Characterization of Neospora caninum macrophage migration inhibitory factor

Abstract

The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.

Keywords: CD74; MIF; Macrophage migration inhibitory factor; NcMIF; Neospora caninum; Tautomerase.

Fig. 1

Sequence alignment (A) and phylohenetic analysis (B) of representative MIF orthologues from Apicomplexan parasites and mammals. The sequences include those encoding the Homo sapiens MIF (NP_002406), Mus musculus MIF (NP_034928), P. falciparum MIF (AAN36370), E. tenella MIF (ABC73371), N. caninum MIF (CBZ51173), T. gondii MIF (ABC69140), and L. major MIF (XP_001685935). The six amino acid residues in mammalian MIF previously predicted to interact with the tautomerase substrate are marked by asterisks, the chemokine motif of CXXC present in mammalian MIF is marked by filled triangles and the adjacent Cys2 and Cys3 present in P. falciparum MIF are labeled with open triangles.

Fig. 2

Purification of rNcMIF and rNcMIFm by size exclusion chromatography on HPLC and analysis by SDS-PAGE. The chromatograph for both purified rNcMIF (first peak in solid line) and rNcMIFm (second peak in broken line) obtained at the same conditions is shown. The coomassie blue stained SDS-PAGE gel for the analysis of purified rNcMIFs is shown in the insert. M, molecular weight ladder; Lane 1, purified rNcMIF; Lane 2, purified rNcMIFm.

Fig. 3

Chemical crosslinking of rNcMIF (A), rNcMIFm, and rNcMIFhis (C) and analysis by silver-stained 4–12% NuPAGE. Recombinant protein (2ug) was chemically crosslinked, precipitated by the TCA method prior to separation on NuPAGE. Lane 1, protein fixed by NaBH4 without 1% glutaraldehyde; Lane 2, protein treated with 1% glutaraldehyde without NaBH4 fixation; Lane 3, protein treated with 1% glutaraldehyde followed by NaBH4 fixation; and Lane 4, protein without any treatment. M, monomer; D, dimer; T, trimer. Molecaulr weight ladder is shown to the left. The results represent 3 experiment.

Fig. 4

Analysis of the presence and relative levels of NcMIF in whole tachyzoite lysate (NcAG) and secretory products (ESAg) prepared from the NC1 and Ncts-8 isolates. ESAg was prepared from purified tachyzoites and concentrated 20-fold using the TCA method and concentrated ESAg in the amount of 2 x 10⁷ tachyzoites equivalent was subjected to analysis by Western blotting using a sheep anti-rNcMIF serum at 1:500 dilution. A, Western blotting of N. caninum ESAg; and B, integrated optical density analysis of the NcMIF band (Lanes 4 through 7) in NcESAg from NC1 and Ncts8 in the absence or presence of calcium ionosphere. Lane 1, rNcMIFhis (25 ng/lane); Lane 2, rNcMIF (25 ng/lane); Lane 3, rNcMIFm (25 ng/lane); Lane 4, NC1 EsAg at 37°C; Lane 5, Ts-8 EsAg at 32°C; Lane 6, NC1 EsAg in the presence of 5 μM calcium ionophore at 37°C; Lane 7, Ts-8 EsAg in the presence of 5 μM calcium ionophore at 32°C; Lane 8, Nc1 EsAg at 4°C; Lane 9, Ts-8 EsAg at 4°C; and Lane 10, NcAg of Nc1 (5 μg/lane). C, Western blotting of NcAg and Ncts-8Ag; and D, integrated optical density analysis of the NcMIF band in NcAg from NC1 and Ncts8 isolates. Lane 1, NcAg (10 ug/ml); Lane 2, Ncts-8Ag (10 ugm/ml). The level of NcMIF was assessed by integrated optical density using an imaging system and data represent the mean ± standard error of 3 independent experiments.

Fig. 5

Immunolocalization of NcMIF in Neospora tachyzoites by indirect fluorescence assay (IFA) (A) and immunoelectron microscopy (B). A, IFA using a rabbit anti-NcMIF serum as first antibody and goat anti-rabbit IgG-FITC as secondary reagent. A: Localization of NcMIF by IFA. a, PBS alone without application of antibodies; b, second antibody (1:100) alone; c, pre-immune rabbit serrum (1:100 dilution); d, rabbit antisera to a non-N. caninum recombinant protein (beta-giardin of Giardia lamblia) (1:100 dilution) as an irrelevant antibody control; e, rabbit antisera to recombinent Neospora profilin (1:100 dilution) as a positive control; f, rabbit antisera against rNcMIF (1:100). B, localization of NcMIF by immunoelectron microscopy using sheep antiNcMIF as first antibody and rabbit anti-sheep IgG-gold as secondary antibody. R, rhoptry; M, microneme; A, apical; N, nucleus; DG, dense granule. a, 10,000x; b-f, 50,000x.

Fig. 6

Dopachrome tautomerase activity of mMIF (A) and rNcMIFs (B). mMIF and rNcMIF tautomerase activities were assayed using the dopachrome substrate by a spectrophotometer using a wavelength of 574 nm. The reaction was monitored at a 30s interval for a total of 20 min. mMIF was used as a positive control in the dopachrome tautomerase activity at a final concentration of 0.5ug/ml (A). rNcMIF and rNcMIFm at a final concentration of 10 μg/ml were assayed for the dopachrome tautomerase activity (B). An equivalent volume of PBS was used in place of MIF to determine the spontaneous substrate decay. Data represent 3 independent experiments.

Fig. 7

Comparison of binding by rNcMIF, and rNcMIFm, rhMIF to the human MIF receptor ectodomain (sCD74) in an in vitro capture assay using biotinylated-rhMIF as competitor. The data represent three independent experiments. Each data point represents mean ± SEM.