Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

Abstract

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.

Expression and alternative processing of IL‐18 inhuman neutrophils

“…We note, however, that an 11.4-kDa product (positions 1-98 in the mature IL-18 sequence) retains this residue and the generation of a functional product cannot be ruled out. Moreover, as Glu-42, in conjunction with Lys-89 (Lys-59 in the mature IL-18 sequence), is implicated in IL-18BP interactions [49] and as this residue is retained in several defined processing products, it is possible that some decoy action on IL-18BP effects could operate. Detailed analyses are now required to formally evaluate the functional contribution of the products predicted on our analyses to be present in an inflammatory environment in which neutrophils are processing IL-18-derived molecules either in an autocrine manner or indeed from adjacent dendritic cells or macrophages.…”

A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity

“…IL-18 binds to IL-18BP with a high affinity (K d ϭ 400 pM) and neutralizes its activity (20,21). In a previous report, we demonstrated that two charged amino acids in the sequence of IL-18 (Glu-42 and Lys-89) are crucial for the interaction of IL-18 both with the IL-18BP and with the IL-18R␣ (22,23).…”

Identification of a Critical Ig-Like Domain in IL-18 Receptor α and Characterization of a Functional IL-18 Receptor Complex

“…The six pPROEX HTa-IL-18R␣ separate domain plasmids were transformed into the competent E. coli strain DH5␣ (Invitrogen) and expressed as described previously (27). An overnight culture of 5 ml was added to 200 ml of Luria-Bertani medium containing 100 g/ml ampicillin and grown to a density of 0.6 -1 OD 600 .…”

“…The cDNA of both IL-18 and IL-18R␣ were cloned as previously described (26,27). The cDNA of actin probe for Northern blot normalization was prepared as follows; the sense primer (5Ј-ACCGCGAGAAGATGAC CCAG) and the reverse primer (5Ј-CCATCTCGTTCTCGAAGTCCA) were used for RT-PCR with the total RNA from human A549 cell line.…”