title: Role of cysteine-58 and cysteine-95 residues in the thiol di-sulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti.
abstract: 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.
authors.affiliation: Division of Microbiology and Immunology, Vector Control Research Centre, Indian Council of Medical Research, Indira Nagar, Medical Complex, Pondicherry 605006, India.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Chauhan
authors.affiliation: Regional Medical Research Centre, Indian Council of Medical Research, Nehru Nagar, Belagavi, Karnataka 590 010, India. Electronic address: slhoti@yahoo.com.
authors.forename: S L
authors.initials: SL
authors.lastname: Hoti
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000276:immunology* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism*; D014958:Wuchereria bancrofti / Q000201:enzymology* / Q000276:immunology*
chemical_list: D008263:Macrophage Migration-Inhibitory Factors; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D003545:Cysteine
keywords: CXXC motif; Filarial parasite; Homology modeling; Macrophage Migration Inhibitory Factor; Oxidoreductase activity; Site directed mutagenesis