Molecular characterization of a coccidian parasite cGMP dependent protein kinase.
The cGMP-dependent protein kinase (PKG) of Eimeria tenella and Toxoplasma gondii is the target of a novel coccidiostat that is effective against coccidiosis and toxoplasmosis in animal models. Preparations of native PKG enzyme from Toxoplasma and Eimeria contain a membrane-associated polypeptide (isoform-I) of about 110 kDa and a slightly smaller soluble polypeptide (isoform-II). Expression of T. gondii and E. tenella PKG cDNA clones in Toxoplasma yield similarly sized recombinant polypeptides, which co-migrate on SDS-polyacrylamide gels with the corresponding native isoforms. Results of targeted mutagenesis of potential translational initiation sites suggest that parasite isoform-II is a product of alternative translational initiation from an internal initiator methionine codon. Exclusive expression of isoform-II or isoform-I can be achieved by preventing initiation at the respective primary or secondary sites. Immunofluorescence analysis indicates that recombinant isoform-I localizes primarily to the parasite plasma membrane, while isoform-II remains cytosolic. Mutagenesis and metabolic labeling studies reveal that the observed membrane-association of full-length recombinant PKG is mediated by N-terminal myristoylation and palmitoylation at amino acids G2 and C4. We also confirm the functional significance of a putative third PKG allosteric site, common to apicomplexan PKGs but absent from vertebrate or insect PKGs. In assays with transiently transfected parasites, constructs harboring a mutation at this site express markedly lower levels of cGMP-dependent PKG activity, while a triple mutant bearing mutations in all three sites reduces kinase activity to background levels.
Donald
Robert G K
RG
Department of Human and Animal Infectious Disease Research, Merck Research Laboratories, Merck and Co Inc, PO Box 2000, R80Y-260, Rahway, NJ 07065-09000, USA. robert_donald@merck.com
Liberator
Paul A
PA
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015153:Blotting, Western; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016786:Eimeria tenella / Q000201:enzymology* / Q000235:genetics; D005347:Fibroblasts; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics
D009842:Oligopeptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; C037642:kemptide; D000255:Adenosine Triphosphate; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP