FEBS 21942
`
`FEBS Letters 450 (1999) 117^122
`
`A fusion protein of interleukin-11 and soluble interleukin-11 receptor acts
`as a superagonist on cells expressing gp130
`
`Stefan P£anza, Ingrid Tackena, Joachim Gro«tzingera, Yannick Jacquesb, Heike Dahmena,
`Peter C. Heinricha, Gerhard Mu«ller-Newena;*
`aInstitut fu«r Biochemie, Rheinisch-Westfa«lische Technische Hochschule Aachen, PauwelsstraMe 30, D-52057 Aachen, Germany
`bGroupe de Recherche Cytokines et Re¨cepteurs, Unite¨ INSERM 463, 9 Quai Moncousu, 44035 Nantes Cedex 1, France
`
`Received 22 March 1999
`
`IL-11 belongs to a subfamily of the 4-helix bundle cytokines
`including interleukin-6 (IL-6),
`leukemia inhibitory factor
`(LIF), oncostatin M (OSM), ciliary neurotrophic factor
`(CNTF) and cardiotrophin-1 (CT-1) that signal via the com-
`mon signal transducing receptor protein gp130 (for review see
`[8]). IL-11 ¢rst binds to its speci¢c K-receptor subunit (IL-
`11R) [9,10] and subsequently, the complex of IL-11 and IL-
`11R triggers the dimerization of gp130 [11] leading to activa-
`tion of the Janus kinase (Jak)/signal transducer and activator
`of transcription (STAT) signal transduction pathway [12].
`Both receptor components belong to the family of hemato-
`poietic cytokine receptors which are characterized by the pres-
`ence of at least one cytokine binding module (CBM) [13]. A
`CBM consists of two ¢bronectin type III-like domains. The
`N-terminal domain is characterized by four conserved cysteine
`residues, the C-terminal domain contains a WSXWS motif.
`The extracellular part of the IL-11R is proposed to consist
`of an Ig-like domain (D1) followed by a single CBM (D2 and
`D3) [9]. Whereas gp130 is expressed ubiquitously [14], IL-11R
`expression is more restricted [9,10]. Cells expressing gp130 but
`lacking membrane-bound IL-11R can be stimulated by the
`combination of IL-11 and soluble IL-11R (sIL-11R) indicat-
`ing that the cytoplasmic and transmembrane parts of IL-11R
`are not required for signal transduction [15].
`In this study, we show that a fusion protein of human IL-11
`and a fragment of human sIL-11R acts as a superagonist to
`which gp130 expressing cells respond more sensitive than to
`the combination of IL-11 and sIL-11R. Superagonistic activ-
`ity has also been described for a recombinant fusion protein
`consisting of a section of sIL-6R connected to IL-6 by a
`polypeptide linker [16]. The covalent linkage of two soluble
`receptor activating components exists naturally as well: the
`two separately encoded subunits of IL-12 (p35 and p40)
`undergo disul¢de-stabilized heterodimerization to constitute
`its biologically active form [17]. Our results support the idea
`that the covalent linkage of two soluble components required
`for receptor activation potentiates their bioactivity.
`
`2. Materials and methods
`
`Interleukin-11 is a hematopoietic cytokine that
`Abstract
`signals via the signal transducer gp130. Although gp130 is
`ubiquitously expressed,
`interleukine-11 responsiveness is re-
`stricted to cells that express the interleukine-11 receptor KK-
`subunit. The interleukine-11 receptor KK-subunit can be function-
`ally replaced by its soluble form indicating that the transmem-
`brane and cytoplasmic parts are not required for signal
`transduction. Here, we show that a recombinant fusion protein
`of a fragment of the human interleukine-11 receptor KK-subunit
`ectodomain linked to human interleukine-11 acts as a super-
`agonist on cells expressing gp130 but lacking the membrane-
`bound interleukine-11 receptor KK-subunit. It induces acute phase
`protein synthesis in hepatoma cells and efficiently promotes
`proliferation of Ba/F3 cells stably, transfected with gp130. In
`these bioassays, the fusion protein of a fragment of the human
`interleukine-11 receptor KK-subunit ectodomain linked to human
`interleukine-11 is 50 times more potent than the combination of
`interleukine-11 and the soluble interleukine-11 receptor KK-
`subunit. Thus, our findings support the concept that covalent
`fusion of two soluble proteins required for receptor activation
`dramatically increases their bioactivity.
