Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein.
IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.
University of Colorado Health Sciences Center, Denver, CO 80262, USA.
S H
SH
Kim
T
T
Azam
D Y
DY
Yoon
L L
LL
Reznikov
D
D
Novick
M
M
Rubinstein
C A
CA
Dinarello
D002478:Cells, Cultured; D015951:Factor Xa / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D020382:Interleukin-18 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016209:Interleukin-8 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D020382:Interleukin-18; D016209:Interleukin-8; D011993:Recombinant Fusion Proteins; C118370:interleukin-18 binding protein; D015951:Factor Xa