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• W2044483798 abstract "The NH2-terminal somatomedin B (SMB) domain (residues 1–44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction andS-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH2-terminal portion of active rSMB were identified as Cys5–Cys9 and Cys19–Cys21. Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys25–Cys31 and Cys32–Cys39 by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins. The NH2-terminal somatomedin B (SMB) domain (residues 1–44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction andS-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH2-terminal portion of active rSMB were identified as Cys5–Cys9 and Cys19–Cys21. Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys25–Cys31 and Cys32–Cys39 by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins. vitronectin type 1 plasminogen activator inhibitor somatomedin B recombinant SMB monoclonal antibody N-ethylmaleimide 4-vinylpyridine Tris (2-carboxyethyl) phosphine hydrochloride phenylthiohydantoin S-ethylsuccinimidocysteine S-pyridylethylcysteine epidermal growth factor Vitronectin (VN)1 is a 75-kDa adhesive glycoprotein that is present in plasma and the extracellular matrix, and it plays a significant role in a number of biological processes (1Tomasini B.R. Mosher D.F. Coller B.S. Progress in Hemostasis and Thrombosis. W. B. Saunders, Philadelphia, PA1991: 269-305Google Scholar). For example, VN is anchored to the extracellular matrix via collagen or proteoglycan binding, and it promotes cell attachment, spreading, and migration through specific interactions of its single Arg-Gly-Asp (R-G-D) sequence with cellular integrins, such as αvβ3 (2Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2716) Google Scholar, 3Seiffert D. Smith J.W. J. Biol. Chem. 1997; 272: 13705-13710Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), αvβ5 (4Smith J.W. Vestal D.J. Irwin S.V. Burke T.A. Cheresh D.A. J. Biol. Chem. 1990; 265: 11008-11013Abstract Full Text PDF PubMed Google Scholar), and αIIbβ3 (3Seiffert D. Smith J.W. J. Biol. Chem. 1997; 272: 13705-13710Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 5Thiagarajan P. Kelly K. Thromb. Haemostasis. 1988; 60: 514-517Crossref PubMed Scopus (11) Google Scholar). Upon binding to VN, these integrins activate signaling pathways and regulate cytoskeletal reorganization and kinase activation (6Meredith J.E., Jr. Winitz S. Lewis J.M. Hess S. Ren X.D. Renshaw M.W. Schwartz M.A. Endocr. Rev. 1996; 17: 207-220Crossref PubMed Scopus (145) Google Scholar). VN also binds to urinary-type plasminogen activator receptor on the cell surface, an interaction that also promotes cell adhesion (7Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 8Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar, 9Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (430) Google Scholar, 10Sidenius N. Blasi F. FEBS Lett. 2000; 470: 40-46Crossref PubMed Scopus (77) Google Scholar). In addition to its functions in cell adhesion and migration, VN was shown to act as an inhibitor of the cytolytic reactions of complement by binding to complement factor C5b-7 (1Tomasini B.R. Mosher D.F. Coller B.S. Progress in Hemostasis and Thrombosis. W. B. Saunders, Philadelphia, PA1991: 269-305Google Scholar). In this regard, it is now clear that VN is identical to the S protein of complement (11Podack E.R. Muller-Eberhard H.J. J. Biol. Chem. 1979; 254: 9808-9814Abstract Full Text PDF PubMed Google Scholar, 12Jenne D. Stanley K.K. EMBO J. 1985; 4: 3153-3157Crossref PubMed Scopus (156) Google Scholar). Finally, VN interacts with several critical proteins that regulate thrombosis and fibrinolysis. For example, it protects thrombin from rapid, heparin-dependent inactivation by antithrombin III, possibly because it acts as a heparin scavenger (13Podack E.R. Griffin J.H. J. Biol. Chem. 1986; 261: 7387-7392Abstract Full Text PDF PubMed Google Scholar). It also binds to type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of both tissue- and urinary-type plasminogen activators, and stabilizes its biological activity (14Declerck P.J., De Mol M. Alessi M.-C. Baudner S. Paques E.-P. Preissner K. Müller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar, 15Wiman B. Almquist A. Sigurdardottir O. Lindahl T. FEBS Lett. 1988; 242: 125-128Crossref PubMed Scopus (130) Google Scholar, 16Mimuro J. Loskutoff D.J. J. Biol. Chem. 1989; 264: 936-939Abstract Full Text PDF PubMed Google Scholar, 17Salonen 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). Moreover, all active PAI-1 in plasma circulates in complex with VN (18Wiman B. Lindahl T. Almqvist A. Thromb. Haemostasis. 1988; 59: 392-395Crossref PubMed Scopus (27) Google Scholar). Taken together, these observations suggest that VN may be a cofactor for PAI-1 (19van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (142) Google Scholar). The interaction between VN and PAI-1 is potentially important because the plasminogen activation system plays a role in physiological processes such as fibrinolysis and cell migration (20Chapman H.A. Curr. Opin. Cell Biol. 1997; 9: 714-724Crossref PubMed Scopus (421) Google Scholar) and in pathological processes such as tumor growth and metastasis (21Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2291) Google Scholar). PAI-1 not only regulates the above biological processes by inhibiting plasminogen activation, but it also can directly affect integrin- and urinary-type plasminogen activator receptor-mediated cell attachment to and migration on VN (9Deng G. Curriden S.A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (430) Google Scholar, 22Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (605) Google Scholar,23Loskutoff D.J. Curriden S.A., Hu, G. Deng G. Acta. Pathol. Microbiol. Immunol. Scand. 1999; 107: 54-61Crossref PubMed Scopus (144) Google Scholar).The mature human VN molecule contains 459 amino acid residues and is composed of a number of functionally unique domains (12Jenne D. Stanley K.K. EMBO J. 1985; 4: 3153-3157Crossref PubMed Scopus (156) Google Scholar, 24Suzuki 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, 25Suzuki S. Oldberg A. Hayman E.G. Pierschbacher M.D. Ruoslahti E. EMBO J. 1985; 4: 2519-2524Crossref PubMed Scopus (277) Google Scholar). The domain at the very NH2 terminus (residues 1–44) of VN is the Cys-rich somatomedin B (SMB) domain, and this domain has been isolated from human serum as a separate soluble protein (26Fryklund L. Sievertsson H. FEBS Lett. 1978; 87: 55-60Crossref PubMed Scopus (34) Google Scholar, 27Standker L. Enger A. Schulz-Knappe P. Wohn K.D. Germer M. Raida M. Forssmann W.G. Preissner K.T. Eur. J. Biochem. 1996; 241: 557-563Crossref PubMed Scopus (23) Google Scholar). The single R-G-D sequence in VN is located immediately COOH-terminal to the SMB domain (residues 45–47) in the so-called connecting region, a region that also contains a putative collagen-binding site (28Izumi M. Shimo-Oka T. Morishita N., Ii, I. Hayashi M. Cell Struct. Funct. 1988; 13: 217-225Crossref PubMed Scopus (45) Google Scholar). Finally, the two most COOH-terminal domains in VN have, respectively, three and four tandem repeat sequences with weak homology to sequences in hemopexin (1Tomasini B.R. Mosher D.F. Coller B.S. Progress in Hemostasis and Thrombosis. W. B. Saunders, Philadelphia, PA1991: 269-305Google Scholar). The second hemopexin homology domain contains the positively charged heparin-binding segment (residues 348–370) (29Kost C. Stuber W. Ehrlich H.J. Pannekoek H. Preissner K.T. J. Biol. Chem. 1992; 267: 12098-12105Abstract Full Text PDF PubMed Google Scholar).The high affinity PAI-1-binding site in VN has been localized to the SMB domain (30Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 31Seiffert D. Ciambrone G. Wagner N.V. Binder B.B. Loskutoff D.J. J. Biol. Chem. 1994; 269: 2659-2666Abstract Full Text PDF PubMed Google Scholar, 32Sigurdardottir O. Wiman B. Biochim. Biophys. Acta. 1994; 1208: 104-110Crossref PubMed Scopus (28) Google Scholar, 33Deng 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, 34Okumura Y. Kamikubo Y. Curriden S.A. Wang J. Kiwada T. Futaki S. Kitagawa K. Loskutoff D.J. J. Biol. Chem. 2002; 11: 9395-9404Abstract Full Text Full Text PDF Scopus (65) Google Scholar, 35Philips M. Johnsen H. Thorsen S. Fibrinolysis Proteolysis. 2000; 14: 22-34Crossref Scopus (5) Google Scholar). This domain contains eight Cys residues arranged in four disulfide bonds, suggesting that it is folded into a compact structure that is stabilized by the disulfide bonds. Seiffert and Wagner (36Seiffert D. Wagner N.V. Biochimie (Paris). 