SMART MODE:

NORMAL GENOMIC

• Pickford AR, Smith SP, Staunton D, Boyd J, Campbell ID
• The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding.
• EMBO J. 2001; 20: 1519-29
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• The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.

• Cota E, Clarke J
• Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module.
• Protein Sci. 2000; 9: 112-20
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• An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.

• Redick SD, Settles DL, Briscoe G, Erickson HP
• Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.
• J Cell Biol. 2000; 149: 521-7
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• Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.

• Fattorusso R, Pellecchia M, Viti F, Neri P, Neri D, Wuthrich K
• NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis.
• Structure Fold Des. 1999; 7: 381-90
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• BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.

• Johnson KJ, Sage H, Briscoe G, Erickson HP
• The compact conformation of fibronectin is determined by intramolecular ionic interactions.
• J Biol Chem. 1999; 274: 15473-9
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• Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

• Bocquier AA, Potts JR, Pickford AR, Campbell ID
• Solution structure of a pair of modules from the gelatin-binding domain of fibronectin.
• Structure Fold Des. 1999; 7: 1451-60
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• BACKGROUND: Fibronectin has a role in vital physiological processes such as cell migration during embryogenesis and wound healing. It mediates the attachment of cells to extracellular matrices that contain fibrous collagens. The affinity of fibronectin for native collagen and denatured collagen (gelatin) is located within a 42 kDa domain that contains four type 1 (F1) and two type 2 (F2) modules. A putative ligand-binding site has been located on an isolated F2 module, but the accessibility of this site in the intact domain is unknown. Thus, structural studies of module pairs and larger fragments are required for a better understanding of the interaction between fibronectin and collagen. RESULTS: The solution structure of the 101-residue 6F1 1F2 module pair, which has a weak affinity for gelatin, has been determined by multidimensional NMR spectroscopy. The tertiary structures determined for each module conform to the F1 and F2 consensus folds established previously. The experimental data suggest that the two modules interact via a small hydrophobic interface but may not be tightly associated. Near-random-coil 1H NMR chemical shifts and fast dynamics for backbone atoms in the linker indicate that this region is unlikely to be involved in the overall stabilisation of the module pair. CONCLUSIONS: The modules in the 6F1 1F2 module pair interact with each other via a flexible linker and a hydrophobic patch, which lies on the opposite side of the 1F2 module to the putative collagen-binding site. The intermodule interaction is relatively weak and transient.

• Potts JR, Bright JR, Bolton D, Pickford AR, Campbell ID
• Solution structure of the N-terminal F1 module pair from human fibronectin.
• Biochemistry. 1999; 38: 8304-12
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• Multiple sites within the N-terminal domain (1-5F1) of fibronectin have been implicated previously in fibronectin matrix assembly, heparin binding, and binding to cell surface proteins of pathogenic bacteria. The solution structure of 1F1(2)F1, the N-terminal F1 module pair from human fibronectin, has been determined using NMR spectroscopy. Both modules in the pair conform to the F1 consensus fold. In 4F1(5)F1, the only other F1 module pair structure available, there is a well-defined intermodule interface; in 1F1(2)F1, however, there is no detectable interface between the modules. Comparison of the backbone 15N-?1H? NOE values for both module pairs confirms that the longer intermodule sequence in 1F1(2)F1 is flexible and that the stabilization of the 4F1 C-D loop observed in 4F1(5)F1, as a result of the intermodule interface, is not observed in 1F1(2)F1.

• Hamburger ZA, Brown MS, Isberg RR, Bjorkman PJ
• Crystal structure of invasin: a bacterial integrin-binding protein.
• Science. 1999; 286: 291-5
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• The Yersinia pseudotuberculosis invasin protein promotes bacterial entry by binding to host cell integrins with higher affinity than natural substrates such as fibronectin. The 2.3 angstrom crystal structure of the invasin extracellular region reveals five domains that form a 180 angstrom rod with structural similarities to tandem fibronectin type III domains. The integrin-binding surfaces of invasin and fibronectin include similarly located key residues, but in the context of different folds and surface shapes. The structures of invasin and fibronectin provide an example of convergent evolution, in which invasin presents an optimized surface for integrin binding, in comparison with host substrates.

