Limits...
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.

Show MeSH

Related in: MedlinePlus

Primary structure of P. falciparum SM/LCPL-PLC. (A) Deduced amino acid sequence of P. falciparum SM/LCPL-PLC. (B) Hydropathy profile of the deduced PfNSM product obtained using Kite-Doolittle algorithm with a window size of nineteen (reference 35). (C) Intracellular distribution of GST-PfNSMase expressed in E. coli. After centrifugation at 105 g for 1 h, E. coli cell lysates transfected with pGEX-PfNSM (lanes 1 and 2) or pGEX-PfNSMd(2/68) (lanes 3 and 4), were analyzed by Western blotting using anti-PfNSMase antibodies. Lanes 1 and 3, supernatant; lanes 2 and 4, pellet fraction. 2 μg protein was loaded in each lane. (D) Multiple sequence alignment of the SMases from different species. Sequence alignments were performed with CLUSTAL W (reference 47) and refined with GeneAlign program (reference 48) using amino acid sequences in the regions where significant homology was observed through the dotplot analysis. The regions in each amino acid sequence used are PFAL (P. falciparum), 182–278; BCER (B. cereus), 129–225; SAUR (S. aureus), 134–230; LINT (L. interrogans), 179–275; HSAP1 (Homo sapiens), 81–181; and HSAP2 (Homo sapiens), 406–513. Amino acid residues conserved in all species and more than four species are highlighted in black and gray, respectively. (E) Phylogenetic tree of SMases. The multiple sequence alignment shown in D was used to make the phylogenetic tree through neighbor-joining algorithm with MEGA version 2.2 (reference 38). Scale bar indicates the number of substitutions per site. Bootstrap values are percentages of 1,000 replications and are shown at the nodes. GenBank/EMBL/DDBJ accession no. for each SMase is in parentheses. UPGMA also gave similar phylogenetic tree with similar topology.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196011&req=5

fig1: Primary structure of P. falciparum SM/LCPL-PLC. (A) Deduced amino acid sequence of P. falciparum SM/LCPL-PLC. (B) Hydropathy profile of the deduced PfNSM product obtained using Kite-Doolittle algorithm with a window size of nineteen (reference 35). (C) Intracellular distribution of GST-PfNSMase expressed in E. coli. After centrifugation at 105 g for 1 h, E. coli cell lysates transfected with pGEX-PfNSM (lanes 1 and 2) or pGEX-PfNSMd(2/68) (lanes 3 and 4), were analyzed by Western blotting using anti-PfNSMase antibodies. Lanes 1 and 3, supernatant; lanes 2 and 4, pellet fraction. 2 μg protein was loaded in each lane. (D) Multiple sequence alignment of the SMases from different species. Sequence alignments were performed with CLUSTAL W (reference 47) and refined with GeneAlign program (reference 48) using amino acid sequences in the regions where significant homology was observed through the dotplot analysis. The regions in each amino acid sequence used are PFAL (P. falciparum), 182–278; BCER (B. cereus), 129–225; SAUR (S. aureus), 134–230; LINT (L. interrogans), 179–275; HSAP1 (Homo sapiens), 81–181; and HSAP2 (Homo sapiens), 406–513. Amino acid residues conserved in all species and more than four species are highlighted in black and gray, respectively. (E) Phylogenetic tree of SMases. The multiple sequence alignment shown in D was used to make the phylogenetic tree through neighbor-joining algorithm with MEGA version 2.2 (reference 38). Scale bar indicates the number of substitutions per site. Bootstrap values are percentages of 1,000 replications and are shown at the nodes. GenBank/EMBL/DDBJ accession no. for each SMase is in parentheses. UPGMA also gave similar phylogenetic tree with similar topology.

Mentions: To identify the gene for plasmodial SMase, in silico sequence analysis was performed by searching the NCBI Malaria Genetics and Genomics databases (http://www.ncbi.nlm.nih.gov/Malaria) through the BLAST program with the amino acid sequence of Staphylococcus aureus SMase (GenBank/EMBL/DDBJ accession no. X13404) as a query. One EST clone PF1350C (GenBank/EMBL/DDBJ accession no. N97823) and DNA contig fragment (GenBank/EMBL/DDBJ accession no. AC005505) in chromosome 12 of P. falciparum was found to encode a candidate gene for SMase. DNA sequence of the 1.7 kbp PF1350C clone matches the corresponding genomic sequence perfectly except for the poly(A) tail. However, comparison of the genome and PF1350C sequences suggested that the longest ORF encoded in PF1350C was not full-length, because there is a Met codon in the genomic sequence at 13 bp upstream of the 5′ terminus of PF1350C in the same frame of the ORF. To know the structure of the full-length ORF, we performed 5′-RACE experiment by using the 3D7 line of P. falciparum as RNA source. The nucleotide sequence analysis of the DNA fragment obtained from 5′-RACE reveals a ∼450 base noncoding sequence before the first Met codon of the putative ORF. Taken together, the 2.18 kbp cDNA sequence reconstituted from PF1350C and the 5′-RACE product was regarded as a cDNA encoding the full-length ORF (GenBank/EMBL/DDBJ accession no. AF323591). For simplicity, we refer to the locus in the genomic DNA encoding this cDNA as the PfNSM. Sequence analysis of PfNSM and its cDNA predicts that PfNSM is an intronless gene that encodes a protein of 393 amino acid residues (Fig. 1 A) with a molecular mass of 46,013 and has 80% A/T content typical of plasmodial genes (31).


