DISULFIDE ANALYSIS REVEALS A ROLE FOR MACROPHAGE-MIGRATION INHIBITORYFACTOR (MIF) AS THIOL-PROTEIN OXIDOREDUCTASE

The molecular mechanism of action of macrophage migration inhibitory f actor (MIF), a cytokine with a critical role in the immune and inflamm atory response, has not yet Seen identified. Here we report that MIF c an function as an enzyme exhibiting thiol-protein oxidoreductase activ ity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys --> Ser mutant s (C57S MIF, COS MIF, and C57S/C60S MIF) of human MIF ( wtMIF), and al leviated wtMIF, we show that this activity is mediated by the CALC reg ion and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfi de bridge. Using two common oxidoreductase assays, MIF was shown to en zymatically catalyze the reduction of insulin and 2-hydroxyethyldisulf ide (HED). Examination of wtMIF and the mutants by far-UV circular dic hroism spectroscopy (CD) together with denaturation studies showed tha t substituting or reducing the cysteine residues of CALC led to a redu ced conformational stability of MIF but did not significantly change i ts overall conformation. A functional role for the CALC region was rev ealed by subjecting the mutants and alkylated wtMIF to the enzymatic a ssays. Mutant C60S did not have any enzymatic activity while mutant C5 7S had a reduced activity. Thiol-modified wtMIF that was alkylated und er oxidizing conditions was found to have full enzymatic activity, whe reas alkylation of wtMIF under reducing conditions completely eliminat ed MIF-mediated redox activity. Importantly, further physiological rel evance of the disulfide motif was obtained by examining the mutants an d alkylated MIF in an immunological assay that involved the macrophage -activating properties of MIF. Ln this test, mutant C60S was essential ly inactive and mutant C57S was partly active, indicating together tha t at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60.Again, use of the alky lated MIF species confirmed the role of the cysteine motif for this MI F activity. Ln conclusion, our results argue (a) that MIF exhibits enz ymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine resid ues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism. (C) 1998 Academic Press.