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Disulfide Bond Structure and N-Glycosylation Sites of the Extracellular Domain of the Human Interleukin-6 Receptor

1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês

10.1074/jbc.274.11.7207

ISSN

1083-351X

Autores

Adam R. Cole, Nathan E. Hall, Herbert Treutlein, James S. Eddes, Gavin E. Reid, Robert L. Moritz, Richard J. Simpson,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular “soluble” part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potentialN-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226. The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular “soluble” part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potentialN-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226. Interleukin-6 (IL-6) 1The abbreviations used are: IL, interleukin; sIL-6R, extracellular or soluble domain of the IL-6 receptor; FN III, fibronectin-type III; Ig, immunoglobulin; CBD, cytokine-binding domain; s, soluble; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel; GlcNAc, N-acetylglucosamine; TCEP, tris-(2-carboxyethyl)-phosphine; MS, mass spectrometry; CID, collision-induced dissociation; RP-HPLC, reversed-phase high performance liquid chromatography; EPO, erythropoietin; GH, growth hormone; PRL, prolactin; ESI, electrospray ionization; IT-MS, ion trap mass spectrometry is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities, as well as its potent ability to induce the acute phase response (1Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar, 2Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (303) Google Scholar, 3Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. J. Biochem. 1998; 334: 297-314Crossref Scopus (1757) Google Scholar). Since overexpression of IL-6 has been implicated in the pathology of a number of diseases (for reviews, see Refs. 4Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar and 5Klein B. Zhang X.G. Lu Z.Y. Bataille R. Blood. 1995; 85: 863-872Crossref PubMed Google Scholar), it is anticipated that selective antagonists of IL-6 action may offer therapeutic benefits in the treatment of IL-6-related diseases. The biological activities of IL-6 are mediated by the IL-6 receptor system which comprises two receptor proteins: the specific ligand-binding α-subunit receptor (IL-6R) and the signal transducing β-subunit, gp130. Gp130 also forms part of the receptor complexes of leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, cardiotrophin-1, and IL-11 (6Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1250) Google Scholar) which, in part, provides a molecular basis for the functional redundancy of these cytokines. IL-6 first binds the IL-6R with an affinity of ∼1 nm and the IL-6·IL-6R complex then binds gp130 with a resulting affinity of ∼10 pm (7Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar). The ternary complex of the IL-6 receptor system is a hexamer, comprising two molecules each of IL-6, IL-6R, and gp130 (8Ward 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, 9Paonessa 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). The cDNA of the human IL-6R encodes a protein of 468 amino acids (7Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar), including a signal peptide of 19 amino acids, an extracellular region of 339 amino acids, a transmembrane domain of 28 amino acids, and a short cytoplasmic domain of 82 amino acids. This sequence shows 54 and 57% overall amino acid identity with the cDNA sequences for mouse (10Sugita T. Totsuka T. Saito M. Yamasaki K. Taga T. Hirano T. Kishimoto T. J. Exp. Med. 1990; 171: 2001-2009Crossref PubMed Scopus (80) Google Scholar) and rat (11Baumann M. Baumann H. Fey G.H. J. Biol. Chem. 1990; 265: 19853-19862Abstract Full Text PDF PubMed Google Scholar) IL-6R, respectively. The mature 80-kDa IL-6R is a glycosylated form of the predicted 50-kDa precursor (12Hirata Y. Taga T. Hibi M. Nakano N. Hirano T. Kishimoto T. J. Immunol. 1989; 143: 2900-2906PubMed Google Scholar) and contains six potential N-linked glycosylation sites. The extracellular region has a modular structure, consisting of three domains of approximately 100 amino acids. The amino acid sequence of the NH2-terminal domain is characteristic of the immunoglobulin superfamily (Ig-like) (13Bork P. Holm L. Sander C. J. Mol. Biol. 1994; 242: 309-320PubMed Google Scholar, 14Vaughn D.E. Bjorkman P.J. Neuron. 