Functional and Structural Characterization of a Thermostable

Article pubs.acs.org/JAFC

Functional and Structural Characterization of a Thermostable Phospholipase A2 from a Sparidae Fish (Diplodus annularis) Nabil Smichi,*,†,‡ Houcemeddine Othman,§ Neila Achouri,† Alexandre Noiriel,∥ Vincent Arondel,⊥ Najet Srairi-Abid,§ Abdelkarim Abousalham,∥ Youssef Gargouri,† Nabil Miled,† and Ahmed Fendri† †

Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS, 3038 Sfax, Tunisia Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR7282, CNRS, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France § Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Université Tunis-El Manar, Institut Pasteur de Tunis, Tunis 1002, Tunisia ∥ Univ Lyon, Université Lyon 1, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), UMR 5246 CNRS, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM2, F-69622 Villeurbanne cedex, France ⊥ Univ Bordeaux, UMR 5200, Laboratoire de Biogenèse Membranaire, Bat. A3 Campus INRA de Bordeaux 71 avenue E., Bourlaux CS 2003233140 Villenave d’Ornon, France ‡

S Supporting Information *

ABSTRACT: Novel phospholipase (PLA2) genes from the Sparidae family were cloned. The sequenced PLA2 revealed an identity with pancreatic PLA2 group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA2 (DaPLA2) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA2 was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA2 was constructed to gain insights into the functional properties of sparidae PLA2. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA2. Using the monolayer technique, the purified DaPLA2 was found to be active on various phospholipids ranging from 10 to 20 mN·m−1, which explained the absence of the hemolytic activity for DaPLA2. KEYWORDS: Sparidae, phospholipase A2, E. coli expression, refolding and purification, monolayer properties, structural characterization, thermostability

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been studied intensively in the larvae of several fish species.10−12 Actually, there is considerable information about the importance of phospholipids for marine fish growth;13−15 however, it remains unclear which digestive enzymes are responsible for dietary lipid digestion.14,16,17 Recently, Ben Bacha et al.18 biochemically characterized a pancreatic PLA2 from a cartilaginous fish stingray.18 This pancreatic PLA2 shows similar properties with classical PLA2 (human and porcine PLA2). The Sparidae, so-called seabreams, are economically important species in the Mediterranean region where they have a high market value.19,20 Despite the economic importance of this fish species, they were studied only with regards to their behavior and reproduction. Unlike cartilaginous fish, the osteichtyens possess a pyloric ceca as a digestive organ, which replaces the pancreas in the classical system.21 The present study gives a more detailed analysis of the lipolytic system in captive groups of five seabreams. We report, for the first time, the isolation of genes encoding mature

INTRODUCTION Phospholipases are a group of enzymes that hydrolyze one of the ester bonds on phospholipids to release a variety of products, including lysophospholipids, free fatty acids, diacylglycerols, choline phosphate, and phosphatides.1 The phospholipase A2 (EC 3.1.1.4, PLA2) is a representative and essential lipolytic enzyme that specifically recognizes the sn-2 acyl bond of phospholipids. Numerous PLA2 enzymes have been isolated from a wide variety of organisms such as mammals, insects, plants, fungi, and bacteria.2,3 The secretory PLA2 (sPLA2) and the calcium-dependent cytosolic PLA2 are the most notable forms of PLA2.4 Among them, sPLA2 forms are the largest group, characterized by their secreted nature, small size, and requirement for millimolar concentrations of Ca2+.5 Important common structural features of the sPLA2’s are well-conserved: the central α-helices with the catalytic (His− Asp) dyad, the hydrogen-bonding network connecting the interfacial binding site, and the catalytic and Ca2+-binding sites.3,6 sPLA2-IB are also known as the pancreatic-type PLA2. They have been isolated from the pancreas of various mammal and bird species.7−9 The aquatic world contains a wide variety of living species and, hence, represents a great potential for discovering new enzymes. Proteolytic and amylolytic digestive processes have © XXXX American Chemical Society

Received: January 1, 2017 Revised: February 27, 2017 Accepted: February 28, 2017

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DOI: 10.1021/acs.jafc.6b05810 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

QIAquick and then ligated into pCR-Blunt II-TOPO vector at 4 °C overnight. Finally, the cloned products were sequenced by Eurofins (Ebersberg, Germany). Different genes encoding for mature Sparidae phospholipases were verified and deposited on GenBank under the following numbers: KU199233 (DaPLA2), KU199234 (DsPLA2), KU199235 (SpPLA2), KU199236 (LmPLA2), and KU199237 (SrPLA2). Expression Procedure, Refolding, and Purification. DNA sequence coding for the mature phospholipase from the annular seabream (D. annularis) was amplified using primers with specific sites for the restriction enzymes NcoI and NotI (Table 1, Supporting Information). Genes were amplified by KOD high-fidelity polymerase (New England Biolabs, New England) using DaPLA2-pCR-Blunt IITOPO vector as template. The gene was cloned into the pET28b vector, using NcoI and NotI sites, under the transcriptional control of the T7 promoter for its expression in E. coli. The pET28b construct, containing a 6-His-tag in the C-terminus DaPLA2, was transformed into E. coli BL21 (DE3) cells. Expression was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, using lysates from single colonies grown at 37 °C in LB with kanamycin (50 μg/mL) and chloramphenicol (30 μg/mL) until optical density at 600 nm reached the value of 0.8. Then, the culture was induced with IPTG (1 mM) and grown at different temperatures (37 or 16 or 4 °C) for 18 h. After centrifugation at 7000g for 5 min, cells were resuspended in 5 mL of lysis buffer (10 mM Tris-HCl, pH 8), sonicated, and checked for protein solubility using 15% polyacrylamide gel. The expressed protein was exclusively found in the insoluble fraction as inclusion bodies. Frozen cells were thawed and resuspended in Tris-HCl buffer 20 mM (pH 8) containing 10 mM NaCl and 20 mM ethylenediaminetetraacetic acid (EDTA) for 20 min at 4 °C, followed by sonication and centrifugation at 7000g for 20 min. The soluble fraction was removed, and the pellet containing the inclusion bodies (unfolded proteins) was resuspended in denaturing buffer containing 20 mM Tris-HCl (pH 8), 5 mM L-cysteine, and 8 M urea by stirring overnight at 4 °C. The denatured PLA2 was refolded by dialyzing, against 2 L of the same buffer containing 0.8 M urea, by mild stirring overnight at 4 °C. Any precipitate was removed by filtration through a 0.45 μm filter. Prior to further purification, the urea was removed from the sample containing the refolded protein by dialyzing against 2 L of buffer devoid of urea (25 mM Tris-HCl, pH 8.0, 10 mM NaCl) overnight at 4 °C. Upon complete removal of urea, insoluble proteins were removed by centrifugation at 12 000g for 30 min at 4 °C, concentration of refolded protein was estimated by the Bradford assay, and the phospholipase activity was assessed by pH-stat method.23 The active fraction recovered from the dialysis was loaded onto nickel affinity chromatography column equilibrated with Tris buffer (20 mM Tris-HCl, pH 8, 10 mM NaCl). The wash was performed with the same buffer. Under these conditions, DaPLA2 was bound to the support and then eluted with a linear gradient of imidazole (between 50 and 350 mM) at a flow rate of 1 mL/min. The purified fractions were analyzed by SDS-PAGE and used for further biochemical characterization. DaPLA2 Activity Measurement. PLA2 activity was measured potentiometrically by titrating the free fatty acids released from a mechanically stirred emulsion of phospholipid substrate, using 0.1 N NaOH and a pH-stat apparatus (Metrohm 718 Stat Titrino, Zofingen, Switzerland). Each assay was carried out in a thermostated (50 °C) vessel containing 0.5% of phosphatidylcholine (PC) substrate and 30 mL of 150 mM NaCl, 8 mM CaCl2, and 6 mM sodium deoxycholate (NaDOC) at pH 8.5.23 One phospholipase activity unit corresponds to l μmol of fatty acid released per minute. Monomolecular Film Technique for Kinetic Measurements on DaPLA2. All experiments were performed using the KSV 2000 Baro-stat equipment (KSV, Helsinki, Finland).24 A zero-order Teflon trough with two compartments, which were connected to each other by a small surface channel, equipped with a mobile Teflon barrier, which was used to compensate for the substrate molecules removed from the film by enzyme hydrolysis, maintained the surface pressure constant. The aqueous subphase contained 10 mM Tris-HCl, pH 8,

Sparidae PLA2. The study of catalytic and biochemical properties of a purified marine PLA2 from bony fishes allowed us to gain more insights into their mode of action on phospholipids. The annular seabream PLA2 tentatively named Diplodus annularis PLA2 (DaPLA2) was used as a model to study Sparidae PLA2 structure−activity relationships. The DaPLA2 was overexpressed in E. coli. The refolded DaPLA2 was purified and biochemically characterized using the emulsified and monolayer system substrates. Molecular modeling, docking, and molecular dynamics were performed in order to investigate, at the molecular level, the mode of action of DaPLA2 with its substrates.

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MATERIALS AND METHODS

Strains, Plasmids, and Reagents. Standard supplies were obtained from the following suppliers: phospholipids (1,2-dilauroylsn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), and 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). All other chemicals were of at least analytical grade and were obtained from either Fisher Scientific or Sigma. Nickel gel affinity was purchased from BioRad. Bacterial cultures were routinely maintained on Luria−Bertani (LB) medium with appropriate antibiotic selection. PCR Purification kit, DNA Gel Extraction kit, primers for cloning and expression, restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs and were used according to the manufacturers’ protocols. IPTG (isopropyl β-D-1-thiogalactopyranoside) and Kanamycin were obtained from Invitrogen (France). E. coli Top 10 and BL21-DE3 strains (from Life Technologies, France) were used for Sparidae PLA2 cloning and expression of annular seabream tagged-PLA2, respectively. The plasmid pCR-Blunt II-TOPO vector (Invitrogen) was used as a cloning vector. For enzyme expression, the genes were cloned into the pET28b vector (Merck, Darmstadt, Germany at NcoI and NotI sites fused with a sequence coding 6 His at the C-terminus and then transformed in BL21-DE3 cells. Recombinant cells were grown in LB media supplemented with kanamycin (50 μg/mL) when required. Biological Material. Fresh-caught fish from the Sparidae family: annular seabream (Diplodus annularis), white seabream (Diplodus sargus), gilthead seabream (Sparus aurata), sand steenbras (Lithognathus mormyrus), and salema (Sarpa salpa) were bought from a local fish market (Sfax, Tunisia). Fresh pyloric ceca from each species were removed immediately after dissection and directly used for total RNA extractions. cDNA Synthesis and Phospholipases Cloning. The total RNA was extracted from each sample with RNA Trizol Plus reagent (Invitrogen) according to the manufacturer’s instructions.22 Quality and quantity of the extracted RNA were estimated using agarose gel electrophoresis (1.5%) and Nanodrop 2000 spectrophotometer (Thermo-Scientific, Wilmington, DE, U.S.A.). Equal amounts of total RNA of six specimens were used for phospholipases cDNA synthesis using the superscript II (Invitrogen) as a reverse transcriptase according to the manufacturer’s protocol. The first strand for each fish was amplified by Phusion DNA polymerase using a RT Reagent kit (New England Biolabs, New England). cDNA was synthesized using oligonucleotide GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT, as a primer for reverse transcriptase; therefore, 3′ RACE was subsequently carried out using the adaptor oligonucleotide GACTCGAGTCGACATCGA and the forward primer deduced from the peptide signal of a precursor of Pagrus major sPLA2 (GenBank Accession number, AB050632.1) (Table 1, Supporting Information). Polymerase chain reaction (PCR) was run under the following cycling conditions: 98 °C for 30 s, followed by 30 cycles of 98 °C for 30 s; 55 °C for 30 s; 72 °C for 2 min; and a final extension at 72 °C for 10 min. PCR fragments for each species, analyzed on 1% agarose gel electrophoresis, were extracted from gel using Gel Extraction kit B


