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• W1972497890 abstract "Interleukin-13 (IL-13), a predominantly Th2-derived cytokine, appears to play a central pathological role in asthma, atopic dermatitis, allergic rhinitis, some parasitic infections, and cancer. We hypothesized that an IL-13 antagonist may have profound therapeutic utility in these conditions. We, therefore, mutagenized human IL-13 in which Glu at position 13 was substituted by a Lys residue. This highly purified recombinant IL-13 variant, IL-13E13K, bound with 4-fold higher affinity to the IL-13 receptor than wild-type IL-13 but retained no detectable proliferative activity on the TF-1 hematopoietic cell line. IL-13E13K competitively inhibited IL-13- and IL-4-dependent TF-1 proliferation. It also inhibited IL-13-induced STAT-6 (signal transduction and activator of transducer-6) activation in immune cells and cancer cells and reversed IL-13-induced inhibition of CD14 expression on human primary monocytes. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand receptor interaction. These results also suggest that IL-13E13K may be a useful antagonist for the treatment of allergic, inflammatory, and parasitic diseases or even malignancies in which IL-13 plays a central role. Interleukin-13 (IL-13), a predominantly Th2-derived cytokine, appears to play a central pathological role in asthma, atopic dermatitis, allergic rhinitis, some parasitic infections, and cancer. We hypothesized that an IL-13 antagonist may have profound therapeutic utility in these conditions. We, therefore, mutagenized human IL-13 in which Glu at position 13 was substituted by a Lys residue. This highly purified recombinant IL-13 variant, IL-13E13K, bound with 4-fold higher affinity to the IL-13 receptor than wild-type IL-13 but retained no detectable proliferative activity on the TF-1 hematopoietic cell line. IL-13E13K competitively inhibited IL-13- and IL-4-dependent TF-1 proliferation. It also inhibited IL-13-induced STAT-6 (signal transduction and activator of transducer-6) activation in immune cells and cancer cells and reversed IL-13-induced inhibition of CD14 expression on human primary monocytes. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand receptor interaction. These results also suggest that IL-13E13K may be a useful antagonist for the treatment of allergic, inflammatory, and parasitic diseases or even malignancies in which IL-13 plays a central role. interleukin (human, otherwise noted) interleukin-13 human interleukin-13E13K mutein human interleukin-13R112D mutein wild-type IL-13 signal transduction and activator of transducer-6 IL-13 receptor(s) (human wild-type, otherwise noted in the text) interleukin-4 IL-4 receptor(s) IL-4 receptor α subunit IL-13 receptor α1 subunit IL-13 receptor α subunit human interleukin-2 receptor γ subunit cytokine receptor homology domain granulocyte-macrophage colony-stimulating factor murine hGM-CSF, human GM-CSF human IL-5 IL-131 binds to its plasma membrane receptors on various cell types (1Debinski W. Obiri N.I. Powers S.K. Pastan I. Puri R.K. Clin. Cancer Res. 1995; 1: 1253-1258PubMed Google Scholar, 2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Puri R.K. Leland P. Obiri N.I. Husain S.R. Kreitman R.J. Haas G.P. Pastan I. Debinski W. Blood. 1996; 87: 4333-4339Crossref PubMed Google Scholar, 4Murata T. Noguchi P.D. Puri R.K. J. Immunol. 1996; 156: 2972-2978PubMed Google Scholar, 5Murata T. Obiri N.I. Debinski W. Puri R.K. Biochem. Biophys. Res. Commun. 1997; 238: 90-94Crossref PubMed Scopus (124) Google Scholar, 6Husain S.R. Obiri N.I. Gill P. Zheng T. Pastan I. Debinski W. Puri R.K. Clin. Cancer Res. 1997; 3: 151-156PubMed Google Scholar, 7Murata T. Obiri N.I. Puri R.K. Int. J. Cancer. 1997; 70: 230-240Crossref PubMed Scopus (61) Google Scholar, 8Obiri N.I. Leland P. Murata T. Debinski W. Puri R.K. J. Immunol. 