1996; 16: 261-273Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Members of this family share a common β-sheet folding topology called a Greek Key (15Richardson J.S. Nature. 1977; 268: 495-500Crossref PubMed Scopus (468) Google Scholar), whereby neighboring β-strands form hydrogen bonds in an anti-parallel fashion to form a β-pleated sheet. Two β-sheets are then packed against each other to produce a hydrophobic core. Similarly, the two COOH-terminal domains of the IL-6R are classified as fibronectin type III-like (FN III) modules, a subclass of the β-sandwich fold (14Vaughn 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 Ig-like modules, with the notable exception of 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. Together, the two FN III domains form a cytokine-binding domain (CBD) which is characteristic of class I cytokine receptors (16Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1881) Google Scholar) (e.g. receptors for interleukins-3, -5, -6, -11, gp130, erythropoietin (EPO), ciliary neurotrophic factor, granulocyte-colony stimulating factor, growth hormone (GH), and prolactin (PRL)). Generally, these receptors are characterized by two conserved disulfide bonds located in the NH2-terminal FN III domain and a conserved WSXWS motif located in the COOH-terminal FN III domain. The cytoplasmic and transmembrane domains of the IL-6R are not required for IL-6 signaling (17Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (181) Google Scholar) and biologically active soluble forms of IL-6R (sIL-6R) are naturally found in low concentrations in human urine (18Novick D. Engelmann H. Wallach D. Rubinstein M. J. Exp. Med. 1989; 170: 1409-1414Crossref PubMed Scopus (357) Google Scholar) and serum (19Narazaki 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, 20Frieling J.T. Sauerwein R.W. Wijdenes J. Hendriks T. van der Linden C.J. Cytokine. 1994; 6: 376-381Crossref PubMed Scopus (65) Google Scholar) of healthy individuals. In contrast to many other soluble cytokine receptors that act as inhibitors by competing for ligand binding with cellular receptors (e.g. tumor necrosis factor, IL-1, -2, -4, interferon-γ, nerve growth factor, leukemia inhibitory factor, granulocyte-stimulating factor and granulocyte macrophage-colony stimulating factor) (for reviews, see Refs. 21Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (693) Google Scholar and 22Heaney M.L. Golde D.W. Blood. 1996; 87: 847-857Crossref PubMed Google Scholar), the sIL-6R acts as an agonist of IL-6 activity (17Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (181) Google Scholar). It is not clear whether sIL-6R is generated by proteolytic shedding of membrane-bound IL-6R (23Mullberg J. Schooltink H. Stoyan T. Gunther M. Graeve L. Buse G. Mackiewicz A. Heinrich P.C. Rose-John S. Eur. J. Immunol. 1993; 23: 473-480Crossref PubMed Scopus (440) Google Scholar), or from an alternatively spliced mRNA species (24Lust J.A. Donovan K.A. Kline M.P. Greipp P.R. Kyle R.A. Maihle N.J. Cytokine. 1992; 4: 96-100Crossref PubMed Scopus (299) Google Scholar, 25Horiuchi S. Koyanagi Y. Zhou Y. Miyamoto H. Tanaka Y. Waki M. Matsumoto A. Yamamoto M. Yamamoto N. Eur. J. Immunol. 1994; 24: 1945-1948Crossref PubMed Scopus (189) Google Scholar), or both. In certain disease states, for example, patients with human immunodeficiency virus infection or multiple myeloma, increased levels of sIL-6R have been reported (26Honda M. Yamamoto S. Cheng M. Yasukawa K. Suzuki H. Saito T. Osugi Y. Tokunaga T. Kishimoto T. J. Immunol. 1992; 148: 2175-2180PubMed Google Scholar, 27Gaillard J.P. Bataille R. Brailly H. Zuber C. Yasukawa K. Attal M. Maruo N. Taga T. Kishimoto T. Klein B. Eur. J. Immunol. 1993; 23: 820-824Crossref PubMed Scopus (216) Google Scholar). Therefore, inhibition of the IL-6·sIL-6R complex has been labeled as a key target to antagonize the in vivo action of IL-6 (28Gaillard J.P. Liautard J. Mani J.C. Fernandez Suarez J.M. Klein B. Brochier J. Immunology. 