Publikationsserver der RWTH Aachen University
Entwicklung und Charakterisierung eines IL-6-Inhibitors basierend auf Funktionsanalysen des murinen IL-6R alpha
Abstract
dc:descriptionAmong other humoral factors, cytokines get secreted by a variety of cell types in response to different external stimuli and induce a multitude of biological effects. IL-6-type cytokine family members activate the Jak/STAT signalling pathway and hence modulate processes such as apoptosis, proliferation, angiogenesis and migration of cells. The cytokine IL-6 possesses great clinical relevance because dysregulation of IL-6 expression can be observed in many chronic inflammatory diseases as well as in cancer. A particular concept for IL-6 inhibition was achieved by preventing receptor activation through sequestration of the cytokine. In a previous study, an IL-6 inhibitor was constructed by a fusion of the ligand binding domains of the human IL-6 receptor components, namely domains D1 to D3 of gp130 and D2-D3 of IL-6R alpha. The present study aimed at the generation of an IL-6-inhibitor based on the murine receptor proteins for the future validation of the fusion protein concept in aminal studies. Although murine and human IL-6R alpha and gp130 proteins show a very high degree of homology, no IL-6 binding could be achieved by an analogous fusion of murine receptor domains. This unexpected result illustrates functional differences in respect of ligand binding between human and murine IL-6R alpha. Based on a model structure of murine IL-6R alpha derived from the solved crystal structure of the human IL-6 receptor complex, a set of amino acids was identified which could potentially influence IL-6 binding and account for the observed differences in the human and murine systems. By generation of specific deletion- and point-mutations within the immunoglobulin-like domain of the full-length murine IL-6R alpha protein the functional relevance of particular amino acid residues within a 12 amino acid stretch (preD1-region) was investigated. This region at the amino-terminus of the protein is connected to domain D2 via a disulfide bond linking the cysteine residues C6 and C171. The disulfide bond is important for the positioning of several hydrophobic N-terminal amino acids (LVL-motif) which interact with amino acid residues located in the beta-strands C and D of domain D2, a region which is in the immediate vicinity of the so-called site I-interface between IL-6R alpha and IL-6. Using life cell imaging and flow cytometry, it was shown that the deletion or substitution of Cysteine C6 within the preD1-region of murine IL-6R alpha shows a dominant negative effect both on localization (surface expression) and protein function (IL-6 binding). By the mutation of this particular cysteine a mutation outside the „classical“ CBM was generated which impairs IL-6 binding. Another motif of functional relevance in murine IL-6R alpha consists of three hydrophobic N-terminal amino acid residues (L1V2L3-motif). By the generation of a triple mutant where the hydrophobic amino acids were replaced by amino acids with small polar side chains, the cooperative effect of these residues in regard to IL-6 binding was characterized. Substitution of this motif resulted in a significantly diminished activation of the JAK/STAT-signal transduction pathway despite a comparable surface expression of the receptor constructs. At a later stage, it could also be shown that this mutation showed reduced IL-6 binding in the context of a mIL-6R alpha/gp130 fusion protein (inhibitor concept). Based on these findings, the protein domains which are crucial for ligand binding in the murine receptor system were defined and a functional IL-6 inhibitor was generated. For this, the domains D1 to D3 of murine gp130 were connected to domains D1 to D3 of murine IL-6R alpha. This mIL-6-R fusion protein (mIL-6-RFP) was capable of neutralizing IL-6 of different species (human, mouse, rat) with comparable efficiency, whereas no inhibition of structurally related cytokines of the IL-6-type-cytokine-family such as IL-11 and OSM was detectable. In vitro, mIL-6-RFP showed a 30-times stronger inhibition of IL-6-dependent proliferation of cells than an antagonistic IL-6R alpha antibody. Moreover, it was demonstrated that mIL-6-RFP efficiently inhibited the so-called trans-signalling which is elicited by IL-6 in combination with a soluble form of IL-6 receptor. In the course of this study a strategy for optimization of protein secretion was established, resulting in a considerable increase of protein amount in mammalian expression systems. The generated mIL-6-RFP also displayed a satisfactory stability in plasma samples of animals if expressed in-vivo in rats. With the generation of mIL-6-RFP a highly potent and specific IL-6 inhibitor is available for the use in animal studies (mouse/ rat). This will permit a further characterization of the role of IL-6 in various disease models and could ultimately lead to the use of this macromolecular inhibitor in anti-IL-6 therapy.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2008
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Wiesinger, Monique Yvonne
- Contributors dc:contributor
-
- Heinrich, Peter C.
Subjects
dc:subject × 11Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger