Receptor Recognition Sites of Cytokines Are Organized as Exchangeable Modules
1999; Elsevier BV; Volume: 274; Issue: 17 Linguagem: Inglês
10.1074/jbc.274.17.11859
ISSN1083-351X
AutoresKarl‐Josef Kallen, Joachim Grötzinger, Eric Lelièvre, Petra Vollmer, Dorthe Aasland, Christoph Renné, Jürgen Müllberg, Karl‐Hermann Meyer zum Büschenfelde, Hugues Gascan, Stefan Rose‐John,
Tópico(s)Immune Cell Function and Interaction
ResumoInterleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4–helical bundle" cytokines of the IL–6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology. Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4–helical bundle" cytokines of the IL–6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology. ciliary neurotrophic factor interleukin-6 IL-6 receptor CNTF receptor leukemia inhibitory factor LIF receptor lipopoysaccharide monoclonal antibody polymerase chain reaction polyacrylamide gel electrophoresis Ciliary neurotrophic factor (CNTF)1 is a survival and differentiation factor for a variety of neuronal and glial cells (1Sendtner M. Gotz R. Holtmann B. Escary J.L. Masu Y. Carroll P. Wolf E. Brem G. Brulet P. Thoenen H. Curr. Biol. 1996; 6: 686-694Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Several groups have demonstrated its ability to prevent or slow down neuronal degeneration in animal models of neuropathic diseases (2Anderson K.D. Panayotatos N. Corcoran T.L. Lindsay R.M. Wiegand S.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7346-7351Crossref PubMed Scopus (122) Google Scholar, 3Sendtner M. Schmalbruch H. Stöckli K.A. Carroll P. Kreutzberg G.W. Thoenen H. Nature. 1992; 358: 502-504Crossref PubMed Scopus (510) Google Scholar, 4Clatterbuck R.E. Price D.L. Koliatsos V.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2222-2226Crossref PubMed Scopus (130) Google Scholar). Non-neuronal effects of CNTF include maintenance of embryonic stem cells in an undifferentiated state (5Koshimizu U. Taga T. Watanabe M. Saito M. Shirayoshi Y. Kishimoto T. Nakatsuji N. Development. 1996; 122: 1235-1242PubMed Google Scholar), initiation of an acute-phase response in liver cells (6Baumann H. Ziegler S.F. Mosley B. Morella K.K. Pajovic S. Gearing D.P. J. Biol. Chem. 1993; 268: 8414-8417Abstract Full Text PDF PubMed Google Scholar), and a myotrophic effect on denervated skeletal muscles of mice (7Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Abstract Full Text PDF PubMed Scopus (213) Google Scholar). CNTF belongs to the IL-6 type family of hematopoietic and neurotrophic cytokines that also encompasses interleukin 6 (IL-6), leukemia inhibitory factor (LIF), oncostatin M, cardiotrophin-1, and interleukin 11 (IL-11). All IL-6 type cytokines use a membrane spanning 130-kDa glycoprotein, gp130, as a signal transducing subunit (8Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar, 9Robledo O. Fourcin M. Chevalier S. Guillet C. Auguste P. Pouplard-Barthelaix A. Pennica D. Gascan H. J. Biol. Chem. 1997; 272: 4855-4863Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Some IL-6 type cytokines also use the LIF receptor (LIFR) and the oncostatin M receptor for signaling. Despite very limited sequence homology, the IL-6 type cytokines were predicted to share a common structure consisting of four anti-parallel α-helices (A, B, C, and D) connected by two long cross-over loops (AB, CD) and one short loop (BC) (10Bazan J.F. Neuron. 1991; 7: 197-208Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 11Sprang S.R. Bazan J.F. Curr. Opin. Struct. Biol. 1993; 3: 815-827Crossref Scopus (240) Google Scholar). This so-called "four-helix bundle" structure represents a fundamental protein fold characteristic of most cytokines (11Sprang S.R. Bazan J.F. Curr. Opin. Struct. Biol. 1993; 3: 815-827Crossref Scopus (240) Google Scholar). Crystallographic and NMR studies have confirmed this structure for IL-6, CNTF, and LIF (12Somers W. Stahl M. Seehra J.S. EMBO J. 1997; 16: 989-997Crossref PubMed Scopus (226) Google Scholar, 13McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (129) Google Scholar, 14Robinson R.C. Grey L.M. Staunton D. Vankelecom H. Vernallis A.B. Moreau J.F. Stuart D.I. Heath J.K. Jones E.Y. Cell. 1994; 77: 1101-1116Abstract Full Text PDF PubMed Scopus (194) Google Scholar). The biological response to CNTF is elicited by formation of a multiunit receptor complex (15Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (593) Google Scholar). CNTF first binds in a 1:1 stoichiometry to a glycosylphosphatidylinositol-anchored ligand binding α-unit, CNTF receptor (CNTFRα), which is not involved in signal transduction (16Davis S. Aldrich T.H. Valenzuela D.M. Wong V.V. Furth M.E. Squinto S.P. Yancopoulos G.D. Science. 1991; 253: 59-63Crossref PubMed Scopus (539) Google Scholar). This is followed by the recruitment of gp130 and LIFR as membrane spanning signal transducing β-units (15Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (593) Google Scholar), which in turn form a disulfide-linked heterodimer that activates the JAK/STAT and the Ras/MAP kinase pathways (8Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Signaling of cardiotrophin-1, LIF, and oncostatin M also occurs via a gp130/LIFR heterodimer. Similar to CNTF, cardiotrophin-1 first binds to non-signaling receptor α-unit (8Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar), whereas LIF and oncostatin M directly bind to LIFR and gp130, respectively (17Gearing D.P. Ziegler S.F. Comeau M.R. Friend D. Thoma B. Cosman D. Park L. Mosley B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1119-1123Crossref PubMed Scopus (123) Google Scholar). In contrast, binding of IL-6 to a non-signaling α-unit, gp80 (IL-6Rα), induces gp130 homodimerization and subsequent activation of Jak/Tyk kinases (8Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Immunoprecipitation experiments in solution as well as biophysical evidence suggested that the IL-6 and CNTF receptor complexes are hexamers consisting of IL-6, IL-6Rα, and gp130 in a 2:2:2 stoichiometry (18Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A.L. Toniatti C. Ciliberto G. EMBO J. 1995; 14: 1942-1951Crossref PubMed Scopus (210) Google Scholar, 19Ward L.D. Howlett G.J. Discolo G. Yasukawa K. Hammacher A. Moritz R.L. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Abstract Full Text PDF PubMed Google Scholar) or CNTF, CNTFRα, gp130, and LIFR in a 2:2:1:1 ratio (20De Serio A. Graziani R. Laufer R. Ciliberto G. Paonessa G. J. Mol. Biol. 1995; 254: 795-800Crossref PubMed Scopus (49) Google Scholar). However, recent analyses of crystallographic and mutagenesis data of CNTF suggested a tetrameric complex as the simplest model of the CNTFR complex (13McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (129) Google Scholar, 21Panayotatos N. Radziejewska E. Acheson A. Somogyi R. Thadani A. Hendrickson W.A. McDonald N.Q. J. Biol. Chem. 1995; 270: 14007-14014Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Furthermore, an arrangement of cytokine and cytokine receptors as in the hexameric models of Paonessa and de Serio (18Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A.L. Toniatti C. Ciliberto G. EMBO J. 1995; 14: 1942-1951Crossref PubMed Scopus (210) Google Scholar, 20De Serio A. Graziani R. Laufer R. Ciliberto G. Paonessa G. J. Mol. Biol. 1995; 254: 795-800Crossref PubMed Scopus (49) Google Scholar) has been considered to be inconsistent with the known dimensions of IL-6 and CNTF (22Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). Other authors argued that structural constraints of the above hexameric models would lead to contradictions with the hitherto observed general conservation of the structural arrangement of cytokine receptors (23Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (303) Google Scholar). A new hexameric model for the IL-6 receptor complex could alleviate these structural objections (23Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (303) Google Scholar). Adapted to the CNTFR receptor complex it nevertheless implies, as does the model suggested by de Serio et al. (20De Serio A. Graziani R. Laufer R. Ciliberto G. Paonessa G. J. Mol. Biol. 1995; 254: 795-800Crossref PubMed Scopus (49) Google Scholar) that the same site of CNTF is able to contact either gp130 or LIFR which rules out the existence of a genuine and specific LIFR-binding site on CNTF. Mutagenesis studies of CNTF, IL-6, and LIF have identified contact sites of these cytokines with the subunits of their respective receptor complexes. For IL-6 and CNTF the contact site with the receptor α-unit could be mapped to a site that includes residues of the C-terminal AB loop and the C-terminal D-helix (13McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (129) Google Scholar, 21Panayotatos N. Radziejewska E. Acheson A. Somogyi R. Thadani A. Hendrickson W.A. McDonald N.Q. J. Biol. Chem. 1995; 270: 14007-14014Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 22Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar, 24Leebeek F.W. Kariya K. Schwabe M. Fowlkes D.M. J. Biol. Chem. 1992; 267: 14832-14838Abstract Full Text PDF PubMed Google Scholar, 25Toniatti C. Cabibbo A. Sporena E. Salvati A.L. Cerretani M. Serafini S. Lahm A. Cortese R. Ciliberto G. EMBO J. 1996; 15: 2726-2737Crossref PubMed Scopus (51) Google Scholar, 26Saggio I. Gloaguen I. Poiana G. Laufer R. EMBO J. 1995; 14: 3045-3054Crossref PubMed Scopus (55) Google Scholar, 27Ehlers M. Grötzinger J. deHon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar, 28Krüttgen A. Grötzinger J. Kurapkat G. Weis J. Simon R. Thier M. Schroder M. Heinrich P. Wollmer A. Comeau M. Müllberg J. Rose-John S. Biochem. J. 1995; 309: 215-220Crossref PubMed Scopus (28) Google Scholar). This site corresponds to site I of growth hormone in its receptor complex (29De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2029) Google Scholar). Residues of the A- and C-helices of CNTF, LIF, and IL-6 constitute a gp130-binding site which is equivalent to site II of growth hormone in its receptor complex (21Panayotatos N. Radziejewska E. Acheson A. Somogyi R. Thadani A. Hendrickson W.A. McDonald N.Q. J. Biol. Chem. 1995; 270: 14007-14014Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 30Hudson K.R. Vernallis A.B. Heath J.K. J. Biol. Chem. 1996; 271: 11971-11978Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Vernallis A.B. Hudson K.R. Heath J.K. J. Biol. Chem. 1997; 272: 26947-26952Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 32Savino R. Ciapponi L. Lahm A. Demartis A. Cabibbo A. Toniatti C. Delmastro P. Altamura S. Ciliberto G. EMBO J. 1994; 13: 5863-5870Crossref PubMed Scopus (129) Google Scholar). In IL-6, a second gp130-binding site consists of amino acids residues of the N-terminal AB loop, the C-terminal CD loop, and the N-terminal D-helix (27Ehlers M. Grötzinger J. deHon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar, 33Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This site is now termed site III in continuation of the growth hormone terminology. Crystallographic and mutagenesis studies of CNTF and LIF indicated that residues of the C-terminal B-helix, possibly the BC loop, CD loop, and the N-terminal D-helix constitute site III in these cytokines (13McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (129) Google