A 16-Residue Peptide Fragment of Macrophage Migration Inhibitory Factor, MIF-(50–65), Exhibits Redox Activity and Has MIF-like Biological Functions
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m301735200
ISSN1083-351X
AutoresMai Tuyet Nguyen, Jürgen Beck, Hongqi Lue, Helge Fünfzig, Robert Kleemann, Pieter Koolwijk, Aphrodite Kapurniotu, Jürgen Bernhagen,
Tópico(s)Nuclear Receptors and Signaling
ResumoMacrophage migration inhibitory factor (MIF) is a cytokine that participates in the host inflammatory response. A Cys-Xaa-Xaa-Cys (CXXC)-based thiol-protein oxidoreductase activity of MIF is associated with certain biological functions. Peptides spanning the CXXC region of thiol-protein oxidoreductases retain some biochemical properties of the full-length protein. We report on the characterization of CXXC-spanning MIF-(50–65) and its serine variant, C57S/C60S-MIF-(50–65). Following disulfide-mediated cyclization, MIF-(50–65) adapted a β-turn conformation comparable with that of β-turn-containing cyclo-57,60-[Asp57,Dap60]MIF-(50–65). MIF-(50–65) had a redox potential E′0 of –0.258 V and formed mixed disulfides with glutathione and cysteine. MIF-(50–65) but not C57S/C60S-MIF-(50–65) had oxidoreductase activity in vitro. Intriguingly, MIF-(50–65) exhibited MIF-like cellular activities. The peptide but not its variant had glucocorticoid overriding and proliferation-enhancing activity and stimulated ERK1/2 phosphorylation. MIF-(50–65) and its variant bound to the MIF-binding protein JAB1 and enhanced cellular levels of p27Kip1. As the peptide and its variant were endocytosed at similar efficiency, sequence 50–65 appears sufficient for the JAB1-related effects of MIF, whereas other activities require CXXC. Cyclo-57,60-[Asp57,Dap60]MIF-(50–65) activated ERK1/2, indicating that CXXC-dependent disulfide and β-turn formation is associated with an activity-inducing conformation. We conclude that CXXC and sequence 50–65 are critical for the activities of MIF. MIF-(50–65) is a surprisingly short sequence with MIF-like functions that could be an excellent molecular template for MIF therapeutics. Macrophage migration inhibitory factor (MIF) is a cytokine that participates in the host inflammatory response. A Cys-Xaa-Xaa-Cys (CXXC)-based thiol-protein oxidoreductase activity of MIF is associated with certain biological functions. Peptides spanning the CXXC region of thiol-protein oxidoreductases retain some biochemical properties of the full-length protein. We report on the characterization of CXXC-spanning MIF-(50–65) and its serine variant, C57S/C60S-MIF-(50–65). Following disulfide-mediated cyclization, MIF-(50–65) adapted a β-turn conformation comparable with that of β-turn-containing cyclo-57,60-[Asp57,Dap60]MIF-(50–65). MIF-(50–65) had a redox potential E′0 of –0.258 V and formed mixed disulfides with glutathione and cysteine. MIF-(50–65) but not C57S/C60S-MIF-(50–65) had oxidoreductase activity in vitro. Intriguingly, MIF-(50–65) exhibited MIF-like cellular activities. The peptide but not its variant had glucocorticoid overriding and proliferation-enhancing activity and stimulated ERK1/2 phosphorylation. MIF-(50–65) and its variant bound to the MIF-binding protein JAB1 and enhanced cellular levels of p27Kip1. As the peptide and its variant were endocytosed at similar efficiency, sequence 50–65 appears sufficient for the JAB1-related effects of MIF, whereas other activities require CXXC. Cyclo-57,60-[Asp57,Dap60]MIF-(50–65) activated ERK1/2, indicating that CXXC-dependent disulfide and β-turn formation is associated with an activity-inducing conformation. We conclude that CXXC and sequence 50–65 are critical for the activities of MIF. MIF-(50–65) is a surprisingly short sequence with MIF-like functions that could be an excellent molecular template for MIF therapeutics. Macrophage migration inhibitory factor (MIF) 1The abbreviations used are: MIF, macrophage migration inhibitory factor; MIF-(50–65), sequence region 50–65 of human MIF; biotin-MIF-(50–65), analog of MIF-(50–65) with N-terminally linked biotinamidocaproate moiety; biotin-C57S/C60S-MIF-(50–65), analog of C57S/C60S-MIF-(50–65) with N-terminally linked biotinamidocaproate moiety; C57S/C60S-MIF-(50–65) or Ser-MIF-(50–65), bis-serine variant of