Surface cysteine to serine substitutions in IL-18 reduce aggregation and enhance activity

PeerJ. 2022 Jul 5:10:e13626. doi: 10.7717/peerj.13626. eCollection 2022.

Abstract

Background: Interleukin-18 (IL-18) is prone to form multimers resulting in inactive aggregates, making this cytokine unstable for clinical use. Therefore, mutations have been introduced into recombinant IL-18 to overcome this issue.

Methods: To prevent the formation of disulfide bonds between the IL-18 molecules, multiple mutations targeting surface cysteines (C38, C68, C76, and C127) were introduced into our previously modified human IL-18 double mutant E6K+T63A (IL-18 DM) by direct gene synthesis. The open reading frames of IL-18 wild-type (WT), IL-18 DM, and IL-18 multiple mutant E6K+T63A+C38S+C68S+C76S+C127S (IL-18 DM1234) were inserted in the pET28a expression vector and transformed into Escherichia coli Rosetta2 (DE3) pLysS cells for protein production. The inclusion bodies of WT and mutated IL-18 were extracted by sonication and refolded by stepwise dialysis using 8 M urea as the starting concentration. The refolded IL-18 proteins were tested for aggregation using the ProteoStat protein aggregation assay. Their activity was also investigated by treating NK-92MI cells with each IL-18 at concentrations of 75, 150, and 300 ng/ml with 0.5 ng/ml of human IL-12 and interferon-gamma (IFN-γ) levels in the supernatant were evaluated using ELISA. The structure of modified IL-18 was visualized using molecular dynamics (MD) simulations.

Results: IL-18 DM1234 exhibited the lowest aggregation signal, approximately 1.79- and 1.63-fold less than that of the WT and IL-18 DM proteins. Additionally, the IFN-γ inducing activity of IL-18 DM1234 was about 10 and 2.8 times higher than that of the WT and IL-18 DM, respectively. MD simulations revealed that binding site I of IL-18 DM1234 was altered mainly due to surface cysteine replacement with serine (C-to-S substitution). This is the first report showing that C-to-S substitutions in IL-18 improved its activity and stability, suggesting the use of this modified IL-18 for medical purposes in the future.

Keywords: Aggregation; Interferon-γ; Interleukin-18; Molecular dynamic simulation; Surface cysteine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cysteine* / genetics
  • Escherichia coli / genetics
  • Humans
  • Interferon-gamma / genetics
  • Interleukin-18* / genetics
  • Recombinant Proteins / genetics
  • Renal Dialysis
  • Serine / genetics

Substances

  • Cysteine
  • Interferon-gamma
  • Interleukin-18
  • Recombinant Proteins
  • Serine
  • IL18 protein, human

Grants and funding

This work was supported by the National Research Council of Thailand (Grant/Award Number: N41A640071) and the Innovative Multidisciplinary Research in Medical Devices Project from the Thailand Research Fund (Grant/Award Number: SCI6101034M). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.