1997; 79: 205-210Crossref PubMed Scopus (5) Google Scholar) demonstrated previously that treatment of the isolated SMB domain with reducing agents abolished PAI-1 binding. Deng et al. (33Deng 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) also reported that conversion of any single Cys residue in the SMB domain into alanine destroyed its PAI-1 binding activity. These observations indicate that correct disulfide linkages in the SMB domain are required for PAI-1 binding. However, the identification and arrangement of the individual disulfide bonds in the SMB domain remain unknown. All eight Cys residues are strictly conserved in the VN molecules isolated from a number of other species, including rabbit (33Deng 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) and mouse (37Seiffert D. Keeton M. Eguchi Y. Sawdey M. Loskutoff D.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9402-9406Crossref PubMed Scopus (103) Google Scholar), and in other SMB-like proteins, such as plasma cell membrane glycoprotein PC-1 (38Buckley M.F. Loveland K.A. McKinstry W.J. Garson O.M. Goding J.W. J. Biol. Chem. 1990; 265: 17506-17511Abstract Full Text PDF PubMed Google Scholar) and autotaxin (39Murata J. Lee H.Y. Clair T. Krutzsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Abstract Full Text PDF PubMed Google Scholar). The disulfide-linked structure of these SMB-like proteins also is unknown. Because disulfide bonds contribute to the native structure, biological activity, and stability of folded proteins, the identity of the disulfide bonds in SMB must be determined to begin to understand its tertiary structure.In the present study, we expressed the SMB domain of human VN in transformed Escherichia coli as a fusion protein containing 97 amino acid residues linked to the COOH terminus of thioredoxin (VN1–97). We then isolated the active recombinant SMB (rSMB) domain from the resulting mixture of active and inactive rSMBs by affinity chromatography using a conformation-specific monoclonal antibody (mAb) and characterized its PAI-1 binding activity. We also mapped the four disulfide bonds in the purified active rSMB domain and evaluated their functional importance. The disulfide linkages present in active rSMB were identified by partial reduction and S-alkylation under acidic conditions, chemical and proteolytic digestions, mass spectrometry, and protein sequencing. These results indicate that the disulfide bonds in active rSMB are arranged consecutively in a linear uncrossed pattern and suggest that the disulfide linkages within the SMB domain of human VN are similarly arranged.DISCUSSIONThe active form of PAI-1 in solution is relatively unstable and rapidly changes into the inactive or latent conformation (50Hekman C.M. Loskutoff D.J. J. Biol. Chem. 1985; 260: 11581-11587Abstract Full Text PDF PubMed Google Scholar). This conformational change is characterized by a dramatic structural rearrangement, with the reactive center loop of the inhibitor being inserted into β-sheet A as the central β-strand s4A (51Mottonen 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). This alteration in the structure of active PAI-1 is accompanied by a decrease in the availability of its reactive site for tissue- and urinary-type plasminogen activators, resulting in the loss of inhibitory activity. Interestingly, the active conformation of PAI-1 can be stabilized by binding to VN, an interaction that results in a 2–3-fold increase in the half-life of the inhibitor (19van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (142) Google Scholar). VN binding is thought to stabilize PAI-1 by restricting movement of the central β-sheet A of the inhibitor and preventing insertion of the reactive center loop (52Lawrence D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar).The Cys-rich domain at the very NH2 terminus of VN (i.e. residues 1–44; the SMB domain) contains the high affinity PAI-1-binding site (30Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 31Seiffert D. Ciambrone G. Wagner N.V. Binder B.B. Loskutoff D.J. J. Biol. Chem. 1994; 269: 2659-2666Abstract Full Text PDF PubMed Google Scholar, 32Sigurdardottir O. Wiman B. Biochim. Biophys. Acta. 1994; 1208: 104-110Crossref PubMed Scopus (28) Google Scholar, 33Deng 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, 34Okumura Y. Kamikubo Y. Curriden S.A. Wang J. Kiwada T. Futaki S. Kitagawa K. Loskutoff D.J. J. Biol. Chem. 2002; 11: 9395-9404Abstract Full Text Full Text PDF Scopus (65) Google Scholar, 35Philips M. Johnsen H. Thorsen S. Fibrinolysis Proteolysis. 