- Open Access
Proteolytic cleavage of stingray phospholipase A2: Isolation and biochemical characterization of an active N-terminal form
© Ben Bacha and Mejdoub; licensee BioMed Central Ltd. 2011
Received: 18 May 2011
Accepted: 26 July 2011
Published: 26 July 2011
Mammalian GIB-PLA2 are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. The aim of this study was to check some biochemical and structural properties of a marine stingray phospholipase A2 (SPLA2).
The effect of some proteolytic enzymes on SPLA2 was checked. Chymotrypsin and trypsin were able to hydrolyze SPLA2 in different ways. In both cases, only N-terminal fragments were accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic and chymotryptic attack generated 13 kDa and 12 kDa forms of SPLA2, respectively. Interestingly, the SPLA2 13 kDa form was inactive, whereas the SPLA2 12 kDa form conserved almost its full phospholipase activity. In the absence of bile slats both native and 12kDa SPLA2 failed to catalyse the hydrolysis of PC emulsion. When bile salts were pre-incubated with the substrate, the native kinetic protein remained linear for more than 25 min, whereas the 12 kDa form activity was found to decrease rapidly. Furthermore, The SPLA2 activity was dependent on Ca2+; other cations (Mg2+, Mn2+, Cd2+ and Zn2+) reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+.
Although marine and mammal pancreatic PLA2 share a high amino acid sequence homology, polyclonal antibodies directed against SPLA2 failed to recognize mammal PLA2 like the dromedary pancreatic one. Further investigations are needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of phospholipases.
Phopholipases A2 (PLA2s) are esterases that catalyse the hydrolysis of acyl groups at the sn-2 position of glycero-phospholipids (PL) and produce free fatty acids, such as arachidonic acid, and lyso-PL by an interfacial activation catalytic mechanism .
A large number of distinct PLA2s have been characterized and classified into the broad categories of intracellular and secreted forms of the enzyme. Intracellular (cytosolic) PLA2s participate in cellular eicosanoid metabolism and signal transduction. Numerous isoforms of secretory phospholipases (sPLA2s) have been identified and divided into several groups, based on their amino acid sequences, structures, catalytic mechanisms, tissue distributions and evolutionary relationships [1, 2]. Ten human secreted PLA2s have been cloned: group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX, and GXIIA PLA2s and the GXII PLA2-like protein that is devoid of catalytic activity  and . sPLA2s are low molecular weight enzymes (14 kDa) and are abundant in various mammalian tissues and fluids. These enzymes are involved in various normal and pathological cell functions . A Group IB enzyme was first found in mammalian pancreatic exudates, and therefore is known as pancreatic PLA2. The main physiologic function of GIB PLA2 is to digest dietary phospholipids. GIB PLA2 is synthesized in pancreatic acinar cells and secreted into the duodenal lumen as an enzymatically inactive PLA2 that is activated by cleavage of a 7-amino acid activation peptide by trypsin .
The sPLA2s from mammalian pancreas and snake venoms have been used as diagnostic biochemical reagents. In fact snake venom sPLA2s were employed to analyse the position of fatty acids in phospholipids from guina pig and pic cardiac membranes . Moreover, porcine PLA2 (PPLA2) was used in the examination of the structural and functional changes of egg yolk low density lipoproteins (LDL) by modifying its phospholipids . In addition, PPLA2 was used in industrial processes in the food industry to produce lyso PC which is an excellent emulsifier for food . The biology and the biochemistry of mammalian and venom PLA2 are well documented. In contrast, few studies were reported on the enzymology and application of PLA2 from marine organisms . Therefore, little information is available on marine gastropod mollusc's sPLA2[10–16]. To date, few studies exist on phospholipase from the digestive gland of marine organisms. Recently, we have purified stingray PLA2 (SPLA2) from the common stingray Dasyatis pastinaca and some of its catalytic properties were determined . High similarity was found between the N-terminal amino acid residues of SPLA2 and those of other known pancreatic PLA2. In the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature, SPLA2 stability makes it a good candidate for its application in food industry. It seems therefore of interest to check some other catalytic and structural properties of SPLA2 to gain more insights into their action mode on phospholipids. We have therefore performed the limited proteolysis experiments on SPLA2, using trypsin and chymotrypsin. Profiles regarding proteolysis and activity are reported.
