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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 and PPMP on the intraerythrocytic development of Plasmodium falciparum. Parasite cells were treated with 1 μM scyphostatin or 5 μM PPMP at different stages. Lane 1, control medium containing 0.1% DMSO; lanes 2, 4, 6, 8, 10, and 12, medium containing 1 μM scyphostatin; lanes 3, 5, 7, 9, 11 and 13, medium containing 5 μM PPMP. The parasite culture was transferred from the control medium into the medium containing inhibitor in place of DMSO at 24 h (lanes 4 and 5), 28 h (lanes 6 and 7), 32 h (lanes 8 and 9), 36 h (lanes 10 and 11), and 40 h (lanes 12 and 13). Each panel shows the major morphology of the intraerythrocytic parasite in each treatment at the indicated time. Inset shows the other morphology that when observed, comprises >50% of the major one. However, in some occasions, no major morphology was observed. The percentage indicated at the left is the parasitemia of newly formed rings at 52 h. Mean values from duplicate slides are shown, results are reproducible based on two independent experiments.
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fig6: Effect of scyphostatin and PPMP on the intraerythrocytic development of Plasmodium falciparum. Parasite cells were treated with 1 μM scyphostatin or 5 μM PPMP at different stages. Lane 1, control medium containing 0.1% DMSO; lanes 2, 4, 6, 8, 10, and 12, medium containing 1 μM scyphostatin; lanes 3, 5, 7, 9, 11 and 13, medium containing 5 μM PPMP. The parasite culture was transferred from the control medium into the medium containing inhibitor in place of DMSO at 24 h (lanes 4 and 5), 28 h (lanes 6 and 7), 32 h (lanes 8 and 9), 36 h (lanes 10 and 11), and 40 h (lanes 12 and 13). Each panel shows the major morphology of the intraerythrocytic parasite in each treatment at the indicated time. Inset shows the other morphology that when observed, comprises >50% of the major one. However, in some occasions, no major morphology was observed. The percentage indicated at the left is the parasitemia of newly formed rings at 52 h. Mean values from duplicate slides are shown, results are reproducible based on two independent experiments.

Mentions: To examine the effect of scyphostatin on the intraerythrocytic development of P. falciparum, we monitored the morphological changes of tightly synchronized parasites within 4 h life span, which were treated with scyphostatin at different stages. PPMP was used for comparison. Honduras-1 line was chosen as the representative parasite since all parasite lines tested so far showed similar ID50 values. A serum-free medium supplemented with 1 μM scyphostatin or 5 μM PPMP (∼ID90s from the microscopic assay in Fig. 5 B) was used to obtain a reproducible intraerythrocytic development. As shown in Fig. 6 , in an inhibitor-free control culture medium containing vehicle solvent (0.1% DMSO), the parasite developed normally and entered the next cycle at 52 h with ∼5.6% parasitemia of newly formed ring (lane 1). When 1 μM scyphostatin was added at the initial ring stage (0 h), parasite cells developed to mature trophozoites, but could not develop to schizonts, exhibiting a slightly bigger but less stained trophozoite-like morphology. These trophozoite-like structures neither developed further nor formed new ring stage parasites even after 52 h cultivation (lane 2). A similar effect on parasite morphology was observed when the scyphostatin was added at the early-trophozoite stage (24 h; lane 4). Addition of scyphostatin at the mid- and late-trophozoite stages (28 and 32 h) resulted in an increase in the parasitemia of newly formed rings to 1–3% (lanes 6 and 8). Conversely, when added at the schizont (36 h) and segmented schizont (40 h) stages, the parasitemia of rings at 52 h reached the level comparable to that of the inhibitor-free control (lanes 10 and 12). These results indicate that 1 μM scyphostatin has no or little effect on the stage progression from ring to trophozoite as well as that from schizont to the next ring stage, but impaired specifically the maturation of trophozoite into schizont. Interestingly, the time for scyphostatin to start exerting the effect on the intraerythrocytic development is consistent with the time to start transcribing the PfNSM (Figs. 2 C and 6).