`z 1999 Federation of European Biochemical Societies.
`
`Key words: Fusion protein; Interleukin-11; Superagonist;
`Gp130
`
`1. Introduction
`
`Interleukin-11 (IL-11) is a hematopoietic cytokine that ex-
`erts various biological responses on di¡erent cells and tissues
`(for review see [1]). IL-11 acts synergistically with other
`growth factors to stimulate various stages of hematopoiesis
`both in vitro and in vivo [2^4]. In a phase II clinical trial, it
`has been shown that due to its thrombopoietic activity, re-
`combinant human IL-11 ameliorates severe chemotherapy-in-
`duced thrombocytopenia therefore representing a valuable
`therapeutical protein [4]. More recently, anti-in£ammatory
`acitivities of IL-11 have been described [5,6]. Female mice
`that do not respond to IL-11 due to targeted deletion of a
`critical receptor component are infertile as a consequence of
`impaired trophoblast implantation [7].
`
`*Corresponding author. Fax: (49) (241) 88 88428.
`E-mail: mueller-newen@rwth-aachen.de
`
`Abbreviations: IL, interleukin; s, soluble; R, receptor; IL-11/R-FP,
`interleukin-11/receptor fusion protein; Jak, Janus kinase; STAT, sig-
`nal transducer and activator of transcription; CBM, cytokine binding
`module; D, domain
`
`2.1. Enzymes, antibodies and cell culture
`Enzymes were purchased from Boehringer Mannheim (Mannheim,
`Germany). The hIL-11 polyclonal antibody used in this study was
`obtained from R and D systems (Wiesbaden-Nordenstadt, Germany).
`K[32P]dATP was purchased from Amersham International (Amer-
`sham, UK) and tran[35S]-label metabolic labelling reagent from ICN
`(Meckenheim, Germany). Other chemicals were obtained from Roth
`(Karlsruhe, Germany) and media components for the cultivation of
`Pichia pastoris were purchased from Sigma-Aldrich (Deisenhofen,
`Germany) or Gibco (Eggenstein, Germany). HepG2 cells were main-
`tained in DMEM/F12 (Life Technologies, Eggenstein, Germany). Re-
`
`Singapore Exhibit 2009
`Lassen v. Singapore et al.
`PGR2019-00053
`
`0014-5793 / 99 / $20.00 ß 1999 Federation of European Biochemical Societies. All rights reserved.
`PII: S 0 0 1 4 - 5 7 9 3 ( 9 9 ) 0 0 4 7 7 - 9
`
`

`

`FEBS 21942
`
`FEBS Letters 450 (1999) 117^122
`
`A fusion protein of interleukin-11 and soluble interleukin-11 receptor acts
`as a superagonist on cells expressing gp130
`
`Stefan P£anza, Ingrid Tackena, Joachim Gro«tzingera, Yannick Jacquesb, Heike Dahmena,
`Peter C. Heinricha, Gerhard Mu«ller-Newena;*
`aInstitut fu«r Biochemie, Rheinisch-Westfa«lische Technische Hochschule Aachen, PauwelsstraMe 30, D-52057 Aachen, Germany
`bGroupe de Recherche Cytokines et Re¨cepteurs, Unite¨ INSERM 463, 9 Quai Moncousu, 44035 Nantes Cedex 1, France
`
`Received 22 March 1999
`
`Interleukin-11 is a hematopoietic cytokine that
`Abstract
`signals via the signal transducer gp130. Although gp130 is
`ubiquitously expressed,
`interleukine-11 responsiveness is re-
`stricted to cells that express the interleukine-11 receptor KK-
`subunit. The interleukine-11 receptor KK-subunit can be function-
`ally replaced by its soluble form indicating that the transmem-
`brane and cytoplasmic parts are not required for signal
`transduction. Here, we show that a recombinant fusion protein
`of a fragment of the human interleukine-11 receptor KK-subunit
`ectodomain linked to human interleukine-11 acts as a super-
`agonist on cells expressing gp130 but lacking the membrane-
`bound interleukine-11 receptor KK-subunit. It induces acute phase
`protein synthesis in hepatoma cells and efficiently promotes
`proliferation of Ba/F3 cells stably, transfected with gp130. In
`these bioassays, the fusion protein of a fragment of the human
`interleukine-11 receptor KK-subunit ectodomain linked to human
`interleukine-11 is 50 times more potent than the combination of
`interleukine-11 and the soluble interleukine-11 receptor KK-
`subunit. Thus, our findings support the concept that covalent
`fusion of two soluble proteins required for receptor activation
`dramatically increases their bioactivity.
`z 1999 Federation of European Biochemical Societies.
`
`Key words: Fusion protein; Interleukin-11; Superagonist;
`Gp130
`
`1. Introduction
`
`Interleukin-11 (IL-11) is a hematopoietic cytokine that ex-
`erts various biological responses on di¡erent cells and tissues
`(for review see [1]). IL-11 acts synergistically with other
`growth factors to stimulate various stages of hematopoiesis
`both in vitro and in vivo [2^4]. In a phase II clinical trial, it
`has been shown that due to its thrombopoietic activity, re-
`combinant human IL-11 ameliorates severe chemotherapy-in-
`duced thrombocytopenia therefore representing a valuable
`therapeutical protein [4]. More recently, anti-in£ammatory
`acitivities of IL-11 have been described [5,6]. Female mice
`that do not respond to IL-11 due to targeted deletion of a
`critical receptor component are infertile as a consequence of
`impaired trophoblast implantation [7].
`
`*Corresponding author. Fax: (49) (241) 88 88428.
`E-mail: mueller-newen@rwth-aachen.de
`
`Abbreviations: IL, interleukin; s, soluble; R, receptor; IL-11/R-FP,
`interleukin-11/receptor fusion protein; Jak, Janus kinase; STAT, sig-
`nal transducer and activator of transcription; CBM, cytokine binding
`module; D, domain
`
`IL-11 belongs to a subfamily of the 4-helix bundle cytokines
`including interleukin-6 (IL-6),
`leukemia inhibitory factor
`(LIF), oncostatin M (OSM), ciliary neurotrophic factor
`(CNTF) and cardiotrophin-1 (CT-1) that signal via the com-
`mon signal transducing receptor protein gp130 (for review see
`[8]). IL-11 ¢rst binds to its speci¢c K-receptor subunit (IL-
`11R) [9,10] and subsequently, the complex of IL-11 and IL-
`11R triggers the dimerization of gp130 [11] leading to activa-
`tion of the Janus kinase (Jak)/signal transducer and activator
`of transcription (STAT) signal transduction pathway [12].
`Both receptor components belong to the family of hemato-
`poietic cytokine receptors which are characterized by the pres-
`ence of at least one cytokine binding module (CBM) [13]. A
`CBM consists of two ¢bronectin type III-like domains. The
`N-terminal domain is characterized by four conserved cysteine
`residues, the C-terminal domain contains a WSXWS motif.
`The extracellular part of the IL-11R is proposed to consist
`of an Ig-like domain (D1) followed by a single CBM (D2 and
`D3) [9]. Whereas gp130 is expressed ubiquitously [14], IL-11R
`expression is more restricted [9,10]. Cells expressing gp130 but
`lacking membrane-bound IL-11R can be stimulated by the
`combination of IL-11 and soluble IL-11R (sIL-11R) indicat-
`ing that the cytoplasmic and transmembrane parts of IL-11R
`are not required for signal transduction [15].
