Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF

Macrophage migration inhibitory factor (MIF) displays both cytokine and enz yme activities, but its molecular mode of action is still unclear. MIF cont ains three cysteine residues and we showed recently that the conserved Cys5 7-AlaLeu-Cys60 (CALC) motif is critical for the oxidoreductase and macropha ge-activating activities of MIE Here we probed further the role of this cat alytic centre by expression, purification, and characterization of the cyst eine-->serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MI F and of mutants Ala58Glyl Leu59Pro and Ala58Gly/Leu59His, containing a thi oredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gl y/Leu59His were only soluble in organic solvent or 6 M GdmHCl when reconsti tuted at concentrations above 1 mu g . mL(-1). This made it necessary to de vise new purification methods. By contrast, mutant Cys81Ser was soluble. Ef fects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stabil ity was examined by denaturant-induced unfolding. The mutants, except for m utant CysX1Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disul phide bond formation, yet had decreased stability. Analysis in the oxidored uctase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIE Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activ ity but was less active than wtMIF; PDI-like MIF was enzymatically inactive . However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the c ytokine activity of MIF may not be tightly regulated by redox effects or th at a distinguishable receptor mechanism exists. This study provides evidenc e for a role of the CALC motif in the oxidoreductase and cytokine activitie s of MIF, and suggests that Cys81 could mediate conformational effects. Ava ilability and characterization of the mutants should greatly aid in the fur ther elucidation of the mechanism of action of the unusual cytokine MIF.