Determination of the Disulfide Structure andN-Glycosylation Sites of the Extracellular Domain of the Human Signal Transducer gp130
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m009979200
ISSN1083-351X
AutoresRobert L. Moritz, Nathan E. Hall, Lisa Connolly, Richard J. Simpson,
Tópico(s)Immune Response and Inflammation
Resumogp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys279 and Cys469), and one (Cys397) is modified byS-cysteinylation. Of the 11 potentialN-glycosylation sites, Asn21, Asn61, Asn109, Asn135, Asn205, Asn357, Asn361, Asn531, and Asn542 are glycosylated but not Asn224 and Asn368. The disulfide bonds, Cys112–Cys122 and Cys150–Cys160, are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys6–Cys32 and Cys26–Cys81) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys436–Cys444. We have created a model predicting that this loop maintains Cys469 in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130. gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys279 and Cys469), and one (Cys397) is modified byS-cysteinylation. Of the 11 potentialN-glycosylation sites, Asn21, Asn61, Asn109, Asn135, Asn205, Asn357, Asn361, Asn531, and Asn542 are glycosylated but not Asn224 and Asn368. The disulfide bonds, Cys112–Cys122 and Cys150–Cys160, are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys6–Cys32 and Cys26–Cys81) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys436–Cys444. We have created a model predicting that this loop maintains Cys469 in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130. interleukin granulocyte colony-stimulating factor GCSF receptor fibronectin type III cytokine-binding region soluble viral mass spectrometry collision-induced dissociation electrospray ionization polyacrylamide gel electrophoresis reverse phase high-performance liquid chromatography Pec,S-β-(4-pyridylethyl)-cysteine leukemia inhibitory factor isoelectrofocusing IL-6 receptor Chinese hamster ovary dithiothreitol matrix-assisted laser desorption ionization time-of-flight phenylthiohydantoin The family of cytokines that signal through the common receptor subunit gp130 (referred to as the gp130 cytokines) comprises interleukin (IL)1-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, oncostatin M, cardiotrophin-1, herpesvirus IL-6, interleukin-11 (IL-11), and neurotrophin-1/B cell-stimulatory factor-3/cardiotrophin-like cytokine. These cytokines play a pivotal role in the immune, hematopoietic and nervous systems, as well as in bone metabolism, inflammation, and the acute-phase response (1Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1996; 6: 929-955Crossref Scopus (303) Google Scholar, 2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar, 3Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1757) Google Scholar, 4Elson G.C. Lelievre E. Guillet C. Chevalier S. Plun-Favreau H. Froger J. Suard I. de Coignac A.B. Delneste Y. Bonnefoy J.Y. Gauchat J.F. Gascan H. Nat. Neurosci. 2000; 3: 867-872Crossref PubMed Scopus (220) Google Scholar). The gp130 cytokine family are characterized by a four-α-helix bundle structure, the helices being connected in an up-up-down-down arrangement by three polypeptide loops (3Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1757) Google Scholar, 5Bravo J. Heath J.K. EMBO J. 2000; 19: 2399-2411Crossref PubMed Google Scholar). These cytokines signal through their respective receptor systems that comprise a ligand-specific α-subunit and the common signal-transducing β-subunit, gp130. The shared use of gp130, in part, provides a molecular basis for the functional redundancy of the gp130 cytokines. The result of gp130-mediated signaling is the regulation of a variety of complex cellular processes such as proliferation, differentiation, and gene activation in a wide variety of adult tissue systems, owing to the ubiquitous expression of gp130 (6Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar). Insights into the possible in vivo role of gp130 have come from transgenic and knock-out animal studies. Targeted inactivation of the gp130 gene results in a prenatal lethal phenotype that includes defects in the cardiac and hematopoietic systems (7Yoshida K. Taga T. Saito M. Suematsu S. Kumanogoh A. Tanaka T. Fujiwara H. Hirata M. Yamagami T. Nakahata T. Hirabayashi T. Yoneda Y. Tanaka K. Wang W.Z. Mori C. Shiota K. Yoshida N. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 407-411Crossref PubMed Scopus (574) Google Scholar, 8Betz U.A.K. Bloch W. van den, B. M. Yoshida K. Taga T. Kishimoto T. Addicks K. Rajewsky K. Muller W. J. Exp. Med. 1998; 188: 1955-1965Crossref PubMed Scopus (188) Google Scholar, 9Hirota H. Chen J. Betz U.A. Rajewsky K. Gu Y. Ross Jr., J. Muller W. Chien K.R. Cell. 1999; 97: 189-198Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). Chronic activation of gp130 signaling in a transgenic mouse model results in cardiac hypertrophy (10Hirota H. Yoshida K. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4862-4866Crossref PubMed Scopus (451) Google Scholar). Intracellular signaling by the gp130 cytokines is initiated by the ligand first making low affinity (∼1 nm) contact with its cognate receptor α-subunit, which then recruits the signal transducing gp130 subunit with a resulting ligand-binding affinity of ∼10 pm. Importantly, receptor activation depends on homodimerization or heterodimerization of gp130 (1Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1996; 6: 929-955Crossref Scopus (303) Google Scholar, 3Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1757) Google Scholar). gp130 was initially cloned as a component of the IL-6 receptor complex (11Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1104) Google Scholar). The binding of IL-6 to its cognate receptor subunit (IL-6R) induces the dimerization of gp130 (12Murakami M. Hibi M. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. Science. 1993; 260: 1808-1810Crossref PubMed Scopus (645) Google Scholar) and the formation of a hexameric complex comprising two molecules each of IL-6, IL-6R, and gp130 (13Ward 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, 14Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A.L. Toniatti C. Ciliberto G. EMBO. 1995; 14: 1942-1951Crossref PubMed Scopus (210) Google Scholar). The IL-11 receptor complex is similar (15Barton V.A. Hall M.A. Hudson K.R. Heath J.K. J. Biol. Chem. 2000; 275: 36197-36203Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), whereas in the case of LIF (16Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (611) Google Scholar), oncostatin M (17Gearing D.P. Bruce A.G. New Biol. 1992; 4: 61-65PubMed Google Scholar, 18Liu J. Modrell B. Aruffo A. Marken J.S. Taga T. Yasukawa K. Murakami M. Kishimoto T. Shoyab M. J. Biol. Chem. 1992; 267: 16763-16766Abstract Full Text PDF PubMed Google Scholar), cardiotrophin-1 (19Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar), and ciliary neurotrophic factor (16Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (611) Google Scholar), ligand binding induces heterodimerization of gp130 and the LIF receptor, another signal transducing subunit (20Gearing D.P. Thut C.J. VandeBos T. Gimpel S.D. Delaney P.B. King J. Price V. Cosman D. Beckmann M.P. EMBO J. 1991; 10: 2839-2848Crossref PubMed Scopus (520) Google Scholar). Soluble forms of many of the gp130 cytokine receptors, including gp130, have been found in body fluids of different mammalian species (3Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1757) Google Scholar). Soluble gp130 (sgp130) has been detected in human serum (21Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar) and is most likely translated from alternatively spliced mRNA (22Diamant M. Rieneck K. Mechti N. Zhang X.G. Svenson M. Bendtzen K. Klein B. FEBS Lett. 1997; 412: 379-384Crossref PubMed Scopus (67) Google Scholar). sgp130 can neutralize IL-6·sIL-6R complexes, thereby acting as an antagonist (21Narazaki M. Yasukawa K. Saito T. Ohsugi Y. Fukui H. Koishihara Y. Yancopoulos G.D. Taga T. Kishimoto T. Blood. 1993; 82: 1120-1126Crossref PubMed Google Scholar). The cDNA of human gp130 encodes a protein of 918 amino acids (11Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1104) Google Scholar), including a signal peptide of 22 amino acids, an extracellular domain of 597 amino acids (sgp130), a transmembrane domain of 22 amino acids, and an intracellular domain of 277 amino acids. The amino acid sequence of murine (23Saito M. Yoshida K. Hibi M. Taga T. Kishimoto T. J. Immunol. 1992; 148: 4066-4071PubMed Google Scholar) and rat gp130 (24Wang Y. Nesbitt J.E. Fuentes N.L. Fuller G.M. Genomics. 