`In this study, we show that a fusion protein of human IL-11
`and a fragment of human sIL-11R acts as a superagonist to
`which gp130 expressing cells respond more sensitive than to
`the combination of IL-11 and sIL-11R. Superagonistic activ-
`ity has also been described for a recombinant fusion protein
`consisting of a section of sIL-6R connected to IL-6 by a
`polypeptide linker [16]. The covalent linkage of two soluble
`receptor activating components exists naturally as well: the
`two separately encoded subunits of IL-12 (p35 and p40)
`undergo disul¢de-stabilized heterodimerization to constitute
`its biologically active form [17]. Our results support the idea
`that the covalent linkage of two soluble components required
`for receptor activation potentiates their bioactivity.
`
`2. Materials and methods
`
`2.1. Enzymes, antibodies and cell culture
`Enzymes were purchased from Boehringer Mannheim (Mannheim,
`Germany). The hIL-11 polyclonal antibody used in this study was
`obtained from R and D systems (Wiesbaden-Nordenstadt, Germany).
`K[32P]dATP was purchased from Amersham International (Amer-
`sham, UK) and tran[35S]-label metabolic labelling reagent from ICN
`(Meckenheim, Germany). Other chemicals were obtained from Roth
`(Karlsruhe, Germany) and media components for the cultivation of
`Pichia pastoris were purchased from Sigma-Aldrich (Deisenhofen,
`Germany) or Gibco (Eggenstein, Germany). HepG2 cells were main-
`tained in DMEM/F12 (Life Technologies, Eggenstein, Germany). Re-
`
`0014-5793 / 99 / $20.00 ß 1999 Federation of European Biochemical Societies. All rights reserved.
`PII: S 0 0 1 4 - 5 7 9 3 ( 9 9 ) 0 0 4 7 7 - 9
`
`

`

`118
`
`S. P£anz et al./FEBS Letters 450 (1999) 117^122
`
`combinant Trx/IL-11 and sIL-11R were prepared from bacteria or
`insect cells, respectively, as described previously [18]. Ba/F3-gp130
`cells [19] were cultured in DMEM containing 5% (v/v) conditioned
`medium from X63Ag8-653 BPV-mIL-3 myeloma cells (as a source of
`IL-3) in the presence of 1 mg/ml G418 in a water-saturated atmos-
`phere containing 5% CO2. Both media were supplemented with 10%
`(v/v) fetal calf serum, streptomycin (100 Wg/ml) and penicillin (60 Wg/
`ml).
`
`2.2. Plasmid construction
`First, a section of the hIL-11R cDNA, encoding domains 2 and 3
`(amino acids (aa) 109^318), was ampli¢ed by standard PCR methods.
`EcoRI/S¢I restriction sites (underlined) were introduced using the
`IL-11R-sense 5P-ACTCTCGGATCCCT-
`following primer pair:
`GGGCTACCCTCCAGC-3P and IL-11R-antisense 5P-TGAGAGGG-
`CCGGCTGGGCCCCCAGTGCTCGGAGTTCC-3P. Second, a frag-
`ment of hIL-11 cDNA encoding the proposed functional part of hIL-
`11 (aa 29^199) was ampli¢ed. Asp718/SacII restriction sites were in-
`troduced using the following primer pair: IL-11-sense 5P-AGA-
`CACGGTACCCCTCGAGTTTCCCCAGAC-3P and IL-11-antisense
`5P-ACTGTGCCGCGGTCACAGCCGAGTCTTCAG-3P. The plas-
`mid pPICZKA (Invitrogen) for secreted expression of recombinant
`proteins in the methylotrophic yeast P. pastoris was digested with
`the restriction endonucleases EcoRI and S¢I. Then, the ¢rst amplicon
`was ligated (pPICZKA/IL-11R). The resulting plasmid was digested
`with Asp718 and SacII and the second amplicon was ligated, which
`led to a plasmid encoding both proteins but lacking a peptide linker
`(pPICZKA/IL-11R/IL-11). The two following oligonucleotides, encod-
`ing the linker sequence, were annealed and subsequently ligated into
`the S¢I/Asp718-digested pPICZKA/IL-11R/IL-11 plasmid:
`linker-
`sense 5P-CGGCCAATCAGGAGGTGGAGGAGGCTCCGGAGG-
`AGGTTCTGGTGGAGGATCG-3P and linker-antisense 5P-GTAC-
`CGATCCTCCACCAGAACCTCCTCCGGAGCCTCCTCCACCT-
`CCTGATTGGCCGGCT-3P
`(pPICZKAIL-11/R-FP). All plasmids
`were sequenced using an ABI Prism automated sequencer (Perkin
`Elmer).
`
`2.3. Transfection of P. pastoris GS115 and expression of IL-11/R-FP
`Competent P. pastoris GS115 were transfected with pPICZKA (con-
`
`trol cells) or with pPICZKAIL-11/R-FP applying the LiCl method
`according to the manufacturer's instructions. Transfected cells were
`selected in YPD medium supplied with 0.1 mg/ml zeocin. Before in-
`duction of protein synthesis by supplementation of methanol, cells
`were grown in standard BMGY medium until A600 s 1.5 was reached.
`The expression conditions for IL-11/R-FP were optimized in a stand-
`ard BMGY medium containing 2% v/v methanol and 1% w/v cas-
`amino acids. For protein expression, the GS115 culture was incubated
`at 29³C for 36 h.
`
`2.4. Immunoblotting and enhanced chemiluminescence (ECL) detection
`Proteins separated by SDS-PAGE were transferred to a PVDF
`membrane by a semi-dry electroblotting procedure. PVDF mem-
`branes were blocked in a solution of 20 mM Tris-HCl (pH 7.6), 137
`mM NaCl, 0.1% Nonidet-P40 containing 10% bovine serum albumin
`and probed with antibody, followed by incubation with horseradish
`peroxidase-conjugated secondary antibody. Immunoreactive proteins
`were detected by chemiluminescence using the ECL-kit (Amersham,
`UK) following the manufacturer's instructions.
`
`2.5. Induction of K1-antichymotrypsin synthesis in HepG2 cells
`HepG2 cells were stimulated with Trx/IL-11 and sIL-11R or IL-11/
`R-FP as indicated in Fig. 3. After 18 h, cells were metabolically pulse-
`labelled with [35S]methionine/cysteine for 4 h. Induction of K1-anti-
`chymotrypsin was measured in cell culture supernatants by immuno-
`precipitation. Immunocomplexes were separated on 10% SDS poly-
`acrylamide gels, visualized by autoradiography and quanti¢ed using a
`Phosphorimager.
`
`2.6. Proliferation assay
`Ba/F3-gp130 cells (20 000 cells/well) were plated on 96 well plates
`and stimulated as indicated in Fig. 4. After 72 h of incubation, viable
`and metabolically active cells were quanti¢ed using a colorimetric
`assay based on the Cell Proliferation kit II (XTT) (Boehringer Mann-
`heim, Germany).
`
`2.7. Electrophoretic mobility shift assay (EMSA)
`Ba/F3-gp130 cells were incubated at 37³C in the presence of 0.5 nM
`IL-11/R-FP or 5 nM of each Trx/IL-11 and sIL-11R for periods of
`
`Fig. 1. Schematic representations of the fusion protein IL-11/R-FP. (A) Schematic representation of the IL-11/R-FP primary structure. The pro-
`tein starts with domains 2 and 3 of human IL-11R (Leu-109^Gly-318), followed by a 21 amino acid linker and human IL-11 (Pro-29^Leu-199).
`Due to the cloning strategy, the fusion protein contains two additional N-terminal amino acids, shown in italic letters. (B) Schematic represen-
`tation of the IL-11/R-FP tertiary structure. The linker (black line) should allow binding of IL-11 to domains 2 (D2) and 3 (D3) of IL-11R. N-
`and C-termini are indicated by N and C, respectively.
