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Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti

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Nikhil Chauhan, Rohit Sharma, S.L. Hoti ∗ Division of Microbiology and Immunology, Vector Control Research Centre, Indian Council of Medical Research, Indira Nagar, Medical Complex, Pondicherry 605006, India

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Article history: Received 14 April 2014 Received in revised form 24 August 2014 Accepted 12 October 2014 Available online xxx

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Keywords: Wuchereria bancrofti Macrophage migration inhibitory factor Oxido-reductase activity Gene annotation CXXC motif

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1. Introduction

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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 ␮mol/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 vicinal cysteine residues. © 2014 Published by Elsevier B.V.

Helminthic parasites are major cause of tropical diseases such as lymphatic filariasis, onchocerciasis, schistosomiasis and intesti25 nal infections. Generally, these infections are of long lasting in 26 nature and the parasites adopt various strategies for ensuring their 27 prolonged survival inside the host (Van et al., 2007). Prominent 28 29Q2 among such strategies is the immune evasion strategy (Maizels et al., 2001a,b). Nematode parasites evade the host immune sys30 tem by secreting immunomodulatory molecules such as proteases 31 and their inhibitors, antioxidant proteins, and orthologs of mam32 malian cytokines and their receptors (Bungiro and Cappello, 2004; 33 Harnett and Harnett, 2006). Prominent among these are trans34 forming growth factor ␤ (TGF ␤), abundant larval transcript (ALT), 35 venom allergen-like proteins (VALT) and homologues of human 36 24

∗ Corresponding author. Tel.: +918105536970; fax: +91 413 2272041. E-mail address: [email protected] (S.L. Hoti).

macrophage migration inhibitory factor (MIF) (Maizels et al., 2001a,b). Human MIF, a pleotropic cytokine reported over 50 years ago, is secreted by several types of cells such as macrophages, eosinophills, ˇ-cells of the islets of Langerhans, keratinocytes and T-cells (Prieto-Lafuente et al., 2009). It plays a very important role in immunoregulation mediated through Th1 type of immune response. It is also a ligand for CD74–CD44 receptor complex present at the surface of target cells. It induces the expression of these receptors leading to the modulation of cytokine expression and counter-regulation of the anti-inflammatory and immunosuppressive effects of glucocorticoid steroids (Flaster et al., 2007). It is also involved in the innate and adoptive immunity and stimulates the production of matrix metalloproteases, cyclooxygenase 2 and prostaglandin E2 (Leng and Bucala, 2006; Mitchell et al., 2002). Presence of MIF is reported to be essential for resistance against bacterial infections as MIF deficient mice succumbed to low dose of Salmonella typhimurium infection. MIF deficient mice show increased susceptibility to Leishmania major, Taenia crassiceps and Toxoplasma gondii (Koebernick et al., 2002; Satoskar et al., 2001;

http://dx.doi.org/10.1016/j.actatropica.2014.10.009 0001-706X/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Chauhan, N., et al., Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.009

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Flores et al., 2008). The hMIF, apart from having cytokine functions, also has two enzymatic activities viz., tautomerase and oxidoreductase activities (Rosengren et al., 1996; Bendrat et al., 1997). The hMIF has generated a lot of interest recently as it is found to be involved in inflammatory disorders such as rheumatoid arthritis, atherosclerosis, diabetes, sepsis and inflammatory bowel diseases (Lue et al., 2002; Morand et al., 2006). Several MIF homologues have also been reported from a range of organism’s viz., bacteria, protozoan, nematode parasites, mammals and plants (Vermeire et al., 2008). The functional role of MIF homologues in these organisms is not clearly understood although several reports indicated that they play important role in immunomodulation in the host, e.g., differential activation of macrophages (Martinez et al., 2009). Filarial parasites viz., Onchocerca volvulus, Brugia malayi and Loa loa are known to produce MIF homologues during different developmental stages. Two homologs of MIF (Bma-MIF-1 and Bma-MIF-2) have been reported from B. malayi, that share 26% homology (Zang et al., 2002). Similar to hMIF the Bma-MIF-1 also possesses two enzymatic activities viz., tautomerase and oxidoreductase, in which conserved proline residue at N-terminal end and CXXC motif are essential for the two activities, respectively (Pastrana et al., 1998). Presumably, BmaMIF-2 lacks the oxidoreductase activity unlike MIF-1, as it lacks the CXXC motif (Zang et al., 2002). Recently, we have described the characterization of Wba-mif-1 from Wuchereria bancrofti (Sharma et al., 2012), a major lymphatic filarial parasite responsible for 90% of the disease burden. In the present study we report the molecular and biochemical characterization of another homologue from W. bancrofti, Wba-mif-2. Interestingly, we found that Wba-MIF-2 possessed the oxidoreductase activity, although it lacked the common CXXC motif, possibly because of the presence of other vicinal cysteine residues. 2. Material and methods