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 and PPMP on the intraerythrocytic development of Plasmodium falciparum. Parasite cells were treated with 1 μM scyphostatin or 5 μM PPMP at different stages. Lane 1, control medium containing 0.1% DMSO; lanes 2, 4, 6, 8, 10, and 12, medium containing 1 μM scyphostatin; lanes 3, 5, 7, 9, 11 and 13, medium containing 5 μM PPMP. The parasite culture was transferred from the control medium into the medium containing inhibitor in place of DMSO at 24 h (lanes 4 and 5), 28 h (lanes 6 and 7), 32 h (lanes 8 and 9), 36 h (lanes 10 and 11), and 40 h (lanes 12 and 13). Each panel shows the major morphology of the intraerythrocytic parasite in each treatment at the indicated time. Inset shows the other morphology that when observed, comprises >50% of the major one. However, in some occasions, no major morphology was observed. The percentage indicated at the left is the parasitemia of newly formed rings at 52 h. Mean values from duplicate slides are shown, results are reproducible based on two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196011&req=5

fig6: Effect of scyphostatin and PPMP on the intraerythrocytic development of Plasmodium falciparum. Parasite cells were treated with 1 μM scyphostatin or 5 μM PPMP at different stages. Lane 1, control medium containing 0.1% DMSO; lanes 2, 4, 6, 8, 10, and 12, medium containing 1 μM scyphostatin; lanes 3, 5, 7, 9, 11 and 13, medium containing 5 μM PPMP. The parasite culture was transferred from the control medium into the medium containing inhibitor in place of DMSO at 24 h (lanes 4 and 5), 28 h (lanes 6 and 7), 32 h (lanes 8 and 9), 36 h (lanes 10 and 11), and 40 h (lanes 12 and 13). Each panel shows the major morphology of the intraerythrocytic parasite in each treatment at the indicated time. Inset shows the other morphology that when observed, comprises >50% of the major one. However, in some occasions, no major morphology was observed. The percentage indicated at the left is the parasitemia of newly formed rings at 52 h. Mean values from duplicate slides are shown, results are reproducible based on two independent experiments.
Mentions: To examine the effect of scyphostatin on the intraerythrocytic development of P. falciparum, we monitored the morphological changes of tightly synchronized parasites within 4 h life span, which were treated with scyphostatin at different stages. PPMP was used for comparison. Honduras-1 line was chosen as the representative parasite since all parasite lines tested so far showed similar ID50 values. A serum-free medium supplemented with 1 μM scyphostatin or 5 μM PPMP (∼ID90s from the microscopic assay in Fig. 5 B) was used to obtain a reproducible intraerythrocytic development. As shown in Fig. 6 , in an inhibitor-free control culture medium containing vehicle solvent (0.1% DMSO), the parasite developed normally and entered the next cycle at 52 h with ∼5.6% parasitemia of newly formed ring (lane 1). When 1 μM scyphostatin was added at the initial ring stage (0 h), parasite cells developed to mature trophozoites, but could not develop to schizonts, exhibiting a slightly bigger but less stained trophozoite-like morphology. These trophozoite-like structures neither developed further nor formed new ring stage parasites even after 52 h cultivation (lane 2). A similar effect on parasite morphology was observed when the scyphostatin was added at the early-trophozoite stage (24 h; lane 4). Addition of scyphostatin at the mid- and late-trophozoite stages (28 and 32 h) resulted in an increase in the parasitemia of newly formed rings to 1–3% (lanes 6 and 8). Conversely, when added at the schizont (36 h) and segmented schizont (40 h) stages, the parasitemia of rings at 52 h reached the level comparable to that of the inhibitor-free control (lanes 10 and 12). These results indicate that 1 μM scyphostatin has no or little effect on the stage progression from ring to trophozoite as well as that from schizont to the next ring stage, but impaired specifically the maturation of trophozoite into schizont. Interestingly, the time for scyphostatin to start exerting the effect on the intraerythrocytic development is consistent with the time to start transcribing the PfNSM (Figs. 2 C and 6).

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