1996; 89: 135-141Crossref PubMed Scopus (6) Google Scholar). To elucidate the tertiary structure of the IL-6R extracellular region, we have purified human sIL-6R using a Chinese hamster ovary (CHO) cell expression system (29Yasukawa K. Saito T. Fukunaga T. Sekimori Y. Koishihara Y. Fukui H. Ohsugi Y. Matsuda T. Yawata H. Hirano T. Taga T. Kishimoto T. J. Biochem. (Tokyo). 1990; 108: 673-676Crossref PubMed Scopus (144) Google Scholar, 30Ward L.D. Howlett G.J. Hammacher A. Weinstock J. Yasukawa K. Simpson R.J. Winzor D.J. Biochemistry. 1995; 34: 2901-2907Crossref PubMed Scopus (75) Google Scholar). This form of the sIL-6R contains four potential N-linked glycosylation sites and 10 cysteine residues. Three cysteines are located in the Ig-like domain, six in the NH2-terminal FN III domain, and one in the COOH-terminal FN III domain. Previously, we have shown that the ligand affinity purified sIL-6R bound IL-6 and gp130 with a 2:2:2 stoichiometry of IL-6, sIL-6R, and sgp130 (8Ward 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) and was bioactive as determined by the ability of the IL-6·sIL-6R complex to prevent the differentiation of embryonic stem cells (31Ward L.D. Hammacher A. Chang J. Zhang J.G. Discolo G. Moritz R.L. Yasukawa K. Simpson R.J. Crabb J.W. Techniques in Protein Chemistry V. Academic Press, New York1994: 331-338Google Scholar). Here, reversed-phase HPLC peptide mapping under nonreducing and reducing conditions, in combination with mass spectrometric and NH2-terminal sequence analysis, was used to determine the disulfide structure and carbohydrate attachment sites of sIL-6R. On the basis of these results, we have created a model that depicts the topology of the extracellular region of the IL-6R and predicts its interactions with IL-6 and gp130. Trypsin (sequencing grade) and neuraminidase (EC3.2.1.18) were obtained from Boehringer-Mannheim. An endoglycosylase preparation obtained from Flavobacterium meningosepticum(32Elder 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 (33Plummer 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, 34Trimble 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). Tris-(2-carboxyethyl)-phosphine (TCEP) was obtained from Pierce. 4-Vinylpyridine was purchased from Sigma. All other chemicals were HPLC grade. sIL-6R was purified from the conditioned medium of CHO cells transfected with an expression vector (pECEdhfr344) which encodes the extracellular binding domain of the IL-6R (truncated at residue 345) (29Yasukawa K. Saito T. Fukunaga T. Sekimori Y. Koishihara Y. Fukui H. Ohsugi Y. Matsuda T. Yawata H. Hirano T. Taga T. Kishimoto T. J. Biochem. (Tokyo). 1990; 108: 673-676Crossref PubMed Scopus (144) Google Scholar). The sIL-6R was concentrated from CHO cell conditioned media using a Sartocon Miniapparatus (Sartorius, Goettingen, Germany) equipped with a 30,000 molecular weight cut-off membrane and purified by ligand affinity chromatography using an IL-6-Sepharose column (30Ward L.D. Howlett G.J. Hammacher A. Weinstock J. Yasukawa K. Simpson R.J. Winzor D.J. Biochemistry. 1995; 34: 2901-2907Crossref PubMed Scopus (75) Google Scholar). SDS-PAGE analysis of sIL-6R samples was performed using pre-cast 4–20% polyacrylamide/SDS gels (Novex) according to the method of Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Isoelectrofocusing (IEF-PAGE) was performed on pre-cast linear pH gradient (pH 3–10), 5% polyacrylamide gels (Novex, San Diego, CA). Protein bands were visualized by staining with Coomassie Brilliant Blue. Recombinant sIL-6R (100 μg, 2 mg/ml) in 50 mm Tris-HCl buffer, pH 7.4, containing 5 mm EDTA was treated with 2% (v/v) neuraminidase (37 °C, overnight) and/or endoglycosylase mixture (37 °C, 3 h). Prior to disulfide determination, free cysteine residues in the sIL-6R (200 μg) were treated with a 5-fold molar excess of 4-vinylpyridine in 50 mm Tris-HCl buffer, pH 8.4, for 1 h at 25 °C in the dark. The modified protein was purified by gel permeation chromatography on a 100 × 10-mm inner diameter fast desalting column (G-25 Sephadex, Pharmacia). Deglycosylated sIL-6R (200 μg, 4 nmol) was digested with trypsin (1:20 w/w, 0.05 m NaH2PO4, pH 6.0) overnight at 37 °C. The tryptic peptide mixture was fractionated by reversed-phase HPLC using a Hewlett-Packard Liquid Chromatograph (model HP 1090A) and a Vydac C18 column (250 × 4.6-mm inner diameter). The column was