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Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris
Accepted Manuscript Title: Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris Authors: Aida Karray, Madiha Bou Ali, Nedia Kharrat, Youssef Gargouri, Sofiane Bezzine PII: DOI: Reference:
S0141-8130(17)30377-X https://doi.org/10.1016/j.ijbiomac.2017.11.045 BIOMAC 8525
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
29-1-2017 7-11-2017 8-11-2017
Please cite this article as: Aida Karray, Madiha Bou Ali, Nedia Kharrat, Youssef Gargouri, Sofiane Bezzine, Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.11.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris
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Karray Aida, Bou Ali Madiha, Kharrat Nedia, Gargouri Youssef and Bezzine Sofiane*
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra,
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université de Sfax-Tunisia. *
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E-mail: [email protected]
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: + 21674675055
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Tel /Fax
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Correspondence address : Pr. Sofiane Bezzine, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra, université de Sfax-Tunisia.
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Highlights
This study describes the expression of group V secreted PLA2 in Pichia pastoris.
Purification, biochemical and biological characterization of PLA2-V is studied.
Antibacterial, antifungal and anticoagulant activities of ChPLA2-V
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Abstract
Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart 1
polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger
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anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be
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considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent.
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Key words: group V secreted Phospholipase A2; antimicrobial activity; anticoagulant activity;
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1.Introduction
Phospholipases A2 (PLA2) EC 3.1.1.4 are a family of enzymes that hydrolyze the ester bond
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at the sn-2 position of phospholipids generating free fatty acids and lysopho-spholipids [1].
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This family includes a number of secreted PLA2s (sPLA2s) referred to as group IB (GIB), GII (subgroups A-F), GIII, GV, GX and GXII (subgroupsA–B)[2]. Clearly, the different mammalian sPLA2s are not isoforms as their sequence identities are only around 15%[3; 4]
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they have distinct enzymatic properties [5] and show different tissue distribution patterns in both mice and humans. Consequently, in various tissues, the different sPLA2s may exert
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distinct biological functions that may be dependent or independent of their enzymatic activities [3; 6; 7].
Much less is known about the regulation and biological roles of phospholipases A2 from
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birds. So that, we focused our current study on the identification of novel phospholipases A2 from chicken using biochemical and molecular techniques. Group V sPLA2 has been cloned from chicken [8], human, rat, and mouse species [9]. Unlike group I and II sPLA2s, this sPLA2 has only six disulfides and does not have the group I- or group II-specific disulfides, thus defining a novel group of sPLA2s [10]. This sPLA2 has a higher level of identity with group IIA sPLA2s, as compared to group IB sPLA2. It neither has a propeptide sequence, indicating its closer relationship with group II sPLA2s. In a previous work, quantitative PCR 2
shows that the expression patterns of chicken PLA2-V resemble to that of mammals with a high expression level in the heart (6.83), intestine (5.235) and liver (3.01). Thus, chicken sPLA2-V is preferentially expressed in heart and intestine, at lower extent in liver, but it wasn’t detected in lung, spleen and colon [8]. Whereas, mammalian sPLA2-V has a preferentially expression level in heart and a much lower expression level in lungs and liver [11]. The relative expression level of ChPLA2-V, has a marked increase in its expression level in the viral infected lungs of chicken [8]. In fact, no or very weak sPLA2 expression was
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observed in normal chicken lungs and spleen suggesting a protective role of this group of sPLA2.
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Because of its up regulation during lung inflammation, and contrary to the variations observed
in the expression level of sPLA2-IB, -IIA and -X, only PLA2-V can be considered as a potential biomarker of the infectious bronchitis disease in chicken.
Diverse biological functions have been attributed to groupV sPLA2. These functions are
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attributed to the ability of this enzyme to provide arachidonic acid for the synthesis of
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eicosanoid. [12]. Additional functions have also been demonstrated; including regulation of
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phagocytosis, foam cell formation, and antibacterial activities [13; 14].
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However, the group V sPLA2 has been isolated and purified from few tissues or cells and its biochemical properties and structural features have not been explored. To understand the
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structure, function and regulation of this important sPLA2, we have synthesized the cDNA by RT-PCR from chicken heart polyA. mRNA, constructed a bacterial expression vector for the chicken group V sPLA2, over-expressed it in Pichia pastoris, and purified it to homogeneity
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using classical technique of chromatography. The active ChPLA2-V was characterized as to its calcium dependence, pH optimum, substrate preferences and biological activities.
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2.Materials and Methods
2.1. Strains, Cell Culture and Vectors The Escherichia coli strain DH5 Alpha was used as a host for cloning the ChPLA2-V PCR fragment in the P.pastoris transfer vector pGAPZA (Invitrogen). The P.pastoris host strain
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was X33 (wild type strain from Invitrogen). The P.pastoris transfer vector pGAPZA (Invitrogen) was used for yeast transformation. The Pfu DNA polymerase, T4 DNA ligase, PCR purification kit and Midi-Prep Kit were purchased from Promega. Pichia pastoris liquid cell cultures were grown in YPD medium containing 10 g yeast extract, 20 g Bacto-peptone and 20 g D-glucose.
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The YPDS medium was YPD medium to which 18.2 g sorbitol per liter were added. To prepare plates for solid cell cultures, 2% agar (w/v) were added to the YPD medium. The Deoxycholic acid sodium salt (NaDC) (purity 99%) was purchased from Bio Basic Inc. 2.2. Construction of the DNA Encoding the ChPLA2-V cDNA synthesis and amplification Total mRNAs were isolated from chicken heart using the single step guanidine isothiocyanate/phenol/chloroform isolation method as described by
acid guanidinium thiocyanate-phenol-chloroform extraction [15].
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Chomczynski and Sacchi Chomczynski P, Sacchi N: Single-step method of RNA isolation by
ChPLA2-V cDNA was obtained from total mRNAs by the reverse transcription procedure
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(Promega). First strand cDNAs were prepared using 10 μg of total mRNAs as template (heatdenaturated for 5 min at 70°C,) 200 U MMLV reverse transcriptase (Invitrogen), 20 pmol of each deoxynucleoside triphosphate, and 20 pmol of each primer: forward primer, 5’-
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GTGTGTGTGAATTCATGAGCCTCTGGCAGCTGCAGGAG -3’ and reverse primer, 5’GTGTGTCTCGAGTCACCTGCACTTGCACCTGGG -3’. The N-and C-terminal primer
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were deduced from the genome of Gallus gallus (GenBank accession number: JF 411004).
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Reverse transcription was carried out in a total reaction volume of 20 μl for 5 min at room
for 15 min and cooled on ice.
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temperature and 60 min at 42°C. The cDNA/RNA heteroduplex was then denaturated at 70°C
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2.3. Cloning of the mature PLA2 gene
Amplification of the specific ChPLA2-IIA cDNA was carried out by PCR using the single strand cDNAs as template with the forward and reverse primers previously described. PCR
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was performed in a 0.2 ml Eppendorf tube with a Gene Amp® PCR System 2700. The PCR mixture contained 20 pmol of both primers, 20 pmol of each deoxynucleoside triphosphate, 5
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U pfx polymerase and polymerization buffer in final volume of 100 μl. The single strand DNAs were directly used as template. The thermal profile involved 35 cycles of denaturation at 94°C for 1 min, primer annealing at 60° C for 1 min, and extension at 72°C for 3 min. The PCR product (500 pb) was isolated and ligated into the EcoRI and XhoI linearized and
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dephosphorylated pGAPZA vector, according to the manufacturer’s protocol (Promega). Protoplasts of E. coli DH10B were transformed with the ligation mixture. The presence of the appropriated insert was verified by restriction analysis. DNA sequences were elucidated by the dideoxynucleotide chain termination method according to a cycle sequencing protocol using thermosequenase (Amersham Pharmacia Biotech). The sequencing reactions were analyzed with the DNA sequencer ABI PRISM 3100/3100-Avant Genetic Analyzer 4
(California, USA). It was performed three times, using the recombinant vector pChPLA2 as template with PGAPZ promoter primer and the AOX reverse primer (Invitrogen). 2.4. Transformation of P.pastoris and Screening of ChPLA2-V Secreting Transformants Electrocompetent P.pastoris X-33 cells were prepared using standard methods [16] and their transformation was performed by electroporation according to Invitrogen manual. Prior to the yeast transformation procedure, recombinant vector (pGAPZaA/ChPLA2-v) was linearized by the restriction enzyme BspHI. The recombinant
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yeast clones were selected on YPDS plates containing 100 mg/ml Zeocin. The colonies were subsequently screened by performing PCR reaction using as template the genomic DNA of
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the selected clones to confirm the integration of the pGAPZaA/ChPLA2-V vector into the yeast genomic DNA [17]. Selected transformants were grown in 50 mL of YPD medium with 100 mg/ml Zeocin at 30°C under shaking at 160 rpm. Time course of ChPLA2-V secretion in
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the culture media was determined for various clones.
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2.5. Expression of ChPLA2-V in P. pastoris
The most efficient ChPLA2-V secreting selected transformant was pre-grown at 30°C in 250
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ml shake flasks containing 50 ml YPD medium with 100 mg/ml Zeocin for 24 h to an OD600
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of 4–6. This cell culture was further used to inoculate 500 ml shake flask containing 100 ml YPD medium without Zeocin. The production of ChPLA2-V was conducted at 30°C for 48
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hours with shaking at 150 rpm.
2.6. Purification of the ChPLA2-V Protein
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The culture was harvested by centrifugation (9500 rpm, 10 min, 4°C). The supernatant was subjected to ammonium sulphate fractionation (20–75%). The precipitates obtained after centrifugation at 15000 rpm for 30 min were resuspended in the
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buffer A and loaded on a column of sephadex G-75 equilibrated with buffer A (20 mM TrisHCl, pH 8, 20 mM CaCl2). The resulting sample containing PLA2 activity (4 ml, 240 U) was poured into a Mono-S Sepharose column equilibrated with buffer A. The pooled fractions
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were concentrated using an Amicon® stirred cells. Gas pressure is applied directly to the ultrafiltration cell. Solutes above the membrane's molecular weight cut-off of 5 kDa, are retained in the cell, while water and solutes below the cut-off pass into the filtrate and out of the cell. Gentle magnetic stirring minimizes concentration polarization and shear denaturation.
Active fractions were analyzed with 15%-SDS-PAGE and the ChPLA2-V activity was measured as described elsewhere. 2.7. Phospholipase activity 5
PLA2 activity was measured titrimetrically at pH 9.5 and at 37 °C with a pH-stat (Metrhom), under the standard assay conditions described previously[18] using Phosphatidyl-Choline (0.5% w⁄ v) in 30 ml of 150 mM NaCl, 4 mM CaCl2, and 1 mM NaTDC. One PLA2 activity unit corresponds to one µmole of fatty acid liberated per minute. 2.8. SDS-PAGE Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed by the method of Laemmli [19]. The proteins were
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tained with Coomassie brilliant blue. 2.9. Amino acid sequencing
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For N-terminal sequencing, the purified enzyme was blotted (60 min, 50 mA, 4 °C) onto a PVDF (polyvinylidene difluoride) membrane (Applied Biosystems, ProBlotTM) in 20 mM CAPS buffer (pH 11) containing 10% methanol using a mini trans-blot cell (BioRad, Hercules, USA). The N-terminal sequence was determined by automated Edman's degradation, using an Applied Biosystems Protein Sequencer Procise 492 equipped with 140 C HPLC system (Roissy, France). [20]
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2.10. Antimicrobial Activity
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2.10.1. Determination of zone of inhibition method
method [21; 22; 23].
