FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2092257114> ?p ?o ?g. }

• W2092257114 endingPage "1506" @default.
• W2092257114 startingPage "1493" @default.
• W2092257114 abstract "We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A2 (sPLA2s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA2s in malaria, we investigated the interactions between bee venom phospholipase A2 (bvPLA2), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100× IC50), bvPLA2 binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA2/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA2s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria. We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A2 (sPLA2s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA2s in malaria, we investigated the interactions between bee venom phospholipase A2 (bvPLA2), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100× IC50), bvPLA2 binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA2/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA2s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria. Malaria is a widespread parasitic disease occurring in over 100 countries. Its annual incidence has been estimated at 350 to 500 million clinical cases with 1.5 to 2.7 million deaths (1The world malaria report 2005. World Health Organization and UNICEF, Geneva, Switzerland2005at http://rbm.who.int/wmr2005Date accessed: May 11, 2006Google Scholar). Plasmodium falciparum and, to a lesser extent, Plasmodium vivax are the main causes of disease and death from malaria. The erythrocytic stage of the parasite life cycle is responsible for the malaria symptoms. The burden of malaria is increasing, especially in sub-Saharan Africa, because of drug and insecticide resistance, as well as social and environmental changes (2Greenwood B. Mutabingwa T. Malaria in 2002.Nature. 2002; 415: 670-672Crossref PubMed Scopus (529) Google Scholar). Thus, there is an urgent need for new drugs, vaccines, and insecticides, as well as for a better understanding of the pathophysiological processes at play in malaria. Phospholipases A2 enzymes (EC 3.1.1.4) exhibit a variety of physiological activities in addition to intrinsic lipolytic action. Those enzymes catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids, leading to the production of NEFAs and lysophospholipids (lysoPLs). Secreted phospholipases A2 (sPLA2s) form a large family of low-molecular-mass (13–19 kDa), water soluble, and structurally conserved enzymes that have primarily been identified in animal venoms, but are also distributed in mammalian tissues, fluids, and secretions (3Kudo I. Murakami M. Phospholipase A2 enzymes.Prostaglandins Other Lipid Mediat. 2002; 68–69: 3-58Crossref PubMed Scopus (659) Google Scholar), plants (4Lee H.Y. Bahn S.C. Kang Y.M. Lee K.H. Kim H.J. Noh E.K. Palta J.P. Shin J.S. Ryu S.B. Secretory low molecular weight phospholipase A2 plays important roles in cell elongation and shoot gravitropism in Arabidopsis.Plant Cell. 2003; 15: 1990-2002Crossref PubMed Scopus (102) Google Scholar), bacteria (5Sugiyama M. Ohtani K. Izuhara M. Koike T. Suzuki K. Imamura S. Misaki H. A novel prokaryotic phospholipase A2. Characterization, gene cloning, and solution structure.J. Biol. Chem. 2002; 277: 20051-20058Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and viruses (6Canaan S. Zadori Z. Ghomashchi F. Bollinger J. Sadilek M. Moreau M.E. Tijssen P. Gelb M.H. Interfacial enzymology of parvovirus phospholipases A2.J. Biol. Chem. 2004; 279: 14502-14508Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 7Nagiec M.J. Lei B. Parker S.K. Vasil M.L. Matsumoto M. Ireland R.M. Beres S.B. Hoe N.P. Musser J.M. Analysis of a novel prophage-encoded group A Streptococcus extracellular phospholipase A2.J. Biol. Chem. 2004; 279: 45909-45918Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Interestingly, despite common catalytic properties, venom sPLA2s differ greatly in their pharmacological effects, such as neurotoxic, myotoxic, cardiotoxic, or anticoagulant properties, and some sPLA2s have been shown to display antibacterial (8Buckland A.G. Wilton D.C. The antibacterial properties of secreted phospholipases A2.Biochim. Biophys. Acta. 2000; 1488: 71-82Crossref PubMed Scopus (115) Google Scholar, 9Koduri R.S. Gronroos J.O. Laine V.J. Le Calvez C. Lambeau G. Nevalainen T.J. Gelb M.H. Bactericidal properties of human and murine group I, II, V, X, and XII secreted phospholipases A2.J. Biol. Chem. 2002; 277: 5849-5857Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), antiviral (HIV) (10Fenard D. Lambeau G. Valentin E. Lefebvre J.C. Lazdunski M. Doglio A. Secreted phospholipases A(2), a new class of HIV inhibitors that block virus entry into host cells.J. Clin. Invest. 1999; 104: 611-618Crossref PubMed Scopus (128) Google Scholar), or anti-Plasmodium properties (11Deregnaucourt C. Schreével J. Bee venom phospholipase A2 induces stage-specific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components.J. Biol. Chem. 2000; 275: 39973-39980Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Guillaume C. Deregnaucourt C. Clavey V. Schreével J. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.Toxicon. 2004; 43: 311-318Crossref PubMed Scopus (31) Google Scholar). We previously showed that sPLA2s from snake, scorpion, or bee venoms are potent inhibitors of the in vitro intraerythrocytic development of P. falciparum (11Deregnaucourt C. Schreével J. Bee venom phospholipase A2 induces stage-specific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components.J. Biol. Chem. 2000; 275: 39973-39980Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Guillaume C. Deregnaucourt C. Clavey V. Schreével J. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.Toxicon. 2004; 43: 311-318Crossref PubMed Scopus (31) Google Scholar). Inhibition at low enzyme concentration occurs only in the presence of serum phospholipids (12Guillaume C. Deregnaucourt C. Clavey V. Schreével J. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.Toxicon. 2004; 43: 311-318Crossref PubMed Scopus (31) Google Scholar), suggesting that hydrolysis of exogenous phospholipids, rather than hydrolysis of the infected red blood cell (RBC) membrane phospholipids, is required for sPLA2s' toxicity. Here, we were interested in deciphering the molecular and cellular interplays between sPLA2 enzyme, human lipoproteins, and infected erythrocytes, as an approach to a better understanding of what may occur in pathophysiological situations such as malaria, in which alterations of the host lipoproteinogram (13Mohanty S. Mishra S.K. Das B.S. Satpathy S.K. Mohanty D. Patnaik J.K. Bose T.K. Altered plasma lipid pattern in falciparum malaria.Ann. Trop. Med. Parasitol. 1992; 86: 601-606Crossref PubMed Scopus (50) Google Scholar) and sPLA2 production (14Vadas P. Keystone J. Stefanski E. Scott K. Pruzanski W. Induction of circulating group II phospholipase A2 expression in adults with malaria.Infect. Immun. 1992; 60: 3928-3931Crossref PubMed Google Scholar, 15Vadas P. Taylor T.E. Chimsuku L. Goldring D. Stefanski E. Pruzanski W. Molyneux M. Increased serum phospholipase A2 activity in Malawian children with falciparum malaria.Am. J. Trop. Med. Hyg. 1993; 49: 455-459Crossref PubMed Scopus (22) Google Scholar) are encountered. To understand what governs the indirect toxicity of sPLA2s, the distribution of the bee venom phospholipase A2 (bvPLA2) between the infected erythrocytes and the triglyceride-rich fraction [chylomicrons (chyls) and VLDLs] of human lipoproteins was analyzed. Incidence of lipoprotein oxidation on bvPLA2 particle binding and anti-Plasmodium activity was also analyzed, because peroxidation has been shown to increase the susceptibility of lipoproteins to hydrolysis by sPLA2 (16Eckey R. Menschikowski M. Lattke P. Jaross W. Minimal oxidation and storage of low density lipoproteins result in an increased susceptibility to phospholipid hydrolysis by phospholipase A2.Atherosclerosis. 