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W1985219728 abstract "The homodimeric form of a recombinant cytokine interleukin-6 (IL-6D) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6D were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 15N-labeled and unlabeled (14N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6D was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [15N/15N] + [15N/14N] + [14N/15N] + [14N/14N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [15N] + [14N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated “mixed isotope cross-linking” MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6D having a domain-swapped fold similar to IL-10 and interferon-γ. This fold implies that IL-6D-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6D, rather than direct blocking of gp-130-binding sites on IL-6D. The homodimeric form of a recombinant cytokine interleukin-6 (IL-6D) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6D were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 15N-labeled and unlabeled (14N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6D was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [15N/15N] + [15N/14N] + [14N/15N] + [14N/14N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [15N] + [14N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated “mixed isotope cross-linking” MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6D having a domain-swapped fold similar to IL-10 and interferon-γ. This fold implies that IL-6D-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6D, rather than direct blocking of gp-130-binding sites on IL-6D. The interleukin-6 (IL-6) 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. cytokine plays a critical role in host defense mechanisms such as T-cell activation, stimulation of B-cell differentiation, acute phase induction in hepatocytes, nerve cell differentiation, and osteoclast turnover (1Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar). Abnormal IL-6 production is associated with a variety of diseases (2Jones S.A. Horiuchi S. Topley N. Yamamoto N. Fuller G.M. FASEB J. 2001; 15: 43-58Crossref PubMed Scopus (530) Google Scholar) such as rheumatoid arthritis (3Hirano T. Matsuda T. Turner M. Miyasaka N. Buchan G. Tang B. Sato K. Shimizu M. Maini R. Feldmann M. Kishimoto T. Eur. J. Immunol. 1988; 18: 1797-1801Crossref PubMed Scopus (656) Google Scholar), AIDS (4Nakajima K. Martinez Maza O. Hirano T. Breen E.C. Nishanian P.G. Salazar Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 531-536PubMed Google Scholar, 5Poli V. Balena R. Fattori E. Markatos A. Yamamoto M. Tanaka H. Ciliberto G. Rodan G.A. Costantini F. EMBO J. 1994; 13: 1189-1196Crossref PubMed Scopus (655) Google Scholar), osteoporosis (6Poli G. Bressler P. Kinter A. Duh E. Timmer W.C. Rabson A. Justement J.S. Stanley S. Fauci A.S. J. Exp. Med. 1990; 172: 151-158Crossref PubMed Scopus (420) Google Scholar, 7Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1274) Google Scholar), psoriasis (8Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (731) Google Scholar), multiple myeloma (9Bataille R. Jourdan M. Zhang X.G. Klein B. J. Clin. Invest. 1989; 84: 2008-2011Crossref PubMed Scopus (436) Google Scholar, 10Kawano M. Hirano T. Matsuda T. Taga T. Horii Y. Iwato K. Asaoku H. Tang B. Tanabe O. Tanaka H. Kuramoto A. Kishimoto T. Nature. 1988; 332: 83-85Crossref PubMed Scopus (1444) Google Scholar) and Kaposi's sarcoma (11Rettig M.B. Ma H.J. Vescio R.A. Pold M. Schiller G. Belson D. Savage A. Nishikubo C. Wu C. Fraser J. Said J.W. Berenson J.R. Science. 1997; 276: 1851-1854Crossref PubMed Scopus (412) Google Scholar). Thus the interactions between IL-6 and its associated receptors, the transmembrane glycoproteins IL-6R and gp130 (12Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (296) Google Scholar), present an attractive target for therapeutic antagonists (13Nishimoto N. Kishimoto T. Yoshizaki K. Ann. Rheum. Dis. 2000; 59: i21-i27Crossref PubMed Google Scholar). IL-6 signaling is known to proceed via initial binding of IL-6 to the IL-6R to form a binary 1:1 complex. This binary complex interacts with gp130, later forming a signaling hexameric 2:2:2 complex comprising IL-6, IL-6R, and gp130 (14Ward 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, 15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar).Previously, we have shown that a dimeric form of recombinant IL-6 (IL-6D) is a potent antagonist