MIF-(50–65) with Cys → Ser changes at positions 57 and 60; C60SMIF, MIF mutant with a Cys → Ser mutation at residue 60; CALC, Cys-Ala-Leu-Cys motif; CXXC, Cys-Xaa-Xaa-Cys motif; CSN5, COP9 signalosome subunit 5; cyclo-MIF-(50–65), cyclo-57,60-[Asp57,Dap60]MIF-(50–65); DTT, dithiothreitol; Fluo-MIF-(50–65), analog of MIF-(50–65) with N-terminally linked carboxyfluorescein moiety; Fluo-C57S/C60S-MIF-(50–65), analog of C57S/C60S-MIF-(50–65) with N-terminally linked carboxyfluorescein moiety; Grx, glutaredoxin; HED, bis-(2-hydroxyethyl)-disulfide; JAB1, c-Jun activation domain binding protein 1; LPS, lipopolysaccharide; PDI, protein-disulfide isomerase; rMIF, biologically active recombinant wild-type human MIF; TPOR, thiol-protein oxidoreductase; Trr, thioredoxin reductase; Trx, thioredoxin; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase HPLC; MPAK, mitogen-activated protein kinase; MALDI-TOF-MS, matrix-assisted laser desorption ionization/time-of-flight-mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; FCS, fetal calf serum; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; VEGF, vascular endothelial cell growth factor; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; MHC, major histocompatibility complex; CFSE, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; SPPS, solid phase synthetic protocols; DEX, dexamethasone; TNF, tumor necrosis factor; hMVEC, human microvascular endothelial cells; GCOR, glucocorticoid overriding; GIF, glycosylation-inhibiting factor; Dap, diaminopropionic acid.1The abbreviations used are: MIF, macrophage migration inhibitory factor; MIF-(50–65), sequence region 50–65 of human MIF; biotin-MIF-(50–65), analog of MIF-(50–65) with N-terminally linked biotinamidocaproate moiety; biotin-C57S/C60S-MIF-(50–65), analog of C57S/C60S-MIF-(50–65) with N-terminally linked biotinamidocaproate moiety; C57S/C60S-MIF-(50–65) or Ser-MIF-(50–65), bis-serine variant of MIF-(50–65) with Cys → Ser changes at positions 57 and 60; C60SMIF, MIF mutant with a Cys → Ser mutation at residue 60; CALC, Cys-Ala-Leu-Cys motif; CXXC, Cys-Xaa-Xaa-Cys motif; CSN5, COP9 signalosome subunit 5; cyclo-MIF-(50–65), cyclo-57,60-[Asp57,Dap60]MIF-(50–65); DTT, dithiothreitol; Fluo-MIF-(50–65), analog of MIF-(50–65) with N-terminally linked carboxyfluorescein moiety; Fluo-C57S/C60S-MIF-(50–65), analog of C57S/C60S-MIF-(50–65) with N-terminally linked carboxyfluorescein moiety; Grx, glutaredoxin; HED, bis-(2-hydroxyethyl)-disulfide; JAB1, c-Jun activation domain binding protein 1; LPS, lipopolysaccharide; PDI, protein-disulfide isomerase; rMIF, biologically active recombinant wild-type human MIF; TPOR, thiol-protein oxidoreductase; Trr, thioredoxin reductase; Trx, thioredoxin; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase HPLC; MPAK, mitogen-activated protein kinase; MALDI-TOF-MS, matrix-assisted laser desorption ionization/time-of-flight-mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; FCS, fetal calf serum; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; VEGF, vascular endothelial cell growth factor; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; MHC, major histocompatibility complex; CFSE, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; SPPS, solid phase synthetic protocols; DEX, dexamethasone; TNF, tumor necrosis factor; hMVEC, human microvascular endothelial cells; GCOR, glucocorticoid overriding; GIF, glycosylation-inhibiting factor; Dap, diaminopropionic acid. was discovered 40 years ago as a lymphocyte mediator that inhibited the random migration of macrophages (1David J.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 72-77Crossref PubMed Scopus (1080) Google Scholar). Later on, MIF was rediscovered as a pituitary factor with hormone-like properties (2Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracey K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. Nature. 