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Plasmodium falciparum phospholipase C hydrolyzing sphingomyelin and lysocholinephospholipids is a possible target for malaria chemotherapy.

Hanada K, Palacpac NM, Magistrado PA, Kurokawa K, Rai G, Sakata D, Hara T, Horii T, Nishijima M, Mitamura T - J. Exp. Med. (2002)

Bottom Line: Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites.In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC).Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Japan Science and Technology Corporation, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. hanak@nih.go.jp

ABSTRACT
Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is approximately 25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

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LCPL-PLC activity in the membrane fraction of E. coli cells transfected with pGEX-PfNSM. (A) LysoPtdCho-, lysoPAF-, and PAF-PLC activities. Membranes (5 μg protein) from E. coli cells transfected with the indicated plasmids were incubated in 50 μl HM buffer (50 mM Hepes-NaOH, pH 7.5, and 10 mM MgCl2) containing 10 μM of various radioactive substrates at 37°C for 30 min. The amount of hydrolyzed substrates was determined as described under Materials and Methods. The data are means ± SD from three experiments. (B) Competition of lysoPtdCho-PLC activity with various lipids. The membrane fraction from pGEX-PfNSM-transfected E. coli (5 μg protein) was incubated in 50 μl of 50 mM Hepes-NaOH (pH 7.5) containing 10 mM MgCl2, 10 μM [palmitoyl-1-14C]lysoPtdCho, and various concentrations of nonradioactive competitors at 37°C for 30 min. The radioactivity of monopalmitoylglycerol produced was determined as described under Materials and Methods. The data shown are the percentages of the mean activity determined in the absence of competitors. Filled circles, lysoPtdCho; open circles, lysoPAF, filled squares, PAF; open squares, sphingosylphosphocholine; filled triangles, lysoPtdSer; open triangles, lysophosphatidylinositol.
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fig3: LCPL-PLC activity in the membrane fraction of E. coli cells transfected with pGEX-PfNSM. (A) LysoPtdCho-, lysoPAF-, and PAF-PLC activities. Membranes (5 μg protein) from E. coli cells transfected with the indicated plasmids were incubated in 50 μl HM buffer (50 mM Hepes-NaOH, pH 7.5, and 10 mM MgCl2) containing 10 μM of various radioactive substrates at 37°C for 30 min. The amount of hydrolyzed substrates was determined as described under Materials and Methods. The data are means ± SD from three experiments. (B) Competition of lysoPtdCho-PLC activity with various lipids. The membrane fraction from pGEX-PfNSM-transfected E. coli (5 μg protein) was incubated in 50 μl of 50 mM Hepes-NaOH (pH 7.5) containing 10 mM MgCl2, 10 μM [palmitoyl-1-14C]lysoPtdCho, and various concentrations of nonradioactive competitors at 37°C for 30 min. The radioactivity of monopalmitoylglycerol produced was determined as described under Materials and Methods. The data shown are the percentages of the mean activity determined in the absence of competitors. Filled circles, lysoPtdCho; open circles, lysoPAF, filled squares, PAF; open squares, sphingosylphosphocholine; filled triangles, lysoPtdSer; open triangles, lysophosphatidylinositol.

Mentions: When assayed in the presence of 0.1% Triton X-100, hydrolysis of [14C]lysoPtdCho by GST-PfNSMase was negligible (data not shown). However, under detergent-free assay conditions, pronounced activity to release radioactive monopalmitoylglycerol from [palmitoyl-1-14C]lysoPtdCho was detected in the membrane fraction of E. coli cells transfected with pGEX-PfNSM, but not with the empty vector (Fig. 3 A). The lysoPtdCho-PLC activity was Mg2+ dependent like SMase activity. The Km value for lysoPtdCho was determined to be ∼25 μM based on double reciprocal plots (data not shown). Surprisingly, the lysoPtdCho-PLC activity in intact membranes did not require the addition of exogenous PtdSer. LysoPtdCho-PLC activity was also detected in the membrane fraction from parasite cells under detergent-free conditions, but not under detergent-mixed micelle conditions (data not shown).