(Merck, India) and 50 ng of template DNA. The PCR cycle parameters were as follows: initial denaturation at 94 ◦ C for 3 min, followed by 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 56 ◦ C for 30 s, and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min. The reaction mixture (50 ␮l) for the amplification of full gene sequence contained 10 pmol of each primer, 0.2 mM dNTPs (Finnzyme, Finland), 2× PrimeSTAR Max DNA polymerase master mix (Takara, Japan) and 50 ng of genomic DNA. The PCR cycle parameters were: initial denaturation at 95 ◦ C for 5 min, followed 35 cycles of denaturation at 98 ◦ C for 15 s, annealing at 54 ◦ C for 20 s, extension at 72 ◦ C for 2 min, with final extension at 72 ◦ C for 10 min. All reactions were performed in a thermal cycler (Eppendorf, Germany).

2.3. Gene annotation and sequence analysis The amplified cDNA of Wba-mif-2 was run on 1.5% agarose gel and a band of 363 bp was excised and purified using gel purification kit (Nucleospin extract II, Macherey-Nagel, Germany). The purified cDNA was sequenced using Big Dye terminator (ABI, USA) in 3100xl ABI sequencer. The identity was confirmed by BLASTN search in GenBank and submitted to GenBank database (accession no. KJ939449). In order to obtain full nucleotide sequence of Wba-mif-2 gene, the Wba-mif-2 cDNA sequence generated was searched against genomic contigs of W. bancrofti available in “Filarial worms Sequencing Project, Broad Institute of Harvard and MIT (http://www.broadinstitute.org/)”. The nucleotide sequence of Wba-mif-2 gene thus obtained was aligned with cDNA sequence and annotated using BioEdit sequence alignment editor (Hall, 1999). Phylogenetic analysis of deduced amino acid sequences of MIF from different organisms was performed by Neighbor joining method with Poisson model using MEGA5 software (http://www.megasoftware.net/) (Tamura et al., 2011), with 1000 boot straps.

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2.1. Sample collection and DNA extraction Blood smear collected on glass slides from W. bancrofti microfilaraemic patients were kindly provided by the Director, National Vector Borne Diseases Control Program (NVBDCP), New Delhi and ␭-ZAP cDNA library of infective stage L3 was provided by Dr. Steven Williams, Filarial Genome Project Resource Centre (Smith College, Northampton, MA, USA). Microfilariae (mf) were isolated from blood smears as reported earlier (Bisht et al., 2006). The genomic DNA was extracted from the isolated mf using QiAmp DNA micro kit (Qiagen, Germany) as per the instructions of the manufacturer and quantified using a spectrophotometer (GenQuant, Amersham Bioscience, USA). 2.2. Amplification of Wba-mif-2 cDNA and genomic DNA Specific primers were designed in Primer 3 software for Wba-mif-2 cDNA amplification, using Bma-mif-2 mRNA sequence (AY004865.1) retrieved from NCBI database. The primer sequences are: forward – 5 -AGATCTGCAGCTATGCCGCTGATAACGCTTG-3 and reverse – 5 -AAAAAGCTTTTATTTCTTCATAAGCTCT-3 , which included restriction sites (underlined) for Pst I and Hind III restriction enzymes, respectively. Primers used for Wba-mif-2 full gene amplification are: forward – 5 -ATGCCGCTGATAACGCTTGC- 3 and reverse – 5 – TTATTTCTTCATAAGCTCTTTCATTG – 3 . The ␭-Zap L3 stage specific cDNA library of W. bancrofti and mf genomic DNA was used as templates for the amplification of cDNA and complete gene respectively. The reaction mixture (50 ␮l) for the amplification of Wba-mif-2 cDNA contained 10 pmol of each primer, 0.2 mM dNTPs (Finnzyme, Finland), 10× PR assay buffer, 1.0 unit of PR polymerase

