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• W1996343089 abstract "The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys5–Cys21, Cys9–Cys39, Cys19–Cys32, and Cys25–Cys31 connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541–544). The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys5–Cys21, Cys9–Cys39, Cys19–Cys32, and Cys25–Cys31 connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541–544). Vitronectin is a multidomain plasma glycoprotein involved in a variety of biological processes such as cell adhesion, cell migration, modulation of the immune system, and regulation of blood coagulation and fibrinolysis (1Tomasini B.R. Mosher D.F. Prog. Hemostasis Thromb. 1991; 10: 269-305PubMed Google Scholar, 2Preissner K.T. Seiffert D. Thromb. Res. 1998; 89: 1-21Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Many regulatory functions of vitronectin result from its ability to interact with plasminogen activator inhibitor 1 (PAI-1), 2The abbreviations used are: PAI-1, plasminogen activator inhibitor 1; SMB, somatomedin B; RP-HPLC, reversed phase-high performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; Acm, acetamidomethyl. 2The abbreviations used are: PAI-1, plasminogen activator inhibitor 1; SMB, somatomedin B; RP-HPLC, reversed phase-high performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; Acm, acetamidomethyl. a member of the serine protease inhibitor superfamily that inhibits both tissue- and urinary-type plasminogen activators (3Mimuro J. Loskutoff D.J. J. Biol. Chem. 1989; 264: 936-939Abstract Full Text PDF PubMed Google Scholar, 4Salonen E.M. Vaheri A. Pollanen J. Stephens R. Andreasen P. Mayer M. Dano K. Gailit J. Ruoslahti E. J. Biol. Chem. 1989; 264: 6339-6343Abstract Full Text PDF PubMed Google Scholar, 5Wiman B. Almquist A. Sigurdardottir O. Lindahl T. FEBS Lett. 1988; 242: 125-128Crossref PubMed Scopus (130) Google Scholar, 6Declerck P.J. De Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Muller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar). PAI-1 plays important roles in thrombosis and fibrinolysis and has been implicated in hemostasis, angiogenesis, and tumor metastasis (7Andreasen P.A. Egelund R. Petersen H.H. Cell Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (826) Google Scholar, 8Dellas C. Loskutoff D.J. Thromb. Haemostasis. 2005; 93: 631-640Crossref PubMed Scopus (230) Google Scholar, 9Wiman B. Thromb. Haemostasis. 1995; 74: 71-76Crossref PubMed Scopus (183) Google Scholar, 10Kohler H.P. Grant P.J. N. Engl. J. Med. 2000; 342: 1792-1801Crossref PubMed Scopus (0) Google Scholar, 11McMahon G.A. Petitclerc E. Stefansson S. Smith E. Wong M.K. Westrick R.J. Ginsburg D. Brooks P.C. Lawrence D.A. J. Biol. Chem. 2001; 276: 33964-33968Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Low abundant PAI-1 circulates in blood complexed with vitronectin (12Wiman B. Lindahl T. Almqvist A. Thromb. Haemostasis. 1988; 59: 392-395Crossref PubMed Scopus (26) Google Scholar); unliganded PAI-1 undergoes rapid “self-inactivation” into an inactive “latent” form in which the inhibitory reactive-site loop inserts as a new strand into the main β-sheet of the molecule (13Shore J.D. Day D.E. Francis-Chmura A.M. Verhamme I. Kvassman J. Lawrence D.A. Ginsburg D. J. Biol. Chem. 1995; 270: 5395-5398Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 14Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (520) Google Scholar, 15Hekman C.M. Loskutoff D.J. J. Biol. Chem. 1985; 260: 11581-11587Abstract Full Text PDF PubMed Google Scholar). Ample evidence suggests that vitronectin regulates the activity of PAI-1 and PAI-1-mediated cellular events by stabilizing the active form of the inhibitor and delaying its conformational transition to the latent state (16Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar, 17Lawrence D.A. Palaniappan S. Stefansson S. Olson S.T. Francis-Chmura A.M. Shore J.D. Ginsburg D. J. Biol. Chem. 1997; 272: 7676-7680Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (219) Google Scholar). Conversely, PAI-1 mediates, via binding, vitronectin-dependent cell adhesion and migration (19Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (603) Google Scholar, 20Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (429) Google Scholar, 21Loskutoff D.J. Curriden S.A. Hu G. Deng G. APMIS. 1999; 107: 54-61Crossref PubMed Scopus (144) Google Scholar, 22Kjoller L. Kanse S.M. Kirkegaard T. Rodenburg K.W. Ronne E. Goodman S.L. Preissner K.T. Ossowski L. Andreasen P.A. Exp. Cell Res. 1997; 232: 420-429Crossref PubMed Scopus (216) Google Scholar, 23Deng G. Curriden S.A. Hu G. Czekay R.P. Loskutoff D.J. J. Cell Physiol. 2001; 189: 23-33Crossref PubMed Scopus (97) Google Scholar). Thus, molecular recognition between vitronectin and PAI-1 is of great importance in biology. Loskutoff and colleagues (24Deng G. Royle G. Wang S. Crain K. Loskutoff D.J. J. Biol. Chem. 1996; 271: 12716-12723Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 25Okumura Y. Kamikubo Y. Curriden S.A. Wang J. Kiwada T. Futaki S. Kitagawa K. Loskutoff D.J. J. Biol. Chem. 2002; 277: 9395-9404Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 26Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 27Seiffert D. Ciambrone G. Wagner N.V. Binder B.R. Loskutoff D.J. J. Biol. Chem. 1994; 269: 2659-2666Abstract Full Text PDF PubMed Google Scholar) first pinpointed a high affinity PAI-1-binding site in vitronectin to the N-terminal somatomedin B (SMB) domain of the adhesive glycoprotein. The SMB domain consists of 44 amino acid residues (28Fryklund L. Sievertsson H. FEBS Lett. 1978; 87: 55-60Crossref PubMed Scopus (34) Google Scholar, 29Jenne D. Stanley K.K. EMBO J. 1985; 4: 3153-3157Crossref PubMed Scopus (154) Google Scholar, 30Suzuki S. Pierschbacher M.D. Hayman E.G. Nguyen K. Ohgren Y. Ruoslahti E. J. Biol. Chem. 1984; 259: 15307-15314Abstract Full Text PDF PubMed Google Scholar, 31Suzuki S. Oldberg A. Hayman E.G. Pierschbacher M.D. Ruoslahti E. EMBO J. 1985; 4: 2519-2524Crossref PubMed Scopus (277) Google Scholar), including eight conserved cysteines that form four functionally indispensable intramolecular disulfide bridges (24Deng G. Royle G. Wang S. Crain K. Loskutoff D.J. J. Biol. Chem. 1996; 271: 12716-12723Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Kamikubo et al. (32Kamikubo Y. Okumura Y. Loskutoff D.J. J. Biol. Chem. 2002; 277: 27109-27119Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) reported that an N-terminal fragment of VN of 97 amino acid residues, expressed in the cytoplasm of Escherichia coli and purified by immuno-affinity chromatography, showed activity in PAI-1 binding and antibody recognition similar to urea-activated vitronectin (uVN) 3Vitronectin is commonly purified from blood plasma under two different conditions, denaturing and non-denaturing. Denaturing chromatography or treatment with urea of natively purified vitronectin (nVN) results in a protein (uVN, standing for urea-treated, -denatured, or -activated vitronectin) often with altered conformational and/or functional properties. However, both nVN and uVN are capable of binding PAI-1 with similar, if not identical, activity. 3Vitronectin is commonly purified from blood plasma under two different conditions, denaturing and non-denaturing. Denaturing chromatography or treatment with urea of natively purified vitronectin (nVN) results in a protein (uVN, standing for urea-treated, -denatured, or -activated vitronectin) often with altered conformational and/or functional properties. However, both nVN and uVN are capable of binding PAI-1 with similar, if not identical, activity. purified from human blood. CNBr cleavage of the recombinant VN fragment released the SMB domain elongated C-terminally by an extra seven-residue RGD motif (we term rVN1–51-1). Partial reduction and S-alkylation of rVN1–51-1 coupled with proteomics techniques identified a consecutively arranged, uncrossed pattern of disulfide bridges in the SMB domain, i.e. Cys5–Cys9, Cys19–Cys21, Cys25–Cys31, and Cys32–Cys39 (32Kamikubo Y. Okumura Y. Loskutoff D.J. J. Biol. Chem. 2002; 277: 27109-27119Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). As rVN1–51-1 was functionally indistinguishable from intact uVN, the linear disulfide topology was thought to exist in native vitronectin (32Kamikubo Y. Okumura Y. Loskutoff D.J. J. Biol. Chem. 2002; 277: 27109-27119Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Read and colleagues (18Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (219) Google Scholar) subsequently reported the crystal structure of PAI-1 complexed with a similarly obtained recombinant VN1–51 (we term rVN1–51-2), which was shown to contain a crossed pattern of disulfides, i.e. Cys5–Cys21, Cys9–Cys39, Cys19–Cys32, and Cys25–Cys31. As Zhou et al. (18Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (219) Google Scholar) did not address the difference in disulfide topology between rVN1–51-1 and rVN1–51-2, the question of which disulfide linkages represent the topology in native vitronectin was left unanswered. Added to the brewing controversy was a recent finding by Peterson and colleagues (33Horn N.A. Hurst G.B. Mayasundari A. Whittemore N.A. Serpersu E.H. Peterson C.B. J. Biol. Chem. 2004; 279: 35867-35878Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) that the first 51 amino acid residues cleaved by CNBr from natively purified vitronectin3 (we term nVN1–51-3) had a third type of disulfide topology (Cys5–Cys9, Cys19–Cys31, Cys21–Cys32, and Cys25–Cys39), which matched neither the pattern reported for rVN1–51-1 nor for rVN1–51-2. Horn et al. (33Horn N.A. Hurst G.B. Mayasundari A. Whittemore N.A. Serpersu E.H. Peterson C.B. J. Biol. Chem. 2004; 279: 35867-35878Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) further suggested that rVN1–51-1 and rVN1–51-2 were stable folding intermediates with non-native disulfide linkages, a view hotly contested in a latest report by Kamikubo et al. (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar). While sharply pointing out the lack of any reported functional data for nVN1–51-3 and the possibility of disulfide scrambling during the handling of nVN1–51-3, Kamikubo et al. (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar) showed that a fully reduced, denatured rVN1–51-1 could be efficiently folded in vitro into a conformationally homogeneous, thermodynamically stable, and functionally active molecule. Furthermore, none of the folding intermediates accumulated showed any biological activity as judged by PAI-1 binding and antibody recognition (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar). The conflicting reports by several laboratories have raised a series of important questions that remain to be answered. However, first and foremost, what is the native disulfide topology in vitronectin? To resolve this increasingly controversial issue that has so far clouded the understanding of PAI-1-vitronectin recognition, we chemically synthesized, by using native chemical ligation and orthogonal protection of selected Cys residues, three topologically different forms of the SMB domain, sSMB-1, sSMB-2, and sSMB-3, with predefined disulfide connectivities corresponding to those reported for rVN1–51-1, rVN1–51-2, and nVN1–51-3, respectively. We also oxidatively folded a fully reduced SMB domain in aqueous solution using the protocol recently published by Kamikubo et al. (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar), yielding sSMB-4. In addition, we prepared, by CNBr cleavage, the SMB-containing fragments nVN1–51 and uVN1–51 from natively purified as well as urea-activated vitronectin of human blood origin. Biochemical and functional characterization of sSMB-1–4, nVN1–51, and uVN1–51 reported here, while fully accounting for various published discrepancies, unequivocally establish the native and the only functional disulfide topology in vitronectin as Cys5–Cys21, Cys9–Cys39, Cys19–Cys32, and Cys25–Cys31, in support of the earlier findings by Read and colleagues (18Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (219) Google Scholar). Peptide Synthesis, Native Chemical Ligation, and Oxidative Folding—Stepwise chemical synthesis of the 44-residue SMB domain on solid phase by t-butoxycarbonyl chemistry turned out to be extremely difficult if not impossible. We took advantage of the native chemical ligation technique pioneered by Kent and colleagues (35Dawson P.E. Muir T.W. Clark-Lewis I. Kent S.B. Science. 1994; 266: 776-779Crossref PubMed Scopus (3064) Google Scholar, 36Dawson P.E. Kent S.B. Annu. Rev. Biochem. 