Cys74 and Cys163 are necessary for IL-18 to elicit IFN-gamma 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-gamma and activating NF-kappaB from ConA-stimulated PBMC. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our results showed that the mutant rhIL-18C74S and C163S induced much less amount of IFN-gamma from PBMC and the decrement of NF-kappaB 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.
Pei
Dong-Sheng
DS
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China.
Fu
Yi
Y
Sun
Ya-Feng
YF
Hu
Shu-Qun
SQ
Zhou
Hu
H
Lu
Liang
L
Zhao
Hui-Ren
HR
D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000096:biosynthesis*; D020382:Interleukin-18 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology; D008214:Lymphocytes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D018076:DNA, Complementary; D020382:Interleukin-18; D016328:NF-kappa B; D011994:Recombinant Proteins; D007371:Interferon-gamma; D003545:Cysteine