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Journal of Agricultural and Food Chemistry 150 mM NaCl, 21 mM CaCl2, and 1 mM EDTA prepared with MilliQ water and filtered through a 0.45 μm Millipore membrane. The monomolecular film was then formed at the air/water interface by spreading the different phospholipids solutions (1 mg/mL in chloroform containing 0.4% methanol) and waiting for at least 15 min for chloroform evaporation. The enzyme solution (30 nM) was injected into the subphase of the reaction compartment only when the 1,2-dilauroyl-sn-glycero-3phospholipid film covered both compartments. Residual surface-active impurities were removed before each experiment by simultaneous sweeping and suction of the surface.24 The surface pressure was measured on the reservoir compartment with a Wilhelmy plate (perimeter 3.94 cm) attached to an electromicrobalance, which was connected in turn to a microprocessor programmed to regulate the mobile barrier movement. The reaction compartment was stirred at 250 rpm using magnetic stirrers. The surface area of the reaction compartment was 120 cm2, and its volume was 120 mL. The reservoir compartment was 147 mm wide and 249 mm long. All activity measurements were performed after 20 min of enzyme injection. Activities were expressed as the number of moles of substrate hydrolyzed by unit time (min) and unit of surface (cm2) of the reaction compartment for an arbitrary phospholipase concentration of 1 M. DaPLA2 Stereospecificity. To determine the stereospecificity of DaPLA2, 50 μg of purified enzyme was incubated with POPC dispersion in 25 mL of buffer (10 mM Tris-HCl, pH 8, 8 mM NaDOC, and 6 mM CaCl2) at 50 °C for 30 min. The reaction was stopped by adding 100 μL of 1 M HCl, and lipids were extracted immediately using Folch’s procedure.25 After separation of the phases, the organic phase was analyzed by thin-layer chromatography on silica 60 (F254) previously activated at 60 °C for 30 min. Spots corresponding to free fatty acids were scraped, methylated, and analyzed by gas chromatography equipped with nonpolar DANI-SPA column (50 m length, 0.32 mm i.d.) and a flame ionization detector. Nitrogen was used as a carrier gas. The temperatures of the column oven, the injection ports, and the detector were maintained at 165 and 250 °C, respectively. Chromatographic peaks identified were compared with the retention times of known standards. Gel Electrophoresis, Mass Spectrometry, and NH2-Terminal Sequence Analysis. The purified DaPLA2 was analyzed by SDSPAGE (15%) and stained with Coomassie brilliant blue (BioRad) according to the Laemmli method.26 The MALDI-TOF analysis of the DaPLA2 was done by an Applied Biosystems voyager DE-RO mass spectrometer. In most cases, samples were desalted by purification on Zip-Tip C18 microcolumns (Millipore). Purified protein (100 μg) was mixed with sinapinic acid matrix solution (1:1, v/v), directly placed onto a target plate, and dried at room temperature. The average mass of the protein was obtained in positive linear mode, after external calibration, using Protein Calibration standard. The DaPLA2 was eluted with an H2O/acetonitrile/trifluoroacetic acid solution (20/80/ 0.1, v/v/v). To verify the NH2-terminal sequencing, the purified enzyme was blotted (1 h, 30 mA, 4 °C) onto a PVDF membrane (Applied Biosystems) in 20 mM Tris-HCl buffer (pH 8) containing 10% methanol using a mini trans-blot cell (BioRad, U.S.A.). The NH2terminal sequence was determined by automated Edman degradation, using an Applied Biosystems protein sequencer (Roissy, France).27 Hemolytic Assay. Hemolytic activity of DaPLA2 was checked as described previously,28 with slight modification. Briefly, hemolysis of erythrocytes from rat was assessed by the expressed and purified Sparidae phospholipases. Fresh blood was placed in Eppendorf tubes containing 3.8% sodium citrate (pH 7.4) as anticoagulant. To obtain a pure suspension of erythrocytes, 1 mL of blood was added to 20 mL of phosphate-buffered saline (PBS, pH 7.4). After centrifugation at 500g for 3 min at 4 °C, the red blood cell package was washed three times with PBS buffer, pH 7.4. Erythrocytes were finally suspended in PBS to make 1% solution for hemolytic assay. Purified enzyme was added to the suspension of red blood cells at final concentrations ranging from 0.05 to 1 mg/mL. Tubes containing the complex erythrocyte-enzymes were incubated for 1 h at 37 °C with mild stirring every 20 min. After centrifugation at 500g for 3 min, supernatants were transferred to 96-

well microplates and the absorbance at 540 nm was measured by using a spectrophotometric microplate reader to measure the extent of red blood cell lysis. Controls included erythrocytes incubated with PBS alone (0% hemolysis), and erythrocytes incubated with 1% Triton X100 in water (100% hemolysis) were also performed. Experiments were performed in triplicate. Sequences and Phylogenetic Analysis. The deduced Sparidae PLA2 amino-acid sequences were compared with those of digestive PLA2 from the NCBI Protein Database using the BLASTP algorithm.29 The amino-acid sequences of all the Sparidae sPLA2 were aligned and analyzed for the conserved regulatory and catalytically important motifs, using ESPript 3 software.30 The deduced amino-acid sequence of DaPLA2 was used as a template to identify homologous phospholipase sequences in PSI-BLAST research in the NCBI Protein Database. Homologous sequences from mammals, birds, reptiles, and fishes were used for multiple sequences alignment by program ClustalW31 from BioEdit v. 7.2.5 using default settings.32 Phylogenetic trees were constructed using the maximum parsimony method (Molecular Evolutionary Genetics Analysis MEGA6).33 The robustness of branches was assessed by bootstrap analysis of 100 replicates of resampling, and only values that were highly significant (=70) are shown.34 Homology Modeling. To establish the structural model for PLA2 from Sparidae species, we started by searching a candidate template in the Protein Data Bank (PDB). Beside the sequence identity and the quality of the structural experimental data, we considered sequence coverage of the entire mature peptide as the main criterion for selecting the template. The optimal pairwise alignment between target and template sequences was obtained by applying the Needleman− Wunsch algorithm implemented in the needle program from EMBOSS.35 MODELER 9.11 program was used to construct the 3D model of the PLA2 structure including the Ca2+ coordinating the catalytic dyad.36 MODELER was set to generate 20 conformers of the protein starting from random seeds. To select the one with the best quality, the ensemble was assessed with the discrete optimized protein energy (DOPE) implemented in the program. Among the first three favored models, we selected the one presenting the better Ramachandran profile. Molecular Docking and Dynamic Simulation. The coordinates of 1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine, an analogue of PLA2 substrates, cocrystallized with bee venom PLA2 (1POC), were used to guide the construction of the coordinates of three ligands, POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac(1-glycerol) (POPG), and POPC inside the substrate-binding site. The atom type assignment was in respect to the Gromos 53a6 force field from Gromacs v. 5.0.1.37 The complexes refinement consists of a 1 ns production run of molecular dynamics. To simulate dynamics of the generated complexes generated from the docking, the molecular system was solvated in an implicit simple point charge (SPC) water model,38 and the appropriate counterions were added to neutralize the system. The assignment of the topology and the parameters was set up using the Gromos 53a6 force field.37 To remove the steric artifacts resulting from the construction of the simulated system, we employed two cycles of energy minimization in implicit water using a restraining force on the headgroup atoms of the ligand as well as the protein atoms for 1000 of the steepest decent steps in the first cycle, then we removed the restraining forces and proceeded with a conjugate gradient minimization for 200 additional steps. A time step of 2 fs is chosen to generate the trajectory. For this reason, the LINCS algorithm39 was applied to constrain the hydrogen atom bonds. In addition, we used particle mesh Ewald (PME)40 approximation and a finite nonbonded cutoff of 12 Å to model the periodicity of the system and for the efficient treatment of the nonbonded calculations, respectively. The equilibration phase consists of two molecular dynamics stages in NVT ensemble. For the first stage, restraining forces were applied for the headgroup atoms of the ligand and the protein residues for a lapse of 40 ps. The nonrestrained molecular dynamics was then applied for the second stage over 20 ps until the system reached the desired temperature. Before starting the collection of snapshots, an additional 100 ps simulation in the NPT C


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Figure 1. Multiple sequence alignment of the deduced Sparidae PLA2 with homologue sequences from various animal groups including porcine and turkey pancreatic PLA2. Similar amino acids are shown in brown. Different amino acids were shown with black shaded boxes. Sequences were extracted by Esprint 3 software. Conserved cysteine residues are shown in yellow. The catalytic dyad (His48/Asp49) is indicated in blue. ensemble was performed using Parrinello−Rahman pressure barostat with a coupling time of 2 ps.41 The production runs for 5 ns were conducted after the correct pressure was attained. Snapshots were saved every 100 ps and analyzed with the build in tool provided in the Gromacs package.

binding loop and the catalytic site share 71.4% and 100% identities, respectively. The sequence alignment also revealed that Sparidae PLA2 have features that are typical of sPLA2’s of other mammals and birds that belong to subgroup IB. Phylogenetic Analysis of Sparidae PLA2. To study the relationship with pancreatic PLA2, a phylogenetic tree of sparidae PLA2 was built (Figure 2). Annotated sparidae PLA2 were aligned to various pancreatic PLA2. Several independent groups were identified, as indicated in Figure 2. Twenty-seven PLA2 sequences were selected from mammals (human, dog, polar bear, panda, macaque, cow, horse, bat, goat, and sheep), birds (turkey, eagle, and chicken), reptiles (turtle and lizard), and fish (Australian ghost shark, sole, Croceine croaker, and the red seabream). The generated alignment was subjected to FastTree 2.1.8 for inferring the phylogenetic tree with the maximum parsimony method.33 The Sparidae PLA2’s are indicated with a brown square. The phylogenetic tree of the deduced amino-acid sequences for Sparidae PLA2 identified in the present study fall into subgroup IB, which also contains PPLA2 and HuPLA2, as well as several sPLA2’s from avian species, including chicken and turkey PLA2-IB (Figure 2). PLA2 sequences from sole and Australian ghost shark are closely related marine pancreatic PLA214 and are grouped in the same branch as Sparidae PLA2. The pancreatic PLA2 from birds (chicken, turkey, and eagle) are the closest to the fish PLA2 (Figure 2). In addition, the neighbor-joining phylogenetic tree may be helpful to understand the mechanisms of evolutionary relationships between different Sparidae species. Figure 2 shows that, among Sparidae sPLA2, the digestive PLA2 of the red seabream (Pagrus major) is likely to be the closest species to the common ancestor of the studied fishes (Figure 2). Among the phylogenetic subgroup of the Sparidae PLA2, DaPLA2, identified as a pancreatic PLA2, was selected on the basis of comparative sequence analysis to expand the knowledge on marine PLA2 properties and structure−activity features.

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RESULTS AND DISCUSSION Cloning and Sequence Analysis of Sparidae PLA2 Genes. We successfully obtained the cDNA’s encoding phospholipases from five Sparidae species by reverse transcription using total RNA from the pyloric ceca from each species. The sequence length of recombinant PLA2 is 378 bp. These sequences are homologous to gene fragments encoding pancreatic PLA2 (sPLA2-IB) (Figure 1). The cDNAs were cloned, and sequences were deposited into the GenBank database. The alignment of Sparidae PLA2 sequences shows that they share a high identity, which ranges from 90 to 97% (Figure 1). Proteins homologous to Sparidae PLA2 were searched using BLAST on the UniProt database. Figure 1B shows the alignment of the amino-acid sequences and the conserved regions of Sparidae PLA2 with sequences of pancreatic sPLA2 from Sus scrofa (PPLA2, NM_001004037.1) and Meleagris gallopavo (TPLA2, NM_001004037.1). The amino-acid sequences share identities of 40−46% with pancreatic PLA2. Each sequence is composed of 125 amino acids with the predicted prepropeptide of 20 amino acids. Highly conserved regions were identified through comparisons with sPLA2’s (PPLA2 and TPLA2): (i) the signal peptide for secretion in the N-terminal region (20 amino acids) followed by a pro-peptide that must be cleaved by trypsin to produce the mature active enzyme; (ii) the PLA2 signature domain that includes the Ca2+-binding loop, the pancreatic loop (positions 62−67), and the catalytic site (His48/Asp49); and (iii) the 14 cysteine residues that potentially form seven intramolecular disulfide bridges (especially C11−C76). These features strongly suggest that fish enzyme would adopt a similar sPLA2 tertiary structure. The conserved regions that correspond to the Ca2+D


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Figure 2. Neighbor-joining phylogenetic tree showing the relationships between the Sparidae PLA2 and homologues in selected species from mammals, birds, and fishes. The analysis was performed using the MEGA (v. 5.0) program. The number of times a clade (sequences common to a node or branch) occurred in the bootstrap replicates are shown. Only replicate values of 70 or more, which are significant, are shown. The numbers at the branches indicate bootstrap values (100 replications). The lengths of the branches represent the amount of change that is estimated to have occurred between a pair of nodes. Sparidae PLA2’s are marked by a brown square. Neighbor-joining phylogenetic tree of five amino-acid PLA2 sequences from different Sparidae species (time tree). The evolutionary history was inferred using the neighbor-joining method.32 Divergence times for all branching points in the topology were calculated with the RelTime method33 using the branch lengths contained in the inferred tree. All positions containing gaps and missing data were eliminated. Evolutionary analyses of different PLA2’s were conducted in MEGA6.32

Expression, Refolding, and Purification of DaPLA2. The sequence corresponding to the DaPLA2 form, without the residues of the signal peptide obtained by PCR-mediated primers but containing NcoI/NotI restriction sites (at the 5′and 3′-extremities, respectively) (Table 1, Supporting Information), was overexpressed in E. coli as a C-terminal 6 × His-tag protein, which greatly accelerated the proteinpurification step. The E. coli BL21 (DE3) cells were transformed with the plasmid pET28b-DaPLA2, which allows the expression of a Cterminal tagged DaPLA2. Overexpression of the annular seabream PLA2 was maximal after a 4-h induction by IPTG

(final concentration of 1 mM). After centrifugation of the disrupted cells, the expressed protein was exclusively found in the precipitate as inclusion bodies (data not shown). The precipitate was solubilized using 8 M urea and then refolded in buffer containing L-cysteine to promote the refolding in a soluble form. Optimal renaturation of the DaPLA2 recombinant protein was achieved by dialyzing this protein against the refolding buffer. This buffer contained 10 mM NaCl and 5 mM L-cysteine, in order to reduce protein aggregation, to stabilize the refolded proteins, and to promote the formation of the correct disulfide bonds during the refolding. E


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Figure 3. Enzymatic properties of DaPLA2. (A) Effect of pH on DaPLA2 activity. DaPLA2 activity was measured at maintained temperature (50 °C) in the presence of substrate at different pH values. (B) pH stability of DaPLA2. The enzyme was incubated during 2 h at different pH values. After centrifugation, the supernatants of different samples were used to measure the activity of DaPLA2 at optimal conditions (pH 8.5 and 50 °C). (C) Effect of temperature on DaPLA2 activity. DaPLA2 activity was measured at various temperature (30−60 °C) in the presence of substrate at pH 8.5 and stability. (D) Thermostability of DaPLA2. The enzyme was preincubated at different temperatures (10−80 °C) for 15 min, and the residual activity was measured at 50 °C and pH 8.5 using PC as substrate, in the presence of 8 mM CaCl2 and 8 mM NaDOC. (E) Effect of Ca2+ concentration on DaPLA2 activity. Enzyme activity was measured at various concentrations of Ca2+ using PC emulsion as substrate at pH 8.5 and at 50 °C in the presence of 8 mM NaDOC. (F) Bile salts dependence of DaPLA2 activity. The enzyme activity was measured using PC emulsion as substrate at pH 8.5 and at 50 °C in the presence of 8 mM Ca2+.

Nickel metal affinity resin column was used for a single-step purification of His6-DaPLA2 (Figure 1A, Supporting Informa-

tion). Under these conditions, the enzyme was adsorbed to the support and then eluted by a linear imidazole gradient ranging F


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Figure 4. . (A) Variation with surface pressure in DaPLA2 activity using pure DLPG (△), DLPE (◊), DLPS (□), and DLPC (×). Assays were carried out at room temperature in a “zero-order” trough. The final enzyme concentration was 30 nM. Buffer: 10 mM Tris−HCl, pH 8.0, 100 mM NaCl, 21 mM CaCl2, and 1 mM EDTA. (B) Gas chromatography profile showing the main free fatty acid (C18:1) released after hydrolysis of POPC by DaPLA2.

from 50 to 350 mM imidazole. SDS-PAGE analysis of the purified DaPLA2 eluted from the nickel gel affinity column showed one homogeneous band corresponding to an apparent molecular mass of ∼15 kDa (Figure 1B, Supporting Information). This molecular mass seems to be similar to those of mammalian pancreatic PLA2 (∼14 kDa)42 and marine invertebrate species PLA2, such as crab digestive PLA2,43 but lower than that of the hepatopancreatic PLA2 of A. pectinifera (∼40 kDa)40 and the marine snail (30 kDa).44 The NH2terminal sequencing of the PVDF transferred band from an

electrophoresis gel allowed the identification of N-terminal residues (MALWQF) of the expressed DaPLA2. Native PAGE was performed to check oligomerization of DaPLA2 (data not shown), and no indications for oligomerization could be evidenced. The molecular mass of DaPLA2 was confirmed by MALDI-TOF mass spectrometry analysis. sPLA2 showed only one peak, with a molecular mass of 15127.02 Da (Figure 1C, Supporting Information). Enzymatic Properties of DaPLA2. Purified recombinant DaPLA2 was used to determine its substrate specificity and G