1997; 158: 756-764PubMed Google Scholar, 9Maini A. Hillman G. Haas G.P. Wang C.Y. Montecillo E. Hamzavi F. Pontes J.E. Leland P. Pastan I. Debinski W. Puri R.K. J. Urol. 1997; 158: 948-953Crossref PubMed Scopus (57) Google Scholar, 10Murata T. Husain S.R. Mohri H. Puri R.K. Int. Immunol. 1998; 10: 1103-1110Crossref PubMed Scopus (90) Google Scholar). We have reported that IL-13Rs are overexpressed on a variety of human solid cancer cell lines including renal cell carcinoma, AIDS-associated Kaposi's sarcoma, ovarian carcinoma, prostate cancer, and malignant glioma (1Debinski W. Obiri N.I. Powers S.K. Pastan I. Puri R.K. Clin. Cancer Res. 1995; 1: 1253-1258PubMed Google Scholar, 2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Puri R.K. Leland P. Obiri N.I. Husain S.R. Kreitman R.J. Haas G.P. Pastan I. Debinski W. Blood. 1996; 87: 4333-4339Crossref PubMed Google Scholar, 4Murata T. Noguchi P.D. Puri R.K. J. Immunol. 1996; 156: 2972-2978PubMed Google Scholar, 5Murata T. Obiri N.I. Debinski W. Puri R.K. Biochem. Biophys. Res. Commun. 1997; 238: 90-94Crossref PubMed Scopus (124) Google Scholar, 6Husain S.R. Obiri N.I. Gill P. Zheng T. Pastan I. Debinski W. Puri R.K. Clin. Cancer Res. 1997; 3: 151-156PubMed Google Scholar, 7Murata T. Obiri N.I. Puri R.K. Int. J. Cancer. 1997; 70: 230-240Crossref PubMed Scopus (61) Google Scholar, 8Obiri N.I. Leland P. Murata T. Debinski W. Puri R.K. J. Immunol. 1997; 158: 756-764PubMed Google Scholar, 9Maini A. Hillman G. Haas G.P. Wang C.Y. Montecillo E. Hamzavi F. Pontes J.E. Leland P. Pastan I. Debinski W. Puri R.K. J. Urol. 1997; 158: 948-953Crossref PubMed Scopus (57) Google Scholar, 11Debinski W. Obiri N.I. Pastan I. Puri R.K. J. Biol. Chem. 1995; 270: 16775-16780Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 12Obiri N.I. Husain S.R. Debinski W. Puri R.K. Clin. Cancer Res. 1996; 2: 1743-1749PubMed Google Scholar, 13Obiri N.I. Murata T. Debinski W. Puri R.K. J. Biol. Chem. 1997; 272: 20251-20258Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Murata T. Obiri N.I. Puri R.K. Int. J. Mol. Med. 1998; 1: 551-557PubMed Google Scholar, 15Debinski W. Gibo D.M. Obiri N.I. Kealiher A. Puri R.K. Nat. Biotechnol. 1998; 16: 449-453Crossref PubMed Scopus (98) Google Scholar, 16Joshi B.H. Plautz G.E. Puri R.K. Cancer Res. 2000; 60: 1168-1172PubMed Google Scholar). We have proposed that the IL-13R complex may exist as three different types. Type I IL-13R appears to be composed of IL-4Rα, IL-13Rα1, and IL-13Rα2 subunits (2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 5Murata T. Obiri N.I. Debinski W. Puri R.K. Biochem. Biophys. Res. Commun. 1997; 238: 90-94Crossref PubMed Scopus (124) Google Scholar, 8Obiri N.I. Leland P. Murata T. Debinski W. Puri R.K. J. Immunol. 1997; 158: 756-764PubMed Google Scholar, 14Murata T. Obiri N.I. Puri R.K. Int. J. Mol. Med. 1998; 1: 551-557PubMed Google Scholar, 17Debinski W. Miner R. Leland P. Obiri N.I. Puri R.K. J. Biol. Chem. 1996; 271: 22428-22433Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Murata T. Taguchi J. Puri R.K. Blood. 1998; 91: 3884-3891Crossref PubMed Google Scholar, 19Aman M.J. Tayebi N. Obiri N.I. Puri R.K. Modi W.S. Leonard W.J. J. Biol. Chem. 1996; 271: 29265-29270Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Type II IL-13R is composed of IL-4Rα and IL-13Rα1, and this arrangement also forms the Type II IL-4R (2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 5Murata T. Obiri N.I. Debinski W. Puri R.K. Biochem. Biophys. Res. Commun. 1997; 238: 90-94Crossref PubMed Scopus (124) Google Scholar, 8Obiri N.I. Leland P. Murata T. Debinski W. Puri R.K. J. Immunol. 1997; 158: 756-764PubMed Google Scholar, 14Murata T. Obiri N.I. Puri R.K. Int. J. Mol. Med. 1998; 1: 551-557PubMed Google Scholar, 17Debinski W. Miner R. Leland P. Obiri N.I. Puri R.K. J. Biol. Chem. 1996; 271: 22428-22433Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Murata T. Taguchi J. Puri R.K. Blood. 