2.4. Cloning and expression of Wba-mif-2 The Wba-mif-2 cDNA was treated with restriction enzymes Pst I and Hind III (New England Biolabs, USA) in a double digestion reaction for 3 h at 37 ◦ C and ligated into expression vector pRSETB using Quick ligation kit (New England Biolabs, USA) as per the manufacturer’s instructions. The ligation was confirmed by PCR using specific primers for T7 promoter. The ligated products were transformed into E. coli DH5␣ competent cells and the transformants were selected on LB plates supplemented with 100 ␮g/ml ampicillin. The colonies appeared were cultured in 5 ml LB broth supplemented with 100 ␮g/ml ampicillin, at 37 ◦ C for 16 h with 200 rpm on a shaker incubator. The recombinant plasmids were isolated using Nucleospin plasmid kit (Macherey-Nagel, Germany) and checked for insert by restriction digestion with Pst I and Hind III enzymes. Construct from the positive clone was transformed into competent salt inducible E. coli strain GJ1158 for expression of Wba-MIF-2 recombinant protein. The recombinant clone was inoculated into 5 ml LBON medium supplemented with 100 ␮g/ml ampicillin. The culture was grown overnight and inoculated to 1 l of fresh LBON medium at 0.5% level. The culture was grown to O.D.600 of 0.6 and induced with 300 mM sterilized NaCl for 3 h for expression of the recombinant protein. The cell mass was harvested by centrifugation at 10,000 rpm for 10 min at 4 ◦ C. The pellet was re-suspended in lysis buffer containing 50 mM Tris, 50 mM of Na2 HPO4, 400 mM NaCl, 0.3 mg/ml lysozyme and sonicated with 50 pulsar duty cycle of amplitude 4.0 (Ultrasonic processor XL 2015, Heat System, New York, USA). The sonicated suspension was centrifuged at 12,000 rpm for 20 min at 4 ◦ C and the clear supernatant

Please cite this article in press as: Chauhan, N., et al., Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.009

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was checked for expression of Wba-MIF-2 recombinant protein on 12% SDS-PAGE (Laemmli, 1970).

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2.5. Purification of Wba-MIF-2 recombinant protein

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The clear supernatant containing recombinant fusion protein with Histidine tag was purified by Ni+ -charged column (GE Healthcare, Sweden) chromatography, using imidazole gradient (50–200 mM). The imidazole concentration of 200 mM was found optimum for elution of the protein and the purity of the eluted protein was checked by 12% SDS-PAGE. Among the eluted fractions, those containing low concentration of protein were concentrated by molecular weight cut-off filter of 10 kDa (Vivaspin 2, GE Healthcare, Sweden). The concentration of purified recombinant protein fractions was determined using Bradford reagent (Sigma, USA) at 595 nm.

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2.6. Western blotting

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The purified Wba-MIF-2 recombinant protein was resolved on a 12% SDS-PAGE gel and transferred on to 0.2 ␮m nitrocellulose membrane (Sigma, USA) using an electro blotting apparatus (BioRad, USA), at 20 V for 3 h. Transfer of protein on to the membrane was confirmed by staining with Ponceau S (MERCK, India). Mouse anti-His HRP tagged antibody (Sigma, USA) and TMB (Genei, India) were used for the detection of the recombinant protein.

