Recombinant expression and initial characterisation of two Plasmodium falciparum copper binding proteins: Cox11 and Cox19.
Salman, Abdumalik Abdullahi.
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Emerging drug resistance hinders the efforts to control malaria and so novel antimalarial drugs are required. Copper is essential for the survival of plasmodial parasites but the proteins involved in copper homeostasis are not well characterised. This study looked at plasmodial copper homeostasis by identifying and partially characterising two P. falciparum copper metallochaperones, Cox11 and Cox19. The Basic Local Alignment Search Tool (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified Cox11 and Cox19 gene orthologues in nine Plasmodium spp. The plasmodial Cox11 amino acid sequence contained a single N-terminus membrane-spanning region and three conserved cysteine residues, two of which are in a CFCF motif. These features are found in mammalian and yeast Cox11 amino acid sequences. The plasmodial Cox19 amino acid sequence has a domain containing a twin Cx9C motif, and a conserved Tyr-Leu dipeptide between the pair of cysteine of one Cx9C motif, similar to the amino acid sequences of human and yeast Cox19. The cloned and expressed recombinant MBP-PfCox11Ct and MBP-PfCox19 fusion proteins resolved on SDS-PAGE gels as ~62 kDa and ~66 kDa proteins respectively. Polyclonal IgY antibodies raised in chickens against rMBP-PfCox11Ct and rMBP-PfCox19 detected the native murine parasite, P. berghei, proteins on a western blot. Both recombinant proteins bound copper in the form of the cuprous ion in vitro and in vivo using the: bicinchoninic acid release, ascorbic acid oxidation, atomic absorption spectroscopy, and differential scanning fluorimetry assays. Three P. falciparum Cox11 mutants (two single- and a double-mutant) were engineered with site-directed mutagenesis, where an alanine replaced the corresponding cysteine residue and the mutant proteins were expressed as MBP fusion proteins. The two P. falciparum Cox11 cysteines, Cys155 and Cys157, in a CFCF motif were shown to be essential for the binding of copper in several assays. P. falciparum Cox11 and Cox19 bind copper in vitro and in an in vivo environment. Both rMBP-PfCox11Ct and rMBP-PfCox19 bound copper in an in vivo environment, enabling the growth of E. coli host cells expressing the proteins in the presence of toxic concentrations of copper. The localisation of the plasmodial Cox11 and Cox19 proteins suggested by proteomic data to be mitochondrial requires experimental confirmation. This study provides the foundation for further experiments to study P. falciparum Cox11 and Cox19 biochemistry and the evaluation of the two proteins as possible drug targets.