Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase.

The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi (S. cervi). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi. Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi. The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~50kDa while the optimum pH and temperature were found to be 7.0 and 35°C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29±1.40kcal/mol. The Km and Vmax were determined to be 0.27±0.045mM; 30.30±1.30U/ml with NADPH and 0.59±0.060mM; 4.16±0.095U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1µM concentration. The inhibition of ScGR activity with NAI (IC50 0.71mM), NEM (IC50 0.50mM) and DEPC (IC50 0.27mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.

Identification of Setaria cervi heat shock protein 70 by mass spectrometry and its evaluation as diagnostic marker for lymphatic filariasis.

Using mass spectrometry and immunological approaches, a heat shock protein 70 associated with lymphatic filariasis (LF) has been identified from a bovine filarial parasite Setaria cervi. A heat shock protein was detected in different life stages of S. cervi when exposed to an elevated temperature of 44 degrees C. A combination of ATP-agarose column chromatography and electro-elution was used for its purification from adult female extract. On closer examination, it migrated as a single band at 68 kDa on 10% SDS-PAGE. Peptide sequences TTPSYVAFTDTER, DSGAIAGLNVLR, IINEPTAAAIAYGLDK, NALESYAFNMK and LLSDFFSGK were obtained through MALDI-LC/MS analysis. Confirmation of peptides was accomplished by MASCOT database which showed substantial sequence homology with S. digitata, Wuchereria bancrofti, and Caenorhabditis elegans. Multiple sequence alignment using Clustal W showed 98% identity with W. bancrofti and only 28% with human HSP70. Furthermore, the antigenicity plot has shown that the highly antigenic amino acid residues are constituted within the conserved peptides. These observations suggest a plausible biological connection of ScHSP70 with the disease and its strong immunogenic nature. ScHSP70 showed antigenic cross-reactivity with IgG class of antibody in different categories of filarial sera. However, when IgG subclasses were tested, IgG4 showed high specificity and sensitivity with asymptomatic microfilaraemic sera.