Characterization of protein Ser/Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex.
Two major protein phosphatase (PP) activities were purified from cytosolic extracts of the erythrocytic stage of the malaria parasite, Plasmodium falciparum. Both enzymes were specific for phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. The biochemical properties of the enzymes suggested their strong similarity with eukaryotic PP2A and PP2B protein phosphatases. Both enzymes preferentially dephosphorylated the alpha subunit of phosphorylase kinase, and were resistant to inhibitor-1. The PP2A-like enzyme required Mn2+ for activity and was inhibited by nanomolar concentrations of okadaic acid (OA). The cDNA sequence of the PP2A-like enzyme was identified through a match of its predicted amino acid sequence with the N-terminal sequence of the catalytic subunit. The PP2B-like (calcineurin) enzyme was stimulated by calmodulin and Ca2+ or Ni2+, but was resistant to OA. Malarial calcineurin was strongly and specifically inhibited by cyclosporin A (CsA) only in the presence of wild type P. falciparum cyclophilin but not a mutant cyclophilin. The inhibition was noncompetitive, and provides a potential explanation for the cyclosporin-sensitivity of the parasite. There was no significant quantitative difference in the total protein Ser/Thr phosphatase activity among the ring, trophozoite, and schizont stages.
Dobson
S
S
Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, Mobile 36688, USA.
May
T
T
Berriman
M
M
Del Vecchio
C
C
Fairlamb
A H
AH
Chakrabarti
D
D
Barik
S
S
D000595:Amino Acid Sequence; D000818:Animals; D065095:Calcineurin Inhibitors; D016572:Cyclosporine / Q000494:pharmacology*; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D065095:Calcineurin Inhibitors; D018076:DNA, Complementary; D015800:Protozoan Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; D016572:Cyclosporine; D010744:Phosphoric Monoester Hydrolases; D019696:Peptidylprolyl Isomerase