Structure-guided Optimization of the Interleukin-6 Trans-signaling Antagonist sgp130
2008; Elsevier BV; Volume: 283; Issue: 40 Linguagem: Inglês
10.1074/jbc.m803694200
ISSN1083-351X
AutoresStephanie Tenhumberg, Georg H. Waetzig, Athena Chalaris, Björn Rabe, Dirk Seegert, Jürgen Scheller, Stefan Rose‐John, Joachim Grötzinger,
Tópico(s)Chemokine receptors and signaling
ResumoBinding of interleukin-6 (IL-6) to its specific receptor IL-6R is a prerequisite for the activation of the signal-transducing receptor glycoprotein 130 (gp130). A soluble form of the IL-6R (sIL-6R) in complex with IL-6 can activate cells lacking membrane-bound IL-6R (trans-signaling). IL-6-trans-signaling is counterbalanced by a naturally occurring, soluble form of gp130 (sgp130), whereby signaling via the membrane-bound IL-6R is not affected. Many inflammatory and neoplastic disorders are driven by IL-6 trans-signaling. By analysis of the three-dimensional structure of gp130 in complex with IL-6 and sIL-6R, we identified amino acid side chains in gp130 as candidates for the generation of sgp130 muteins with increased binding affinity to IL-6/sIL-6R. In addition, with information from modeling and NMR analysis of the membrane proximal domain of gp130, we generated a more stable variant of sgp130Fc. Proteins were tested for binding to the IL-6/sIL-6R-complex, for inhibition of IL-6/sIL-6R-induced cell proliferation and of acute phase gene expression. Several mutations showed an additive effect in improving the binding affinity of human sgp130 toward human IL-6/sIL-6R. Finally, we demonstrate the species specificity of these mutations in the optimal triple mutein (T102Y/Q113F/N114L) both in vitro and in a mouse model of acute inflammation. Binding of interleukin-6 (IL-6) to its specific receptor IL-6R is a prerequisite for the activation of the signal-transducing receptor glycoprotein 130 (gp130). A soluble form of the IL-6R (sIL-6R) in complex with IL-6 can activate cells lacking membrane-bound IL-6R (trans-signaling). IL-6-trans-signaling is counterbalanced by a naturally occurring, soluble form of gp130 (sgp130), whereby signaling via the membrane-bound IL-6R is not affected. Many inflammatory and neoplastic disorders are driven by IL-6 trans-signaling. By analysis of the three-dimensional structure of gp130 in complex with IL-6 and sIL-6R, we identified amino acid side chains in gp130 as candidates for the generation of sgp130 muteins with increased binding affinity to IL-6/sIL-6R. In addition, with information from modeling and NMR analysis of the membrane proximal domain of gp130, we generated a more stable variant of sgp130Fc. Proteins were tested for binding to the IL-6/sIL-6R-complex, for inhibition of IL-6/sIL-6R-induced cell proliferation and of acute phase gene expression. Several mutations showed an additive effect in improving the binding affinity of human sgp130 toward human IL-6/sIL-6R. Finally, we demonstrate the species specificity of these mutations in the optimal triple mutein (T102Y/Q113F/N114L) both in vitro and in a mouse model of acute inflammation. The members of the interleukin (IL) 2The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FACS, fluorescent-activated cell sorting; ELISA, enzyme-linked immunosorbent assay. -6 cytokine family include, besides IL-6, leukemia inhibitory factor (LIF), IL-11, IL-27, IL-31, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-like related cytokine (CLC), neurotrophin-1/B-cell-stimulating factor 3 (NNT-1), neuropoietin (NPN), and cardiotrophin-1 (CT-1) (1.Hibi M. Nakajima K. Hirano T. J. Mol. Med. 1996; 74: 1-12Crossref PubMed Scopus (201) Google Scholar, 2.Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar). The transmembrane protein gp130 is the common β-receptor subunit and signal transducer of the whole family (3.Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1104) Google Scholar). The usage of gp130 together with specific α-receptors (IL-6R, IL-11R, CNTFR, CLF-1) and other β-receptors (LIFR, OSMR, GPL, and Wsx1) as signal transducers is responsible for the overlapping and in part distinct cellular functions described for IL-6 type cytokines (for detailed reviews see Refs. 1.Hibi M. Nakajima K. Hirano T. J. Mol. Med. 1996; 74: 1-12Crossref PubMed Scopus (201) Google Scholar, 2.Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar). gp130 belongs to the class of tall cytokine receptors, which exhibit three fibronectin-type III-like domains between their ligand binding domains and transmembrane domains (4.Sprang S.R. Bazan J.F. Curr. Opin. Struct. Biol. 1993; 3: 815-827Crossref Scopus (240) Google Scholar). Soluble forms of gp130 are generated by alternative splicing and are found in the plasma of healthy individuals at concentrations of 100-200 ng/ml (5.Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). IL-6 signals via a complex of IL-6, IL-6-α-receptor (IL-6R), and two gp130 molecules (2.Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar) that leads to the transcription of target genes, such as acute phase response genes in hepatocytes (6.Scheller J. Grötzinger J. Rose-John S. Signal Transduct. 2006; 6: 240-259Crossref Scopus (44) Google Scholar, 7.Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Muller-Newen G. Schaper F. Biochem. J. 2003; 374: 1-20Crossref PubMed Scopus (2544) Google Scholar). IL-6-type cytokines are involved in proliferation and differentiation processes, predominantly in the hematopoietic system, in neural cells and in the immune response (1.Hibi M. Nakajima K. Hirano T. J. Mol. Med. 1996; 74: 1-12Crossref PubMed Scopus (201) Google Scholar, 2.Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar). IL-6 has major functions in inflammatory reactions of the body (1.Hibi M. Nakajima K. Hirano T. J. Mol. Med. 1996; 74: 1-12Crossref PubMed Scopus (201) Google Scholar, 2.Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar). Mice with a targeted inactivation of the IL-6 gene are protected of rheumatoid arthritis (8.Alonzi T. Fattori E. Lazzaro D. Costa P. Probert L. Kollias G. De Benedetti F. Poli V. Ciliberto G. J. Exp. Med. 1998; 187: 461-468Crossref PubMed Scopus (519) Google Scholar, 9.Boe A. Baiocchi M. Carbonatto M. Papoian R. Serlupi-Crescenzi O. Cytokine. 1999; 11: 1057-1064Crossref PubMed Scopus (118) Google Scholar) and multiple sclerosis (10.Okuda Y. Sakoda S. Bernard C.C. Fujimura H. Saeki Y. Kishimoto T. Yanagihara T. Int. Immunol. 1998; 10: 703-708Crossref PubMed Scopus (184) Google Scholar). Furthermore, regenerative reactions like wound healing and liver regeneration are severely compromised in IL-6-/- mice (11.Cressman D.E. Greenbaum L.E. DeAngelis R.A. Ciliberto G. Furth E.E. Poli V. Taub R. Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1325) Google Scholar). The expression of the IL-6R is limited mainly to hepatocytes and some leukocytes (12.Rose-John S. Scheller J. Elson G. Jones S. J. Leuk. Biol. 2006; 80: 227-236Crossref PubMed Scopus (527) Google Scholar). Cells lacking IL-6R expression are not responsive to the cytokine IL-6. A soluble form of the IL-6R, however, can bind IL-6 with the same affinity as the membrane-bound form, and the complex of IL-6 and the soluble IL-6R (sIL-6R) can induce signaling in a process called trans-signaling (13.Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (693) Google Scholar, 14.Peters M. Müller A. Rose-John S. Blood. 1998; 92: 3495-3504Crossref PubMed Google Scholar). Because the IL-6R is only sparely expressed, IL-6 trans-signaling dramatically increases the number of potential IL-6 target cells (12.Rose-John S. Scheller J. Elson G. Jones S. J. Leuk. Biol. 2006; 80: 227-236Crossref PubMed Scopus (527) Google Scholar). We have shown that the naturally occurring soluble form of gp130 selectively inhibits IL-6 responses mediated by the soluble IL-6R without affecting responses via the membrane-bound IL-6R (15.Jostock T. Müllberg J. Özbek S. Atreya R. Blinn G. Voltz N. Fischer M. Neurath M.F. Rose-John S. Eur. J. Biochem. 2001; 268: 160-167Crossref PubMed Scopus (508) Google Scholar, 16.Atreya R. Mudter J. Finotto S. Müllberg J. Jostock T. Wirtz S. Schütz M. Bartsch B. Holtmann M. Becker C. Strand D. Czaja J. Schlaak J.F. Lehr H.A. Autschbach F. Schürmann G. Nishimoto N. Yoshizaki K. Ito H. Kishimoto T. Galle P.R. Rose-John S. Neurath M.F. Nat. Med. 2000; 6: 583-588Crossref PubMed Scopus (1074) Google Scholar). Recently it has been found that inhibition of the inflammatory cytokines TNFα (17.Feldmann M. Maini R.N. Annu. Rev. Immunol. 2001; 19: 163-196Crossref PubMed Scopus (1155) Google Scholar) and IL-6 (18.Nishimoto N. Yoshizaki K. Miyasaka N. Yamamoto K. Kawai S. Takeuchi T. Hashimoto J. Azuma J. Kishimoto T. Arth. Rheum. 2004; 50: 1761-1769Crossref PubMed Scopus (693) Google Scholar) in rheumatoid arthritis and other inflammatory diseases can have dramatic therapeutic effects. More than 1 million patients have been treated so far with TNFα-neutralizing agents (19.Feldmann M. Steinman L. Nature. 2005; 435: 612-619Crossref PubMed Scopus (240) Google Scholar). Global blockade of cytokines, however, is hampered by side effects including recurrent infections, highlighting the fact that cytokines exhibit pro- and anti-inflammatory properties (20.Steinman L. Nat. Med. 2007; 13: 139-145Crossref PubMed Scopus (1123) Google Scholar). A fusion protein consisting of the extracellular region of gp130 and the Fc part of a human IgG1 antibody (sgp130Fc) selectively inhibits IL-6 trans-signaling without affecting responses via the membrane-bound IL-6R. Therefore, this protein preferentially inhibits pro-inflammatory activities of IL-6, as it has been postulated that IL-6 signaling via the membrane-bound receptor mainly contributes to anti-inflammatory reactions, whereas IL-6-trans-signaling mediates pro-inflammatory and chronic inflammatory states (21.Rose-John S. Schooltink H. Rec. Res. Cancer Res. 2007; 174: 57-66Crossref PubMed Scopus (51) Google Scholar). The structure of the extracellular portion of gp130 (domains 1-3) in complex with IL-6 and the extracellular part of the IL-6R (domains 2 and 3) has been solved by x-ray crystallography (22.Chow D.-C. He X.-L. Snow A.L. Rose-John S. Garcia K.C. Science. 2001; 291: 2150-2155Crossref PubMed Scopus (232) Google Scholar, 23.Boulanger M.J. Chow D.-C. Brevnova E.E. Garcia K.C. Science. 2003; 300: 2101-2104Crossref PubMed Scopus (494) Google Scholar). We have inspected the contact areas between IL-6 and gp130 and have identified several amino acid side chains as candidates for site-directed mutagenesis to improve the ligand binding properties of gp130. It turned out that only mutations within the contact area to site III of IL-6 improved binding of gp130 to the IL-6/sIL-6R complex, whereas changes within the contact region to site II of IL-6 resulted in decreased binding. In a previous study, we have shown that sgp130Fc tends to form large amounts of aggregates (24.Schroers A. Hecht O. Kallen K.J. Pachta M. Rose-John S. Grötzinger J. Protein Sci. 2005; 14: 783-790Crossref PubMed Scopus (69) Google Scholar). Based on the three-dimensional model structure of the extracellular domain 6 of gp130 (25.Pachta M. Dissertation. University of Kiel, Kiel, Germany2004Google Scholar), we generated an optimized version with a higher stability and a much lower tendency to form aggregates. Here, we demonstrate that one of the mutated sgp130Fc proteins with only three amino acid exchanges is far more effective in blocking different biologic responses mediated by the IL-6/sIL-6R complex in vitro. Moreover, using an animal model of acute inflammation, which is driven by the IL-6/sIL-6R complex (26.Chalaris A. Rabe B. Paliga K. Lange H. Laskay T. Fielding C.A. Jones S.A. Rose-John S. Scheller J. Blood. 2007; 110: 1748-1755Crossref PubMed Scopus (269) Google Scholar, 27.Rabe B. Chalaris A. May U. Waetzig G.H. Seegert D. Williams A.S. Jones S.A. Rose-John S. Scheller J. Blood. 2008; 111: 1021-1028Crossref PubMed Scopus (209) Google Scholar), we show that the sgp130Fc mutein is effective in blocking the progression of the inflammation from an acute to a chronic state. Our study therefore provides a general strategy to improve the efficacy of proteins, which are candidates for therapeutic applications. Constructs—The sgp130Fc protein was produced as described (15.Jostock T. Müllberg J. Özbek S. Atreya R. Blinn G. Voltz N. Fischer M. Neurath M.F. Rose-John S. Eur. J. Biochem. 2001; 268: 160-167Crossref PubMed Scopus (508) Google Scholar). The optimized sgp130Fc containing a C-terminal extended part of the extracellular region of gp130 (amino acids 1-595) was fused to the Fc part (15.Jostock T. Müllberg J. Özbek S. Atreya R. Blinn G. Voltz N. Fischer M. Neurath M.F. Rose-John S. Eur. J. Biochem. 2001; 268: 160-167Crossref PubMed Scopus (508) Google Scholar). Muteins with single amino acid substitutions or combinations thereof were generated by overlapping primer PCR within three cassettes flanked by endonuclease restriction sites (XhoI/SspI, SspI/BclI, BclI/KpnI, respectively). These constructs were subcloned into the expression vector pcDNA3.1(+) (Invitrogen, Karlsruhe, Germany). Cell Culture—COS-7, HepG2, and CHO-K1 were from DSMZ (Braunschweig, Germany). BAF3 cells (murine pre-B-cells) stably transfected with human gp130 (BAF3/gp130 cells) have been described previously (28.Fischer M. Goldschmitt J. Peschel C. Brakenhoff J.P. Kallen K.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 145-148Crossref Scopus (433) Google Scholar, 29.Vollmer P. Oppmann B. Voltz N. Fischer M. Rose-John S. Eur .J. Biochem. 1999; 263: 438-446Crossref PubMed Scopus (46) Google Scholar). All cells except CHO-K1 were grown in DMEM High Glucose Culture Medium (PAA Laboratories, Cölbe, Germany) supplemented with 10% fetal bovine serum (FBS), penicillin (60 mg/liter), and streptomycin (100 mg/liter) at 37 °C with 5% CO2 in a humidified atmosphere. BAF3/gp130 medium was supplemented with 10 ng/ml Hyper-IL6 (28.Fischer M. Goldschmitt J. Peschel C. Brakenhoff J.P. Kallen K.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 145-148Crossref Scopus (433) Google Scholar). After transient DEAE transfection, COS7 and HepG2 cells were grown with 0.5% FBS, and supernatants were harvested after 3 and 6 days (30.McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan C.I. Copeland N.G. Huebner K. Croce C.M. Cannizzarro L.A. Benjamin D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (622) Google Scholar). CHO-K1 cells were grown in Ham's F12 medium (PAA) supplemented with 5% IgG-stripped FBS (PAA), 60 mg/liter penicillin and 100 mg/liter streptomycin. CHO-K1 cells were stably transfected with sgp130Fc cDNAs using Lipofectamine 2000 (Invitrogen), and clones were