Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay.
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| Abstract |
Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Ralpha1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Ralpha1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Ralpha1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Ralpha1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.
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| Authors | Xiaoke Yang, Julie Lee, Jonathan Brooks, James Wilhelm, David Myszka, Marion T Kasaian, Samuel Goldman, Stanley Wolf, Lori J Fitz |
| Journal | Analytical biochemistry (Anal Biochem) Vol. 376 Issue 2 Pg. 206-12 (May 15 2008) ISSN: 1096-0309 [Electronic] United States |
| PMID | 18328249 (Publication Type: Journal Article) |
| Chemical References |
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