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The antibacterial and antifungal activities of ChPLA2-V was checked by well diffusion
Briefly, bacteria were cultivated in BHI medium at 37°C for 3 h. A basal layer of Brain Heart
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Infusion (BHI) containing 2-5% agar, was poured in Petri dishes. When plates were dried, 5 ml of soft BHI (0-7% agar) containing 107 cells of the indicator strain were overlaid. BHI was
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from Hispanlab, S.A (Madrid).
Then, wells were punched in the agar plate and filled with 50 μg of test samples. The zones of growth inhibition around the well were measured after 18 hours to 24 hours of in incubation at 37°C for bacteria and 48 hours to 96 hours for fungi at 30°C. The sensitivities of
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the microorganism species to the ChPLA2-V were determined by measuring the sizes of inhibitory zones (including the diameter of disk) on the agar surface around the well, and values <8 mm were considered as not active against microorganisms. One arbitrary unit (AU) of antibacterial activity was defined as the amount of ChPLA2-V sufficient to give a zone of inhibition around the well. The bacteria used were Staphylococcus. aureus (SA), Bacillus cereus (BC), Bacillus subtilis (BS), Pseudomonas aeruginosa, (Ps), Salmonella (S), Klebsielle 6
pneumoniae (KP) and E. coli (ECo). The zones of growth inhibition around the disks were measured after 18 to 24 hours of incubation at 37°C for bacteria and 48 to 96 hours for fungi at 30°C. 2.10.2. Determination of the minimum inhibitory concentration (MIC) The Minimal Inhibitory Concentrations (MICs) of ChPLA2-V against the tested microorganisms were determined by the broth microdilution method [24]. All tests were performed in LB. Strains were cultured overnight at 37°C in LB. Test strains were suspended
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in LB (The initial absorbance measured at 600 nm was approximately adjusted to 0.2 Optical
density (OD) value). Geometric dilutions ranging from 100 μg/ml to 1.5 μg/ml of ChPLA2-V
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were prepared in 96-well microtiter plate, including one growth control (LB only). Plates
were incubated under normal atmospheric conditions at 37°C for 24 h. Absorbance was then measured at 600 nm and MICs values were determined as the lowest ChPLA2-V concentrations inhibiting visible growth of bacterial strains. Tests were performed in
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duplicates.
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2.11. Hemolysis assay
Hemolytic activity of the pure ChPLA2-V was tested using erythrocytes from human, rabbit
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and rat. Freshly collected, blood samples were immediately mixed with anticoagulant,
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Alsever’s solution (pH 7.4) to prevent blood coagulation. To obtain a pure suspension of erythrocytes, 1 ml of whole blood was made up to 20 ml in phosphate buffered saline (PBS,
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pH 7.4), and centrifuged at 250 rcf for 5 min at 4 °C. The supernatant and buffy coats were removed by gentle aspiration, and the above process was repeated two more times.
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Erythrocytes were finally resuspended in PBS to make 1% solution for hemolytic assay. For this, various concentrations of pure ChPLA2-V (0.05–1 mg/ml) were added to the suspension of red blood cells obtained from different species (human, rabbit and rat). The pure ChPLA2-
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V erythrocyte mixtures were incubated at 37 °C for 30 min in water bath and then centrifuged at 250 rcf for 5 min at 4°C. The absorbance of the supernatants was determined at 545 nm to measure the extent of red blood cell lyses. Positive control (100% hemolytic) and negative
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control (0% hemolysis) were also run by incubating erythrocytes in PBS containing 1% Triton X-100 and PBS alone, respectively [25]. 2.12.Anticoagulant activity Sheep citrated plasma was centrifuged at 1000 g at 4ºC and the anticoagulant effect was assessed as described by Gutiérrez et al. (1986a) [26]. Subsequently, 0.25 ml aliquots of plasma were incubated with 50 μl of solutions containing the following quantities of PLA2, from 0.39; 0.78; 1.56; 3.13; 6.25; 12.5 and 25μg) dissolved in phosphate buffered saline 7
(PBS, pH 7.5) for 10 min at 37ºC. Then, 0.1 ml of a solution of 0.25 M CaCl2 was added and the clotting time determined. Control tubes contained only PBS incubated plasma, while CaCl2 was added as described above. Observations were carried out for a maximum period of 60 min.
3.Results
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3.1. Construction of ChPLA2-V Expression Vector
The DNA fragment encoding for the ChPLA2-V (500 bp) was amplified by PCR using
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plasmid DNA (pGAPZαA/ChPLA2-V) as template with specific primers containing the
EcoRI site upstream of its first codon and Xho site downstream of its last codon. The amplified fragment was digested with EcoRI/Xho and inserted into the pGAPZαA vector
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previously digested with the same enzymes, in frame with the yeast α-factor signal sequence at the N-terminal side for protein secretion. The integrity of the construction (pGAPZαA/Ch-
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PLA2-V) was confirmed by DNA sequencing (data not shown).
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3.2. Generation of Recombinant Clones Expressing ChPLA2-V The P.pastoris X33 strain was transformed by electroporation using (pGAPZαA/ ChPLA2-V) plasmid DNA linearized by the BspHI restriction enzyme. P.pastoris tranformants were
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selected on YPDS-Zeocin plates and incubated at 30°C for 3 days.
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3.3. Selection of ChPLA2-V -secreting Transformants Transformants carrying the ChPLA2-V gene (C1-C6) were grown on a YPD medium in 250
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ml Erlen-meyer’s flasks with shaking at 150 rpm, 30°C to select the clone showing the highest phospholipase activity level. The time-course of the ChPLA2-V secretion by the P.pastoris clones was performed (data not shown). The C3 clone exhibited the highest activity level reaching about 0.8 U/ml after 48 hours of culture. This clone was then selected
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for the production and purification of the recombinant ChPLA2-V 3.4. Purification of the ChPLA2-V ChPLA2-V was purified successively by the steps procedure described in material and methods. The results of the purification procedure are summarized in Table 1. Figure 1A represents the elution profile of gel filtration through a Sephadex G-75 column (95 cm × 2.6 8
cm) equilibrated with buffer A. Elution of proteins was performed with the same buffer at 30 ml/h. The fractions containing the PLA2 activity was detected in a fraction eluted as a single peak between 1.5 and 2 void volumes. The second chromatographic step used is Mono-S Sepharose column equilibrated with buffer A. The column (5 cm × 2 cm)was then washed with the same buffer containing 0.1 M NaCl. Linear salt gradient (0.1 to 0.5 M NaCl, dotted line) was applied to the column. Results are shown in the figure 1B.
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After the final purification step, ChPLA2-V was purified 9 fold, with a recovery of 48%. It has a specific activity of 157 U/mg, using egg yolk emulsion as substrate in the presence of 4
mM NaDC and 4 mM CaCl2. The fractions containing the ChPLA2-V activity were pooled
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and analyzed by SDS-PAGE (Figure 1C). This figure shows that group V-ChPLA2 is homogenously pure and has an apparent molecular mass of about 14 kDa. These data obtained in the native and denaturized conditions suggested that ChPLA2-V was a monomeric protein
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like pancreatic and intestinal ones described in our previous works [27; 28].
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The molar absorption coefficient of ChPLA2-V was determined using Expazy tool: ProtParam tool. Once the protein sequence is copied, the value of the extinction coefficients
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in units of M-1 cm-1, at 280 nm measured in water was calculate. In these conditions, the
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molar absorption coefficient was 37650 M-1 cm-1. The experimental results using the formula OD280=£*L*C confirmed the bioinformatics results. (Concentration C was determined with
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Bradford reactif and expressed on mg/ml).
3.5. Ca2+dependence and pH optima of ChPLA2-V
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The Ca2+ dependence of enzymatic activity is one of the major defining characteristics for sPLA2. The Ca2+ dependence of the ChPLA2-V was examined by measuring enzymatic
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activity at deferent concentrations of calcium. In the presence of 1 mM EDTA and the absence of calcium, the enzyme was completely inactive. Figure. 2A shows the Ca2+ dependence of the ChPLA2-V. A typical saturation curve was obtained and indicated a
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Specific Activity of 157 U/mg for Ca2+ of about 6 mM. All crystal structures of sPLA2 have a `calcium binding loop' in the protein [29; 30; 31] and the calcium dependence of the group V PLA2 is similar to that of the human group IIA PLA2 [32]. The optimum pH for ChPLA2V was examined with 50 mM buffer. It had a broad pH optimum, but the maximal enzymatic activity occurred at pH 8.5 with 50 mM Tris-HCl (Figure 2B).
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6. Microbial activity of ChPLA2-V The antimicrobial activity of the ChPLA2-V were studied in different concentrations (3-50 µg/ml) against several Gram+ and Gram- bacterial strains and three fungal strains (Aspergillus niger MTCC 282 Trichoderma citrino- viride MTCC 1323 and Fusarium solani MTCC 227. Antibacterial and antifungal potential of ChPLA2-V were assessed in terms of zone of inhibition of microbial growth. The results of the antibacterial and antifungal
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activities are presented in table 2, 3 and 4. [33]. ChPLA2V was tested against several Grampositive bacteria: Bacillus cereus (BC), Staphylococcus aureus (SA), Bacillus subtilis (BS), Micrococcus luteus (ML), Brevibacterium flavum (BF), Enterococcus faecalis (EF),
and Gram-negative bacteria: Pseudomonas Aeruginosa,
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Staphylococcus epidermidis (Sep)
(Ps) Enterobacter cloacae (EC), Klebsielle pneumoniae (KP), Salmonella (S) and E. coli (ECo). Bacteria were incubated for 24 hours. ChPLA2-V was active against closely related Bacillus
aeruginosa and E. coli
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species, Staphylococcus aureus but also some other Gram-negatif bacteria : Pseudomonas (Tables 2 and 3). Indeed, the enzyme exhibited an important
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bactericidal effect against BC, BS and SA with a diameter of inhibition higher than 20 mm. A
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moderate bactericidal effect was obtained with a diameter of inhibition between 15 and 20
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mm against E coli and Pseudomonas aeruginosa. The bactericidal effects of ChPLA2-V were also tested by measuring the minimum inhibition concentration (MIC) of ChPLA2-V for SA, BS, BC, PA and Eco was also investigated. Table 3 shows that ChPLA2-V has the most
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potent effect on Gram + strains. It exhibits the lowest MIC values towards the three tested strains (25 μg/ml against staphylococcus strains, while MIC values towards Eco (gram
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negative bacteria) were estimated to more than 50 μg/ml. Finally, ChPLA2-V does not exhibit any antimicrobial effect up to 5 mg/ml against S. epidermidis, Micrococcus Luteus (ML),
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Enterococcus faecalis (Ef.), E. faecium (EF), Enterobacter cloacae (Ec), Brevibacterium flavum (BF), Salmonella (S) and Klebsielle pneumonia (K). The antifungal activities of the ChPLA2-V increased linearly with increase in concentration
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of enzyme extracts (μg/ml). The results summarized in table 4 revealed that for antifungal activity, Aspergillus niger shows good result as compare with Trichodorma citrino virid and Fusarium solani. The growth inhibition zone measured ranged from 11 to 20 mm for all the sensitive bacteria, and ranged from 2 to 9 mm for fungal strains 3.7. Hemolytic activity of ChPLA2V
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Our results show that pure ChPLA2V used in this study did not exhibit any significant hemolytic activity. 3.8. Anticoagulant activity of ChPLA2V and ChPLA2IIA The recalcification time of sheep platelet-poor plasma was prolonged after incubation with isolated phospholipase A2. Clotting time showed a direct dependence according with the amount of enzyme considered, from 0.08 to 0.800 µmol (Figure. 3). The anticoagulant
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activity of the ChPLA2V was evaluated by measuring the partial thromboplastin time
activator (TTA) using Kaolin as an activator. This is to take a delay of coagulation of the
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plasma, based on the addition of an increasing concentration of enzyme. The results are
compared with the value of a normal report 1.2. Figure 3A shows that the anticoagulant activity was positive for ChPLA2V. It is noted that a low concentration of the ChPLA2-V (0.08 µmol) possesses anticoagulant activity, compared with the CPLA2-IIA, which shows an anticoagulant
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activity at a concentration of 0, 2 µmol. In addition, with the ChPLA2V, the ratio TTA of the sample/ normal TTA reaches a value of 4, compared with the ChPLA2-IIA which shows a ratio of 1.6 (for the
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same enzyme concentrations used).
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Discussion
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Contrary to the previously reported expression of the sPLA2 using E.coli system accumulated in inclusion bodies [34], this study describes, and for the first time, the expression of group V
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secreted PLA2 using Pichia pastoris. Unlike the classical prokaryotic system, in which the solubilization of inclusion bodies with guanidine hydrochloride was essential for the purification and folding to obtain the active enzyme, the protein expression in Pichia pastoris
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is directly obtained from the culture medium. Knowing that pancreatic PLA2-IB and intestinal PLA2-IIA are the most abundant enzymes
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and the best documented in mammals among all secreted PLA2. In order to gain more informations about their bird’s orthologs, we have recently purified and characterized PLA2IB from chicken pancreas (delipidated powder) than PLA2-IIA from intestinal mucosa. Very few studies were done on group V PLA2 enzyme, it is consequently interesting for us to
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study, for the first time, a newly purified chicken secreted PLA2-V from the heart of the bird and to provide basic information about its main biochemical and biological characteristics. Six Zeocin-resistant clones picked on the solid selective medium were selected and the integration of ChPLA2-V gene was analyzed by PCR using the pGAPZαA universal primers (pGAP Forward and AOX1 primers). C3 and C6 clones showed the amplification of the expected size (1040 bp). This fragment corresponded to the size of the ChPLA2-V gene (500 11
bp) plus a portion of the vector (540 bp). This result confirms the integration of the expression cassette (pGAPZαA/ ChPLA2-V) in P. pastoris genomic DNA. . Actually, there is no solid evidence that each integration event on the yeast genome contributes equally to the levels of the expressed protein. The multiple integration events have little detrimental effect on the expression of secreted protein in Pichia pastoris since other factors can also affect the
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expression level [35].
The purification flow sheet presented in Table.1 shows that the specific activity (SA) of pure
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ChPLA2-V reached 157 U/mg when egg yolk was used as substrate at pH 8.5 and 37°C, in the presence of 4 mM NaTDC and 4 mM CaCl2, value which is comparable to what observed
with the PLA2-V from stingray Dasyatis pastinaca recently studied wich shows a SA of 56U/mg on the presence of 6 mM NaTDC and 4 mM CaCl2. [36]. The ChPLA2-V
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purification yield was around 48% of the total initial activity. The procedure described and
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summarized in table 1 is more rapid than those used previously to purify other group V phospholipase A2 [37]. In fact, chicken PLA2 was purified after only two chromatographic
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steps whereas in the case of dromedary or porcine PLA2 four chromatographic steps were
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needed [18].
Starting with the whole P. pastoris cell extract, 9-fold purification was achieved and the
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overall recovery of enzyme activity was 48%. From 800 ml of P. pastoris cell culture media, about 1.12 mg of pure ChPLA2-V was obtained with a specific activity of 157 U/mg (Table
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1). Specific activity of 156 U/mg for Ca2+ of about 4mM and the maximal enzymatic activity occurred at pH 8.5 with 50 mM Tris-HCl. The apparent molecular mass value of the
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recombinant PLA2-V was found to be 14.000 Da, The N-terminal amino acid sequence of recombinant PLA2-V was found to be SLWQLQEVVTK. Because of its up regulation in infected chicken lungs, and the negative variations observed in the expression levels of ChPLA2-IB, -IIA and –X in the lungs [8], we can considerate ChPLA2-V as a specific
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biomarker of the infectious bronchitis disease in chicken . Lapointe et al. [38] investigated the effect of sPLA2-V on lipopolysaccharide mediated leukocyte recruitment supporting the contribution of sPLA2-V in the development of inflammatory innate immune response and its ability to modulate adhesion molecule expression. In the present work, the antimicrobial activity of the pure enzyme was performed. Firstly, we have demonstrated that only 25 μg/ml (final concentration) of ChPLA2-V is able to kill 100% 12
of BS, BC, and SA and. The same amount of enzyme kills 60% of Gram negative bacteria PA and E.coli. Whereas, even at a final concentration of 50 μg/ml, ChPLA2-V was inactive against all the other stains tested. The differences observed are due to the cell’s membrane composition. The cell wall of gram positive bacteria contains a dense peptidoglycan array, which bears a high anionic charge due to the presence of phosphate diester units of lipoteichoic acid. The sPLA2 must go through the highly anionic cell wall of Gram positive bacteria to reach the phospholipids membrane target. ChPLA2-V, with an electrostatic
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potential of +14(+22; -8) based on the total number of Lys, Arg, His, Asp and Glu, can bind highly anionic bacterial cell walls [39] and could explain the antibacterial activity observed.
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Secondly, the antifungual activity was tested against several fungi. In this work we demonstrated that the most important inhibition grouth was observed against Aspergillus
niger with a diameter of 9 mm using 75 µg of pure enzyme. Furthermore, in vivo studies done
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on mammals, (Balboa et al., 2004) [40] have shown that PLA2V are secreted by immune cells during phagocytosis of bacteria and fungi. The presence of these antimicrobial activities may
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be due to hydrolysis of phospholipid membranes of bacteria and fungi by the enzyme.
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With reference to the anticoagulant activity of PLA2, we observed that when
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increasing the concentration of the enzyme the clotting time is prolonged. Díaz and coworkers [41] suggest that the anticoagulant effect of myotoxic phospholipases A2 from Bothrops
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venoms is dependent on enzymatic degradation of phospholipids necessary for the adherence and activation of coagulation protein factors. This hypothesis is supported by the observation that myotoxins prolong both recalcification time and prothrombin time, but not thrombin
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time, since only the former two depend on plasma phospholipids [42] Figure 3 shows an important anticoagulant activity of the ChPLA2V observed at a low
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concentration: 0,08 μM, compared with the ChPLA2-IIA. The latest didn’t express any anticoagulant activity at a concentration lower than 0,2 µM. Moreover, the ratio’s value of TTA /TTA control) in presence of ChPLA2V reaches 4, compared with that observed in presence of ChPLA2-IIA, which reaches the value of 1,6 (for the same enzyme concentration
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tested). As sets for Group IIA of PLA2, the anticoagulant activity is supported by the basic residues located at the site of interaction with the substrate (IBS) that govern the interaction with factor Xa clotting [43]. These basic residues constituting the IBS are well conserved in the ChPLA2V, (shown in purple in Figure 4), based on the sequence alignment of chicken’s group IIA PLA2 and group V PLA2. Basic clusters of the IBS of the enzyme are indicated in:
13
cyan for K10/K16 /R115/K116/K74/ R92; and K38. Hydrophobic residues of the IBS of the ChPLA2-V are indicated in purple: L2, , A19, L20, and V31(Figure 4). This test is still in the preliminary study of the anticoagulant activity of chicken sPLA2V. Indeed, it is necessary to test the anti-prothrombinase activity in presence of purified fractions of prothrombinase, namely factors Va and Xa, phospholipids and calcium. 4. Conclusion This study describes, and for the first time, the expression of group V secreted PLA2 using
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Pichia pastoris. Biochemical and biological characterizations of the pure enzyme was performed. Thus, chicken group V PLA2, may be considered as a future therapeutic agents
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against fungal and bacterial infections and as an anticoagulant agent.
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REFERENCES
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[1] R.H. Schaloske, and E.A. Dennis, The phospholipase A2 superfamily and its group numbering system. Biochim Biophys Acta 1761 (2006) 1246-59. [2] C.M. Mounier, D. Wendum, E. Greenspan, J.F. Flejou, D.W. Rosenberg, and G. Lambeau, Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate. Br J Cancer 98 (2008) 587-95. [3] E. Valentin, and A.G. Singer, Ghomashchi, F., Lazdunski, M., Gelb, M. H., and Lambeau, G, Cloning and recombinant expression of human group IIF secreted phospholipase A2. Biochem. Biophys. Res. Commun 279 (2000) 223-228. [4] M. Rouault, J.G. Bollinger, M. Lazdunski, M.H. Gelb, and G. Lambeau, Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity. Biochemistry 42 (2003) 11494503. [5] A.G. Singer, F. Ghomashchi, C. Le Calvez, J. Bollinger, S. Bezzine, M. Rouault, M. Sadilek, E. Nguyen, M. Lazdunski, G. Lambeau, and M.H. Gelb, Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2. J Biol Chem 277 (2002) 48535-49. [6] K.F. Scott, G.G. Graham, and K.J. Bryant, Secreted phospholipase A2 enzymes as therapeutic targets. Expert Opin Ther Targets 7 (2003) 427-40. [7] J. Ishizaki, N. Suzuki, K. Higashino, Y. Yokota, T. Ono, K. Kawamoto, N. Fujii, H. Arita, and K. Hanasaki, Cloning and characterization of novel mouse and human secretory phospholipase A(2)s. J Biol Chem. 274 (1999) 24973-9. [8] B.A.Y. Karray A, Boujelben J, Amara S, Carrière F, Gargouri Y, Bezzine S., Drastic changes in the tissue-specific expression of secreted phospholipases A2 in chicken pulmonary disease. . Biochimie 94(2) (2012 Feb) 451-60. [9] H. Yang, S.G. Cao, L. Ma, Z.T. Ding, S.D. Liu, and Y.H. Cheng, A new kind of immobilized lipase inorganic solvent and its structure model. Biochem Biophys Res Commun 200 (1994) 83-88.
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[10] F. Ferrato, F. Carrière, L. Sarda, and R. Verger, A critical re-evaluation of the phenomenon of "interfacial activation". in: E. Dennis, and B. Rubin, (Eds.), Methods in Enzymology., Academic Press, New York, 1997, pp. 327-347. [11] L. Touqui, and Y.Z. Wu, Interaction of secreted phospholipase A2 and pulmonary surfactant and its pathophysiological relevance in acute respiratory distress syndrome. Acta Pharmacol Sin 24 (2003) 1292-6. [12] Y.J.K. Nilda M. Muñoz‡, Angelo Y. Meliton‡, Kwang Pyo Kim§, Sang-Kyou Han§, Evan Boetticher‡, Eileen O'Leary¶, Shigeharu Myou‡, Xiangdong Zhu‡, Joseph V. Bonventre¶, Alan R. Leff‡∥ and Wonhwa Cho, . Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils. J. Biol. Chem 278 (2003) 7.
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[13] A.M. Barbara Balestrieri, Wei Xing, Michael H. Gelb, Howard R. Katz, and Jonathan P. Arm, Group V Secretory Phospholipase A2 Modulates Phagosome Maturation and Regulates the Innate Immune Response against Candida albicans. J. Immunol 182 (2009) 7. [14] Y.L. Eric Boilard, Katherine Larabee, Barbara Balestrieri, Farideh Ghomashchi, Daisuke Fujioka, Reuben
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Gobezie, Jonathan S. Coblyn, Michael E. Weinblatt, Elena M. Massarotti, Thomas S. Thornhill, Maziar Divangahi, Heinz Remold, Gérard Lambeau, Michael H. Gelb, Jonathan P. Arm, David M. Lee , A novel anti‐inflammatory role for secretory phospholipase A2 in immune complex‐mediated arthritis. . EMBO Mol. Med 2 (2010) 15.
