Cys74 and Cys163 are necessary for IL-18 to elicit IFN-γ production from peripheral blood lymphoid mononuclear cells

There are four cysteines (Cys74, Cys104, Cys112 and Cys163) in mature human IL-18 (hIL-18). These cysteines are highly conserved in IL-18s of 11 species cloned so far, suggesting that one or more of the cysteines may be important for hIL-18 function. In this study, each cysteine residue was individually replaced with serine by site-directed mutagenesis. The wild type and mutant IL-18s were expressed in Escherichia coli and renatured by two renaturing methods. The purified wild type and mutant rhIL-18s were assayed for their capacity of inducing IFN-γ and activating NF-κB from ConA-stimulated PBMC. DNA binding activity of NF-κB was performed by electrophoretic mobility-shift analysis. Our results showed that the mutant rhIL-18C74S and C163S induced much less amount of IFN-γ from PBMC and the decrement of NF-κB DNA binding activity was also observed from C74S and C163S treated PBMC. These results indicate that functional hIL-18 has an absolute requirement for residues Cys74 and Cys163.