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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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Effect of scyphostatin on SM/LCPL-LPC activity. Membrane fraction prepared from GST-PfNSMase-expressing E. coli and bovine brain (0.5 mg protein/ml each) was incubated with various concentrations of scyphostatin in HSEI buffer for 30 min on ice. SMase activity of plasmodial (filled circles) and mammalian enzyme (open circles) was determined in the presence of 0.1% Triton X-100, and activities of lysoPtdCho-PLC (filled squares) and lysoPAF-PLC (filled triangles) were determined under detergent-free conditions as described under Materials and Methods. The values of the activity are shown as the percentage of the control activity determined in the absence of the drug.
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fig4: Effect of scyphostatin on SM/LCPL-LPC activity. Membrane fraction prepared from GST-PfNSMase-expressing E. coli and bovine brain (0.5 mg protein/ml each) was incubated with various concentrations of scyphostatin in HSEI buffer for 30 min on ice. SMase activity of plasmodial (filled circles) and mammalian enzyme (open circles) was determined in the presence of 0.1% Triton X-100, and activities of lysoPtdCho-PLC (filled squares) and lysoPAF-PLC (filled triangles) were determined under detergent-free conditions as described under Materials and Methods. The values of the activity are shown as the percentage of the control activity determined in the absence of the drug.

Mentions: Previously we have shown that neutral SMase activity associated with the membrane fraction from isolated P. falciparum parasite was inhibited by scyphostatin (27), a compound that was originally found as an inhibitor of mammalian neutral SMase (39, 40). The PfNSM encodes the enzyme exhibiting PLC activity toward SM and LCPL. We tested if the PLC activity of this enzyme was inhibited by scyphostatin. Scyphostatin inhibited PLC activity toward SM in a dose-dependent manner with an ID50 value of ∼3 μM (Fig. 4) , similar to the SMase activity detected in isolated parasite membranes (27). Likewise, scyphostatin inhibited PLC activities toward lysoPtdCho and lysoPAF with ID50s of 3–5 μM (Fig. 4). The ID50 value of scyphostatin to neutral SMase activity of bovine brain membrane was ∼15 μM (Fig. 4), which is ∼5-fold higher than that of plasmodial SMase.


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)

Effect of scyphostatin on SM/LCPL-LPC activity. Membrane fraction prepared from GST-PfNSMase-expressing E. coli and bovine brain (0.5 mg protein/ml each) was incubated with various concentrations of scyphostatin in HSEI buffer for 30 min on ice. SMase activity of plasmodial (filled circles) and mammalian enzyme (open circles) was determined in the presence of 0.1% Triton X-100, and activities of lysoPtdCho-PLC (filled squares) and lysoPAF-PLC (filled triangles) were determined under detergent-free conditions as described under Materials and Methods. The values of the activity are shown as the percentage of the control activity determined in the absence of the drug.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196011&req=5

fig4: Effect of scyphostatin on SM/LCPL-LPC activity. Membrane fraction prepared from GST-PfNSMase-expressing E. coli and bovine brain (0.5 mg protein/ml each) was incubated with various concentrations of scyphostatin in HSEI buffer for 30 min on ice. SMase activity of plasmodial (filled circles) and mammalian enzyme (open circles) was determined in the presence of 0.1% Triton X-100, and activities of lysoPtdCho-PLC (filled squares) and lysoPAF-PLC (filled triangles) were determined under detergent-free conditions as described under Materials and Methods. The values of the activity are shown as the percentage of the control activity determined in the absence of the drug.
Mentions: Previously we have shown that neutral SMase activity associated with the membrane fraction from isolated P. falciparum parasite was inhibited by scyphostatin (27), a compound that was originally found as an inhibitor of mammalian neutral SMase (39, 40). The PfNSM encodes the enzyme exhibiting PLC activity toward SM and LCPL. We tested if the PLC activity of this enzyme was inhibited by scyphostatin. Scyphostatin inhibited PLC activity toward SM in a dose-dependent manner with an ID50 value of ∼3 μM (Fig. 4) , similar to the SMase activity detected in isolated parasite membranes (27). Likewise, scyphostatin inhibited PLC activities toward lysoPtdCho and lysoPAF with ID50s of 3–5 μM (Fig. 4). The ID50 value of scyphostatin to neutral SMase activity of bovine brain membrane was ∼15 μM (Fig. 4), which is ∼5-fold higher than that of plasmodial SMase.

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