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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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Parasite growth inhibition by scyphostatin. (A) In vitro susceptibility of parasite lines to scyphostatin in a standard medium. The values are expressed as the percentage of the [3H]hypoxanthine incorporation into parasites treated with scyphostatin over those without treatment. The DMSO content in the assay media did not exceed 0.6%, which did not show any effect on the [3H]hypoxanthine incorporation into parasites. The mean values of triplicates from two independent experiments were used for each plot. Filled circles, 3D7; filled triangle, Honduras-1; filled squares, FCR3. (B) In vitro susceptibility of Honduras-1 line to scyphostatin (circles) or PPMP (triangles) in a serum-free medium was examined through either [3H]hypoxanthine incorporation assay (filled symbols) or microscopic assay (open symbols).
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fig5: Parasite growth inhibition by scyphostatin. (A) In vitro susceptibility of parasite lines to scyphostatin in a standard medium. The values are expressed as the percentage of the [3H]hypoxanthine incorporation into parasites treated with scyphostatin over those without treatment. The DMSO content in the assay media did not exceed 0.6%, which did not show any effect on the [3H]hypoxanthine incorporation into parasites. The mean values of triplicates from two independent experiments were used for each plot. Filled circles, 3D7; filled triangle, Honduras-1; filled squares, FCR3. (B) In vitro susceptibility of Honduras-1 line to scyphostatin (circles) or PPMP (triangles) in a serum-free medium was examined through either [3H]hypoxanthine incorporation assay (filled symbols) or microscopic assay (open symbols).

Mentions: We then examined the effect of scyphostatin on the in vitro culture of P. falciparum to implicate the PLC activities associated with PfNSM product to the intraerythrocytic proliferation of parasite cells. For this, three parasite lines were used: FCR3 line resistant to chloroquine, Honduras-1 line resistant to pyrimethamine and cycloguanyl, and 3D7 line sensitive to those conventional antimalarial drugs. As shown in Fig. 5 A, the growth of all parasite lines tested was inhibited by scyphostatin in a dose-dependent manner with 50% inhibition observed at 8, 6, and 6.5 μM for 3D7, Honduras-1, and FCR3, respectively. ID50s for the parasite growth are close to those for SM/LCPL-PLC activities (Fig. 4). PPMP, a ceramide-related compound that inhibits the SM synthase activity as well as intraerythrocytic proliferation of P. falciparum (7, 27), showed 50% growth inhibition at ∼6 μM (data not shown). Difference in ID50 values from the previous report (7) may be partly due to the purity of PPMP and/or the culture condition used.


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)

Parasite growth inhibition by scyphostatin. (A) In vitro susceptibility of parasite lines to scyphostatin in a standard medium. The values are expressed as the percentage of the [3H]hypoxanthine incorporation into parasites treated with scyphostatin over those without treatment. The DMSO content in the assay media did not exceed 0.6%, which did not show any effect on the [3H]hypoxanthine incorporation into parasites. The mean values of triplicates from two independent experiments were used for each plot. Filled circles, 3D7; filled triangle, Honduras-1; filled squares, FCR3. (B) In vitro susceptibility of Honduras-1 line to scyphostatin (circles) or PPMP (triangles) in a serum-free medium was examined through either [3H]hypoxanthine incorporation assay (filled symbols) or microscopic assay (open symbols).
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Related In: Results  -  Collection

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

fig5: Parasite growth inhibition by scyphostatin. (A) In vitro susceptibility of parasite lines to scyphostatin in a standard medium. The values are expressed as the percentage of the [3H]hypoxanthine incorporation into parasites treated with scyphostatin over those without treatment. The DMSO content in the assay media did not exceed 0.6%, which did not show any effect on the [3H]hypoxanthine incorporation into parasites. The mean values of triplicates from two independent experiments were used for each plot. Filled circles, 3D7; filled triangle, Honduras-1; filled squares, FCR3. (B) In vitro susceptibility of Honduras-1 line to scyphostatin (circles) or PPMP (triangles) in a serum-free medium was examined through either [3H]hypoxanthine incorporation assay (filled symbols) or microscopic assay (open symbols).
Mentions: We then examined the effect of scyphostatin on the in vitro culture of P. falciparum to implicate the PLC activities associated with PfNSM product to the intraerythrocytic proliferation of parasite cells. For this, three parasite lines were used: FCR3 line resistant to chloroquine, Honduras-1 line resistant to pyrimethamine and cycloguanyl, and 3D7 line sensitive to those conventional antimalarial drugs. As shown in Fig. 5 A, the growth of all parasite lines tested was inhibited by scyphostatin in a dose-dependent manner with 50% inhibition observed at 8, 6, and 6.5 μM for 3D7, Honduras-1, and FCR3, respectively. ID50s for the parasite growth are close to those for SM/LCPL-PLC activities (Fig. 4). PPMP, a ceramide-related compound that inhibits the SM synthase activity as well as intraerythrocytic proliferation of P. falciparum (7, 27), showed 50% growth inhibition at ∼6 μM (data not shown). Difference in ID50 values from the previous report (7) may be partly due to the purity of PPMP and/or the culture condition used.

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