2000; 69: 923-960Crossref PubMed Scopus (917) Google Scholar, 37Muir T.W. Dawson P.E. Kent S.B. Methods Enzymol. 1997; 289: 266-298Crossref PubMed Scopus (105) Google Scholar), assembled the following four full-length peptides in high purity (hereinafter referred to as peptides 1–4; C denotes Acm-protected Cys; ↓ depicts the site for native chemical ligation). Specifically, the C-terminal peptides were assembled on t-butoxycarbonyl-Thr(benzyl)-OCH2-phenylacetamidomethyl resin using the n,n-diisopropylethylamine in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation protocol developed by Kent and colleagues (38Schnolzer M. Alewood P. Jones A. Alewood D. Kent S.B. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (936) Google Scholar). The N-terminal peptides were synthesized on custom-made thioester resin (trityl)SCH2CH2COO-Leu-OCH2-phenylacetamidomethyl using an otherwise identical chemistry. After HF cleavage and deprotection, crude peptides were purified to homogeneity by preparative C18 HPLC, and their molecular masses were verified by electrospray ionization mass spectrometry (ESI-MS). Two-segment native chemical ligation was performed as described previously (39Wu Z. Alexandratos J. Ericksen B. Lubkowski J. Gallo R.C. Lu W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11587-11592Crossref PubMed Scopus (42) Google Scholar, 40Li X. de Leeuw E. Lu W. Biochemistry. 2005; 44: 14688-14694Crossref PubMed Scopus (21) Google Scholar), resulting in the four full-length SMB peptides with correct molecular masses. To obtain sSMB-1, sSMB-2, and sSMB-3, Acm-protected peptides 1–3 were first oxidized at 0.25 mg/ml in phosphate-buffered saline buffer, pH 7.4, by 20% (v/v) Me2SO. An overnight oxidation of each peptide at room temperature yielded three chromatographically distinct species. After disulfide mapping, three desired folding intermediates with Cys19–Cys21/Cys32–Cys39 (for sSMB-1), Cys5–Cys21/Cys25–Cys31 (for sSMB-2), and Cys5–Cys9/Cys21–Cys32 (for sSMB-3) were selected for Acm deprotection/disulfide formation. Specifically, the two-disulfide-bridged peptides at 0.5 mg/ml in an acidic solution containing 0.1 m citric acid, 0.2 m HCl, and 20% methanol were treated by 1 mm iodine for 15 min, each resulting in three fully oxidized and topologically different SMB domains, from which sSMB-1, sSMB-2, or sSMB-3 was eventually decoded. Spontaneous folding of the fully unprotected peptide 4 in aqueous solution under redox control of reduced and oxidized glutathione was carried out essentially as described for rVN1–51-1 (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar), giving rise to a predominant folding species termed sSMB-4. All four synthetic SMB domains were purified to homogeneity on RP-HPLC and verified by ESI-MS. Quantification of SMB domains was carried out by UV absorbance measurements at 280 nm using molar extinction coefficients calculated according to a published algorithm (41Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3362) Google Scholar). Dissection of Two-disulfide-bridged Oxidation Intermediates—We strategically selected Cys residues to be protected by Acm in the SMB sequence so that discerning the pattern of the first two disulfides after Me2SO oxidation could be achieved by one-step proteolysis coupled with liquid chromatography-MS analysis. Bovine chymotrypsin and trypsin and Staphyloccocus aureus Glu-specific V8 protease were purchased from Worthington Biochemical Co. Enzymatic digestion was carried out for 1 h at 37°C in 50 mm Tris, 20 mm CaCl2, 0.005% Triton X-100, pH 8.3. The desired oxidation intermediate derived from peptide 1 contained Cys19–Cys21/Cys32–Cys39, and was readily identified by digestion with chymotrypsin that cleaved (Tyr27-Tyr28 or Tyr28-Gln29) between Cys21 and Cys32. The desired oxidation intermediates derived from peptides 2 and 3 both contained two linear and uncrossed disulfides, Cys5–Cys21/Cys25–Cys31, and Cys5–Cys9/Cys21–Cys32, respectively. For the peptide 2 derivative, Glu-specific V8 protease made a clean split (Glu23-Leu24) between Cys21 and Cys25, whereas trypsin cleaved (Lys17-Lys18 or Lys18-Cys19) between Cys9 and Cys21 of the peptide 3 derivative. Using this highly simplified approach, all undesired Me2SO-oxidized intermediates with crossed patterns of disulfide bonding were readily excluded. Dissection of Fully Oxidized sSMB-1, sSMB-2, and sSMB-3—To confirm the linear and uncrossed disulfide pattern in sSMB-1, the peptide was treated with chymotrypsin/Glu-specific V8 protease for 1 h at 37°C, generating, as judged by ESI-MS, the following four major fragments: [SC5KGRC9TE], [NVDKKC19QC21DE], [LC25SYY][QSC31C32TDY][C39KPQVT], and [LC25SYYQSC31C32TDYTAEC39KPQVT]. For sSMB-2, cleavage by chymotrypsin/trypsin yielded three major fragments: [DQESC5K][C19QC21DELC25SY][QSC31C32TDY], [C9TEGF][TAEC39KPQVT], and [DQESC5K][C19QC21DELC25-SYYQSC31C32TDY], thus confirming the presence of Cys19–Cys32 and Cys9–Cys39 in sSMB-2. A combination cleavage of sSMB-3 by chymotrypsin and Glu-specific V8 protease resulted in [SC5KGRC9TE], [NVDKKC19QC21DE][YQSC31C32TDY], [NVDKKC19QC21DE][QSC31C32TDY], and [LC25SY] [C39KPQVT], thus confirming the presence of Cys19–Cys31 and Cys25–Cys39 in the molecule. Isolation of VN1–51 from Intact Vitronectin—Natively purified and urea-activated vitronectins of human blood source were purchased from Molecular Innovations, Inc. The SMB-containing VN1–51 was released from vitronectin (0.5 mg/ml in 2.5% trifluoroacetic acid) by CNBr (20 mg/ml). After an overnight cleavage at room temperature, VN1–51 was purified by analytical C18 RP-HPLC. Two products resulted, the major component containing a C-terminal homoserine lactone and the minor species ending with homoserine. At basic pH, spontaneous hydrolysis quickly converts homoserine lactone to homoserine. Proteolytic Fingerprinting of sSMB-2, sSMB-4, nVN1–51, and uVN1–51—50 μg of sSMB-2 or sSMB-4 was dissolved at 1 mg/ml in 50 mm Tris/HCl buffer containing 20 mm CaCl2 and 0.005% Triton X-100, pH 8.3, to which 1 μg of each enzyme was added in one of the following three binary combinations: chymotrypsin/trypsin, chymotrypsin/V8 protease, and trypsin/V8 protease. After an 18-h incubation at 37 °C, the cleavage reaction was terminated by addition of 50 μl of 10% acetic acid, followed by liquid chromatography-MS analyses. Due to limitations in quantities, much less nVN1–51 or uVN1–51 was used in otherwise identical experiments. Surface Plasmon Resonance Spectroscopy—The ability of synthetic SMB domains and VN1–51 to interact with PAI-1 (purchased from Oxford Biomedical Research) in solution was quantified on a Biacore 3000 surface plasmon resonance instrument according to a competition assay protocol developed by Loskutoff and colleagues (32Kamikubo Y. Okumura Y. Loskutoff D.J. J. Biol. Chem. 2002; 277: 27109-27119Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar, 42Kamikubo Y. De Guzman R. Kroon G. Curriden S. Neels J.G. Churchill M.J. Dawson P. Oldziej S. Jagielska A. Scheraga H.A. Loskutoff D.J. Dyson H.J. Biochemistry. 2004; 43: 6519-6534Crossref PubMed Scopus (33) Google Scholar). Briefly, 1100 resonance units of urea-activated vitronectin were immobilized (in 10 mm acetate buffer, pH 4.0) to a CM5 sensor chip using the 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide coupling chemistry and procedures recommended by the manufacturer. Kinetic analysis of the binding to vitronectin of PAI-1, either alone or in the presence of sSMB, was carried out at 25 °C in HBS-EP buffer (10 mm HEPES, 150 mm NaCl, 3 mm EDTA, 0.005% surfactant P20, pH 7.4). For competition, 25 nm PAI-1 was incubated at room temperature for 15 min with varying concentrations of synthetic SMB and injected at a flow rate of 20 μl/min for 5 min, followed by 5-min dissociation. The concentration of free PAI-1 in solution (not complexed with sSMB) was deduced, based on the initial rate (slope) of VN association, from a calibration curve established by resonance unit measurements of different concentrations of PAI-1 injected alone. Non-linear regression analysis was performed using GraphPad Prism 4 to give rise to IC50 values, concentrations of SMB at which 50% of PAI-1 was sequestered in SMB-PAI-1 complexes, thus unavailable for VN binding. The SMB domain with eight Cys residues possesses 105 unique disulfide connectivities. We selectively protected four Cys residues in SMB with the orthogonal protecting agent Acm and oxidatively folded the resultant peptide containing four free cysteines, yielding three two-disulfide-bridged intermediates separable on reversed phase HPLC. After the disulfide linkages were discerned for all three species by mass mapping of peptide fragments generated by proteolysis, the folding intermediate with desired disulfide bridges was selected for Acm deprotection and simultaneous formation of two remaining disulfide bonds, also in three unique combinations. Mass mapping aided by enzymatic digestion was performed again to definitively establish disulfide connectivities in three fully oxidized products. The strategy for the synthesis of sSMB-1, sSMB-2, and sSMB-3 with predefined disulfide connectivities corresponding to the ones previously reported for rVN1–51-1, rVN1–51-2, and nVN1–51-3, respectively, is illustrated in Fig. 1. For comparison, a fully reduced and unprotected synthetic SMB was oxidatively folded in aqueous solution according to the published protocol (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar), yielding conformationally homogeneous and thermodynamically stable sSMB-4. In addition, nVN1–51 and uVN1–51, which contain the SMB domain C-terminally connected to a seven-residue RGD motif (RGDVFTM), were prepared by CNBr cleavage, at the Met51-Pro52 peptide bond, of natively purified and urea-treated vitronectin from human blood, respectively. All synthetic SMB domains as well as nVN1–51 and uVN1–51 were purified by RP-HPLC to homogeneity, and their molecular masses ascertained by ESI-MS. Shown in Fig. 2 are sSMB-1, sSMB-2, sSMB-3, and sSMB-4 analyzed by C18 RP-HPLC and ESI-MS. The molecular masses of synthetic sSMB domains were found to be 5003.2 ± 0.3 Da, in agreement with the expected value of 5003.5 Da calculated on the basis of average isotopic compositions of fully oxidized SMB. High resolution analytical RP-HPLC is a powerful tool for chromatographically differentiating peptides with different disulfide connectivities (43Wu Z. Hoover D.M. Yang D. Boulegue C. Santamaria F. Oppenheim J.J. Lubkowski J. Lu W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8880-8885Crossref PubMed Scopus (363) Google Scholar). As shown in Fig. 2, the three topological analogs sSMB-1, sSMB-2 and sSMB-3, at least 1.5 min apart from each other, were fully separated on analytical C18 RP-HPLC. One immediate anomaly was that sSMB-4 had different retention from sSMB-1 (1.6 min apart), suggesting that sSMB-4 and sSMB-1 were topologically different. This finding was surprising because, based on the recent report by Kamikubo et al. (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar), we had expected sSMB-4 to be identical to sSMB-1. Proteolytic digestion of sSMB-1 and sSMB-4 by Glu-specific V8 protease confirmed their difference in disulfide bonding pattern (data not shown). Notably, sSMB-4 and sSMB-2 showed identical retention on RP-HPLC (Fig. 2), and, in fact, co-eluted when injected as a mixture. We therefore hypothesized that folding of fully reduced SMB under redox control in aqueous solution had produced, in contrast to the published assumption (34Kamikubo Y. Kroon G. Curriden S.A. Dyson H.J. Loskutoff D.J. Biochemistry. 2006; 45: 3297-3306Crossref PubMed Scopus (11) Google Scholar), a non-linear disulfide-bonding pattern identical to the one reported by Zhou et al. (18Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (219) Google Scholar) for the crystal structure of rVN1–51-2 complexed with PAI-1. Two powerful lines of evidence support this hypothesis. First, sSMB-2 and sSMB-4 were structurally identical. We incubated sSMB-2 and sSMB-4 with three different binary combinations of chymotrypsin, trypsin, and Glu-specific V8 protease and generated identical proteolytic “fingerprints” for the two SMB domains (Fig. 3). Results from mass spectroscopic analyses of all peptide" @default.
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• W1996343089 title "Defining the Native Disulfide Topology in the Somatomedin B Domain of Human Vitronectin" @default.
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