Plasmodium falciparum phospholipase C hydrolyzing sphingomyelin and lysocholinephospholipids is a possible target for malaria chemotherapy.

Hanada K, Palacpac NM, Magistrado PA, Kurokawa K, Rai G, Sakata D, Hara T, Horii T, Nishijima M, Mitamura T - J. Exp. Med. (2002)

LCPL-PLC activity in the membrane fraction of E. coli cells transfected with pGEX-PfNSM. (A) LysoPtdCho-, lysoPAF-, and PAF-PLC activities. Membranes (5 μg protein) from E. coli cells transfected with the indicated plasmids were incubated in 50 μl HM buffer (50 mM Hepes-NaOH, pH 7.5, and 10 mM MgCl2) containing 10 μM of various radioactive substrates at 37°C for 30 min. The amount of hydrolyzed substrates was determined as described under Materials and Methods. The data are means ± SD from three experiments. (B) Competition of lysoPtdCho-PLC activity with various lipids. The membrane fraction from pGEX-PfNSM-transfected E. coli (5 μg protein) was incubated in 50 μl of 50 mM Hepes-NaOH (pH 7.5) containing 10 mM MgCl2, 10 μM [palmitoyl-1-14C]lysoPtdCho, and various concentrations of nonradioactive competitors at 37°C for 30 min. The radioactivity of monopalmitoylglycerol produced was determined as described under Materials and Methods. The data shown are the percentages of the mean activity determined in the absence of competitors. Filled circles, lysoPtdCho; open circles, lysoPAF, filled squares, PAF; open squares, sphingosylphosphocholine; filled triangles, lysoPtdSer; open triangles, lysophosphatidylinositol.
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Related In: Results  -  Collection

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fig3: LCPL-PLC activity in the membrane fraction of E. coli cells transfected with pGEX-PfNSM. (A) LysoPtdCho-, lysoPAF-, and PAF-PLC activities. Membranes (5 μg protein) from E. coli cells transfected with the indicated plasmids were incubated in 50 μl HM buffer (50 mM Hepes-NaOH, pH 7.5, and 10 mM MgCl2) containing 10 μM of various radioactive substrates at 37°C for 30 min. The amount of hydrolyzed substrates was determined as described under Materials and Methods. The data are means ± SD from three experiments. (B) Competition of lysoPtdCho-PLC activity with various lipids. The membrane fraction from pGEX-PfNSM-transfected E. coli (5 μg protein) was incubated in 50 μl of 50 mM Hepes-NaOH (pH 7.5) containing 10 mM MgCl2, 10 μM [palmitoyl-1-14C]lysoPtdCho, and various concentrations of nonradioactive competitors at 37°C for 30 min. The radioactivity of monopalmitoylglycerol produced was determined as described under Materials and Methods. The data shown are the percentages of the mean activity determined in the absence of competitors. Filled circles, lysoPtdCho; open circles, lysoPAF, filled squares, PAF; open squares, sphingosylphosphocholine; filled triangles, lysoPtdSer; open triangles, lysophosphatidylinositol.
Mentions: When assayed in the presence of 0.1% Triton X-100, hydrolysis of [14C]lysoPtdCho by GST-PfNSMase was negligible (data not shown). However, under detergent-free assay conditions, pronounced activity to release radioactive monopalmitoylglycerol from [palmitoyl-1-14C]lysoPtdCho was detected in the membrane fraction of E. coli cells transfected with pGEX-PfNSM, but not with the empty vector (Fig. 3 A). The lysoPtdCho-PLC activity was Mg2+ dependent like SMase activity. The Km value for lysoPtdCho was determined to be ∼25 μM based on double reciprocal plots (data not shown). Surprisingly, the lysoPtdCho-PLC activity in intact membranes did not require the addition of exogenous PtdSer. LysoPtdCho-PLC activity was also detected in the membrane fraction from parasite cells under detergent-free conditions, but not under detergent-mixed micelle conditions (data not shown).

Bottom Line: Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites.In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC).Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Japan Science and Technology Corporation, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. hanak@nih.go.jp

ABSTRACT
Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is approximately 25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Show MeSH
Related in: MedlinePlus