Production of a Biologically Active Human Interleukin 18 Requires Its Prior Synthesis as Pro-Il-18

Liu, Bianling; Novick, Daniela; Kim, Soohyun; Rubinstein, Menachem

doi:10.1006/cyto.2000.0749

Cited by 41 publications

(25 citation statements)

References 19 publications

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“…In addition, the caspase-1 cleavage site of proIL-18 was mutated into a Factor Xa site as described (23). The following oligonucleotide primers were used: primer 1 (sense), 5Ј-ATATGAATTCATGGCTGCTGAACCA-GTAG-3Ј; primer 2 (reverse), 5Ј-TAATTTAGATTTAAGCTTGCC-3Ј; primer 3 (sense), 5Ј-GGCAAGCTTAAATCTAAATTA-3Ј; primer 4 (reverse), 5Ј-ATATGGATCCTAGTCTTCGTTTTGAACAGTG-3Ј; primer 5 (sense), 5Ј-GGCAAGCTTTCTAAATTA-3Ј; primer 6 (sense), 5Ј-AGTAT-GTATGAAGATAGCCAG-3Ј; primer 7 (sense), 5Ј-AGTATGTATGATA-GCCAG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Identification of Amino Acid Residues Critical for Biological Activity in Human Interleukin-18

Kim

Azam

Novick

et al. 2002

Journal of Biological Chemistry

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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 ␣ chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor ␣ 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 I-B␣, 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.

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Section: Methodsmentioning

confidence: 99%

Identification of Amino Acid Residues Critical for Biological Activity in Human Interleukin-18

Kim

Azam

Novick

et al. 2002

Journal of Biological Chemistry

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“…The human IL-18 cDNA was isolated as described (11). The cDNA of the human IL-18 precursor (proIL-18) was used for generating a mutation of IL-1␤ converting enzyme (ICE, caspase-1) cleavage site into a factor Xa site.…”

Section: Methodsmentioning

confidence: 99%

“…Each of the four pPROEX HTa͞IL-18 plasmids was transformed into the competent E. coli strain DH␣ (GIBCO͞BRL) and expressed as described (11). An overnight culture of 25 ml was added to 450 ml of LB medium containing 100 g͞ml ampicillin and grown until it reached a density of 0.6-1 OD 600 .…”

Section: Methodsmentioning

confidence: 99%

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

Kim

Azam

Yoon

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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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.

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“…The mature IL-18 is then secreted not through the endoplasmic reticulum but via an alternate pathway (37). It is important to note that in some cases the expression of mature IL-18 in a transfected cell results in a molecule with no or little IL-18 activity, possibly due to problems with protein folding (38). It is also known that expression of proIL-18 in cells that do not also produce ICE (most cells do not) results in a nonsecreted proIL-18 (37,39).…”

Section: Discussionmentioning

confidence: 99%

Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity

Acres

Gantzer²,

Remy³

et al. 2005

Cancer Research

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To redress the immune imbalances created by pathologies such as cancer, it would be beneficial to create novel cytokine molecules, which combine desired cytokine activities with reduced toxicities. Due to their divergent but complementary activities, it is of interest to combine interleukin-2 (IL-2) and IL-18 into one recombinant molecule for immunotherapy. Evaluation of a fusokine protein that combines murine IL-2/IL-18 shows that it is stable, maintains IL-2 and IL-18 bioactivities, has notably reduced IL-2 associated toxicities, and has a novel lymphocyte-stimulating activity. An adeno-viral expression system was used to explore the biology of this ''fusokine''. Inclusion of the IL-18 prosequence (proIL-18) increases the expression, secretion, and potency of this fusokine. In vivo gene transfer experiments show that Ad-IL-2/proIL-18 dramatically outdoes Ad-IL-2, Ad-proIL-18, or the combination of both, by inducing high rates of tumor rejection in several murine models. Both innate and adaptive effector mechanisms are required for this antitumor activity. (Cancer Res 2005; 65(20): 9536-46)

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Production of a Biologically Active Human Interleukin 18 Requires Its Prior Synthesis as Pro-Il-18

Cited by 41 publications

References 19 publications

Identification of Amino Acid Residues Critical for Biological Activity in Human Interleukin-18

Identification of Amino Acid Residues Critical for Biological Activity in Human Interleukin-18

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

Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity

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