1992; 14: 666-672Crossref PubMed Scopus (44) Google Scholar) share an overall sequence identity of 85 and 88%, respectively, with that of human gp130. There are 11 potential N-linked glycosylation sequons in the extracellular domain of the predicted 101-kDa human gp130 (11Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1104) Google Scholar), suggesting that the mature ∼130-kDa gp130 (11Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1104) Google Scholar) is highly glycosylated. The extracellular region of gp130 has a modular structure consisting of six domains of ∼100 amino acids each. The NH2-terminal domain (domain 1) is predicted to be a member of the immunoglobulin superfamily (Ig-like) (25Bork P. Holm L. Sander C. J. Mol. Biol. 1994; 242: 309-320PubMed Google Scholar, 26Vaughn D.E. Bjorkman P.J. Neuron. 1996; 16: 261-273Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), which has "Greek key" β-sheet topology (27Richardson J.S. Nature. 1977; 268: 495-500Crossref PubMed Scopus (468) Google Scholar). In the Ig-like fold, neighboring β-strands form hydrogen bonds in an antiparallel fashion to form a β-pleated sheet, and two β-sheets pack against each other to produce a hydrophobic core. Domains 2–6 of the gp130 are classified as fibronectin type III (FN III)-like modules, a subclass of the β-sandwich fold (26Vaughn D.E. Bjorkman P.J. Neuron. 1996; 16: 261-273Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The topology of these domains is similar to those of the Ig-like modules, the notable exception being the "sheet switching" of β-strand D from the first β-sheet of an Ig-like domain to form β-strand C′ on the second β-sheet of FN III domains. The two membrane distal FN III domains (domains 2 and 3) form the cytokine-binding region (CBR) that is characteristic of class I cytokine receptors (28Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1881) Google Scholar, 29Nicola N.A. Guidebook to Cytokines and Their Receptors. Oxford Press, Oxford1994: 1-16Google Scholar). CBRs are characterized by two conserved disulfide bonds in their NH2-terminal domain and a WSXWS motif in their COOH-terminal domain. The functional anatomy of the extracellular region of gp130 is still poorly understood. A truncated form of gp130 lacking the membrane-proximal FN III modules and the cytoplasmic and transmembrane domains has been shown to bind a complex of either IL-6·sIL-6R or LIF·sLIF receptor (30Horsten U. Schmitz-Van de Leur H. Müllberg J. Heinrich P.C. Rose-John S. FEBS Lett. 1995; 360: 43-46Crossref PubMed Scopus (44) Google Scholar, 31Zhang J.G. Zhang Y. Owczarek C.M. Ward L.D. Moritz R.L. Simpson R.J. Yasukawa K. Nicola N.A. J. Biol. Chem. 1998; 273: 10798-10805Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Val252 in the BC loop of the COOH-terminal domain of the CBR (domain 3 of gp130) has been implicated in the interaction of gp130 with IL-6·IL-6R (32Horsten U. Muller-Newen G. Gerhartz C. Wollmer A. Wijdenes J. Heinrich P.C. Grotzinger J. J. Biol. Chem. 1997; 272: 23748-23757Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and IL-11·IL-11 receptor (33Dahmen H. Horsten U. Kuster A. Jacques Y. Minvielle S. Kerr I.M. Ciliberto G. Paonessa G. Heinrich P.C. Muller-Newen G. Biochem. J. 1998; 331: 695-702Crossref PubMed Scopus (69) Google Scholar). The location of this residue corresponds to a tryptophan residue in the growth hormone receptor that has been shown to be critical for ligand binding (34Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1793) Google Scholar), suggesting a conserved mode of ligand binding among the cytokine receptor superfamily. The Ig-like module of gp130 (domain 1) has been shown to be involved in the interaction of gp130 with ligands that induce homodimerization of gp130 (IL-6 (35Moritz R.L. Ward L.D. Tu G.-F. Fabri L.J. Ji H. Yasukawa K. Simpson R.J. Growth Factors. 1999; 16: 265-278Crossref PubMed Scopus (24) Google Scholar, 36Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and IL-11 (37Kurth I. Horsten U. Pflanz S. Dahmen H. Kuster A. Grotzinger J. Heinrich P.C. Muller-Newen G. J. Immunol. 1999; 162: 1480-1487PubMed Google Scholar)) and essential for the formation of high affinity hexameric complexes (15Barton V.A. Hall M.A. Hudson K.R. Heath J.K. J. Biol. Chem. 2000; 275: 36197-36203Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 35Moritz R.L. Ward L.D. Tu G.