developed at 0.5 ml/min using a 60-min linear gradient of 0–100% B, where solvent A was aqueous 0.1% (v/v) trifluoroacetic acid and solvent B was 60% acetonitrile in aqueous 0.09% trifluoroacetic acid (45 °C). The column eluent was split (∼1:160), post-detector, using a stainless steel Tee-union (Upchurch catalog number U428, Upchurch, Oak Harbor, WA) directing 0.6% of the total flow at 3 μl/min to the mass spectrometer, while the remainder (99.4%) was collected into polypropylene microcentrifuge tubes (Eppendorf) for further analysis. To identify disulfide-containing fractions, 25% of the digest (1 nmol) was reduced with an equal volume of 10 mm TCEP in 0.2 msodium citrate buffer, pH 6.0 (65 °C, 10 min) (36Gray W.R. Protein Sci. 1993; 2: 1732-1748Crossref PubMed Scopus (236) Google Scholar), and then re-chromatographed under identical conditions. On-line MS analysis of peptide fractions was performed on a Finnigan-MAT LCQ quadrupole ion trap mass spectrometer equipped with an ESI source (San Jose, CA). “Triple play” experiments, consisting of MS/zoom scan/and MS/MS, were performed as described elsewhere (37Courchesne P.L. Jones M.D. Robinson J.H. Spahr C.S. McCracken S. Bently D.L. Luethy R. Patterson S.D. Electrophoresis. 1998; 19: 956-967Crossref PubMed Scopus (39) Google Scholar). Source CID/single ion monitoring (sCID/SIM) was employed to identify S-pyridylethyl cysteine-containing peptides (38Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). For sCID/SIM, the relative collision energy in the source region was set at 70% (arbitrary value) and the mass range was scanned from m/z 104.5–107.5 to detectS-pyridylethyl fragment ions (m/z 106). Peptides were identified using the Finnigan PEPMAP™ program, and from their CID product ion spectra using the MS-Tag and MS-Product algorithms (Prospector pacific rim mirror site, http://jpsl.ludwig.edu.au). Automated Edman degradation of proteins and peptides was performed using a HP-G1005A biphasic NH2-terminal protein sequencer (Hewlett-Packard, version 3.0 chemistry) (38Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). The Ig-like domain of the IL-6R was modeled using the structure of the mouse monoclonal antibody FAB D44.1 VL domain (39Braden B.C. Souchon H. Eisele J.L. Bentley G.A. Bhat T.N. Navaza J. Poljak R.J. J. Mol. Biol. 1994; 243: 767-781Crossref PubMed Scopus (130) Google Scholar) which showed 23% sequence identity, the highest for all known Ig three-dimensional structures. The coordinates for the template were taken from the Protein Data Bank (40Bernstein F.C. Koetzle T.F. Williams G.J. Meyer Jr., E.E. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Crossref PubMed Scopus (8183) Google Scholar), entry 1MLB(chain A, residues 1–109). Template structures for the CBD of IL-6R were the CBD of gp130 (41Bravo J. Staunton D. Heath J.K. Jones E.Y. EMBO J. 1998; 17: 1665-1674Crossref PubMed Scopus (119) Google Scholar), Protein Data Bank entry 1BQU; GHR, chain B (first binding receptor) of Protein Data Bank entry 3HHR (42de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2029) Google Scholar); EPOR, Protein Data Bank entry 1EBP (43Livnah O. Stura E.A. Johnson D.L. Middleton S.A. Mulcahy L.S. Wrighton N.C. Dower W.J. Jolliffe L.K. Wilson I.A. Science. 1996; 273: 464-471Crossref PubMed Scopus (558) Google Scholar); and PRLR, Protein Data Bank entry1BP3 (44Somers W. Ultsch M. de Vos A.M. Kossiakoff A.A. Nature. 1994; 372: 478-481Crossref PubMed Scopus (360) Google Scholar). The sequence alignment was prepared in two parts. The Ig-like domain of IL-6R was manually aligned with the template FAB D44.1 VL structure. For the CBD, a structure-based multiple sequence alignment was performed manually. The β-sheets of the known CBD structures were superimposed to provide the basis of the alignment. The remaining sequences of unknown structure were manually aligned with these structures, conserving the disulfide patterns, WSXWS motif, and hydrophobic patterns of the β-sheets. The MODELLER program (45Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar) was used to generate separate models of the Ig-like domain and CBD. The quality of the models was assessed as described previously (46Smith D.K. Treutlein H.R. Protein Sci. 1998; 7: 886-896Crossref