Abstract
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-6Ralpha 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-6Ralpha, 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.
Highlights
Cytokines are important mediators in the regulation of immune responses and inflammation
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
We have shown that IL-6-RFP is a highly specific IL-6 inhibitor that does not interfere with the bioactivity of the related cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM)
Summary
Cytokines are important mediators in the regulation of immune responses and inflammation. The apparent molecular mass of the IL-61⁄7IL-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. Based on the structural features of the IL-6 receptor complex and the well characterized antagonistic activity of the combination of sIL-6R␣ and sgp130, we designed a fusion protein (interleukin-6 receptor fusion protein, IL-6-RFP) consisting of the ligand binding domains of gp130 (D1–D3) and IL-6R␣ (D2– D3) fused with an appropriate peptide linker (see Fig. 7A) [3].
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