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 occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1beta 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 beta-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 approximately 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.
Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0445, USA.
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Xiang
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Moss
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002846:Chromatography, Affinity; D000939:Epitopes / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D020382:Interleukin-18 / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000939:Epitopes; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D020382:Interleukin-18; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C118370:interleukin-18 binding protein; D000409:Alanine