Conversion of Interleukin-13 into a High Affinity Agonist by a Single Amino Acid Substitution

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 fou...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-05, Vol.275 (19), p.14375-14380
Main Authors: Oshima, Yasuo, Joshi, Bharat H., Puri, Raj K.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Arginine - chemistry
Arginine - metabolism
Bone Marrow Cells - cytology
Cell Division
Down-Regulation
Humans
Interleukin-13 - agonists
Interleukin-13 - chemistry
Interleukin-13 - genetics
Interleukin-13 - metabolism
Lipopolysaccharide Receptors - immunology
Molecular Sequence Data
Monocytes - immunology
Mutagenesis, Site-Directed
Radioligand Assay
Sequence Homology, Amino Acid
STAT6 Transcription Factor
Trans-Activators - metabolism
Tumor Cells, Cultured
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Summary: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 Pseudomonasexotoxin 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.275.19.14375