`
`

`

`S. P£anz et al./FEBS Letters 450 (1999) 117^122
`
`119
`
`time as indicated in Fig. 5. Preparation of nuclear extracts and EM-
`SAs were performed as described [20]. A double-stranded oligonucleo-
`tide derived from the c-fos promoter (m67SIE: 5P-GGGAGGGATT-
`TACGGGGAAATGCTG-3P) was used as 32P-labelled probe [21].
`The protein-DNA complexes were separated on a 4.5% polyacryl-
`amide gel containing 7.5% glycerol. The electrophoresis was per-
`formed using 0.25-fold TBE bu¡er at 260 V.
`
`3. Results
`
`3.1. Expression of IL-11/R-FP in the methylotrophic yeast
`P. pastoris
`Using a PCR-based methodology, we constructed an ex-
`pression vector encoding a fusion protein of the human IL-
`11R CBM (domains 2 and 3) and human IL-11. In order to
`allow binding of IL-11 to the binding epitope of IL-11R, both
`proteins were connected by a £exible polypeptide linker (Fig.
`1A). In Fig. 1B, the anticipated ternary structure of the fusion
`protein IL-11/R-FP is schematically represented in which the
`CBM of IL-11R accommodates IL-11. This arrangement of
`the two fusion partners is based on the analogy to the struc-
`ture of the growth hormone/growth hormone receptor com-
`plex [22]. Similarly, fusion proteins of the two IL-12 subunits
`p35 and p40 [23] as well as IL-6 and sIL-6R [16] were con-
`structed and shown to be biologically active. The methylotro-
`phic yeast P. pastoris was transfected with the vector pPICZ-
`KAIL-11/R-FP allowing the expression and secretion of the
`fusion protein (for details see Section 2). Supernatants of
`mock- or pPICZKAIL-11/R-FP-transfected cells were ana-
`lyzed by SDS-PAGE, followed by electroblotting and immu-
`nostaining with a hIL-11 polyclonal antibody (Fig. 2). A
`prominent band corresponding to a protein of approximately
`50 kDa was detected in the IL-11/R-FP supernatants but not
`
`Fig. 2. Detection of IL-11/R-FP in the supernatants of transfected
`yeasts. Supernatants of mock-transfected (control SN) and pPICZ-
`KAIL-11/R-FP-transfected yeasts (IL-11/R-FP SN) were concen-
`trated, treated with endoglycosidase H as indicated and, after sepa-
`ration of proteins on a 10% polyacrylamide SDS gel, analyzed by
`immunoblotting using a human IL-11 polyclonal antibody. Electro-
`phoretic mobilities of molecular mass marker proteins are indicated
`on the left.
`
`in the supernatants of mock-transfected cells. The electropho-
`retic mobility of the 50 kDa protein was increased after pre-
`treatment with endoglycosidase H resulting in a 44 kDa pro-
`tein. This is in good agreement with the calculated molecular
`
`Fig. 3. Induction of the K1-ACT synthesis in HepG2 cells. HepG2 cells were stimulated with equilibrated supernatants containing IL-11/R-FP
`or the combination of Trx/IL-11 and sIL-11R, each protein at molar concentrations as indicated. Cells incubated with supernatants of mock-
`transfected yeast cells served as a control. Newly synthesized proteins were metabolically labelled using [35S]methionine/cysteine. After immuno-
`precipitation from cell supernatants and separation by SDS-PAGE, radioactively-labelled K1-ACT was visualized by autoradiography (upper
`panel). Quanti¢cation of radioactivity was performed using a Phosphorimager (lower panel). The S.D.s were calculated from two independent
`experiments.
`
`

`

`120
`
`S. P£anz et al./FEBS Letters 450 (1999) 117^122
`
`mass of 43.5 kDa. Thus, the glycoprotein detected by the
`antibody can be envisaged as IL-11/R-FP. Band intensities
`of IL-11/R-FP and serial dilutions of an IL-11 preparation
`of a known concentration were quanti¢ed using a Lumi-Im-
`ager (not shown). From these data, the concentration of
`IL-11/R-FP in the yeast supernatant was calculated to be
`200 ng/ml. Yeast supernatants were equilibrated with PBS
`previous to application in the following bioassays.
