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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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Stage-specific transcription of PfNSMase in the intraerythrocytic parasite P. falciparum. (A) Northern blotting of asynchronous parasite culture. 10 μg of total RNA prepared from asynchronous cultures of three P. falciparum lines was loaded in each lane. The ethidium bromide-stained gel (lanes 1–3) shows the comparable loadings of RNA. Lane 1 and 4, 3D7; lane 2 and 5, Honduras-1; lane 3 and 6, Dd2. The position of the standard RNA marker (GIBCO BRL) is shown at the left. The stage distribution for each line is indicated: 3D7, 63% ring, 26% trophozoite, 11% schizont; Honduras-1, 71% ring, 22% trophozoite, 7% schizont; and Dd2, 48% ring, 26% trophozoite, 26% schizont. (B) Northern blotting of synchronous parasite culture. 4 μg (lanes 1, 2, 5, and 6) and 10 μg (lanes 3, 4, 7, and 8) of total RNA prepared from different stages of tightly synchronized culture of HB3 line was loaded. The ethidium bromide–stained gel (lanes 1–4) indicates the comparable loadings of RNA from the different stages at two different concentrations. Lanes 1, 3, 5, and 7, ring-rich culture (99% ring, 1% trophozoite, 0% schizont); lanes 2, 4, 6, and 8, trophozoite- and schizont-rich culture (0% ring, 86% trophozoite, 14% schizont). The position of the standard RNA marker is shown at the left. (C) RT-PCR experiment. PCR products obtained from different concentrations of first strand cDNA from various stages of tightly synchronized parasite culture of Honduras-1 line were analyzed in 0.8% agarose gel. Lanes 1 and 2, 3 and 4, 5–9, 10–14, and 15–19 are products obtained from ring, young trophozoite, mature trophozoite, schizont, and segmented-schizont, respectively. Parasite morphology at each stage used is shown on top. Dilution factors of the first strand cDNA solution are as follows: lanes 1, 2, 3, 5, 10, and 15, no dilution; lanes 4, 6, 11, and 16, 10-fold; lanes 7, 12, and 17, 100-fold; lanes 8, 13, and 18, 1,000-fold; lanes 9, 14, and 19, 10,000-fold.
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fig2: Stage-specific transcription of PfNSMase in the intraerythrocytic parasite P. falciparum. (A) Northern blotting of asynchronous parasite culture. 10 μg of total RNA prepared from asynchronous cultures of three P. falciparum lines was loaded in each lane. The ethidium bromide-stained gel (lanes 1–3) shows the comparable loadings of RNA. Lane 1 and 4, 3D7; lane 2 and 5, Honduras-1; lane 3 and 6, Dd2. The position of the standard RNA marker (GIBCO BRL) is shown at the left. The stage distribution for each line is indicated: 3D7, 63% ring, 26% trophozoite, 11% schizont; Honduras-1, 71% ring, 22% trophozoite, 7% schizont; and Dd2, 48% ring, 26% trophozoite, 26% schizont. (B) Northern blotting of synchronous parasite culture. 4 μg (lanes 1, 2, 5, and 6) and 10 μg (lanes 3, 4, 7, and 8) of total RNA prepared from different stages of tightly synchronized culture of HB3 line was loaded. The ethidium bromide–stained gel (lanes 1–4) indicates the comparable loadings of RNA from the different stages at two different concentrations. Lanes 1, 3, 5, and 7, ring-rich culture (99% ring, 1% trophozoite, 0% schizont); lanes 2, 4, 6, and 8, trophozoite- and schizont-rich culture (0% ring, 86% trophozoite, 14% schizont). The position of the standard RNA marker is shown at the left. (C) RT-PCR experiment. PCR products obtained from different concentrations of first strand cDNA from various stages of tightly synchronized parasite culture of Honduras-1 line were analyzed in 0.8% agarose gel. Lanes 1 and 2, 3 and 4, 5–9, 10–14, and 15–19 are products obtained from ring, young trophozoite, mature trophozoite, schizont, and segmented-schizont, respectively. Parasite morphology at each stage used is shown on top. Dilution factors of the first strand cDNA solution are as follows: lanes 1, 2, 3, 5, 10, and 15, no dilution; lanes 4, 6, 11, and 16, 10-fold; lanes 7, 12, and 17, 100-fold; lanes 8, 13, and 18, 1,000-fold; lanes 9, 14, and 19, 10,000-fold.

Mentions: Northern blotting experiment with three different parasite lines (Honduras-1, 3D7, and Dd2) showed a single hybridized signal at 2.2 kb (Fig. 2 A), indicating that the PfNSM is indeed transcribed in intraerythrocytic parasite cells, and ubiquitously expressed in various P. falciparum strains. The 2.2 kb size detected in Northern blotting is consistent with that of the PfNSM cDNA described above.


