The online version of this article (doi:10.1186/1475-2875-11-397) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
All authors contributed to planning the experimental strategy, analysing the data and writing the paper. DLC conducted the experiments with the guidance of JPDG. DLC conducted the immunofluorescence experiments with help from JMP. All authors read and approved the final manuscript.
Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated.
PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy.
Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein.
Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds.
Additional file 1: Important features of the PF14_0369 amino acid sequence. Important features of the PF14_0369 amino acid sequence include a predicted N-terminal signal peptide (underlined), three transmembrane domains (black boxes and numbered 1,2,3), an essential methionine residue M, 20 amino acids N-terminal of the first transmembrane domain, and the MX3M and GX3G motifs. Features thought to contribute to protein trafficking include a partial PEXEL motif (LAD) in the signal peptide and an enrichment of phenylalanine residues (F) in the third transmembrane domain. (PNG 16 KB)12936_2012_2556_MOESM1_ESM.png
Additional file 2: Expression and purification of recombinant MBP- Pf Ctr211Nt -S . Expression of MBP-Pf Ctr211Nt-S was targeted to the E. coli periplasm. Steps in the isolation of recombinant MBP-Pf Ctr211Nt-S were analysed on a 10% reducing SDS-PAGE. Lane 1 and 2, total E. coli lysate; lane 3, periplasmic proteins; lane 4 represents proteins that did not bind and lanes 5–10 show protein eluted off the amylose resin. Fermentas unstained protein marker standards are shown to the left of each image. (PNG 115 KB)12936_2012_2556_MOESM2_ESM.png
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- A Plasmodium falciparum copper-binding membrane protein with copper transport motifs
David L Choveaux
Jude M Przyborski
JP Dean Goldring
- BioMed Central
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