Identification of amino acid residues critical for biological activity in human interleukin-18.
Interleukin-18 (IL-18) is a pro-inflammatory cytokine, and IL-18-binding protein (IL-18BP) is a naturally occurring protein that binds IL-18 and neutralizes its biological activities. Computer modeling of human IL-18 identified two charged residues, Glu-42 and Lys-89, which interact with oppositely charged amino acid residues buried in a large hydrophobic pocket of IL-18BP. The cell surface IL-18 receptor alpha chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor alpha chain does not share significant homology to IL-18BP. In the present study, Glu-42 was mutated to Lys and Lys-89 to Glu; Glu-42 and Lys-89 were also deleted separately. The deletion mutants (E42X and K89X) were devoid of biological activity, and the K89E mutant lost 95% of its activity. In contrast, compared with wild-type (WT) IL-18, the E42K mutant exhibited a 2-fold increase in biological activity and required a 4-fold greater concentration of IL-18BP for neutralization. The binding of WT IL-18 and its various mutants to human natural killer cells was evaluated by competition assays. The mutant E42K was more effective than WT IL-18 in inhibiting the binding of (125)I-IL-18 to natural killer cells, whereas the three inactive mutants E42X, K89E, and K89X were unable to compete with (125)I-IL-18 for binding. Similarly, WT IL-18 and the E42K mutant induced degradation of Ikappa-Balpha, whereas the three biologically inactive mutants did not induce degradation. The present study reveals that Glu-42 and Lys-89 are critical amino acid residues for the integrity of IL-18 structure and are important for binding to cell surface receptors, for signal transduction, and for neutralization by IL-18BP.
Kim
Soo-Hyun
SH
University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. soohyun.kim@UCHSC.edu
Azam
Tania
T
Novick
Daniela
D
Yoon
Do-Young
DY
Reznikov
Leonid L
LL
Bufler
Philip
P
Rubinstein
Menachem
M
Dinarello
Charles A
CA
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D020672:I-kappa B Proteins; D020382:Interleukin-18 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000072000:NF-KappaB Inhibitor alpha; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D017931:DNA Primers; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D020382:Interleukin-18; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha