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• W2049013912 abstract "Tenascin-C is a large, multimeric extracellular matrix protein that is found in a variety of tissues and can have profound effects on cell adhesion. It is secreted from cells as a hexamer of six identical chains called a hexabrachion. Disulfide bonding among tenascin subunits mediates intracellular assembly into hexamers. The amino-terminal assembly domain consists of heptad repeats and at least six cysteine residues (Cys-64, -111, -113, -140, -146, -147) that could be involved in multimerization. We have now determined the requirements for these cysteine residues during hexamer assembly. Our results show that only Cys-64 is required to form the hexameric structure. Mutation of Cys-64 to glycine resulted in release of trimer intermediates, which probably form via the heptad repeats, but no hexamers were secreted. In contrast, individual or pairs of mutations of each of the other cysteines had no effect on tenascin hexamer formation, and inclusion of any other cysteine mutations along with C64G did not further disrupt the multimer pattern. However, when all six cysteines were mutated, monomers were the major extracellular form. Together, these results show that trimers are an intermediate of tenascin-C assembly and that Cys-64 is essential for formation of hexabrachions. Tenascin-C is a large, multimeric extracellular matrix protein that is found in a variety of tissues and can have profound effects on cell adhesion. It is secreted from cells as a hexamer of six identical chains called a hexabrachion. Disulfide bonding among tenascin subunits mediates intracellular assembly into hexamers. The amino-terminal assembly domain consists of heptad repeats and at least six cysteine residues (Cys-64, -111, -113, -140, -146, -147) that could be involved in multimerization. We have now determined the requirements for these cysteine residues during hexamer assembly. Our results show that only Cys-64 is required to form the hexameric structure. Mutation of Cys-64 to glycine resulted in release of trimer intermediates, which probably form via the heptad repeats, but no hexamers were secreted. In contrast, individual or pairs of mutations of each of the other cysteines had no effect on tenascin hexamer formation, and inclusion of any other cysteine mutations along with C64G did not further disrupt the multimer pattern. However, when all six cysteines were mutated, monomers were the major extracellular form. Together, these results show that trimers are an intermediate of tenascin-C assembly and that Cys-64 is essential for formation of hexabrachions. Tenascin-C is a large extracellular matrix glycoprotein that functions during embryogenesis, in the nervous system, and in tumors (1Erickson H.P. Bourdon M.A. Annu. Rev. Cell Biol. 1989; 5: 71-92Crossref PubMed Scopus (525) Google Scholar, 2Erickson H.P. Curr. Opin. Cell Biol. 1993; 5: 869-876Crossref PubMed Scopus (355) Google Scholar, 3Chiquet-Ehrismann R. Experientia (Basel). 1995; 51: 853-862Crossref PubMed Scopus (136) Google Scholar, 4Crossin K.L. J. Cell. Biochem. 1996; 61: 592-598Crossref PubMed Scopus (50) Google Scholar). Tenascin is able to interact with cell surface receptors including integrins (5Zisch A.H. D'Alessandri L. Ranscht B. Falchetto R. Winterhalter K.H. Vaughan L. J. Cell Biol. 1992; 119: 203-213Crossref PubMed Scopus (153) Google Scholar, 6Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar, 7Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar, 8Yokosaki Y. Palmer E.L. Prieto A.L. Crossin K.L. Bourdon M.A. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar, 9Deryugina E.I. Bourdon M.A. J. Cell Sci. 1996; 109: 643-652PubMed Google Scholar, 10Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and also binds to extracellular matrix proteins such as fibronectin (11Chiquet-Ehrismann R. Matsuoka Y. Hofer U. Spring J. Bernasconi C. Chiquet M. Cell Regul. 1991; 2: 927-938Crossref PubMed Scopus (135) Google Scholar, 12Chung C.Y. Zardi L. Erickson H.P. J. Biol. Chem. 1995; 270: 29012-29017Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). It is secreted as a large hexabrachion composed of six identical subunits disulfide-bonded together at their amino termini, yielding a hexamer as large as 2 MDa. Subunits range in size from 180 to 320 kDa depending on species and on alternative splicing within the type III repeats. Each subunit consists of an amino-terminal assembly domain containing heptad repeats and multiple cysteine residues, a domain of epidermal growth factor like repeats, fibronectin type III repeats, and a terminal knob that is homologous to fibrinogen (13Jones F.S. Hoffman S. Cunningham B.A. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1905-1909Crossref PubMed Scopus (152) Google Scholar, 14Spring J. Beck K. Chiquet-Ehrismann R. Cell. 1989; 59: 325-334Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 15Saga Y. Tsukamoto T. Jing N. Kusakabe M. Sakakura T. Gene. 1991; 104: 177-185Crossref PubMed Scopus (90) Google Scholar, 16Siri A. Carnemolla B. Saginati M. Leprini A. Casari G. Baralle F. Zardi L. Nucleic Acids Res. 1991; 19: 525-531Crossref PubMed Scopus (161) Google Scholar, 17Weller A. Beck S. Ekblom P. J. Cell Biol. 1991; 112: 355-362Crossref PubMed Scopus (148) Google Scholar). Tenascin-C is a member of a family of related proteins containing varying numbers of type III and epidermal growth factor repeats. All family members, TN-C, 1The abbreviations used are: TN, tenascin; TN-S, small alternatively spliced form of tenascin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BHK, baby hamster kidney.1The abbreviations used are: TN, tenascin; TN-S, small alternatively spliced form of tenascin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BHK, baby hamster kidney. TN-R, TN-X, and apparently TN-Y (18Hagios C. Koch M. Spring J. Chiquet M. Chiquet-Ehrismann R. J. Cell Biol. 1996; 134: 1499-1512Crossref PubMed Scopus (82) Google Scholar) are multimeric but can differ in the numbers of subunits per multimer (2Erickson H.P. Curr. Opin. Cell Biol. 1993; 5: 869-876Crossref PubMed Scopus (355) Google Scholar).The conservation of the amino-terminal assembly domain among all family members suggests that the multimeric structure of the tenascin family is important for function. During tenascin-C biosynthesis, the hexamer is detected very rapidly without accumulation of intermediates, suggesting that tenascin multimers form by association of nascent polypeptides during translation (19Redick S.D. Schwarzbauer J.E. J. Cell Sci. 1995; 108: 1761-1769PubMed Google Scholar). Within the assembly domain reside at least six cysteine residues that are available for interchain disulfide bonding in the hexabrachion. Some or all of these cysteines are likely to function in maintaining the disulfide-bonded structure. To determine which cysteines are required for hexamer assembly and to identify any disulfide-bonded assembly intermediates, we have mutagenized these cysteine residues and have used a transient expression system based on COS-1 cells to analyze the requirements for each of these cysteines and for combinations of cysteines in the formation of tenascin hexamers. Our results clearly show that only Cys-64 is essential for hexamer formation. Mutant recombinant tenascins lacking Cys-64 are assembled into trimers but not hexamers, demonstrating that trimers are an intermediate in hexamer assembly. In addition, mutation of four cysteine residues flanking the heptad repeat region does not prevent hexamer formation, indicating that trimers might form by noncovalent interactions mediated by the heptad repeats, and these trimer intermediates are then assembled into hexamers by disulfide bonding via Cys-64. The disulfide bonds surrounding the heptad repeats probably function to stabilize the trimeric intermediate structure.DISCUSSIONThe multimeric structure is conserved among the tenascin family of proteins. Each type of tenascin has a set of heptad repeats within the amino-terminal assembly domain, and these are predicted to form a triple coiled-coil structure. We have analyzed the roles of each of the six cysteines in and around the heptad repeats and have found that only one cysteine, Cys-64, is essential for hexamer formation. Mutation of the other cysteines did not prevent the secretion of hexamers, and mutation of Cys-64 resulted in assembly and secretion of trimers but no higher multimers. Our results also demonstrate that tenascin is assembled through a trimer intermediate. Apparently, tenascin follows a stepwise process of assembly into hexamers beginning with trimer formation among subunits followed quickly by linking of two trimers into a hexabrachion.Previously we had shown that the rapid intracellular assembly of tenascin did not involve detectable intermediates (19Redick S.D. Schwarzbauer J.E. J. Cell Sci. 1995; 108: 1761-1769PubMed Google Scholar). Only hexamers were found at even the earliest time points, suggesting cotranslational assembly. Based on our mutational analyses, we propose that noncovalent trimers are formed via the heptad repeats as soon as the chains are sufficiently long to contact each other. Upon further translation elongation, the trimers would then be assembled into disulfide-bonded hexamers via the essential Cys-64 residue. At some point during this process, interchain disulfide bonds form between the other cysteines in this domain and help to stabilize the trimer structure. However, not all cysteines flanking the heptad repeats are essential for hexamer or trimer secretion as evidenced by efficient production, even with multiple cysteine mutations.Analyses of secreted products composed of 4×C and 5×C polypeptides showed a significant level of dimers and monomers. These might represent tenascins that are secreted as dimers and monomers. An alternative explanation seems more likely, i.e. that these intermediates actually represent subunits dissociated from secreted hexamers that are not completely disulfide-bonded. Tenascin trimers probably form by association of three subunits through their heptad repeats. Such noncovalent coiled-coil interactions could be sufficient for trimer or hexamer secretion. However, if they are not stabilized by disulfide bonds, the interchain interactions would not withstand SDS denaturation before SDS-PAGE. Therefore, multimers lacking stabilizing disulfide bonds among all chains would dissociate into smaller intermediates upon denaturation. In addition to dimers and monomers, SDS-PAGE of 4×C and 5×C mutant tenascins also reveals a subfraction of disulfide-bonded multimers containing four or more subunits. Covalent interactions between a subset of subunits and involving one other cysteine in addition to Cys-64 would be sufficient to produce these higher multimers. Cysteines 161 or 172 downstream of the heptad repeats could participate in their formation.Apparently, not all chains are disulfide-bonded together in intact tenascin, as SDS-PAGE of HxB.S tenascin showed some multimers consisting of five, four, and three subunits, although only hexamers were seen by electron microscopy of purified protein (21Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar). Disulfide bonding at both ends of the coiled-coil is also not necessary for efficient expression of hexamers. Both C146,147G and C111,113G mutants had structures indistinguishable from native tenascin. Thus, within a population of normal or mutant tenascins, there can be multimers with interchain disulfide bonds between different numbers and combinations of subunits. In intact tenascin, one hexamer can have six disulfide-bonded chains, whereas another might have only four or five. This indicates that assembly of specific disulfide bonding partners is not a fixed process but is stochastic.Fibrinogen is a major secreted hexameric protein that shows several structural similarities with tenascin, including coiled-coil regions and interchain disulfide bridges (24Budzynski A.Z. Crit. Rev. Oncol. Hematol. 1986; 6: 97-146Crossref PubMed Scopus (44) Google Scholar). Additionally, both have cysteines flanking the coiled-coil region as well as one or more cysteines near the amino terminus that are important for hexamer formation (25Huang S. Cao Z. Davie E.W. Biochem. Biophys. Res. Commun. 1993; 190: 488-495Crossref PubMed Scopus (37) Google Scholar, 26Zhang J.-Z. Kudryk B. Redman C.M. J. Biol. Chem. 1993; 268: 11278-11282Abstract Full Text PDF PubMed Google Scholar). One major difference between these two proteins is in the roles of the disulfides that flank the coiled-coil domains. In tenascin, we have shown that these disulfides are dispensible, and hexamer assembly and secretion proceed very efficiently without them. In contrast, the disulfide rings of fibrinogen, particularly the carboxyl-terminal ring, must be properly formed in order for hexamers to be secreted (27Zhang J.-Z. Redman C.M. J. Biol. Chem. 1994; 269: 652-658Abstract Full Text PDF PubMed Google Scholar, 28Xu W. Chung D.W. Davie E.W. J. Biol. Chem. 1996; 271: 27948-27953Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Zhang J.-Z. Redman C. J. Biol. Chem. 1996; 271: 30083-30088Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The differing roles of the disulfides used to assemble fibrinogen versus tenascin might reflect differences in the mechanism of assembly of these hexamers. As fibrinogen is formed from three different gene products (α, β, γ), assembly must occur post-translationally. Therefore, it is possible that proper alignment of the cysteines is important for proper register of the coiled-coil. In addition, the coiled-coil of fibrinogen is significantly longer than that of tenascin, which could make molecular register more critical. The complex disulfide bonding pattern of fibrinogen would also assure that all three chains are present in the hexamer. As all six tenascin chains are identical, this is not a concern with this hexamer.The crystal structures of three- and five-chain parallel coiled-coils have been reported (30Harbury P.B. Kim P.S. Alber T. Nature. 1994; 371: 80-83Crossref PubMed Scopus (419) Google Scholar, 31Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (267) Google Scholar). Comparison of the positioning of cysteine residues in and around the heptad repeats of tenascin to trimeric coiled-coil peptides did not reveal any obvious orientiation of cysteine side chains that might predict disulfide bonding partners. In the case of the five- stranded coiled-coil of cartilage oligomeric matrix protein, there are two cysteines at the carboxyl terminus of the heptad repeats, and these form the interchain disulfide bonds (31Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (267) Google Scholar). Although these residues lie at the end of the heptad repeats, they are not part of the coiled-coil structure. In fact, the coiled-coil is disrupted just before these residues, and they actually reside within a type III β turn. Thus, other determinants bring the cysteine residues into a favorable position for disulfide bridge formation. Using the Multicoils 2Internet address: ostrich.lcs.mit.edu/cgi-bin/score program (32Berger B. Wilson D.B. Wolf E. Tonchev T. Milla M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263Crossref PubMed Scopus (592) Google Scholar) to predict heptad repeats in the assembly domain of tenascin indicated that Cys-111,-113 and Cys-146,-147 are just outside of the heptad repeats and not likely to be involved in coiled-coil interactions. 3J. A. Luczak, S. D. Redick, and J. E. Schwarzbauer, unpublished observations. Therefore, tenascin may resemble cartilage oligomeric matrix protein and use structures in addition to a coiled-coil to bring cysteines into proximity for disulfide bonding.Whereas tenascin-C is a hexamer, tenascin-R has only been isolated as trimers or dimers (33Pesheva P. Spiess E. Schachner M. J. Cell Biol. 1989; 109: 1765-1778Crossref PubMed Scopus (231) Google Scholar), and tenascin-X (34Matsumoto K.-I. Saga Y. Ikemura T. Sakakura T. Chiquet-Ehrismann R. J. Cell Biol. 1994; 125: 483-493Crossref PubMed Scopus (143) Google Scholar) and tenascin-Y (18Hagios C. Koch M. Spring J. Chiquet M. Chiquet-Ehrismann R. J. Cell Biol. 1996; 134: 1499-1512Crossref PubMed Scopus (82) Google Scholar) contain unknown numbers of chains. Comparison of the assembly domains from these three shows that they are highly conserved. However, there are several differences that could account for the different numbers of subunits in the multimers. We have shown that tenascin-C uses Cys-64 to form hexamers. This cysteine is absent from tenascin-X (35Bristow J. Tee M.K. Gitelman S.E. Mellon S.H. Miller W.L. J. Cell Biol. 1993; 122: 265-278Crossref PubMed Scopus (252) Google Scholar), indicating that this tenascin is probably trimeric. However, a cysteine equivalent to Cys-64 (at position 79) is present in tenascin-R (36Norenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 37Fuss B. Wintergerst E.-S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar, 38Carnemolla B. Leprini A. Borsi L. Querze G. Urbini S. Zardi L. J. Biol. Chem. 1996; 271: 8157-8160Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Why have only trimers and dimers been observed? In addition to Cys-79, mammalian tenascin-R has an additional cysteine 48 amino acids upstream (37Fuss B. Wintergerst E.-S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar, 38Carnemolla B. Leprini A. Borsi L. Querze G. Urbini S. Zardi L. J. Biol. Chem. 1996; 271: 8157-8160Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Perhaps these two cysteines can form an intrachain disulfide bond, preventing them from participating in hexamer assembly and thus yielding only trimers. This potential mechanism could also hold for chicken tenascin-R, which has three extra cysteines in the first 78 amino acids (36Norenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar). All have cysteines flanking the heptad repeats, suggesting a functional role for these residues. Tenascins probably need to withstand a certain amount of tension within the extracellular matrix, and disulfide bonding around the coiled-coil could serve to prevent unraveling of the trimers in these situations. Clearly, nature has evolved a very complex set of interactions to produce tenascin family members consisting of different numbers of chains and has added extra cysteines for stability, probably to strengthen the hexabrachion for its structural role within the extracellular matrix. Tenascin-C is a large extracellular matrix glycoprotein that functions during embryogenesis, in the nervous system, and in tumors (1Erickson H.P. Bourdon M.A. Annu. Rev. Cell Biol. 1989; 5: 71-92Crossref PubMed Scopus (525) Google Scholar, 2Erickson H.P. Curr. Opin. Cell Biol. 1993; 5: 869-876Crossref PubMed Scopus (355) Google Scholar, 3Chiquet-Ehrismann R. Experientia (Basel). 1995; 51: 853-862Crossref PubMed Scopus (136) Google Scholar, 4Crossin K.L. J. Cell. Biochem. 1996; 61: 592-598Crossref PubMed Scopus (50) Google Scholar). Tenascin is able to interact with cell surface receptors including integrins (5Zisch A.H. D'Alessandri L. Ranscht B. Falchetto R. Winterhalter K.H. Vaughan L. J. Cell Biol. 1992; 119: 203-213Crossref PubMed Scopus (153) Google Scholar, 6Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (215) Google Scholar, 7Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar, 8Yokosaki Y. Palmer E.L. Prieto A.L. Crossin K.L. Bourdon M.A. Pytela R. Sheppard D. J. Biol. Chem. 1994; 269: 26691-26696Abstract Full Text PDF PubMed Google Scholar, 9Deryugina E.I. Bourdon M.A. J. Cell Sci. 1996; 109: 643-652PubMed Google Scholar, 10Yokosaki Y. Monis H. Chen J. Sheppard D. J. Biol. Chem. 1996; 271: 24144-24150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and also binds to extracellular matrix proteins such as fibronectin (11Chiquet-Ehrismann R. Matsuoka Y. Hofer U. Spring J. Bernasconi C. Chiquet M. Cell Regul. 1991; 2: 927-938Crossref PubMed Scopus (135) Google Scholar, 12Chung C.Y. Zardi L. Erickson H.P. J. Biol. Chem. 1995; 270: 29012-29017Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). It is secreted as a large hexabrachion composed of six identical subunits disulfide-bonded together at their amino termini, yielding a hexamer as large as 2 MDa. Subunits range in size from 180 to 320 kDa depending on species and on alternative splicing within the type III repeats. Each subunit consists of an amino-terminal assembly domain containing heptad repeats and multiple cysteine residues, a domain of epidermal growth factor like repeats, fibronectin type III repeats, and a terminal knob that is homologous to fibrinogen (13Jones F.S. Hoffman S. Cunningham B.A. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1905-1909Crossref PubMed Scopus (152) Google Scholar, 14Spring J. Beck K. Chiquet-Ehrismann R. Cell. 1989; 59: 325-334Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 15Saga Y. Tsukamoto T. Jing N. Kusakabe M. Sakakura T. Gene. 1991; 104: 177-185Crossref PubMed Scopus (90) Google Scholar, 16Siri A. Carnemolla B. Saginati M. Leprini A. Casari G. Baralle F. Zardi L. Nucleic Acids Res. 1991; 19: 525-531Crossref PubMed Scopus (161) Google Scholar, 17Weller A. Beck S. Ekblom P. J. Cell Biol. 1991; 112: 355-362Crossref PubMed Scopus (148) Google Scholar). Tenascin-C is a member of a family of related proteins containing varying numbers of type III and epidermal growth factor repeats. All family members, TN-C, 1The abbreviations used are: TN, tenascin; TN-S, small alternatively spliced form of tenascin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BHK, baby hamster kidney.1The abbreviations used are: TN, tenascin; TN-S, small alternatively spliced form of tenascin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BHK, baby hamster kidney. TN-R, TN-X, and apparently TN-Y (18Hagios C. Koch M. Spring J. Chiquet M. Chiquet-Ehrismann R. J. Cell Biol. 1996; 134: 1499-1512Crossref PubMed Scopus (82) Google Scholar) are multimeric but can differ in the numbers of subunits per multimer (2Erickson H.P. Curr. Opin. Cell Biol. 1993; 5: 869-876Crossref PubMed Scopus (355) Google Scholar). The conservation of the amino-terminal assembly domain among all family members suggests that the multimeric structure of the tenascin family is important for function. During tenascin-C biosynthesis, the hexamer is detected very rapidly without accumulation of intermediates, suggesting that tenascin multimers form by association of nascent polypeptides during translation (19Redick S.D. Schwarzbauer J.E. J. Cell Sci. 1995; 108: 1761-1769PubMed Google Scholar). Within the assembly domain reside at least six cysteine residues that are available for interchain disulfide bonding in the hexabrachion. Some or all of these cysteines are likely to function in maintaining the disulfide-bonded structure. To determine which cysteines are required for hexamer assembly and to identify any disulfide-bonded assembly intermediates, we have mutagenized these cysteine residues and have used a transient expression system based on COS-1 cells to analyze the requirements for each of these cysteines and for combinations of cysteines in the formation of tenascin hexamers. Our results clearly show that only Cys-64 is essential for hexamer formation. Mutant recombinant tenascins lacking Cys-64 are assembled into trimers but not hexamers, demonstrating that trimers are an intermediate in hexamer