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Journal of Agricultural and Food Chemistry activity dependency on bile salts and Ca2+ ions. The DaPLA2 was active on PC liposomes, and its specific activity was determined to be 100 U/mg in the presence of 8 mM Ca2+ and 8 mM NaDOC using PC as substrate (Table 2, Supporting Information). It is worth noticing that the specific activity of the purified DaPLA2 is higher than that of Solaster paxillatus PLA2 (26 U/mg) reported in previous works,41 but it was lower than those of mammal and bird pancreatic sPLA2.8,45 The pH activity profile of the pure DaPLA2 is shown in Figure 3A. The DaPLA2 was found to be active between pH 7 and 10, with an optimum around pH 8 (Figure 3A). At low and high pH values, the DaPLA2 activity decreases, and this might be attributed to an irreversible protein denaturation. These results are in coherence with those described by Ben Bacha et al. for stingray sPLA2-IB, which presents an optimum pH activity at 8.5.18 The effect of pH on DaPLA2 stability was carried out, and residual activities were measured at 50 °C (Figure 3B). The DaPLA2 was found to be stable at a wide pH interval ranging from 6 to 10 (Figure 4B). This optimum was also similar to that of PLA2 purified from the marine snail,44 which is an alkaline enzyme with an optimum activity at pH 8.5. The effect of temperature (30−70 °C) on DaPLA2 activity was studied. The enzyme was found to be thermoactive, since the optimum activity was observed between 45 and 50 °C with steep decreases below and above this optimal temperature (Figure 3C). The relative activities at 37 and 55 °C were about 70% and 65%, respectively. The optimal temperature for DaPLA2 activity was similar to that of H. trunculus PLA2 (50 °C).44 The enzyme was preincubated at different temperatures (30−75 °C) for 15 min before its activity was determined at 50 °C, under standard conditions. The resulting curve showed a strong thermal inactivation above 65 °C (Figure 3D). Unlike dromedary8 and crab PLA2’s,41 which were found to be stable up to 40 °C, the DaPLA2 was stable at temperatures ranging from 20 to 60 °C. It retained 80% of its maximal activity after incubation for 15 min at 60 °C. DaPLA2 displayed the same behavior as the stingray pancreatic PLA2.18 Several studies provided evidence that Ca2+ is essential for both catalysis and enzyme binding to the substrate.45 The role of calcium as cofactor/activator of the catalytic activity is a common feature of sPLA2 from different sources.46 Therefore, we measured the enzyme activity at pH 8.5 and 50 °C using PC as a substrate in the presence of increasing Ca2+ concentrations (Figure 4E). The DaPLA2 activity could not be detected in the presence of Ca2+ chelator such as EDTA. Assessment of Ca2+ effect on PLA2 activity demonstrates that calcium is required for the catalytic activity. The specific activity of the enzyme increased to reach its maximum in the presence of 6 mM Ca2+ (Figure 3E). Similar results were observed with previously purified PLA2 from stingray,18 marine crab, and snail,43,44 which showed an absolute Ca2+ ions requirement for their activities. Several studies showed that bile salts are tensioactive agents that engage in micelle formation and ensure dispersion of the hydrolysis products increasing the activity of PLA2.7,47 To investigate the effect of bile salts on DaPLA2 activity, the rate of PC hydrolysis by the pure enzyme was recorded in the presence of increasing concentrations of bile salts, at pH 8.5 and 50 °C (Figure 3F). Bile salts (NaDOC) were required for the DaPLA2 activity. The maximal phospholipase activity was observed in the presence of 6 mM NaDOC. These findings corroborate previous observations with mammal and marine pancreatic PLA2’s.8,18

Substrate Specificity of DaPLA2 Using Monomolecular Film Technique. It is well-known that venom sPLA2 are able to hydrolyze monolayer films of phospholipids at a high surface pressure (30−40 mN·m−1), which allows them to attack biological membranes.47 To check the ability of fish PLA2 to hydrolyze phospholipid monolayer films, we reported in this study the rate of hydrolysis of different dilauroyl phospholipids films by DaPLA2 as a function of the surface pressure (Figure 4A). The activities of DaPLA2 toward DLPC, DLPE, DLPS, and DLPG were measured at various surface pressures using the Barostat technique to keep the interfacial quality constant during the reaction. To allow comparison, we reported in the same figure the rates of hydrolysis of different phospholipids by DaPLA2. Figure 4A shows that DaPLA2 presents an activity maximum, with bell-shaped surface pressure curves for all used phospholipids. On the basis of the classification established by Verheij et al.,48 DaPLA2 belongs to group III of PLA2, which includes pancreatic sPLA2. The optimum surface pressures that lead to the maximal DaPLA2 activities were dependent on the type of phospholipids used. The hydrolysis rates were negligible at low surface pressure, except for DLPG, and increased continuously until the highest pressures. It is clear that the DaPLA2 activities measured on DLPG and DLPE films are the highest as compared to those measured on DLPC and DLPS. The DaPLA2 had a strong preference for DLPG (Figure 4A). The same behavior was observed with chicken PLA2.9 This result is in agreement with the fact that PG is considered as the best substrate for all known sPLA2’s. These results suggest a higher penetration capacity of the DaPLA2 into DLPG films as compared to other phospholipids. DaPLA2 activity increased continuously between surface pressures of 10 and 15 mN·m−1, on DLPC, DLPS, and DLPE monolayers (Figure 4A). Beyond 15 mN·m−1, a drop in DaPLA2 activity on all phospholipids was recorded, and the enzyme was partially inactive at 30 mN·m−1 (Figure 4A). In a previous work, Singer et al.49 used anionic vesicles of POPG and 1-palmitoyl-2-oleoyl-phosphatidyl-Lserine (POPS) and showed that the hydrolysis reaction started immediately after the addition of sPLA2 with no discernible lag. In our study, we have observed a lag time before DaPLA2 hydrolyzes the substrates. This could be explained by the fact that DaPLA2 takes more times to penetrate to the oil/water interface.24 DaPLA2 Positional Specificity. To determine the stereospecificity of the DaPLA2, the enzyme was incubated with POPC as substrate, and the fatty acid products obtained after the hydrolysis were analyzed by gas chromatography (Figure 4B). As shown in Figure 4B, oleic acid (C18:1) could be seen as a main peak with a retention time of ∼35 min. However, a small proportion of palmitic (C16:0) could be observed at a retention time of ∼10 min. This might be a result of an intramolecular process of acyl migration during the lipolysis.50 This result indicates that the purified DaPLA2 belongs to the PLA2 family (E.C. 3.1.1.4) and is able to hydrolyze mainly the sn-2 position of phospholipids, because the PC used as substrate contains an unsaturated fatty acid only at the sn-2 position. Hemolytic Activity of DaPLA2. sPLA2 can play digestive and toxic roles, like venom PLA2’s that have similar threedimensional structures and that exert an amazing variety of proinflammatory, platelet-aggregation inhibitory, cytotoxic, and bactericidal activities.51 We measured the hemolytic activity of DaPLA2 with human erythrocytes using various enzyme concentrations (data not shown). The DaPLA2 showed no H