1997; 132: 165-176Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and oxidized lipoproteins have been found in malaria patients (17Sibmooh N. Yamanont P. Krudsood S. Leowattanah W. Brittenham G. Looareesuwan S. Udomsangpetch R. Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules.Lipids Health Dis. 2004; 25: 3-15Google Scholar). Finally, we attempted to establish which of the lipolyzed particles or lipid products are responsible for parasite killing. LysoPLs and NEFAs generated by the bvPLA2 activity were identified, and their individual involvement in parasite killing was established, exemplifying the major role of PUFAs. Our results illustrate the capacity of sPLA2s to be active on biological targets via the generation of exogenous NEFAs and raise the question of a potential role for oxidized lipoproteins and/or endogenous sPLA2s in the host defense against malaria. Phospholipase A2 from Apis mellifera venom (bvPLA2), BSA (fraction V), fatty acid (FA)-free BSA, and butylated hydroxytoluene (BHT) (2,6-di-tert-butyl-p-cresol), as well as individual FAs and lysoPLs were purchased from Sigma (St Quentin-Fallavier, France). 3H-hypoxanthine (37 MBq/ml) was from Amersham Biosciences (Orsay, France). The Bio-Rad DC protein assay and Affi-Gel10® gel were from Bio-Rad. Anti-bvPLA2 polyclonal antibody was a gift from Dr. G. Lambeau (Centre National de la Recherche Scientifque; Sophia-Antipolis, France). Anti-human apolipoprotein B (apoB) polyclonal antibodies were from Dade Behring S. A. (Paris, France). The 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-py-C10-HPC) fluorogenic substrate was obtained from Molecular Probes (Invitrogen SARL; Cergy Pontoise, France). The Colombian strain FcB1 of P. falciparum was used throughout the work. Cultures were grown in complete medium consisting of RPMI 1640 (Life Technologies, Inc.) supplemented with 11 mM glucose, 27.5 mM NaHCO3, 100 UI/ml penicillin, 100 μg/ml streptomycin, adjusted to pH 7.4 before addition of heat-inactivated human serum (8% final), according to the procedure of Trager and Jensen (18Trager W. Jensen J.B. Human malaria parasites in continuous culture.Science. 1976; 193: 673-677Crossref PubMed Scopus (6185) Google Scholar). Parasites were grown at 37°C in human O+ or A+ RBCs at a 2% hematocrit and a 3–6% parasitemia, in a 3% CO2, 6% O2, and 91% N2 atmosphere. Cell cultures were synchronized by successive Plasmagel® (19Pasvol G. Wilson R.J.M. Smalley M.E. Brown J. Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood.Ann. Trop. Med. Parasitol. 1978; 72: 87-88Crossref PubMed Scopus (302) Google Scholar) and sorbitol (20Lambros C. Vanderberg J.P. Synchronization of Plasmodium falciparum erythrocytic stages in culture.J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2840) Google Scholar) treatments. The d<1.006 g/ml lipoprotein fraction comprising chyls and VLDLs was purified by centrifugation (24 h, 160,000 g, 4°C) of nonfasted human serum diluted 1:5 in sterile NaCl, 9 g/l, according to the technique by Havel, Eder, and Bragdon (21Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar). The protein content of the fraction was determined using the Bio-Rad DC protein assay according to the manufacturer’s recommendations. Coupling of bvPLA2 to Affi-Gel 10 beads (Bio-Rad) was achieved following the instructions of the manufacturer. Efficiency of the coupling and enzymatic activity of the immobilized bvPLA2 were measured as in (12Guillaume C. Deregnaucourt C. Clavey V. Schreével J. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.Toxicon. 