Biological activities and functional analysis of macrophage migration inhibitory factor in Oncomelania hupensis, the intermediate host of Schistosoma japonicum.

Schistosomiasis is a destructive parasitic zoonosis caused by agents of the genus Schistosoma, which afflicts more than 250 million people worldwide. The freshwater amphibious snail Oncomelania hupensis serves as the obligate intermediate host of Schistosoma japonicum. Macrophage migration inhibitory factor (MIF) has been demonstrated to be a pleiotropic immunoregulatory cytokine and a key signaling molecule involved in adaptive and innate immunity. In the present study, we obtained the full-length cDNA of OhMIF and analyzed the characteristics of the ORF and the peptide sequence in O. hupensis. Next we have successfully expressed and purified the recombinant OhMIF protein (rOhMIF) together with a site-directed mutant rOhMIFP2G, in which the N-terminal Proline (Pro2) was substituted by a Gly. Our results indicated that rOhMIF displayed the conserved D-dopachrome tautomerase activity which is dependent on Pro2, and this enzymatic activity can be significantly inhibited by the MIF antagonist ISO-1. Moreover, we also measured and compared the steady state kinetic values for D-dopachrome tautomerase activity of rOhMIF and rHsMIF, and the results showed that the reaction rate, catalytic efficiency and substrate affinity of rOhMIF are significantly lower than those of rHsMIF. Additionally, we also showed that rOhMIF had the oxidoreductase activity which can utilize DTT as reductant to reduce insulin. Furthermore, the results obtained from the in vitro injection assay demonstrated that rOhMIF and its mutant rOhMIFP2G can also induce the phosphorylation and activation of ERK1/2 pathway in O. hupensis circulating hemocytes, indicating that the tautomerase activity is not required for this biological function. These results are expected to produce a better understanding of the internal immune defense system in O. hupensis, and help to further explore the interaction between O. hupensis and its natural parasite S. japoniucm.