2000; 14: 22-34Crossref Scopus (5) Google Scholar). Alanine scanning mutagenesis of the SMB domain demonstrated that each of the eight Cys residues are critical for PAI-1 binding and are required for VN to stabilize PAI-1 activity (33Deng 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). The specific binding of PAI-1 to VN also requires the correct disulfide linkages in SMB (36Seiffert D. Wagner N.V. Biochimie (Paris). 1997; 79: 205-210Crossref PubMed Scopus (5) Google Scholar), further pointing to the importance of the Cys residues. Despite this, the identity of the disulfide linkages in the SMB domain and the exact location of the essential disulfide bonds for PAI-1 binding remain unknown. In fact, very little is known about the three-dimensional structure of VN because no NMR or x-ray crystallography data are available. These considerations emphasize the importance of identifying the disulfide linkages in the SMB domain to begin to understand its tertiary structure.To determine the disulfide bonds present in the SMB domain of human VN, the correctly folded and biologically active form of rSMB was purified to homogeneity and characterized biochemically. For this purpose, rSMB was expressed in transformed E. coli as a fusion protein with thioredoxin, and the active form was purified from the mixture of active and inactive forms by immunoaffinity chromatography using a column of mAb 153-conjugated gel. This mAb recognizes an immunoepitope within the SMB domain and inhibits the binding of PAI-1 to VN (31Seiffert D. Ciambrone G. Wagner N.V. Binder B.B. Loskutoff D.J. J. Biol. Chem. 1994; 269: 2659-2666Abstract Full Text PDF PubMed Google Scholar). Moreover, this mAb only weakly recognizes VN in Western blots, and treatment of VN with reducing agents destroys the ability of both PAI-1 and mAb 153 to bind to the SMB domain (36Seiffert D. Wagner N.V. Biochimie (Paris). 1997; 79: 205-210Crossref PubMed Scopus (5) Google Scholar). These results indicate that mAb 153 recognizes a conformational epitope that is in close proximity to the PAI-1-binding site and is created by correctly formed disulfide linkages. This epitope is presumably masked or altered in the inactive form of SMB. Taken together, these observations indicate that mAb 153 is a useful tool for distinguishing between the active and inactive forms of SMB. This conclusion is supported by the observation that the PAI-1 binding activity of the purified rSMB was similar to that of urea-activated VN itself (Fig. 4). However, the form of rSMB that did not bind to the mAb 153-conjugated gel lacked PAI-1 binding activity (Fig. 4), even though it had the same molecular mass and NH2-terminal amino acid sequence as the active form (TableI).A variety of approaches were taken to identify the four pairs of disulfide bonds present in the active human SMB domain. We initially attempted to cleave the intact and unreduced SMB domain using trypsin, endoproteinase Glu-C, pepsin, and thermolysin, but the recombinant peptide was relatively resistant to these proteinases (data not shown). We next employed the partial reduction and S-alkylation method, an approach that has been widely used for determining the disulfide linkages present in small disulfide bond-rich proteins (45Gray W.R. Protein Sci. 1993; 2: 1749-1755Crossref PubMed Scopus (53) Google Scholar, 46White C.E. Hunter M.J. Meininger D.P. Garrod S. Komives E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10177-10182Crossref PubMed Scopus (27) Google Scholar, 47Katsumi A. Tuley E.A. Bodo I. Sadler J.E. J. Biol. Chem. 2000; 275: 25585-25594Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 48Bures E.J. Hui J.O. Young Y. Chow D.T. Katta V. Rohde M.F. Zeni L. Rosenfeld R.D. Stark K.L. Haniu M. Biochemistry. 