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[15] P. Chomczynski, and N. Sacchi, Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 (1987) 156-159. [16] R.K. Cregg JM1, Transformation. Methods Mol Biol. 103 (1998) 12. [17] B. Sias, F. Ferrato, P. Grandval, D. Lafont, P. Boullanger, A. De Caro, B. Leboeuf, R. Verger, and F. Carriere, Human pancreatic lipase-related protein 2 is a galactolipase. Biochemistry 43 (2004) 10138-48. [18] G.H. de Haas, N.M. Postema, W. Nieuwenhuisen, and L.L.M. van Deenen, Purification and properties of phospholipase A from porcine pancreas. Biochim. Biophys. Acta 159 (1968) 103-117. [19] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. [20] R.M. Hewick, M.W. Hunkapiller, L.E. Hood, and W.J. Dreyer, A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 256 (1981) 7990-7. [21] B.S. Paik HD, Park SH, Pan JG, Identification and partial characterization of tochicin, a bacteriocin offduced by Bacillus thuringiensis subsp tochigiensis. J Ind Microbiol Biotechnol 19 (1997) 4. [22] T.J. Jack RW, Ray B, Bacteriocins of gram-positive bacteria. Microbiol Rev 59 (1995) 19. [23] R.M. Rios JL, Villar AJ Screening methods for natural products with antimicrobial activity: a review of the literature. Ethnopharmacol 23 (1988) 22. [24] NCCLS, Performance standards for antimicrobial disk susceptibility testing National Committee for Clinical Laboratory Standards, 6th :l International Supplement M2-A6 (1997). [25] M. Malagoli, A Full-length Protocol to Test Hemolytic Activity of Palytoxin on Human Erythrocytes. (2007). [26] L.B. Gutierrez JM, Cerdas L Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer. Toxicon 24 (1986a) 9. [27] A. Karray, F. Frikha, A. Ben Bacha, Y. Ben Ali, Y. Gargouri, and S. Bezzine, Biochemical and molecular characterization of purified chicken pancreatic phospholipase A2. Febs J 276 (2009) 4545-54. [28] A. Karray, F. Frikha, Y. Ben Ali, Y. Gargouri, and S. Bezzine, Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2. Lipids Health Dis 10 27. [29] E.A. Dennis, Diversity of group types, regulation, and function of phospholipase A(2). J Biol Chem 269 (1994) 13057-13060. 15
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[30] E.A. Dennis, The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22 (1997) 1-2. [31] J. Tischfield, A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem. 272 (1997) 17247-50. [32] A. Karray, Y. Ben Ali, J. Boujelben, S. Amara, F. Carriere, Y. Gargouri, and S. Bezzine, Drastic changes in the tissue-specific expression of secreted phospholipases A2 in chicken pulmonary disease. Biochimie 94 (2012) 9. [33] N.R.B.a.V.J. Shukla, Antibacterial and antifungal activities from leaf extracts of Cassia fistula l.: An ethnomedicinal plant. Adv Pharm Technol Res 2 (2011) 5. [34] G. Lambeau, and M.H. Gelb, Biochemistry and physiology of mammalian secreted phospholipases A2. Annu Rev Biochem 77 (2008) 495-520. [35] H.M. Daly R, Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18 (2005) 19. [36] b. Abir Ben Bachaa, ∗ , Islem Abidc, Habib Horchanid, Hafedh Mejdoub Enzymatic properties of stingray Dasyatis pastinaca group V, IIA and IB phospholipases A2: A comparative study International Journal of Biological Macromolecules 62 (2013) 5. [37] A.I. Ben Bacha A, Horchani H, Mejdoub H., Enzymatic properties of stingray Dasyatis pastinaca group V, IIA and IB phospholipases A(2): a comparative study. Int J Biol Macromol 62 (2013) 537-42. [38] P.G. Wilson, M. Manji, and J.P. Neoptolemos, Acute pancreatitis as a model of sepsis. J Antimicrob Chemother 41 Suppl A (1998) 51-63. [39] W.D. Buckland AG, The antibacterial properties of secreted phospholipases A(2). Biochim Biophys Acta 1488 ( 2000, ) 9. [40] S.Y. Balboa MA1, Gaietta G, Ellisman MH, Balsinde J, Dennis EA, . , Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells. J Biol Chem 278 (2003) 6. [41] G.J. Díaz C, Lomonte B, Gené JA, The effect of myotoxins isolated from Bothrops snake venoms on multilamellar liposomes: relationship to phospholipase A2, anticoagulant and myotoxic activities. Biochim Biophys Acta 1070 (1991) 5. [42] S.S. Stefansson et al., Kini RM, Evans HJ, The inhibition of clotting complexes of the extrinsic coagulation cascade by the phospholipase A2 isoenzymes from Naja nigricollis venom. Thromb Res 55 (1989) 10. [43] C. Mounier, P. Luchetta, C. Lecut, R. Koduri, G. Faure, G. Lambeau, E. Valentin, A. Singer, F. Ghomashchi, S. Beguin, M. Gelb, and C. Bon, Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2. Eur. J. Biochem. 267 (2000) 4960-9.
Legend Fig.1: Purification of ChPLA2-V (A) Gel filtration chromatography of recombinant ChPLA2-V on Sephadex G-75. The column (1.5 cm ×34 cm) equilibrated in 20 mM Tris-HCl buffer pH 8.5 containing 20 mM CaCl2. Elution was performed with the same buffer at a flow rate of 30 ml.h-1 and 3
16
ml samples were collected. ChPLA2-V activity was measured as described previously using egg yolk emulsion as substrate. The pooled fractions containing the phosphlipase activity were indicated by horizontal line. (B) Mono-S Sepharose chromatography. The column (5 cm × 2 cm) was equilibrated with 20 mM Tris HCl buffer pH 8.5 containing 20 mM CaCl2; and then washed with the same buffer containing 0.1 M NaCl. Linear salt gradient (0.1 to 0.5 M NaCl, dotted line) was applied
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to the column; gradient chamber 50 ml; 3 ml fraction; flow rate, 10 ml/h. The pooled fractions containing the PLA2 activity were indicated by horizontal line. SDS-PAGE (15%) analysis of pure ChPLA2-V was inserted in Figure 1B. Lane 1, molecular mass markers (MM); Lane 2, 15 μg of
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proteins obtained after Sephadex G-75 chromatography; Lane 3, 15 μg of purified ChPLA2-V, obtained after Mono-S chromatography.
Fig.2: Effect of Ca2+ and pH on ChPLA2-V activity. (A) Ca2+ dependence of the group V
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phospholipase A2 activity using egg yolk emulsion as substrate at pH 8.5 and at 37°C. The star
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indicates the phospholipase activity measured in the absence of CaCl2 and in the presence of 10 mM EDTA. (B) pH optimum of the group V phospholipase A2 activity on egg yolk emulsion at pH 8.5 and
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at 37°C.
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Fig.3: Coagulation tests with human blood plasma of ChPLA2-V (black square) and ChPLA2IIA (black triangle): the anticoagulant effect of ChPLA2-V (black square) and ChPLA2-IIA (black
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triangle) on human normal plasma. The value of partial thromboplastin time activator (TTA, using Kaolin as an activator) is 29 second in absence of PLA2. Mesurments are expressed as a ratio of (TTA
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in presence of ChPLA2 )/ (TTA without ChPLA2).
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Fig.4: Amino acid sequence of the ChPLA2-IIA and ChPLA2-V. For ChPLA2-V: Y24-G30 highlighted, Ca2+ loop; and H47-D48 with stars, active site. Residues implicated in the anticoagulant activity of ChPLA2-V: Basic clusters of the IBS of the enzyme are indicated in: cyan for K11/K15 / K38/ R74/ R92/R115 and K117. Hydrophobic residues of the IBS of the ChPLA2-V are indicated in
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pink: L2, , V18, L19, and V30.
Table 1: Flow sheet of chicken group V phospholipase A2 purification Table 2: Minimum inhibitory concentration of ChPLA2-V on bacteria Table 3: Inhibitory spectrum of ChPLA2-V on several Gram-positive and Gram-negative bacteria Table 4: Antifungal effects of ChPLA2-V 17
FIGURE 1 A 3.5
2.4
A
2.2 2 1.8
2.5
---- OD 280
1.6 2
1.4
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1.2 1.5
1 0.8
1
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0.6 0.4 0.2 0 10
20
30
40
50
60
70
80
90
0.5 0 100
U
0
M
A
N
Fraction N°
C
0.25
97
0.2
67 43 30
0.15
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20 14
0.1
2.5 2
PT
110
1.5 1
0.05
0.5
A
0
0
10
20
0 30
40
50
60
Fraction N°
18
70
[NaCl ]mM
3
B
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0.3
80
---- PLA2 Activity (U/ml)
FIGURE 1B
---- OD 280
---- PLA2 Activity (U/ml)
3
- 0.5 - 0.4 - 0.3 - 0.2 - 0.1
FIGURE 2A
A
120
IP T
80
40
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Specific activity (U/mg)
160
0 0
2
4
6
U
[CaCl2] mM
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FIGURE 2B
B
M
A
160
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120
80
40
PT
Specific activity (U/mg)
8
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0
6
7
8 pH
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5
FIGURE 3
19
9
10
4.4 3.6 3.2 2.8 2.4 2 1.6 1.2
The normal TTA limit
0.8 0.4 0 0.2
0.4
0.6
0.8
U
[Enzyme] µmol
1
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0
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TTA of the sample / TTA control
4
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FIGURE 4
NIAQFGIMIKEKTGK-PALSYNGYGCHCGLGGSKYPVDATDWCCHAHDCCYKRLSSVTCV 59 SLWQLQEVVTKMTGKNAVLNYSSYGCYCGVGGHGQPKDATDRCCQLHDTCYDSLQRYHCN 60 .: *: ::.: *** ..*.*..***:**:** * **** **: ** **. *. *
Ch-IIA Ch-V
PHLATYKFSIKRGQITCGSGSSCQRAACECDKKAAECFKRNLRTFNKSYQNYLNFKCKGS119 AKKQRYKYSWHSGRLTCNRDSWCAQLSCECDRSLGLCLQRNRGSYNWRYVLYPRCKCR— 118 .: **:*: *::**. .* * : :****:. . *::** ::* * * . **:
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RPSC 123 ----
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Ch-IIA Ch-V
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Ch-IIA Ch-V
A
Table 1: Flow sheet of chicken group V phospholipase A2 purification
Purification Step
Total Proteine (mg) (b)
Total activity (U)(a)
Specific activity (U/mg)
Yield (%)
Purification factor
Supernatant
20.7
360
18
100
1
20
13.5
320
23.7
88
1.05
Sephadex G-75
9.28
240
25
66
3.08
Mono-S
2.38
185
77
51
4.27
Concentration
1.12
176
157
48
9
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(NH4)2SO4 precipitation
(a)
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1 Unit: µmol of fatty acid released per min using egg yolk emulsion as a substrate in the presence of 6 mM NaDC and 6 mM CaCl2. (b) Proteins were estimated using the Bradford method. The experiments were conducted three times.
ChPLA2-V (µg)
50 µg
25 µg
12.5 µg
6.25 µg
3.12 µg
MIC
-
-
+
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Table 2: Minimum inhibitory concentration of ChPLA2-V on bacteria. Symbol (-) means the absence of bacterial growth, symbol (+) means the presence of bacterial growth
+
25 ug
-
-
+
+
+
25 ug
-
-
+
+
+
25 ug
-
-
+
+
+
25 ug
+
+
+
50 ug
Bacteria Stain
aeruginosa Escherichia coli.
+
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-
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Pseudomonas
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aureus Bacillus subtilis
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Staphylococcus
+
A
Bacillus cereus
Table 3: Inhibitory spectrum of ChPLA2-V on several Gram-positive and Gram-negative bacteria
Bacteria Strain
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Bacillus cereus Bacillus subtilis Micrococcus luteus Brevibacterium flavum Enterococcus faecalis Staphylococcus aureus Staphylococcus xylosus Staphylococcus epidermidis Pseudomonas aerigenosa Enterobacter cloacae
Gram
Sensibility
+ + ++ + + + + + -
++ ++ ++ + 21
Klebsielle pneumoniae Escherchia coli Salmonella
-
+ -
Table 4 : Antifungal effects of ChPLA2-V ChPLA2-V Concentration (µg)
inhibition zone (mm)
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The bactericidal level was estimated by measuring the size of inhibition zone of the indicator strain. Insensitivity (-), low sensitivity (+: Diameter of inhibition < 15 mm), high sensitivity (++: Diameter of inhibition between 15 et 20 mm).
Trichodorma citrino viride
Fusarium solani
25
-
-
-
50
2
-
100
9
-
A
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PT
ED
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A
N
U
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Aspergillus niger
22
-
S0141-8130(17)30377-X https://doi.org/10.1016/j.ijbiomac.2017.11.045 BIOMAC 8525
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
29-1-2017 7-11-2017 8-11-2017
Please cite this article as: Aida Karray, Madiha Bou Ali, Nedia Kharrat, Youssef Gargouri, Sofiane Bezzine, Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.11.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris
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Karray Aida, Bou Ali Madiha, Kharrat Nedia, Gargouri Youssef and Bezzine Sofiane*
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra,
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université de Sfax-Tunisia. *
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E-mail: [email protected]
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: + 21674675055
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Tel /Fax
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Correspondence address : Pr. Sofiane Bezzine, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra, université de Sfax-Tunisia.
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Highlights
This study describes the expression of group V secreted PLA2 in Pichia pastoris.
Purification, biochemical and biological characterization of PLA2-V is studied.
Antibacterial, antifungal and anticoagulant activities of ChPLA2-V
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Abstract
Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart 1
polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger
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anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be
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considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent.
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Key words: group V secreted Phospholipase A2; antimicrobial activity; anticoagulant activity;
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1.Introduction
Phospholipases A2 (PLA2) EC 3.1.1.4 are a family of enzymes that hydrolyze the ester bond
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at the sn-2 position of phospholipids generating free fatty acids and lysopho-spholipids [1].
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This family includes a number of secreted PLA2s (sPLA2s) referred to as group IB (GIB), GII (subgroups A-F), GIII, GV, GX and GXII (subgroupsA–B)[2]. Clearly, the different mammalian sPLA2s are not isoforms as their sequence identities are only around 15%[3; 4]
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they have distinct enzymatic properties [5] and show different tissue distribution patterns in both mice and humans. Consequently, in various tissues, the different sPLA2s may exert
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distinct biological functions that may be dependent or independent of their enzymatic activities [3; 6; 7].
Much less is known about the regulation and biological roles of phospholipases A2 from
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birds. So that, we focused our current study on the identification of novel phospholipases A2 from chicken using biochemical and molecular techniques. Group V sPLA2 has been cloned from chicken [8], human, rat, and mouse species [9]. Unlike group I and II sPLA2s, this sPLA2 has only six disulfides and does not have the group I- or group II-specific disulfides, thus defining a novel group of sPLA2s [10]. This sPLA2 has a higher level of identity with group IIA sPLA2s, as compared to group IB sPLA2. It neither has a propeptide sequence, indicating its closer relationship with group II sPLA2s. In a previous work, quantitative PCR 2
shows that the expression patterns of chicken PLA2-V resemble to that of mammals with a high expression level in the heart (6.83), intestine (5.235) and liver (3.01). Thus, chicken sPLA2-V is preferentially expressed in heart and intestine, at lower extent in liver, but it wasn’t detected in lung, spleen and colon [8]. Whereas, mammalian sPLA2-V has a preferentially expression level in heart and a much lower expression level in lungs and liver [11]. The relative expression level of ChPLA2-V, has a marked increase in its expression level in the viral infected lungs of chicken [8]. In fact, no or very weak sPLA2 expression was
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observed in normal chicken lungs and spleen suggesting a protective role of this group of sPLA2.
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Because of its up regulation during lung inflammation, and contrary to the variations observed
in the expression level of sPLA2-IB, -IIA and -X, only PLA2-V can be considered as a potential biomarker of the infectious bronchitis disease in chicken.
Diverse biological functions have been attributed to groupV sPLA2. These functions are
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attributed to the ability of this enzyme to provide arachidonic acid for the synthesis of
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eicosanoid. [12]. Additional functions have also been demonstrated; including regulation of
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phagocytosis, foam cell formation, and antibacterial activities [13; 14].
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However, the group V sPLA2 has been isolated and purified from few tissues or cells and its biochemical properties and structural features have not been explored. To understand the
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structure, function and regulation of this important sPLA2, we have synthesized the cDNA by RT-PCR from chicken heart polyA. mRNA, constructed a bacterial expression vector for the chicken group V sPLA2, over-expressed it in Pichia pastoris, and purified it to homogeneity
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using classical technique of chromatography. The active ChPLA2-V was characterized as to its calcium dependence, pH optimum, substrate preferences and biological activities.
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2.Materials and Methods
2.1. Strains, Cell Culture and Vectors The Escherichia coli strain DH5 Alpha was used as a host for cloning the ChPLA2-V PCR fragment in the P.pastoris transfer vector pGAPZA (Invitrogen). The P.pastoris host strain
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was X33 (wild type strain from Invitrogen). The P.pastoris transfer vector pGAPZA (Invitrogen) was used for yeast transformation. The Pfu DNA polymerase, T4 DNA ligase, PCR purification kit and Midi-Prep Kit were purchased from Promega. Pichia pastoris liquid cell cultures were grown in YPD medium containing 10 g yeast extract, 20 g Bacto-peptone and 20 g D-glucose.
3
The YPDS medium was YPD medium to which 18.2 g sorbitol per liter were added. To prepare plates for solid cell cultures, 2% agar (w/v) were added to the YPD medium. The Deoxycholic acid sodium salt (NaDC) (purity 99%) was purchased from Bio Basic Inc. 2.2. Construction of the DNA Encoding the ChPLA2-V cDNA synthesis and amplification Total mRNAs were isolated from chicken heart using the single step guanidine isothiocyanate/phenol/chloroform isolation method as described by
acid guanidinium thiocyanate-phenol-chloroform extraction [15].
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Chomczynski and Sacchi Chomczynski P, Sacchi N: Single-step method of RNA isolation by
ChPLA2-V cDNA was obtained from total mRNAs by the reverse transcription procedure
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(Promega). First strand cDNAs were prepared using 10 μg of total mRNAs as template (heatdenaturated for 5 min at 70°C,) 200 U MMLV reverse transcriptase (Invitrogen), 20 pmol of each deoxynucleoside triphosphate, and 20 pmol of each primer: forward primer, 5’-
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GTGTGTGTGAATTCATGAGCCTCTGGCAGCTGCAGGAG -3’ and reverse primer, 5’GTGTGTCTCGAGTCACCTGCACTTGCACCTGGG -3’. The N-and C-terminal primer
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were deduced from the genome of Gallus gallus (GenBank accession number: JF 411004).
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Reverse transcription was carried out in a total reaction volume of 20 μl for 5 min at room
for 15 min and cooled on ice.
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temperature and 60 min at 42°C. The cDNA/RNA heteroduplex was then denaturated at 70°C
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2.3. Cloning of the mature PLA2 gene
Amplification of the specific ChPLA2-IIA cDNA was carried out by PCR using the single strand cDNAs as template with the forward and reverse primers previously described. PCR
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was performed in a 0.2 ml Eppendorf tube with a Gene Amp® PCR System 2700. The PCR mixture contained 20 pmol of both primers, 20 pmol of each deoxynucleoside triphosphate, 5
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U pfx polymerase and polymerization buffer in final volume of 100 μl. The single strand DNAs were directly used as template. The thermal profile involved 35 cycles of denaturation at 94°C for 1 min, primer annealing at 60° C for 1 min, and extension at 72°C for 3 min. The PCR product (500 pb) was isolated and ligated into the EcoRI and XhoI linearized and
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dephosphorylated pGAPZA vector, according to the manufacturer’s protocol (Promega). Protoplasts of E. coli DH10B were transformed with the ligation mixture. The presence of the appropriated insert was verified by restriction analysis. DNA sequences were elucidated by the dideoxynucleotide chain termination method according to a cycle sequencing protocol using thermosequenase (Amersham Pharmacia Biotech). The sequencing reactions were analyzed with the DNA sequencer ABI PRISM 3100/3100-Avant Genetic Analyzer 4
(California, USA). It was performed three times, using the recombinant vector pChPLA2 as template with PGAPZ promoter primer and the AOX reverse primer (Invitrogen). 2.4. Transformation of P.pastoris and Screening of ChPLA2-V Secreting Transformants Electrocompetent P.pastoris X-33 cells were prepared using standard methods [16] and their transformation was performed by electroporation according to Invitrogen manual. Prior to the yeast transformation procedure, recombinant vector (pGAPZaA/ChPLA2-v) was linearized by the restriction enzyme BspHI. The recombinant
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yeast clones were selected on YPDS plates containing 100 mg/ml Zeocin. The colonies were subsequently screened by performing PCR reaction using as template the genomic DNA of
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the selected clones to confirm the integration of the pGAPZaA/ChPLA2-V vector into the yeast genomic DNA [17]. Selected transformants were grown in 50 mL of YPD medium with 100 mg/ml Zeocin at 30°C under shaking at 160 rpm. Time course of ChPLA2-V secretion in
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the culture media was determined for various clones.
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2.5. Expression of ChPLA2-V in P. pastoris
The most efficient ChPLA2-V secreting selected transformant was pre-grown at 30°C in 250
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ml shake flasks containing 50 ml YPD medium with 100 mg/ml Zeocin for 24 h to an OD600
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of 4–6. This cell culture was further used to inoculate 500 ml shake flask containing 100 ml YPD medium without Zeocin. The production of ChPLA2-V was conducted at 30°C for 48
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hours with shaking at 150 rpm.
2.6. Purification of the ChPLA2-V Protein
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The culture was harvested by centrifugation (9500 rpm, 10 min, 4°C). The supernatant was subjected to ammonium sulphate fractionation (20–75%). The precipitates obtained after centrifugation at 15000 rpm for 30 min were resuspended in the
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buffer A and loaded on a column of sephadex G-75 equilibrated with buffer A (20 mM TrisHCl, pH 8, 20 mM CaCl2). The resulting sample containing PLA2 activity (4 ml, 240 U) was poured into a Mono-S Sepharose column equilibrated with buffer A. The pooled fractions
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were concentrated using an Amicon® stirred cells. Gas pressure is applied directly to the ultrafiltration cell. Solutes above the membrane's molecular weight cut-off of 5 kDa, are retained in the cell, while water and solutes below the cut-off pass into the filtrate and out of the cell. Gentle magnetic stirring minimizes concentration polarization and shear denaturation.