• Bisig D, Weber P, Vaughan L, Winterhalter KH, Piontek K
• Purification, crystallization and preliminary crystallographic studies of a two fibronectin type-III domain segment from chicken tenascin encompassing the heparin- and contactin-binding regions.
• Acta Crystallogr D Biol Crystallogr. 1999; 55: 1069-73
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• A fragment of chicken tenascin consisting of fibronectin type-III domains 5 and 6 has been expressed in Escherichia coli. After modifying a previously reported purification protocol, an electrophoretically homogeneous recombinant protein was obtained from which various crystal forms could be grown under identical conditions. Only one form was suitable for structure determination. These crystals belong to space group P21, with unit-cell parameters a = 45.2, b = 57.9, c = 72.2 A, beta = 91.4 degrees, and diffract to at least 2.6 A resolution using synchrotron radiation. From density measurements of the crystals, it was found that there are two molecules in the asymmetric unit. Diffraction data of native, two platinum-derivative and one palladium-derivative crystals were collected.

• Sharma A, Askari JA, Humphries MJ, Jones EY, Stuart DI
• Crystal structure of a heparin- and integrin-binding segment of human fibronectin.
• EMBO J. 1999; 18: 1468-79
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• The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

• Ward CW
• Members of the insulin receptor family contain three fibronectin type III domains.
• Growth Factors. 1999; 16: 315-22
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• The insulin receptor is a large transmembrane dimer, comprised of several domains. Detailed 3D structural information is available for the L1-cys-rich-L2 domains in the extracellular region (ectodomain) and for the tyrosine kinase catalytic domain in the cytoplasmic portion of the receptor. In addition, previous sequence analyses have identified two fibronectin type III domains in the C-terminal half of each ectodomain monomer. In this report, evidence is provided to show that a third fibronectin type III module exists between the L2 domain and the two previously described fibronectin type III domains.

• de Pereda JM, Wiche G, Liddington RC
• Crystal structure of a tandem pair of fibronectin type III domains from the cytoplasmic tail of integrin alpha6beta4.
• EMBO J. 1999; 18: 4087-95
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• The integrin alpha6beta4 is an essential component of hemidesmosomes but it also plays a dynamic role in invasive carcinoma cells. The cytoplasmic tail of the beta4 subunit is uniquely large among integrins and includes two pairs of fibronectin type III domains separated by a connecting segment. Here we describe the crystal structure of the first tandem domain pair, a module that is critical for alpha6beta4 function. The structure reveals a novel interdomain interface and candidate protein-binding sites, including a large acidic cleft formed from the surfaces of both domains and a prominent loop that is reminiscent of the RGD integrin-binding loop of fibronectin. This is the first crystal structure of either a hemidesmosome component or an integrin cytoplasmic domain, and it will enable the intracellular functions of alpha6beta4 to be dissected at the atomic level.

• Krammer A, Lu H, Isralewitz B, Schulten K, Vogel V
• Forced unfolding of the fibronectin type III module reveals a tensile molecular recognition switch.
• Proc Natl Acad Sci U S A. 1999; 96: 1351-6
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• The 10th type III module of fibronectin possesses a beta-sandwich structure consisting of seven beta-strands (A-G) that are arranged in two antiparallel sheets. It mediates cell adhesion to surfaces via its integrin binding motif, Arg78, Gly79, and Asp80 (RGD), which is placed at the apex of the loop connecting beta-strands F and G. Steered molecular dynamics simulations in which tension is applied to the protein's terminal ends reveal that the beta-strand G is the first to break away from the module on forced unfolding whereas the remaining fold maintains its structural integrity. The separation of strand G from the remaining fold results in a gradual shortening of the distance between the apex of the RGD-containing loop and the module surface, which potentially reduces the loop's accessibility to surface-bound integrins. The shortening is followed by a straightening of the RGD-loop from a tight beta-turn into a linear conformation, which suggests a further decrease of affinity and selectivity to integrins. The RGD-loop therefore is located strategically to undergo strong conformational changes in the early stretching stages of the module and thus constitutes a mechanosensitive control of ligand recognition.

