Characterization of Plasmodium falciparum CDP-diacylglycerol synthase, a proteolytically cleaved enzyme

Cytidine diphosphate-diacylglycerol (CDP-DAC), an obligatory intermediate c ompound in the biosynthesis of the major anionic and zwitterionic phospholi pids, is synthesized by CDP-DAG synthase (CDS). The gene encoding CDS was i solated from the human malaria parasite Plasmodium falciparum, based on seq uence conservation to CDS from other organisms. The P. falciparum gene is l ocated as a single copy on chromosome 14. The open reading frame (ORF) of P fCDS gene encodes a putative protein of 667 amino acids and 78 kDa. Only th e C-terminal 422 amino acids share 40% homology with eukaryotic CDSs, The v ery long and non-conserved N-terminal region of 245 amino acids is hydrophi lic and contains asparagine-rich and repetitive sequences. Two mRNA of 3.5 and 4 kb were detected. Transcription is developmentally regulated during t he asexual intraerythrocytic cycle, being the weakest in the ring-stage. Pf CDS enzyme activities in infected erythrocytes correlates with the transcri ption pattern, consistent with an increased synthesis of phospholipids in t rophozoites and schizonts. Antisera raised against two synthetic peptides f rom the C-terminal region of PfCDS detected a single protein of 51 kDa in W estern blot analysis, specific for parasitized erythrocytes. A protein of 2 8 kDa was recognized by an antiserum against an N-terminal peptide, indicat ing that PfCDS is proteolytically processed. Expression of 51- and 28-kDa p roteins was developmentally regulated similar to regulation of the transcri pts and the enzyme activity. The conserved C-terminal region of PfCDS, clon ed into a eukaryote expression vector and transfected in COS-7 cells, showe d a two-fold increase CDP-DAG synthase activities, indicating that the isol ated gene most likely encoded the P. falciparum CDS enzyme. (C) 2000 Elsevi er Science B.V, All rights reserved.