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Figure 5. Comparative modeling and structural analysis of DaPLA2. (A) The homology models of DaPLA2 (red) and HuPLA2 (transparent gray) were superposed. (B) The nonidentical residues of DaPLA2 compared to HuPLA2 are mapped on the catalytic surface of the homology model structure. The residues in orange and blue colors correspond, respectively, to nonhomologous amino acids. (C) Hydrophobicity mapping at the DaPLA2 and HuPLA2 protein surfaces using the Kyte and Doolittle scale implemented in Chimera molecular viewer.

each of these residues are at 4.4 and 7.6 Å distance from the catalytic Ca2+ ion, respectively. Compared to the human PLA2 (HuPLA2, P04054), DaPLA2 shares 47% of sequence identity (Figures 1 and 5B). The hydrophobic surface of DaPLA2 is very different from the HuPLA2, presenting more hydrophobic patches on the accessible surface of the protein. In fact, the apolar accessible surface areas, calculated over the ensemble of conformations from the molecular dynamics simulations, correspond to 658.3 ± 10 Å2 and 621.0 ± 12 Å2 for DaPLA2 and HuPLA2, respectively (Figure 5C). Hydrophobic residues W3, L19, F30, L66, and V69 are placed in the same plane, suggesting that these residues might play an important role in the enzyme binding to phospholipids extracellular layer, explaining the preference of the enzyme for neutral substrates (Figure 2, Supporting Information). Molecular Docking. The DaPLA2/POPG and DaPLA2/ POPE complexes were refined using 1 ns explicit molecular dynamics simulations (Figure 6). Only the last frame of each trajectory was analyzed (Figure 6). For both complexes, the sn2 aliphatic chain lays inside a long cavity constituted by several hydrophobic amino acids, F5, I9, and F105, whereas L2 and F30 residues have exposed lateral chains and stabilize the sn-1 aliphatic chain. The polar moiety of the two ligands establishes contacts with a cluster of polar residues belonging to H2 helix and the amino acids from the long loop separating H2 and S1 (the first strand of the β sheet). We noticed that the packing of the polar headgroup of POPG ligand (Figure 6A) is improved compared to the corresponding atoms in POPE (Figure 6A). The examination of the surrounding environment revealed the

hemolytic activity with human erythrocytes even at the highest concentration tested. Unlike venoms PLA2’s, DaPLA2 was mainly active at surface pressures ranging from 12 to 20 mN· m−1 (See Figure 4A). This kinetic property can be correlated to its low capacity to attack biological membranes.48 These observations agree with earlier results reporting that full-length human IB pancreatic PLA2 had no hemolytic activity toward human cells.52 Molecular Modeling of DaPLA2 Structure. Up to now, there is no solved X-ray crystal structure for fish PLA2. Thus, the wild boar (Sus scrofa) PLA2 structure (PDB code: 1FX9) was identified to be the best template to construct the 3D structure model of DaPLA2. The sequence identity of 50% is covering the protein length. The final selected model is second ranked according to the DOPE score in which 96%, 3%, and 1% of the corresponding dihedral angles are situated in the highly favored domain, the allowed regions, and the outlier regions of the Ramachandran plot, respectively. The protein displayed the hallmark of a PLA2 fold (Figure 5A,B). Most of the protein residues make part of either the three major helices H1 (2−12), H2 (39−56), and H3 (89−107); the three singleturned helices H4 (18−21), H5 (67−69), H6 (112−114), and H7 (119−122); or the long loops separating the rigid secondary structure elements. In addition, two segments corresponding to residues 74−77 and 80−83 are involved in the beta wing formation. Seven disulfide bonds (C11−C76, C83−C95, C60−C90, C50−C97, C43−C104, C28−C44, and C26−C123) are stabilizing the structure of PLA2. The catalytic dyad that constitutes the most important structure element of pancreatic PLA2 is also conserved (H48 and D49) (Figure 5A). The Cα atoms of I


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Figure 6. Binding and stability study of the substrate/PLA2 complexes using docking and molecular dynamics. (A) Molecular docking of POPG and POPE ligands to the DaPLA2 model. (B) Molecular dynamics of POPC/DaPLA2 and POPC/HuPLA2 using different temperatures. The positions of S1, S2, and S3 segments are marked with blue lines. The position of the 69 loop is marked with gray shaded boxes. HuPLA2 was simulated at 324 K only. (C) Mapping the S1, S2, and S3 segments on the structure of DaPLA2.

ns) were run with POPC to explain the enzyme’s thermostability in the in vitro assay using PC liposomes (Figure 6B,C). We calculated the evolution of the secondary structure during the 5 ns molecular dynamics simulations for the three temperatures. Differences were noted between the three profiles representing the different simulation temperatures. The H2 loop, particularly its 51−55 segment, loses the helical secondary structure at 324 K, while the same region is stable at 311 and 333 K. The D49, which constitutes the catalytic dyad, is still within the α-helical structure of H2, appearing to maintain a functional geometry. In addition, at the high temperature of 333 K, the C-terminal part of H1 seems to be less stable compared to the other temperatures (Figures 3 and 4, Supporting Information). At the end of the simulation, the nearby short helix (18−22) appears to block the interaction hydrophobic cleft of the enzyme. The sn-1 and sn-2 lose contact with this interaction site and become more exposed. This behavior was not observed for the two other temperatures. We

presence of two positively charged residues, K52 and R55. It seems that the electrostatic repulsion between POPE headgroup and these two residues is effective because of the positive charge carried by the amine group of the ligand, which might add an energetic penalty upon the binding or affect the substrate/enzyme-recognition process or the stability of the substrate after reaching the catalytic binding site. The POPG headgroup is neutral. The electrostatic environment does not seem to disrupt the ligand packing. In fact, in one of the collected snapshots from the molecular dynamics refinement, R55 was capable of forming a hydrogen bond with one of the hydroxyl groups from the polar moiety of POPG. The key feature of an enzyme function is the maintenance of an adequate balance between molecular stability and structural flexibility. To explain the thermostability of the DaPLA2, we used molecular dynamics (MD) simulations. The MD simulations at three temperatures (311, 324, and 333 K for 5 J