Scholar, 21Panayotatos N. Radziejewska E. Acheson A. Somogyi R. Thadani A. Hendrickson W.A. McDonald N.Q. J. Biol. Chem. 1995; 270: 14007-14014Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 30Hudson K.R. Vernallis A.B. Heath J.K. J. Biol. Chem. 1996; 271: 11971-11978Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 34Di Marco A. Gloaguen I. Graziani R. Paonessa G. Saggio I. Hudson K.R. Laufer R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9247-9252Crossref PubMed Scopus (51) Google Scholar, 35Owczarek C.M. Layton M.J. Metcalf D. Lock P. Willson T.A. Gough N.M. Nicola N.A. EMBO J. 1993; 12: 3487-3495Crossref PubMed Scopus (41) Google Scholar). These experiments also suggested site III as a potential LIFR binding epitope in LIF and CNTF. Considering the conserved four-helical bundle structure of most cytokines we reasoned that receptor recognition sites of cytokines might have evolved as discontinuous modules which should principally be exchangeable between different cytokines. A comparison of the homology based IL-6 model and the x-ray structure of CNTF (13McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (129) Google Scholar, 27Ehlers M. Grötzinger J. deHon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar) prompted us to define boundaries of the potential LIFR binding epitope of CNTF which encompasses residues of the C-terminal A-helix, the N-terminal AB loop, the BC loop, the C-terminal CD-loop, and the N-terminal D-helix. The transfer of this putative "LIFR binding module" from CNTF to IL-6 resulted in a chimeric IL-6/CNTF molecule that binds to IL-6Rα and signals via a heterodimer of gp130 and LIFR. Effectively, this "module swap" created a new cytokine with LIF-like, but IL-6R dependent activity on cells expressing gp130, IL-6Rα, and LIFR. On a more general basis, our results indicate that cytokines are organized as a set of modules, making specific contacts to different receptors. Human SK-N-MC neuroblastoma, HepG2 and Hep3B hepatoma and COS-7 cells (bought from ATCC (Manassas, VA)) were routinely grown in RPMI or Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. BAF/3 cells transfected with human gp130 were a kind gift from Immunex (Seattle, WA). BAF/3-(gp130) cells were stably transfected with cDNAs coding for human IL-6R, human CNTFR, or human LIFR as described elsewhere (17Gearing D.P. Ziegler S.F. Comeau M.R. Friend D. Thoma B. Cosman D. Park L. Mosley B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1119-1123Crossref PubMed Scopus (123) Google Scholar). Thus four different cell lines were generated: BAF/3-(gp130,IL-6R) cells, BAF/3-(gp130,LIFR) cells, BAF/3-(gp130,LIFR,IL-6R) cells, and BAF/3-(gp130,LIFR,CNTFR) cells. The relative expression of the receptors of the IL-6 family on the different BAF/3 cell lines as well as the SK-N-MC cells was analyzed by fluorescence-activated cell sorter analysis using the murine monoclonal antibodies B-S12 (anti-gp130), B-R6 (anti-IL-6R), AN-E1 (anti-LIFR), and AN-D3 (anti-CNTFR) and is shown in Table I. B-S12 and B-R6 have been described in detail before (36Fourcin M. Chevalier S. Guillet C. Robledo O. Froger J. PouplardBarthelaix A. Gascan H. J. Biol. Chem. 1996; 271: 11756-11760Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), AN-E1 and AN-D3 are newly developed murine monoclonal anti-LIFR and anti-CNTFR antibodies. 