Asp(126), Asp(130) and Asp(134) are Necessary for Human IL-18 to Elicit IFN-gamma Production from PBMC.
To identify the amino acid residues which are critical to interleukin 18 (IL-18) function, three highly-conserved amino acids (Asp(126), Asp(130) and Asp(134)) were mutated to Asn, Lys and Lys. The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E.coli, renatured by stepwise dilution and purified by Sephadex G-75 chromatography. The purity of the recombinant proteins was over 95% and Western blot showed that the mutant rhIL-18 had the same immunogenicity as that of wild type rhIL-18. The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma(IFN-gamma) production from human peripheral blood mononuclear cells(PBMC). The results showed that the three mutants induced significantly less amount of IFN-gamma from PBMC(32%, 8% and 10% of wild type for hIL-18D(126)N, hIL-18D(130)K and hIL-18Df(134)K, respectively) indicating that the three highly conserved amino acids are necessary for human IL-18 function.
Fu
Yi
Y
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, China. zhaohuir@yahoo.com
Pei
Dong-Sheng
DS
Zhao
Hui-Ren
HR