2. Material and methods
2.2. Determination of phospholipase activity
The stingray PLA2 activity was measured titrimetrically at pH 8.5 and at 40°C with a pH-stat, under the optimum conditions, using purified egg phosphatidylcholine (PC), phosphatidylserine (PS) or phosphatidylethanolamine (PE) emulsions as substrate in the presence of 4 mM NaTDC and 8 mM CaCl2 . One unit of phospholipase activity was defined as 1 μmole of fatty acid liberated under standard conditions.
2.3. Limited proteolysis
SPLA2 (1 mg) was dissolved in 1 ml of 50 mM Tris-HCl buffer, pH 8.5 without benzamidine. The PLA2 solution was digested at 4, 30 and 37°C with the selected endopeptidase. The endopeptidase/PLA2 molar ratio varied from 0.01 to 0.1. Samples (50 μl) were withdrawn from the incubation mixture at various times to assess the residual activity and the electrophoretic profile. The reaction was stopped by addition of benzamidine (4 mM final concentration).
2.4. Analytical methods
Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed by the method of Laemmli . Samples for sequencing or immunoblotting were electrotransferred onto polyvinylidene difluorid and a nitrocellulose membrane respectively . Protein transfer was performed during 2 h at 4 mA/cm2 at room temperature.
2.5. Amino acid sequencing
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 .
2.6. Production of polyclonal antibodies
Polyclonal antibodies directed against purified SPLA2 were produced on rabbits after subcutaneous and intra-muscular injections every 3 weeks of 0.5 mg of pure SPLA2. The first injection included complete Freund's adjuvant, while the last two injections contained incomplete adjuvant.
2.7. Immunoblotting technique
The reactivity of anti-SPLA2 serum with PLA2 (SPLA2 or DrPLA2) was checked using immunoblotting technique. After protein transfer, membranes were rinsed three times with PBS (phosphate buffer saline: 10 mM phosphate pH 7.2, 150 mM NaCl), then saturated with 3% of milk powder in PBS (saturating buffer) for 1 h at room temperature. Thereafter, anti-SPLA2 serum diluted at 1:1000 with PBS containing 0.05% Tween-20 (PBS/Tween-20) were incubated with the membranes for 1 h at room temperature. Afterwards, membranes were washed three times with PBS/Tween-20 then incubated for 1 h at room temperature with a 1:2000 dilution of alkaline phosphatase-conjugated anti-rabbit immunoglobulin (Sigma). After washing, as mentioned above, membranes were incubated with a phosphatase substrate solution containing 0.3 mg/ml of nitroblue tetrazolium chloride (Sigma), 0.2 mg/ml of 5-bromo-4-chloro-3 indolyl-phosphate (Sigma) and 0.2 mg/ml of MgCl2 to reveal the specific immunoreactivity.
2.8. Enzyme linked immunosorbent assay (ELISA) analysis
The immunoreactivity of anti-SPLA2 polyclonal antibodies with phospholipases (SPLA2 or DrPLA2) was checked, using the ELISA technique. Purified phospholipases (SPLA2 or DrPLA2) were diluted using coating buffer (PBS) to obtain a final concentration of 1 μg/ml. Aliquots (100 μl) were coated onto polyvinyl chloride microtiter wells and incubated overnight at 4°C. The wells were then saturated by adding 100 μl of saturating buffer (3% of powder milk in PBS) for 2 h at 37°C. Thereafter, 100 μl of serum, diluted at 1:500 with saturating buffer, were added to each well and the plates were incubated for 1 h at 37°C. Afterwards, 100 μl of peroxidase-conjugated anti-rabbit immunoglobulin (Sigma) diluted at 1:2000 with saturating buffer were added to each well and the plates were kept at 37°C for an additional hour. Then, 100 μl of freshly prepared peroxidase substrate solution (an o-phenylenediamine tablet (Sigma) was solubilized in 50 mM sodium phosphate/citrate, pH 5 containing 0.4% of fresh hydrogen peroxide) were added to each well. The plates were incubated in the dark for 30 min at room temperature. The enzymatic reaction was then stopped by adding 50 μl of 0.5 M H2SO4. The absorbance was read at 490 nm in a micro-ELISA reader (Dynatech).
3. Results and discussion
3.1. Level of expression of SPLA2 activity
3.2. Annual distribution of the SPLA2 activity levels
3.3. Effect of metal ions on SPLA2 activity
Effects of different metal ions on the activity of stingray PLA2
PLA2 Activity (%)
0 mM Ca2+
1 mM Ca2+
3.5 ± 1.1
60 ± 5.4
2 ± 1
57 ± 2.2
3 ± 0.5
15 ± 2.4
2 ± 0.8
12 ± 1.8
3.4. Immunochemical properties
3.5. Cleavage of SPLA2
Sequences comparison of protein fragments isolated after chymotryptic and tryptic proteolysis of SPLA2 with PPLA2
Our results showed that the deletion of the N-terminal fragment of SPLA2 (residue A1 to K10) resulted in a loss of the enzyme activity and, thus, provided evidence that the N-terminal fragment was a crucial structural domain necessary for the activity of the enzymes. These findings collaborate with previous findings demonstrating that Helix 1 at the N-terminal of human group 1B PLA2 (hG1B) play an important role in enzyme function. An engineered hG1B lacking the N-terminal helix 1 bound to membranes with weaker affinity exhibited ~100-fold lower enzymatic activity compared with that of the full-length hG1B. It is inferred that this helix 1 facilitates the membrane binding, thus enhances the enzymatic activity based on polarized infrared spectroscopic experiments . Experiments using semi-synthetic hG1B demonstrated that helix 1 residues act as a regulatory domain and mediate interfacial activation .
The 12-kDa form was further purified by filtration on Bio-sil SEC-125 HPLC gel filtration column (300 × 7.8 mm) equilibrated with phosphate 0.1 M buffer pH 7.0 containing 0.15 M NaCl. Elution of proteins was performed with the same buffer at 30 ml/h and SPLA2 emerged 9 min after injection (Figure 4A). The fractions containing the PLA2 activity were pooled, and SDS⁄PAGE analysis revealed only one band corresponding to 12 kDa SPLA2 (Figure 4B).
Apparent kinetic parameters of SPLA2, 12 kDa SPLA2 and DrPLA2
Kcat/Km (mM-1 s-1)
2850 ± 200
750 ± 50
290 ± 15
13 ± 0.7
17.33 ± 0.5
20 ± 1.3
669 ± 5.2
187 ± 2.65
68 ± 2.7
51.5 ± 3.7
11.5 ± 0.5
3.4 ± 0.8
12 kDa SPLA2
2730 ± 150
700 ± 80
300 ± 17
13.8 ± 1
16.5 ± 0.9
22 ± 0.7
640.8 ± 7
146.3 ± 3.8
70.42 ± 3.3
47.4 ± 2.2
9.96 ± 0.3
3.2 ± 0.3
2300 ± 120
600 ± 45
190 ± 12
16 ± 1.2
22 ± 0.9
28 ± 1.5
539.9 ± 10
140 ± 2.7
44.6 ± 1.9
33.74 ± 2.8
6.36 ± 0.7
2.03 ± 0.1
From these values, one can say that the two marine PLA2s, showing essentially the same activity toward all the tested substrates, hydrolyse the different phospholipids more efficiently than mammalian one since the ratio representing the catalytic efficiency (Kcat/Km) is about 1.5-2 times higher with native SPLA2 and 12 kDa SPLA2 than with DrPLA2.
Several studies have provided evidence that bile salts are tensioactive agents ensuring in their micellar form, the dispersion of the lipolytic products (of hydrolysis) [30, 31]. Along the same line, De Haas et al. reported that micellar forms of the substrate were hydrolyzed at a much higher rate than substrates molecularly dispersed by PLA2 . As previously reported SPLA2 and DrPLA2 failed to catalyze the hydrolysis of pure PC. To trigger the PLA2 activity on PC, NaTDC was added prior to the enzyme injection [17, 18]. In its presence, the kinetics remained linear for more than 25 min (Figure 5). Similar result was obtained with 12 kDa SPLA2 (data not shown), whereas, the pancreatic chicken PLA2 (ChPLA2) was found to hydrolyze efficiently PC in the absence of NaTDC and its maximum specific activity was found to be nearly independent of NATDC . This difference between SPLA2, DrPLA2 and ChPLA2 might be explained by structural variation of exposed residues between the different phospholipases.
Previous work reported that the overall structural and functional perturbations caused by deleting nine C-terminus residues of bovine pancreatic PLA2 over expressed in Escherichia coli were modest, but the C-terminus deletion mutant displayed an interesting and significant property. It functioned well at the anionic interface, but its activity decreased substantially at the zwitterionic interface possibly due to the uncoupling between calcium binding and substrate (inhibitor) binding .
Marine and mammal pancreatic PLA2 share a high amino-acid sequence homology. However; the absence of cross-immunoreactivity between DrPLA2, taken as mammal model, and anti-SPLA2 serum strengthens the idea that SPLA2 could be structurally different from mammalian pancreatic PLA2. Further investigations are needed to better establish the structure-function relationship of this class of enzyme and to identify key residues involved in substrate recognition responsible for biochemical differences between the classes of PLA2.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No: RGP-VPP-070.
- Kudo I, Murakami M: Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat. 2002, 68-69: 3-58.View ArticlePubMedGoogle Scholar
- Burke JE, Dennis EA: Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Research. 2009, 50: S237-S242.View ArticleGoogle Scholar
- Six DA, Dennis EA: The expanding superfamily of phospholipase A2 enzymes: classification and characterization. Biochim Biophys Acta. 2000, 1488: 1-19.View ArticlePubMedGoogle Scholar
- Rouault M, Bollinger JG, Lazdunski M, Gelb MH, Lambeau G: Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity. Biochemistry. 2003, 42: 11494-11503. 10.1021/bi0349930View ArticlePubMedGoogle Scholar
- de Haas GH, Postema NM, Nieuwenhuizen W, van Deenen LLM: Purification and properties of an anionic zymogen of phospholipase A2 from porcine pancreas. Biochim Biophys Acta. 1968, 159: 118-129.View ArticlePubMedGoogle Scholar
- Stoll U: The analysis of phospholipids from cardiac membranes by phospholipase A. Fett/Lipid. 1996, 98: 26-30. 10.1002/lipi.19960980108.View ArticleGoogle Scholar
- Yoshinori M: Structural and Functional Changes of Hen's Egg Yolk Low-Density Lipoproteins with Phospholipase A2. J Agric Food Chem. 1997, 45: 4558-4563. 10.1021/jf9704064.View ArticleGoogle Scholar
- Aoi N: Soy lysolecithin. Yukagaku J. 1990, 39: 10-15.Google Scholar
- Rana RL, Sarath G, Stanley DW: digestive phospholipase A2 in midguts of tobacco hornworms, Manduca sexta L. J Insect Physiol. 1998, 44: 297-303. 10.1016/S0022-1910(97)00118-2View ArticlePubMedGoogle Scholar
- Audley MA, Shetty KJ, Kinsella JE: Isolation and properties of phospholipase A from pollock muscle. J Food Sci. 1978, 43: 771-1775.View ArticleGoogle Scholar
- Neas NP, Hazel JR: Partial purification and kinetic characterization of the microsomal phospholipase A2 from thermally acclimated rainbow trout (Salmo gairdneri). J Comp Physiol B. 1985, 155: 461-469. 10.1007/BF00684676View ArticlePubMedGoogle Scholar
- Aaen B, Jessen F, Jensen B: Partial purification and characterization of a cellular acidic phospholipase A2 from cod (Gadus morhua). Comp Biochem Physiol B. 1995, 110: 547-554. 10.1016/0305-0491(94)00185-W.View ArticleGoogle Scholar
- Zambonino Infante JL, Cahu CL: High dietary lipid levels enhance digestive tract maturation and improve Dicentrarchus labrax larval development. J Nutr. 1999, 129: 1195-1200.PubMedGoogle Scholar
- Cherif S, Ben Bacha A, Ben Ali Y, Horchani H, Rekik W, Gargouri Y: Crab digestive phospholipase: a new invertebrate member. Bioresour Technol. 2010, 101: 366-71. 10.1016/j.biortech.2009.07.031View ArticlePubMedGoogle Scholar
- Pattus F, Slotboom AJ, de Haas GH: Regulation of Phospholipase A2 Activity by the Lipid-Water Interface: a Monolayer Approach. Biochemistry. 1979, 18: 2703-2707. 10.1021/bi00580a003View ArticlePubMedGoogle Scholar
- Zarai Z, Ben Bacha A, Horchani H, Bezzine S, Gargouri Y, Mejdoub H: A novel marine hepatopancreatic phospholipase A2 with digestive and toxic activities. Archives of Biochemistry and Biophysics. 2010, 494: 121-129. 10.1016/j.abb.2009.11.020View ArticlePubMedGoogle Scholar
