Biochemical and molecular characterization of purified chicken pancreatic phospholipase A 2 Aida Karray, Fakher Frikha, Abir Ben Bacha, Yassine Ben Ali, Youssef Gargouri and Sofiane Bezzine Laboratoire de Biochimie et de Ge ´ nie Enzymatique des Lipases, ENIS, Sfax, Tunisia Introduction Phospholipases A 2 (PLA 2 s; EC 3.1.1.4) hydrolyze stereospecifically the 2-acyl ester bond of 1,2-diacyl- sn-3-phosphoglycerides, generating fatty acids and lysophospholipids. Several PLA 2 s have been identified on the basis of their gene sequences. They have been classified mainly into three groups: (a) cytosolic PLA 2 ; (b) Ca 2+ -independent intracellular PLA 2 ; and (c) Ca 2+ -dependent secreted PLA 2 (sPLA 2 ). They differ from each other in terms of substrate specificity, Ca 2+ requirement, and lipid modification. There is also a PLA 2 class that hydrolyzes platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and oxidized lipids, called platelet-activating factor acetyl- hydrolases [1,2]. sPLA 2 s form one of the main classes within the PLA 2 superfamily, and some of their members are well characterized. Important common structural features of the sPLA 2 s are well conserved: the central a-helices with the catalytic His–Asp dyad, and the hydrogen- bonding network connecting the interfacial binding site, the catalytic site and the Ca 2+ -binding site [3]. Eleven sPLA 2 s have been identified in mammals (groups IB, IIA, IIC, IID, IIE, IIF, V, X, III, XIIA, and XIIB). They display partially overlapping tissue Keywords bile salts; chicken; modeling; phospholipase A 2 ; purification Correspondence S. Bezzine, Laboratoire de Biochimie et de Ge ´ nie Enzymatique des Lipases, ENIS, Route de Soukra, 3038 Sfax, Tunisia Fax: +216 74675055 Tel: +216 74675055 E-mail: sofiane_bezzine@yahoo.com Database The nucleotide sequence of ChPLA2 deter- mined in this study has been deposited in the GenBank database under the accession number EU617018 (Received 13 March 2009, revised 7 June 2009, accepted 17 June 2009) doi:10.1111/j.1742-4658.2009.07160.x Chicken pancreatic phospholipase A 2 (ChPLA 2 ) was purified from delipi- dated pancreases using ammonium sulfate and ethanol precipitation, followed by sequential column chromatography steps on MonoQ Sepha- rose and size exclusion HPLC columns. ChPLA 2 was found to be a non- glycosylated monomeric protein with a molecular mass of 14 kDa and a specific activity of 400 UÆmg )1 in the presence of 1 mm sodium taur- odeoxycholate and 4 mm CaCl 2 with phosphatidylcholine as substrate. The N-terminal sequence of the first 15 amino acids of ChPLA 2 was deter- mined, and showed a high degree of homology with known mammal pan- creatic phospholipases A 2 . The gene encoding the mature ChPLA 2 was cloned and sequenced. The deduced amino acid sequence of the mature ChPLA 2 confirmed the high level of identity with mammal pancreatic PLA 2 . To investigate the structure–activity relationships, a 3D model of group IB ChPLA 2 was built using the porcine pancreatic phospholipase A 2 structure as template. Abbreviations ChPLA 2 , chicken pancreatic phospholipase A 2 ; DrPLA 2 , dromedary pancreatic phospholipase A 2 ; NaTDC, sodium taurodeoxycholate; OPLA 2 , ostrich pancreatic phospholipase A 2 ; PC, phosphatidylcholine; PLA 2 , phospholipase A 2 ; PPLA 2 , porcine pancreatic phospholipase A 2 ; sPLA 2 , secreted pancreatic phospholipase A 2 ; TPLA 2 , turkey pancreatic phospholipase A 2 . FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS 4545 distributions [3,4]. Only sPLA 2 -IB and sPLA 2 -X have an N-terminal prepropeptide, and the proteolytic cleavage of this prepropeptide is the regulatory step for the generation of an active enzyme [5]. sPLA 2 -IB is found in large amounts in the pancreas, and its princi- pal function is the digestion of dietary lipids [5]. How- ever, sPLA 2 -IB is also found in nondigestive tissues, including the lung, spleen, gonad, and kidney [4,6]. Receptors of this enzyme have been identified in vari- ous tissues, and sPLA 2 -IB is reported to play a role in cell proliferation and hormone release via these recep- tors [6,7]. Group IB sPLA 2 s have been isolated from the pan- creases of various mammal species [8–13]. In contrast, few studies have been performed on the bird PLA 2 [14,15]. Recently, Ben Bacha et al. [15] biochemically characterized an active thermostable PLA 2 from ostrich pancreas (OPLA 2 ). It is therefore interesting to investigate more biochemical and structural properties of other purified bird PLA 2 s to gain more insights into their mode of action on phospholipids. We describe in this study the purification and some biochemical prop- erties of an sPLA 2 from chicken pancreas (ChPLA 2 ). This article also reports, for the first time, the cloning of a bird PLA 2 cDNA and a comparison of the corre- sponding amino acid sequence with that of known mammal PLA 2 s. Molecular modeling was also pro- posed to explain some biochemical differences between ChPLA 2 and other pancreatic PLA 2 . Results and discussion Purification of ChPLA 2 Thirty grams of delipidated chicken pancreas was sus- pended in 300 mL of buffer A (10 mm Tris ⁄ HCl, pH 8.5, 10 mm CaCl 2 , 0.15 m NaCl) and ground mechani- cally twice for 30 s at room temperature using the Waring Blendor System. The mixture was then stirred for 30 min at room temperature and centrifuged for 30 min at 12 000 g. The endogenous trypsin was found to be sufficient to achieve PLA2 activation, as the total PLA 2 activity obtained (2240 U) did not increase when trypsin was added at different ratios to the PLA 2 solu- tion (data not shown). Ammonium sulfate precipitation Chicken pancreatic extract was brought to 65% satu- ration with solid ammonium sulfate under stirring and maintained for 30 min at 4 °C. After centrifugation (30 min, 12 000 g 4 °C), the precipitate was resus- pended in minimum volume of buffer A containing 2mm benzamidine. Insoluble proteins were removed by centrifugation (15 min, 24 000 g). The recovery of PLA 2 activity was about 49%. Ethanol fractionation Resuspended precipitate (50 mL) was subjected to frac- tionation using ethanol, 21.5 mL of ethanol 30% (v ⁄ v) being added at 0 °C. Insoluble proteins were removed by centrifugation (30 min, 24 000 g 4 °C), and ethanol was again added slowly at 0 °C to the supernatant, increasing the alcohol concentration to 80% (v ⁄ v). Pre- cipitated proteins, which contained about 43% of the starting amount of PLA 2 , were solubilized in a mini- mum volume of buffer A containing 2 mm benzami- dine. In the present study, we have found that this step is of prime importance for eliminating the residual lipids and facilitating the ChPLA 2 purification. The resulting sample was dialyzed overnight against 20 mm Tris ⁄ HCl buffer (pH 8) containing 20 mm NaCl and 2mm benzamidine (buffer B). Anion exchange and gel filtration chromatography The dialyzed sample (40 mL, 970 U) of ChPLA 2 was centrifuged (10 min, 12 000 g at 4 °C) and poured into a MonoQ Sepharose column (3 · 7.5 cm) equili- brated with buffer B. Under these conditions, the enzyme is adsorbed to the cationic support. The col- umn was washed with 200 mL of buffer B containing 100 mm NaCl to eliminate some contaminant. ChPLA 2 was eluted from the MonoQ Sepharose col- umn upon a single wash with the same buffer con- taining 200 mm NaCl. Fractions containing ChPLA 2 activity were pooled and concentrated (Fig. 1A). The recovery of ChPLA 2 after the MonoQ step was  25%, with a specific activity of 50 UÆmg )1 . The concentrated proteins were loaded on a Bio-sil SEC- 125 size exclusion HPLC column (300 · 7.8 mm) equilibrated in buffer C [0.1 m phosphate buffer (pH 6.8) containing 0.15 m NaCl]. Elution was performed with phosphate buffer at 1 mLÆmin )1 , and ChPLA 2 emerged 7 min after injection (Fig. 1B). The fractions containing the PLA 2 activity were pooled, and SDS ⁄ PAGE analysis revealed only one band corre- sponding to ChPLA 2 . The molecular mass of ChPLA 2 as estimated by gel filtration on HPLC was 14 kDa (Fig. 1B). Oligosaccharide analysis of the pure ChPLA 2 indicated that the enzyme was not gly- cosylated, similarly to all previously described pancre- atic PLA 2 s (data not shown). Altogether, these results suggest that ChPLA 2 is a monomeric protein, like the PLA 2 s from the ostrich and all mammals. Chicken pancreatic PLA 2 A. Karray et al. 4546 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS The purification flow sheet given in Table 1 shows that the specific activity of pure ChPLA 2 reached 400 UÆmg )1 when phosphatidylcholine (PC) was used as substrate, at pH 9.5 and 37 °C, in the presence of 1mm sodium taurodeoxycholate (NaTDC) and 4 mm CaCl 2 . The ChPLA 2 purification yield was  16%, which is comparable to that obtained for the dromedary PLA 2 (DrPLA 2 ) and porcine PLA 2 (PPLA 2 ) [5,13]. Moreover, the procedure described here is more rapid than those used to purify mammal pancreatic PLA 2 .In fact, ChPLA 2 was purified after only two chromato- graphy steps, whereas in the case of DrPLA 2 or PPLA 2 , four chromatography steps were needed [5,13]. Enzymatic properties of the purified ChPLA 2 Ca 2+ dependence It is well established that Ca 2+ is essential for both catalysis and enzyme binding to the substrate [16,17]. In this study, we measured ChPLA 2 activity at pH 9.5 and at 37 °C, using PC as substrate, in the presence of increasing concentrations of Ca 2+ (Fig. 2A). No PLA 2 Fraction no 0 0.2 0.4 0.6 0.8 1 1.2 A B 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 PLA2 activity (U·mL –1 ) Absorbance (280 nm) [NaCl] mM 0.2 0.3 0.1 Molecular mass markers; MM (kDa) 1 10 30 100 Active fraction 0 2 4 6 8 10 12 14 16 18 Absorbance (280) Time ( min ) 0.064 0.054 0.044 0.034 0.024 0.014 0.0043 14 20 30 43 67 97 MM (kDa) 1 2 3 Fig. 1. (A) MonoQ chromatography. A Mono-Q column (3 · 7.5 cm) was prepared and equilibrated with buffer B (20 m M Tris ⁄ HCl, 20 mM NaCl, 2 mM benzamidine). The dialyzed sample from ethanol precipita- tion was poured into this column, and fixed proteins were eluted with 200 mL of the same buffer containing 100 m M NaCl fol- lowed by 150 mL containing 200 m M NaCl. Fractions containing ChPLA 2 were eluted at a flow rate of 45 mLÆh )1 . The horizontal bold line represents pooled samples from frac- tion no. 36 to fraction no. 48. (B) Filtration on HPLC column and SDS-PAGE (15%) analysis of ChPLA 2 . Left panel: filtration on a Bio-sil SEC-125 HPLC column (300 cm · 7.8 mm) equilibrated in buffer C (0.1 M phosphate buffer, pH 6.8, containing 0.15 M NaCl). Elution was performed at room temperature within 20 min, with the same buffer at a flow rate of 1 mLÆmin )1 . Molecular mass markers were used to esti- mate the molecular masses of eluted pro- teins. Right panel: SDS ⁄ PAGE (15%). Lane 1: molecular mass markers. Lane 2: 10 lg of fractions eluted and concentrated from Mono-Q chromatography. Lane 3: 10 lgof fractions eluted from HPLC filtration and containing ChPLA 2 activity. The gel was stained with Coomassie blue. Table 1. Flow sheet of ChPLA 2 purification; 30 g of defatted pancreas obtained from 100 g of fresh tissue was homogenized in 300 mL of buffrer (10 m M Tris ⁄ HCl, pH 8.5, 10 mM CaCl 2 , 0.15 M NaCl). Total activity: 1 U is lmole of fatty acid released per minute with PC as sub- strate in the presence of 1 m M NaTDC and 4 mM CaCl 2 . Protein concentrations were estimated by the Bradford method [26]. The experi- ments were conducted