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)

Primary structure of P. falciparum SM/LCPL-PLC. (A) Deduced amino acid sequence of P. falciparum SM/LCPL-PLC. (B) Hydropathy profile of the deduced PfNSM product obtained using Kite-Doolittle algorithm with a window size of nineteen (reference 35). (C) Intracellular distribution of GST-PfNSMase expressed in E. coli. After centrifugation at 105 g for 1 h, E. coli cell lysates transfected with pGEX-PfNSM (lanes 1 and 2) or pGEX-PfNSMd(2/68) (lanes 3 and 4), were analyzed by Western blotting using anti-PfNSMase antibodies. Lanes 1 and 3, supernatant; lanes 2 and 4, pellet fraction. 2 μg protein was loaded in each lane. (D) Multiple sequence alignment of the SMases from different species. Sequence alignments were performed with CLUSTAL W (reference 47) and refined with GeneAlign program (reference 48) using amino acid sequences in the regions where significant homology was observed through the dotplot analysis. The regions in each amino acid sequence used are PFAL (P. falciparum), 182–278; BCER (B. cereus), 129–225; SAUR (S. aureus), 134–230; LINT (L. interrogans), 179–275; HSAP1 (Homo sapiens), 81–181; and HSAP2 (Homo sapiens), 406–513. Amino acid residues conserved in all species and more than four species are highlighted in black and gray, respectively. (E) Phylogenetic tree of SMases. The multiple sequence alignment shown in D was used to make the phylogenetic tree through neighbor-joining algorithm with MEGA version 2.2 (reference 38). Scale bar indicates the number of substitutions per site. Bootstrap values are percentages of 1,000 replications and are shown at the nodes. GenBank/EMBL/DDBJ accession no. for each SMase is in parentheses. UPGMA also gave similar phylogenetic tree with similar topology.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Primary structure of P. falciparum SM/LCPL-PLC. (A) Deduced amino acid sequence of P. falciparum SM/LCPL-PLC. (B) Hydropathy profile of the deduced PfNSM product obtained using Kite-Doolittle algorithm with a window size of nineteen (reference 35). (C) Intracellular distribution of GST-PfNSMase expressed in E. coli. After centrifugation at 105 g for 1 h, E. coli cell lysates transfected with pGEX-PfNSM (lanes 1 and 2) or pGEX-PfNSMd(2/68) (lanes 3 and 4), were analyzed by Western blotting using anti-PfNSMase antibodies. Lanes 1 and 3, supernatant; lanes 2 and 4, pellet fraction. 2 μg protein was loaded in each lane. (D) Multiple sequence alignment of the SMases from different species. Sequence alignments were performed with CLUSTAL W (reference 47) and refined with GeneAlign program (reference 48) using amino acid sequences in the regions where significant homology was observed through the dotplot analysis. The regions in each amino acid sequence used are PFAL (P. falciparum), 182–278; BCER (B. cereus), 129–225; SAUR (S. aureus), 134–230; LINT (L. interrogans), 179–275; HSAP1 (Homo sapiens), 81–181; and HSAP2 (Homo sapiens), 406–513. Amino acid residues conserved in all species and more than four species are highlighted in black and gray, respectively. (E) Phylogenetic tree of SMases. The multiple sequence alignment shown in D was used to make the phylogenetic tree through neighbor-joining algorithm with MEGA version 2.2 (reference 38). Scale bar indicates the number of substitutions per site. Bootstrap values are percentages of 1,000 replications and are shown at the nodes. GenBank/EMBL/DDBJ accession no. for each SMase is in parentheses. UPGMA also gave similar phylogenetic tree with similar topology.
Mentions: To identify the gene for plasmodial SMase, in silico sequence analysis was performed by searching the NCBI Malaria Genetics and Genomics databases (http://www.ncbi.nlm.nih.gov/Malaria) through the BLAST program with the amino acid sequence of Staphylococcus aureus SMase (GenBank/EMBL/DDBJ accession no. X13404) as a query. One EST clone PF1350C (GenBank/EMBL/DDBJ accession no. N97823) and DNA contig fragment (GenBank/EMBL/DDBJ accession no. AC005505) in chromosome 12 of P. falciparum was found to encode a candidate gene for SMase. DNA sequence of the 1.7 kbp PF1350C clone matches the corresponding genomic sequence perfectly except for the poly(A) tail. However, comparison of the genome and PF1350C sequences suggested that the longest ORF encoded in PF1350C was not full-length, because there is a Met codon in the genomic sequence at 13 bp upstream of the 5′ terminus of PF1350C in the same frame of the ORF. To know the structure of the full-length ORF, we performed 5′-RACE experiment by using the 3D7 line of P. falciparum as RNA source. The nucleotide sequence analysis of the DNA fragment obtained from 5′-RACE reveals a ∼450 base noncoding sequence before the first Met codon of the putative ORF. Taken together, the 2.18 kbp cDNA sequence reconstituted from PF1350C and the 5′-RACE product was regarded as a cDNA encoding the full-length ORF (GenBank/EMBL/DDBJ accession no. AF323591). For simplicity, we refer to the locus in the genomic DNA encoding this cDNA as the PfNSM. Sequence analysis of PfNSM and its cDNA predicts that PfNSM is an intronless gene that encodes a protein of 393 amino acid residues (Fig. 1 A) with a molecular mass of 46,013 and has 80% A/T content typical of plasmodial genes (31).

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