• Su XD, Gastinel LN, Vaughn DE, Faye I, Poon P, Bjorkman PJ
• Crystal structure of hemolin: a horseshoe shape with implications for homophilic adhesion.
• Science. 1998; 281: 991-5
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• Hemolin, an insect immunoglobulin superfamily member, is a lipopolysaccharide-binding immune protein induced during bacterial infection. The 3.1 angstrom crystal structure reveals a bound phosphate and patches of positive charge, which may represent the lipopolysaccharide binding site, and a new and unexpected arrangement of four immunoglobulin-like domains forming a horseshoe. Sequence analysis and analytical ultracentrifugation suggest that the domain arrangement is a feature of the L1 family of neural cell adhesion molecules related to hemolin. These results are relevant to interpretation of human L1 mutations in neurological diseases and suggest a domain swapping model for how L1 family proteins mediate homophilic adhesion.

• Oberhauser AF, Marszalek PE, Erickson HP, Fernandez JM
• The molecular elasticity of the extracellular matrix protein tenascin.
• Nature. 1998; 393: 181-5
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• Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.

• Berry H, Pauthe E, Gallet O, Larreta-Garde V
• Proteolysis of aggregated fibronectin. A model for in vivo matrix degradation.
• Ann N Y Acad Sci. 1998; 864: 198-202

• Magnusson MK, Mosher DF
• Fibronectin: structure, assembly, and cardiovascular implications.
• Arterioscler Thromb Vasc Biol. 1998; 18: 1363-70

• Akke M, Liu J, Cavanagh J, Erickson HP, Palmer AG 3rd
• Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain.
• Nat Struct Biol. 1998; 5: 55-9
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• A novel off-resonance rotating-frame 15N NMR spin relaxation experiment is used to characterize conformational fluctuations with correlation times between 32 and 175 microseconds in the third fibronectin type III domain of human tenascin-C. Conformational fluctuations of contiguous regions of the beta-sandwich structure of the type III domain may represent collective motions, such as transient twisting or breathing of the beta-sheets. Flexibility of the loop containing the Arg-Gly-Asp (RGD) tripeptide may affect the accessibility of this motif in protein-protein interactions.

• Copie V et al.
• Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure.
• J Mol Biol. 1998; 277: 663-82
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• We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 A or 3 degrees. Although the beta-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) A, respectively, the rmsd of the core atom coordinates increases to 3.63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration, Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(+/-0.5) A, agrees with the average Rg calculated for the NMR structures, 20.4 A, and is ca 1 A less than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy, D ||/D perpendicular, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.

• Tsyguelnaia I, Doolittle RF
• Presence of a fibronectin type III domain in a plant protein.
• J Mol Evol. 1998; 46: 612-4
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• A hidden Markov model (HMM) approach was used to identify potential candidates in sequence databases for fibronectin type III domains in plants, a kingdom heretofore bereft of these structures. Fortuitously, one of the proteins uncovered had already had a crystal structure published, allowing direct structural confirmation of the existence of this domain in plants.