selected with G418 (0.4 mg/ml, PAA). Protein Purification—Cell supernatants were subjected to affinity chromatography (protein A-Sepharose (Roche Applied Sciences, Mannheim, Germany)) and eluted by 5 column volumes of 50 mm citric acid. Relevant fractions were concentrated, and the buffer exchanged for phosphate-buffered saline (PBS) using PD10 columns (GE Healthcare, Freiburg, Germany). The proteins were further purified by gel filtration on a Sephacryl S-200 HR (16/60) column (GE Healthcare) at a flow rate of 0.5 ml/min PBS. Fractions of 2.5 ml were collected, pooled, and concentrated. Protein concentrations were determined by recording absorption spectra in the range from 240 to 320 nm (31.Waxman E. Rusinova E. Hasselbacher C.A. Schwartz G.P. Laws W.R. Ross J.B. Anal. Biochem. 1993; 210: 425-428Crossref PubMed Scopus (36) Google Scholar). sgp130Fc ELISA and Hyper-IL-6 Titration Assay—The concentration of sgp130Fc and muteins derived from cell supernatants was measured by ELISA. Plates (Maxisorp, Nunc, Wiesbaden, Germany) were coated with 100 μl of 10 μg/ml protein A (Sigma) for 5 h at room temperature and blocked overnight at 4 °C with 3% BSA and 5% sucrose in PBS. Supernatants were diluted 1:20 in 1% BSA plus 0.5% FBS in PBS, and 200 μl of the dilution were incubated for 2 h at room temperature. Wells were blocked with 5% human serum (PAA) for 30 min to saturate residual protein A binding sites. Plates were then washed with PBS containing 0.05% Tween 20 (PBST) and incubated with 100 μl of anti-human CD130 antibody (B-P4, Diaclone Research, Stamford, CT) diluted 1:5,000 in 1% BSA plus 1% FBS in PBS for 2 h. Detection was performed with anti-mouse IgG POD (ImmunoPure, Pierce) at 1:5,000 in 1% BSA plus 1% FBS in PBS for 1 h, the assay was developed with BM blue POD substrate (Roche Applied Science) and finally terminated by adding 1 m H2SO4 followed by measurement at 450 nm. In a second round, equal amounts of sgp130Fc proteins in the supernatant were loaded and incubated with 1 ng/well Hyper-IL-6 for 1 h. Hyper-IL-6 was detected with the biotinylated anti-human IL-6R antibody BAF277 (R&D Systems, Minneapolis, MN) at a 1:2,500 dilution and streptavidin-POD conjugate (Roche Applied Science) at 1:5,000 in 1% BSA in PBS. Proliferation Assay—Proliferation of BAF3/gp130 cells in response to Hyper-IL-6 or the human or murine IL-6/IL-6R complex (all components purchased from R&D Systems) and its inhibition by sgp130Fc, and the muteins was measured by [3H]thymidine incorporation (32.Kallen K.-J. Grötzinger J. Lelièvre E. Vollmer P. Aasland D. Renné C. Müllberg J. Meyer zum Büschenfelde K.-H. Gascan H. Rose-John S. J. Biol. Chem. 1999; 274: 11859-11867Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), Cell Titer Blue fluorescence assay or Cell Titer MTS colorimetric assay (both from Promega, Mannheim, Germany). For this purpose, 5 × 103 BAF3/gp130 cells were seeded per well in 96-well plates and cultured in a final volume of 100 μl. Proliferation was induced by adding 1 ng/ml Hyper-IL-6 or 100 ng/ml human IL-6 plus 50 ng/ml human sIL-6R or 300 ng/ml murine IL-6 plus 150 ng/ml murine sIL-6R. The assay was performed with each value being determined in quadruplicate. Native Polyacrylamide Gel Analysis—Native PAGE was carried out in 7.5% polyacrylamide gels with a Tris/HCl buffer system (pH 6.8 in the glycerol sample buffer and the stacking gel, pH 8.8 in the separating gel). Gels were run in a buffer of 25 mm Tris and 192 mm glycine at a constant voltage of 60 V until the samples had completely entered the stacking gel, then at 80 V for 4-5 h. Proteins were subsequently fixed and silver-stained according to standard procedures. Western