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2.7. Enzyme assays

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The tautomerase activity of Wba-MIF-2 recombinant protein was determined by measuring the rate of formation of intermediate compound 5,6-dihydroxyindole-2-carboxylic acid (DHICA) from the l-dopachrome methyl ester at pH ∼7.4 and 308 nm (Aroca et al., 1990). Sodium periodate (20 mM) was used as an oxidant for l-dopachrome methyl ester (10 mM) at a ratio of 3:2. The tautomerase activity of the Wba-MIF-2 recombinant protein was also checked for spontaneous tautomerisation of ldopachrome methyl ester at 475 nm (Rosengren et al., 1997). Assuming a molar absorption coefficient (ε) of 3000 M−1 cm−1 at 475 nm and 10,960 M−1 cm−1 at 308 nm, the stoichiometric rate was calculated after adjusting the path length based on the final reaction volume in the microtitre wells. The specific activity of enzyme was measured according to the standard formula; Specific activity of enzyme = (Asample − Acontrol ) * reaction volume (ml) * 106 /ε * pathlength (cm) * t * protein sample (mg). The oxidoreductase activity was checked by insulin reduction assay (Kleemann et al., 1998). Dithiothretiol (DTT) was used as a reductant in the assay. The precipitation of insulin beta chain in the presence of DTT alone and with Wba-MIF-2 recombinant protein was measured at 650 nm. 2.8. Inhibition of tautomerase activity The effect of three inhibitors of hMIF viz., (S,R)-3-(4Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), 4iodo-6-phenypyrimidine (4-IPP) (Calbiochem, Germany), and Curcumin (Sigma, USA), against tautomerase activity of Wba-MIF-2 recombinant protein was tested as reported earlier (Winner et al., 2008). The recombinant protein (20 ␮g) was pre-incubated with each inhibitor (10 ␮M) in separate reactions for 30 min and then reacted with the substrate l-dopachrome methyl ester in the reaction mix. The tautomerization activity was recorded after 30 min using end point method under the UV (308 nm). The organic solvent dimethyl sulfoxide (DMSO) was used as control in the reaction.

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Fig. 1. Alignment of derived amino acid sequences of MIF homologues filarial parasites and human MIF. Wba-MIF-2: Wuchereria bancrofti (KJ939449), Bma-MIF-2: Brugia malayi (AAF91074.1), Ovo-MIF-2: Onchocerca volvulus (AF384028 1), WbaMIF-1: Wuchereria bancrofti (EJW88743.1), Hsa-MIF-1: Homo sapiens (CAG46452.1). Sequence shaded in black show complete identity; similar sequences are in gray, boxes with a sign of star represent conserved residues involved in tautomerase catalytic site, box with triangle show CXXC catalytic site, and box with circle show glycosylation motif residues and box with rectangle shows difference of five extra amino acids.

3. Results

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The full length of Wba-mif-2 cDNA was PCR amplified from L3 stage specific ␭-Zap cDNA library of W. bancrofti. The amplicon was sequenced and the nucleotide sequence of 363 bp obtained was aligned with Bma-mif-2 cDNA available in NCBI. The alignment showed a difference of four nucleotide bases of which three are non-synonymous (at positions 30th, 59th and 79th). The overall nucleotide sequence similarity of cDNA of Wba-mif-2 with Bmamif-2 and hmif was 99% and 36%, respectively. The comparison of Wba-mif-1and Wba-mif-2 cDNA sequences showed a difference of 15 bases. Search using the Wba-mif-2 cDNA sequence generated in the filarial genome database yielded a complete gene sequence of 4.275 kb (ADBV01000282.1). Annotation of the Wba-mif-2 gene thus revealed the presence of two exons of 183 and 180 bp, interspersed with a large intron of 3.912 kb. The intron was separated from exons with known splicing sites GT at 5 and AG at 3 . The Wba-mif-2 gene of 4.275 kb was amplified using genomic DNA of microfilariae as template in the PCR and its identity was confirmed by partial sequencing. Similarly, the amplified Bma-mif-2 gene was also found to be about 4.275 kb upon agarose gel electrophoresis and its identity was confirmed by sequencing. The deduced amino acid sequence of Wba-mif-2 cDNA contains the tautomerase motif with conserved proline residue at the position 2 and glycosylation motif at the positions 110–114, but the common CXXC motif for oxido-reductase activity was found absent (Fig. 1). 3.2. Phylogenetic analysis of MIF homologues from different organisms We assessed the phylogenetic relationship of Wba-mif-2, based on its derived amino acid sequence with that of other organisms MIF homologues available in the GenBank, using Neighborhood joining algorithm. The phylogenetic tree formed two branches; one comprising of MIF-1 of nematodes and MIF of bacteria and algae, and the other comprising MIF-2 of nematodes (Fig. 2). The hMIF clustered along with the MIF-1 type sequences. Thus, the analysis indicated that the two homologues of MIF formed two distinct branches and MIF-2 homologues are completely diverged from the hMIF when compared to MIF-1 and could have different

Please cite this article in press as: Chauhan, N., et al., Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.009

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Fig. 2. Evolutionary relationships of MIF homologs of different organisms based on deduced amino acid sequences. The phylogenetic tree was constructed using MEGA5. Accession numbers-Wba-MIF-2 (KJ939449), Bma-MIF-2 (AAF91074.1), Ovo-MIF-2 (AF384028 1), Asi-MIF-2 (ABM30179.1), Asu-MIF-2 (ERG80126.1), Llo-MIF-2 (XP 003139697.1), Cel-MIF-2 (NP 001256386.1), Sra-MIF-2 (ACH88456.1), Bma-MIF-1 (XP 001897136.1), Ovo-MIF-1 (AF384027 1), Wba-MIF-1 (EJW88743.1), Llo-MIF1 (XP 003143739.1), Tsp-MIF-1 (XP 003378412.1), Pma-MIF-1 (YP 397762.1), Cst-MIF-1 (YP 007165515.1), Oac-MIF-1 (YP 007088871.1), Tmo-MIF-1 (YP 007244276.1), Mme-MIF-1 (YP 004511513.1), Dal-MIF-1 (YP 002432275.1), Hsa-MIF-1 (CAG46452.1). Wba – Wuchereria bancrofti, Bma – Brugia malayi, Ovo – Onchocerca volvulus, Asi – Anisakis simplex, Asu – Ascaris summ, Llo – Loa loa, Cel – Caenorhabditis elegans, Sra – Strongyloides ratti, Tsp – Trichinella spiralis, Pma – Prochlorococcus marinus, Cst – Cyanobacterium stanieri, Oac – Oscillatoria acuminata, Tmo – Thioflavicoccus mobilis, Mme – Methylomonas methanica, Dal – Desulfatibacillum alkenivorans, Hsa – Homo sapiens.

3.4. Enzyme activity assays

Fig. 3. Expression of recombinant Wba-MIF-2 protein on SDS PAGE. Lane 1. Molecular weight markers (175k–7 kDa); Lane 2. Uninduced GJ1158 culture; Lane 3. NaCl induced (300 mM) culture; Lane 4. GJ1158 culture without transformation; Lane 5. Purified Wba-MIF-2 recombinant protein by Ni affinity column chromatography (IMAC); Lane 6. Recombinant Wba-MIF-2 protein detected by TMB substrate using mouse anti-histidine HRP tagged polyclonal antibodies in western blotting; Lane 7. Molecular weight markers (175 kDa–7 kDa).

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functional properties. There is no relationship between the phylogenetic clades of nematodes and the phylogeny of MIF.

3.3. Expression of Wba-MIF-2 recombinant protein Sequencing the recombinant plasmid pRSETB + Wba-mif-2 confirmed that Wba-mif-2 cDNA sequence is in correct open reading frame. The recombinant plasmid was transformed into the salt inducible host E. coli GJ1158. The Wba-MIF-2 fusion protein expressed was found to be 17 kDa on 12% SDS-PAGE (Fig. 3). It was purified using immobilized metal affinity chromatography (IMAC), using Ni+ charged sepharose column and 200 mM concentration of Imidazole. The purified Wba-MIF-2 recombinant protein showed reactivity against mouse anti-His antibody in western blot at the expected position of 17 kDa (Fig. 3).

