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Ito, Takachika*; Suzuki, Shoichi*; Kanaji, Sachiko*; Shiraishi, Hiroshi*; Ota, Shoichiro*; Arima, Kazuhiko*; Tanaka, Go*; Tamada, Taro; Honjo, Eijiro*; Garcia, K. C.*; et al.
Journal of Biological Chemistry, 284(36), p.24289 - 24296, 2009/09
Times Cited Count：24 Percentile：46.00(Biochemistry & Molecular Biology)Both IL-4 and IL-13 can bind to the shared receptor composed of the IL-4 receptor 
chain and the IL-13 receptor -1 chain (IL-13R1); however, the assembly mechanisms of these ligands to the receptor is different, enabling the principal functions of these ligands to be different. We have previously shown that the N-terminal Ig-like domain in IL-13R1, called the D1 domain, is the specific and critical binding unit for IL-13. However, it has still remained obscure which the amino acid has specific binding capacity to IL-13 and why the D1 domain acts as the binding site for IL-13, but not IL-4. To address these questions, in this study, we performed the mutational analyses for the D1 domain, combining the structural data to identify the amino acids critical for binding to IL-13. Mutations of Lys76, Lys77, or Ile78 in c' strand in which the crystal structure showed interact with IL-13 and those of Trp65 and Ala79 adjacent to the interacting site, resulted in significant impairment of IL-13 binding, demonstrating that these amino acids generate the binding site. Furthermore, mutations of Val35, Leu38, or Val42 at N-terminal 
-strand also resulted in loss of IL-13 binding, probably from decrease structural stability. None of the mutations employed here affected IL-4 binding. These results demonstrate that the hydrophobic patch composed of Lys76, Lys77, and Ile78 is the IL-13 recognition site and solidify our understanding that the differential requirements of the D1 domain in IL-13R1 allows the shared receptor to respond differentially to IL-4 and IL-13.
chain and interleukin-13 receptor 1 chain by a silkworm-baculovirus systemHonjo, Eijiro; Shoyama, Yoshinari; Tamada, Taro; Shigematsu, Hideki*; Hatanaka, Takaaki*; Kanaji, Sachiko*; Arima, Kazuhiko*; Ito, Yuji*; Izuhara, Kenji*; Kuroki, Ryota
Protein Expression and Purification, 60(1), p.25 - 30, 2008/07
Times Cited Count：13 Percentile：32.93(Biochemical Research Methods)The receptor binding to Interleukin (IL)-13 is composed of the IL-13 receptor 1 chain (IL-13R 1) and the IL-4 receptor chain (IL-4R ). In order to investigate the interaction of IL-13 with IL-13R 1 and IL-4R , the DNA fragments coding the extracellular regions of human IL-13R 1 and the IL-4R were fused with mouse Fc and expressed by a silkworm-baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13R1 showed predominant affinity to IL-4R, although neither detectable affinity of IL-13 nor IL-13R1 was observed against IL-4R. Combining these data with the moderate affinity of IL-13 to IL-13R1, this indicates that IL-13 first binds to IL-13R1 and recruits consequently to IL-4R.
N NMR relaxation measurementsYoshida, Yuichiro*; Okuri, Takatoshi*; Takeda, Chika*; Kuroki, Ryota; Izuhara, Kenji*; Imoto, Taiji*; Ueda, Tadashi*
Biochemical and Biophysical Research Communications, 358(1), p.292 - 297, 2007/06
Times Cited Count：4 Percentile：9.71(Biochemistry & Molecular Biology)The single nucleotide polymorphism interleukin-13 (IL-13) R110Q is associated with severe bronchial asthma because its lower affinity leads to the augmentation of local IL-13 concentration, resulting in an increase in the signal transduction via IL-13R. Since the mutation site does not directly bind to IL-13 R2, we carried out NMR relaxation analyses of the wild-type IL-13 and IL-13 R110Q in order to examine whether the R110Q mutation affects the internal motions in IL-13 molecules. The results showed that the internal motion in the micro- to millisecond time scale on helix D, which is suggested to be important for the interaction between IL-13 and IL-13R2, is increased in IL-13-R110Q compared with that in the wild-type IL-13. It therefore appears that the difference in the internal motions on helix D between the wild-type IL-13 and IL-13-R110Q may be involved in their affinity differences with IL-13R2.
