Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica - PubMed (original) (raw)

Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica

Mary Sekiya et al. FEBS Lett. 2006.

Abstract

The parasitic helminth Fasciola hepatica secretes a 2-Cys peroxiredoxin (Prx) that may play important functions in host-parasite interaction. Recombinant peroxiredoxin (FhePrx) prevented metal-catalyzed oxidative nicking of plasmid DNA and detoxified hydrogen peroxide when coupled with Escherichia coli thioredoxin and thioredoxin reductase (k(cat)/K(m)=5.2 x 10(5)M(-1)s(-1)). Enzyme kinetic analysis revealed that the catalytic efficiency of FhePrx is similar to other 2-Cys peroxiredoxins; the enzyme displayed saturable enzyme Michaelis-Menten type kinetics with hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide, and is sensitive to concentrations of hydrogen peroxide above 0.5 mM. Like the 2-Cys peroxiredoxins from a related helminth, Schistosoma mansoni, steady-state kinetics indicate that FhePrx exhibits a saturable, single displacement-like reaction mechanism rather than non-saturable double displacement (ping-pong) enzyme substitution mechanism common to other peroxiredoxins. However, unlike the schistosome Prxs, FhePrx could not utilise reducing equivalents supplied by glutathione or glutathione reductase.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Production and purification of FhePrx under reducing (A) and non-reducing (B) conditions. (A) Total soluble bacterial extract from induced FhePrx culture (1); run-through from Ni-NTA resin column (2), and eluted recombinant FhePrx (3). (B) Recombinant FhePrx (1), recombinant FhePrx incubated with 0.5 mM DTT for 20 min before loading (2), recombinant FhePrx incubated with 0.5 mM DTT for 10 min before addition of 0.5 mM hydrogen peroxide for a further 10 min (3), recombinant FhePrx incubated with 0.5 mM hydrogen peroxide for 10 min before addition of 0.5 mM DTT for a further 10 min (4), and recombinant FhePrx incubated with 0.5 mM hydrogen peroxide for 20 min. MW, molecular weight markers.

Fig. 2

Fig. 2

Expression of FhePrx by parasitic stages in SDS–PAGE (A), immunoblotting (B) and RT-PCR (C). (A) Soluble extract of adult F. hepatica (1), adult F. hepatica ES products (2), and recombinant FhePrx (3). (B) Arrangement of lanes as for (A), anti-Prx sera was raised in sheep. (C) Expression of FhePrx and actin (control) in infective larvae (1) and adult F. hepatica (2) was analysed by RT-PCR with gene-specific primers.

Fig. 3

Fig. 3

Enzymatic activity of recombinant FhePrx, FhePrx protects plasmid DNA from oxidative nicking (A) and specific activity determined by NADPH consumption assay (B). (A) Molecular weight markers (1), plasmid pGEM 3zf only (2), pGEM 3zf with Fe3+ and DTT (3), pGEM 3zf, Fe3+, DTT and recombinant FhePrx [880 ng] (4), pGEM 3zf, Fe3+, Ascorbic acid and recombinant FhePrx [880 ng] (5), pGEM 3zf, Fe 3+, DTT, and recombinant F. hepatica cathepsin L [1.4 μg] (6). The nicked (n) and supercoiled (s.c) form of plasmid are indicated by arrows. (B) Specific activity of FhePrx determined by NADPH (0.25 mM) consumption. Hydrogen peroxide at a concentration of 0.25 mM was provided as substrate. FhePrx, recombinant FhePrx, TR, E. coli thioredoxin and Trx, E. coli thioredoxin reductase. Values are the means of three independent experiments.

Fig. 4

Fig. 4

Steady-state kinetics for FhePrx at varying concentrations of thioredoxin (A) and susceptibility of FhePrx to inactivation by H2O2 (B). (A) The enzyme-normalized reciprocal initial velocities are plotted against the reciprocal molarities of hydrogen peroxide. Thioredoxin concentrations are 5 μm (diamond), 7.5 μm (square), 10 μm (triangle) and 20 μm (X). (B) The time course of NADPH (0.25 mM) oxidation by recombinant FhePrx coupled with E. coli thioredoxin (10 μM) and thioredoxin reductase (2 U) is shown for H2O2 concentrations of 0 mM (plus sign), 0.5 mM (diamond), 1.0 mM (square), 2.5 mM (triangle), 5.0 mM (×), 7.5 mM (× with bar), and 10 mM (closed circle). (C) FhePrx is sensitive to over-oxidation. The reaction rates from (B) were plotted over time at the varying hydrogen peroxide concentrations, then slopes of this data were plotted against reciprocal substrate (hydrogen peroxide) concentrations.

References

    1. Barrett J. Peroxide metabolism in the liver fluke. J. Parasitol. 1980;66:697. - PubMed
    1. Callahan H.L., Crouch R.K., James E.R. Helminth anti-oxidant enzymes – a protective mechanism against host oxidants. Parasitol. Today. 1988;4:218–225. - PubMed
    1. Carmona C., Dowd A., Smith A., Dalton J.P. Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents the antibody-mediated eosinophil attachment to newly excysted juveniles. Mol. Biochem. Parasitol. 1993;62:9–18. - PubMed
    1. Chae H.Z., Chung S.J., Rhee S.G. Thioredoxin-dependent peroxide reductase from yeast. J. Biol. Chem. 1994;269:27670–27678. - PubMed
    1. Chang J.W., Lee S.H., Jeong J.Y., Chae H.Z., Kim Y.C., Park Z.Y., Yoo Y.J. Peroxiredoxin-I is an autoimmunogenic tumor antigen in non-small cell lung cancer. FEBS Lett. 2005;579:2873–2877. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources