UPR 9025 du CNRS, Laboratoire de Lipolyse Enzymatique,31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France
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Abstract |
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Keywords: baculovirus/calcium ions/C2 domain/insect cells/octyl-Sepharose/phospholipase D
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Introduction |
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A eukaryotic PLD cDNA was first cloned and sequenced from castor bean (Wang et al., 1994
). The PLD
from maize (Ueki et al., 1995
), rice (Ueki et al., 1995
; Morioka et al., 1997
), cabbage (Kim et al., 1999
), cowpea (Vigna unguiculata) (El Maarouf et al., 1999
), Pimpinella brachycarpa (Cha et al., 1997
), Arabidopsis (Dyer et al., 1995
; Pappan et al., 1997a
; Qin et al., 1997
) and yeast PLD (Rose et al., 1995
) have since been cloned. In mammalian cells, two isozymes have been cloned and designated PLD1 and PLD2 (Hammond et al., 1995
; Colley et al., 1997
).
Three forms of PLD, known as PLD, PLDß and PLD
, have been described in Arabidopsis thaliana. They are encoded by distinct genes and differ in their catalytic properties and regulatory processes (Pappan et al., 1997b
, 1998
; Qin et al., 1997
). PLDß and PLD
require phosphoinositide as a cofactor and they are most active in vitro at micromolar calcium concentrations. PLD
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
from several plant species have been studied and found to show considerable similarities. The enzyme was purified from cabbage leaves (Lambrecht and Ulbrich-Hohhmann, 1992
; Abousalham et al., 1993
), castor bean endosperm (Wang et al., 1993
), rice (Ueki et al., 1995
), soybean cells (Abousalham et al., 1995
) and germinating sunflower seeds (Abousalham et al., 1997
).
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 cDNAs, from Ricinus communis and cabbage, were found to be expressed in Escherichia coli JM109 (Wang et al., 1994
; Kim et al., 1999
). These rPLD
showed a comparable low level of activity to that of the purified enzyme. Furthermore, the rPLD
were able to catalyze the hydrolytic and transphosphatidylation reactions. No rPLD
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 from cowpea in insect cells, along with the corresponding one-step purification procedure.
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Materials and methods |
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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 from cowpea (Vigna unguiculata) was obtained as described previously (El Maarouf et al., 1999
). The PLD
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
Ziplox plasmid (Invitrogen). A new EcoRI site was inserted by PCR upstream of the PLD
start codon in order to remove the 5' non-coding region. A 2700 bp EcoRI/BamHI DNA fragment containing the entire PLD
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., 1989
) 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 using the baculovirus expression system
Recombinant baculovirus production and rPLD expression were performed as described previously (Bezzine et al., 1999
). The pVL1392 baculovirus transfer vector containing the PLD
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 antibioticantimycotic 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
. An additional cell infection stage was performed to amplify the virus and generate the high-titer virus stocks (107109 pfu/ml or plaque-forming units per ml of culture) required for the recombinant protein production. The production of the rPLD
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 antibioticantimycotic solution. An inoculum of the recombinant baculovirus encoding rPLD
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
and to determine whether any proteolytic degradation had occurred.
PLD assay
PLD activity was assayed spectrophotometrically (Abousalham et al., 1997
) 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 TrisHCl, 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 were tested simultaneously. The amount of free choline released was quantified based on a calibration curve obtained with pure choline.
One unit of PLD 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 TrisHCl (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 chloroformmethanol (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 chloroformmethanolacetic 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
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 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
activity were pooled and concentrated 1020-fold by ultrafiltration in an Amicon cell with a PM 30 membrane. The pure rPLD
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, 1976) with Bio-Rad Dye Reagent and bovine serum albumin as the standard. Samples were separated by 10% SDSPAGE 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
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 TBSTween 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 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 (1046%) 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 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% acetonitrile60% water containing 0.1% trifluoroacetic acid).
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Results and discussion |
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rPLD was expressed in High Five insect cells using the baculovirus expression system. The rPLD
was found to be secreted by insect cells grown in monolayers. The first 34 amino acids encoded by the PLD
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., 1999
) and gastric lipase (Dupuis et al., 1997
). The active fraction of the rPLD
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
activity. The time course of rPLD
expression is shown in Figure 1
. rPLD
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
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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Most plant PLD bound strongly to a hydrophobic gel in the presence of Ca2+ ions (Lambrecht and Ulbrich-Hohhmann, 1992
; Abousalham et al., 1993
). We used this property to purify the main form of rPLD
(rPLD
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
b was eluted by decreasing the concentration of Ca2+ ions in the buffer from 50 to 30 mM (Figure 3
). Highly purified rPLD
b devoid of any detectable contaminants gives a single protein band at a position corresponding to a molecular mass of 88 kDa (Figure 3
inset, lane 2). The specific activities and recovery yield of the enzyme after the purification step are summarized in Table I
. 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
b was obtained with a specific activity of 25 U/mg (Table I
).
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The molecular masses obtained using mass spectrometric analysis were found to be 88 020 and 87 150 Da for rPLDa and rPLD
b, respectively (Table II
), which is consistent with the apparent molecular mass values, ranging from 87 000 to 90 000, obtained with other purified plant PLD
. The N-terminal amino acid sequence of rPLD
a was QNIEETVGIGKGVTK, whereas it was IGKGVTK in the case of rPLD
b. rPLD
b may result from either a proteolytic degradation of rPLD
a with the cleavage site occurring between Gly8 and Ile9 (Table II
) or an alternative cleavage of the signal peptide. The sequential appearance of rPLD
a first and rPLD
b second, as shown from the electophoretic analysis in Figure 2
, is in favor of the first hypothesis, however. Both rPLD
a and rPLD
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
(Lambrecht and Ulbrich-Hohhmann, 1992
; Abousalham et al., 1993
, 1995
: Wang et al., 1993
).
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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 described here will constitute a valuable source of enzyme for the study of the structurefunction relationship of this key lipolytic enzyme.
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Notes |
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() |
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Abousalham,A., Teissère,M., Gardies,A.-M., Verger,R. and Noat,G. (1995) Plant Cell Physiol., 36, 989996.[ISI][Medline]
Abousalham,A., Nari,J., Teissère,M., Ferté,N., Noat,G. and Verger,R. (1997) Eur. J. Biochem., 248, 374379.[Abstract]
Abousalham,A., Nari,J. and Noat,G. (1999) Curr. Top. Plant Biol., 1, 123132.
Ball,A., Nielsen,R., Gelb,M.H. and Robinson,B.H. (1999) Proc. Natl Acad. Sci. USA, 96, 66376642.
Bezzine,S., Ferrato,F., Lopez,V., de Caro,A., Verger,R. and Carrière,F. (1999) Methods Mol. Biol., 109, 187202.[Medline]
Bittova,L., Sumandea,M. and Cho,W. (1999) J. Biol. Chem., 274, 96659672.
Bradford,M.M. (1976) Anal. Biochem., 72, 248254.[ISI][Medline]
Cha,Y.Y., Lee,K.W., Kim,J.C., Han,T.G., Lee,W.S. and Cho,S.S. (1997) Plant Physiol., 114, 11351136.
Colley,W.C., Sung,T.C., Roll,R., Jenco,J., Hammond,S.M., Altshuller,Y., Bar-Sagi,D., Morris,A.J. and Frohmann,M.A. (1997) Curr. Biol., 7, 191201.[ISI][Medline]
Dupuis,L., Canaan,S., Riviere,M. and Wicker-Planquart,C. (1997) Methods Enzymol., 284, 261272.[ISI][Medline]
Dyer,J.H., Zheng,X. and Wang,X. (1995) Plant Physiol., 109, 1497.
Eibl,H. and Kovatchev,S. (1981) Methods Enzymol., 72, 632639.[Medline]
El Maarouf,H., Zuily-Fodil,Y., Gareil,M., d'Arcy-Lameta,A. and Pham-Thi,A.T. (1999) Plant Mol. Biol., 39, 12571265.[ISI][Medline]
Essen,L.-O., Perisic,O., Cheung,R., Katan,M. and Williams,R.L. (1996) Nature, 380, 595602.[ISI][Medline]
Essen,L.-O., Perisic,O., Lynch,D.E., Katan,M. and Williams,R.L. (1997) Biochemistry, 36, 27532762.[ISI][Medline]
Exton,J.H. (1999) Biochim. Biophys. Acta, 1439, 121133.[ISI][Medline]
Frohman,M.A., Sung,T.C. and Morris,A.J. (1999) Biochim. Biophys. Acta, 1439, 175186.[ISI][Medline]
Hammond,S.M., Altshuller,Y.M., Sung,T.C., Rudge,S.A., Rose,K., Engebrecht,J., Morris,A.J. and Frohman,M.A. (1995) J. Biol. Chem., 270, 2964029643.