Active fractions were analyzed with 15%-SDS-PAGE and the ChPLA2-V activity was measured as described elsewhere. 2.7. Phospholipase activity 5
PLA2 activity was measured titrimetrically at pH 9.5 and at 37 °C with a pH-stat (Metrhom), under the standard assay conditions described previously[18] using Phosphatidyl-Choline (0.5% w⁄ v) in 30 ml of 150 mM NaCl, 4 mM CaCl2, and 1 mM NaTDC. One PLA2 activity unit corresponds to one µmole of fatty acid liberated per minute. 2.8. SDS-PAGE Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed by the method of Laemmli [19]. The proteins were
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tained with Coomassie brilliant blue. 2.9. Amino acid sequencing
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For N-terminal sequencing, the purified enzyme was blotted (60 min, 50 mA, 4 °C) onto a PVDF (polyvinylidene difluoride) membrane (Applied Biosystems, ProBlotTM) in 20 mM CAPS buffer (pH 11) containing 10% methanol using a mini trans-blot cell (BioRad, Hercules, USA). The N-terminal sequence was determined by automated Edman's degradation, using an Applied Biosystems Protein Sequencer Procise 492 equipped with 140 C HPLC system (Roissy, France). [20]
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2.10. Antimicrobial Activity
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2.10.1. Determination of zone of inhibition method
method [21; 22; 23].
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The antibacterial and antifungal activities of ChPLA2-V was checked by well diffusion
Briefly, bacteria were cultivated in BHI medium at 37°C for 3 h. A basal layer of Brain Heart
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Infusion (BHI) containing 2-5% agar, was poured in Petri dishes. When plates were dried, 5 ml of soft BHI (0-7% agar) containing 107 cells of the indicator strain were overlaid. BHI was
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from Hispanlab, S.A (Madrid).
Then, wells were punched in the agar plate and filled with 50 μg of test samples. The zones of growth inhibition around the well were measured after 18 hours to 24 hours of in incubation at 37°C for bacteria and 48 hours to 96 hours for fungi at 30°C. The sensitivities of
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the microorganism species to the ChPLA2-V were determined by measuring the sizes of inhibitory zones (including the diameter of disk) on the agar surface around the well, and values <8 mm were considered as not active against microorganisms. One arbitrary unit (AU) of antibacterial activity was defined as the amount of ChPLA2-V sufficient to give a zone of inhibition around the well. The bacteria used were Staphylococcus. aureus (SA), Bacillus cereus (BC), Bacillus subtilis (BS), Pseudomonas aeruginosa, (Ps), Salmonella (S), Klebsielle 6
pneumoniae (KP) and E. coli (ECo). The zones of growth inhibition around the disks were measured after 18 to 24 hours of incubation at 37°C for bacteria and 48 to 96 hours for fungi at 30°C. 2.10.2. Determination of the minimum inhibitory concentration (MIC) The Minimal Inhibitory Concentrations (MICs) of ChPLA2-V against the tested microorganisms were determined by the broth microdilution method [24]. All tests were performed in LB. Strains were cultured overnight at 37°C in LB. Test strains were suspended
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in LB (The initial absorbance measured at 600 nm was approximately adjusted to 0.2 Optical
density (OD) value). Geometric dilutions ranging from 100 μg/ml to 1.5 μg/ml of ChPLA2-V
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were prepared in 96-well microtiter plate, including one growth control (LB only). Plates
were incubated under normal atmospheric conditions at 37°C for 24 h. Absorbance was then measured at 600 nm and MICs values were determined as the lowest ChPLA2-V concentrations inhibiting visible growth of bacterial strains. Tests were performed in
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duplicates.
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2.11. Hemolysis assay
Hemolytic activity of the pure ChPLA2-V was tested using erythrocytes from human, rabbit
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and rat. Freshly collected, blood samples were immediately mixed with anticoagulant,
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Alsever’s solution (pH 7.4) to prevent blood coagulation. To obtain a pure suspension of erythrocytes, 1 ml of whole blood was made up to 20 ml in phosphate buffered saline (PBS,
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pH 7.4), and centrifuged at 250 rcf for 5 min at 4 °C. The supernatant and buffy coats were removed by gentle aspiration, and the above process was repeated two more times.
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Erythrocytes were finally resuspended in PBS to make 1% solution for hemolytic assay. For this, various concentrations of pure ChPLA2-V (0.05–1 mg/ml) were added to the suspension of red blood cells obtained from different species (human, rabbit and rat). The pure ChPLA2-
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V erythrocyte mixtures were incubated at 37 °C for 30 min in water bath and then centrifuged at 250 rcf for 5 min at 4°C. The absorbance of the supernatants was determined at 545 nm to measure the extent of red blood cell lyses. Positive control (100% hemolytic) and negative
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control (0% hemolysis) were also run by incubating erythrocytes in PBS containing 1% Triton X-100 and PBS alone, respectively [25]. 2.12.Anticoagulant activity Sheep citrated plasma was centrifuged at 1000 g at 4ºC and the anticoagulant effect was assessed as described by Gutiérrez et al. (1986a) [26]. Subsequently, 0.25 ml aliquots of plasma were incubated with 50 μl of solutions containing the following quantities of PLA2, from 0.39; 0.78; 1.56; 3.13; 6.25; 12.5 and 25μg) dissolved in phosphate buffered saline 7
(PBS, pH 7.5) for 10 min at 37ºC. Then, 0.1 ml of a solution of 0.25 M CaCl2 was added and the clotting time determined. Control tubes contained only PBS incubated plasma, while CaCl2 was added as described above. Observations were carried out for a maximum period of 60 min.
3.Results
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3.1. Construction of ChPLA2-V Expression Vector
The DNA fragment encoding for the ChPLA2-V (500 bp) was amplified by PCR using
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plasmid DNA (pGAPZαA/ChPLA2-V) as template with specific primers containing the
EcoRI site upstream of its first codon and Xho site downstream of its last codon. The amplified fragment was digested with EcoRI/Xho and inserted into the pGAPZαA vector
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previously digested with the same enzymes, in frame with the yeast α-factor signal sequence at the N-terminal side for protein secretion. The integrity of the construction (pGAPZαA/Ch-
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PLA2-V) was confirmed by DNA sequencing (data not shown).
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3.2. Generation of Recombinant Clones Expressing ChPLA2-V The P.pastoris X33 strain was transformed by electroporation using (pGAPZαA/ ChPLA2-V) plasmid DNA linearized by the BspHI restriction enzyme. P.pastoris tranformants were
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selected on YPDS-Zeocin plates and incubated at 30°C for 3 days.
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3.3. Selection of ChPLA2-V -secreting Transformants Transformants carrying the ChPLA2-V gene (C1-C6) were grown on a YPD medium in 250
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ml Erlen-meyer’s flasks with shaking at 150 rpm, 30°C to select the clone showing the highest phospholipase activity level. The time-course of the ChPLA2-V secretion by the P.pastoris clones was performed (data not shown). The C3 clone exhibited the highest activity level reaching about 0.8 U/ml after 48 hours of culture. This clone was then selected
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for the production and purification of the recombinant ChPLA2-V 3.4. Purification of the ChPLA2-V ChPLA2-V was purified successively by the steps procedure described in material and methods. The results of the purification procedure are summarized in Table 1. Figure 1A represents the elution profile of gel filtration through a Sephadex G-75 column (95 cm × 2.6 8
cm) equilibrated with buffer A. Elution of proteins was performed with the same buffer at 30 ml/h. The fractions containing the PLA2 activity was detected in a fraction eluted as a single peak between 1.5 and 2 void volumes. The second chromatographic step used is Mono-S Sepharose column equilibrated with buffer A. The column (5 cm × 2 cm)was then washed with the same buffer containing 0.1 M NaCl. Linear salt gradient (0.1 to 0.5 M NaCl, dotted line) was applied to the column. Results are shown in the figure 1B.
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After the final purification step, ChPLA2-V was purified 9 fold, with a recovery of 48%. It has a specific activity of 157 U/mg, using egg yolk emulsion as substrate in the presence of 4
mM NaDC and 4 mM CaCl2. The fractions containing the ChPLA2-V activity were pooled
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and analyzed by SDS-PAGE (Figure 1C). This figure shows that group V-ChPLA2 is homogenously pure and has an apparent molecular mass of about 14 kDa. These data obtained in the native and denaturized conditions suggested that ChPLA2-V was a monomeric protein
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like pancreatic and intestinal ones described in our previous works [27; 28].
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The molar absorption coefficient of ChPLA2-V was determined using Expazy tool: ProtParam tool. Once the protein sequence is copied, the value of the extinction coefficients
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in units of M-1 cm-1, at 280 nm measured in water was calculate. In these conditions, the
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molar absorption coefficient was 37650 M-1 cm-1. The experimental results using the formula OD280=£*L*C confirmed the bioinformatics results. (Concentration C was determined with
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Bradford reactif and expressed on mg/ml).
3.5. Ca2+dependence and pH optima of ChPLA2-V
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The Ca2+ dependence of enzymatic activity is one of the major defining characteristics for sPLA2. The Ca2+ dependence of the ChPLA2-V was examined by measuring enzymatic
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activity at deferent concentrations of calcium. In the presence of 1 mM EDTA and the absence of calcium, the enzyme was completely inactive. Figure. 2A shows the Ca2+ dependence of the ChPLA2-V. A typical saturation curve was obtained and indicated a
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Specific Activity of 157 U/mg for Ca2+ of about 6 mM. All crystal structures of sPLA2 have a `calcium binding loop' in the protein [29; 30; 31] and the calcium dependence of the group V PLA2 is similar to that of the human group IIA PLA2 [32]. The optimum pH for ChPLA2V was examined with 50 mM buffer. It had a broad pH optimum, but the maximal enzymatic activity occurred at pH 8.5 with 50 mM Tris-HCl (Figure 2B).
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6. Microbial activity of ChPLA2-V The antimicrobial activity of the ChPLA2-V were studied in different concentrations (3-50 µg/ml) against several Gram+ and Gram- bacterial strains and three fungal strains (Aspergillus niger MTCC 282 Trichoderma citrino- viride MTCC 1323 and Fusarium solani MTCC 227. Antibacterial and antifungal potential of ChPLA2-V were assessed in terms of zone of inhibition of microbial growth. The results of the antibacterial and antifungal
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activities are presented in table 2, 3 and 4. [33]. ChPLA2V was tested against several Grampositive bacteria: Bacillus cereus (BC), Staphylococcus aureus (SA), Bacillus subtilis (BS), Micrococcus luteus (ML), Brevibacterium flavum (BF), Enterococcus faecalis (EF),
and Gram-negative bacteria: Pseudomonas Aeruginosa,
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Staphylococcus epidermidis (Sep)
(Ps) Enterobacter cloacae (EC), Klebsielle pneumoniae (KP), Salmonella (S) and E. coli (ECo). Bacteria were incubated for 24 hours. ChPLA2-V was active against closely related Bacillus
aeruginosa and E. coli
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species, Staphylococcus aureus but also some other Gram-negatif bacteria : Pseudomonas (Tables 2 and 3). Indeed, the enzyme exhibited an important
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bactericidal effect against BC, BS and SA with a diameter of inhibition higher than 20 mm. A
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moderate bactericidal effect was obtained with a diameter of inhibition between 15 and 20
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mm against E coli and Pseudomonas aeruginosa. The bactericidal effects of ChPLA2-V were also tested by measuring the minimum inhibition concentration (MIC) of ChPLA2-V for SA, BS, BC, PA and Eco was also investigated. Table 3 shows that ChPLA2-V has the most
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potent effect on Gram + strains. It exhibits the lowest MIC values towards the three tested strains (25 μg/ml against staphylococcus strains, while MIC values towards Eco (gram
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negative bacteria) were estimated to more than 50 μg/ml. Finally, ChPLA2-V does not exhibit any antimicrobial effect up to 5 mg/ml against S. epidermidis, Micrococcus Luteus (ML),
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Enterococcus faecalis (Ef.), E. faecium (EF), Enterobacter cloacae (Ec), Brevibacterium flavum (BF), Salmonella (S) and Klebsielle pneumonia (K). The antifungal activities of the ChPLA2-V increased linearly with increase in concentration
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of enzyme extracts (μg/ml). The results summarized in table 4 revealed that for antifungal activity, Aspergillus niger shows good result as compare with Trichodorma citrino virid and Fusarium solani. The growth inhibition zone measured ranged from 11 to 20 mm for all the sensitive bacteria, and ranged from 2 to 9 mm for fungal strains 3.7. Hemolytic activity of ChPLA2V
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Our results show that pure ChPLA2V used in this study did not exhibit any significant hemolytic activity. 3.8. Anticoagulant activity of ChPLA2V and ChPLA2IIA The recalcification time of sheep platelet-poor plasma was prolonged after incubation with isolated phospholipase A2. Clotting time showed a direct dependence according with the amount of enzyme considered, from 0.08 to 0.800 µmol (Figure. 3). The anticoagulant
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activity of the ChPLA2V was evaluated by measuring the partial thromboplastin time
activator (TTA) using Kaolin as an activator. This is to take a delay of coagulation of the
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plasma, based on the addition of an increasing concentration of enzyme. The results are
compared with the value of a normal report 1.2. Figure 3A shows that the anticoagulant activity was positive for ChPLA2V. It is noted that a low concentration of the ChPLA2-V (0.08 µmol) possesses anticoagulant activity, compared with the CPLA2-IIA, which shows an anticoagulant
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activity at a concentration of 0, 2 µmol. In addition, with the ChPLA2V, the ratio TTA of the sample/ normal TTA reaches a value of 4, compared with the ChPLA2-IIA which shows a ratio of 1.6 (for the
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same enzyme concentrations used).
