TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxides, is localized to the endoplasmic reticulum

Abstract

Until recently, it had been thought that trypanosomes lack glutathione peroxidase activity. Here we report the subcellular localization and biochemical properties of a second glutathione-dependent peroxidase from Trypanosoma cruzi (TcGPXII). TcGPXII is a single-copy gene which encodes a 16 kDa protein that appears to be specifically dependent on glutathione as the source of reducing equivalents. Recombinant TcGPXII was purified and shown to have peroxidase activity towards a narrow substrate range, restricted to hydroperoxides of fatty acids and phospholipids. Analysis of the pathway revealed that TcGPXII activity could be readily saturated by glutathione and that the peroxidase functioned by a Ping Pong mechanism. Enzyme reduction was shown to be the rate-limiting step in this pathway. Using immunofluorescence, TcGPXII was shown to co-localize with a homologue of immunoglobulin heavy-chain binding protein (BiP), a protein restricted to the endoplasmic reticulum and Golgi. As the smooth endoplasmic reticulum is the site of phospholipid and fatty acid biosynthesis, this suggests that TcGPXII may play a specific role in the T. cruzi oxidative defence system by protecting newly synthesized lipids from peroxidation.

Vitamin C biosynthesis in trypanosomes: A role for the glycosome

“…1). Previously, we have noted that other components of enzyme-mediated oxidative defense in trypanosomes share surprising similarity with their plant counterparts, including an ascorbate-dependent hemoperoxidase from T. cruzi (6,36). Genome analysis now strongly suggests that the presence of these and other ''plant-like'' enzymes, notably those involved in carbohydrate metabolism, represent evidence that trypanosomatids possessed a plastid at some point in their evolution (37).…”

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

“…Thus, GzmB both induces generation of superoxide anions and destroys parasite mechanisms to detoxify it. To assess the importance of crippling oxidative defense in parasite killing, we treated T. cruzi epimastigotes overexpressing SodB, APX, MPX, CPX or a glutathione peroxidase (GPX1 or GPX2) [17][18][19][20] with GzmB and GNLY and found that overexpression of any of these enzymes, but not their inactive mutants, significantly rescued T. cruzi from death both in vitro (Fig. 2g) and in vivo (Fig.…”

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

“…However, other peroxidases may also complement the tryparedoxin peroxidases system in trypanosomatids, with non-selenium GPX homologues recently being characterized in T. cruzi (18) and T. brucei (14). Interestingly, the T. cruzi GPX II was reported to be reduced directly by GSH and not by TryX or T[SH] 2 (20). Moreover, GPX II appeared to be specific for fatty acid and lipid hydroperoxides and to be localized to the ER.…”

“…Initial studies showed the complex in the anterior of the parasite, particularly around the nucleus and kinetoplast. This pattern is characteristic of proteins located to the endoplasmic reticulum (ER) (20,39). Double labeling of parasites with anti-C. fasciculata eEF1B antiserum and an antiserum raised against the T. brucei homologue of the ER-localized chaperone BiP (39) was therefore performed.…”

“…These are homologues of the selenoprotein glutathione peroxidase (GPX), with the active site selenocysteine being replaced by a cysteine residue. However, with the exception of the T. cruzi GPX II (20), these enzymes use TryX, rather than GSH as their preferred reductant (14,19).…”