Higgins,D.G., Thompson,J.D. and Gibson,T.J. (1996) Methods Enzymol., 266, 383402.[ISI][Medline]
Jones,D., Morgan,C. and Cockcroft,S. (1999) Biochim. Biophys. Acta, 1439, 229244.[ISI][Medline]
Kim,D.U., Roh,T.Y., Lee,J., Noh,J.Y., Jang,Y.J., Hoe,K.L., Yoo,H.S. and Choi,M.U. (1999) Biochim. Biophys. Acta, 1437, 409414.[ISI][Medline]
Kobayashi,M. and Kanfer,J.N. (1987) J. Neurochem., 48, 15971603.[ISI][Medline]
Laemmli,U.K. (1970) Nature, 227, 680685.[ISI][Medline]
Lambrecht,R. and Ulbrich-Hohhmann,R. (1992) Biol. Chem. Hoppe-Seyler, 373, 8188.[ISI][Medline]
Morioka,S., Ueki,J. and Komari,T. (1997) Plant Physiol., 114, 396.[ISI]
Nalefski,E.A. and Falke,J.J. (1996) Protein Sci., 5, 23752390.
Pappan,H., Qin,W., Dyer,J.H., Zheng,L. and Wang,X. (1997a) J. Biol. Chem., 272, 70557061.
Pappan,K., Zheng,S. and Wang,X. (1997b) J. Biol. Chem., 272, 70487054.
Pappan,K., Austin-Brown,S., Chapman,K.D. and Wang,X. (1998) Arch. Biochem. Biophys., 353, 131140.[ISI][Medline]
Perisic,O., Fong,S., Lynch,D.E., Bycroft,M. and Williams,R.L. (1998) J. Biol. Chem., 273, 15961604.
Ponting,C.P. and Kerr,I.D. (1996) Protein Sci., 5, 914922.
Ponting,C.P. and Parker,P.J. (1996) Protein Sci., 5, 162166.
Qin,W., Pappan,H. and Wang,X. (1997) J. Biol. Chem., 272, 2826728273.
Ritchie,S. and Gilroy,S. (1998) Proc. Natl Acad. Sci. USA, 95, 26972702.
Rose,K., Rudge,S.A., Frohman,M.A., Morris,A.J. and Engebrecht,J. (1995) Proc. Natl Acad. Sci. USA, 92, 1215112155.[Abstract]
Sambrook,J., Fritsch,E.F. and Maniatis,T. eds. (1989) Molecular Cloning. A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Shao,X., Davletov,B.A., Sutton,R.B., Sudhof,T.C. and Rizo,J. (1996) Science, 273, 248251.[Abstract]
Sutton,R.B., Davletov,B.A., Berghuis,A.M., Südhof,T.C. and Sprang,S.R. (1995) Cell, 80, 929938.[ISI][Medline]
Takrama,J.F. and Taylor,K.E. (1991) Biochem. Biophys. Methods, 23, 217226.[ISI][Medline]
Ueki,J., Morioka,S., Komari,T. and Kumashiro,T. (1995) Plant Cell Physiol., 36, 903914.[ISI][Medline]
Wang,X. (1999) Plant Physiol., 120, 645651.
Wang,X., Dyer,J.H. and Zheng,L. (1993) Arch. Biochem. Biophys., 306, 486494.[ISI][Medline]
Wang,X., Xu,L. and Zheng,L. (1994) J. Biol. Chem., 269, 2031220317.
Xu,G.-Y., McDonagh,T., Yu,H.-A., Nalefski,E., Clark,J.D. and Cumming,D.A. (1998) J. Mol. Biol., 280, 485500.[ISI][Medline]
Received June 1, 2000; revised August 30, 2000; accepted September 8, 2000.