2004; 43: 311-318Crossref PubMed Scopus (31) Google Scholar). For enzymatic lipolysis, 1 ml chyl/VLDLs (approximately 0.15 mg/ml in PBS), either freshly prepared (F-chyl/VLDL) or oxidized (Ox-chyl/VLDL), was mixed with 130 pM active immobilized bvPLA2 in the presence of 1 mM CaCl2 and incubated at 37°C for 17 h. To induce air/light minimal oxidation of lipoproteins, chyl/VLDLs in PBS or in NaCl (9 g/l) were stored in a transparent flask at ambient temperature and under sterile air exchange for 18 days. Lipid peroxides were measured in terms of thiobarbituric acid-reactive substances (TBARS) according to the method of Morlieère et al. (22Morlieère P. Moysan A. Santus R. Huppe G. Mazieère J.C. Dubertret R. UVA-induced lipid peroxidation in cultured human fibroblasts.Biochim. Biophys. Acta. 1991; 1084: 261-268Crossref PubMed Scopus (144) Google Scholar). Results are expressed in malondialdehyde equivalents. In experiments comparing properties of fresh and oxidized lipoproteins, chyl/VLDLs were isolated from plasma that had been aliquotted and frozen at −20°C just after blood drawing. One aliquot was thawed for chyl/VLDL purification and air/light oxidation. At the end of the 2 1/2 week oxidation period and just prior to the experiment, a second aliquot was thawed for purification of F-chyl/VLDLs. Binding of bvPLA2 to RBCs was quantified by sedimenting the cells and measuring the fraction of enzyme remaining in the supernatant. RBCs were sedimented by centrifugation at 90 g for 3 min. The amount of bvPLA2 in the supernatant was measured using the fluorimetric assay with β-py-C10-HPC as substrate (23Radvanyi F. Jordan L. Russo-Marie F. Bon C. A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of albumin.Anal. Biochem. 1989; 177: 103-109Crossref PubMed Scopus (235) Google Scholar). Binding experiments were performed either in PBS (10 mM NaPO4, 150 mM NaCl, pH 7.4) or in RPMI. Binding reactions (200 μl) contained 8.0 ng (2.5 nM) bvPLA2 and 2.5 × 107 RBCs, either normal or parasitized by mature forms of P. falciparum. Parasitized erythrocytes were highly enriched (70–80%) in schizont forms (36–48 h of age) recovered by Plasmagel® treatment. The procedure was as follows: RBCs in PBS were distributed in the BSA-coated wells of a 96-well microplate at the rate of 100 μl/ per well, then 100 μl bvPLA2 (5 nM) in PBS or in PBS + 0.15 mg/ml chyl/VLDLs was added. In the experiment with oxidized lipoproteins, bvPLA2 + chyl/VLDLs were incubated for 45 min at 37°C prior to addition. After 45 min incubation at 37°C, the plate was gently centrifuged at 90 g and supernatants were collected for the spectrofluorimetric assay. The reaction medium was 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 6 mM CaCl2, 0.1% BSA, and 2 μM β-py-C10-HPC. Two hundred microliters of substrate was added to 100 μl of supernatant. The pyrene monomer fluorescence, corresponding to the phospholipid hydrolysis upon bvPLA2 activity, was measured continuously for 10 min with a luminescence spectrometer (AMINCO; Bowman, Series 2) using 345 nm (excitation) and 398 nm (emission) wavelengths. PLA2 activity is expressed in fluorescence variation per second. BvPLA2 100% activity in PBS and in PBS + lipoproteins, and in the absence of cells, was measured at time zero of the incubation. Residual activity after 45 min incubation in the absence of cells was measured to estimate bvPLA2 binding to coated BSA. Negative controls were without bvPLA2. Detection limits were 25 pM enzyme in the absence and 250 pM enzyme in the presence of chyl/VLDLs. Two milliliters of chyl/VLDLs (0.15 mg/ml in PBS, 250 μM CaCl2) was incubated with 130 pM active Affi-Gel-immobilized bvPLA2 for 17 h at 37°C. After a brief centrifugation to pellet Affi-Gel beads, the supernatant was mixed with FA-free BSA (20 mg/ml) for a 2 h incubation at 37°C under gentle agitation. PBS was added up to 8 ml and chyl/VLDLs were separated from the lipid-charged BSA by ultracentrifugation (160,000 g, 20 h, 4°C). The buoyant lipoproteins were recovered from the top fraction, and the pelleted BSA was recovered from the bottom fraction. Both fractions were tested in dose-response assays toward the in vitro intraerythrocytic development of P. falciparum. Lipid-enriched BSA was supplemented with 50 μM BHT and