MOLECULAR AND ENZYMATIC CHARACTERIZATION OFSCHISTOSOMA MANSONITHIOREDOXIN PEROXIDASE

Abstract

The ability of Schistosoma mansoni to escape oxidative damage from immune system-generated reactive oxygen intermediates has been extensively documented. The limiting step in the parasite's detoxification process appears to be at the level of hydrogen peroxide neutralization. In the present study, the possible role of a novel class of antioxidant enzymes, thioredoxin peroxidase (TPx), in hydrogen peroxide neutralization by schistosomes was investigated. An expressed sequence tag was characterized from the Schistosoma Genome Initiative with high similarity to TPx from other organisms. The gene encodes a polypeptide containing 2 conserved active-site cysteines and flanking amino acids, and 60-70% identity with previously characterized TPx proteins. Recombinant schistosome TPx was enzymatically active and found to have thioredoxin-dependent hydrogen peroxide reducing activity of 4500 nmol hydrogen peroxide/min/mg protein. Native TPx activity was determined to be 48.1 nmol hydrogen peroxide/min/mg protein in adult worm homogenates compared with 46.9 for glutathione peroxidase. TPx activity was precipitated from adult worm homogenates with antibodies prepared against the recombinant protein. Western blotting with antibodies made against recombinant protein showed that TPx was expressed in both male and female adult worms. This is the first demonstration of a TPx activity in schistosomes and our results suggest that TPx plays a significant role in schistosome-host interactions.

Biochemical Characterization of 2-Cys Peroxiredoxins from Schistosoma mansoni

“…In our previous work we described a Prx (referred to as thioredoxin peroxidase) in schistosomes with biochemical properties similar to known Prx enzymes using thioredoxin as an electron donor (12). This enzyme was reasonably abundant in all stages tested and was found at elevated levels in the parasite egg and egg secretions (12,13).…”

“…Identification of Novel S. mansoni Peroxiredoxin Genes-To identify novel Prx genes, the GenBank TM EST data base was queried by tblastn with the open reading frame of Prx1 (12). A novel Prx sequence (Prx2) was identified in EST MAAD0093.RAR, which was obtained from Dr. Jucara Para, Belo Horizonte, Brazil and sequenced in its entirety.…”

“…Recombinant Protein Production and Purification-The entire open reading frame of each S. mansoni Prx was amplified by PCR (21) using oligonucleotide primers modified to contain BamHI and HindIII restriction sites to facilitate subcloning afterward into pRSETA expression plasmid (12) and TaqDNA polymerase (Promega, Madison, WI). Primers used were Prx2F (5Ј-CATGGATCCATGTTGTTACCAAATCAACC-3Ј), Prx2R (5Ј-CTGAAGCTTTCAGTGACAAGAGGAGAA-3Ј), Prx3F (5Ј-CTCAGGATCCGTTATGTCGTCATTATGCAGC-3Ј), and Prx3R (5Ј-CT-GCAAGCTTAATTAACCTTATGAAAATATTC-3Ј).…”

“…Open reading frames in pRSETA were verified by nucleotide sequencing. The induction of recombinant proteins in E. coli strain BLR(DE3)pLysS (Novagen, Madison, WI) was performed as described (12). In the case of Prx2 and Prx3 cells were sonicated in 5% monothioglycerol (3-mercapto-1,2-propanediol) and 10 mM imidazole in start buffer (0.07 M Na 2 HPO 4 , 0.01 M NaH 2 PO 4 , and 0.15 M NaCl, pH 7.4).…”

Redox Balance Mechanisms in Schistosoma mansoni Rely on Peroxiredoxins and Albumin and Implicate Peroxiredoxins as Novel Drug Targets

“…Monoclonal Antibody Production-Recombinant S. mansoni Prxs were produced as His 6 proteins and prepared as described (9,12). Monoclonal antibodies against recombinant S. mansoni Prxs were generated using standard protocols (23).…”