for IL-6 signaling (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Recombinant IL-6D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6D(sIL-6R)2 complex (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In contrast to the binary IL-6·sIL-6R complex, IL-6D(sIL-6R)2 binds gp130 weakly and does not show significant biological activity in the signal transducer and activator of transcription 3 (STAT3) phosphorylation assay (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Natural (glycosylated) human IL-6 is also known to form a dimer that makes up a substantial part of IL-6 in blood or fibroblast secretions (17May L.T. Santhanam U. Sehgal P.B. J. Biol. Chem. 1991; 266: 9950-9955Abstract Full Text PDF PubMed Google Scholar, 18Fong Y. Moldawer L.L. Marano M. Wei H. Tatter S.B. Clarick R.H. Santhanam U. Sherris D. May L.T. Sehgal P.B. J. Immunol. 1989; 142: 2321-2324PubMed Google Scholar, 19Jablons D.M. Mule J.J. McIntosh J.K. Sehgal P.B. May L.T. Huang C.M. Rosenberg S.A. Lotze M.T. J. Immunol. 1989; 142: 1542-1547PubMed Google Scholar) and has also been shown to interact with membrane-bound IL-6R (15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar, 20Rose-John S. Hipp E. Lenz D. Legres L.G. Korr H. Hirano T. Kishimoto T. Heinrich P.C. J. Biol. Chem. 1991; 266: 3841-3846Abstract Full Text PDF PubMed Google Scholar, 59Wijdenes J. Clement C. Klein B. Morel-Fourrier B. Vita N. Ferrara P. Peters A. Mol. Immunol. 1991; 28: 1183-1192Crossref PubMed Scopus (46) Google Scholar). Recently, glycosylated natural human IL-6D, identified by immunoblotting and size exclusion chromatography, was shown to be a survival factor secreted by epithelial cells that inhibited the apoptosis of B-chronic lymphocytic leukemia cells (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Significantly, recombinant human IL-6D fromEscherichia coli acted as a survival factor in a similar way (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Taken together, these results suggest that natural and recombinant IL-6D may have similar biological activity.Elucidation of the IL-6D structure will be critical to understanding the basis of its antagonistic properties. Whereas the structure of IL-6 is known to be a 4-α-helical bundle (22Xu 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 (65) Google Scholar), the structure of IL-6D is unknown. Previous biophysical studies of the sedimentation properties and the unfolding-dissociation relationship of IL-6D (23Matthews J.M. Hammacher A. Howlett G.J. Simpson R.J. Biochemistry. 1998; 37: 10671-10680Crossref PubMed Scopus (10) Google Scholar) have shown it is likely to form a metastable domain-swapped dimer (24Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Crossref PubMed Scopus (590) Google Scholar, 25Bennett M.J. Schlunegger M.P. Eisenberg D. Protein Sci. 1995; 4: 2455-2468Crossref PubMed Scopus (681) Google Scholar) in which adjacent subunits have the IL-6 structure, but contain interchanged α-helical bundle domain elements.Here, we investigate the arrangement of domain-swapped IL-6 chains within IL-6D using a technique based on cross-linking and mass spectrometry. Although the established techniques of x-ray crystallography and NMR spectroscopy yield high resolution data, often this takes months or years to obtain (26Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar, 27Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2775) Google Scholar). Techniques in mass spectrometry (MS) combined with cross-linking (28Henry C.M. Chem. Eng. News. 2001; 78: 22-36Crossref Scopus (7) Google Scholar, 29Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar, 30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar) or other chemical labeling techniques such as hydrogen/deuterium exchange (31Apuy J.L. Park Z.Y. Swartz P.D. Dangott L.J. Russell D.H. Baldwin T.O. Biochemistry. 2001; 40: 15153-15163Crossref PubMed Scopus (31) Google Scholar,32Yamada N. Suzuki E. Hirayama K. Rapid Commun. Mass Spectrom. 2002; 16: 293-299Crossref PubMed Scopus (36) Google Scholar) have been evaluated for rapid low-resolution three-dimensional study of proteins (30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar) or protein complexes (33Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar, 34Sinz A. Wang K. Biochemistry. 2001; 40: 7903-7913Crossref PubMed Scopus (64) Google Scholar). Cross-linking/MS methods involve chemically or photochemically cross-linking a protein complex (35Means G.E. Feeney R.E. Bioconjugate Chem. 1990; 1: 2-12Crossref PubMed Scopus (171) Google Scholar), followed by digestion of the cross-linked complex and MS analysis of the resulting peptide mixture (36Kuster B. Mann M. Curr. Opin. Struct. Biol. 1998; 8: 393-400Crossref PubMed Scopus (94) Google Scholar). Cross-linked peptides can be identified by parent ion mass and/or the fragmentation pattern produced by tandem mass spectrometry (MS/MS), thereby locating adjacent protein regions and enabling assembly of low-resolution models of proteins or protein complexes. Cross-linking/MS experiments are generally fast and do not require large quantities of protein (30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar). For these reasons, cross-linking/MS methods have potential as a low-resolution counterpart to x-ray or NMR methods for rapid determination of interacting surfaces between proteins (29Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar) and the topology of proteins within complexes (33Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar).Despite the advantages of cross-linking methods, namely high molecular weight capability, speed, and the small quantities of protein required, the large number of peptide species that are seen from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. This problem has been partially addressed by “tagging” methodologies that allow rapid visual MS location of cross-linked species within complex peptide mixtures (34Sinz A. Wang K. Biochemistry. 2001; 40: 7903-7913Crossref PubMed Scopus (64) Google Scholar, 37Chen X. Chen Y.H. Anderson V.E. Anal. Biochem. 1999; 273: 192-203Crossref PubMed Scopus (63) Google Scholar, 38Bennett K.L. Kussmann M. Bjork P. Godzwon M. Mikkelsen M. Sorensen P. Roepstorff P. Protein Sci. 2000; 9: 1503-1518Crossref PubMed Scopus (132) Google Scholar, 39Muller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M.O. Anal. Chem. 2001; 73: 1927-1934Crossref PubMed Scopus (186) Google Scholar). For example, the use of a 1:1 mixture of undeuterated and deuterated (d 0/d 4-labeled) cross-linking reagent readily allows mass spectrometric detection of all cross-linked species by the presence ofd 0/d 4-isotope tags (39Muller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M.O. Anal. Chem. 2001; 73: 1927-1934Crossref PubMed Scopus (186) Google Scholar). However, for studying interactions between proteins, even these tagging methodologies fall short in that they fail to distinguishinter- and intra-cross-linked peptides. This results in cross-linked peptide species being tagged that do not yield useful information on intermolecular interactions, such as intramolecular cross-linked peptides or peptides modified by partially hydrolyzed cross-linking reagent (30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar, 40Pearson K.M. Pannell L.K. Fales H.M. Rapid Commun. Mass Spectrom. 2002; 16: 149-159Crossref PubMed Scopus (102) Google Scholar). Moreover, these tagging methodologies make cross-linking studies of oligomeric proteins, such as IL-6D, ambiguous because identical cross-linked peptides can in principle arise from inter- orintramolecular origins (38Bennett K.L. Kussmann M. Bjork P. Godzwon M. Mikkelsen M. Sorensen P. Roepstorff P. Protein Sci. 2000; 9: 1503-1518Crossref PubMed Scopus (132) Google Scholar).Here we present a method for visualizing intermolecular cross-linked peptides in the IL-6 homodimer that we have designated mixed isotope cross-linking (MIX). Applied to IL-6D, the MIX method requires preparation of uniformly 15N-labeled and unlabeled (14N) IL-6, combined as a 1:1 mixture and re-associated to form a population of 14N-, mixed14N/15N-, and 15N-labeled IL-6D. Cross-linking and mass spectrometric peptide mapping on this mixture allows intermolecular cross-linked peptides to be identified easily as they form distinctive triplet or quadruplet mass spectrum peaks because of the distribution of 14N- and15N-labeled peptides within these cross-linked peptides. In contrast, all intramolecular cross-linked and noncross-linked peptides are seen as doublet mass spectrum peaks. This ability to discriminate between inter- and intra- cross-linked species makes the MIX technique a uniquely useful new tool for studying intermolecular interactions. We describe the application of this technique to determine proximal intermolecular residues within the homodimeric form of IL-6 and to deduce the mode of three-dimensional domain swapping, based on the known structure of monomeric human interleukin-6 (22Xu 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 (65) Google Scholar). The interleukin-6 (IL-6) 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. 1The abbreviations used for: IL, interleukin; CID, collision-induced dissociation; ESI, electrospray ionization; gp130, IL-6 receptor signaling subunit β chain; IL-6M, monomeric IL-6; IL-6D, dimeric IL-6; IL-6R, IL-6 receptor α-chain; MIX, mixed isotope cross-linking; MS, mass spectrometry; RP-HPLC, reversed-phase high-performance liquid chromatography; SEC, size-exclusion chromatography; BS3, bis(sulfosuccinimidyl)suberate. cytokine plays a critical role in host defense mechanisms such as T-cell activation, stimulation of B-cell differentiation, acute phase induction in hepatocytes, nerve cell differentiation, and osteoclast turnover (1Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar). Abnormal IL-6 production is associated with a variety of diseases (2Jones S.A. Horiuchi S. Topley N. Yamamoto N. Fuller G.M. FASEB J. 2001; 15: 43-58Crossref PubMed Scopus (530) Google Scholar) such as rheumatoid arthritis (3Hirano T. Matsuda T. Turner M. Miyasaka N. Buchan G. Tang B. Sato K. Shimizu M. Maini R. Feldmann M. Kishimoto T. Eur. J. Immunol. 1988; 18: 1797-1801Crossref PubMed Scopus (656) Google Scholar), AIDS (4Nakajima K. Martinez Maza O. Hirano T. Breen E.C. Nishanian P.G. Salazar Gonzalez J.F. Fahey J.L. Kishimoto T. J. Immunol. 1989; 142: 531-536PubMed Google Scholar, 5Poli V. Balena R. Fattori E. Markatos A. Yamamoto M. Tanaka H. Ciliberto G. Rodan G.A. Costantini F. EMBO J. 1994; 13: 1189-1196Crossref PubMed Scopus (655) Google Scholar), osteoporosis (6Poli G. Bressler P. Kinter A. Duh E. Timmer W.C. Rabson A. Justement J.S. Stanley S. Fauci A.S. J. Exp. Med. 1990; 172: 151-158Crossref PubMed Scopus (420) Google Scholar, 7Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1274) Google Scholar), psoriasis (8Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D.P. May L.T. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6367-6371Crossref PubMed Scopus (731) Google Scholar), multiple myeloma (9Bataille R. Jourdan M. Zhang X.G. Klein B. J. Clin. Invest. 1989; 84: 2008-2011Crossref PubMed Scopus (436) Google Scholar, 10Kawano M. Hirano T. Matsuda T. Taga T. Horii Y. Iwato K. Asaoku H. Tang B. Tanabe O. Tanaka H. Kuramoto A. Kishimoto T. Nature. 1988; 332: 83-85Crossref PubMed Scopus (1444) Google Scholar) and Kaposi's sarcoma (11Rettig M.B. Ma H.J. Vescio R.A. Pold M. Schiller G. Belson D. Savage A. Nishikubo C. Wu C. Fraser J. Said J.W. Berenson J.R. Science. 1997; 276: 1851-1854Crossref PubMed Scopus (412) Google Scholar). Thus the interactions between IL-6 and its associated receptors, the transmembrane glycoproteins IL-6R and gp130 (12Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (296) Google Scholar), present an attractive target for therapeutic antagonists (13Nishimoto N. Kishimoto T. Yoshizaki K. Ann. Rheum. Dis. 2000; 59: i21-i27Crossref PubMed Google Scholar). IL-6 signaling is known to proceed via initial binding of IL-6 to the IL-6R to form a binary 1:1 complex. This binary complex interacts with gp130, later forming a signaling hexameric 2:2:2 complex comprising IL-6, IL-6R, and gp130 (14Ward 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, 15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar). Previously, we have shown that a dimeric form of recombinant IL-6 (IL-6D) is a potent antagonist for IL-6 signaling (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Recombinant IL-6D binds tightly to soluble IL-6R (sIL-6R) to form a 1:2 IL-6D(sIL-6R)2 complex (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In contrast to the binary IL-6·sIL-6R complex, IL-6D(sIL-6R)2 binds gp130 weakly and does not show significant biological activity in the signal transducer and activator of transcription 3 (STAT3) phosphorylation assay (16Ward L.D. Hammacher A. Howlett G.J. Matthews J.M. Fabri L. Moritz R.L. Nice E.C. Weinstock J. Simpson R.J. J. Biol. Chem. 1996; 271: 20138-20144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Natural (glycosylated) human IL-6 is also known to form a dimer that makes up a substantial part of IL-6 in blood or fibroblast secretions (17May L.T. Santhanam U. Sehgal P.B. J. Biol. Chem. 1991; 266: 9950-9955Abstract Full Text PDF PubMed Google Scholar, 18Fong Y. Moldawer L.L. Marano M. Wei H. Tatter S.B. Clarick R.H. Santhanam U. Sherris D. May L.T. Sehgal P.B. J. Immunol. 1989; 142: 2321-2324PubMed Google Scholar, 19Jablons D.M. Mule J.J. McIntosh J.K. Sehgal P.B. May L.T. Huang C.M. Rosenberg S.A. Lotze M.T. J. Immunol. 1989; 142: 1542-1547PubMed Google Scholar) and has also been shown to interact with membrane-bound IL-6R (15Onishi M. Nosaka T. Kitamura T. Int. Rev. Immunol. 1998; 16: 617-634Crossref PubMed Scopus (20) Google Scholar, 20Rose-John S. Hipp E. Lenz D. Legres L.G. Korr H. Hirano T. Kishimoto T. Heinrich P.C. J. Biol. Chem. 1991; 266: 3841-3846Abstract Full Text PDF PubMed Google Scholar, 59Wijdenes J. Clement C. Klein B. Morel-Fourrier B. Vita N. Ferrara P. Peters A. Mol. Immunol. 1991; 28: 1183-1192Crossref PubMed Scopus (46) Google Scholar). Recently, glycosylated natural human IL-6D, identified by immunoblotting and size exclusion chromatography, was shown to be a survival factor secreted by epithelial cells that inhibited the apoptosis of B-chronic lymphocytic leukemia cells (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Significantly, recombinant human IL-6D fromEscherichia coli acted as a survival factor in a similar way (21Moreno A. Villar M.L. Camara C. Luque R. Cespon C. Gonzalez-Porque P. Roy G. Lopez-Jimenez J. Bootello A. Santiago E.R. Blood. 2001; 97: 242-249Crossref PubMed Scopus (46) Google Scholar). Taken together, these results suggest that natural and recombinant IL-6D may have similar biological activity. Elucidation of the IL-6D structure will be critical to understanding the basis of its antagonistic properties. Whereas the structure of IL-6 is known to be a 4-α-helical bundle (22Xu 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 (65) Google Scholar), the structure of IL-6D is unknown. Previous biophysical studies of the sedimentation properties and the unfolding-dissociation relationship of IL-6D (23Matthews J.M. Hammacher A. Howlett G.J. Simpson R.J. Biochemistry. 1998; 37: 10671-10680Crossref PubMed Scopus (10) Google Scholar) have shown it is likely to form a metastable domain-swapped dimer (24Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Crossref PubMed Scopus (590) Google Scholar, 25Bennett M.J. Schlunegger M.P. Eisenberg D. Protein Sci. 1995; 4: 2455-2468Crossref PubMed Scopus (681) Google Scholar) in which adjacent subunits have the IL-6 structure, but contain interchanged α-helical bundle domain elements. Here, we investigate the arrangement of domain-swapped IL-6 chains within IL-6D using a technique based on cross-linking and mass spectrometry. Although the established techniques of x-ray crystallography and NMR spectroscopy yield high resolution data, often this takes months or years to obtain (26Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar, 27Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2775) Google Scholar). Techniques in mass spectrometry (MS) combined with cross-linking (28Henry C.M. Chem. Eng. News. 2001; 78: 22-36Crossref Scopus (7) Google Scholar, 29Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar, 30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar) or other chemical labeling techniques such as hydrogen/deuterium exchange (31Apuy J.L. Park Z.Y. Swartz P.D. Dangott L.J. Russell D.H. Baldwin T.O. Biochemistry. 2001; 40: 15153-15163Crossref PubMed Scopus (31) Google Scholar,32Yamada N. Suzuki E. Hirayama K. Rapid Commun. Mass Spectrom. 2002; 16: 293-299Crossref PubMed Scopus (36) Google Scholar) have been evaluated for rapid low-resolution three-dimensional study of proteins (30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar) or protein complexes (33Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar, 34Sinz A. Wang K. Biochemistry. 2001; 40: 7903-7913Crossref PubMed Scopus (64) Google Scholar). Cross-linking/MS methods involve chemically or photochemically cross-linking a protein complex (35Means G.E. Feeney R.E. Bioconjugate Chem. 1990; 1: 2-12Crossref PubMed Scopus (171) Google Scholar), followed by digestion of the cross-linked complex and MS analysis of the resulting peptide mixture (36Kuster B. Mann M. Curr. Opin. Struct. Biol. 1998; 8: 393-400Crossref PubMed Scopus (94) Google Scholar). Cross-linked peptides can be identified by parent ion mass and/or the fragmentation pattern produced by tandem mass spectrometry (MS/MS), thereby locating adjacent protein regions and enabling assembly of low-resolution models of proteins or protein complexes. Cross-linking/MS experiments are generally fast and do not require large quantities of protein (30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar). For these reasons, cross-linking/MS methods have potential as a low-resolution counterpart to x-ray or NMR methods for rapid determination of interacting surfaces between proteins (29Wallon G. Rappsilber J. Mann M. Serrano L. EMBO J. 2000; 19: 213-222Crossref PubMed Scopus (60) Google Scholar) and the topology of proteins within complexes (33Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar). Despite the advantages of cross-linking methods, namely high molecular weight capability, speed, and the small quantities of protein required, the large number of peptide species that are seen from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. This problem has been partially addressed by “tagging” methodologies that allow rapid visual MS location of cross-linked species within complex peptide mixtures (34Sinz A. Wang K. Biochemistry. 2001; 40: 7903-7913Crossref PubMed Scopus (64) Google Scholar, 37Chen X. Chen Y.H. Anderson V.E. Anal. Biochem. 1999; 273: 192-203Crossref PubMed Scopus (63) Google Scholar, 38Bennett K.L. Kussmann M. Bjork P. Godzwon M. Mikkelsen M. Sorensen P. Roepstorff P. Protein Sci. 2000; 9: 1503-1518Crossref PubMed Scopus (132) Google Scholar, 39Muller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M.O. Anal. Chem. 2001; 73: 1927-1934Crossref PubMed Scopus (186) Google Scholar). For example, the use of a 1:1 mixture of undeuterated and deuterated (d 0/d 4-labeled) cross-linking reagent readily allows mass spectrometric detection of all cross-linked species by the presence ofd 0/d 4-isotope tags (39Muller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M.O. Anal. Chem. 2001; 73: 1927-1934Crossref PubMed Scopus (186) Google Scholar). However, for studying interactions between proteins, even these tagging methodologies fall short in that they fail to distinguishinter- and intra-cross-linked peptides. This results in cross-linked peptide species being tagged that do not yield useful information on intermolecular interactions, such as intramolecular cross-linked peptides or peptides modified by partially hydrolyzed cross-linking reagent (30Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (389) Google Scholar, 40Pearson K.M. Pannell L.K. Fales H.M. Rapid Commun. Mass Spectrom. 2002; 16: 149-159Crossref PubMed Scopus (102) Google Scholar). Moreover, these tagging methodologies make cross-linking studies of oligomeric proteins, such as IL-6D, ambiguous because identical cross-linked peptides can in principle arise from inter- orintramolecular origins (38Bennett K.L. Kussmann M. Bjork P. Godzwon M. Mikkelsen M. Sorensen P. Roepstorff P. Protein Sci. 2000; 9: 1503-1518Crossref PubMed Scopus (132) Google Scholar). Here we present a method for visualizing intermolecular cross-linked peptides in the IL-6 homodimer that we have designated mixed isotope cross-linking (MIX). Applied to IL-6D, the MIX method requires preparation of uniformly 15N-labeled and unlabeled (14N) IL-6, combined as a 1:1 mixture and re-associated to form a population of 14N-, mixed14N/15N-, and 15N-labeled IL-6D. Cross-linking and mass spectrometric peptide mapping on this mixture allows intermolecular cross-linked peptides to be identified easily as they form distinctive triplet or quadruplet mass spectrum peaks because of the distribution of 14N- and15N-labeled peptides within these cross-linked peptides. In contrast, all intramolecular cross-linked and noncross-linked peptides are seen as doublet mass spectrum peaks. This ability to discriminate between inter- and intra- cross-linked species makes the MIX technique a uniquely useful new tool for studying intermolecular interactions. We describe the application of this technique to determine proximal intermolecular residues within the homodimeric form of IL-6 and to deduce the mode of three-dimensional domain swapping, based on the known structure of monomeric human interleukin-6 (22Xu 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 (65) Google Scholar). We thank D. Frecklington, E. A. Kapp, L. Mao, R. L. Moritz, J. Hong, and G. F. Tu for expert advice on various aspects of the project, and A. W. Burgess and G. E. Reid for critical comments on the manuscript." @default.
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W1985219728 title "Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry" @default.
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