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J. Biol. Chem., Vol. 282, Issue 2, 1238-1248, January 12, 2007

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Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP

FUSED RECEPTOR DOMAINS ACT AS HIGH AFFINITY CYTOKINE-BINDING PROTEINS^*

Silke Metz, Monique Wiesinger, Michael Vogt, Heike Lauks, Günther Schmalzing, Peter C. Heinrich, and Gerhard Müller-Newen¹

From the Institut für Biochemie, Pauwelsstrasse 30 and the Institut für Molekulare Pharmakologie, Universitätsklinikum RWTH Aachen, Wendlingweg 2, 52074 Aachen, Germany

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6R and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6R, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6·IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.

Received for publication, July 19, 2006 , and in revised form, October 17, 2006.

^* This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich SFB 542 and Graduiertenkolleg "Biointerface" GRK 1035) and the Fonds der Chemischen Industrie (Frankfurt am Main). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. Tel.: 49-241-80-88860; Fax: 49-241-80-2428; E-mail: mueller-newen{at}rwth-aachen.de.

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This article has been cited by other articles:

				S. Metz, G. Naeth, P. C. Heinrich, and G. Muller-Newen Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors J. Biol. Chem., March 7, 2008; 283(10): 5985 - 5995. [Abstract] [Full Text] [PDF]