Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl p eroxides is achieved by the NADPH-dependent trypanothione peroxidase system , whose components are trypanothione reductase (TRYR), trypanothione, trypa redoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the clon ing of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma b rucei brucei encoding a protein with two catalytic VCP motifs similar to th e cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2). TRYP2 shows 51% similarity to TRYP1, possesses a putative mitochondrial imp ort sequence at its N-terminus and has a variant IPC motif replacing the se cond VCP motif implicated in catalysis in other 2-Cys peroxiredoxins. TRYP1 and TRYP2 were expressed in Escherichia coli, and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, tryp anothione, TRYR and TRYX from T. brucei, similar to the C. fasciculata cyto plasmic system. Western blots showed that TRYX, TRYP1 and TRYP2 are express ed in both bloodstream and procyclic forms of the life cycle. To determine the precise localisation of TRYX, TRYP1 and TRYP2 in the parasite, polyclon al antibodies to purified recombinant TRYX and TRYP1 and monoclonal antibod y to TRYP2 were generated in mice. In-situ immunofluorescence and immunoele ctron microscopy revealed a colocalisation of TRYX and TRYP1 in the cytosol , whereas TRYP2 was principally localised in the mitochondrion. (C) 2001 El sevier Science B.V. All rights reserved.