- Bacha A, Karray A, Bouchaala E, Gargouri Y, Ben Ali Y: Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca. Lipids in Health and Disease. 2011, 10: 32- 10.1186/1476-511X-10-32PubMed CentralView ArticlePubMedGoogle Scholar
- Ben Bacha A, Gargouri Y, Bezzine S, Mejdoub H: Purification and biochemical characterization of phospholipase A2 from dromedary pancreas. Biochim Biophys Acta. 2006, 1760: 1202-View ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3View ArticlePubMedGoogle Scholar
- Abousalham A, Verger R: Egg yolk lipoproteins as substrates for lipases. Biochim Biophys Acta. 2000, 1485: 56-62.View ArticlePubMedGoogle Scholar
- Laemmli UK: Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0View ArticlePubMedGoogle Scholar
- Bergman H, Jörnvall T: Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis. Eur J Biochem. 1987, 169: 912-View ArticleGoogle Scholar
- Hewick RM, Hunkapiller MW, Hood LE: A gaz-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981, 256: 7990-7997.PubMedGoogle Scholar
- ISMEN A: Age, Growth, Reproduction and Food of Common Stingray (Dasyatis pastinaca L., 1758) in Iskenderun Bay, the Eastern Mediterranean. Fisheries Research. 2003, 60: 169-176. 10.1016/S0165-7836(02)00058-9.View ArticleGoogle Scholar
- Scott DL, Otwinowski Z, Gelb MH, Sigler PB: Crystal Structure of Bee-Venom Phospholipase A2 in a Complex with a Transition-State Analogue. Science. 1990, 250: 1563-1566. 10.1126/science.2274788View ArticlePubMedGoogle Scholar
- Yu BZ, Berg OG, Jain MK: The divalent cation is obligatory for the binding of ligands to the catalytic site of secreted phospholipase A2. Biochemistry. 1993, 32: 6485-6492. 10.1021/bi00076a024View ArticlePubMedGoogle Scholar
- Mezna M, Ahmad T, Chettibi S, Drainas D, Lawrence AJ: Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms. Biochem J. 1994, 301: 503-508.PubMed CentralView ArticlePubMedGoogle Scholar
- Qin S, Pande AH, Nemec KN, Tatulian SA: The N-terminal α-helix of pancreatic phospholipase A2 determines productive-mode orientation of the enzyme at the membrane surface. J Mol Biol. 2004, 344: 71-89. 10.1016/j.jmb.2004.09.034View ArticlePubMedGoogle Scholar
- Qin S, Pande AH, Nemec KN, He X, Tatulian SA: Evidence for the regulatory role of the N-terminal helix of secretory phospholipase A(2) from studies on native and chimeric proteins. J Biol Chem. 2005, 280: 36773-36783. 10.1074/jbc.M506789200View ArticlePubMedGoogle Scholar
- Evenberg A, Meyer H, Verheij HM, DeHaas GH: Isolation and properties of prophospholipase A2 from horse pancreas and horse pancreatic juice. Biochim Biophys Acta. 1977, 491: 265-274.View ArticlePubMedGoogle Scholar
- Nieuwenhuizen W, Steenbergh P, De Haas GH: The isolation and properties of two prephospholipases A2 from porcine pancreas. Eur J Biochem. 1973, 40: 1-7. 10.1111/j.1432-1033.1973.tb03161.xView ArticlePubMedGoogle Scholar
- De Haas GH, Bonsen PP, Pieterson WA, Van Deenen LLM: Studies on phospholipase A2 and its zymogen from porcine pancreas action of the enzyme on short-chain lecithins. Biochim Biophys Acta. 1971, 239: 252-266.View ArticlePubMedGoogle Scholar
- Karray A, Frikha F, Ben Bacha A, Ben Ali Y, Gargouri Y, Bezzine S: Biochemical and molecular characterization of purified chicken pancreatic phospholipase A2. FEBS J. 2009, 276: 4545-4554. 10.1111/j.1742-4658.2009.07160.xView ArticlePubMedGoogle Scholar
- Huang B, Yu BZ, Rogers J, Byeon IJ, Sekar K, Chen X, Sundaralingam M, Tsai MD, Jain MK: Phospholipase A2 Engineering. Deletion of the C-Terminus Segment Changes Substrate Specificity and Uncouples Calcium and Substrate Binding at the Zwitterionic Interface. Biochemistry. 1996, 35: 12164-12174. 10.1021/bi960234oView ArticlePubMedGoogle Scholar
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