title: Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti.
abstract: Homologues of human macrophage migration inhibitory factor (hMIF) have been reported from vertebrates, invertebrates and prokaryotes, as well as plants. Filarial parasites produce two homologues of hMIF viz., MIF-1 and MIF-2, which play important role in the host immune modulation. Earlier, we have characterized MIF-1 (Wba-mif-1) from Wuchereria bancrofti, the major causal organism of human lymphatic filariasis. Here, we are reporting the molecular and biochemical characterization of MIF-2 from this parasite (Wba-mif-2). The complete Wba-mif-2 gene and its cDNA were amplified, cloned and sequenced. The size of Wba-mif-2 gene and cDNA were found to be 4.275 kb and 363 bp, respectively. The gene annotation revealed the presence of a large intron of 3.912 kb interspersed with two exons of 183 bp and 180 bp. The alignment of derived amino acid sequences of Wba-MIF-2 with Wba-MIF-1 showed 44% homology. The conserved CXXC oxido-reductase catalytic site present in Wba-mif-1 was found absent in Wba-mif-2 coding sequence. The amplified Wba-mif-2 cDNA was cloned into an expression vector pRSET-B and transformed into salt inducible Escherichia coli strain GJ1158. The expressed recombinant Wba-MIF-2 protein showed tautomerase activity against L-dopachrome methyl ester and the specific activity was determined to be 18.57±0.77 μmol/mg/min. Three known inhibitors of hMIF tautomerase activity significantly inhibited the tautomerase activity of recombinant Wba-MIF-2. Although the conserved CXXC oxido-reductase motif is absent in Wba-mif-2, the recombinant protein showed significant oxido-reductase activity in the insulin reduction assay, possibly because of the presence of vicinal cysteine residues.
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: Division of Microbiology and Immunology, Vector Control Research Centre, Indian Council of Medical Research, Indira Nagar, Medical Complex, Pondicherry 605006, India.
authors.forename: Rohit
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Regional Medical Research Centre (ICMR), Nehru Nagar, Belgaum, Karnataka, India. Electronic address: slhoti@yahoo.com.
authors.forename: S L
authors.initials: SL
authors.lastname: Hoti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004605:Elephantiasis, Filarial / Q000469:parasitology*; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D014958:Wuchereria bancrofti / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008263:Macrophage Migration-Inhibitory Factors
keywords: CXXC motif; Gene annotation; Macrophage migration inhibitory factor; Oxido-reductase activity; Wuchereria bancrofti