Role of cysteine-58 and cysteine-95 residues in the thiol di-sulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti.

Macrophage Migration Inhibitory Factor (MIF) is the first human cytokine reported and was thought to have a central role in the regulation of inflammatory responses. Homologs of this molecule have been reported in bacteria, invertebrates and plants. Apart from cytokine activity, it also has two catalytic activities viz., tautomerase and di-sulfide oxidoreductase, which appear to be involved in immunological functions. The CXXC catalytic site is responsible for di-sulfide oxidoreductase activity of MIF. We have recently reported thiol-disulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti (Wba-MIF-2), although it lacks the CXXC motif. We hypothesized that three conserved cysteine residues might be involved in the formation of di-sulfide oxidoreductase catalytic site. Homology modeling of Wba-MIF-2 showed that among the three cysteine residues, Cys58 and Cys95 residues came in close proximity (3.23Å) in the tertiary structure with pKa value 9, indicating that these residues might play a role in the di-sulfide oxidoreductase catalytic activity. We carried out site directed mutagenesis of these residues (Cys58Ser & Cys95Ser) and expressed mutant proteins in Escherichia coli. The mutant proteins did not show any oxidoreductase activity in the insulin reduction assay, thus indicating that these two cysteine residues are vital for the catalytic activity of Wba-MIF-2.

Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization.

BACKGROUND: The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. OBJECTIVE: The objective of this study was to investigate the cross-reactivity between a major glutathione-S transferase allergen of cockroach (Bla g 5) and the glutathione-S transferase of Wuchereria bancrofti (WbGST), a major lymphatic filarial pathogen of humans. METHODS: We compared the molecular and structural similarities between Bla g 5 and WbGST by in silico analysis and by linear epitope mapping. The levels of IgE, IgG, and IgG(4) antibodies were measured in filarial-infected and filarial-uninfected patients. Mice were infected with Heligmosomoides bakeri, and their skin was tested for cross-reactive allergic responses. RESULTS: These 2 proteins are 30% identical at the amino acid level with remarkable similarity in the N-terminal region and overall structural conservation based on predicted 3-dimensional models. Filarial infection was associated with IgE, IgG, and IgG(4) anti-Bla g 5 antibody production, with a significant correlation between antibodies (irrespective of isotype) to Bla g 5 and WbGST (P< .0003). Preincubation of sera from cockroach-allergic subjects with WbGST partially depleted (by 50%-70%) anti-Bla g 5 IgE, IgG, and IgG(4) antibodies. IgE epitope mapping of Bla g 5 revealed that 2 linear N-terminal epitopes are highly conserved in WbGST corresponding to Bla g 5 peptides partially involved in the inhibition of WbGST binding. Finally, mice infected with H bakeri developed anti-HbGST IgE and showed immediate-type skin test reactivity to Bla g 5. CONCLUSION: These data demonstrate that helminth glutathione-S transferase and the aeroallergen Bla g 5 share epitopes that can induce allergic cross-sensitization.

Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm.

A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine pi-class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.

Brugia malayi and Wuchereria bancrofti: gene comparison and recombinant expression of pi-class related glutathione S-transferases.

The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867.