four times. Purification step Total activity (U) Total protein (mg) Specific activity (UÆmg )1 ) Yield (%) Purification factor Extraction (pH 8.5) 2240 11 200 0.2 100 1 (NH 4 ) 2 SO 4 precipitation (25–65%) 1100 1833 0.6 49 3 Ethanol precipitation (30–80%) 970 323 3 43 15 MonoQ Sepharose 600 12 50 26 250 Filtration (HPLC) 360 0.9 400 16 2000 A. Karray et al. Chicken pancreatic PLA 2 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS 4547 activity was determined in the presence of chelators such as EDTA or EGTA at 10 mm. In the absence of chelators, the specific activity of ChPLA 2 increased to reach 400 UÆmg )1 at 4 mm CaCl 2 (Fig. 2A). In agree- ment with previous findings regarding mammal pancre- atic PLA 2 [8–10,12–14,18], our study also attempted to show that bird PLA 2 requires the presence of Ca 2+ to trigger the hydrolysis of phospholipids. Bile salts dependence Several studies have provided evidence that micellar concentration of bile salts ensure the dispersion of the hydrolysis products [12,19]. In order to investigate the effect of bile salts on ChPLA 2 activity, the rate of hydrolysis of PC by ChPLA 2 in the presence of vari- ous concentrations of bile salts, at pH 9.5 and at 37 °C, was studied. As shown in Fig. 2B, in contrast to all other known PLA 2 s, which are bile salt dependent [8–10,12–14,18], ChPLA 2 displays 50% of its maximum activity in the absence of this detergent. The maximum phospholipase activity was measured at 1 mm NaTDC. Effect of temperature on ChPLA 2 activity and stability As shown in Fig. 3A, ChPLA 2 maximal activity was measured at 37 °C, using PC as substrate in the pres- ence of 4 mm Ca 2+ and 1 mm NaTDC. The purified enzyme was found to lose more then 50% of its activ- ity at temperatures higher than 40 °C (Fig. 3B). Simi- lar results were obtained with turkey pancreatic PLA 2 (TPLA 2 ), which is completely inactivated at 60 °C [14]. In contrast, PPLA 2 , taken as a model of mammal PLA 2 , can tolerate incubation at high temperature (Fig. 3B). Effect of pH on ChPLA 2 activity and stability The maximal activity of ChPLA 2 was measured at pH 9.5, using PC as substrate in the presence of 4 mm Ca 2+ and 1 mm NaTDC. ChPLA 2 was inactivated after incubation for 30 min at pH values lower than 4 (Fig. 3D) [14]. For further comparison, we report the results obtained with PPLA 2 . As shown in Fig. 3D, PPLA 2 was found to be more resistant at pH 3 than ChPLA 2 . N-terminal sequence analysis of ChPLA 2 ChPLA 2 N-terminal sequencing allowed the unambigu- ous identification of 15 residues of the pure enzyme. The results presented in Table 2 show the alignment of the N-terminal sequence of ChPLA 2 with those of TPLA 2 [14], OPLA 2 [15], DrPLA 2 [13], PPLA 2 [20], bovine PLA 2 [20], and human PLA 2 [21]. The N-termi- nal sequences of bird PLA 2 s exhibit more than 66% identity with those of mammal PLA 2 s. Cloning and sequencing of the gene coding for the mature PLA 2 The cDNA encoding ChPLA 2 was selectively amplified by RT-PCR from total mRNA extracted from chicken pancreas, as described in Experimental procedures. The corresponding DNA was ligated into PCR-Blunt vector and used for transformation into Escherichia coli 0 100 200 300 400 500 0 2 4 6 [NaTDC] (mM) Specific activity (U·mg –1 ) 0 100 200 300 400 500 0 2 4 6 8 10 12 Specific activity (U·mg –1 ) [CaCl 2 ] (mM) * A B Fig. 2. (A) Effect of Ca 2+ concentration on ChPLA 2 activity. Enzyme activity was measured at increasing concentrations of Ca 2+ , using PC as substrate, at pH 9.5 and at 37 °C in the presence of 1 m M NaTDC. The star indicates the phospholipase activity measured in the absence of CaCl 2 and in the presence of 10 mM EDTA or EGTA. (B) Effect of increasing concentrations of bile salts (NaTDC) on ChPLA 2 activity. PLA 2 activity was measured using PC as sub- strate, at pH 9.5 and at 37 °C in the presence of 4 m M Ca 2+ . Chicken pancreatic PLA 2 A. Karray et al. 4548 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS DH5a cells. Several clones were selected, and some of them contained a recombinant plasmid with a 500 bp EcoRI insert. The cDNA sequencing confirmed that the PCR product corresponded to the gene coding for the mature ChPLA 2 (Fig. 4). The deduced polypeptide sequence of ChPLA 2 , corresponding to the mature protein, comprises 124 amino acids and has a calcu- lated molecular mass of 14 167 Da. As revealed by sequence similarity with pancreatic PLA 2 , ChPLA 2 shares 65% identity with PPLA 2 . Residues of the cata- lytic diad and the pancreatic loop are conserved in ChPLA 2 . The 14 cysteines involved in disulfide bridges in PLA 2 are also conserved, suggesting the presence of seven disulfide bridges in the avian pancreatic phos- pholipase structure. Homology modeling of ChPLA 2 As previously described, the overall folds of PLA 2 are highly conserved, and the primary sequences also exhibit reasonable homology [22,23]. In order to investigate the biochemical properties of ChPLA 2 , a structural model was built. The 3D structure of ChPLA 2 was modeled, using the crystal structure of PPLA 2 as template (Pro- tein Data Bank code: 1l8sB), as these two proteins share 65% amino acid identity. A model of ChPLA 2 in com- plex with a substrate analog was also generated, using the crystal structure of PPLA 2 in complex with the MJ33 inhibitor as template (Protein Data Bank code: 1fx9A). The generated model of ChPLA 2 was then subjected to molecular mechanics optimization using Table 2. Alignment of the N-terminal sequences of ChPLA 2 , PPLA 2 , TPLA 2 , OPLA 2 DrPLA 2 , bovine PLA 2 and human PLA 2 . Identical amino acids are in bold, and homologous amino acids are in italic. 11015 ChPLA 2 :ALWEF RSMIK CAIPH This study PPLA 2 :ALWQFRSMIK CTIPG [20] TPLA 2 :ALFE FRSMIK CTIPG [14] OPLA 2 :AV WQFREMIK CTIPP [15] DrPLA 2 :ALWQFRDMIK CKIPD [13] Bovine PLA 2 :ALWQF NGMIK CKIPS [20] Human PLA 2 :AV WQFRKMIK CV IPG [21] 0 200 400 600 800 1000 A C D B 25 30 35 40 45 50 55 Specific activity (U·mg –1 ) Temperature (°C) Specific activity (U·mg –1 ) 0 200 400 600 800 1000 78 9 10111213 pH 0 20 40 60 80 100 120 0 102030405060 Temperature (°C) Residual activity (%) 0 20 40 60 80 100 120 0 2 4 6 8 10 12 Residual activity (%) pH Fig. 3. Effect of temperature and pH on ChPLA 2 activity (A,C) and stability (B,D). ChPLA 2 ( ) and PPLA 2 ( ), used as controls, were tested for activity at various temperatures (A) and pH values (C), as described in Experimental procedures. For stability studies, 1 mg ⁄ mL ChPLA 2 or PPLA 2 was incubated for 30 min at various temperatures (B) or pH values (D). Residual activity was measured as described in Experimental procedures. For temperature stability studies, enzymes were incubated in 10 m M Tris (pH 8), 150 mM NaCl, and 10 mM CaCl 2 . For pH stability studies, Tris buffer was replaced with the appropriate buffer for the pH range. A. Karray et al. Chicken pancreatic PLA 2 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS 4549 the CHARMM27 forcefield and hyperchem soft- ware. Energy minimization (geometry optimization) was performed until a gradient of 0.02 kcal ⁄ (A ˚ .mol) was reached. The rmsd between the initial and the optimized model was  1.4 A ˚ (calculated on 124 a- carbons). The Ramachandran plot statistics of ChPLA 2 and the complex ChPLA 2 –substrate analog model showed that 97% and 94.6–97.3% of the resi- dues were in the most favored or in the additional allowed regions, respectively. Overall 3D structure model of ChPLA 2 The ChPLA 2 model structure showed seven disulfide bridges like those found in other group IB PLA 2 s, and has the following structural features: (a) an N-terminal a-helix (aA, Leu2–Ala12); (b) a ‘short’ helix (aB, Leu19–Phe22); (c) a Ca 2+ -binding loop (Trp28– Gly33); (d) two antiparallel a-helices (aC, Glu40– Lys56; and aD, Ala90–Lys108); (e) two short strands of an antiparallel b-sheet (b-wing) (b2, Ser74–Ser78; and b3, Glu81–Asn85); and (f) a C-terminal loop con- taining a short b4-strand (His115–Asn117) antiparallel to the b1-strand (Asn24–Gly26) (Fig. 5A). The cata- lytic network for class IB PLA2s, formed by His48, Tyr52, Tyr73, and Asp99, is fully conserved in ChPLA 2 . Ca 2+ site Ca 2+ binding, required for PLA 2 activity, involves Asp49 and Tyr28. The Asp49–Ca 2+ diatnace is in the range 2.6–2.8 A ˚ [23]. Therefore, the low catalytic activ- ity of ChPLA 2 may be due to distortion of the Ca 2+ - binding loop that places the Ca 2+ away from the d1 atom of Asp49. The ChPLA 2 models show that the catalytic Ca 2+ would be coordinated in a tetrahedral environment formed by the oxygen atoms of Tyr28, Gly30, Gly32 and Asp49 at an interaction distance in the range 2.4–2.7 A ˚ , as was observed with PPLA 2 Fig. 4. Nucleotide sequence of the cDNA of ChPLA 2 and the deduced amino acid sequence. Sequencing was performed in triplicate with three independent PCRs; no difference was observed. The amino acid sequence obtained by N-terminal sequenc- ing of the pure ChPLA 2 is shown in italic. M1–A14 in bold, signal peptide; A15–R22, propeptide; C49–G54 in square, Ca 2+ loop; and H70–D71 with stars, active site. Chicken pancreatic PLA 2 A. Karray et al. 4550 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS (Fig. 5). These results are in line with our finding showing that ChPLA 2 requires the presence of Ca 2+ to trigger the hydrolysis of phospholipids. For PPLA 2 , a second Ca 2+ is bound to three residues: Glu71, Ser72, and Glu92 (Fig. 5). In the case of ChPLA 2 , the second Ca 2+ -binding site is conserved and is formed by four residues: Glu71, Ile72, Asn89, and Glu92. It is worth noticing that both ChPLA 2 and PPLA 2 require Ca 2+ for their activities. The optimal Ca 2+ concentra- tions are about 8 m m and 4 mm for PPLA 2 and ChPLA 2 , respectively (data not shown). Surface binding and catalytic pocket The interfacial binding step is crucial for enzyme func- tion. Initially, the enzyme is present in bulk in its free form (E). In the presence of substrate, the enzyme binds to the interface as an active form (E*). This cru- cial step is mediated by a planar surface region of the protein composed of approximately 20 amino acids [22]. For PPLA 2 (Protein Data Bank code: 1l8sB), a surface of about 1028 A ˚ 2 , composed mainly of hydro- phobic residues, has been identified and is likely to be involved in interactions with the interface. In the case of ChPLA 2 , the corresponding hydrophobic surface (827 A ˚ 2 ) also involves hydrophobic residues (Leu2, Trp3, Phe19, Leu20, Leu31, Ile64, Leu65, Tyr69, Ile72, Tyr75, and Leu118). When PPLA 2 and ChPLA 2 are in complex with substrate analogs, the hydrophobic sur- faces involved in lipid binding increase by 40 A ˚ 2 and 270 A ˚ 2 , respectively, as compared with the free enzymes. This surface increases when the enzyme passes from the free (E) form to the active (E*) form, as has been described for the lipases [24]. Conclusions The group IB ChPLA 2 was purified to homogeneity from delipidated pancreases. It has a molecular mass of about 14 kDa and it is not a glycosylated enzyme. Bile salts and Ca 2+ are required for the PLA 2 to express its maximum activity. Unlike the known pan- creatic PLA 2 s, ChPLA 2 displayed 50% of its activity in the absence of NaTDC. The maximal PLA 2 specific activity of 400 UÆmg )1 was measured at pH 9.5 and at 37 °C in the presence of 1 mm NaTDC and 4 mm CaCl 2 . Pure ChPLA 2 lost  80% of its activity after 20 min of incubation at 40 °C, and it was found to be