assembly. In addition, mutation of four cysteine residues flanking the heptad repeat region does not prevent hexamer formation, indicating that trimers might form by noncovalent interactions mediated by the heptad repeats, and these trimer intermediates are then assembled into hexamers by disulfide bonding via Cys-64. The disulfide bonds surrounding the heptad repeats probably function to stabilize the trimeric intermediate structure. DISCUSSIONThe multimeric structure is conserved among the tenascin family of proteins. Each type of tenascin has a set of heptad repeats within the amino-terminal assembly domain, and these are predicted to form a triple coiled-coil structure. We have analyzed the roles of each of the six cysteines in and around the heptad repeats and have found that only one cysteine, Cys-64, is essential for hexamer formation. Mutation of the other cysteines did not prevent the secretion of hexamers, and mutation of Cys-64 resulted in assembly and secretion of trimers but no higher multimers. Our results also demonstrate that tenascin is assembled through a trimer intermediate. Apparently, tenascin follows a stepwise process of assembly into hexamers beginning with trimer formation among subunits followed quickly by linking of two trimers into a hexabrachion.Previously we had shown that the rapid intracellular assembly of tenascin did not involve detectable intermediates (19Redick S.D. Schwarzbauer J.E. J. Cell Sci. 1995; 108: 1761-1769PubMed Google Scholar). Only hexamers were found at even the earliest time points, suggesting cotranslational assembly. Based on our mutational analyses, we propose that noncovalent trimers are formed via the heptad repeats as soon as the chains are sufficiently long to contact each other. Upon further translation elongation, the trimers would then be assembled into disulfide-bonded hexamers via the essential Cys-64 residue. At some point during this process, interchain disulfide bonds form between the other cysteines in this domain and help to stabilize the trimer structure. However, not all cysteines flanking the heptad repeats are essential for hexamer or trimer secretion as evidenced by efficient production, even with multiple cysteine mutations.Analyses of secreted products composed of 4×C and 5×C polypeptides showed a significant level of dimers and monomers. These might represent tenascins that are secreted as dimers and monomers. An alternative explanation seems more likely, i.e. that these intermediates actually represent subunits dissociated from secreted hexamers that are not completely disulfide-bonded. Tenascin trimers probably form by association of three subunits through their heptad repeats. Such noncovalent coiled-coil interactions could be sufficient for trimer or hexamer secretion. However, if they are not stabilized by disulfide bonds, the interchain interactions would not withstand SDS denaturation before SDS-PAGE. Therefore, multimers lacking stabilizing disulfide bonds among all chains would dissociate into smaller intermediates upon denaturation. In addition to dimers and monomers, SDS-PAGE of 4×C and 5×C mutant tenascins also reveals a subfraction of disulfide-bonded multimers containing four or more subunits. Covalent interactions between a subset of subunits and involving one other cysteine in addition to Cys-64 would be sufficient to produce these higher multimers. Cysteines 161 or 172 downstream of the heptad repeats could participate in their formation.Apparently, not all chains are disulfide-bonded together in intact tenascin, as SDS-PAGE of HxB.S tenascin showed some multimers consisting of five, four, and three subunits, although only hexamers were seen by electron microscopy of purified protein (21Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar). Disulfide bonding at both ends of the coiled-coil is also not necessary for efficient expression of hexamers. Both C146,147G and C111,113G mutants had structures indistinguishable from native tenascin. Thus, within a population of normal or mutant tenascins, there can be multimers with interchain disulfide bonds between different numbers and combinations of subunits. In intact tenascin, one hexamer can have six disulfide-bonded chains, whereas another might have only four or five. This indicates that assembly of specific disulfide bonding partners is not a fixed process but is stochastic.Fibrinogen is a major secreted hexameric protein that shows several structural similarities with tenascin, including coiled-coil regions and interchain disulfide bridges (24Budzynski A.Z. Crit. Rev. Oncol. Hematol. 1986; 6: 97-146Crossref PubMed Scopus (44) Google Scholar). Additionally, both have cysteines flanking the coiled-coil region as well as one or more cysteines near the amino terminus that are important for hexamer formation (25Huang S. Cao Z. Davie E.W. Biochem. Biophys. Res. Commun. 1993; 190: 488-495Crossref PubMed Scopus (37) Google Scholar, 26Zhang J.-Z. Kudryk B. Redman C.M. J. Biol. Chem. 1993; 268: 11278-11282Abstract Full Text PDF PubMed Google Scholar). One major difference between these two proteins is in the roles of the disulfides that flank the coiled-coil domains. In tenascin, we have shown that these disulfides are dispensible, and hexamer assembly and secretion proceed very efficiently without them. In contrast, the disulfide rings of fibrinogen, particularly the carboxyl-terminal ring, must be properly formed in order for hexamers to be secreted (27Zhang J.-Z. Redman C.M. J. Biol. Chem. 1994; 269: 652-658Abstract Full Text PDF PubMed Google Scholar, 28Xu W. Chung D.W. Davie E.W. J. Biol. Chem. 1996; 271: 27948-27953Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Zhang J.