• Kishore R, Samuel M, Khan MY, Hand J, Frenz DA, Newman SA
• Interaction of the NH2-terminal domain of fibronectin with heparin. Role of the omega-loops of the type I modules.
• J Biol Chem. 1997; 272: 17078-85
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• Determinants of the interaction of the 29-kDa NH2-terminal domain of fibronectin with heparin were explored by analysis of normal and mutant recombinant NH2-terminal fibronectin fragments produced in an insect cell Baculovirus host vector system. A genomic/cDNA clone was constructed that specified a secretable human fibronectin NH2 fragment. With the use of site-directed mutagenesis a set of 29 kDa fragments was obtained that contained glycine or glutamic acid residues in place of basic residues at various candidate sites for heparin binding in the five type I modules that make up the domain. The recombinant fragment containing the wild type sequence had a nearly normal circular dichroic spectra and a melting profile, as assayed by loss of ellipticity at 228 nm, that was indistinguishable from that of the native fragment obtained by trypsinization of plasma fibronectin. A substantial proportion of the wild type recombinant fragment bound to heparin-Sepharose, where it was eluted at the same NaCl concentration as the native fragment. The wild type fragment was capable of promoting matrix-driven translocation, a morphogenetic effect in artificial extracellular matrices that depends on the interaction of the fibronectin NH2 terminus with heparin-like molecules on the surfaces of particles. Mutant fragments in which arginines predicted to be most exposed in the folded fragment were converted to glycines retained the same affinity for heparin as the wild type fragment. In contrast, a mutant fragment in which the single basic residue (Arg99) in the minor loop ("Omega-loop") of the second type I module was converted to a glycine had an essentially normal melting profile but exhibited no binding to heparin and failed to promote matrix-driven translocation. A mutant fragment in which the single basic residue (Arg52) of the first type I module was converted to a glycine also completely lacked heparin binding activity, but one in which the single basic residue (Arg191) the fourth type I module was converted to a glycine retained the ability to bind heparin. A mutant fragment in which the single basic residue (Lys143) in the Omega-loop of the third type I module was converted to a glutamic acid lacked heparin binding activity but had a CD spectrum similar to the heparin-liganded native protein and was capable of promoting matrix-driven translocation. The results indicate that multiple residues in the Omega-loops of the fibronectin NH2-terminal domain participate in its interactions with heparin. In addition, the conformation of one of the nonbinding mutants may mimic the heparin-induced structural alteration in this fibronectin domain required for certain morphogenetic events.

• Chen Y, Zardi L, Peters DM
• High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils.
• Scanning. 1997; 19: 349-55
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• High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.

• Pickford AR, Potts JR, Bright JR, Phan I, Campbell ID
• Solution structure of a type 2 module from fibronectin: implications for the structure and function of the gelatin-binding domain.
• Structure. 1997; 5: 359-70
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• BACKGROUND: Fibronectin is an extracellular matrix glycoprotein involved in cell adhesion and migration events in a range of important physiological processes. Aberrant adhesion of cells to the matrix may contribute to the breakdown of normal tissue function associated with various diseases. The adhesive properties of fibronectin may be mediated by its interaction with collagen, the most abundant extracellular matrix protein. The collagen-binding activity of fibronectin has been localized to a 42 kDa proteolytic fragment on the basis of this fragment's affinity for denatured collagen (gelatin). This gelatin-binding domain contains the only type 2 (F2) modules found in the protein. The F2 modules of the matrix metalloproteinases MMP2 and MMP9 are responsible for the affinity of these proteins for gelatin. Knowledge of the structure of fibronectin will provide insights into its interactions with other proteins, and will contribute to our understanding of the structure and function of the extracellular matrix, in both normal and disease-altered tissues. RESULTS: We have determined the solution structure of the first F2 (1F2) module from human fibronectin by two-dimensional NMR spectroscopy. The tertiary structure of the 1F2 module is similar to that of a shorter F2 module, PDC-109b, from the bovine seminal plasma protein PDC-109. The 1F2 module has two double-stranded antiparallel beta sheets oriented approximately perpendicular to each other, and enclosing a cluster of highly conserved aromatic residues, five of which form a solvent-exposed hydrophobic surface. The N-terminal extension in 1F2 brings the N and C termini of the module into close proximity. CONCLUSIONS: The close proximity of the N and C termini in 1F2 allows for interactions between non-contiguous modules in the gelatin-binding domain. Thus, instead of forming an extended, linear chain of modules, the domain may have a more compact, globular structure. A pocket in the module's solvent-exposed hydrophobic surface may bind nonpolar residues in the putative fibronectin-binding site of the extracellular matrix component type I collagen.