unstable at extreme pH. The N-terminal sequence of ChPLA 2 shows a high degree of identity with that of PPLA 2 . This latter enzyme is twice as active as ChPLA 2 . Three-dimensional structure models of Fig. 5. Cartoon representation 3D model of the mature ChPLA 2 . Secondary structure elements labels are indicated. a-Helices and b-strands are colored in red and yellow, respectively. The catalytic network and the Ca 2+ -binding residues are indicated and shown as sticks. (B) The Ca 2+ -binding resi- dues in the case of PPLA 2 are indicated and shown as sticks. The Ca 2+ is represented by a blue sphere. This figure was generated using PYMOL software. A. Karray et al. Chicken pancreatic PLA 2 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS 4551 ChPLA 2 in its free form or in complex with a substrate analog were built on the basis of the PPLA 2 structures. These models allowed us to identify ChPLA 2 key resi- dues involved in Ca 2+ and substrate binding. Experimental procedures Material Benzamidine was from Fluka (Buchs, Switzerland), BSA, NaTDC and PC were from Sigma Chemical (St Louis, MO, USA), acrylamide and bis-acrylamide (electrophoresis grade) were from BDH (Poole, UK), marker proteins and MonoQ Sephacryl were from Pharmacia (Uppsala, Sweden), poly(vinylidene difluoride) membrane and protein sequencer Procise 492 equipped with a 140 C HPLC system were from Applied Biosystems (Roissy, France), and the pH-stat was from Metrohm (Herisau, Switzerland). All enzymes and reagents used in DNA manipulations were from Promega and Invitrogen (Paris, France). Oligonuclotides were synthesized by Invitrogen. E. coli strain DH5a was used as cloning host for the gene part encoding the mature phospholipase. PCR products were purified using the Wizard PCR Preps DNA purification System (Promega). Delipidation of pancreases Pancreases from chicken were collected from a local slaugh- terhouse (Chahiya, Sfax, Tunisia) immediately after slaugh- ter, and kept at )20 °C. After being defrosted, pancreases were cut into small pieces (1–2 cm 2 ) and delipidated accord- ing to the method described previously [25]. After delipida- tion, about 20 g of delipidated pancreas powder was obtained from 100 g of fresh tissue. Phospholipase activity PLA 2 activity was measured titrimetrically at pH 9.5 and at 37 °C with a pH-stat (metrhom), under the standard assay conditions described previously [5], using PC (0.5% w ⁄ v) in 30 mL of 150 mm NaCl, 4 mm CaCl 2 , and 1 mm NaTDC. One PLA 2 activity unit corresponds to one lmole of fatty acid liberated per minute. Determination of protein concentration Protein concentration was determined as described by Bradford et al. [26], using BSA ðE 1% 1 cm ¼ 6:7Þ as reference: Oligosaccharide content The presence of glycan chains in the purified ChPLA 2 was checked by the anthrone ⁄ sulfuric acid method, using glucose as standard [27]. One milliliter of pure ChPLA 2 (1 mg ⁄ mL in Tris ⁄ HCl buffer) was mixed with 4 mL of dis- tilled water in a screwcap-type culture tube. The tube was then placed on ice, and 10 mL of cold anthrone reagent (0.2 g in 100 mL of concentrated H 2 SO 4 ), prepared fresh daily, was added. After mixing, a marble was placed on top of the tube to prevent evaporation, and the mixture was incubated in a boiling water bath for 16 min. The tube was then cooled on ice for 2–3 min, and at room temperature for 5–10 min, and the absorbance was read at 620 nm. Bacterial strains, plasmids, and media E. coli strain DH5a was used as cloning host for the gene part encoding for the mature PLA 2 . The E. coli strain was grown in LB medium, supplemented with 100 lgÆmL )1 kanamicin whenever plasmid maintenance was required. The plasmid PCR-Blunt (Invitrogen) was used as cloning vector. cDNA synthesis and amplification The coding sequence of ChPLA 2 