2 chain (IL-13R2)Matsumoto, Fumiko; Tamada, Taro; Honjo, Eijiro*; Ota, Shoichiro*; Izuhara, Kenji*; Kuroki, Ryota
no journal, ,
The allergic disease has dramatically increased in recent years. Interleukin-13 (IL-13) is a key cytokine critical to the development of T-cell-mediated humoral immune responses which are associated with allergy and asthma. IL-13 possesses two types of receptor: the heterodimer, composed of IL-13R1 and IL-4R, transducing the IL-13 signals; and the IL-13R2, acting as a nonsignaling "decoy" receptor. Although the extracellular region of IL-13R1 and IL-13R2 are composed of a common structure of the class 1 cytokine receptor superfamily and they have similar amino acid sequence, the affinity of IL-13R2 with IL-13 is more than ten-fold higher than that of IL-13R1. In order to investigate the reason for this higher ligand affinity to IL-13R2, extra cellular region of IL-13R2 was expressed by silkworm-baculovirus expression system after fusing the DNA cording the extracellular region of human IL-13R2 with the Fc derived from mouse IgG2a. The expressed fusion protein IL-13R2-Fc was purified by a protein G column, and the affinity to IL-13 was confirmed by gel filtration followed by light scattering analysis. After Fc region was removed by thrombin digestion, the resulting extra cellular region of IL-13R2 receptor was confirmed to retain strong affinity to IL-13 with 1:1 ratio.
1 chain and its interaction with its ligandKuroki, Ryota; Honjo, Eijiro; Tamada, Taro; Arima, Kazuhiko*; Izuhara, Kenji*
no journal, ,
Interleukin-13 (IL-13) interacts with two types of receptors, the IL-13R1 chain (IL-13R1) and the IL-4R chain (IL-4R), that transduce the IL-13 signals. In order to investigate the interaction of IL-13 with IL-13R1 and IL-4R, the genes coding the extracellular regions of human IL-13R1 and the IL-4R containing cytokine receptor homologous region (CRH), were fused with human Fc, and expressed by silk worm. After further purification with anion-exchange chromatography, the receptors were used to investigate the ligand-receptor interaction. The size exclusion chromatography showed that association between IL-13 and IL-13Ra1 could not be observed in this condition whereas the clear association of IL-13 and IL-13Ra1 was observed after adding IL-4R. It indicates that the association between IL-13 and IL-4R increase the IL-13 affinity to IL-13R1.
2 chainMatsumoto, Fumiko; Hatanaka, Takaaki*; Tamada, Taro; Honjo, Eijiro*; Ota, Shoichiro*; Ito, Yuji*; Izuhara, Kenji*; Kuroki, Ryota
no journal, ,
Interleukin-13 (IL-13) is a key cytokine critical to the development of T-cell-mediated humoral immune responses which are associated with allergy and asthma. IL-13 possesses two types of receptor: the heterodimer, composed of IL-13R1 and IL-4R, transducing the IL-13 signals; and the IL-13R2, it has high affinity rather than IL-13R1 against IL-13, acting as a nonsignaling "decoy" receptor. In order to investigate the inhibition mechanism of IL-13 signal by IL-13R2, extra cellular region of IL-13R2 was expressed by silkworm-baculovirus expression system after fusing the DNA cording the extracellular region of human IL-13R2 with the Fc derived from mouse IgG2a. The expressed fusion protein (IL-13R2)2-Fc was purified by a protein A column followed by an ion exchange column. The affinities of the ligand complexes, IL-13/IL-13R1 and IL-13/IL-13R2 to (IL-4R)2-Fc were investigated to explore the inactivation mechanism of the IL-13R1 signal with IL-13R2 by surface plasmon resonance analysis. IL-13/IL-13R1 complex has an affinity (KD=1.20
10
M) with (IL-4Ra)2-Fc, whereas the IL-13/IL-13R2 complex had no affinity with (IL-4R)2-Fc. This observation suggests that IL-13R2 does not inactivate the IL-13R1 through formation of a ternary complex with IL-13 and IL-4R, but removes the IL-13 with its higher affinity to IL-13 without forming a ternary complex.
1 chain and IL-4 receptor Honjo, Eijiro; Shoyama, Yoshinari; Tamada, Taro; Arima, Kazuhiko*; Kanaji, Sachiko*; Izuhara, Kenji*; Kuroki, Ryota
no journal, ,
The receptor binding to Interleukin (IL)-13 is composed of the IL-13 receptor 1 chain (IL-13R1) and the IL-4 receptor chain (IL-4R). In order to investigate the interaction of IL-13 with IL-13R1 and IL-4R, the DNA fragments coding the extracellular regions of human IL-13R1 and the IL-4R, containing a cytokine receptor homologous region, were fused with human Fc and expressed by silkworm-baculovirus system. Size exclusion chromatography did not reveal association between IL-13 and IL-13R1 under these conditions, but the clear association of IL-13 and IL-13R1 was observed after adding IL-4R. This indicates that both extracellular regions of IL-13R1 and IL-4R have functions, and the association between IL-13 and IL-4R increases the IL-13 affinity to IL-13R1.