extracted twice with chloroform-methanol-water (2.5:2.5:1.25; v/v/v) using the Bligh and Dyer protocol (24Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). Extracted lipids were dried in a rotary evaporator and stored at −20°C under argon for further analysis. Extracted lipids in chloroform-methanol (1:1, v/v) were separated by TLC on silica gel 60G plates. Samples were duplicated, and migration was realized in chloroform-methanol-water (65:25:4; v/v/v) with standard markers. Lipids were fractionated into lysophosphatidylcholine (lysoPC) (RF 0.20), lysophosphatidylethanolamine (lysoPE) (RF 0.40), and NEFAs (RF 0.91). They were either revealed by 2′-7′-dichlorofluorescein, 0.02% in ethanol-water (95:5; v/v) vaporization, or left unstained for recovery before gas chromatography (GC) analysis. Lipids were collected by plate scraping. LysoPLs were resuspended in 1 ml H2SO4 (5% in MeOH), and incubated for 1 h 30 at 100°C in a dry bath. The reaction was stopped by transfer at 0°C, lipids were neutralized by the addition of 1.5 ml K2CO3 (5% in H2O) and extracted by the addition of 2 ml isooctane. The organic phase was collected upon centrifugation at 900 g for 10 min and dried, and lipids were resuspended in isooctane (2 ml) for GC analysis. NEFAs were extracted twice with 1 ml diethyl ether-methanol (9:1; v/v). The organic phase was collected after centrifugation (10 min, 450 g), dried under argon, and derivatized by a 15 min incubation in the dark with 250 μl diazomethane. Upon derivatization, the fraction was dried under argon and resuspended in 250 μl isooctane for GC analysis. GC separation of FA methyl esters was carried out on a Supelco SP2380 capillary column (30 m × 0.25 mm) and analyzed on an HP 6890 G1530 chromatograph. Heptadecanoic acid (C17:0) and heptadecanoyl-lysoPC were used as internal standards. FAs were quantified according to the known amount of added internal standard. The diverse lipid preparations described above, as well as commercial lipids, were tested for their capacity to inhibit the in vitro intraerythrocytic development of P. falciparum. Dry preparations of commercial lysoPLs and NEFAs, to be tested either individually or upon mixing, were solubilized in 20 μl ethanol; then 1 ml RPMI 8% serum was added, and the lipid solution was shaken for 2 h at ambient temperature before being tested for parasite growth inhibition. Dose-response assays based upon 3H-hypoxanthine incorporation by growing parasites were performed as in (12Guillaume C. Deregnaucourt C. Clavey V. Schreével J. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.Toxicon. 2004; 43: 311-318Crossref PubMed Scopus (31) Google Scholar). Radioactivity was measured with a 1450 Microbeta counter (Wallac, Perkin Elmer). Percentage of growth inhibition was calculated from the parasite-associated radioactivity compared with the control (25Desjardins R.S. Canfield C.J. Haynes J.D. Chulay J.D. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.Antimicrob. Agents Chemother. 1979; 16: 710-718Crossref PubMed Scopus (2261) Google Scholar). Values for the IC50 and IC100 minimum (IC100min) were determined from dose-response curves. Five milliliters of PBS containing bvPLA2 (3 μg/ml), BSA fraction V (1 mg/ml), and CaCl2 (1 mM) were supplemented with chyl/VLDLs, either fresh or oxidized (70 μg/ml final concentration), or supplemented by the same volume of PBS. Each preparation was centrifuged at 160,000 g for 24 h. Fractions (0.6 ml) were collected from the top to the bottom of each tube. They were analyzed by SDS-PAGE on an 8% polyacrylamide gel under reducing conditions. The gel was then stained by Coomassie blue or blotted for immunodetection of bvPLA2 with anti-bvPLA2 antibodies (1:5,000). F-chyl/VLDLs or Ox-chyl/VLDLs were hydrolyzed for 45 min at 37°C by 6 nM bvPLA2 in a 300 μl final volume of PBS containing 1 mM CaCl2, or treated equally in the absence of enzyme. Three hundred microliters of a 6% hematocrit suspension