Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis

The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occ urring antagonist of IL-18, a proinflammatory cytokine that is related to I L-1 beta and has an important role in defense against microbial invaders. A s its name implies, the hIL-18BP binds to IL-18 with high affinity and prev ents the interaction of IL-18 with its receptor. We genetically modified th e C terminus of hIL-18BP by appending a 15-amino acid biotinylation recogni tion site and a six-histidine tag and then performed site-directed mutagene sis 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 immobiliz ed on streptavidin-coated chips. Using surface plasmon resonance, we identi fied seven amino acids of hIL-18BP which, when changed individually to alan ine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted P-s trand c and d of hIL-18BP immunoglobulin-like domain, and most had hydropho bic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, c ontributed similar to 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.