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Discussion
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Contrary to the previously reported expression of the sPLA2 using E.coli system accumulated in inclusion bodies [34], this study describes, and for the first time, the expression of group V
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secreted PLA2 using Pichia pastoris. Unlike the classical prokaryotic system, in which the solubilization of inclusion bodies with guanidine hydrochloride was essential for the purification and folding to obtain the active enzyme, the protein expression in Pichia pastoris
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is directly obtained from the culture medium. Knowing that pancreatic PLA2-IB and intestinal PLA2-IIA are the most abundant enzymes
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and the best documented in mammals among all secreted PLA2. In order to gain more informations about their bird’s orthologs, we have recently purified and characterized PLA2IB from chicken pancreas (delipidated powder) than PLA2-IIA from intestinal mucosa. Very few studies were done on group V PLA2 enzyme, it is consequently interesting for us to
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study, for the first time, a newly purified chicken secreted PLA2-V from the heart of the bird and to provide basic information about its main biochemical and biological characteristics. Six Zeocin-resistant clones picked on the solid selective medium were selected and the integration of ChPLA2-V gene was analyzed by PCR using the pGAPZαA universal primers (pGAP Forward and AOX1 primers). C3 and C6 clones showed the amplification of the expected size (1040 bp). This fragment corresponded to the size of the ChPLA2-V gene (500 11
bp) plus a portion of the vector (540 bp). This result confirms the integration of the expression cassette (pGAPZαA/ ChPLA2-V) in P. pastoris genomic DNA. . Actually, there is no solid evidence that each integration event on the yeast genome contributes equally to the levels of the expressed protein. The multiple integration events have little detrimental effect on the expression of secreted protein in Pichia pastoris since other factors can also affect the
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expression level [35].
The purification flow sheet presented in Table.1 shows that the specific activity (SA) of pure
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ChPLA2-V reached 157 U/mg when egg yolk was used as substrate at pH 8.5 and 37°C, in the presence of 4 mM NaTDC and 4 mM CaCl2, value which is comparable to what observed
with the PLA2-V from stingray Dasyatis pastinaca recently studied wich shows a SA of 56U/mg on the presence of 6 mM NaTDC and 4 mM CaCl2. [36]. The ChPLA2-V
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purification yield was around 48% of the total initial activity. The procedure described and
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summarized in table 1 is more rapid than those used previously to purify other group V phospholipase A2 [37]. In fact, chicken PLA2 was purified after only two chromatographic
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steps whereas in the case of dromedary or porcine PLA2 four chromatographic steps were
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needed [18].
Starting with the whole P. pastoris cell extract, 9-fold purification was achieved and the
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overall recovery of enzyme activity was 48%. From 800 ml of P. pastoris cell culture media, about 1.12 mg of pure ChPLA2-V was obtained with a specific activity of 157 U/mg (Table
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1). Specific activity of 156 U/mg for Ca2+ of about 4mM and the maximal enzymatic activity occurred at pH 8.5 with 50 mM Tris-HCl. The apparent molecular mass value of the
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recombinant PLA2-V was found to be 14.000 Da, The N-terminal amino acid sequence of recombinant PLA2-V was found to be SLWQLQEVVTK. Because of its up regulation in infected chicken lungs, and the negative variations observed in the expression levels of ChPLA2-IB, -IIA and –X in the lungs [8], we can considerate ChPLA2-V as a specific
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biomarker of the infectious bronchitis disease in chicken . Lapointe et al. [38] investigated the effect of sPLA2-V on lipopolysaccharide mediated leukocyte recruitment supporting the contribution of sPLA2-V in the development of inflammatory innate immune response and its ability to modulate adhesion molecule expression. In the present work, the antimicrobial activity of the pure enzyme was performed. Firstly, we have demonstrated that only 25 μg/ml (final concentration) of ChPLA2-V is able to kill 100% 12
of BS, BC, and SA and. The same amount of enzyme kills 60% of Gram negative bacteria PA and E.coli. Whereas, even at a final concentration of 50 μg/ml, ChPLA2-V was inactive against all the other stains tested. The differences observed are due to the cell’s membrane composition. The cell wall of gram positive bacteria contains a dense peptidoglycan array, which bears a high anionic charge due to the presence of phosphate diester units of lipoteichoic acid. The sPLA2 must go through the highly anionic cell wall of Gram positive bacteria to reach the phospholipids membrane target. ChPLA2-V, with an electrostatic
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potential of +14(+22; -8) based on the total number of Lys, Arg, His, Asp and Glu, can bind highly anionic bacterial cell walls [39] and could explain the antibacterial activity observed.
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Secondly, the antifungual activity was tested against several fungi. In this work we demonstrated that the most important inhibition grouth was observed against Aspergillus
niger with a diameter of 9 mm using 75 µg of pure enzyme. Furthermore, in vivo studies done
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on mammals, (Balboa et al., 2004) [40] have shown that PLA2V are secreted by immune cells during phagocytosis of bacteria and fungi. The presence of these antimicrobial activities may
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be due to hydrolysis of phospholipid membranes of bacteria and fungi by the enzyme.
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With reference to the anticoagulant activity of PLA2, we observed that when
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increasing the concentration of the enzyme the clotting time is prolonged. Díaz and coworkers [41] suggest that the anticoagulant effect of myotoxic phospholipases A2 from Bothrops
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venoms is dependent on enzymatic degradation of phospholipids necessary for the adherence and activation of coagulation protein factors. This hypothesis is supported by the observation that myotoxins prolong both recalcification time and prothrombin time, but not thrombin
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time, since only the former two depend on plasma phospholipids [42] Figure 3 shows an important anticoagulant activity of the ChPLA2V observed at a low
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concentration: 0,08 μM, compared with the ChPLA2-IIA. The latest didn’t express any anticoagulant activity at a concentration lower than 0,2 µM. Moreover, the ratio’s value of TTA /TTA control) in presence of ChPLA2V reaches 4, compared with that observed in presence of ChPLA2-IIA, which reaches the value of 1,6 (for the same enzyme concentration
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tested). As sets for Group IIA of PLA2, the anticoagulant activity is supported by the basic residues located at the site of interaction with the substrate (IBS) that govern the interaction with factor Xa clotting [43]. These basic residues constituting the IBS are well conserved in the ChPLA2V, (shown in purple in Figure 4), based on the sequence alignment of chicken’s group IIA PLA2 and group V PLA2. Basic clusters of the IBS of the enzyme are indicated in:
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cyan for K10/K16 /R115/K116/K74/ R92; and K38. Hydrophobic residues of the IBS of the ChPLA2-V are indicated in purple: L2, , A19, L20, and V31(Figure 4). This test is still in the preliminary study of the anticoagulant activity of chicken sPLA2V. Indeed, it is necessary to test the anti-prothrombinase activity in presence of purified fractions of prothrombinase, namely factors Va and Xa, phospholipids and calcium. 4. Conclusion This study describes, and for the first time, the expression of group V secreted PLA2 using
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Pichia pastoris. Biochemical and biological characterizations of the pure enzyme was performed. Thus, chicken group V PLA2, may be considered as a future therapeutic agents
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against fungal and bacterial infections and as an anticoagulant agent.
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REFERENCES
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[10] F. Ferrato, F. Carrière, L. Sarda, and R. Verger, A critical re-evaluation of the phenomenon of "interfacial activation". in: E. Dennis, and B. Rubin, (Eds.), Methods in Enzymology., Academic Press, New York, 1997, pp. 327-347. [11] L. Touqui, and Y.Z. Wu, Interaction of secreted phospholipase A2 and pulmonary surfactant and its pathophysiological relevance in acute respiratory distress syndrome. Acta Pharmacol Sin 24 (2003) 1292-6. [12] Y.J.K. Nilda M. Muñoz‡, Angelo Y. Meliton‡, Kwang Pyo Kim§, Sang-Kyou Han§, Evan Boetticher‡, Eileen O'Leary¶, Shigeharu Myou‡, Xiangdong Zhu‡, Joseph V. Bonventre¶, Alan R. Leff‡∥ and Wonhwa Cho, . Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils. J. Biol. Chem 278 (2003) 7.
