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J Biol Chem, Vol. 275, Issue 19, 14375-14380, May 12, 2000
From the Laboratory of Molecular Tumor Biology, Division of
Cellular and Gene Therapies, Center for Biologics Evaluation and
Research, Food and Drug Administration, Bethesda, Maryland 20892
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.
Conversion of Interleukin-13 into a High Affinity Agonist by a
Single Amino Acid Substitution*
*
This work was supported in part by a subsidy from the
Ryoichi Naito Foundation for Medical Research to Dr. Yasuo Oshima.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug
Administration, 29 Lincoln Dr., NIH Bldg. 29B, Rm. 2NN10, Bethesda, MD
20892. Tel.: 301-827-0471; Fax: 1-301-827-0449; E-mail:
Puri@cber.fda.gov.
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