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• W2004226937 abstract "Secreted phospholipases A2(sPLA2s) from snake and insect venoms and from mammalian pancreas are structurally related enzymes that have been associated with several toxic, pathological, or physiological processes. We addressed the issue of whether toxic sPLA2s might exert specific effects on the Plasmodium falciparumintraerythrocytic development. We showed that both toxic and non-toxic sPLA2s are lethal to P. falciparum grownin vitro, with large discrepancies between respective IC50 values; IC50 values from toxic PLA2s ranged from 1.1 to 200 pm, and IC50 values from non-toxic PLA2s ranged from 0.14 to 1 μm. Analysis of the molecular mechanisms responsible for cytotoxicity of bee venom PLA2 (toxic) and hog pancreas PLA2 (non-toxic) demonstrated that, in both cases, enzymatic hydrolysis of serum phospholipids present in the culture medium was responsible for parasite growth arrest. However, bee PLA2-lipolyzed serum induced stage-specific inhibition ofP. falciparum development, whereas hog PLA2-lipolyzed serum killed parasites at either stage. Sensitivity to bee PLA2-treated serum appeared restricted to the 19–26-h period of the 48 h parasite cycle. Analysis of the respective role of the different lipoprotein classes as substrates of bee PLA2 showed that enzyme treatment of high density lipoproteins, low density lipoproteins, and very low density lipoproteins/chylomicrons fractions induces cytotoxicity of either fraction. In conclusion, our results demonstrate that toxic and non-toxic PLA2s 1) are cytotoxic to P. falciparum via hydrolysis of lipoprotein phospholipids and 2) display different killing processes presumably involving lipoprotein by-products recognizing different targets on the infected red blood cell. Secreted phospholipases A2(sPLA2s) from snake and insect venoms and from mammalian pancreas are structurally related enzymes that have been associated with several toxic, pathological, or physiological processes. We addressed the issue of whether toxic sPLA2s might exert specific effects on the Plasmodium falciparumintraerythrocytic development. We showed that both toxic and non-toxic sPLA2s are lethal to P. falciparum grownin vitro, with large discrepancies between respective IC50 values; IC50 values from toxic PLA2s ranged from 1.1 to 200 pm, and IC50 values from non-toxic PLA2s ranged from 0.14 to 1 μm. Analysis of the molecular mechanisms responsible for cytotoxicity of bee venom PLA2 (toxic) and hog pancreas PLA2 (non-toxic) demonstrated that, in both cases, enzymatic hydrolysis of serum phospholipids present in the culture medium was responsible for parasite growth arrest. However, bee PLA2-lipolyzed serum induced stage-specific inhibition ofP. falciparum development, whereas hog PLA2-lipolyzed serum killed parasites at either stage. Sensitivity to bee PLA2-treated serum appeared restricted to the 19–26-h period of the 48 h parasite cycle. Analysis of the respective role of the different lipoprotein classes as substrates of bee PLA2 showed that enzyme treatment of high density lipoproteins, low density lipoproteins, and very low density lipoproteins/chylomicrons fractions induces cytotoxicity of either fraction. In conclusion, our results demonstrate that toxic and non-toxic PLA2s 1) are cytotoxic to P. falciparum via hydrolysis of lipoprotein phospholipids and 2) display different killing processes presumably involving lipoprotein by-products recognizing different targets on the infected red blood cell. phospholipase A2 p-bromophenacyl bromide IC100 minimum low density lipoprotein very LDL high density lipoprotein Phospholipases A2(PLA2s)1 are enzymes that catalyze the hydrolysis of the sn-2 acyl bond of glycerophospholipids to produce free fatty acids and lysophospholipids. They are important for signal transduction processes, general lipid metabolism, and membrane remodeling (for review, see Refs. 1Dennis E.A. Biotechnology. 