Copper is an essential component of cuproproteins but can be toxic to cells, therefore copper metabolism is very carefully regulated within cells. To gain insight into trypanosome copper metabolism, Trypanosoma spp. genomic databases were screened for the presence of copper-containing and -transporting proteins. Among other genes encoding copper-binding proteins, a copper-transporting P-type ATPase (CuATPase) gene was identified. Sequence and phylogenetic analyses suggest that the gene codes for a Cu+ transporter belonging to the P1B-1 ATPase subfamily that has an N-terminal domain with copper binding motifs. The N-terminal cytosolic domains of the proteins from Trypanosoma congolense and Trypanosoma brucei brucei were recombinantly expressed in Escherichia coli as maltose binding protein (MBP) fusion proteins. These N-terminal domains bound copper in vitro and within E. coli cells, more than the control MBP fusion partner alone. The copper binding properties of the recombinant proteins were further confirmed when they inhibited copper catalysed ascorbate oxidation. Native CuATPases were detected in a western blot of lysates of T. congolense IL3000 and T. b. brucei ILTat1.1 bloodstream form parasites using affinity purified IgY antibodies against N-terminal domain peptides. The CuATPase was also detected by immunofluorescence in T. b. brucei bloodstream form parasites where it was associated with subcellular vesicles. In conclusion, Trypanosoma species express a copper-transporting P1B-1-type ATPase and together with other copper-binding proteins identified in the genomes of kinetoplastid parasites may constitute potential targets for anti-trypanosomal drug discovery.
Keywords: Animal trypanosomiasis; Copper-binding; Copper-transporting P-type ATPase; Trypanosoma.
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