PubMed Scopus (17) Google Scholar); in particular using the ProsaII program (47Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1792) Google Scholar). MODELLER was also used to determine the relative orientations of the Ig-like domain and CBD. A disulfide restraint between Cys6 and Cys174 of the Ig-like domain and FN III domain, respectively, was introduced in accordance with our experimental results. Fifty models were generated with a range of orientations between the Ig-like domain and the CBD. The final two models were chosen on the basis of the quality checks described above and agreement with experimental data. The model of the sIL-6R, complexed with the crystal structures of IL-6 (48Somers W. Stahl M. Seehra J.S. EMBO J. 1997; 16: 989-997Crossref PubMed Scopus (226) Google Scholar) (Protein Data Bank entry 1ALU) and the gp130 CBD (41Bravo J. Staunton D. Heath J.K. Jones E.Y. EMBO J. 1998; 17: 1665-1674Crossref PubMed Scopus (119) Google Scholar), was constructed by superimposing these moieties over the human GH receptor complex (42de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2029) Google Scholar). NH2-terminal sequence analysis of the first 20 residues of the purified sIL-6R was in agreement with the published sequence (7Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar) (data not shown). The purified sIL-6R yielded a single broad band on SDS-PAGE with an apparent molecular mass of ∼52,000 (Fig. 1A, lane 1), a value significantly higher than 36,368 Da calculated from the amino acid composition (7Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar). Upon treatment with either neuraminidase or a combination of neuraminidase and an endoglycosidase mixture, the Mr of the sIL-6R was reduced to ∼50,000 and ∼40,000, respectively (Fig. 1A, lanes 2 and 3). These data suggest that the increasedMr of sIL-6R (∼12,000) is due to glycosylation of the CHO cell-derived protein. Pronounced charge heterogeneity of the mature sIL-6R was observed upon IEF (Fig. 1B, lane 1). Treatment with neuraminidase (Fig. 1B, lane 2) and neuraminidase plus an endoglycosidase mixture (Fig. 1B, lane 3) reduced this complexity to one or two major bands, respectively, indicating that the heterogeneity of the sIL-6R preparation is primarily due to differential N-linked glycosylation. Free cysteine residues in sIL-6R (∼200 μg) were modified with 4-vinylpridine at pH 8.5. Following enzymatic deglycosylation, the treated sIL-6R was digested with trypsin at pH 6.0, and subjected to RP-HPLC/ESI-IT-MS analysis as described under “Experimental Procedures.” The total ion current profiles of nonreduced and reduced tryptic digest of sIL-6R are shown in Fig. 2 (panels A and B, respectively). sCID/SIM of the nonreduced digest forS-pyridylethyl ions (m/z 106) revealed a single peak at retention time 31.42 min (Fig. 2C). The mass spectrum of the peak at retention time 31.42 min (Fig. 2D) revealed the presence of two peptides: peptides T1a and T1b with calculated masses of 2007.3 Da and 1992.5 Da, respectively. Automated CID MS/MS of the doubly charged ion (m/z 1003.8) of peptide T1a (Fig. 2E) identified this peptide as residues 253–268 of the sIL-6R (DLQHHCVIHDAWSGLR) containing an additional mass of 118.9 Da located at Cys258. Upon reduction with TCEP, the mass of peptide T1a decreased by 119.3 Da and was located at 32.20 min (peptide T9, Fig. 2B; see Table I), consistent with cysteinylation of Cys258.Table ICalculated and observed masses of nonreduced and reduced tryptic peptides of the deglycosylated extracellular domain of the human interleukin-6 receptorPeptide fractionaTryptic peptide fractions are labeled according to their order of retention as shown in Fig. 2A.SequencebPeptide sequences identified from MS/MS spectral data using the programs MS-Tag, MS-Product, and manual assignment. Underlined sequences were confirmed by NH2-terminal sequencing. One-letter abbreviations were used for amino acids. Tryptic peptides shown in parentheses refer to peptides produced upon reduction of the tryptic digest prior to RP-HPLC (Fig. 2B).Residue numbercNumbers denote amino acid positions in the sequence of the mature protein (Fig. 6).Nonreduced massdNumbers refer to observed mass while those in parentheses are calculated from the amino acid sequence minus 2 daltons for each disulfide bond.Reduced masseNumbers refer to observed mass while those in parentheses are calculated from the amino acid sequence.Δ massfDifference in mass between the sum of observed reduced masses and the observed nonreduced mass.Cysteine connectivityDaT1 aDLQHHCVIHDAWSGLR (T9)253–2682007.31888.0 (1887.1)−119.3gThe difference in mass between the nonreduced and reduced peptide is consistent with S-cysteinylated cysteine.‖CysbDLQHHCVIHDAWSGLR (T1b)253–2681992.51992.5 (1887.1)+0.0hThe difference in mass between the nonreduced and reduced peptide is consistent with S-pyridylethylated cysteine.‖PecT2TQTFQGCGILQPDPPANITVTAVAR (T11)186–2102718.82599.9 (2599.0)−118.9gThe difference in mass between the nonreduced and reduced peptide is consistent with S-cysteinylated cysteine.‖CysRCPAQEVAR (T6)5–131029.0 (1029.2)‖T3FSCQLAVPEGDSSFYIVSMCVASSVGSK (T14)155–1826544.0 (6543.3)2898.5 (2899.3)+2.26–174, 146–157‖KFQNSPAEDFQEPCQYSQESQK (T8)133–1542618.7 (2618.8)GVLTSLPGDSVTLTCPGVEPEDNATVHWVLR (T13)14–443264.8 (3263.7)T4‖4893.6 (4890.5)+0.828–77SVQLHDSGNYSCYR (T7)66–791629.6 (1628.8)AGRPAGTVHLLVDVPPEEPQLSCFR (T12)80–1042689.8 (2689.1)T5‖4259.9 (4258.9)+1.7102–113KSPLSNVVCEWGPR (T10)105–1181571.8 (1571.8)a Tryptic peptide fractions are labeled according to their order of retention as shown in Fig. 2A.b Peptide sequences identified from MS/MS spectral data using the programs MS-Tag, MS-Product, and manual assignment. Underlined sequences were confirmed by NH2-terminal sequencing. One-letter abbreviations were used for amino acids. Tryptic peptides shown in parentheses refer to peptides produced upon reduction of the tryptic digest prior to RP-HPLC (Fig. 2B).c Numbers denote amino acid positions in the sequence of the mature protein (Fig. 6).d Numbers refer to observed mass while those in parentheses are calculated from the amino acid sequence minus 2 daltons for each disulfide bond.e Numbers refer to observed mass while those in parentheses are calculated from the amino acid sequence.f Difference in mass between the sum of observed reduced masses and the observed nonreduced mass.g The difference in mass between the nonreduced and reduced peptide is consistent with S-cysteinylated cysteine.h The difference in mass between the nonreduced and reduced peptide is consistent with S-pyridylethylated cysteine. Open table in a new tab Sequence data of peptide T1b was not available due to the low abundance of this ion (∼20% compared with peptide T1a). However, the 14.8-Da difference observed between peptides T1a and T1b is consistent with the difference in mass between cysteinylated (+119 Da) and S-pyridylethylated (+105 Da) Cys258 of peptide Asp253-Arg268. These data suggest that the peak observed in the sCID/SIM profile (Fig. 2C) emanates from anS-pyridylethylated form (Cys258) of peptide T1b. Peptide T1b was also observed (in low abundance) at approximately the same retention time in the TCEP-reduced total ion current profile (Fig. 2B). Taken together, these data indicate that the majority of sIL-6R purified from CHO cell conditioned medium contain a modified Cys258 (cysteinylated), and only a small portion (∼20%) remains as the unmodified Cys258 (free sulfhydryl). A portion (25%) of the tryptic digest of deglycosylated/S-pyridylethylated sIL-6R was reduced with 10 mm TCEP at pH 6.0 and subjected to on-line RP-HPLC/ESI-IT-MS analysis (Fig. 2B) using the same chromatographic conditions described for the nonreduced digest. Inspection of the nonreduced and reduced tryptic peptide maps of sIL-6R (Fig. 2, panels A and B) revealed that upon reduction of the digest, the retention times of five peptide fractions (T1-T5) in the nonreduced tryptic map (Fig. 2A) changed with the concomitant appearance of nine peptide fractions (T6-T14) in the reduced tryptic map (Fig. 2B). A summary of peptide masses found in fractions T1-T14 is shown in Table I. As mentioned above, MS analysis of peptide fraction T1 revealed tryptic peptide Asp253-Arg268 (Fig. 2, A,D, and E) containing a cysteinyl-cysteine at position 258 in the sequence (T1a), as well as the small proportion of the peptide that had been reacted with 4-vinylpyridine (T1b). Similarl

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