of normal or Plasmodium-infected RBCs (trophozoites/schizonts at a 70–80% parasitemia) were added, then 50 μl aliquots were taken at time zero and after 0.5, 1, 2, 3, 5, 10, 15, and 30 min incubation at room temperature, and immediately centrifuged (briefly) to pellet the cells. The supernatants were boiled in SDS/β-mercaptoethanol buffer and subjected to SDS-PAGE on an 8% polyacrylamide gel. Proteins from gels were blotted onto nitrocellulose and analyzed for the presence of apoB-48 using anti-apoB antibodies diluted 1:10,000. ApoB-48 (molecular mass ≈ 250 kDa) is the main and specific apolipoprotein of chyls. Because of its high molecular mass (about 500 kDa), apoB-100, the main apolipoprotein of the VLDLs, was confined to the stacking gel, and could not be visualized on the Western blot under our experimental conditions. A P. falciparum culture was synchronized on a 4 h time window. After 16 h of growth under classical culture conditions, 100 μl of culture (parasite age: 16–20 h; parasitemia: 1%; hematocrit: 4%) in RPMI + 16% serum was distributed in a 96-well plate. PLA2-hydrolyzed F- and Ox-chyl/VLDLs diluted in RPMI alone were added at their respective IC100mins (100 μl/well). As a control, the effect of nonhydrolyzed lipoproteins (fresh and oxidized) at the same concentration was analyzed. Culture was carried out in a candle jar at 37°C. Aliquots of the culture were taken every 2 h for 20 h (until parasites were 36–40 h old), then after 24 h of culture (40–44 h-old parasites) and 48 h (16–20 h-old reinvaded parasites). The parasitemia, stage distribution, and morphological development of the parasites were followed by optical examination of Giemsa-stained smears and by the counting of 4,000 cells. Images were captured by the Canon Power Shot S40 camera coupled to the Canon Utilities Remote Capture 2.1.0.10 software (magnification ×1140). To determine why the anti-Plasmodium activity of most bvPLA2s results from hydrolysis of exogenous phospholipids and not from a direct action on the infected erythrocyte membrane, we analyzed the distribution of bvPLA2 between the lipoproteins and the erythrocytes. Experiments were carried out at an enzyme-to-cells ratio 100-fold higher than the IC50 toxic ratio (enzyme-to-cells ratio at the bvPLA2 IC50: 1.2 ng bvPLA2:4.5 × 108 RBCs). Noninfected or infected erythrocytes with mature forms of the parasite were incubated with bvPLA2 with or without chyl/VLDLs in BSA-coated plastic wells; then enzymatic activity in the supernatant was measured. Initial velocity at time zero of the incubation (100% enzymatic activity), as well as residual activity after incubation in the BSA-coated wells in the absence of cells, was determined. The initial velocity in the presence of chyl/VLDLs could not be determined without preincubation, because phospholipids from lipoproteins competed strongly with β-py-C10-HPC for hydrolysis by bvPLA2. In PBS alone, a net decrease (by approximately 56%) in enzyme activity was observed after the bvPLA2 had been incubated in the BSA-coated wells, indicating that the enzyme had adsorbed to the BSA (see Fig. 1A). By contrast, when incubation was performed in the presence of healthy or parasitized erythrocytes, 100% activity was recovered in the supernatant, showing that under these conditions, the enzyme does not bind (or not at a detectable rate) to BSA or to erythrocytes. The same results were obtained when experiments were carried out in RPMI (not shown), indicating that in culture medium also, the enzyme does not bind to erythrocytes. When the experiment was performed in the presence of chyl/VLDLs and in the absence of cells, bvPLA2 activity in the supernatant was found to be high (Fig. 1B), suggesting that the lipoproteins had largely prevented the enzyme adsorption to BSA. The presence of erythrocytes, either infected or not, did not lower the activity, indicating that in the presence of lipoproteins, as well as in PBS or RPMI alone, the