2H. Gascan, manuscript in preparation. Anti-STAT3 mAb was obtained from Transduction Laboratories (Lexington, KY), anti-phosphotyrosine mAb 4G10 was bought from UBI (Lake Placid, NY), and the anti-mouse peroxidase-coupled mAb was from BIOSOURCE (Calmarillo, CA). The restriction enzymes NcoI, HindIII, and XbaI were obtained from AGS (Heidelberg, Germany); calf intestinal phosphatase was bought from Boehringer Mannheim (Mannheim, Germany). The restriction enzyme AccI, Vent DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs (Schwalbach, Germany), the T7 sequencing kit was from Pharmacia (Freiburg, Germany). [α-32P]dATP, [thio-α-35S]dATP, and [3H]thymidine were obtained from Amersham International. Oligonucleotides were bought from Eurogentec (Seraing, Belgium), Brij-96 and Nonidet P-40 from Sigma (Munich, Germany).Table IRelative expression of receptors of the IL-6 family present in the BAF/3 cell lines and SK-N-MC cells used in this studyCell lineIL-6RCNTFRgp130LIFRSK-N-MCNegative13.85HepG21Negative6.4PositiveHep3BPositiveNegativePositiveNegativeBAF/3-(gp130,IL-6R)2.5Negative1NegativeBAF/3-(gp130,IL-6R,LIFR)3.3Negative11BAF/3-(gp130,LIFR)NegativeNegative11BAF/3-(gp130,LIFR,CNTFR)Negative811Surface expression of the receptors of the IL-6 family on SK-N-MC and transfected BAF/3 cells was monitored by FACS analysis and related to the receptor with the lowest surface expression which was defined as 1. The antibodies used are listed under "Experimental Procedures." FACS data were also available for expression of IL-6R and gp130 on HepG2, the other information concerning HepG2 and Hep3B cells were taken from the literature (6Baumann H. Ziegler S.F. Mosley B. Morella K.K. Pajovic S. Gearing D.P. J. Biol. Chem. 1993; 268: 8414-8417Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab Surface expression of the receptors of the IL-6 family on SK-N-MC and transfected BAF/3 cells was monitored by FACS analysis and related to the receptor with the lowest surface expression which was defined as 1. The antibodies used are listed under "Experimental Procedures." FACS data were also available for expression of IL-6R and gp130 on HepG2, the other information concerning HepG2 and Hep3B cells were taken from the literature (6Baumann H. Ziegler S.F. Mosley B. Morella K.K. Pajovic S. Gearing D.P. J. Biol. Chem. 1993; 268: 8414-8417Abstract Full Text PDF PubMed Google Scholar). The discrete regions of human IL-6 and CNTF used to construct the chimeras are given in Fig. 1. Construction of chimeras IC1, IC2, and IC3 relied heavily on PCR-ligation-PCR (37Ali S.A. Steinkasserer A. BioTechniques. 1995; 18: 746-750PubMed Google Scholar). cDNAs of human IL-6 and CNTF cloned into the pRSET5d bacterial expression vector via NcoI and HindIII restriction sites served as PCR templates (27Ehlers M. Grötzinger J. deHon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar,28Krüttgen A. Grötzinger J. Kurapkat G. Weis J. Simon R. Thier M. Schroder M. Heinrich P. Wollmer A. Comeau M. Müllberg J. Rose-John S. Biochem. J. 1995; 309: 215-220Crossref PubMed Scopus (28) Google Scholar). To construct chimera IC1, an IL-6 cDNA fragment encoding the N-terminal part of the molecule to Arg40 and a C-terminal CNTF cDNA fragment starting at the codon for Asp36 were amplified by standard PCR. The PCR products were ligated (37Ali S.A. Steinkasserer A. BioTechniques. 1995; 18: 746-750PubMed Google Scholar) and the ligation product subsequently amplified by PCR to produce the fragment IL-6(Pro1-Arg40):CNTF(Glu36-Met56). This fragment was purified from a 1% agarose gel and ligated to the amplified C-terminal IL-6 cDNA fragment starting at the codon for Asn60. The ligation product, IC1, was amplified by PCR and subsequently cloned into the bacterial pRSET5d expression vector after digestion with NcoI and HindIII. Chimeras IC2 and IC3 were constructed analogously. The sequences of all primers used are available on request. Chimeras IC4 to IC6 were produced from chimeras IC1 to IC3 using NcoI, AccI, XbaI, andHindIII restriction enzymes. IC7 was produced from IC1 and IC6. The integrity of all constructs was verified by