1987; 5: 1294-1300Crossref Scopus (131) Google Scholar and 2Dennis E.A. Rhee S.G. Billah M.M. Hannun Y.A. FASEB J. 1991; 5: 2068-2077Crossref PubMed Scopus (471) Google Scholar). From this superfamily of secreted and cytosolic enzymes, classified in 1997 into nine groups (3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar), the groups I, II, and III are all secreted, low molecular mass (13–18 kDa), Ca2+-dependent PLA2s (4Davidson F.F. Dennis E.A. J. Mol. Evol. 1990; 31: 228-238Crossref PubMed Scopus (292) Google Scholar). The pancreatic group IB is expressed at high level in the pancreas but has also been detected in various other tissues (5Sakata T. Nakamura E. Tsuruta Y. Tamaki M. Teraoka H. Tojo H. Ono T. Okamoto M. Biochim. Biophys. Acta. 1989; 1007: 124-126Crossref PubMed Scopus (101) Google Scholar, 6Cupillard L. Koumanov K. Mattei M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), and apart from its role in the digestion of dietary lipids, it has been implicated in biological activities such as cell proliferation (7Arita H. Hanasaki K. Nakano T. Oka S. Teraoka H. Matsumo K. J. Biol. Chem. 1991; 266: 19139-19141Abstract Full Text PDF PubMed Google Scholar), cell migration (8Ohara O. Ishizaki J. Harita H. Prog. Lipid. Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar, 9Kundu G.C. Mukherjee A.B. J. Biol. Chem. 1997; 272: 2346-2353Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and eicosanoid release (10Kishino J. Ohara O. Nomura K. Kramer R.M. Arita H. J. Biol. Chem. 1994; 269: 5092-5098Abstract Full Text PDF PubMed Google Scholar). Snake and insect venoms contain a wide variety of secreted PLA2s (sPLA2s), which can be potent toxins exerting deleterious effects such as myotoxicity, neurotoxicity, cardiotoxicity and inflammation (11Hawgood B. Bon C. Tu A.T. Handbook of Natural Toxins. Marcel Dekker, Inc., New York1991: 3-52Google Scholar, 12Gutierrez J.M. Lomonte B. Toxicon. 1995; 33: 1405-1424Crossref PubMed Scopus (427) Google Scholar). Involvement of enzymatic activity in many of these effects is unclear because not all venomous PLA2s display toxic effects, although they all have similar catalytic activities (13Tischfield J.A. Xia Y.R. Shih D.M. Klisak I. Chem J. Engle S.J. Siakotos A.N. Winstead M.V. Seilhamer J.J. Allamand V. Gyapay G. Lusis A.J. Genomics. 1996; 32: 328-333Crossref PubMed Scopus (90) Google Scholar). In some cases, like the cytotoxicity of nigexine, a PLA2 from cobra venom (14Chwetzoff S. Tsunasawa S. Sakiyama F. Menez A. J. Biol. Chem. 1989; 264: 13289-13297Abstract Full Text PDF PubMed Google Scholar), or the activation of prostaglandin E2 production in rat mesangial cells by the mammalian pancreatic PLA2 (15Kishino J. Kawamoto K. Ishizaki J. Verheij H.M. Ohara O. Arita H. J. Biochem. (Tokyo ). 1995; 117: 420-424Crossref PubMed Scopus (31) Google Scholar), it was demonstrated that catalytic activity and toxicity or biological effects are unrelated. Furthermore, the identification of different membrane proteins that bind secreted PLA2s strongly suggest that these enzymes could behave as ligands for receptors and might be responsible for other physiological functions than the catalytic one (16Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Malaria is a widespread parasitic disease. Its estimated incidence in the world is in the order of 300–500 million clinical cases each year, with mortality estimated to be more than one million (46World Health OrganizationWeekly Epidemiol. Rec. 1998; 72: 269Google Scholar). The causative agent of malaria is an intracellular protozoan from the genusPlasmodium. Of the four human Plasmodium species,Plasmodium falciparum is the one responsible for most of the severe physiopathology. Increasing resistance of the parasite to classical antimalarial drugs calls for new chemotherapeutic approaches based on a better understanding of Plasmodium biology and its interaction with the host. In humans, part of the P. falciparum life cycle takes place inside the erythrocyte, where it develops through the successive stages ring, trophozoite and schizont, within 48 h. During its intraerythrocytic maturation, the parasite undergoes a large re-organization of the host cell membrane by inserting newly synthesized proteins, forming “knob-like” electron-dense structures (17Atkinson C.T. Aikawa M. Blood Cells (N. Y. ). 1990; 16: 351-368PubMed Google Scholar), and remodeling of the molecular species of phospholipid composition (18Hsiao L.L. Howard R.J. Aikawa M. Taraschi T.F. Biochem. J. 1991; 274: 121-132Crossref PubMed Scopus (123) Google Scholar). Secreted PLA2s have been used as tools for studying the phospholipid asymmetry of the Plasmodium-infected erythrocyte membrane (19Joshi P. Dutta G.P. Gupta C.M. Biochem. J. 1987; 246: 103-108Crossref PubMed Scopus (30) Google Scholar, 20Van der Schaft P.H. Beaumelle B. Vial H. Roelofsen B. Op den Kamp J.A.F. Van Deenen L.L.M. Biochim. Biophys. Acta. 1987; 901: 1-14Crossref PubMed Scopus (38) Google Scholar). Apart from these structural studies, one study reported the toxicity of a chemically modified pig pancreatic PLA2 toward Plasmodium knowlesi-parasitized erythrocytes (21Moll G.N. Vial H.J. van der Wiele F.C. Ancelin M.L. Roelofsen B. Slotboom A.J. de Haas G.H. van Deenen L.L.M. Op den Kamp J.A.F. Biochim. Biophys. Acta. 1990; 1024: 189-192Crossref PubMed Scopus (19) Google Scholar); it was demonstrated that the fatty acylated PLA2 acquired an enhanced penetrative power, leading to the selective elimination of the parasitized erythrocytes, which present an altered membrane lipid packing compared with healthy erythrocytes. To our knowledge, no study has investigated the perturbation of Plasmodiumphysiological processes by exogenous PLA2s activity. Owing to the many deleterious effects attributed to these enzymes and the variability of their mode of action, we were interested in testing the possibility that externally added PLA2s might induce specific toxic effects on P. falciparum. In this study, we analyzed the toxicity of bee and snake venom enzymes (toxic PLA2s) and hog and bovine pancreas enzymes (non-toxic PLA2s) toward P. falciparum cultured in vitro. Toxic and non-toxic PLA2s both killed the intraerythrocytic parasite, but with very different efficiencies, allowing a clear discrimination between the two categories of enzymes. Comparative analysis of the development of a synchronized culture ofPlasmodium upon addition at different times of bee venom PLA2 and hog pancreas PLA2, revealed that a low dose of the toxic enzyme is lethal to the young trophozoite stage only, whereas pancreatic PLA2 kills the parasite at each developmental stage. We demonstrate that these specific effects can be reproduced by using human serum previously incubated with the corresponding PLA2, and that purified lipoproteins lipolyzed by the bee venom PLA2 are inhibitory to the parasite growth. Taken together, our results demonstrate that bee PLA2 and hog PLA2 modify serum lipoproteins in different ways, leading to the generation of specific lipoprotein by-products toxic to the intraerythrocytic Plasmodium and presumably acting on different targets. Phospholipases A2s fromAgkistrodon halys venom and from hog pancreas were purchased from Fluka; the PLA2 from Crotalus adamanteusand Naja mossambica venoms were from Sigma; the bee venom PLA2 was purchased from Fluka and from Sigma.p-Bromophenacyl bromide (p-BPB) andl-α-lecithin were purchased from Sigma. [3H]Hypoxanthine (10–30 Ci/mmol) was from ICN Pharmaceuticals, France. Affi-Gel 10 gel was purchased from Bio-Rad. P. falciparum Colombian strain FcB1 was used in all the experiments and was cultured as described previously (22Le Bonniec S. Deregnaucourt C. Redeker V. Banerjee R. Grellier P. Goldberg D.E. Schrével J. J. Biol. Chem. 1999; 274: 14218-14223Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) in a 3% CO2, 6% O2, 91% N2atmosphere with human red blood cells in complete medium. Complete medium consisted of RPMI 1640 (Life Technologies, Inc.) containing 25 mm Hepes, supplemented with 11 mm glucose, 27.5 mm NaHCO3, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 7% (v/v) compatible heat-inactivated human serum. The parasite cell culture was synchronized by Plasmagel (23Pasvol G. Wilson R.J.M. Smalley M.E. Brown J. Ann. Trop. Med. Parasitol. 1978; 72: 87-88Crossref PubMed Scopus (300) Google Scholar) and sorbitol (24Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2774) Google Scholar) treatments. Under our culture conditions, the in vitro life cycle of the FcB1 strain was 48 h. Enzymatic assays on mixed micelles of phosphatidylcholine and Triton X-100 were performed according to Lôbo de Araujo and Radvanyi (25Lôbo de Araujo A. Radvanyi F. Toxicon. 1987; 25: 1181-1188Crossref PubMed Scopus (157) Google Scholar). Briefly, the substrate solution was prepared by stirring 3.5 mml-α-lecithin from egg yolk in 7 mm Triton X-100, 100 mm NaCl, 10 mm CaCl2, and 55 μm phenol red; the pH of the solution was adjusted to 7.5 with 40 mm NaOH so that the absorbance reading at 558 nm was 1.8 to 2. The substrate solution (1 ml) was introduced into the titration cuvette, and the reaction was started by adding the enzyme (resuspended in H20 at a known concentration) in a volume smaller than 50 μl. The difference in absorbance between the reference (substrate solution alone) and the sample cuvettes was monitored continuously at 558 nm using a Uvikon spectrophotometer (Kontron, Zurich, Switzerland). Under these conditions, a decrease in absorbance of 0.1 corresponds to the release of 0.01 μmol of fatty acid. 1 IU of PLA2 hydrolyzes 1 μmol ofl-α-phosphatidylcholine tol-α-lysophosphatidylcholine and a fatty acid per min at room temperature and appropriate pH. An asynchronous culture of P. falciparum at 1–1.5% parasitemia and 4% hematocrit in complete medium was distributed (100 μl/well) in a 96-microwell plate. Increasing concentrations of PLA2s resuspended in complete medium were added (100 μl/well), and the cells were allowed to grow in a candle jar system. After 24 h in culture, [3H]hypoxanthine was added (0.5 μCi/well), and after an additional 24-h incubation period, cells were harvested on filters after a freeze-thawing cycle. Dried filters were submerged in a liquid scintillation mixture (OptiScint Hisafe) and counted in a 1450 Microbeta counter (Wallac). Growth inhibition was calculated from the parasite-associated radioactivity (incorporated into nucleic acids) compared with controls. The IC50 was determined according to Desjardins et al. (26Desjardins R.S. Canfield C.J. Haynes J.D. Chulay J.D. Antimicrob. Agents Chemother. 1979; 16: 710-718Crossref PubMed Scopus (2233) Google Scholar) as was the IC100 minimum (IC100m = minimum enzyme concentration leading to 100% inhibition of the parasite growth). To estimate the concentration in terms of enzymatic units (IU/ml), enzymatic activity of each commercial PLA2 was measured in the mixed micelles assay system. Supernatant of the cell culture was used to determine the extent of hemolysis from its optical density at 420 nm and by comparing this value with that of a 100% lysate prepared from the corresponding batch of cells. The effect of bee and hog PLA2s on Plasmodiumre-invasion rate was analyzed on synchronized cultures, by adding the enzymes at their respective IC100m at different times of the parasite cycle. Re-invasion rate was determined 60 h after time zero of the cycle by counting 2000 cells on Giemsa-stained smears. Chemical modification of bee and hog PLA2s by p-BPB was achieved according to the procedure described in Robertset al. (27Roberts M.F. Deems R.A. Mincey T.C. Dennis E.A. J. Biol. Chem. 1977; 252: 2405-2411Abstract Full Text PDF PubMed Google Scholar) with slight modifications. Enzyme (50 μg/ml in 25 mm Tris, pH 8) was incubated with 1 × 10−4m p-BPB at room temperature for 3 h. The reagent was added from a 2 × 10−3m stock solution in acetone. The reaction mixture was acidified to pH 3 to quench the reaction, then excess p-BPB was removed by extensive dialysis against phosphate-buffered saline. Control samples were prepared in the same way except that p-BPB was omitted from the procedure. Aliquots from p-BPB-treated and control samples were tested for PLA2 activity in the mixed micelles assay system. Coupling of PLA2s to Affi-Gel 10 beads (Bio-Rad) was achieved following instructions recommended by the manufacturer. At the end of the reaction, the efficiency of the coupling was determined by measuring the concentration of free PLA2 in the supernatant using the DC protein assay (Bio-Rad), and enzymatic activity of the Affi-Gel-coupled PLA2 was measured in the mixed micelles assay system. After coupling, 1% of the bee PLA2 had retained activity, but all hog PLA2 activity had been lost. Complete culture medium containing 14% heat-inactivated human serum was incubated at 37 °C with 130 pm Affi-Gel-coupled bee venom PLA2 for various periods of time (1, 3, 6, 17 h). Here, 130 pm PLA2 is the concentration of enzyme still active after immobilization. After a 17-h incubation, an aliquot of the supernatant was tested in the mixed micelles assay system for the presence of released PLA2 to ensure that any effect on parasite growth would be due to modified serum and not to free PLA2 in the supernatant. To check if in the case of hog pancreas PLA2 enzyme toxicity toward the parasites might be due to modification of the serum, complete medium containing 14% heat-inactivated serum was incubated at 37 °C for 30 h with 40 nm enzyme. The 40 nm concentration was chosen since it is the highest PLA2 concentration with no effect on parasite growth for one cell cycle. An asynchronous culture ofP. falciparum was washed twice in culture medium without serum and resuspended at 1–1.5% parasitemia and 4% hematocrit in the same medium. 100 μl per well were distributed in a 96-microwell plate. PLA2-treated culture medium at increasing dilutions in non-treated complete medium containing 14% serum was added (100 μl/well), and parasites were allowed to grow in culture conditions. After 24 h, 0.5 μCi/well [3H]hypoxanthine was added, and cells were harvested after an additional 24-h period. Radioactivity incorporated into macromolecules was counted as described above. Parasite growth inhibition was plotted against dilution of the treated medium, and the highest dilution leading to 100% inhibition was determined. To analyze the respective susceptibility of parasite stages to PLA2-modified serum, synchronized cultures were grown in the presence of modified medium (at the 100% inhibitory dilution) for various periods of time, and the re-invasion rate at the end of the parasite cycle was determined from Giemsa-stained smears. VLDL/chylomicrons, LDL, and HDL were prepared by differential ultracentrifugation from heat-inactivated human serum following the procedure described in Grellier et al. (28Grellier P. Rigomier D. Clavey V. Fruchart J-C. Schrével J. J. Cell Biol. 1991; 112: 267-277Crossref PubMed Scopus (106) Google Scholar). The lipoprotein fractions were extensively dialyzed at 4 °C against phosphate-buffered saline (0.15 m NaCl, 10 mmsodium phosphate buffer, pH 7.2) and filtered through 0.2-μm membrane. The protein content of the different fractions was determined using the Bio-Rad DC protein assay. An aliquot of each fraction was incubated at 37 °C for 17 h with Affi-Gel-immobilized bee PLA2 (130 pm active enzyme). The supernatant was analyzed for parasite growth inhibition by the [3H]hypoxanthine incorporation test. Two non-toxic, non-inflammatory PLA2s from hog and bovine pancreas (group IB) and four toxic PLA2s of venomous origin, the N. mossambica (group IA), A. halys and C. adamanteus (group IIA), and Apis mellifera (group III), were tested for their capacity to inhibit the intraerythrocytic development of P. falciparum. Asynchronous cultures of the FcB1 strain were grown at 1.5% parasitemia for one cell cycle (48 h) in the presence of various concentrations of PLA2. Parasite growth was checked via nucleic acid synthesis by [3H]hypoxanthine incorporation, and extent of hemolysis was measured by reading absorbance of the supernatants of the culture media at 420 nm. IC50 values obtained for each PLA2 as well as minimum PLA2 concentration leading to detectable hemolysis are given in TableI.Table IComparative analysis between the IC50 values of toxic and non-toxic PLA2s toward the P. falciparum in vitro growthPLA2 originA. mellifera group IIIN. mossambica group IAA. halys group IIAC. adamanteus group IIAHog pancreas group IBBovine pancreas group IBIC50(pm)1.12.2100200145 × 103>1 × 106(S.D. ± 1.2)(S.D. ± 2.7)(S.D. ± 50)(S.D. ± 55)(S.D. ± 10.5 × 103)IC50 (IU/ml)0.65 × 10−52.30 × 10−513.50 × 10−519.60 × 10−51.12>0.15(S.D. ± 0.7 × 10−5)(S.D. ± 5.7 × 10−5)(S.D. ± 0.7 × 10−5)(S.D. ± 5.3 × 10−5)(S.D. ± 0.07)Concentration of PLA2 inducing 1% hemolysis>62.5 nm≥47 nm≥7 μmND≥100 μm>1.4 μmAn asynchronous culture of the FcB1 strain of P. falciparumwas grown in the presence of increasing concentrations of various toxic (from A. mellifera, N. mossambica, A. halys, and C. adamanteus) and non-toxic (from hog and bovine pancreas) PLA2s. Respective concentrations leading to 50% parasite growth inhibition (IC50) were determined via the [3H]hypoxanthine incorporation test and expressed either in molarity or in number of enzymatic units per ml. Release of hemoglobin in the culture supernatant was measured spectrophotometrically at 414 