-F. Fabri L.J. Ji H. Yasukawa K. Simpson R.J. Growth Factors. 1999; 16: 265-278Crossref PubMed Scopus (24) Google Scholar). This domain is thought to bind site III in IL-6 and IL-11 (35Moritz R.L. Ward L.D. Tu G.-F. Fabri L.J. Ji H. Yasukawa K. Simpson R.J. Growth Factors. 1999; 16: 265-278Crossref PubMed Scopus (24) Google Scholar), and Ig-like modules in the GCSFR and LIF receptor are also thought to make contact with their cognate ligands (38Hiraoka O. Anaguchi H. Asakura A. Ota Y. J. Biol. Chem. 1995; 270: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 39Owczarek C.M. Zhang Y. Layton M.J. Metcalf D. Roberts B. Nicola N.A. J. Biol. Chem. 1997; 272: 23976-23985Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The three membrane-proximal FN III modules of gp130, although not directly involved in ligand binding, may be required for gp130 dimerization by ligands such as IL-6 (40Hammacher A. Wijdenes J. Hilton D.J. Nicola N.A. Simpson R.J. Layton J.E. Biochem. J. 2000; 345: 25-32Crossref PubMed Scopus (29) Google Scholar) and IL-11 (41Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO. 1994; 13: 4765-4775Crossref PubMed Scopus (256) Google Scholar) or transmembrane signaling events such as stabilization and/or orientation of transmembrane receptor dimers (5Bravo J. Heath J.K. EMBO J. 2000; 19: 2399-2411Crossref PubMed Google Scholar). The structure of the COOH-terminal domain of the gp130 CBR (domain 3) has been determined by NMR (42Kernebeck T. Pflanz S. Muller-Newen G. Kurapkat G. Scheek R.M. Dijkstra K. Heinrich P.C. Wollmer A. Grzesiek S. Grotzinger J. Protein Sci. 1999; 8: 5-12Crossref PubMed Scopus (24) Google Scholar), and the complete CBR (i.e.domains 2 and 3) by x-ray crystallography (43Bravo J. Staunton D. Heath J.K. Jones E.Y. EMBO J. 1998; 17: 1665-1674Crossref PubMed Scopus (119) Google Scholar); however, there is no complete structure for gp130 or any member of the gp130 family of receptors. To elucidate the tertiary structure of the gp130 extracellular region, we have purified human sgp130 using a Chinese hamster ovary (CHO) cell expression system (13Ward 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, 44Yasukawa K. Futatsugi K. Saito M. Yawata H. Narazaki M. Suzuki H. Taga T. Kishimoto T. Immunol. Lett. 1992; 31: 123-130Crossref PubMed Scopus (69) Google Scholar). This form of human sgp130 contains 11 potential N-linked glycosylation sites and 13 cysteine residues. Four cysteines are located in the Ig-like domain (domain 1), four are in the NH2-terminal FN III domain of the CBR (domain 2), one is in the COOH-terminal FN III domain of the CBR (domain 3), one is in domain 4, and three are in domain 5. Previously, we have shown that affinity purified sgp130 bound a binary complex of IL-6·sIL-6R with a 2:2:2 stoichiometry (13Ward 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). Here, reverse phase HPLC peptide mapping under reducing and nonreducing conditions in combination with mass spectrometric and NH2-terminal sequence analysis was used to determine the cysteine connectivities and carbohydrate attachment sites for sgp130. 4-Vinylpyridine was purchased from Aldrich. Trifluoroacetic acid (HPLC/Spectro grade) was from Pierce. Dithiothreitol (DTT, Ultrol grade) was obtained from Calbiochem/Novabiochem. Sequencing grade trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), pepsin (EC 3.4.23.1), and neuraminidase (EC3.2.1.18) were from Roche Molecular Biochemicals. An endoglycosidase preparation from Flavobacterium meningosepticum (45Elder J.H. Alexander S. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4540-4544Crossref PubMed Scopus (487) Google Scholar) containing three β-N-acetylglucosidase F (endo F) activities (F1, F2, and F3) as well as peptide N-glycosidase (46Plummer Jr., T.H. Elder J.H. Alexander S. Phelan A.W. Tarentino A.L. J. Biol. Chem. 1984; 259: 10700-10704Abstract Full Text PDF PubMed Google Scholar, 47Trimble R.B. Tarentino A.L. J. Biol. Chem. 1991; 266: 1646-1651Abstract Full Text PDF PubMed Google Scholar) was a kind gift from Dr. G. E. Norris (Massey University, New Zealand). HPLC grade solvents were from Mallinckrodt, and all other buffers and reagents (Analar grade) were from BDH. Anti-human gp130 monoclonal antibodies AM64, GPZ35, GPX7, and GPX22 (11Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1104) Google Scholar, 48Saito T. Taga T. Miki D. Futatsugi K. Yawata H. Kishimoto T. Yasukawa K. J. Immunol. Methods. 