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)

Stage-specific transcription of PfNSMase in the intraerythrocytic parasite P. falciparum. (A) Northern blotting of asynchronous parasite culture. 10 μg of total RNA prepared from asynchronous cultures of three P. falciparum lines was loaded in each lane. The ethidium bromide-stained gel (lanes 1–3) shows the comparable loadings of RNA. Lane 1 and 4, 3D7; lane 2 and 5, Honduras-1; lane 3 and 6, Dd2. The position of the standard RNA marker (GIBCO BRL) is shown at the left. The stage distribution for each line is indicated: 3D7, 63% ring, 26% trophozoite, 11% schizont; Honduras-1, 71% ring, 22% trophozoite, 7% schizont; and Dd2, 48% ring, 26% trophozoite, 26% schizont. (B) Northern blotting of synchronous parasite culture. 4 μg (lanes 1, 2, 5, and 6) and 10 μg (lanes 3, 4, 7, and 8) of total RNA prepared from different stages of tightly synchronized culture of HB3 line was loaded. The ethidium bromide–stained gel (lanes 1–4) indicates the comparable loadings of RNA from the different stages at two different concentrations. Lanes 1, 3, 5, and 7, ring-rich culture (99% ring, 1% trophozoite, 0% schizont); lanes 2, 4, 6, and 8, trophozoite- and schizont-rich culture (0% ring, 86% trophozoite, 14% schizont). The position of the standard RNA marker is shown at the left. (C) RT-PCR experiment. PCR products obtained from different concentrations of first strand cDNA from various stages of tightly synchronized parasite culture of Honduras-1 line were analyzed in 0.8% agarose gel. Lanes 1 and 2, 3 and 4, 5–9, 10–14, and 15–19 are products obtained from ring, young trophozoite, mature trophozoite, schizont, and segmented-schizont, respectively. Parasite morphology at each stage used is shown on top. Dilution factors of the first strand cDNA solution are as follows: lanes 1, 2, 3, 5, 10, and 15, no dilution; lanes 4, 6, 11, and 16, 10-fold; lanes 7, 12, and 17, 100-fold; lanes 8, 13, and 18, 1,000-fold; lanes 9, 14, and 19, 10,000-fold.
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Related In: Results  -  Collection

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fig2: Stage-specific transcription of PfNSMase in the intraerythrocytic parasite P. falciparum. (A) Northern blotting of asynchronous parasite culture. 10 μg of total RNA prepared from asynchronous cultures of three P. falciparum lines was loaded in each lane. The ethidium bromide-stained gel (lanes 1–3) shows the comparable loadings of RNA. Lane 1 and 4, 3D7; lane 2 and 5, Honduras-1; lane 3 and 6, Dd2. The position of the standard RNA marker (GIBCO BRL) is shown at the left. The stage distribution for each line is indicated: 3D7, 63% ring, 26% trophozoite, 11% schizont; Honduras-1, 71% ring, 22% trophozoite, 7% schizont; and Dd2, 48% ring, 26% trophozoite, 26% schizont. (B) Northern blotting of synchronous parasite culture. 4 μg (lanes 1, 2, 5, and 6) and 10 μg (lanes 3, 4, 7, and 8) of total RNA prepared from different stages of tightly synchronized culture of HB3 line was loaded. The ethidium bromide–stained gel (lanes 1–4) indicates the comparable loadings of RNA from the different stages at two different concentrations. Lanes 1, 3, 5, and 7, ring-rich culture (99% ring, 1% trophozoite, 0% schizont); lanes 2, 4, 6, and 8, trophozoite- and schizont-rich culture (0% ring, 86% trophozoite, 14% schizont). The position of the standard RNA marker is shown at the left. (C) RT-PCR experiment. PCR products obtained from different concentrations of first strand cDNA from various stages of tightly synchronized parasite culture of Honduras-1 line were analyzed in 0.8% agarose gel. Lanes 1 and 2, 3 and 4, 5–9, 10–14, and 15–19 are products obtained from ring, young trophozoite, mature trophozoite, schizont, and segmented-schizont, respectively. Parasite morphology at each stage used is shown on top. Dilution factors of the first strand cDNA solution are as follows: lanes 1, 2, 3, 5, 10, and 15, no dilution; lanes 4, 6, 11, and 16, 10-fold; lanes 7, 12, and 17, 100-fold; lanes 8, 13, and 18, 1,000-fold; lanes 9, 14, and 19, 10,000-fold.
Mentions: Northern blotting experiment with three different parasite lines (Honduras-1, 3D7, and Dd2) showed a single hybridized signal at 2.2 kb (Fig. 2 A), indicating that the PfNSM is indeed transcribed in intraerythrocytic parasite cells, and ubiquitously expressed in various P. falciparum strains. The 2.2 kb size detected in Northern blotting is consistent with that of the PfNSM cDNA described above.

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