nm. ND, not determined. PLA2s are classified according to Dennis (3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). Open table in a new tab An asynchronous culture of the FcB1 strain of P. falciparumwas grown in the presence of increasing concentrations of various toxic (from A. mellifera, N. mossambica, A. halys, and C. adamanteus) and non-toxic (from hog and bovine pancreas) PLA2s. Respective concentrations leading to 50% parasite growth inhibition (IC50) were determined via the [3H]hypoxanthine incorporation test and expressed either in molarity or in number of enzymatic units per ml. Release of hemoglobin in the culture supernatant was measured spectrophotometrically at 414 nm. ND, not determined. PLA2s are classified according to Dennis (3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). All PLA2s appeared toxic to P. falciparum, with IC50 values much lower than the minimum enzyme concentration required for 1% hemolysis. It should be noted, however, that the amount of released hemoglobin would hardly account for lysis being restricted to infected erythrocytes, owing to the low parasitemia in the test. PLA2s from venoms appear much more toxic to P. falciparum than pancreatic PLA2s, with low IC50 values ranging from 1.1 pm to 0.2 nm. Values were translated in terms of enzymatic units after the amount of active enzyme present in each PLA2commercial preparation had been checked by kinetic analysis of mixed micelles (Triton X-100/phosphatidylcholine) hydrolysis. The same differences in magnitude were found (see Table I); IC50s of toxic PLA2s ranged from 0.65 × 10−5 to 19.60 × 10−5 IU/ml, whereas IC50s of non-toxic PLA2s were 1.12 IU/ml (hog) and >0.15 IU/ml (bovine). Several conclusions may be drawn from these data. If added to cell culture, both sets of PLA2 (toxic and non-toxic) are able to stop parasite development; as previously shown (21Moll G.N. Vial H.J. van der Wiele F.C. Ancelin M.L. Roelofsen B. Slotboom A.J. de Haas G.H. van Deenen L.L.M. Op den Kamp J.A.F. Biochim. Biophys. Acta. 1990; 1024: 189-192Crossref PubMed Scopus (19) Google Scholar), PLA2 concentrations effective in killing parasite are clearly much lower than concentrations required to lyse healthy red blood cells; pancreatic PLA2s are very inefficient in parasite killing compared with venom PLA2s. From these results, the bee venom (toxic) and hog pancreas (non-toxic) PLA2s were chosen for further investigations. Because some situations have been reported where cytotoxicity or biological effects due to secreted PLA2s were independent of catalytic activity, we analyzed the importance of PLA2 enzymatic activity inPlasmodium growth arrest by chemical inhibition of the bee venom and hog pancreas enzymes by p-BPB (27Roberts M.F. Deems R.A. Mincey T.C. Dennis E.A. J. Biol. Chem. 1977; 252: 2405-2411Abstract Full Text PDF PubMed Google Scholar). Reaction of PLA2s with p-BPB has been shown to be active site-directed, leaving the overall conformation of the protein mainly unchanged and free to interact with other molecules. Inhibition of the venom and pancreatic enzyme activities was measured in assays using mixed micelles as substrates. In our hands, PLA2 inhibition by p-BPB never reached 100%, and the best inhibition rates that could be obtained varied from 80 to 95%. Bee venom PLA2 and hog pancreas PLA2 used in the experiment presented in Fig. 1 were inhibited by 95 and 94%, respectively. Analysis of the growth inhibition of the FcB1 strain in the presence ofp-BPB-treated enzymes (Fig. 1) shows that their lethal effect depends largely upon enzymatic activity in both cases, since the IC50 of bee PLA2 is increased by more than 2 log units upon enzyme inhibition and the IC50 of hog pancreas PLA2 by more than 1 log unit upon inhibition, even though both enzymes were not totally inactivated. The same results were obtained with different batches of phospholipases A2, making it most unlikely that a p-BPB-sensitive contaminant from the commercial preparation would be responsible for the observed effect. It must be noted that IC50 value of native bee PLA2 in Fig. 1 appeared higher (70 pm) than IC50 value in Table I (1.1 pm), indicating that some enzyme has been lost during the successive steps of the inhibition procedure. The more effective killing of