The tautomerase and oxido-reductase activities of the recombinant Wba-MIF-2 were assessed against l-dopachrome methyl ester and insulin protein, respectively. Tautomerase activity was assessed by two methods; first method involved formation of intermediate DHICA (Fig. 4a) and second method involved spontaneous tautomerization of l-dopachrome methyl ester with the formation of intermediate DHI (Fig. 4b). The recombinant protein showed activity by both the methods, which confirmed its functionality. The tautomerase specific activity of recombinant Wba-MIF-2 protein was determined to be 18.57 ± 0.77 ␮mol/min/mg, which is almost similar to that of Ovo-MIF-2 (21.43 ± 1.47 ␮mol/min/mg) (Ajonina et al., 2013). The tautomerase activity of recombinant Wba-MIF2 was directly proportional to the concentration of Wba-MIF-2 recombinant protein (Fig. 5a and b). The recombinant Wba-MIF2 also showed significant oxidoreductase activity in the insulin reduction assay (Fig. 6a and b). 3.5. Inhibition of tautomerase activity The inhibition of tautomerase activity of the recombinant Wba-MIF-2 by three known inhibitors of hMIF was tested at a concentration of 10 ␮M. All inhibitors were found to inhibit the tautomerase activity of recombinant Wba-MIF-2 at varying levels such as 50.32% (ISO-1), 29.17% (4-IPP), 58.98% (Curcumin) (Fig. 7). Among the three inhibitors, Curcumin exhibited highest inhibition of tautomerase activity, followed by ISO-1. 4. Discussion Nematodes modulate the host immune response to their advantage, thereby ensuring their longer survival inside the host. In order to achieve this, they effectively employ the strategy of mimicking molecules of host immune system, a strategy similar to that known in viral infections. They secrete a variety of homologues of human immune components such as TGF-␤, MIF etc. (Maizels et al., 2001a,b). Immune modulating characteristics of these homologues have been reported in murine studies and in in

Please cite this article in press as: Chauhan, N., et al., Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.009

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Fig. 5. Tautomerase activity of different concentrations of recombinant Wba-MIF-2. (a) Tautomerase activity using three different concentration of recombinant WbaMIF-2 protein (10 ␮g, 20 ␮g, 30 ␮g) at 308 nm. (b) Tautomerase activity using three different concentrations of recombinant Wba-MIF-2 protein (10 ␮g, 20 ␮g, 30 ␮g) at 475 nm.

Fig. 4. Tautomerase activity of recombinant Wba-MIF-2 protein. (a) Tautomerase activity read at wavelength 308 nm: control reaction (dopachrome methyl ester + sodium periodate + elution buffer) and enzyme reaction (dopachrome methyl ester + sodium periodate + recombinant Wba-MIF-2 protein). (b) Tautomerase activity read at wavelength 475 nm: control reactions dopachrome methyl ester + sodium periodate + elution buffer) and enzyme reaction (dopachrome methyl ester + sodium periodate + recombinant Wba-MIF-2 protein).

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vitro cellular studies (Cho et al., 2011). Among parasitic diseases, lymphatic filariasis caused by W. bancrofti, B. malayi and Brugia timori is reported as a disease with most complex immunological reactions. Filarial parasites modulate the immune response by adopting several strategies like stage specific surface alteration, secretion of excretory/secretory (ES) products and by residing in specific location inside the host (Ottesen and Eric, 1980). Among filarial ES products, MIF homologues have been considered as a potential immunomodulators, and act by diverting or modifying important functions of the host derived MIF and hence could contribute significantly to the parasite’s ability to survive and replicate. Both host and pathogen derived MIF are thought to play important role in several infectious diseases such as tuberculosis, malaria, filariasis, schistosomiasis and taeniasis. A better understanding of the nature of these molecules and the way they alter the immune responsiveness will be critical for understanding

