Cloning and characterization of Plasmodium vivax thioredoxin peroxidase-1

Abstract

Reactive oxygen species produced from hemoglobin digestion and the host immune system could have adverse effects on malaria parasites. To protect themselves, malaria parasites are highly dependent on the antioxidant enzymes, including superoxide dismutases and thioredoxin-dependent peroxidases. To date, several thioredoxin peroxidases (TPx) have been characterized in Plasmodium falciparum, but the TPx in Plasmodium vivax has not yet been characterized. The complete sequence of gene coding for thioredoxin peroxidase-1 of P. vivax (PvTPx-1) was amplified by PCR and cloned. Using the recombinant PvTPx-1 (rPvTPx-1), polyclonal antibody was produced in mice for immunolocalization of the enzyme in the parasite. The antioxidant activity of rPvTPx-1 was evaluated by mixed-function oxidation assay. PvTPx-1 has two conserved cysteine residues in the amino acid sequence at the positions 50 and 170 which formed a dimer under a non-reducing condition. Using a thiol mixed-function oxidation assay, the antioxidant activity of rPvTPx-1 was revealed. Indirect immunofluorescence microscopy with the specific antibody indicated that PvTPx-1 was expressed in the cytoplasm of the erythrocytic stage of the parasite in a dots-like pattern. The results suggest that P. vivax uses TPx-1 to reduce and detoxify hydrogen peroxides in order to maintain their redox homeostasis and proliferation in the host body.

Cloning and Characterization of a 2-Cys Peroxiredoxin from <i>Babesia gibsoni</i>

“…3B, BgTPx-1 was observed around the nucleus of the parasite. This cytoplasmic expression pattern was also shown in typical 2-Cys peroxiredoxins of P. falciparum (PfTPx-1) [31], P. vivax (PvTPx-1) [11] and B. bovis (BbTPx-1) [27]. Recently, our group showed that BbTpx-1 gene disruption does not affect in vitro intraerythrocytic growth of B. bovis [2], as previously reported for the gene disrupted in P. berghei [30], indicating that the TPx-1 gene is not essential for the erythrocytic stage of Babesia parasites.…”

“…Since 2-Cys Prxs use electrons provided by the small protein thioredoxin, these enzymes are also called thioredoxin peroxidases (TPx) [15, 19]. In recent years, several Prxs of malaria parasites have been characterized, and the structural and functional properties of the enzymes have been determined as key factors for development of new drugs [3, 11, 13, 16, 21]. Recently, a Prx from the bovine Babesia parasite B. bovis (BbTPx-1) was identified, and its antioxidant activity was demonstrated [27].…”

First Report of a Peroxiredoxin Homologue in Jellyfish: Molecular Cloning, Expression and Functional Characterization of CcPrx4 from Cyanea capillata

“…Therefore, it is thought to be the principal enzyme for the removal of excessive H 2 O 2 and to protect living organisms from such oxidative damages [51]. Moreover, peroxiredoxin also plays a key role in tumor suppression, immune response, signal transduction, as well as maintaining redox homeostasis [52,53,54,55,56,57,58]. Peroxiredoxins do not contain tightly bound metal ions, like other well-known antioxidant enzymes, but they contain highly conserved redox-active cysteine, which is involved in the catalytic mechanism [59].…”

Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate Plasmodium falciparum from P. vivax and P. knowlesi

“…Recombinant proteins were produced as described previously [12, 13]. Briefly, the PCR products were digested with restriction enzymes (Supplementary Table S1) and then inserted into pET-28a expression vector (EMD Biosciences, San Diego, CA) by overnight incubation with ligation mixture (Takara, Otsu, Japan) at 15°C.…”