Identification of Important Amino Acid Residues for Human IL-18
Function by Mutant Construction

FU Yi, PEI Dong-Sheng¹, SUN Bing², SHEN Wan-Hua¹, LU Liang¹, HU Shu-Qun¹, ZHAO Hui-Ren^1*

( Department of Biochemistry, School of Medicine, Yangzhou University, Yangzhou 225001, China;
¹ Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, China;
² Department of Neurosurgery, Affiliated Hospital of Suzhou University, Suzhou 215001, China )

Abstract To study the structure-function relationship of IL-18, two IL-18 mutants, N- and C-terminal mutant (DNC) and IL-1 signature-like sequence mutant S¹⁵⁴A/Y¹⁵⁶F/E¹⁵⁷P/C¹⁶³T (S), were constructed by PCR. The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E. coli, purified by Sephadex G-75 chromatography and renatured by stepwise dilution. The purity of the recombinant proteins was over 95%. The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma (IFN-g) production and NF-kB activation from human peripheral blood mononuclear cells (PBMC). Our results showed that the two mutants induced significantly less amount of IFN-g from PBMC (13%, 48% of wild type rhIL-18 for DNC, S respectively), and the activation of NF-kB also lower than wild type rhIL-18 (69.7%, 89.8% of wild type rhIL-18 respectively), indicating that the deleted or mutated amino acids might be important for IL-18 function.

Key words IL-18; structure-function relationship; interferon-g; nuclear factor-kB; site-directed mutagenesis

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