1993; 365: 756-759Crossref PubMed Scopus (922) Google Scholar, 3Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1045) Google Scholar). Today, MIF is known as a widely expressed pleiotropic cytokine exhibiting a broad range of immune and inflammatory activities, including induction of inflammatory cytokines, nitric oxide and superoxide anion, and regulation of macrophage and lymphocyte proliferation. The immuno-regulatory activities of MIF are based upon transcriptional regulation of inflammatory gene products, modulation of cell proliferation and cell cycle inhibition of p53-mediated apoptosis, and on a number of metabolic effects (summarized in Refs. 4Bucala R. FASEB J. 1996; 10: 1607-1613Crossref PubMed Scopus (181) Google Scholar, 5Mitchell R.A. Bucala R. Semin. Cancer Biol. 2000; 10: 359-366Crossref PubMed Scopus (137) Google Scholar, 6Lue H. Kleemann R. Calandra T. Roger T. Bernhagen J. Microb. Infect. 2002; 4: 449-460Crossref PubMed Scopus (302) Google Scholar). MIF plays a pivotal role in the pathogenesis of a number of immune and inflammatory conditions such as septic shock, rheumatoid arthritis, cancer, and lung diseases. Consequently, MIF-based therapeutic approaches have become of major interest (2Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracey K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. 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In particular, a membrane receptor for MIF has not been identified. MIF modulates the phosphorylation and activity of protein kinases (13Mitchell R.A. Metz C.N. Peng T. Bucala R. J. Biol. Chem. 1999; 274: 18100-18106Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar) and interacts with and regulates the activity of the transcriptional co-activator JAB1/CSN5 (14Kleemann R. Hausser A. Geiger G. Mischke R. Burger-Kentischer A. Flieger O. Johannes F.J. Roger T. Calandra T. Kapurniotu A. Grell M. Finkelmeier D. Brunner H. Bernhagen J. Nature. 2000; 408: 211-216Crossref PubMed Scopus (501) Google Scholar, 15Burger-Kentischer A. Goebel H. Seiler R. Fraedrich G. Schaefer H.E. Dimmeler S. Kleemann R. Bernhagen J. Ihling C. Circulation. 2002; 105: 1561-1566Crossref PubMed Scopus (225) Google Scholar). However, it is currently unclear what the upstream molecular events of these effects are. It has been considered that an observed catalytic thiol-protein oxidoreductase (TPOR) activity of MIF could be responsible, at least in part, for the cellular functions of MIF.The TPOR family of proteins encompasses enzymes such as thioredoxin (Trx), glutaredoxin (Grx), protein-disulfide isomerase (PDI), or the disulfide bond proteins, with Trx being the prototype member of the family. TPORs catalyze the reduction of protein disulfides in a cysteine-based dithiol/disulfide-dependent process, and some family members also serve as potent protein folding catalysts. TPOR proteins share a common Cys-Xaa-Xaa-Cys (CXXC) consensus motif within a homologous so-called Trx-fold, where the CXXC motif is located at the N terminus of an α-helix. MIF contains a CXXC TPOR consensus motif, with the residues Ala and Leu placed between the cysteines and also contains additional conserved residues that are frequently found N-terminally of the CXXC region (16Ellis L.B.M. Saurugger P. Woodward C. Biochemistry. 1992; 31: 4882-4891Crossref PubMed Scopus (29) Google Scholar). Although the overall three-dimensional structure of the MIF monomer shows a remote resemblance to the Trx monomer, MIF is not structurally homologous to the TPOR proteins, and the Cys-Ala-Leu-Cys (CALC) redox motif of MIF lies at the N terminus of a β-strand element with Cys57 located in the preceding loop (17Sun H. Bernhagen J. Bucala R. Lolis E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5191-5196Crossref PubMed Scopus (287) Google Scholar, 18Sugimoto H. Suzuki M. Nakagawa A. Tanaka I. Nishihira J. FEBS Lett. 1996; 389: 145-148Crossref PubMed Scopus (64) Google Scholar) rather than in a Trx-like fold. MIF exhibits TPOR activity in vitro being able to catalyze the reduction of both insulin and small molecular weight compound disulfides (19Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Jüttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar, 20Kleemann R. Mischke R. Kapurniotu A. Brunner H. Bernhagen J. FEBS Lett. 1998; 430: 191-196Crossref PubMed Scopus (61) Google Scholar, 21Potolicchio I. Santambrogio L. Strominger J.L. J. Biol. Chem. 2003; 278: 30889-30895Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Mutation of the CXXC cysteines of MIF results in partly or fully immunologically inactive MIF protein, an observation that has led to the suggestion that the TPOR activity of MIF is responsible in part for its immunological and cellular activities (14Kleemann R. Hausser A. Geiger G. Mischke R. Burger-Kentischer A. Flieger O. Johannes F.J. Roger T. Calandra T. Kapurniotu A. Grell M. Finkelmeier D. Brunner H. Bernhagen J. Nature. 2000; 408: 211-216Crossref PubMed Scopus (501) Google Scholar, 19Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Jüttner S. Brunner H. Bernhagen J. J. Mol. 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Nature. 2000; 408: 167-168Crossref PubMed Scopus (58) Google Scholar).Thus, although MIF is not structurally related to the TPOR proteins, it shares with these proteins catalytic TPOR activity in vitro and the capability to participate in redox regulation in vivo. Moreover, MIF shares additional intriguing properties with Trx. Trx or adult T cell leukemia-derived factor plays a role in the regulation of cellular redox stress (27Tagaya Y. Maeda Y. Mitsui A. Kondo Matsui H. Hamuro J. Brown R. Arai K. Yokota T. Wakasugi N. Yodoi J. EMBO J. 1989; 8: 757-764Crossref PubMed Scopus (514) Google Scholar, 28Tanaka T. Nakamura H. Nishiyama A. Hosoi F. Masutani H. Wada H. Yodoi J. Free Radic. Res. 2000; 33: 851-855Crossref PubMed Scopus (128) Google Scholar) through its TPOR activity. Also, Trx, although originally discovered as a TPOR and ribonucleotide reductase enzyme, has recently been re-defined as a cytokine and immune mediator (29Bertini R. Howard O.M.Z. Dong H.-F. Oppenheim J.J. Bizzarri C. Caselli G. Pagliei S. Romines B. Wilshire J.A. Mengozzi M. Nakamura H. Yodoi J. Pekkari K. Gurunath R. Holmgren A. Herzenberg L.A. Herzenberg L.A. Ghezzi P. J. Exp. Med. 1999; 189: 1783-1789Crossref PubMed Scopus (285) Google Scholar, 30Pekkari K. Gurunath R. Arner E.S. Holmgren A. J. Biol. Chem. 2000; 275: 37474-37480Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).The redox potential of the TPOR proteins is governed by both the overall three-dimensional structural constraints of the respective enzymes and the sequence composition of the CXXC motif together with the surrounding residues of the Trx fold (31Krause G. Lundström J. Barea J.L. Pueyo de la Cuesta C. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 32Chivers P.T. Laboissiere M.C.A. Raines R.T. EMBO J. 1996; 15: 2659-2667Crossref PubMed Scopus (159) Google Scholar, 33Huber-Wunderlich M. Glockshuber R. 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In this respect, it is noteworthy of mentioning that it has been suggested that a mono- or dimeric structure, possibly with slightly different conformational properties, but not the MIF trimer, may be the predominant MIF species under physiological conditions (38Mischke R. Kleemann R. Brunner H. Bernhagen J. FEBS Lett. 1998; 427: 85-90Crossref PubMed Scopus (51) Google Scholar). Interestingly, thioredoxin reductase (Trr), a member of the TPOR family, also does not contain an apparent Trx fold structure but otherwise shares similar redox properties with the TPOR proteins, indicating that it may undergo a marked conformational change during catalysis (39Waksman G. Krishna T.S. Williams C.H.J. Kuriyan J. J. Mol. Biol. 1994; 236: 800-816Crossref PubMed Scopus (188) Google Scholar). Computer-based secondary structure predictions had shown previously (19Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Jüttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (267) Google Scholar) that the CXXC sequence region of MIF has a high β-turn-forming propensity that is comparable with that of the TPOR enzymes. If altered conformational elements were involved in MIF-mediated redox catalysis and biological activity, it thus appeared possible that MIF-derived peptides featuring such properties could function as MIF mimetics.Here we elected a 16-residue MIF peptide fragment spanning the CALC motif of MIF and encompassing the residues that are part of the predicted β-turn. The peptide ranged from residue Phe-50 to Ile-65 and was termed MIF-(50–65). It contained additional turn-stabilizing residues flanking the CXXC motif and covered the aromatic residue (Phe-50) which is part of extended CXXC sequence patterns according to Ellis et al. (16Ellis L.B.M. Saurugger P. Woodward C. Biochemistry. 