Mitochondrial Tryparedoxins Figure 1. Gene-targeted deletion of LiTXN2. A. Map of the LiTXN2 locus i) before and after LiTXN2 replacement by the ii) HYG and the iii) PHLEO constructs. Homologous recombination occurred via the 59 and the 39 flanking regions of the LiTXN2 gene, cloned upstream and downstream of the selectable marker genes. Also depicted are the sizes of the EcoRI (E) fragments and the 39-flanking region of LiTXN2 (39UTR, dashed line) used as probe in the Southern blot analysis. B. Southern blot analysis of EcoRI-digested genomic DNA of wild type parasites and homozygous Dtxn2::HYG/ Dtxn2::PHLEO (txn22) mutants, hybridized with the LiTXN2 ORF and corresponding 39UTR. In txn22 mutants no band was detected when the LiTXN2 ORF was used as probe. In these parasites the 39UTR probe labelled two bands of 1.6 kb and 4.8 kb, corresponding to the HYG and the PHLEO cassettes, respectively. C. Western blot analysis of 25 mg of total protein extracts from wild type and txn22 promastigotes incubated with the anti- LiTXN2 antibody. Upon stripping, the membrane was incubated with the anti-gp63 antibody, as control for loading. D. Analysis of wild type and txn22 promastigotes by indirect immunofluorescence. Parasites were fixed, permeabilized and incubated with the anti-LiTXN2 antibody (green labeling) and with DAPI (blue labeling). Arrows point to DAPI-stained mitochondrial DNA (kDNA). doi:10.1371/journal.pone.0012607.g001 to display activity (LiTXN1, LiTXN2, TbTXN1 and TcTXN1) [3,10,11], as well as to its counterparts in T. brucei and T. cruzi (TbTXN2 and TcTXN2, respectively). An overview of this alignment reveals three major alterations in the amino acidic sequence of LiTXN3 relative to formerly characterized TXNs. First, at position 74 (LiTXN3 numbering), where classical enzymes harbor a Glu or an Asp, LiTXN3 contains a basic Arg residue. An acidic residue at this position was described to be important for TXN interaction with trypanothione [21], as well as with 2-Cys PRXs [22]. Accordingly, when the negative charge at position 74 was reversed in CfTXN2 and TbTXN1, the specific activities of these proteins dropped to 17% and 41% of the wild-type levels, respectively [21,22]. Second, position 40 in LiTXN3 is occupied by an Arg residue, replacing the Ser present in other TXNs. This Ser is in the vicinity of an acidic area proposed to be important for the contact between TXN and 2-Cys PRXs [22]. Finally, LiTXN3 has a Lys at position 112, instead of a Glu residue, reported to be involved in interaction of TXNs with nsGPXs [23], the second family of TXN-dependent peroxidases. Of notice, with the exception of Arg 40, the predicted TXN2 sequences of trypanosomes also present alterations in the positions notified above for LiTXN3 (Figure 4). In addition to the modifications referred to above, there is one sequence feature that readily distinguishes LiTXN3 from all TXN enzymes characterized to date, which is the presence of a C- terminus extension enriched in hydrophobic amino acids. Based on bioinformatics analysis (using TopPred and TMpred) this hydrophobic C-tail is predicted to specify a transmembrane domain. Again, such feature is maintained in the TXN2 enzymes of Trypanosoma (Figure 4). LiTXN3 retains oxidoreductase but not TXN activity To investigate whether LiTXN3 displays TXN activity, the enzyme was expressed in bacteria and purified as an N-terminal His-tagged recombinant protein, however most of it aggregated as inclusion bodies and the yield of soluble enzyme was very poor (data not shown). To overcome this limitation and to maximize the chances of obtaining correctly folded and active LiTXN3, a truncated version of the protein lacking the C-terminal hydro- phobic sequence (6HisLiDTXN3) was produced. Unless otherwise indicated all enzymatic assays were performed with the truncated enzyme, using recombinant LiTXN2 [10] as a positive control for TXN activity. PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12607