restriction fragment analysis and DNA sequencing according to standard methods (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Figure 1The site III gp130 and LIFR binding epitope of IL-6 and CNTF, respectively. A, schematic drawing of the common four-helix bundle cytokine fold. B, ribbon models of the recently solved IL-6 NMR and CNTF x-ray structures. The different parts of site III are color coded: yellow (site IIIA),green (site IIIB), and blue (site IIIC).C, bar representation of IL-6, CNTF, and chimeras IC1 to IC7. Sequence stretches that are part of the exchanged epitopes of IL-6 and CNTF are hatched. On CNTF the N- and C-terminal amino acid residues of the transferred stretches are designated insingle letter code, on IL-6 the residues adjacent to the transferred CNTF stretches are denoted. The symbols locatedleft of the bars are kept throughout the figures of this article to mark the respective mutant or natural cytokine.D, SDS-PAGE (12.5%) of the purified IL-6/CNTF chimeras after staining with Coomassie Blue. IC6 could not be expressed in our bacterial expression systems.View Large Image Figure ViewerDownload (PPT) The boundaries of the IL-6 and CNTF regions exchanged were derived from a molecular model of IL-6 (27Ehlers M. Grötzinger J. deHon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar) and the x-ray structure of CNTF as taken from the Brookhaven data bank (accession code 1cnt). Recently, the x-ray as well as the NMR structure of human IL-6 were solved (12Somers W. Stahl M. Seehra J.S. EMBO J. 1997; 16: 989-997Crossref PubMed Scopus (226) Google Scholar, 39Xu G.Y. Yu H.A. Hong J. Stahl M. McDonagh T. Kay L.E. Cumming D.A. J. Mol. Biol. 1997; 268: 468-481Crossref PubMed Scopus (66) Google Scholar), the regions interchanged are color coded on the ribbon models of the IL-6 NMR and CNTF x-ray structures. Structure comparisons and all computer graphic work were performed with the WHATIF program package running on an SGI-Indigo2 (40Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar). For graphical representation the program Grasp was used (41Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar). Escherichia coli bacteria (strains BL21-DE3 and BL21 pLysS) were transformed with the expression vector pRSET5d containing human IL-6, human CNTF, and chimeric cDNAs. Transformed bacteria were grown to an A 600 of approximately 0.5–0.7 and induced to produce recombinant protein by addition of 0.4 mm isopropyl-1-thio-β-d-galactopyranoside. After 2 h, purification of inclusion bodies and denaturation with 6 m guanidinium chloride was performed as described before (42van Dam M. Müllberg J. Schooltink H. Stoyan T. Brakenhoff J.P. Graeve L. Heinrich P.C. Rose-John S. J. Biol. Chem. 1993; 268: 15285-15290Abstract Full Text PDF PubMed Google Scholar). Refolding of proteins was achieved by dialysis against refolding buffer (1 m guanidinium chloride, 3 mm oxidized glutathione, 0.6 mm reduced glutathione, 12 h) and 20 mm Tris-Cl, pH 6.8 (12 h). The purity of the recombinant proteins was ascertained by 12.5% SDS-PAGE, stained with Coomassie Blue or silver. In addition, protein concentrations were determined by hydrolysis of the proteins in 6 m HCl and subsequent amino acid analysis. LPS concentrations in the purified protein preparations were ascertained with the Limulus amobecyte lysate assay (Biowhittaker, Walkersville, MD). CD spectra of all chimeras were taken on an AVIV CD spectrometer 62DS and on a Jasco J-600 spectropolarimeter. Both instruments were calibrated with an aqueous solution of 10-camphosulfonic acid at 25 °C. The spectral band width was 1.5 nm. Protein samples were dissolved in water, the pH was adjusted to 3.5. Proliferation of at least two different clones of the transfected