Cloning of a Ca2+-ATPase gene of Plasmodium falciparum and comparison with vertebrate Ca2+-ATPases

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Abstract
A Ca2-ATPase gene was cloned from the genomic libraries of Plasmodium falciparum. From the deduced amino acid sequence of the gene, a 139 kDa protein with a total of 1228 amino acids was predicted. Sequence of a partial cDNA clone of the gene identified two introns near the 3-end at the regions identical to the regions assumed for the Ca2-ATPase gene of P. yoelii, a rodent malaria species. As compared with a variety of Ca2+-ATPases, the P. falciparum Ca2+-ATPase had the highest amino acid sequence homology (78%) to the P. yoelii Ca2+-ATPase, moderate homology (45-50%) to vertebrate sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs), and lowest homology (20%) to a plasma membrane Ca2+-ATPase. The P. falciparum protein conserved sequences and residues that are important for the function and/or structure of the organellar type Ca2+-ATPase, such as high affinity Ca2+-binding sites, fluorescein isothiocyanate (FITC)-binding regions, and the phosphorylation site, but the protein did not contain calmodulin-binding regions that occur in the plasma membrane type Ca2+-ATPase. Thus we concluded the cloned gene was the organellar type Ca2+-ATPase of P. falciparum. In a region between the phosphorylation site and FITC-binding region, the P. falciparum protein was about 200 residues longer than the rabbit SERCA and lacked a sequence that binds to phospholamban, a protein that regulates the activity of the rabbit SERCA. Comparison of the two malarial Ca2+-ATPases with site-directed mutants of the rabbit SERCA showed perfect conservation of residues, the mutations of which resulted in dysfunction of the enzyme, but low conservation of residues, the mutations of which affected enzyme activity little. Incorporation of the malarial sequences into wheel diagrams for analysis of 10 transmembrane domains of the organellar Ca2+-ATPase showed a cluster of conserved residues on the surface of each-helix. Our comparisons suggest that the malarial Ca2+-ATPases are useful for identifying residues or regions that are important for the function and/or structure of the organellar Ca2+-ATPase.