with EDTA.



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Fig. 6. Multiple sequence alignment of C2 domains. Amino acid sequences of C2 domain of PLDß and PLD{gamma} from Arabidopsis (ar) and PLD{alpha} from Arabidopsis, castor bean (cb), cabbage (c) and recombinant cowpea (co.rPLD{alpha}a and co.rPLD{alpha}b) were aligned with synC2A and PLC{delta}1 C2 domains. The sequence alignments were produced with the program CLUSTAL W (Higgins et al., 1996Go) and adjusted manually. Positions of the five acidic amino acid residues involved in calcium binding are shown in bold and numbered after alignment with synC2A. The positions of the three loops (CBR1, CBR2 and CBR3) are also indicated. The most highly conserved segments in various C2 domain-containing proteins are underlined. The secondary structures determined by the X-ray structure of type I and type II topologies in synC2A and PLC{delta}1 are shown above and below the sequence, respectively.

 
It has been hypothesized that the calcium binding increases the hydrophobicity of the C2 domain surface, probably by changing the net charge in the vicinity of the CBRs, and that membrane binding of the C2 domains is mainly driven by hydrophobic residues in the CBR1 and CBR3 calcium binding loops (Ball et al., 1999Go; Bittova et al., 1999Go). The calcium-dependent binding of PLD{alpha} to a hydrophobic column suggests that a similar binding process to that observed with C2 domains may be at work. Our data, together with the results of sequence alignment, support the existence of a C2 domain in plant PLD.

There now exists growing evidence that animal PLD may be involved in signal transduction, but this process is still rudimentary in plants. The availability of large amounts of baculovirus-derived recombinant PLD{alpha} described here will constitute a valuable source of enzyme for the study of the structure–function relationship of this key lipolytic enzyme.


    Notes
 
1 To whom correspondence should be addressed. E-mail: abousal{at}ibsm.cnrs-mrs.fr Back


    Acknowledgments
 
The authors are grateful to Dr R.Verger of the Laboratoire de Lipolyse Enzymatique (LLE, Marseille) for helpful discussions, J.Bonicel (IBSM, Marseille) for mass spectrometric analyses, Dr P.Mensuelle (UMR 6560, Marseille) and A.Guevara-Sala (IBSM, Marseille) for N-terminal sequence analysis and Dr J.Blanc for revising the English. This research was carried out with the financial support of the EC FAIR 97 3228 project of the European Union.


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received June 1, 2000; revised August 30, 2000; accepted September 8, 2000.





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