Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay.
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.
Yang
Xiaoke
X
Wyeth Inflammation, 200 Cambridge Park Drive, Cambridge, MA 02140, USA.
Lee
Julie
J
Brooks
Jonathan
J
Wilhelm
James
J
Myszka
David
D
Kasaian
Marion T
MT
Goldman
Samuel
S
Wolf
Stanley
S
Fitz
Lori J
LJ
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D018793:Interleukin-13 / Q000737:chemistry / Q000378:metabolism*; D053661:Interleukin-13 Receptor alpha1 Subunit / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D019948:Receptors, Interleukin-4 / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance
D018793:Interleukin-13; D053661:Interleukin-13 Receptor alpha1 Subunit; D019948:Receptors, Interleukin-4