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[13] A.M. Barbara Balestrieri, Wei Xing, Michael H. Gelb, Howard R. Katz, and Jonathan P. Arm, Group V Secretory Phospholipase A2 Modulates Phagosome Maturation and Regulates the Innate Immune Response against Candida albicans. J. Immunol 182 (2009) 7. [14] Y.L. Eric Boilard, Katherine Larabee, Barbara Balestrieri, Farideh Ghomashchi, Daisuke Fujioka, Reuben
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[15] P. Chomczynski, and N. Sacchi, Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 (1987) 156-159. [16] R.K. Cregg JM1, Transformation. Methods Mol Biol. 103 (1998) 12. [17] B. Sias, F. Ferrato, P. Grandval, D. Lafont, P. Boullanger, A. De Caro, B. Leboeuf, R. Verger, and F. Carriere, Human pancreatic lipase-related protein 2 is a galactolipase. Biochemistry 43 (2004) 10138-48. [18] G.H. de Haas, N.M. Postema, W. Nieuwenhuisen, and L.L.M. van Deenen, Purification and properties of phospholipase A from porcine pancreas. Biochim. Biophys. Acta 159 (1968) 103-117. [19] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. [20] R.M. Hewick, M.W. Hunkapiller, L.E. Hood, and W.J. Dreyer, A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 256 (1981) 7990-7. [21] B.S. Paik HD, Park SH, Pan JG, Identification and partial characterization of tochicin, a bacteriocin offduced by Bacillus thuringiensis subsp tochigiensis. J Ind Microbiol Biotechnol 19 (1997) 4. [22] T.J. Jack RW, Ray B, Bacteriocins of gram-positive bacteria. Microbiol Rev 59 (1995) 19. [23] R.M. Rios JL, Villar AJ Screening methods for natural products with antimicrobial activity: a review of the literature. Ethnopharmacol 23 (1988) 22. [24] NCCLS, Performance standards for antimicrobial disk susceptibility testing National Committee for Clinical Laboratory Standards, 6th :l International Supplement M2-A6 (1997). [25] M. Malagoli, A Full-length Protocol to Test Hemolytic Activity of Palytoxin on Human Erythrocytes. (2007). [26] L.B. Gutierrez JM, Cerdas L Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer. Toxicon 24 (1986a) 9. [27] A. Karray, F. Frikha, A. Ben Bacha, Y. Ben Ali, Y. Gargouri, and S. Bezzine, Biochemical and molecular characterization of purified chicken pancreatic phospholipase A2. Febs J 276 (2009) 4545-54. [28] A. Karray, F. Frikha, Y. Ben Ali, Y. Gargouri, and S. Bezzine, Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2. Lipids Health Dis 10 27. [29] E.A. Dennis, Diversity of group types, regulation, and function of phospholipase A(2). J Biol Chem 269 (1994) 13057-13060. 15
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A
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U
SC R
IP T
[30] E.A. Dennis, The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22 (1997) 1-2. [31] J. Tischfield, A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem. 272 (1997) 17247-50. [32] A. Karray, Y. Ben Ali, J. Boujelben, S. Amara, F. Carriere, Y. Gargouri, and S. Bezzine, Drastic changes in the tissue-specific expression of secreted phospholipases A2 in chicken pulmonary disease. Biochimie 94 (2012) 9. [33] N.R.B.a.V.J. Shukla, Antibacterial and antifungal activities from leaf extracts of Cassia fistula l.: An ethnomedicinal plant. Adv Pharm Technol Res 2 (2011) 5. [34] G. Lambeau, and M.H. Gelb, Biochemistry and physiology of mammalian secreted phospholipases A2. Annu Rev Biochem 77 (2008) 495-520. [35] H.M. Daly R, Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18 (2005) 19. [36] b. Abir Ben Bachaa, ∗ , Islem Abidc, Habib Horchanid, Hafedh Mejdoub Enzymatic properties of stingray Dasyatis pastinaca group V, IIA and IB phospholipases A2: A comparative study International Journal of Biological Macromolecules 62 (2013) 5. [37] A.I. Ben Bacha A, Horchani H, Mejdoub H., Enzymatic properties of stingray Dasyatis pastinaca group V, IIA and IB phospholipases A(2): a comparative study. Int J Biol Macromol 62 (2013) 537-42. [38] P.G. Wilson, M. Manji, and J.P. Neoptolemos, Acute pancreatitis as a model of sepsis. J Antimicrob Chemother 41 Suppl A (1998) 51-63. [39] W.D. Buckland AG, The antibacterial properties of secreted phospholipases A(2). Biochim Biophys Acta 1488 ( 2000, ) 9. [40] S.Y. Balboa MA1, Gaietta G, Ellisman MH, Balsinde J, Dennis EA, . , Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells. J Biol Chem 278 (2003) 6. [41] G.J. Díaz C, Lomonte B, Gené JA, The effect of myotoxins isolated from Bothrops snake venoms on multilamellar liposomes: relationship to phospholipase A2, anticoagulant and myotoxic activities. Biochim Biophys Acta 1070 (1991) 5. [42] S.S. Stefansson et al., Kini RM, Evans HJ, The inhibition of clotting complexes of the extrinsic coagulation cascade by the phospholipase A2 isoenzymes from Naja nigricollis venom. Thromb Res 55 (1989) 10. [43] C. Mounier, P. Luchetta, C. Lecut, R. Koduri, G. Faure, G. Lambeau, E. Valentin, A. Singer, F. Ghomashchi, S. Beguin, M. Gelb, and C. Bon, Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2. Eur. J. Biochem. 267 (2000) 4960-9.
Legend Fig.1: Purification of ChPLA2-V (A) Gel filtration chromatography of recombinant ChPLA2-V on Sephadex G-75. The column (1.5 cm ×34 cm) equilibrated in 20 mM Tris-HCl buffer pH 8.5 containing 20 mM CaCl2. Elution was performed with the same buffer at a flow rate of 30 ml.h-1 and 3
16
ml samples were collected. ChPLA2-V activity was measured as described previously using egg yolk emulsion as substrate. The pooled fractions containing the phosphlipase activity were indicated by horizontal line. (B) Mono-S Sepharose chromatography. The column (5 cm × 2 cm) was equilibrated with 20 mM Tris HCl buffer pH 8.5 containing 20 mM CaCl2; and then washed with the same buffer containing 0.1 M NaCl. Linear salt gradient (0.1 to 0.5 M NaCl, dotted line) was applied
IP T
to the column; gradient chamber 50 ml; 3 ml fraction; flow rate, 10 ml/h. The pooled fractions containing the PLA2 activity were indicated by horizontal line. SDS-PAGE (15%) analysis of pure ChPLA2-V was inserted in Figure 1B. Lane 1, molecular mass markers (MM); Lane 2, 15 μg of
SC R
proteins obtained after Sephadex G-75 chromatography; Lane 3, 15 μg of purified ChPLA2-V, obtained after Mono-S chromatography.
Fig.2: Effect of Ca2+ and pH on ChPLA2-V activity. (A) Ca2+ dependence of the group V
U
phospholipase A2 activity using egg yolk emulsion as substrate at pH 8.5 and at 37°C. The star
N
indicates the phospholipase activity measured in the absence of CaCl2 and in the presence of 10 mM EDTA. (B) pH optimum of the group V phospholipase A2 activity on egg yolk emulsion at pH 8.5 and
A
at 37°C.
M
Fig.3: Coagulation tests with human blood plasma of ChPLA2-V (black square) and ChPLA2IIA (black triangle): the anticoagulant effect of ChPLA2-V (black square) and ChPLA2-IIA (black
ED
triangle) on human normal plasma. The value of partial thromboplastin time activator (TTA, using Kaolin as an activator) is 29 second in absence of PLA2. Mesurments are expressed as a ratio of (TTA
PT
in presence of ChPLA2 )/ (TTA without ChPLA2).
CC E
Fig.4: Amino acid sequence of the ChPLA2-IIA and ChPLA2-V. For ChPLA2-V: Y24-G30 highlighted, Ca2+ loop; and H47-D48 with stars, active site. Residues implicated in the anticoagulant activity of ChPLA2-V: Basic clusters of the IBS of the enzyme are indicated in: cyan for K11/K15 / K38/ R74/ R92/R115 and K117. Hydrophobic residues of the IBS of the ChPLA2-V are indicated in
A
pink: L2, , V18, L19, and V30.
Table 1: Flow sheet of chicken group V phospholipase A2 purification Table 2: Minimum inhibitory concentration of ChPLA2-V on bacteria Table 3: Inhibitory spectrum of ChPLA2-V on several Gram-positive and Gram-negative bacteria Table 4: Antifungal effects of ChPLA2-V 17
FIGURE 1 A 3.5
2.4
A
2.2 2 1.8
2.5
---- OD 280
1.6 2
1.4
IP T
1.2 1.5
1 0.8
1
SC R
0.6 0.4 0.2 0 10
20
30
40
50
60
70
80
90
0.5 0 100
U
0
M
A
N
Fraction N°
C
0.25
97
0.2
67 43 30
0.15
CC E
20 14
0.1
2.5 2
PT
110
1.5 1
0.05
0.5
A
0
0
10
20
0 30
40
50
60
Fraction N°
18
70
[NaCl ]mM
3
B
ED
0.3
80
---- PLA2 Activity (U/ml)
FIGURE 1B
---- OD 280
---- PLA2 Activity (U/ml)
3
- 0.5 - 0.4 - 0.3 - 0.2 - 0.1
FIGURE 2A
A
120
IP T
80
40
SC R
Specific activity (U/mg)
160
0 0
2
4
6
U
[CaCl2] mM
N
FIGURE 2B
B
M
A
160
ED
120
80
40
PT
Specific activity (U/mg)
8
CC E
0
6
7
8 pH
A
5
FIGURE 3
19
9
10
4.4 3.6 3.2 2.8 2.4 2 1.6 1.2
The normal TTA limit
0.8 0.4 0 0.2
0.4
0.6
0.8
U
[Enzyme] µmol
1
SC R
0
IP T
TTA of the sample / TTA control
4
N
FIGURE 4
NIAQFGIMIKEKTGK-PALSYNGYGCHCGLGGSKYPVDATDWCCHAHDCCYKRLSSVTCV 59 SLWQLQEVVTKMTGKNAVLNYSSYGCYCGVGGHGQPKDATDRCCQLHDTCYDSLQRYHCN 60 .: *: ::.: *** ..*.*..***:**:** * **** **: ** **. *. *
Ch-IIA Ch-V
PHLATYKFSIKRGQITCGSGSSCQRAACECDKKAAECFKRNLRTFNKSYQNYLNFKCKGS119 AKKQRYKYSWHSGRLTCNRDSWCAQLSCECDRSLGLCLQRNRGSYNWRYVLYPRCKCR— 118 .: **:*: *::**. .* * : :****:. . *::** ::* * * . **:
M
ED
RPSC 123 ----
CC E
PT
Ch-IIA Ch-V
A
Ch-IIA Ch-V
A
Table 1: Flow sheet of chicken group V phospholipase A2 purification
Purification Step
Total Proteine (mg) (b)
Total activity (U)(a)
Specific activity (U/mg)
Yield (%)
Purification factor
Supernatant
20.7
360
18
100
1
20
13.5
320
23.7
88
1.05
Sephadex G-75
9.28
240
25
66
3.08
Mono-S
2.38
185
77
51
4.27
Concentration
1.12
176
157
48
9
IP T
(NH4)2SO4 precipitation
(a)
SC R
1 Unit: µmol of fatty acid released per min using egg yolk emulsion as a substrate in the presence of 6 mM NaDC and 6 mM CaCl2. (b) Proteins were estimated using the Bradford method. The experiments were conducted three times.
ChPLA2-V (µg)
50 µg
25 µg
12.5 µg
6.25 µg
3.12 µg
MIC
-
-
+
U
Table 2: Minimum inhibitory concentration of ChPLA2-V on bacteria. Symbol (-) means the absence of bacterial growth, symbol (+) means the presence of bacterial growth
+
25 ug
-
-
+
+
+
25 ug
-
-
+
+
+
25 ug
-
-
+
+
+
25 ug
+
+
+
50 ug
Bacteria Stain
aeruginosa Escherichia coli.
+
CC E
PT
-
ED
Pseudomonas
M
aureus Bacillus subtilis
N
Staphylococcus
+
A
Bacillus cereus
Table 3: Inhibitory spectrum of ChPLA2-V on several Gram-positive and Gram-negative bacteria
Bacteria Strain
A
Bacillus cereus Bacillus subtilis Micrococcus luteus Brevibacterium flavum Enterococcus faecalis Staphylococcus aureus Staphylococcus xylosus Staphylococcus epidermidis Pseudomonas aerigenosa Enterobacter cloacae
Gram
Sensibility
+ + ++ + + + + + -
++ ++ ++ + 21
Klebsielle pneumoniae Escherchia coli Salmonella
-
+ -
Table 4 : Antifungal effects of ChPLA2-V ChPLA2-V Concentration (µg)
inhibition zone (mm)
IP T
The bactericidal level was estimated by measuring the size of inhibition zone of the indicator strain. Insensitivity (-), low sensitivity (+: Diameter of inhibition < 15 mm), high sensitivity (++: Diameter of inhibition between 15 et 20 mm).
Trichodorma citrino viride
Fusarium solani
25
-
-
-
50
2
-
100
9
-
A
CC E
PT
ED
M
A
N
U
SC R
Aspergillus niger
22
-