1998; 37: 12172-12177Crossref PubMed Scopus (72) Google Scholar, 49Gray W.R. Protein Sci. 1993; 2: 1732-1748Crossref PubMed Scopus (235) Google Scholar). This method relies on partial reduction by TCEP at low pH, followed by reversed phase chromatography to separate the partially reduced and S-alkylated products. The purified peptides are then analyzed by protein sequencing to determine the location of theS-alkylated Cys residues. By using this approach, two pairs of disulfide bonds, Cys5–Cys9 and Cys19–Cys21, were identified in the NH2-terminal portion of the rSMB domain (Table III). In subsequent experiments, protease treatment of partially reduced andS-alkylated rVN1–51 identified the two other disulfide linkages as Cys25–Cys31 and Cys32–Cys39 (Table IV). Based on these observations, we propose that the active, recombinant SMB domain of human VN has four simple disulfide linkages arranged consecutively in a linear, uncrossed pattern: Cys5–Cys9, Cys19–Cys21, Cys25–Cys31, and Cys32–Cys39 (Fig.9A).Harrison and Sternberg (53Harrison P.M. Sternberg M.J. J. Mol. Biol. 1996; 264: 603-623Crossref PubMed Scopus (112) Google Scholar) reported previously that small disulfide bond-rich proteins or functional domains (which are defined as having less than 100 amino acid residues and two or more disulfide bonds) have a structural motif termed the disulfide β-cross, a structural feature imposed by four closely clustered Cys residues. This structural motif consists of a β-hairpin loop with two disulfide bonds connecting each strand of the hairpin to two adjacent Cys residues. Importantly, the disulfide linkages are generally overlapping and crossed (53Harrison P.M. Sternberg M.J. J. Mol. Biol. 1996; 264: 603-623Crossref PubMed Scopus (112) Google Scholar). This structural motif may provide a folding nucleus in small disulfide bond-rich proteins and thus may play a crucial part in determining both the structure and stability of these proteins. The four disulfide linkages of several toxin-agglutinins such as snake toxin (54Rees B. Bilwes A. Samama J.P. Moras D. J. Mol. Biol. 1990; 214: 281-297Crossref PubMed Scopus (74) Google Scholar), spider toxin (55Yu H. Rosen M.K. Saccomano N.A. Phillips D. Volkmann R.A. Schreiber S.L. Biochemistry. 1993; 32: 13123-13129Crossref PubMed Scopus (54) Google Scholar), scorpion neurotoxin (56Zhao B. Carson M. Ealick S.E. Bugg C.E. J. Mol. Biol. 1992; 227: 239-252Crossref PubMed Scopus (80) Google Scholar), and hevein (57Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar), as well as the disintegrin echistatin (45Gray W.R. Protein Sci. 1993; 2: 1749-1755Crossref PubMed Scopus (53) Google Scholar) are shown in Fig. 9B. This complex crossed pattern of disulfide bonds is strikingly different from the linear uncrossed pattern proposed for the rSMB domain (Fig.9A). However, a similar linear and uncrossed pattern was recently described for the disulfide bond topology of the fifth epidermal growth factor (EGF)-like domain of thrombomodulin (46White C.E. Hunter M.J. Meininger D.P. Garrod S. Komives E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10177-10182Crossref PubMed Scopus (27) Google Scholar, 58Hunter M.J. Komives E.A. Protein Sci. 1995; 4: 2129-2137Crossref PubMed Scopus (21) Google Scholar,59Wood M.J. Sampoli Benitez B.A. Komives E.A. Nat. Struct. Biol. 2000; 7: 200-204Crossref PubMed Scopus (43) Google Scholar). This domain, which is essential for thrombin binding, has three simple disulfide linkages in an uncrossed pattern (Fig. 9C) and thus differs from the crossed pattern of the other EGF-like domains (47Katsumi A. Tuley E.A. Bodo I. Sadler J.E. J. Biol. Chem. 2000; 275: 25585-25594Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It is interesting to note that the thrombin binding affinity of synthetic peptides containing the “uncrossed” fifth EGF-like domain was nearly an order of magnitude higher than that of synthetic peptides containing the “crossed” fifth EGF-like domains (58Hunter M.J. Komives E.A. Protein Sci. 1995; 4: 2129-2137Crossref PubMed Scopus (21) Google Scholar). NMR analysis also demonstrated that the fifth uncrossed EGF-like domain was much more mobile than the fourth EGF-like domain and that the residues in the fifth EGF-like domain, including the Cys residues, showed shifts upon thrombin binding (59Wood M.J. Sampoli Benitez B.A. Komives E.A. Nat. Struct. Biol. 2000; 7: 200-204Crossref PubMed Scopus (43) Google Scholar). These results suggest that the uncrossed disulfide bonds may contribute to the conformational flexibility of the fifth EGF-like domain and that this may be important for interactions with thrombin.During the course of these studies, we also investigated the relationship between the disulfide linkages of the SMB domain and its PAI-1 binding activity. As mentioned previously, Deng et al.