`
`3.2. IL-11/R-FP e¤ciently induces acute phase protein
`synthesis in hepatoma cells
`The combination of IL-11 and sIL-11R induces the acute
`phase protein synthesis in hepatoma cells [24]. In order to
`evaluate the bioactivity of IL-11/R-FP, HepG2 cells were
`stimulated with either the combination of Trx/IL-11 and
`sIL-11R or IL-11/R-FP. Supernatants from mock-transfected
`cells served as a control. Trx/IL-11 is a well-characterized
`recombinant fusion protein of thioredoxin and IL-11 which
`has been shown to have biological properties indistinguishable
`from IL-11 wild-type [18,25]. After metabolic labelling of the
`proteins with [35S]methionine and [35S]cysteine, secreted K1-
`antichymotrypsin (K1-ACT) was precipitated from superna-
`
`Fig. 5. STAT activation in response to IL-11/R-FP and the combi-
`nation of Trx/IL-11 and sIL-11R. Ba/F3-gp130 cells were stimulated
`either with IL-11/R-FP (upper panel) at a concentration of 0.5 nM
`or the combination of Trx/IL-11 and sIL-11R (lower panel), each at
`a concentration of 5 nM. After di¡erent time points (as indicated),
`nuclear extracts were prepared and the protein concentrations were
`quanti¢ed. 10 Wg of nuclear protein was analyzed by EMSA using a
`32P-labelled m67SIE probe derived from the c-fos promotor provid-
`ing a binding site for STAT1 and STAT3. Protein-DNA complexes
`were separated by PAGE and visualized by autoradiography.
`STAT3 and STAT1 homodimers as well as STAT1/3 heterodimers
`are indicated by arrows.
`
`tants, transferred to SDS-PAGE and quanti¢ed using a Phos-
`phorimager (Fig. 3). Whereas no stimulation of the K1-ACT
`synthesis was evident at a concentration of 400 pM Trx/IL-11
`in the presence of equimolar amounts of sIL-11R, the hepa-
`toma cells responded signi¢cantly to 8 pM IL-11/R-FP. Ob-
`viously, IL-11/R-FP is a more potent agonist than the combi-
`nation of Trx/IL-11 and sIL-11R.
`
`3.3. IL-11/R-FP acts as a superagonist on Ba/F3-gp130 cells
`In order to quantify the agonistic activity of IL-11/R-FP
`more precisely, proliferation assays were performed. Ba/F3
`cells, which normally grow IL-3-dependently, are known to
`proliferate in response to various cytokines after transfection
`of the corresponding receptor chains. Ba/F3 cells transfected
`with gp130 (Ba/F3-gp130) proliferate after stimulation with
`Trx/IL-11 and sIL-11R [18]. Half-maximal proliferation was
`achieved with 6^7 nM Trx/IL-11 in the presence of equimolar
`amounts of sIL-11R (Fig. 4A). When Ba/F3-gp130 cells were
`stimulated with IL-11/R-FP, a concentration of 130^140 pM
`was su¤cient for half-maximal proliferation (Fig. 4B). Equil-
`ibrated supernatants from mock-transfected yeast cells did
`not
`induce proliferation. Thus, IL-11/R-FP is a 50-fold
`more potent agonist compared to the combination of Trx/
`IL-11 and sIL-11R. Moreover, an increased sensitivity of
`Ba/F3-gp130 cells to IL-11/R-FP cells is obvious from the
`response to low concentrations of agonist. Signi¢cant prolif-
`eration of Ba/F3-gp130 cells upon incubation with IL-11/R-
`FP is observed already at an 80-fold lower concentration
`
`Fig. 4. Proliferation of Ba/F3-gp130 cells. Ba/F3 cells stably trans-
`fected with gp130 were seeded in a 96 well plate (20 000 cells/well)
`and incubated with increasing amounts of (A) Trx/IL-11 in the pres-
`ence of sIL-11R or (B) IL-11/R-FP as indicated in the diagram.
