Determination of the Functional Epitopes of Human Interleukin-18-binding Protein by Site-directed Mutagenesis

Yan Xiang, Bernard Moss

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1β and has an important role in defense against microbial invaders. As its name implies, the hIL-18BP binds to IL-18 with high affinity and prevents the interaction of IL-18 with its receptor. We genetically modified the C terminus of hIL-18BP by appending a 15-amino acid biotinylation recognition site and a six-histidine tag and then performed site-directed mutagenesis to determine the functional epitopes that mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on streptavidin-coated chips. Using surface plasmon resonance, we identified seven amino acids of hIL-18BP which, when changed individually to alanine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted β-strand c and d of hIL-18BP immunoglobulin-like domain, and most had hydrophobic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, contributed ∼50% of the binding free energy. Information obtained from these studies could contribute to the design of molecular antagonists of IL-18 for treatment of inflammatory diseases.

Original languageEnglish (US)
Pages (from-to)17380-17386
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number20
DOIs
StatePublished - May 18 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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