(33Deng 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) reported that replacing any single Cys residue with alanine severely decreased the PAI-1 binding activity of the SMB domain. However, we could not identify the critical disulfide bond pairs for PAI-1 binding because each of these replacements created a free reactive Cys residue that in turn would be expected to disrupt the overall structure of the molecule. Therefore, in the current study, we compared the PAI-1 binding activity of partially reduced andS-alkylated-rVN1–51 with that of intact and unreduced rVN1–51. As shown in Fig. 7, the activity of the rVN1–51 form containing the selectively reduced Cys5–Cys9disulfide linkage was significantly but not completely destroyed. Denget al. (33Deng 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) also observed that replacement of Cys5 with alanine did not completely destroy PAI-1 binding activity. However, the rVN1–51 form in which both Cys5–Cys9 and Cys19–Cys21 were reduced andS-alkylated, lacked detectable PAI-1 binding activity. Thus, the protein structure created by the Cys5–Cys9and Cys19–Cys21 disulfide linkages in the NH2-terminal portion of the SMB domain is essential for PAI-1 binding. Because the disulfide linkages in the COOH-terminal portion of SMB were more resistant to reduction, we could not prepare rVN1–51 in which these two disulfide linkages were selectively reduced. Thus, their role in PAI-1 binding remains to be determined.Finally, it is important to note that the unusual linear uncrossed pattern of disulfide bonds present in active recombinant SMB may not be present in the SMB domain of urea-activated vitronectin itself. Although this issue cannot be resolved until the structure of native SMB is determined, a number of considerations argue against this possibility. These considerations suggest instead that rSMB and native SMB are substantially similar. For example, both molecules are recognized by mAb 153, a conformationally dependent mAb. The facts that both forms of SMB compete with each other for binding to this mAb (34Okumura Y. Kamikubo Y. Curriden S.A. Wang J. Kiwada T. Futaki S. Kitagawa K. Loskutoff D.J. J. Biol. Chem. 2002; 11: 9395-9404Abstract Full Text Full Text PDF Scopus (65) Google Scholar) and that this mAb does not recognize denatured or misfolded SMB (36Seiffert D. Wagner N.V. Biochimie (Paris). 1997; 79: 205-210Crossref PubMed Scopus (5) Google Scholar) suggest that the SMB domain is correctly and similarly folded in both SMB forms. This conclusion is supported by the observation that both forms of SMB bind active PAI-1 with similar high affinities (34Okumura Y. Kamikubo Y. Curriden S.A. Wang J. Kiwada T. Futaki S. Kitagawa K. Loskutoff D.J. J. Biol. Chem. 2002; 11: 9395-9404Abstract Full Text Full Text PDF Scopus (65) Google Scholar). Because any disruption of disulfide bond pairing in SMB results in a dramatic loss of high affinity PAI-1 binding (33Deng 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, 36Seiffert D. Wagner N.V. Biochimie (Paris). 1997; 79: 205-210Crossref PubMed Scopus (5) Google Scholar, 41Royle G. Deng G. Seiffert D. Loskutoff D.J. Anal. Biochem. 2001; 296: 245-253Crossref PubMed Scopus (10) Google Scholar), the active rSMB must have correct disulfide pairing and, therefore, substantially correct folding. Finally, we have identified five additional mAbs that not only bind to recombinant and native SMB but also compete with PAI-1 for binding to these molecules (41Royle G. Deng G. Seiffert D. Loskutoff D.J. Anal. Biochem. 2001; 296: 245-253Crossref PubMed Scopus (10) Google Scholar). These mAbs recognize three separate, partially overlapping epitopes within SMB. Thus, recombinant SMB and native SMB are recognized by six conformation-dependent mAbs and by PAI-1 itself, and these molecules bind to different regions of SMB (41Royle G. Deng G. Seiffert D. Loskutoff D.J. Anal. Biochem. 2001; 296: 245-253Crossref PubMed Scopus" @default.
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