pathogenesis in humans and development of effective therapies (Kaech et al., 2007). MIF homologues of parasites possess enzymatic activities, which are reported to play a role in the immunomodulation, especially in the activation of Th2 immune response. These molecules have been reported to play important role in immune evasion by parasites, a strategy for their long term persistence inside the host. Recently, MIF-2 of Anisakis simplex has been reported in the suppression of intestinal colitis and in the up regulation of IL-10 production in the intestinal epithelial cell line, through the activation of Toll like receptor-2 (Cho et al., 2011). Human MIF and parasitic MIF homolog share the same receptor, CD74, for binding to the cell, which gives reminiscence of competition with human MIF. The detailed study on this receptor mediated signaling and its effect on the outcome of parasitic diseases will be of great interest. Hence, MIF and its homologs of parasites have generated a lot of interest, especially because of their role in the pathogenesis of major diseases such as malaria, leishmaniasis and other parasitic infections (Malu et al., 2011; Rosado and Rodriguez, 2011; Rodríguez et al., 2006; McDevitt et al., 2006; Kamir et al., 2008). Parasite MIF is also considered as a potential therapeutic molecule in the treatment of inflammatory diseases (Cho et al., 2011; Nishihira, 2012; Sanchez et al., 2010), as also a drug target in parasitic diseases. In the present study, we are reporting the molecular identification and biochemical characterization of Wba-mif-2 and inhibitors

Please cite this article in press as: Chauhan, N., et al., Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.009

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Fig. 7. Inhibition of tautomerase activity of recombinant Wba-MIF-2 by three known hMIF inhibitors ISO-1, 4-IPP, Curcumin with DMSO.

Fig. 6. Oxidoreductase activity of recombinant Wba-MIF-2. (a) Oxidoreducatse activity: blank (insulin), control (insulin + DTT), enzyme reaction (insulin + WbaMIF-2 recombinant protein + DTT). (b) Oxidoreductase activity using three different concentration of Wba-MIF-2 recombinant protein (5 ␮g + 10 ␮g + 20 ␮g) respectively + insulin, control (DDT + insulin), blank (insulin).

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of its tautomerase activity. Unlike in mammals where only one mif gene is present, two members of the gene family, mif-1 and mif-2, have been reported in most of the parasitic organisms (Vermeire et al., 2008), including the lymphatic filarial parasite B. malayi, whereas four types of mif have been reported from the free living nematode Caenorhabditis elegans (Ajonina et al., 2013). The Wbamif-2 gene was found to be considerably larger (4.275 kb) than Wba-mif-1 (1.1 kb) and the former comprises two exons while the latter contains three exons. The size of Wba-mif-2 is much larger when compared to the size of mif-2 of other nematodes and other organisms, which range from 1 to 1.5 kb. This is mainly due to the presence of a single large intron of 3.912 kb in it. The size of introns of mif in different organisms varies and exceptionally MIF of Strongyloides ratti does not have introns at all (Younis et al., 2012). It is now well known that introns play important role in regulation of gene expression (Duncker et al., 1997). The functional significance of exceptionally large intron of Wba-mif-2 needs to be investigated. The number of exons and introns of mif-2 with conserved catalytic residues shows its essential occurrence for certain functions and gradual evolution among different species. The catalytic activities of MIF, the structural similarities to microbial enzymes, and the

