ABSTRACT
Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.
Subject(s)
Interleukin-1/metabolism , Peptides/chemistry , Peptides/pharmacology , Receptors, Interleukin-1/antagonists & inhibitors , Animals , Base Sequence , Binding, Competitive , Cell Line , Cells, Cultured , DNA Primers , Databases, Factual , Dinoprostone/metabolism , ErbB Receptors/biosynthesis , Escherichia coli , Haplorhini , Humans , Interleukin-1/pharmacology , Kinetics , Male , Mice , Molecular Sequence Data , Peptides/chemical synthesis , Polymerase Chain Reaction , Radioligand Assay , Receptors, Interleukin-1/biosynthesis , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/biosynthesis , Skin/drug effects , Skin/immunology , Skin/metabolism , Spleen/immunologyABSTRACT
The gene, XYL1, encoding the major extracellular endo-beta 1,4-xylanase from the maize pathogen Cochliobolus carbonum was cloned using a synthetic, degenerate oligonucleotide based on a tryptic fragment from the purified enzyme. The deduced product of XYL1 has a M(r) of 20,869 and a predicted pI of 9.1, in good agreement with the measured M(r) and pI of the purified enzyme. The XYL1 product has strong amino acid identity to seven endo-beta 1,4-xylanases from six prokaryotes but no obvious similarity to 10 other prokaryotic endoxylanases or a yeast endoxylanase. An internal fragment of the gene was used to create a specific xylanase mutant by transformation-mediated gene disruption via homologous recombination. Total extracellular xylanase activity in the mutant was reduced by 85-94%. When analyzed by cation exchange HPLC, culture filtrates of the mutant and wild type had identical protein profiles, but the mutant lacked the major peak of UV absorption corresponding to the major xylanase activity. Xylanase II activity was also missing in the mutant, but xylanase III activity was still present. The XYL1 mutant grew as well as the wild type on sucrose, on corn cell walls, and on xylan. The pathogenicity of the mutant was indistinguishable from the wild type, indicating that XYL1 is not required for pathogenicity.