Blot Analysis—After SDS-PAGE and transfer to a polyvinylidene difluoride membranes (GE Healthcare), Western blot analysis was performed using a secondary peroxidase-conjugated antibody (ImmunoPure anti-mouse IgG horseradish peroxidase from goat, Pierce). Membranes were developed using the Amersham Biosciences ECL plus kit (GE Healthcare). The gp130 part of sgp130Fc was detected by the monoclonal mouse antibody anti-human CD130 clone B-P4 (Diaclone), which recognizes domain 4 of gp130. Haptoglobin ELISA—An ELISA to detect the human acute phase protein haptoglobin was performed as described (33.Oppmann B. Stoyan T. Fischer M. Voltz N. März P. Rose-John S. J. Immunol. Methods. 1996; 195: 153-159Crossref PubMed Scopus (9) Google Scholar). Briefly, 105 HepG2 cells per well were seeded in 96-well plates and left to adhere overnight. Cells were washed twice with PBS (37 °C) and starved in DMEM without FBS for 2 h. In the mean-time, sgp130Fc and muteins were diluted and mixed with 5 ng/ml Hyper-IL-6 in DMEM without FBS and placed in the incubator for at least 30 min. Cells were incubated with 200 μl of cytokine mixture for 20 h, and the amount of haptoglobin in the cell supernatant was determined by ELISA. Each value was determined in quadruplicate. Surface Plasmon Resonance Studies—Plasmon resonance experiments were carried out with a ProteOn XPR36 protein interaction array system (Bio-Rad). The running buffer was PBS) with 0.005% Tween 20, pH 7.4 (PBST), and experiments were carried out at 25 °C. The surface of a ProteOn GLC sensor chip was activated with the ProteOn amine coupling kit (4 mm EDAC/1 mm sulfo-NHS, flow rate 30 μl/min, volume 40 μl), and proteins were covalently coupled at 10 μg/ml in 10 mm acetate buffer pH 4.5 (flow rate 30 μl/min, volume 150 μl). The respective levels of immobilization were 2,250 RU (resonance units) for sgp130Fc, 1,330 RU for the optimized sgp130Fc and 1,120 RU for mutein XIII. The concentrations of Hyper-IL-6 ranged from 80 to 2.5 nm in 2-fold dilutions in PBST, and the flow rate was 100 μl/min. Association was monitored for 60 s before replacing the sample by PBST and monitoring the dissociation phase for another 600 s. Each sensogram set was referenced using the reference channel and was baseline-aligned. Sensograms were analyzed using the ProteOn Manager 2.0 software. Animal Treatment—All procedures involving animals and their care were conducted in accordance to national and international laws and policies as well as the guidelines for animal care of the University of Kiel (acceptance no.: V 742-72241.121-3 (20-2/04) and (76-7/00)). Mice were maintained in a 12-h light/dark cycle under standard conditions and were provided with food and water ad libitum. Blood was drawn by tail bleeding or by cardiac puncture under general anesthesia. Air Pouch Model—The air pouch model of local inflammation was performed with C57Bl/6 mice (34.Edwards J.C. Sedgwick A.D. Willoughby D.A. J. Pathol. 1981; 134: 147-156Crossref PubMed Scopus (415) Google Scholar). In brief, mice were anesthetized and subcutaneous dorsal pouches were created by injection of 6 ml of sterile air. After 3 days, the pouches were reinjected with 4 ml of air. On day 6, 1 ml of 1% carrageenan (Sigma-Aldrich) in sterile PBS was injected into the pouches. sgp130Fc wild-type protein or muteins (10 μg/mouse) or PBS were administered intraperitoneally 6 h before the carrageenan injection. Seventy-two hours after treatment, animals were sacrificed, and the pouches were washed with 3 ml of PBS. The lavage fluid was immediately cooled on ice and centrifuged at 5,000 rpm for 10 min at 4 °C. The cells contained in the lavage fluid were analyzed