pattern of invariant residues to preserve this site suggest that the catalytic sites are important in the biology of MIF (Vermeire et al., 2008). Mammalian MIF is distinct from other cytokines in that it also catalyzes certain chemical reactions viz., tautomerase and oxidoreductase activities. All MIF homologues in parasites reported so far are also having enzymatic activities, but at lower level compared to human MIF. Also, MIF from different species are common in having tautomerase activity. The N-terminal catalytic Pro-2 residue is essential for tautomerase activity (Thiele and Bernhagen, 2005). This is because mutation of proline residue to glycine significantly reduced both the catalytic and cytokine stimulatory activities in both mammalian and nematode MIF (Zang et al., 2002; Swope et al., 1998). Conversely, S. ratti MIF did not exhibit in vitro tautomerase activity, although its exposure to the host immune system indicated it to activate monocytes/macrophage lineage with consequent release of IL-10 (Younis et al., 2012). Oxidoreductase activity is not the signature enzymatic activity of MIF homologues. Although the common CXXC motif is present in MIF-1 of parasitic nematodes, evidence for oxido-reductase activity is lacking. Prochlorococcus marinus MIF lacks CXXC motif and engineering the motif into it did not result in oxidoreductase activity (Wasiel et al., 2010). We have reported the presence of this motif as well as oxidoreductase activity in Wba-MIF-1 (Sharma et al., 2012). Surprisingly, although this motif was found absent in Wba-MIF-2, we found significant oxidoreductase activity. Motifs other than CXXC motif for oxidoreductase catalytic activity have been reported in MIF homologues and characterized among different organisms. Recently, Alam et al. (2011) reported that mutation of either of the vicinal Cys-3 or Cys-4 of Plasmodium falciparum MIF abolished the thioredoxin like activity, indicating their possible catalytic role. These two cysteine residues are conserved in MIF across species of malarial parasites, but not in filarial parasite MIF. However, the three conserved cysteine residues at 58th, 95th and 109th positions are presented in the primary structure, homology modeling of Wba-MIF-2 showed the presence of two cysteine ˚ in the tertiary structure of the residues in close proximity (3.273YA) protein. The close proximity is crucial parameter to have di-sulfide based oxido-reductase activity. The close association of these two cysteine residues in the tertiary structure might be responsible for the oxido-reductase activity of Wba-MIF-2 recombinant protein, similar to that reported in P. falciparum (Alam et al., 2011). We are currently investigating this through mutating these residues by site-directed mutagenesis. The enzymatic activities of MIF are known to be involved in the regulation of immune responses (Kleemann et al., 2000) and hence can form good targets for anti-parasitic and models for antiinflammatory drug development. The specific nature, in which

Please cite this article in press as: Chauhan, N., et al., Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.009

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the inhibitory compounds selectively bind to human MIF and not to several parasitic MIFs, shows substantial divergences in the tautomerase active site that could potentially be used in the development of new anti-parasite inhibitors (Cho et al., 2007). The inhibitors, ISO-1, 4-IPP and Curcumin significantly inhibited the tautomerase activity of Wba-MIF-2 recombinant protein. Variable tautomerase catalytic sites have been reported in MIF homologues to ISO-1 inhibitor viz., Ancylostoma sp. and T. gondii MIFs do not show any inhibitory effect of ISO-1 (Sommerville et al., 2013; Cho et al., 2007). Similar to other MIF homologues, Wba-MIF-2 could tautomerise specific substrates but the functional substrate for this enzymatic activity in the host and parasite is not known yet. Since the tautomerase activity of MIF is known to be involved in immunoregulation and possibly in pathogenesis, the inhibitors of Wba-MIF-2 identified in the present could be the leads for developing drugs for prevention of filarial pathogenesis. In summary, we account here the gene annotation, cloning, expression and biochemical characterization of a novel MIF-2 homologue of W. bancrofti, the major lymphatic filarial parasite, for the first time. We believe that the expression cloning and characterization of Wba-mif-1 and Wba-mif-2 will pave the way for generating important insights in to the immunomodulation and development of therapeutic tools against this parasite, which is a great important public health problem in the tropical countries. Acknowledgements

The authors are grateful to Dr. P. Jambulingam, Director, Vector control Research Centre, Pondicherry, for his support in the study. 452 Authors are also thankful to Dr. K. Athisaya Mary for her techni453 Q4 cal suggestions. The financial support of Indian Council of Medical 454 Research, New Delhi, for the study is gratefully acknowledged. 455 Q3 451

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Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti.

Homologues of human macrophage migration inhibitory factor (hMIF) have been reported from vertebrates, invertebrates and prokaryotes, as well as plant...
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