bulk of enzyme does not bind to erythrocytes. In a second step, we looked for bvPLA2 association with chyl/VLDLs in a mixture of enzyme, lipoproteins, and BSA. The mixture was ultracentrifuged so that buoyant lipoproteins were separated from proteins, and top to bottom fractions were analyzed for the presence of bvPLA2 by SDS-PAGE and immunoblotting. A large amount of enzyme was found in the top fractions containing lipoproteins (Fig. 2A, B for control), demonstrating that bvPLA2 associates tightly with the lipoprotein particles (affinity is high enough to prevent total enzyme dissociation from the particles under a 160,000 g centrifuge force applied for 24 h), even in the presence of BSA. Also, under the same constraints, BSA was not found associated with the lipoproteins (it is not present in the upper fractions), suggesting that the protein does not exhibit high affinity for chyl/VLDLs. The absence of BSA in the bvPLA2-lipoprotein complexes also demonstrates that the enzyme associates freely with the lipoprotein particles, despite its capability of binding to BSA. From this, we can infer that bvPLA2 displacement from the coated BSA observed previously in the presence of lipoproteins resulted from a higher enzyme affinity for the lipoproteins than for the BSA, rather than from a competition between the enzyme and the chyl/VLDLs for binding to BSA. Taken together, these results indicate that the bvPLA2 associates more readily with lipoproteins than with erythrocytes, and strongly suggest that the indirect toxicity of the enzyme toward infected erythrocytes is mainly dictated by its distribution in favor of lipoproteins. To assess whether lysoPLs and NEFAs are involved in the anti-Plasmodium toxicity of the lipolyzed lipoproteins, lipids were sequestered from particles by using lipid-free BSA, then both the resulting lipid-charged BSA and the delipidated lipoproteins were tested for their capacity to inhibit the parasite intraerythrocytic growth. As can be seen in Fig. 3A, toxicity of the bvPLA2-hydrolyzed chyl/VLDLs was decreased after one round of lipid extraction and almost totally lost after two rounds, indicating that lipid products are obligatory elements in the hydrolyzed lipoproteins' toxicity. In good correlation with this, the lipid-charged BSA became toxic to Plasmodium (Fig. 3B). To improve our understanding of the molecular events at play in the lipoprotein-derived bvPLA2 toxicity, we identified and quantified the molecular species of lipid products. Chyl/VLDLs were hydrolyzed by bvPLA2, then lysoPLs and NEFAs from one BSA extraction cycle were analyzed by TLC/GC. Three independent experiments were performed with chyl/VLDLs from different serums. The results are reported in Table 1.TABLE 1Quantitative analysis of the lipids produced by bvPLA2 digestion of chyl/VLDLsLysoPCLysoPENEFAsTotal LipidsNative chyl/VLDLs0.48 ± 0.236.54 ± 4.282.41 ± 0.869.04 ± 4.59BvPLA2-hydrolyzed chyl/VLDLs15.38 ± 2.875.08 ± 4.3535.48 ± 16.9655.94 ± 19.35Values are in" @default.
• W2092257114 created "2016-06-24" @default.
• W2092257114 creator A5002894950 @default.
• W2092257114 creator A5055859205 @default.
• W2092257114 creator A5073988774 @default.
• W2092257114 creator A5078144972 @default.
• W2092257114 creator A5082058024 @default.
• W2092257114 date "2006-07-01" @default.
• W2092257114 modified "2023-10-16" @default.
• W2092257114 title "Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity" @default.
• W2092257114 cites W1777122173 @default.
• W2092257114 cites W1787204411 @default.
• W2092257114 cites W1891661735 @default.
• W2092257114 cites W1965646898 @default.
• W2092257114 cites W1968008704 @default.
• W2092257114 cites W1969503178 @default.
• W2092257114 cites W1976337474 @default.
• W2092257114 cites W1978628047 @default.
• W2092257114 cites W1978721448 @default.
• W2092257114 cites W1982638309 @default.
• W2092257114 cites W1986872682 @default.
• W2092257114 cites W1992315080 @default.