`Open circles represent the response of cells stimulated with control
`supernatants. After 72 h, a tetrazolium compound was added as a
`substrate and incubated for 5 h at 37³C. Subsequently, the absorb-
`ance at 450 and 690 nm was measured. The di¡erence of absorban-
`ces corresponds to the number of metabolically active cells (XTT
`proliferation assay).
`
`

`

`S. P£anz et al./FEBS Letters 450 (1999) 117^122
`
`121
`
`(50 pM) than in response to Trx/IL-11 and sIL-11R (4 nM,
`Fig. 4). At high agonist concentrations, both stimuli led to an
`almost identical maximal response.
`
`3.4. STAT activation in response to IL-11/R-FP
`Signal transduction of IL-11 and related cytokines via the
`common signal transducer gp130 is mediated by the activation
`of STAT3 and to a lesser extent STAT1 [12,18]. In order to
`test in how far STAT activation is a¡ected by fusion of IL-11
`to the CBM of its receptor, Ba/F3-gp130 cells were stimulated
`either with IL-11/R-FP or the combination of Trx/IL-11 and
`sIL-11R. To obtain comparable levels of STAT activation,
`IL-11R/FP was applied at a 10-fold lower molar concentra-
`tion than Trx/IL-11 and sIL-11R. At di¡erent time points,
`nuclear extracts were prepared and analyzed for STAT bind-
`ing activity by an EMSA using a radioactively-labelled DNA
`probe that contains a binding site for STAT1 and STAT3
`homodimers as well as STAT1/STAT3 heterodimers (Fig. 5).
`Within a few minutes, both stimuli lead to a strong activation
`of STAT3 and to a much lesser extent STAT1 that peaks
`within 30 min and again after 4 h. No signi¢cant discrepancies
`in the pattern as well as the time course of STAT activation in
`response to both stimuli were observed. Thus, superagonism
`of IL-11/R-FP is not due to an altered mechanism of STAT
`activation.
`
`STAT3 that is comparable to the response to IL-11/R-FP.
`Fusion of IL-11 to its K-receptor has no consequence for
`the quality of
`the STAT signal,
`i.e.
`the time course of
`STAT activation and the ratio of STAT3 and STAT1 activa-
`tion suggesting that signaling receptor complexes of similar
`conformation are formed in response to both stimuli.
`How can the superagonistic activity of IL-11/R-FP be ex-
`plained? Receptor activation of gp130 by IL-11 is a two-step
`process requiring two binding events with di¡erent a¤nities.
`First, IL-11 binds to either the membrane-bound or the solu-
`ble IL-11R with a low a¤nity. Subsequently, the complex of
`IL-11 and IL-11R binds gp130 with a high a¤nity (KD = 250
`pM) [30] leading to gp130 oligomerization and signal trans-
`duction. Covalent linkage of IL-11 to IL-11R circumvents the
`¢rst low a¤nity binding step. Thus, the fusion protein IL-11/
`R-FP can be envisaged as an activated cytokine that directly
`binds its signal transducing receptor gp130 via the high a¤n-
`ity interaction. Since IL-11, due to its hematopoietic and anti-
`in£ammatory properties, is of high interest for various medi-
`cal applications, this superagonistic fusion protein may be of
`great value.
`
`Acknowledgements: This work was supported by Grants from the
`European Community (BIO4 CT 972010), the Deutsche Forschungs-
`gemeinschaft (Bonn) and the Fonds der Chemischen Industrie (Frank-
`furt a. M). The authors thank Stephan Hellwig for helpful discussions.
`
`4. Discussion
`
`References
`
`In this study, we describe a fusion protein consisting of the
`human IL-11R CBM fused by a £exible 21 amino acid linker
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`pastoris. Since treatment of the fusion protein with endogly-
`cosidase H led to a decrease in the apparent molecular mass,
`we conclude that at least one of the two potential N-glycosyl-
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`the two individual components. In line with these ¢ndings is
`the observation that a 10-fold higher molar concentration of
`IL-11 and sIL-11R is required to obtain an activation of
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