-Z. Redman C. J. Biol. Chem. 1996; 271: 30083-30088Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The differing roles of the disulfides used to assemble fibrinogen versus tenascin might reflect differences in the mechanism of assembly of these hexamers. As fibrinogen is formed from three different gene products (α, β, γ), assembly must occur post-translationally. Therefore, it is possible that proper alignment of the cysteines is important for proper register of the coiled-coil. In addition, the coiled-coil of fibrinogen is significantly longer than that of tenascin, which could make molecular register more critical. The complex disulfide bonding pattern of fibrinogen would also assure that all three chains are present in the hexamer. As all six tenascin chains are identical, this is not a concern with this hexamer.The crystal structures of three- and five-chain parallel coiled-coils have been reported (30Harbury P.B. Kim P.S. Alber T. Nature. 1994; 371: 80-83Crossref PubMed Scopus (419) Google Scholar, 31Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (267) Google Scholar). Comparison of the positioning of cysteine residues in and around the heptad repeats of tenascin to trimeric coiled-coil peptides did not reveal any obvious orientiation of cysteine side chains that might predict disulfide bonding partners. In the case of the five- stranded coiled-coil of cartilage oligomeric matrix protein, there are two cysteines at the carboxyl terminus of the heptad repeats, and these form the interchain disulfide bonds (31Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (267) Google Scholar). Although these residues lie at the end of the heptad repeats, they are not part of the coiled-coil structure. In fact, the coiled-coil is disrupted just before these residues, and they actually reside within a type III β turn. Thus, other determinants bring the cysteine residues into a favorable position for disulfide bridge formation. Using the Multicoils 2Internet address: ostrich.lcs.mit.edu/cgi-bin/score program (32Berger B. Wilson D.B. Wolf E. Tonchev T. Milla M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263Crossref PubMed Scopus (592) Google Scholar) to predict heptad repeats in the assembly domain of tenascin indicated that Cys-111,-113 and Cys-146,-147 are just outside of the heptad repeats and not likely to be involved in coiled-coil interactions. 3J. A. Luczak, S. D. Redick, and J. E. Schwarzbauer, unpublished observations. Therefore, tenascin may resemble cartilage oligomeric matrix protein and use structures in addition to a coiled-coil to bring cysteines into proximity for disulfide bonding.Whereas tenascin-C is a hexamer, tenascin-R has only been isolated as trimers or dimers (33Pesheva P. Spiess E. Schachner M. J. Cell Biol. 1989; 109: 1765-1778Crossref PubMed Scopus (231) Google Scholar), and tenascin-X (34Matsumoto K.-I. Saga Y. Ikemura T. Sakakura T. Chiquet-Ehrismann R. J. Cell Biol. 1994; 125: 483-493Crossref PubMed Scopus (143) Google Scholar) and tenascin-Y (18Hagios C. Koch M. Spring J. Chiquet M. Chiquet-Ehrismann R. J. Cell Biol. 1996; 134: 1499-1512Crossref PubMed Scopus (82) Google Scholar) contain unknown numbers of chains. Comparison of the assembly domains from these three shows that they are highly conserved. However, there are several differences that could account for the different numbers of subunits in the multimers. We have shown that tenascin-C uses Cys-64 to form hexamers. This cysteine is absent from tenascin-X (35Bristow J. Tee M.K. Gitelman S.E. Mellon S.H. Miller W.L. J. Cell Biol. 1993; 122: 265-278Crossref PubMed Scopus (252) Google Scholar), indicating that this tenascin is probably trimeric. However, a cysteine equivalent to Cys-64 (at position 79) is present in tenascin-R (36Norenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 37Fuss B. Wintergerst E.-S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar, 38Carnemolla B. Leprini A. Borsi L. Querze G. Urbini S. Zardi L. J. Biol. Chem. 1996; 271: 8157-8160Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Why have only trimers and dimers been observed? In addition to Cys-79, mammalian tenascin-R has an additional cysteine 48 amino acids upstream (37Fuss B. Wintergerst E.-S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar, 38Carnemolla B. Leprini A. Borsi L. Querze G. Urbini S. Zardi L. J. Biol. Chem. 1996; 271: 8157-8160Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Perhaps these two cysteines can form an intrachain disulfide bond, preventing them from participating in hexamer assembly and thus yielding only trimers. This potential mechanism could also hold for chicken tenascin-R, which has three extra cysteines in the first 78 amino acids (36Norenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar). All have cysteines flanking the heptad repeats, suggesting a functional role for these residues. Tenascins probably need to withstand a certain amount of tension within the extracellular matrix, and disulfide bonding around the coiled-coil could serve to prevent unraveling of the trimers in these situations. Clearly, nature has evolved a very complex set of interactions to produce tenascin family members consisting of different numbers of chains and has added extra cysteines for stability, probably to strengthen the hexabrachion for its structural role within the extracellular matrix. The multimeric structure is conserved among the tenascin family of proteins. Each type of tenascin has a set of heptad repeats within the amino-terminal assembly domain, and these are predicted to form a triple coiled-coil structure. We have analyzed the roles of each of the six cysteines in and around the heptad repeats and have found that only one cysteine, Cys-64, is essential for hexamer formation. Mutation of the other cysteines did not prevent the secretion of hexamers, and mutation of Cys-64 resulted in assembly and secretion of trimers but no higher multimers. Our results also demonstrate that tenascin is assembled through a trimer intermediate. Apparently, tenascin follows a stepwise process of assembly into hexamers beginning with trimer formation among subunits followed quickly by linking of two trimers into a hexabrachion. Previously we had shown that the rapid intracellular assembly of tenascin did not involve detectable intermediates (19Redick S.D. Schwarzbauer J.E. J. Cell Sci. 1995; 108: 1761-1769PubMed Google Scholar). Only hexamers were found at even the earliest time points, suggesting cotranslational assembly. Based on our mutational analyses, we propose that noncovalent trimers are formed via the heptad repeats as soon as the chains are sufficiently long to contact each other. Upon further translation elongation, the trimers would then be assembled into disulfide-bonded hexamers via the essential Cys-64 residue. At some point during this process, interchain disulfide bonds form between the other cysteines in this domain and help to stabilize the trimer structure. However, not all cysteines flanking the heptad repeats are essential for hexamer or trimer secretion as evidenced by efficient production, even with multiple cysteine mutations. Analyses of secreted products composed of 4×C and 5×C polypeptides showed a significant level of dimers and monomers. These might represent tenascins that are secreted as dimers and monomers. An alternative explanation seems more likely, i.e. that these intermediates actually represent subunits dissociated from secreted hexamers that are not completely disulfide-bonded. Tenascin trimers probably form by association of three subunits through their heptad repeats. Such noncovalent coiled-coil interactions could be sufficient for trimer or hexamer secretion. However, if they are not stabilized by disulfide bonds, the interchain interactions would not withstand SDS denaturation before SDS-PAGE. Therefore, multimers lacking stabilizing disulfide bonds among all chains would dissociate into smaller intermediates upon denaturation. In addition to dimers and monomers, SDS-PAGE of 4×C and 5×C mutant tenascins also reveals a subfraction of disulfide-bonded multimers containing four or more subunits. Covalent interactions between a subset of subunits and involving one other cysteine in addition to Cys-64 would be sufficient to produce these higher multimers. Cysteines 161 or 172 downstream of the heptad repeats could participate in their formation. Apparently, not all chains are disulfide-bonded together in intact tenascin, as SDS-PAGE of HxB.S tenascin showed some multimers consisting of five, four, and three subunits, although only hexamers were seen by electron microscopy of purified protein (21Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar). Disulfide bonding at both ends of the coiled-coil is also not necessary for efficient expression of hexamers. Both C146,147G and C111,113G mutants had structures indistinguishable from native tenascin. Thus, within a population of normal or mutant tenascins, there can be multimers with interchain disulfide bonds between different numbers and combinations of subunits. In intact tenascin, one hexamer can have six disulfide-bonded chains, whereas another might have only four or five. This indicates that assembly of specific disulfide bonding partners is not a fixed process but is stochastic. Fibrinogen is a major secreted hexameric protein that shows several structural similarities with tenascin, including coiled-coil regions and interchain disulfide bridges (24Budzynski A.Z. Crit. Rev. Oncol. Hematol. 1986; 6: 97-146Crossref PubMed Scopus (44) Google Scholar). Additionally, both have cysteines flanking the coiled-coil region as well as one or more cysteines near the amino terminus that are important for hexamer formation (25Huang S. Cao Z. Davie E.W. Biochem. Biophys. Res. Commun. 1993; 190: 488-495Crossref PubMed Scopus (37) Google Scholar, 26Zhang J.-Z. Kudryk B. Redman C.M. J. Biol. Chem. 1993; 268: 11278-11282Abstract Full Text PDF PubMed Google Scholar). One major difference between these two proteins is in the roles of the disulfides that flank the coiled-coil domains. In tenascin, we have shown that these disulfides are dispensible, and hexamer assembly and secretion proceed very efficiently without them. In contrast, the disulfide rings of fibrinogen, particularly the carboxyl-terminal ring, must be properly formed in order for hexamers to be secreted (27Zhang J.-Z. Redman C.M. J. Biol. Chem. 1994; 269: 652-658Abstract Full Text PDF PubMed Google Scholar, 28Xu W. Chung D.W. Davie E.W. J. Biol. Chem. 1996; 271: 27948-27953Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Zhang J.-Z. Redman C. J. Biol. Chem. 1996; 271: 30083-30088Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The differing roles of the disulfides used to assemble fibrinogen versus tenascin might reflect differences in the mechanism of assembly of these hexamers. As fibrinogen is formed from three different gene products (α, β, γ), assembly must occur post-translationally. Therefore, it is possible that proper alignment of the cysteines is important for proper register of the coiled-coil. In addition, the coiled-coil of fibrinogen is significantly longer than that of tenascin, which could make molecular register more critical. The complex disulfide bonding pattern of fibrinogen would also assure that all three chains are present in the hexamer. As all six tenascin chains are identical, this is not a concern with this hexamer. The crystal structures of three- and five-chain parallel coiled-coils have been reported (30Harbury P.B. Kim P.S. Alber T. Nature. 