1998; 91: 3884-3891Crossref PubMed Google Scholar). In type III IL-4R/IL-13R systems, IL-4Rα, IL-13Rα1, and IL-2Rγ subunits are present. Whether IL-4 or IL-13 engage all three subunits simultaneously for signaling is still not known. IL-13 regulates a variety of functions in immune cells (20Minty A. Chalon P. Derocq J.M. Dumont X. Guillemot J.C. Kaghad M. Labit C. Leplatois P. Liauzun P. Miloux B. Minty C. Casellas P. Loison G. Lupker J. Shire D. Ferrara P. Caput D. Nature. 1993; 362: 248-250Crossref PubMed Scopus (854) Google Scholar, 21McKenzie A.N.J. Culpepper J.A. Malefyt R.D. Briere F. Punnonen J. Aversa G. Sato A. Dang W. Cocks B.G. Menon S. Devries J.E. Banchereau J. Zurawski G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3735-3739Crossref PubMed Scopus (537) Google Scholar, 22Wills-Karp M. Luyimbazi J. Xu X. Schofield B. Neben T.Y. Karp C.L. Donaldson D.D. Science. 1998; 282: 2258-2261Crossref PubMed Scopus (2405) Google Scholar, 23Cosentino G. Soprana E. Thienes C.P. Siccardi A.G. Viale G. Vercelli D. J. Immunol. 1995; 155: 3145-3151PubMed Google Scholar, 24Punnonen J. Aversa G. Cocks B.G. McKenzie A.N.J. Menon S. Aurawski G. Malefyt R.D.W. Vries J.E. d. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3730-3734Crossref PubMed Scopus (987) Google Scholar). It has been shown to play a prominent role in atopic dermatitis (25Akdis M. Akdis C.A. Weigl L. Disch R. Blaser K. J. Immunol. 1997; 159: 4611-4619PubMed Google Scholar, 26Katagiri K. Itami S. Hatano Y. Takayasu S. Clin. Exp. Immunol. 1997; 108: 289-294Crossref PubMed Scopus (72) Google Scholar), allergic rhinitis (27Pawankar R.U. Okuda M. Hasegawa S. Suzuki K. Yssel H. Okubo K. Okumura K. Ra C.S. Am. J. Respir. Crit. Care Med. 1995; 152: 2059-2067Crossref PubMed Scopus (110) Google Scholar), pulmonary asthma and related lung injury (22Wills-Karp M. Luyimbazi J. Xu X. Schofield B. Neben T.Y. Karp C.L. Donaldson D.D. Science. 1998; 282: 2258-2261Crossref PubMed Scopus (2405) Google Scholar,28Grunig G. Warnock M. Wakil A.E. Venkayya R. Brombacher F. Rennick D.M. Sheppard D. Mohrs M. Donaldson D.D. Locksley R.M. Corry D.B. Science. 1998; 282: 2261-2263Crossref PubMed Scopus (1743) Google Scholar, 29Zhou Z. Homer R.J. Wang Z.D. Chen Q.S. Geba G.P. Wang J.M. Zhang Y. Elias J.A. J. Clin. Invest. 1999; 103: 779-788Crossref PubMed Scopus (1504) Google Scholar), hepatic fibrosis induced by schistosomiasis (30Chiaramonte M.G. Donaldson D.D. Cheever A.W. Wynn T.A. J. Clin. Invest. 1999; 104: 777-785Crossref PubMed Scopus (515) Google Scholar), and susceptibility to Leishmania major infection (31Matthews D.J. Emson C.L. McKenzie G.J. Jolin H.E. Blackwell J.M. McKenzie A.N.J. J. Immunol. 2000; 164: 1458-1462Crossref PubMed Scopus (130) Google Scholar) and malignancies (2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 12Obiri N.I. Husain S.R. Debinski W. Puri R.K. Clin. Cancer Res. 1996; 2: 1743-1749PubMed Google Scholar, 32Kapp U. Yeh W.C. Patterson B. Elia A.J. Kagi D. Ho A. Hessel A. Tipsword M. Williams A. Mirtsos C. Itie A. Moyle M. Mak T.W. J. Exp. Med. 1999; 189: 1939-1945Crossref PubMed Scopus (229) Google Scholar, 33Fricker J. Mol. Med. Today. 1999; 5: 463Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). Therefore, it has been hypothesized that blocking the effect of IL-13 can provide therapeutic benefit in these pathological conditions. Cytokine receptors for hematopoietic growth factors with a four α-helix structure exist largely as homodimers or heterodimers (34Nicola N.A. Hilton D.J. Adv. Protein Chem. 1999; 52: 1-65Google Scholar). For example, receptors for erythropoietin, thrombopoietin, granulocyte colony-stimulating factor, growth hormone, prolactin, and leptin can exist as homodimers (34Nicola N.A. Hilton D.J. Adv. Protein Chem. 1999; 52: 1-65Google Scholar). In this class of receptors, a single cytokine binds to two identical subunits and causes receptor internalization and signal transduction. Heterodimeric cytokine receptors