1992; 31: 4882-4891Crossref PubMed Scopus (29) Google Scholar).The peptide and its C57S/C60S bis-serine, biotinylated, and fluorescein-modified variants as well as a peptide analog covalently cyclized by a lactam bridge were synthesized by solid phase Fmoc chemistry, HPLC-purified, and investigated for their conformational, redox, and in vitro TPOR catalytic properties. Applying a variety of immunological and cellular assays that are characteristic of the immunological, inflammatory, and cellular functions of MIF in vivo, we then asked whether the 16-meric peptide may have retained MIF-like functional properties and could serve to mimic cellular MIF activities.MATERIALS AND METHODSChemicals, Buffers, Recombinant MIF, and General Cell Culture Reagents—Protected amino acids were purchased from Rapp Polymer (Tübingen, Germany). All other peptide synthesis reagents were obtained from Bachem (Heidelberg, Germany). 5(6)-Carboxyfluorescein-N-hydroxysuccinimide ester (CFSE) was from Roche Diagnostics. Analytical grade acetonitrile was from Mallinckrodt Chemical Works and from Merck. Trifluoroacetic acid, reduced and oxidized glutathione (GSH and GSSG), l-cysteine and l-cystine, 1,4-dithiothreitol (DTT), insulin, bis-(2-hydroxyethyl)-disulfide (HED), β-nicotinamide adenine dinucleotide phosphate (reduced form, NADPH), glutathione reductase, l-3,4-dihydroxyphenylalanine methyl ester, sodium peroxidate, lipopolysaccharide O111:B4 (LPS), and dexamethasone (DEX) were obtained from Sigma. All general cell culture reagents such as media, supplements, antibiotics, and serum (for the latter, see below) were from Invitrogen. NIH 3T3 and THP-1 cells were bought from the German Society for Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cells were cultured by routine protocols at 37 °C in a humidified incubator with 5% CO2. Streptavidin-conjugated magnetic beads (Dynabeads M-280 streptavidin) were bought from Dynal Biotec ASA (Oslo, Norway). Streptavidin-coupled nanoparticles were a kind gift from Dr. G. Tovar (Fraunhofer IGB, Stuttgart, Germany). Biologically active recombinant human MIF (rMIF) was expressed, purified, and refolded as described previously (40Mischke R. Gessner A. Kapurniotu A. Jüttner S. Kleemann R. Brunner H. Bernhagen J. FEBS Lett. 1997; 414: 226-232Crossref PubMed Scopus (33) Google Scholar). Vascular endothelial cell growth factor (VEGF) was purchased from REALITech (Braunschweig, Germany), and basic fibroblast growth factor (bFGF) was bought from PeproTech (Rocky Hill, NJ). Crude endothelial cell growth factor was prepared from bovine hypothalamus as described by Maciag et al. (41Maciag T. Cerundolo J. Ilsley S. Kelley P.R. Forand R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5674-5678Crossref PubMed Scopus (576) Google Scholar). [3H]Thymidine was from Amersham Biosciences. Anti-phospho-ERK1/2 (E4, sc-7383) and anti-ERK1/2 (C16, sc-93) antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Miscellaneous chemicals, solvents, and salts were from Sigma. All reagents were of the highest grade commercially available.Synthesis and Purification of the MIF-derived Peptides MIF-(50–65) and C57S/C60S-MIF-(50–65) and Their Biotinylated, Fluoresceinated, Lactam-bridged Derivatives, and Control Peptides—Peptide synthesis was performed by solid phase synthetic protocols (SPPS) as described by us previously (42Kazantzis A. Waldner M. Taylor J.W. Kapurniotu A. Eur. J. Biochem. 2002; 269: 780-791Crossref PubMed Scopus (29) Google Scholar). The N terminus of the peptides was acetylated and the C terminus amidated. For synthesis of C57S/C60S-MIF-(50–65), serine residues were coupled at positions 57 and 60 instead of the active site cysteines. Biotinylation of MIF-(50–65) and C57S/C60S-MIF-(50–65) was performed at the N terminus of the fully protected and resin-bound peptides using normal coupling protocols in a mixture (2/1) of dimethylformamide and N-methyl-2-pyrrolidone, and an aminocaproate residue was also incorporated as a spacer between biotin and the peptide. N-terminal fluorescein-labeled forms of MIF-(50–65) and C57S/C60S-MIF-(50–65) were obtained by reaction of the side chain-protected, resin-bound peptides with CFSE which was applied in 1.7–2.3 molar excess. Reactions with CFSE were carried out for 3 h in a mixture (3/1) of dimethylformamide and dimethyl sulfoxide (Me2SO). Synthesis and side chain-to-side chain cyclization of cyclo57,60-[Asp57,Dap60]MIF-(50–65) and the linear control peptide [Asp57,Dap60]MIF-(50–65) was performed according to a procedure published previously (42Kazantzis A. Waldner M. Taylor J.W. Kapurniotu A. Eur. J. Biochem. 2002; 269: 780-791Crossref PubMed Scopus (29) Google Scholar).Briefly, as to the SPPS procedure in general, synthesis was performed on Rink resin applying the 9-fluorenylmethoxycarbonyl (Fmoc) group for temporary protection of the α-amino function. Side chains of trifunctional amino acids were protected with t-butyl (Ser), trityl (Cys), Trt (His), and t-butyl (Glu). Deprotection with simultaneous cleavage of the peptide from the resin was performed by treatment with 95% trifluoroacetic acid, 2.5% ethanedithiol, 2.5% water. Following evaporation of trifluoroacetic acid under vacuum at 30 °C, 10% acetic acid was added to the resulting product, and the aqueous phase was extracted three times with diethyl ether. The aqueous phase was then lyophilized. The obtained crude product, which was obtained in its reduced form and in a purity of about 90%, was further purified by C18 reverse phase (RP) HPLC (250 × 8 mm; 100 Å pore size, 7 μm particle size; Grom, Herrenberg, Germany). Elution of the peptides was achieved with two different water/acetonitrile elution programs. The first program consisted of a gradient of 10% B to 90% B between 1 and 31 min; and the second elution program was 0–7 min at 30% B, followed by a gradient from 30 to 60% B for 30 min, with buffer A 0.058% trifluoroacetic acid; buffer B 90% acetonitrile, 0.05% trifluoroacetic acid; flow rate 2 ml/min, and peptides were detected at 214 nm. The identity and purity of the peptides was verified by mass spectrometric analysis (see below and figure legends for details) and analytical HPLC. Following lyophilization, purified reduced peptide was stored at –20 °C until used further. Disulfide bridge-containing forms of the peptides were obtained by air oxidation in 0.1 m ammonium bicarbonate at a peptide concentration of 0.1 mg/ml. The oxidized peptide as well as the mixed disulfide species were collected on dry ice, deep frozen, and lyophilized. After lyophilization, the sample was dissolved in 10% acetic acid and immediately purified by HPLC (see above). For preparation of the purified reduced peptide see below. Mutant peptide C57S/C60S-MIF-(50–65) and the biotinylated, fluoresceinated, and side chain-to-side chain cyclized peptides were purified directly from their corresponding crude synthesis products by HPLC. The biotinylated and fluorescein-labeled wild-type derivatives were applied in the biological assays in their reduced forms.Peptide Verification by Mass Spectrometry, Electrophoresis, and Amino Acid Analysis—The mass spectrometric measurements for peptide verification and determination of the degree of oxidation/reduction and mixed disulfide formation were performed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS; instruments from Shimadzu Kratos Kompact MALDI 3 version 3.0.2, Duisburg, Germany) and G2025A LD-TOF-System Mass Spectrometer from Hewlett-Packard (Böblingen, Germany), and for some preparations by liquid chromatography-coupled electrospray MS (LC-ESMS) on a quadrupole ion trap MS (LCQ from Thermo-Finnigan, San Jose, CA) equipped with an electrospray ion source. Some analyses were performed with an Applied Biosystems API 3000 liquid chromatography-MS. For characterization of some of the side products of the syntheses, fast atom bombardment-MS (VARIAN-MAT 711, Thermo-Finnigan, Bremen, Germany) was applied.In addition, peptide verification and quantification of the fluoresceinated peptide analogs was performed by 4–12% NuPAGE and silver staining analysis (25Nguyen M. Lue H. Kleemann R. Thiele M. Tolle G. Finkelmeier D. Wagner E. Braun A. Bernhagen J. J. Immunol. 2003; 170: 3337-3347Crossref PubMed Scopus (115) Google Scholar) and (quantitative) amino acid analysis on Biotronik amino acid analyzer LC 5001 equipped with fluorescence detection of o-phthalaldehyde (Eppendorf, Hamburg, Germany) as described previously (43Soulimane T. Than M.E. Dewor M. Huber R. Buse G. Protein Sci. 2000; 9: 2
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