was determined by RT- PCR amplification of mRNA from chicken pancreas. Total mRNAs were isolated from chicken pancreas using the sin- gle-step guanidine isothiocyanate ⁄ phenol ⁄ chloroform isola- tion method as described by Chamczynski and Sacchi [28]. ChPLA 2 cDNA was obtained from total mRNAs by the reverse transcription procedure (Promega). First-strand cDNAs were prepared using heat-denaturated (5 min at 70 °C) total mRNAs (10 lg) as template, 200 U of Molo- ney murine leukemia virus reverse transcriptase (Invitro- gen), 20 pmol of each deoxynucleoside triphosphate, and 20 pmol of each primer (forward primer, 5¢-ATGAGAC TCTTGGCGTGCTTTTCTTG-3¢; reverse primer, 5¢-GAC AAGAAGAAATACTGCACCAGTTAA-3¢). The N-termi- nal primer was predicted from the N-terminal sequence of ChPLA 2 ; however, the C-terminal primer was deduced from the genome of Gallus gallus (GenBank accession number: XM 415272). Reverse transcription was carried out in a total reaction volume of 20 lL for 5 min at room temperature and 60 min at 42 °C. The cDNA–RNA heteroduplex was then denaturated at 70 °C for 15 min and cooled on ice. Cloning of the mature PLA 2 gene Amplification of the specific ChPLA 2 cDNA was carried out by PCR using the single-strand cDNAs as template, with the forward and reverse primers previously described. PCR 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 U of pfu polymerase and polymerization buffer in final volume of 100 lL. The single-strand cDNAs were used directly as template. The thermal profile involved Chicken pancreatic PLA 2 A. Karray et al. 4552 FEBS Journal 276 (2009) 4545–4554 ª 2009 The Authors Journal compilation ª 2009 FEBS 35 cycles of denaturating at 94 °C for 1 min, primer anneal- ing at 55 °C for 1 min, and extension at 72 °C for 3 min. The PCR product (500 kbp) was isolated and ligated into the EcoRI-linearized and dephosphorylated PCR-Blunt vec- tor, using the PCR-Blunt-ended cloning kit, according to the manufacturer’s protocol (Promega). Protoplasts of E. coli DH5a were transformed with the ligation mixture. The resulting recombinant plasmid was named pChPLA2. The presence of the appropriate insert was verified by restriction analysis. DNA products were analyzed on a standard 1% agarose gel containing ethidium bromide (1 lgÆmL )1 ). 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 Analyser from Genome Express (Berkley, CA, USA). They were performed three times, using the recombinant vector (pChPLA2) as template with the M13 promoter primer and the M13 reverse primer (Invitrogen). Software and infrastructure Sequence alignment was performed with bioedit version 4.8.4 software. deep view ⁄ swiss-pdb viewer v. 3.7 (SP5) (http://www.expasy.org/spdbv/) (SPDBV) software was used for homology modeling and structure visualization. The models were stereochemically evaluated by the pro- gram procheck [29]. Modeling and molecular mechanics optimization were performed using hyperchem profes- sional 7.52 for Windows molecular modeling system. Visualization was performed and figures were produced with pymol version 0.99beta06 (http://www.pymol.org). excel 2003 was used for data processing and data graph- ics generation. The accessible surface of the model was calculated with surface racer, a computer program for fast calculation of accessible and molecular surface areas [30]. Homology modeling The 3D coordinates of PPLA 2 were extracted from the Pro- tein Data Bank (http://www.rcsb.org/pdb). The PPLA 2 structure was used as template to build a model of the ChPLA 2 structure by using the structure-modeling program deep view ⁄ swiss-pdb viewer v. 3.7. The model was then subjected to molecular mechanics optimization using the CHARMM27 forcefield, until a gradient of 0.02 kcal ⁄ (A ˚ .mol) was reached. 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