1994; 371: 80-83Crossref PubMed Scopus (419) Google Scholar, 31Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (267) Google Scholar). Comparison of the positioning of cysteine residues in and around the heptad repeats of tenascin to trimeric coiled-coil peptides did not reveal any obvious orientiation of cysteine side chains that might predict disulfide bonding partners. In the case of the five- stranded coiled-coil of cartilage oligomeric matrix protein, there are two cysteines at the carboxyl terminus of the heptad repeats, and these form the interchain disulfide bonds (31Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (267) Google Scholar). Although these residues lie at the end of the heptad repeats, they are not part of the coiled-coil structure. In fact, the coiled-coil is disrupted just before these residues, and they actually reside within a type III β turn. Thus, other determinants bring the cysteine residues into a favorable position for disulfide bridge formation. Using the Multicoils 2Internet address: ostrich.lcs.mit.edu/cgi-bin/score program (32Berger B. Wilson D.B. Wolf E. Tonchev T. Milla M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263Crossref PubMed Scopus (592) Google Scholar) to predict heptad repeats in the assembly domain of tenascin indicated that Cys-111,-113 and Cys-146,-147 are just outside of the heptad repeats and not likely to be involved in coiled-coil interactions. 3J. A. Luczak, S. D. Redick, and J. E. Schwarzbauer, unpublished observations. Therefore, tenascin may resemble cartilage oligomeric matrix protein and use structures in addition to a coiled-coil to bring cysteines into proximity for disulfide bonding. Whereas tenascin-C is a hexamer, tenascin-R has only been isolated as trimers or dimers (33Pesheva P. Spiess E. Schachner M. J. Cell Biol. 1989; 109: 1765-1778Crossref PubMed Scopus (231) Google Scholar), and tenascin-X (34Matsumoto K.-I. Saga Y. Ikemura T. Sakakura T. Chiquet-Ehrismann R. J. Cell Biol. 1994; 125: 483-493Crossref PubMed Scopus (143) Google Scholar) and tenascin-Y (18Hagios C. Koch M. Spring J. Chiquet M. Chiquet-Ehrismann R. J. Cell Biol. 1996; 134: 1499-1512Crossref PubMed Scopus (82) Google Scholar) contain unknown numbers of chains. Comparison of the assembly domains from these three shows that they are highly conserved. However, there are several differences that could account for the different numbers of subunits in the multimers. We have shown that tenascin-C uses Cys-64 to form hexamers. This cysteine is absent from tenascin-X (35Bristow J. Tee M.K. Gitelman S.E. Mellon S.H. Miller W.L. J. Cell Biol. 1993; 122: 265-278Crossref PubMed Scopus (252) Google Scholar), indicating that this tenascin is probably trimeric. However, a cysteine equivalent to Cys-64 (at position 79) is present in tenascin-R (36Norenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 37Fuss B. Wintergerst E.-S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar, 38Carnemolla B. Leprini A. Borsi L. Querze G. Urbini S. Zardi L. J. Biol. Chem. 1996; 271: 8157-8160Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Why have only trimers and dimers been observed? In addition to Cys-79, mammalian tenascin-R has an additional cysteine 48 amino acids upstream (37Fuss B. Wintergerst E.-S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar, 38Carnemolla B. Leprini A. Borsi L. Querze G. Urbini S. Zardi L. J. Biol. Chem. 1996; 271: 8157-8160Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Perhaps these two cysteines can form an intrachain disulfide bond, preventing them from participating in hexamer assembly and thus yielding only trimers. This potential mechanism could also hold for chicken tenascin-R, which has three extra cysteines in the first 78 amino acids (36Norenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar). All have cysteines flanking the heptad repeats, suggesting a functional role for these residues. Tenascins probably need to withstand a certain amount of tension within the extracellular matrix, and disulfide bonding around the coiled-coil could serve to prevent unraveling of the trimers in these situations. Clearly, nature has evolved a very complex set of interactions to produce tenascin family members consisting of different numbers of chains and has added extra cysteines for stability, probably to strengthen the hexabrachion for its structural role within the extracellular matrix. We would like to thank Harold Erickson for providing the BHK-HxB.S cell line, Ihor Lemischka for the COS-1 cells and the stromal cell cDNA library, and Peter Yurchenco for laminin-1. We are also grateful to Michael Fitzgerald for technical assistance and Melissa Wenk for critically reading the manuscript." @default.
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• W2049013912 cites W1608511206 @default.
• W2049013912 cites W1854788195 @default.
• W2049013912 cites W1964834956 @default.
• W2049013912 cites W1968209401 @default.
• W2049013912 cites W1972348246 @default.
• W2049013912 cites W1990747139 @default.
• W2049013912 cites W1993017660 @default.
• W2049013912 cites W1998031852 @default.
• W2049013912 cites W1998130142 @default.
• W2049013912 cites W2000265611 @default.
• W2049013912 cites W2011008267 @default.
• W2049013912 cites W2011629288 @default.
• W2049013912 cites W2014195384 @default.
• W2049013912 cites W2019776446 @default.
• W2049013912 cites W2029236303 @default.
• W2049013912 cites W2029774675 @default.
• W2049013912 cites W2045363327 @default.
• W2049013912 cites W2067551535 @default.
• W2049013912 cites W2069007382 @default.
• W2049013912 cites W2077381883 @default.
• W2049013912 cites W2077846360 @default.
• W2049013912 cites W2090226055 @default.
• W2049013912 cites W2099174793 @default.
• W2049013912 cites W2121764325 @default.
• W2049013912 cites W2142376043 @default.
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• W2049013912 cites W2149707830 @default.
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• W2049013912 cites W2169267331 @default.
• W2049013912 cites W2171609018 @default.
• W2049013912 cites W2172208039 @default.
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