1993; 163: 217-223Crossref PubMed Scopus (44) Google Scholar) were from Dr. K. Yasukawa (TOSOH, Tokyo, Japan). All buffers and solutions were prepared with deionized water purified and polished by a tandem Milli-RO and Milli-Q system (Millipore). Reverse phase (RP)-HPLC was performed using either a Vydac 5-μm, 300 Å octadecyl silica column (inner diameter, 250 × 4.6 mm) (Vydac) or a Brownlee RP-300 7-μm, 300 Å octylsilica column (inner diameter, 100 × 4.6 mm) (Applied Biosystems), operated at a flow rate of 0.5 ml/min. For capillary RP-HPLC, a fused silica column (inner diameter, 50 × 0.2 mm) was packed "in-house" with Brownlee RP-300 7-μm, 300 Å octylsilica (49Moritz R.L. Simpson R.J. J. Chromatogr. 1992; 599: 119-130Crossref PubMed Scopus (56) Google Scholar). For SEC, an analytical Superose 12 column (inner diameter, 300 × 10 mm) operated at 0.5 ml/min was employed. Rapid desalting and buffer exchange of proteins was performed using a Fast-desaltingTM column (Sephadex G25; inner diameter, 100 × 10 mm; Amersham Pharmacia Biotech). Chromatography was performed using a HP-1090A Liquid Chromatograph (Agilent Technologies) equipped with a manual injector fitted into the column compartment, and a diode array UV detector was used for real time multiple wavelength monitoring of the column eluent. Samples were collected manually in polypropylene (Eppendorf) tubes. sgp130 was purified from the conditioned medium of CHO (G16) cells stably transfected with a plasmid (pECEdhfrgp620) that encodes the extracellular domain of gp130 truncated at amino acid 621 (44Yasukawa K. Futatsugi K. Saito M. Yawata H. Narazaki M. Suzuki H. Taga T. Kishimoto T. Immunol. Lett. 1992; 31: 123-130Crossref PubMed Scopus (69) Google Scholar). Briefly, transfected CHO cells were grown in a large scale fermentation apparatus with a working volume of 1.25 liters (New Brunswick Celligen plus fermenter). Cell conditioned medium was concentrated (20-fold) by ultrafiltration using a Sartocon Mini apparatus (Sartorius, Germany) fitted with a 30,000 molecular weight cut-off filter. sgp130 was purified from concentrated CHO cell medium by binding to a 10-ml column of AM64-Sepharose and eluting sgp130 with 4 mMgCl2. sgp130 was further purified by preparative SEC (>95% pure, as judged by SDS-PAGE and Western blot analysis), and sgp130-containing fractions were pooled, and buffer was exchanged using a Fast-desaltingTM column prior to storage at −20 °C. Samples were analyzed by SDS-PAGE on precast 4–20% polyacrylamide gels (Novex) using nonreducing or reducing (5.25% (v/v) β-mercaptoethanol) conditions and by isoelectrofocusing (IEF-PAGE) on precast linear pH gradient (pH 3–10) 5% polyacrylamide gels (Novex) according to the manufacturer's instructions. Protein bands were visualized by staining with Coomassie Brilliant Blue as described (50Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). sgp130 (400 μg, 1 mg/ml) in 0.1m sodium phosphate buffer, pH 7.4, containing 0.025m EDTA was treated with 1 unit of neuraminidase and/or endoglycosidase mixture (37 °C, 16 h). Prior to disulfide determination, free cysteine residues in sgp130 (400 μg) were treated with a 5-fold molar excess of 4-vinylpyridine in 0.1m Tris-HCl, pH 8.4, for 1 h at 25 °C in the dark. The modified protein was buffer exchanged using a Fast-desaltingTM column operated at 1 ml/min. 4-Vinylpyridine was chosen as the alkylating agent becauseS-β-(4-pyridylethyl)-cysteine (Pec) containing peptides can be identified by their characteristic absorption spectra at 254 nm (51Friedman M. Krull L.H. Cavins J.F. J. Biol. Chem. 1970; 245: 3868-3871Abstract Full Text PDF PubMed Google Scholar) and are also readily identified during ESI-MS by the presence of an ion of m/z 106 following collision-induced dissociation (CID), characteristic of the protonatedS-pyridylethyl moiety (50Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). sgp130 (400 μg) in 2 ml of 1% (w/v) ammonium bicarbonate, containing 2 mm calcium chloride, was digested at 37 °C for 16 h with either trypsin or chymotrypsin at an enzyme to substrate ratio of 1:20. For pepsin digestion, sgp130 (400 μg, ∼4 nmol) in 5% formic acid was digested for 1 h at 37 °C using an enzyme to substrate ratio of 1:20. Resultant peptides were fractionated by RP-HPLC on either a Vydac C18 column or a Brownlee RP-300 column at 0.5 ml/min at 45 °C. The column eluent was split (∼1:160) post-detector, using a stainless steel Tee-union (52Cole A.R. Hall N.E. Treutlein H.R. Eddes J.S. Reid G.E. Moritz R.L. Simpson R.J. J. Biol. Chem. 1999; 274: 7207-7215Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), whereas the remainder (99.4%) was collected for further analysis. For disulfide-containing peptide identification, a portion of the digest (25%) was reduced with 10 mm DTT at 45 °C for 1 h and rechromatographed under identical conditions. Peaks whose retention times shifted upon reduction were subjected to NH2-terminal sequence analysis. NH2-terminal sequence analyses were performed using a Hewlett-Packard biphasic NH2-terminal protein sequencer (model G1005A, Hewlett-Packard) using version 3.0 chemistry as described (50Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). On-line MS analysis of peptide fractions was performed using either a Finnigan LCQ quadrupole ion trap mass spectrometer or a TSQ-700 triple quadrupole mass spectrometer, both equipped with an ESI source as described (50Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar, 53Moritz R.L. Reid G.E. Ward L.D. Simpson R.J. Methods. 1994; 6: 213-226Crossref Scopus (39) Google Scholar).S-Pyridylethylated peptides were identified by parent-ion scanning using the TSQ mass spectrometer by monitoring all peptides for the labile protonated S-pyridylethyl group (m/z 106) following CID. Source CID/single ion monitoring using the LCQ ion trap mass spectrometer was employed to identify S-pyridylethyl cysteine-containing peptides as described (52Cole A.R. Hall N.E. Treutlein H.R. Eddes J.S. Reid G.E. Moritz R.L. Simpson R.J. J. Biol. Chem. 1999; 274: 7207-7215Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Peptides were identified from their CID product ion spectra using either the Finnigan Xcalibur-Biomass software or Protein Prospector MS-Product algorithm (ProteinProspector Pacific-Rim mirror). Peptides analyzed by MALDI-TOF mass spectrometry (Kompact MALDI-IV fitted with a 337 nm laser, Kratos) were co-crystallized with α-cyano 4-hydroxycinnaminic acid (16 mg of matrix/ml aqueous 60% acetonitrile/0.1% (v/v) trifluoroacetic acid). Matrix (0.5 μl) was deposited onto a clean sample slide immediately followed by 0.5 μl of peptide fraction. Spectra were calibrated using the external standards angiotensin (1297.5 [M+H]1+) and a matrix-derived ion (173.17 [M+H]1+). Domain 5 of sgp130 was modeled using the two FN III domains from the structure of the cytoplasmic tail of human integrin α6β4 (54de Pereda J.M. Wiche G. Liddington R.C. EMBO J. 1999; 18: 4087-4095Crossref PubMed Scopus (49) Google Scholar), which showed 19 and 16% amino acid sequence identity. These particular FN III domains were chosen as templates to minimize the insertions and deletions in the loop regions. The coordinates for the template were taken from the Protein Data Bank entry 1qg3. Domain 5 of sgp130 was manually aligned with the template of the cytoplasmic tail of integrin α6β crystal structure (54de Pereda J.M. Wiche G. Liddington R.C. EMBO J. 1999; 18: 4087-4095Crossref PubMed Scopus (49) Google Scholar), conserving the hydrophobic and sequence patterns of the FN III β-sheets. The MODELLER program (55Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar) was used to generate a model of FN III domain 5. The quality of the model was assessed as described (56Smith D.K. Treutlein H.R. Protein Sci. 1998; 7: 886-896Crossref PubMed Scopus (17) Google Scholar) in particular using the ProsaII program (57Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1792) Google Scholar). A disulfide restraint between Cys436 and Cys444 in FN III domain 5 was introduced in accordance with our experimental results. Over 100 models of domain 5 were generated with the final model being chosen on the basis of the quality checks described. The final model of gp130 domain 5 follows integrin α6β4 domain 1 in the B-C and C′-E loops, and follows domain 2 in the C-C′ loop and G strand. The ProsaII Z-score of the final model is −6.26, comparing favorably with −4.70 and −6.39 for the templates
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