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attractive features, making it a potential candidate for various applications and processes. Stability of DaPLA2 and its tolerance to high temperatures and alkaline pH might open new opportunities for the treatment phospholipid-rich industrial effluents, the production of efficient inter-esterification substances in the food-processing industry, or the synthesis of cost-effective chemical compounds for the food-production industry. With the development of DNA technology, a number of phospholipases could be expressed in different hosts. This could lead to the production of new phospholipases and the development of new industrial applications. Further improvements of the recombinant DaPLA2 characteristics could be obtained by protein engineering.

also checked the evolution of the hydrogen bond number for the three simulation. Compared to 311 and 324 K, the number of hydrogen bonds is more important at 333 K (Figure 4, Supporting Information). This seems to rigidify the structure. It seems that the loss of activity at 60 °C temperature is in relation to accessibility of the ligand to the hydrophobic interaction site in DaPLA2. At high temperature, the hydrophobic interactions increase between the substrate aliphatic chain interacting cleft and the loop between H1 helix and the Ca2+-binding loop, which prevents the substrate from adopting an optimized conformation prior to the catalysis. The Cα root-mean-square deviation (RMSD) time evolution was stable during the first 3.5 ns at 311 K, and then the deviation of the PLA2 structure increased to be stable until the end of the simulation (Figure 6B). At 324 K a first stable phase lasts for 1300 ns followed by another stable evolution of the RMSD during the rest of the trajectory, between 0.25 and 0.3 nm. However, at 333 K, the first plateau lasted only for 2.5 ns; then the RMSD was elevated to reach 0.25 nm and continued to rise until the end of the simulation. Except for a few intervals, the structure of the enzyme at 311 K presented less deviation compared to other trajectories at 324 and 333 K. The correlation of the enzyme stability with the RMSD time evolution does not seem clear for these two temperatures because the second plateau phases are relatively similar in both trajectories. We calculated the root-mean-square fluctuation (RMSF) profiles from the three sets of collected snapshots (Figure 6C). The flexibility of segments S1 (residues 38−44 belonging to the N-terminal end of H2 α helix) and S2 (residues 59−65) does not seem to depend on the temperature increasing because the RMSF per amino acid is enhanced only at 324 K and displayed lesser flexibility at 311 and 333 K (Figure 6C). The temperature seems to enhance the flexibility of the S3 segment composed of amino acids 115−118. This might explain a reduced activity when the temperature increases. The comparison of RMSF profile from DaPLA2 with that of the HuPLA2, used as a control, shows that the loop separating the H2 helix and the first strand of the β sheet element behaves differently between the two enzymes (Figure 6C). Its flexibility is enhanced for the HuPLA2. The difference in flexibility between DaPLA2 and HuPLA2 occurs in the so-called 69-loop (the loop separating the H2 helix and the first beta strand of the protein and containing the key residue Y69 in HuPLA2). It has been demonstrated that this segment plays an important role in the substrate recognition.53 The comparison between the two structures revealed that the conformation of the segment in the crystal structure of HuPLA2 is less ordered as compared to the equivalent residues in DaPLA2, which form the H5 singleturned helix. Y68 (equivalent to Y69 in HuPLA2) side chain is packed toward the core of the protein, while in HuPLA2 the side chain of the residue is exposed. The N-terminal end of HuPLA2 adopting a loop conformation occupies in part the catalytic binding side and remains at this position during the full 5 ns trajectory while the N-terminal of DaPLA2 makes part of the H1 helix. The 69-loop in HuPLA2 seems to be less stable than that in DaPLA2. The rigid structure of 69-loop compared to the equivalent segment in HuPLA2 might be relevant in stabilizing the ligands positioning in the catalytic binding site.54 The highly efficient production of recombinant DaPLA2 provides the basis for the industrial use of the recombinant enzyme. Moreover, this marine phospholipase exhibited several

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05810. Primers used for the cloning and expression of genes encoding mature sparidae phospholipases; flow sheet of DaPLA2 purification; DaPLA2 purification; DaPLA2 membrane contact residues; secondary structure of the DaPLA2/POPC complex during the molecular dynamics simulation; evolution of the hydrogen bond number for the molecular dynamics simulation of DaPLA2/POPC complex at different temperatures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Dr. Nabil Smichi, CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Email: [email protected], [email protected]. ORCID

Nabil Smichi: 0000-0003-1416-5378 Funding

This work received financial support from the Ministry of Higher Education, Scientific Research, in Tunisia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ms. Régine Lebrun (Plateforme Protéomique de l’Institut de Microbiologie de la Méditerranée, Marseille, France) for her help in protein sequence determination and MALDI-TOF analysis.

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ABBREVIATIONS 1,2-DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2DLPE, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine; 1,2DLPG, 1,2-dilauroyl-sn-glycero-3-phosphoglycerol; 1,2-DLPS, 1,2-dilauroyl-sn-glycero-3-phosphoserine; DaPLA2, Diplodus annularis PLA2; DsPLA2, Diplodus sargus PLA2; HuPLA2, human pancreatic sPLA2; IPTG, isopropyl β-1-thiogalactopyranoside; LmPLA2, Lithognathus mormyrus PLA2; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PBS, phosphate-buffered saline; POPC, 1,2-palmitoyloleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol); PC, phosphatidylcholine; PPLA2, porcine pancreatic sPLA2; sPLA2, secreted phospholiK


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pase A2; SsPLA2, Sarpa salpa PLA2; NaDOC, sodium deoxycholate; SaPLA2, Sparus aurata PLA2; TPLA2, turkey pancreatic PLA2

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