generally consist of a major cytokine binding subunit and a signaling subunit (or shared subunit) (34Nicola N.A. Hilton D.J. Adv. Protein Chem. 1999; 52: 1-65Google Scholar). In this class of receptors, signaling subunits are often shared with more than one cytokine. For example, gp130 receptor subunit is shared with receptors for IL-6, IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin-1, and oncostatin M. IL-2Rγ subunit is shared by IL-2, IL-4, IL-9, and IL-15 receptors. The common β-subunit is shared by receptors for IL-3, IL-5, and GM-CSF (34Nicola N.A. Hilton D.J. Adv. Protein Chem. 1999; 52: 1-65Google Scholar). Similarly, IL-4Rα subunit is shared by the IL-4R and IL-13R system (14Murata T. Obiri N.I. Puri R.K. Int. J. Mol. Med. 1998; 1: 551-557PubMed Google Scholar, 35Izuhara K. Harada N. J. Biol. Chem. 1993; 268: 13097-13102Abstract Full Text PDF PubMed Google Scholar, 36Smerz-Bertling C. Duschl A. J. Biol. Chem. 1996; 270: 966-970Abstract Full Text Full Text PDF Scopus (130) Google Scholar, 37Wang L. Keegan A. Paul W. Heidaran M. Gutkind J. Pierce J. EMBO J. 1992; 11: 4899-4908Crossref PubMed Scopus (159) Google Scholar). In heterodimeric receptor systems, usage of intracellular signaling mechanism(s) is generally also shared (34Nicola N.A. Hilton D.J. Adv. Protein Chem. 1999; 52: 1-65Google Scholar). One of the difficulties in understanding the interaction between ligand and the shared receptor subunits is that the subunit itself usually binds its ligand with low affinity. To overcome this problem, numerous cytokine antagonists have been generated by site-directed mutagenesis, which has been shown to utilize heterodimeric receptor systems. These mutants have clarified the crucial role of a particular residue in the ligand-receptor interaction. Among them, various antagonistic muteins including mGM-CSF (38Altmann S.W. Patel N. Kastelein R.A. Growth Factors. 1995; 12: 251-262Crossref PubMed Scopus (1) Google Scholar, 39Altmann S.W. Kastelein R.A. J. Biol. Chem. 1995; 270: 2233-2240Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), hIL-5 (40Tavernier J. Tuypens T. Verhee A. Plaetinck G. Devos R. Heyden J.V.D. Guisez Y. Oefner C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5194-5198Crossref PubMed Scopus (105) Google Scholar), human IL-6 (41Savino R. Ciapponi L. 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Meuller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (117) Google Scholar) have been produced. However, no antagonist of murine or human IL-13 has been produced. To produce an IL-13 antagonist, we created a mutation in the IL-13 molecule. The selection of the residue to be mutated was based on the knowledge of mutants produced for the IL-4 family of lymphokines (e.g. GM-CSF, IL-5, IL-4, and IL-13) (48Boulay J. Paul W. J. Biol. Chem. 1992; 267: 20525-20528Abstract Full Text PDF PubMed Google Scholar, 49Paul W. Blood. 1991; 77: 1859-1870Crossref PubMed Google Scholar). When the amino acid sequence of α-helix A of these molecules was aligned, a conserved structure was identified. Mutation in one of the Glu residue in the conserved helical structure produced molecules with altered interaction with their receptors, suggesting that this region is critical for ligand-receptor interaction and signal transduction. For example, when Glu-21, located in α-helix A of the mGM-CSF molecule, was mutated to Ala, a mGM-CSF with decreased bioactivity was produced. Similarly, when Glu-9, also located in α-helix A of the IL-4 molecule, was mutated to Lys (IL-4E9K), a dramatic inhibition in binding to IL-4Rα chain was observed (47Kruse N. Shen B. Arnold S. Tony H. Meuller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (117) Google Scholar). When amino acid residues in α-helix A of IL-13, IL-4, mGM-CSF, and hIL-5 were aligned, Glu-13 in IL-13 was identified, which is located at the equivalent position in IL-4, mGM-CSF, and hIL-5 (Fig. 1 A). In addition, since