title: Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution.
abstract: We created a novel mutated form of human interleukin-13 (IL-13) in which a positively charged arginine (R) at position 112 was substituted to a negatively charged aspartic acid (D). This mutant, termed IL-13R112D, was expressed in Escherichia coli and purified to near homogeneity. IL-13R112D was found to be a potent IL-13 agonist with 5-10-fold improved binding affinity to IL-13 receptors compared with wild-type IL-13 (wtIL-13). The conclusion of IL-13 agonist activity was drawn on the basis of approximately 10-fold improved activity over wtIL-13 in several assays: (a) inhibition of CD14 expression in primary monocytes; (b) proliferation of TF-1 and B9 cell lines; and (c) activation of STAT6 in Epstein-Barr virus-immortalized B cells, primary monocytes, and THP-1 monocytic cell line. Furthermore, mutant IL-13R112D neutralized the cytotoxic activity of a chimeric fusion protein composed of wtIL-13 and a Pseudomonas exotoxin A (IL-13-PE38) approximately 10 times better than wtIL-13. Based on these results, it was concluded that IL-13R112D interacts with much stronger affinity than wtIL-13 on all cell types tested and that Arg-112 plays an important role in the interaction with its receptors (IL-13R). Thus, these results suggest that IL-13R112D may be a useful ligand for the study of IL-13 interaction with its receptors or, alternatively, in designing specific targeted agents for IL-13R-positive malignancies.
authors.affiliation: Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Oshima
authors.forename: B H
authors.initials: BH
authors.lastname: Joshi
authors.forename: R K
authors.initials: RK
authors.lastname: Puri
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology; D002455:Cell Division; D015536:Down-Regulation; D006801:Humans; D018793:Interleukin-13 / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D050800:STAT6 Transcription Factor; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D018793:Interleukin-13; D018950:Lipopolysaccharide Receptors; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D001120:Arginine