Identification of important amino acid residues for human IL-18 function by mutant construction.
To study the structure-function relationship of IL-18, two IL-18 mutants, N- and C-terminal mutant (Delta NC) and IL-1 signature-like sequence mutant S(154)A/Y(156)F/E(157)P/C(163)T (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-gamma) production and NF-kappa B activation from human peripheral blood mononuclear cells (PBMC). Our results showed that the two mutants induced significantly less amount of IFN-gamma from PBMC (13%, 48% of wild type rhIL-18 for Delta NC, S, respectively), and the activation of NF-kappa B 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.
Fu
Yi
Y
Department of Biochemistry, School of Medicine, Yangzhou University, China.
Pei
Dong-Sheng
DS
Sun
Bing
B
Shen
Wan-Hua
WH
Lu
Liang
L
Hu
Shu-Qun
SQ
Zhao
Hui-Ren
HR
D000595:Amino Acid Sequence; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D020382:Interleukin-18 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D020382:Interleukin-18; D016328:NF-kappa B; D011994:Recombinant Proteins; D018698:Glutamic Acid; D007371:Interferon-gamma