by FACS (see below), and the supernatant was analyzed by ELISA for MCP-1 (DuoSet mouse MCP-1 ELISA Kit, R&D Systems) and sgp130Fc (DuoSet human sgp130 ELISA Kit, R&D Systems). Flow Cytometry Analysis—Aliquots of the air pouch lavage fluid containing 2 × 105 cells were used for FACS analysis (FACS-Canto, Becton-Dickinson, Heidelberg, Germany). The mAbs Ly-6G (BD Bioscience, Heidelberg, Germany) and F4/80 (Invitrogen) were used to count neutrophils and monocytes, respectively. Data were acquired from 10,000 gated events. Molecular Modeling—To construct a model of the mouse IL-6/mouse IL-6R/human gp130 complex, the recently published structure of the human IL-6/IL-6R/gp130 complex was used as a template (23.Boulanger M.J. Chow D.-C. Brevnova E.E. Garcia K.C. Science. 2003; 300: 2101-2104Crossref PubMed Scopus (494) Google Scholar). According to the published alignment, amino acid residues of IL-6 and IL-6R were exchanged in the template (35.Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). Insertions and deletions in the molecules were modeled using a data base search approach included in the software package WHATIF (36.Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar). For graphical representations, the program RIBBONS (37.Carson M. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Scopus (784) Google Scholar) was used. Selection of Amino Acid Exchanges—IL-6 interacts with three receptor subunits (38.Grötzinger J. Kernebeck T. Kallen K.-J. Rose-John S. Biol. Chem. 1999; 380: 803-813Crossref PubMed Scopus (103) Google Scholar, 39.Boulanger M.J. Garcia K.C. Adv. Protein Chem. 2004; 68: 107-146Crossref PubMed Scopus (63) Google Scholar). Site I is occupied by the specific IL-6R and site II is in contact with the cytokine binding module (domains 2 and 3) of gp130, whereas site III interacts with the Ig-like domain 1 (D1) of gp130 (Fig. 1, A and B) (23.Boulanger M.J. Chow D.-C. Brevnova E.E. Garcia K.C. Science. 2003; 300: 2101-2104Crossref PubMed Scopus (494) Google Scholar). When analyzing the three-dimensional structure of gp130 (D1-D3) in complex with IL-6 and the extracellular portion of the IL-6R, we focused on hydrophilic amino acid residues in gp130 participating in the interaction with IL-6 or IL-6R. Such residues were exchanged to hydrophobic side chains to achieve larger hydrophobic interaction areas and thereby increasing the affinity between the two molecules. The amino acid residue S251E was chosen to establish an additional salt bridge between gp130 and IL-6. As indicated in Fig. 1A, the amino acid side chains Ser-251, Ser-279, Thr-285, and Lys-303 of gp130 are in contact with site II of IL-6, whereas amino acid side chains Thr-102, Gly-109, Gln-113 and Asn-114 of gp130 are in the contact region to site III of IL-6. In addition, combinations of all these mutations were generated (Fig. 1A). Binding to Hyper-IL-6—The sgp130Fc cDNA and the mutated cDNAs were cloned into eukaryotic expression vectors and were used to transiently transfect HepG2 cells. For testing protein ability to bind Hyper-IL-6, a fusion protein of IL-6 and IL-6R (28.Fischer M. Goldschmitt J. Peschel C. Brakenhoff J.P. Kallen K.