BAF/3-(gp130) cell lines in response to human IL-6, human CNTF, human LIF, and the chimeras IC1 to IC7 was measured in 96-well microtiter plates. The cells were exposed to test samples for 72 h and subsequently pulse-labeled with [3H]thymidine for 4 h. Proliferation rates were measured by harvesting the cells on glass filters and determination of the incorporated radioactivity by scintillation counting. For each mutant and each cell line the proliferation assay was performed at least three times in triplicate. Haptoglobin production by HepG2 and Hep3B cells in response to stimulation with the above cytokines and chimeras was measured by a sandwich enzyme-linked immunosorbent assay as described recently (43Oppmann B. Stoyan T. Fischer M. Voltz N. März P. Rose-John S. J. Immunol. Methods. 1996; 195: 153-159Crossref PubMed Scopus (9) Google Scholar). COS-7 cells were simultaneously transfected with cDNAs for human gp130, LIFR, IL-6R, and STAT3 with the DEAE-dextran method as described (44Renné C. Kallen K.-J. Müllberg J. Jostock T. Grötzinger J. Rose-John S. J. Biol. Chem. 1998; 273: 27213-27319Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). gp130 and LIFR were subcloned into the p409 expression vector (a kind gift from Immunex). The human IL-6R was in the pCDM8 expression vector, STAT3 in pSVL. After transfection, COS-7 cells were cultured for 2 days in RPMI plus 10% fetal calf serum before starvation in serum-free medium. SK-N-MC, HepG2, and transfected COS-7 cells were starved overnight in serum-free medium before stimulation with cytokines. After stimulation, cells were lysed in 50 mm Tris, pH 7.5, 100 mm NaCl, 50 mm sodium fluoride, and 3 mm sodium orthovanadate containing 1% Brij-96. Insoluble material was pelleted and the supernatants immunoprecipitated overnight with B-S12 anti-gp130 (2 μg/ml) or AN-E1 anti-LIFR mAb (10 μg/ml). The complexes were isolated with Protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) and the supernatants subjected to a second immunoprecipitation with anti-phosphotyrosine mAb 4G10 (5 μg/ml) after addition of 1% Nonidet P-40. The precipitates were subjected to SDS-PAGE and transferred to an Immobilon membrane (Millipore, Bedford, MA). The membranes were incubated with an appropriate primary antibody before being labeled with a secondary antibody coupled to peroxidase. Subsequently, the membranes were developed using the Amersham ECL kit. The four-helix bundle fold (Fig. 1 A) is the characteristic structure of most cytokines (11Sprang S.R. Bazan J.F. Curr. Opin. Struct. Biol. 1993; 3: 815-827Crossref Scopus (240) Google Scholar). A schematic representation of the backbone atoms of the IL-6 NMR and the CNTF x-ray structure is shown in Fig. 1 B. Both structures were superimposed onto each other using the Cα-atoms of the helices to identify putative components of the site III receptor binding epitope. The segments of IL-6 and CNTF which participated in the "epitope shuffle" of site III are color coded and designated as IIIA, IIIB, and IIIC, respectively (Fig. 1, A andB). A structural analysis of CNTF suggested that amino acid residues situated in the C-terminal A-helix (Glu36-Gln42) and N-terminal AB loop (Gly43-Met56) form site IIIA, while the BC loop (His97-Asp104) with adjacent residues of the B- (Leu91-Val96) and C-helix (Phe105-Ile109) represents site IIIB. Together with site IIIC which closely corresponds to Bazan's D1 motif (10Bazan J.F. Neuron. 1991; 7: 197-208Abstract Full Text PDF PubMed Scopus (420) Google Scholar) and consists of the C-terminal CD loop (Gly147-Leu151) and the N-terminal D-helix (Phe152-Leu162) they constitute the putative LIFR-binding epitope on CNTF (site III). Potential site III residue
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