Characterization of Plasmodium falciparum CDP-diacylglycerol synthase, a proteolytically cleaved enzyme.
Cytidine diphosphate-diacylglycerol (CDP-DAG), an obligatory intermediate compound in the biosynthesis of the major anionic and zwitterionic phospholipids, is synthesized by CDP-DAG synthase (CDS). The gene encoding CDS was isolated from the human malaria parasite Plasmodium falciparum, based on sequence conservation to CDS from other organisms. The P. falciparum gene is located as a single copy on chromosome 14. The open reading frame (ORF) of PfCDS gene encodes a putative protein of 667 amino acids and 78 kDa. Only the C-terminal 422 amino acids share 40% homology with eukaryotic CDSs. The very long and non-conserved N-terminal region of 245 amino acids is hydrophilic and contains asparagine-rich and repetitive sequences. Two mRNA of 3.5 and 4 kb were detected. Transcription is developmentally regulated during the asexual intraerythrocytic cycle, being the weakest in the ring-stage. PfCDS enzyme activities in infected erythrocytes correlates with the transcription pattern, consistent with an increased synthesis of phospholipids in trophozoites and schizonts. Antisera raised against two synthetic peptides from the C-terminal region of PfCDS detected a single protein of 51 kDa in Western blot analysis, specific for parasitized erythrocytes. A protein of 28 kDa was recognized by an antiserum against an N-terminal peptide, indicating that PfCDS is proteolytically processed. Expression of 51- and 28-kDa proteins was developmentally regulated similar to regulation of the transcripts and the enzyme activity. The conserved C-terminal region of PfCDS, cloned into a eukaryote expression vector and transfected in COS-7 cells, showed a two-fold increase CDP-DAG synthase activities, indicating that the isolated gene most likely encoded the P. falciparum CDS enzyme.
Martin
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D
UMR 5539, CNRS/Université Montpellier II, cc 107, Place E. Bataillon, 34095 Cedex 05, Montpellier, France.
Gannoun-Zaki
L
L
Bonnefoy
S
S
Eldin
P
P
Wengelnik
K
K
Vial
H
H
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D003001:Cloning, Molecular; D002798:Diacylglycerol Cholinephosphotransferase / Q000235:genetics* / Q000378:metabolism*; D004912:Erythrocytes / Q000469:parasitology / Q000502:physiology; D018628:Gene Dosage; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D010743:Phospholipids; D011994:Recombinant Proteins; D002798:Diacylglycerol Cholinephosphotransferase