parasites by toxic PLA2s compared with non-toxic ones led us to question their respective mechanisms of lethal action. One possible explanation was the higher penetration power of toxic PLA2s into a lipid mono- or bilayer compared with non-toxic PLA2s. Indeed, the penetration power of PLA2 enzymes depends on lipid pressure of the layer (29Demel R.A. Geurts van Kessel W.S.M. Zwaal R.F.A. Roelofsen B. van Deenen L.L.M. Biochim. Biophys. Acta. 1975; 406: 97-107Crossref PubMed Scopus (482) Google Scholar), and disorganization of the plasma membrane lipid bilayer in parasitized cells is expressed in part by a decreased lateral surface pressure compared with intact erythrocytes. Mollet al. (21Moll G.N. Vial H.J. van der Wiele F.C. Ancelin M.L. Roelofsen B. Slotboom A.J. de Haas G.H. van Deenen L.L.M. Op den Kamp J.A.F. Biochim. Biophys. Acta. 1990; 1024: 189-192Crossref PubMed Scopus (19) Google Scholar) report the preferential attack ofPlasmodium-infected erythrocytes by a modified pig pancreas PLA2 with enhanced penetration power. However, if we hypothesize that killing of parasites strictly depends upon enzyme capacity to penetrate the erythrocyte membrane, we must notice that although all PLA2s tested kill parasites, the number of enzymatic units required to kill is very different between toxic and non toxic PLA2s, suggesting that the substrate itself,i.e. the infected erythrocyte, might be “seen” differently by the two types of enzymes. First of all, we analyzed the effects of bee venom and hog pancreas PLA2s on the intraerythrocytic cell cycle of P. falciparum. Parasites from the FcB1 strain were synchronized. Cultures of young rings at 1–1.5% parasitemia and 2% hematocrit were distributed into 96-well plates in RPMI containing 7% heat-inactivated human serum. The minimum concentration of PLA2 enzyme leading to 100% inhibition of the parasite growth (IC100m) was determined from curves of growth inhibition plotted as a function of PLA2concentration (not shown). Bee PLA2 IC100m was set at 30 pm, and hog PLA2 IC100m was set at 500 nm. PLA2s at these concentrations were added to the cell cultures at different times of the 48 h cell cycle, and parasitemia from each well was determined on Giemsa-stained smears after 60 h. 100% re-invasion was determined from control cultures under the same conditions but in the absence of PLA2. Experiments were repeated twice with red blood cells and serums from different donors. Results are presented in Fig. 2. When added to culture medium early in the cell cycle (at the young ring stage), bee PLA2prevented re-invasion, but when added beyond the 25–29-h period (i.e. the young trophozoite stage), bee PLA2 displayed only a slight inhibitory effect on parasite re-invasion. In contrast, almost no re-invasion was observed upon the addition of hog PLA2 to the culture, regardless of the time of addition, demonstrating that killing potencies of the two PLA2s at IC100ms are different. Furthermore, we could notice from the observation of the Giemsa-stained smears that the healthy erythrocytes of the PLA2s-treated cultures were shrunk, with an echinocytic shape, compared with the control erythrocytes. The results obtained with bee venom PLA2 might be explained either by the necessity for a long period of time for the toxic activity to exert its effect (i.e. the longer the time of incubation with PLA2, the more deleterious the effect on cells) or by a specific targeting of the toxic effect to pre-schizogonic forms of the intraerythrocytic Plasmodium, whereas hog pancreas PLA2 would exert its toxic effect on any development stage. Since in vitrocultivation of intraerythrocytic P. falciparum is performed in the presence of human serum, it was possible that phospholipids from serum lipoproteins might be substrates of PLA2s as well as phospholipid" @default.
• W2004226937 created "2016-06-24" @default.
• W2004226937 creator A5055859205 @default.
• W2004226937 creator A5073988774 @default.
• W2004226937 date "2000-12-01" @default.
• W2004226937 modified "2023-10-16" @default.
• W2004226937 title "Bee Venom Phospholipase A2 Induces Stage-specific Growth Arrest of the Intraerythrocytic Plasmodium falciparum via Modifications of Human Serum Components" @default.
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