{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:50273"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:50273","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Entwicklung und Charakterisierung eines IL-6-Inhibitors basierend auf Funktionsanalysen des murinen IL-6R alpha","abstract":"Among 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.","abstract_html":"Among 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.","abstract_has_math":false,"creators":["Wiesinger, Monique Yvonne"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Heinrich, Peter C."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2008,"date_issued":"2008","date_published":"2008","updated_at":"2026-07-30T19:40:16Z","subjects":["info:eu-repo/classification/ddc/570","Inhibitor","Zytokine","Entzündung","Fusionsprotein","IL-6R alpha","Biowissenschaften, Biologie","cytokines","inflammation","fusion protein","IL-6 receptor alpha"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112824%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112824%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112824%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/50273","outbound_label":"Repository record","outbound_source":"dc:identifier"},"source_record":{"url":"https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50273","prefix":"oai_dc"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Heinrich, Peter C."]},{"key":"dc:creator","label":"Author","values":["Wiesinger, Monique Yvonne"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2008"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-25271"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","Inhibitor","Zytokine","Entzündung","Fusionsprotein","IL-6R alpha","Biowissenschaften, Biologie","cytokines","inflammation","fusion protein","IL-6 receptor alpha"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/50273","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112824%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Among 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."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University 131 S. : Ill., graph. Darst. (2008). = Aachen, Techn. Hochsch., Diss., 2008"]},{"key":"dc:title","label":"Title","values":["Entwicklung und Charakterisierung eines IL-6-Inhibitors basierend auf Funktionsanalysen des murinen IL-6R alpha"]}]}],"canonical_facts":{"dc:contributor":["Heinrich, Peter C."],"dc:coverage":["DE"],"dc:creator":["Wiesinger, Monique Yvonne"],"dc:date":["2008"],"dc:description":["Among 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."],"dc:identifier":["https://publications.rwth-aachen.de/record/50273","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112824%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-25271"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University 131 S. : Ill., graph. Darst. (2008). = Aachen, Techn. 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