IL-4 and IL-13 share two receptor chains with each other and Glu-9 in IL-4 molecule has been shown to interact with the IL-4Rα chain, we mutagenized Glu to Lys at this position. This molecule, IL-13E13K was then expressed in Escherichia coli, purified, and further characterized. We demonstrate that IL-13E13K has a 4–8-fold higher binding affinity to IL-13R compared with wtIL-13. Interestingly, IL-13E13K inhibited IL-13-induced signal transduction, cell proliferation, and biological activities in many different cell types. We conclude on the basis of these results that IL-13E13K is a novel powerful antagonist that may have many clinical applications. Sequence-specific oligonucleotide primers were synthesized at Bioserve Biotechnologies (Laurel, MD). The pET based expression vector (Novagen, Madison, WI) was used for construction of mutein clone. Plasmids were amplified in E. coli, DH5α (Life Technologies, Inc.), and DNA was extracted using plasmid purification kits (Qiagen, Chatsworth, CA). Restriction endonucleases and DNA ligase were obtained from New England Biolabs (Beverly, MA), Life Technologies, Inc., Panvera (Madison, WI), and Roche Molecular Biochemicals. TF-1 human erythroleukemia cell line was purchased from ATCC (Manassas, VA). PM-RCC renal cell carcinoma cell line was established in our laboratory (50Obiri N.I. Hillman G.G. Haas G.P. Sud S. Puri R.K. J. Clin. Invest. 1993; 91: 88-93Crossref PubMed Scopus (175) Google Scholar). THP-1, human monocytic cell line, TORY, virus-immortalized B cell, and KSY-1 AIDS-related Kaposi's sarcoma cell line were obtained and maintained as previously described (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The mutagenesis of IL-13 gene was performed using cDNA of wtIL-13 (20Minty A. Chalon P. Derocq J.M. Dumont X. Guillemot J.C. Kaghad M. Labit C. Leplatois P. Liauzun P. Miloux B. Minty C. Casellas P. Loison G. Lupker J. Shire D. Ferrara P. Caput D. Nature. 1993; 362: 248-250Crossref PubMed Scopus (854) Google Scholar) as a template. Sense primer 5′-agg aga tat aca tat gtc ccc agg ccc tgt gcc tcc ctc tac agc cct cag gaa gct cat tga gga-3′ and antisense primer 5′-taa ttt gcc cga att cag ttg aac cgt ccc tcg cg-3′ were used to mutate Glu-13 to Lys and incorporateNdeI and EcoRI restriction enzyme sites at the 5′ and 3′ termini, respectively. Construction of the expression vector for IL-13R112D was described before (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). After subcloning the PCR products, the fragment was restricted by NdeI andEcoRI and inserted into an expression vector. We confirmed the existence of mutation and restriction sites by sequencing of the plasmid. Expression and purification of wtIL-13, IL-13 mutants, and IL-4 was carried out by similar techniques as previously reported (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 52Kreitman R.J. Puri R.K. McPhie P. Pastan I. Cytokine. 1995; 7: 311-318Crossref PubMed Scopus (16) Google Scholar). wtIL-13, IL-13 mutants, and IL-4 were produced in inclusion bodies. Proliferation assays were performed as described previously (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 53Leland P. Obiri N. Aggarwal B.B. Puri R.K. Oncol. Res. 1995; 7: 227-235PubMed Google Scholar). Briefly, 1 × 104 TF-1 cells/well were cultured in 96-well plates in RPMI with 5% fetal bovine serum. Varying concentrations of wtIL-13 or IL-4 and/or IL-13 mutein were added to the wells, and the cells were cultured for ∼2 days. Tritiated thymidine (0.5 μCi) was added to each well 6–12 h before the plates were harvested in a Skatron cell harvester (Skatron, Inc., Sterling, VA). Glass fiber filter mats were counted in a β Plate counter (Wallac, Gaithersburg, MD). wtIL-13 was labeled as previously described (2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The specific activity of radiolabeled IL-13 was 26 μCi/μg. The equilibrium binding studies were performed as described elsewhere (2Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly, 5 × 105cells in 100 μl of binding buffer were incubated at 4 °C for 2 h with 125I-IL-13 (200 or 500 pm) in the absence or presence of various concentrations of unlabeled wtIL-13 or IL-13 mutant. Receptor-bound 125I-IL-13 was separated from unbound 125I-IL-13. The cell pellets were counted in a γ counter (Wallac). EMSA was performed as described before (7Murata T. Obiri N.I. Puri R.K. Int. J. Cancer. 1997; 70: 230-240Crossref PubMed Scopus (61) Google Scholar, 18Murata T. Taguchi J. Puri R.K. Blood. 1998; 91: 3884-3891Crossref PubMed Google Scholar, 51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). After incubation with various concentrations of wtIL-13 or IL-13 mutants for 15 min, THP-1 cells, Ebstein-Barr virus-immortalized B cells, or KSY-1 cells were washed with cold phosphate-buffered saline and solubilized with cold whole-cell extraction buffer (1 mm MgCl2, 20 mm HEPES, pH 7.0, 10 mm KCl, 300 mmNaCl, 0.5 mm dithiothreitol, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mmNa3VO4, and 20% glycerol). DNA-protein interactions were assessed by electrophoretic mobility shift assay using the Bandshift kit (Amersham Pharmacia Biotech) using the32P-labeled double-stranded oligonucleotide probe (4.2 × 109 cpm/μg) SBE-1. Primary monocytes were cultured at 1 × 107 cells/ml for 48 h with 1 ng/ml wtIL-13 with or without 1 μg/ml IL-13E13K. Staining of the cells was performed as described elsewhere (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The fluorescence data were collected on a FACScan/C32 (Becton Dickinson, San Jose, CA). The results were analyzed with the CELLQuest (Becton Dickinson) program. Protein synthesis inhibition assay was performed as previously described (3Puri R.K. Leland P. Obiri N.I. Husain S.R. Kreitman R.J. Haas G.P. Pastan I. Debinski W. Blood. 1996; 87: 4333-4339Crossref PubMed Google Scholar, 51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In brief, 1 × 103 PM-RCC or U251 cells/well were cultured with various concentrations of IL-13PE38QQR incubated for 20–24 h at 37 °C, and then 1 μCi of [3H]leucine (PerkinElmer Life Sciences) was added to each well and cultured for an additional 4 h. For blocking experiments, wtIL-13 or IL-13 mutants and IL-13PE38QQR were added simultaneously. Finally, cells were washed and harvested on a fiberglass filtermat, and cell associated radioactivity was measured in a β Plate counter (Wallac). The concentration of IL-13PE38QQR at which 50% inhibition of protein synthesis (IC50) occurred was calculated. The sequence of CRH domains of various cytokines were aligned by the Bestfit program of GCG software (Genetics Computer Group, Inc., Madison, WI). Helical wheel analyses were also performed using GCG software. Percent similarity and identity of extracellular domains between IL-13Rα1 and IL-2Rγ subunits were 40.5 and 31.9%, respectively. These numbers indicate reasonable sequence similarity justifying the use of IL-2Rγ subunit as a template for modeling IL-13Rα1 subunit. Conserved sequence patterns such as the WSXWS motif and four conserved Cys residues between β-strands of IL-13Rα1 and IL-2Rγ were perfectly aligned. The alignment of sequences between IL-4 and IL-13 was also performed as previously reported (20Minty A. Chalon P. Derocq J.M. Dumont X. Guillemot J.C. Kaghad M. Labit C. Leplatois P. Liauzun P. Miloux B. Minty C. Casellas P. Loison G. Lupker J. Shire D. Ferrara P. Caput D. Nature. 1993; 362: 248-250Crossref PubMed Scopus (854) Google Scholar, 54Bamborough P. Duncan D. Richards W.G. Protein Eng. 1994; 7: 1077-1082Crossref PubMed Scopus (26) Google Scholar). The similarity and identity of α-helix A and D of IL-13 to the known structure of IL-4 was in a similar range, as observed for IL-2Rγ and IL-13Rα1 subunits. However, the similarity of α-helix B and C could not be reasonably determined (54Bamborough P. Duncan D. Richards W.G. Protein Eng. 1994; 7: 1077-1082Crossref PubMed Scopus (26) Google Scholar). The coordinate of the CRH domain of the IL-4Rα subunit was also used in our model that was obtained from protein data bank entry1ILL. The model building and refinement procedures were based on the procedure previously described in detail (55Greer J. Proteins. 1990; 7: 317-334Crossref PubMed Scopus (391) Google Scholar). An initial model was built using the homology module of InsightII (Molecular Simulations Inc., San Diego, CA). Small loops and splices were created and handled such that the energy was kept at minimum for best model. The structures were finally refined using the Discover program (Molecular Simulations Inc., San Diego, CA). When the α-helix A of hIL-13, hIL-4, mGM-CSF, and hIL-5 were aligned, glutamic acid residues were found to be aligned perfectly in all of these molecules. In IL-13, this Glu was located at position 13 (Fig. 1 A). Helical wheel analysis of IL-13, IL-4, and mGM-CSF suggested that hydrophobic residues clustered in one side of the α-helix (Fig. 1 B). These hydrophobic residues may be buried in the core of the molecules, as shown for IL-4 and hGM-CSF (56Hage T. Sebald W. Reinemer P. Cell. 1999; 97: 271-281Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 57Walter M. Cook W. Ealick S. Nagabhushan T. Trotta P. Bugg C. J. Mol. Biol. 1992; 224: 1075-1085Crossref PubMed Scopus (124) Google Scholar). α-Helix A of hIL-5 did not show a cluster of hydrophobic residues (data not shown). Further pattern analysis demonstrated that the aligned critical residues are located in the other side of the hydrophobic buried cluster. Based on these analyses and the fact that Glu in IL-4 and mGM-CSF interact with shared receptor subunits, it is predicted that Glu at position 13 in IL-13 molecule is the “hot residue,” and it may also interact with shared receptor subunit. Recombinant wtIL-13, IL-13E13K, and IL-13R112D in which the 112th Arg (R) residue of IL-13 molecule was substituted for Asp (D) were expressed inE. coli and purified from inclusion bodies as previously described (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). After purification, each recombinant protein was analyzed using SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue. Each protein showed a prominent single band at ∼13 kDa with purity of at least 95% (Fig.2). Binding studies were performed using U251 glioblastoma and PM-RCC renal cell carcinoma cell lines, both of which express type I IL-13 receptors (8Obiri N.I. Leland P. Murata T. Debinski W. Puri R.K. J. Immunol. 1997; 158: 756-764PubMed Google Scholar). As expected, wtIL-13 displaced specific binding of radiolabeled IL-13 (Fig. 3). Interestingly, IL-13E13K also inhibited binding of125I-IL-13 (Fig. 3). IL-13E13K was better in displacing125I-IL-13 binding as compared with wtIL-13. In the experiment shown, the EC50 (concentration causing 50% inhibition of 125I-IL-13 binding) of wtIL-13 and IL-13E13K on U251 cells was ∼20 and 2.5 nm, respectively. On PM-RCC, it was ∼100 and 25 nm, respectively. Thus, IL-13E13K appeared to show ∼4.0–8-fold better binding avidity than wtIL-13 in displacing 125I-IL-13 binding. TF-1 erythroleukemia cells proliferate in response to IL-13 (51Oshima Y. Joshi B.H. Puri R.K. J. Biol. Chem. 2000; 275: 14375-14380Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We, therefore, measured proliferative activity of wtIL-13 and IL-13E13K (Fig.4 A) either alone or in combination of both (Fig. 4, B and C). As expected, wtIL-13 stimulated the grow" @default.
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• W1972497890 title "Characterization of a Powerful High Affinity Antagonist That Inhibits Biological Activities of Human Interleukin-13" @default.
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• W1972497890 doi "https://doi.org/10.1074/jbc.m010159200" @default.
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