• W2092257114 cites W1992733039 @default.
• W2092257114 cites W1995617096 @default.
• W2092257114 cites W2004226937 @default.
• W2092257114 cites W2009088816 @default.
• W2092257114 cites W2011502676 @default.
• W2092257114 cites W2014749976 @default.
• W2092257114 cites W2020069378 @default.
• W2092257114 cites W2020251171 @default.
• W2092257114 cites W2022230162 @default.
• W2092257114 cites W2028467871 @default.
• W2092257114 cites W2046525300 @default.
• W2092257114 cites W2059163718 @default.
• W2092257114 cites W2067421758 @default.
• W2092257114 cites W2069725001 @default.
• W2092257114 cites W2070769983 @default.
• W2092257114 cites W2073079404 @default.
• W2092257114 cites W2073859623 @default.
• W2092257114 cites W2074954129 @default.
• W2092257114 cites W2077065351 @default.
• W2092257114 cites W2078143666 @default.
• W2092257114 cites W2086692004 @default.
• W2092257114 cites W2093963839 @default.
• W2092257114 cites W2105668259 @default.
• W2092257114 cites W2111878207 @default.
• W2092257114 cites W2119818848 @default.
• W2092257114 cites W2121791231 @default.
• W2092257114 cites W2132699773 @default.
• W2092257114 cites W2135099183 @default.
• W2092257114 cites W2135503869 @default.
• W2092257114 cites W2137474950 @default.
• W2092257114 cites W2145459653 @default.
• W2092257114 cites W2159587755 @default.
• W2092257114 cites W2168541231 @default.
• W2092257114 cites W2170316119 @default.
• W2092257114 cites W2171263915 @default.
• W2092257114 cites W2300552860 @default.
• W2092257114 cites W2336622083 @default.
• W2092257114 cites W2441535877 @default.
• W2092257114 cites W2473455258 @default.
• W2092257114 cites W340702771 @default.
• W2092257114 cites W4240264940 @default.
• W2092257114 cites W4296587586 @default.
• W2092257114 doi "https://doi.org/10.1194/jlr.m600111-jlr200" @default.
• W2092257114 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16607035" @default.
• W2092257114 hasPublicationYear "2006" @default.
• W2092257114 type Work @default.
• W2092257114 sameAs 2092257114 @default.
• W2092257114 citedByCount "20" @default.
• W2092257114 countsByYear W20922571142014 @default.
• W2092257114 countsByYear W20922571142015 @default.
• W2092257114 countsByYear W20922571142016 @default.
• W2092257114 countsByYear W20922571142017 @default.
• W2092257114 countsByYear W20922571142019 @default.
• W2092257114 countsByYear W20922571142020 @default.
• W2092257114 countsByYear W20922571142021 @default.
• W2092257114 countsByYear W20922571142023 @default.
• W2092257114 crossrefType "journal-article" @default.
• W2092257114 hasAuthorship W2092257114A5002894950 @default.
• W2092257114 hasAuthorship W2092257114A5055859205 @default.
• W2092257114 hasAuthorship W2092257114A5073988774 @default.
• W2092257114 hasAuthorship W2092257114A5078144972 @default.
• W2092257114 hasAuthorship W2092257114A5082058024 @default.
• W2092257114 hasBestOaLocation W20922571141 @default.
• W2092257114 hasConcept C178790620 @default.
• W2092257114 hasConcept C181199279 @default.
• W2092257114 hasConcept C185592680 @default.
• W2092257114 hasConcept C2776551241 @default.
• W2092257114 hasConcept C2779448229 @default.
• W2092257114 hasConcept C29730261 @default.
• W2092257114 hasConcept C3020806191 @default.
• W2092257114 hasConcept C33070731 @default.
• W2092257114 hasConcept C55493867 @default.
• W2092257114 hasConcept C86803240 @default.
• W2092257114 hasConcept C90856448 @default.
• W2092257114 hasConceptScore W2092257114C178790620 @default.
• W2092257114 hasConceptScore W2092257114C181199279 @default.

• next