ABSTRACT
Interleukin 13 (IL-13) is a pleiotropic cytokine secreted by activated T cells. Both IL-13 and its polymorphic variant (IL-13-R110Q) have been shown to be associated with multiple diseases such as asthma and allergy. Two IL-13 receptors have been identified, IL-13R alpha-1 receptor (IL-13Rα1) and IL-13R alpha-2 receptor (IL-13Rα2). It has been well established that IL-13 binds to IL-13Rα1 alone with low nM affinity while binding to the IL-13Rα1/IL-4R receptor complex is significantly tighter (pM). The affinity between IL-13 and IL-13Rα2, however, remains elusive. Several values have been reported in the literature varying from 20 pM to 2.5 nM. The affinities previously reported were obtained using surface plasmon resonance (SPR) or Scatchard analysis of (125) I-IL-13 binding data. This report presents the results for the kinetics and equilibrium binding analysis studies performed using label-free kinetic exclusion assay (KEA) for the interaction of human IL-13 and IL-13Rα2. KEA equilibrium analysis showed that the affinities of IL-13Rα2 are 107 and 56 pM for IL-13 and its variant (IL-13-R110Q), respectively. KEA kinetic analysis showed that a tight and very stable complex is formed between IL-13Rα2 and IL-13, as shown by calculated dissociation rate constants slower than 5 × 10(-5) per second. Kinetic analysis also showed significant differences in the kinetic behavior of wild type (wt) versus IL-13-R110Q. IL-13-R110Q not only associates to IL-13Rα2 slower than wt human IL-13 (wt-IL-13), as previously reported, but IL-13-R110Q also dissociates slower than wt-IL-13. These results show that IL-13Rα2 is a high affinity receptor and provide a new perspective on kinetic behavior that could have significant implications in the understanding of the role of IL-13-R110Q in the disease state.
Subject(s)
Interleukin-13 Receptor alpha2 Subunit/chemistry , Interleukin-13/chemistry , Amino Acid Substitution , Humans , Immobilized Proteins/chemistry , Interleukin-13/genetics , Interleukin-13 Receptor alpha2 Subunit/isolation & purification , Kinetics , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Surface Plasmon ResonanceABSTRACT
Although mice have nanogram per milliliter serum levels of soluble (s) IL-13Ralpha2, humans lack sIL-13Ralpha2 in serum. Our data provide a mechanism for this biological divergence. In mice, discrete transcripts encoding soluble and membrane forms of IL-13Ralpha2 are generated by alternative splicing. We used small interfering RNA to specifically deplete the transcript encoding membrane (mem) IL-13Ralpha2 (full-length) or sIL-13Ralpha2 (DeltaEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Ralpha2 but had no effect on the level of sIL-13Ralpha2 in cell supernatants at baseline or following cytokine stimulation. Depletion of the DeltaEx10 transcript decreased sIL-13Ralpha2 in supernatants at baseline and following stimulation. In contrast to mice, we were unable to find a transcript encoding sIL-13Ralpha2 in humans and siRNA-mediated depletion of full-length IL-13Ralpha2 decreased both sIL-13Ralpha2 and memIL-13Ralpha2 in human cells. Inhibition of matrix metalloproteinases (MMP)/MMP-8 abolished production of sIL-13Ralpha2 from human cells. Thus, sIL-13Ralpha2 is derived exclusively from the memIL-13Ralpha2 transcript in humans through MMPs/MMP-8 cleavage of memIL-13Ralpha2, supporting a limited role for sIL-13Ralpha2 in humans and highlighting the potential importance of memIL-13Ralpha2 in human immunity. These observations require consideration when results of murine IL-13 studies are applied to humans.
Subject(s)
Interleukin-13 Receptor alpha2 Subunit , Membrane Proteins , Alternative Splicing/immunology , Animals , Cell Line , Humans , Interleukin-13 Receptor alpha2 Subunit/blood , Interleukin-13 Receptor alpha2 Subunit/deficiency , Interleukin-13 Receptor alpha2 Subunit/genetics , Interleukin-13 Receptor alpha2 Subunit/isolation & purification , Membrane Proteins/blood , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Sequence Data , Protein Isoforms/biosynthesis , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Solubility , U937 CellsABSTRACT
Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.