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 145-148Crossref Scopus (433) Google Scholar), crude supernatants from these cells were used (Fig. 2A) and tested by ELISA. Equal amounts of sgp130Fc protein were captured from the supernatants of transfected HepG2 cells by immobilized protein A. After incubation with Hyper-IL-6 and removal of the unbound material, the amount of bound Hyper-IL-6 was determined. Interestingly, mutations in the binding region of site II (muteins I to VI) resulted in reduced binding ability toward Hyper-IL-6 compared with the wild-type sequence. In contrast, all variants involving site III mutations, except for mutation VIII, exhibited similar or even higher binding abilities (muteins VII and IX to XIII) (Fig. 2A). Combination of all seven (site II and site III) mutations from muteins V and XIII in mutein XIV led to reduced binding most likely due to the effect of mutation IV, which was removed from the combination in mutein XV, resulting in a partial rescue of the Hyper-IL-6 binding ability (Fig. 2A). Purification and BAF3/gp130 Cell Assay—To verify these results in a cell-based assay, we purified the proteins by protein A-Sepharose affinity chromatography. The biologic activity of the purified muteins was tested on the cellular trans-signaling model cell line BAF3/gp130, which, due to stable transfection with a human gp130 cDNA, proliferates upon stimulation with IL-6/sIL-6R or Hyper-IL-6 (28.Fischer M. Goldschmitt J. Peschel C. Brakenhoff J.P. Kallen K.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 145-148Crossref Scopus (433) Google Scholar). This proliferation can be inhibited by sgp130Fc (15.Jostock T. Müllberg J. Özbek S. Atreya R. Blinn G. Voltz N. Fischer M. Neurath M.F. Rose-John S. Eur. J. Biochem. 2001; 268: 160-167Crossref PubMed Scopus (508) Google Scholar). As shown in Fig. 2B, proliferation of Hyper-IL-6-stimulated BAF3/gp130 cells was inhibited in a dose-dependent manner by sgp130Fc. Based on their inhibitory potential, the muteins could be divided into three categories. Muteins I to VI, VIII, XIV, and XV exhibited a considerably weaker or no inhibitory activity compared with wild-type sgp130Fc. Mutein VII showed only a slightly enhanced capacity of inhibition, whereas muteins IX to XIII exhibited a significantly higher potency in inhibiting BAF3/gp130 cell proliferation (Fig. 2B). Interestingly, the muteins with lower inhibitory potential all contained mutations in the gp130 contact region for site II of IL-6, whereas the muteins with increased inhibitory potential carried mutations within the gp130 contact region for site III. Moreover, the mutations V and VI abolished the inhibitory effect of sgp130Fc completely, but the activity of the corresponding muteins could partially be rescued by introducing beneficial mutations in the site III binding region (mutein XIV and XV, Fig. 2, A and B). It should be noted that mutein VII and IX showed higher binding to the IL-6/sIL-6R complex than the combined mutein XIII, although mutein XIII was most effective in the trans-signaling inhibition assay (Fig. 2B), which might be explained by differential protein stability (see below). After the initial purification by affinity chromatography, we found that about 50% of the material was present as aggregates. These aggregates could be detected by Western blotting even under denaturing and reducing conditions (Fig. 3A). To examine whether these aggregates were also present under native conditions, we performed size-exclusion chromatography (Fig. 3D). About 50% of the material eluted in the void volume, indicating the presence of aggregated sgp130Fc. An NMR-supported three-dimensional model of the sixth (membrane proximal) domain of gp130 (25.Pachta M. Dissertation. University of Kiel, Kiel, Germany2004Google Scholar) revealed that 11 amino acid residues missing in the original sgp130Fc protein constitute the last β-strand of domain 6 (Fig. 3B). Therefore, in a new construct, we added these 11 amino acid residues to prevent misfolding and subsequent aggregation of this domain. After establishing stably transfected CHO-K1 cell lines expressing the
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