The cytokine mif has enzymatic redox activity: Proof of role for cysteines

We recently re-discovered the classical T cell cytokine migration inhibitory factor (MIF)as a hormone and cytokine that plays a pivotal role in the regulation of host stress and infection. We demonstrated that MIF is a mediator of bacterial and allergic diseases. Of note, we found MIF to be the first counterregulator of the immuno-suppressive activities of glucocortieoids (Nature 365. 1993; Nature 377, 1995). Two Cys residues within the conserved Cys-x-x-Cys motif of MIF have been suggested to form a disulfide bond. We tested this hypothesis by structure activity studies using Cys->Ser mutants, alkylation. and partial sequence peptides. We generated the Cys mutants of huMIF by site-directed mutagenesis and bacterial expression. CD analysis and unfolding studies of wtMIF in comparison with the mutants and alkylated wtMIF revealed a marked stabilizing effect of Cys57 and Cys60 (Ac[GdnHCI]0. 5 = 0. 48 M; AT = 5. 5C). To test the role of the Cys motif for MIF function, we employed the mutants in a eytokine assay and found them to be significantly less active than wtMIF (20% and 55%, resp. ). Importantly, we found that MIF has Cys-mediated redox activity in the insulin reduction and HED transhydrogenase assays. These data were confirmed by the use of MIF peptides spanning the putative redox center, suggesting that both the enzymatic redox and cytokine-like activities of MIF are at least in part Cys-mediated. These data give important clues about the molecular mechanism of MIF function in inflammation and infectious diseases.