• Carr PA, Erickson HP, Palmer AG 3rd
• Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin.
• Structure. 1997; 5: 949-59
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• BACKGROUND: Fibronectin type III domains are found as autonomously-folded domains in a large variety of multidomain proteins, including extracellular matrix proteins. A subset of these domains employ an Arg-Gly-Asp (RGD) tripeptide motif to mediate contact with cell-surface receptors (integrins). This motif mediates protein-protein interactions in a diverse range of biological processes, such as in tissue development, would healing and metastasis. The molecular basis for affinity and specificity of cell adhesion via type III domains has not been clearly established. The tenth type III domain from fibronectin (FNfn10) and the third type III domain from tenascin-C (TNfn3) have 27% sequence identity and share the same overall protein fold, but present the RGD motifs in different structural contexts. The dynamical properties of the RGD motifs may affect the specificity and affinity of the FNfn10 and TNfn3 domains. Structure-dynamics correlations for these structurally homologous proteins may reveal common molecular features which are important to the dynamical properties of proteins. RESULTS: The intramolecular dynamics of the protein backbones of FNfn10 and TNfn3 have been studied by 15N nuclear spin relaxation. The FG loop in FNfn10, which contains the RGD motif, exhibits extensive flexibility on picosecond to nanosecond timescales, but motions on microsecond to millisecond timescales are not observed. The equivalent region in TNfn3 is as rigid as regular elements of secondary structure. The CC' loop also is more flexible on picosecond-nanosecond timescales in FNfn10 than in TNfn3. Conformational exchange, reflecting flexibility on microsecond-millisecond timescales, is observed in beta strands A and B of both FNfn10 and TNfn3. CONCLUSIONS: Comparison of the structures of the FNfn10 and TNfn3 reveals several features related to their different dynamical properties. The larger amplitude motions of loops in FNfn10 are consistent with the hypothesis that flexibility of these regions facilitates induced-fit recognition of fibronectin by multiple receptors. Similarly, the more rigid loops of TNfn3 may reflect greater specificity for particular integrins. The correlations observed between structural features and dynamical properties of the homologous type III domains indicate the influence of hydrogen bonding and hydrophobic packing on dynamical fluctuations in proteins.

• Chi-Rosso G et al.
• Fibronectin type III repeats mediate RGD-independent adhesion and signaling through activated beta1 integrins.
• J Biol Chem. 1997; 272: 31447-52
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• Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of beta1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and beta1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated beta1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.

• Grant RP, Spitzfaden C, Altroff H, Campbell ID, Mardon HJ
• Structural requirements for biological activity of the ninth and tenth FIII domains of human fibronectin.
• J Biol Chem. 1997; 272: 6159-66
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• The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

• Muller YA, Ultsch MH, de Vos AM
• The crystal structure of the extracellular domain of human tissue factor refined to 1.7 A resolution.
• J Mol Biol. 1996; 256: 144-59
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• Exposure of blood to cells expressing tissue factor results in formation of a high-affinity complex with factor VIIa, initiating the extrinsic pathway of blood coagulation by the activation of factors IX and X. The structure of the extracellular portion of tissue factor was refined to a crystallographic R-value of 20.4% to a resolution of 1.69 A against synchroton data collected from a flash-frozen crystal. The structure consists of two fibronectin type III modules whose hydrophobic cores merge in the domain-domain interface, suggesting that the extracellular portion serves as a relatively rigid template for factor VIIa binding. Analysis of the hydrophobic core of each individual module identifies a cluster of residues forming a packing motif centered on Trp25 which appears to be characteristic for fibronectin type III modules. Comparison of the structure to that of the human growth hormone receptor, which belongs to a different class (class I) of the same cytokine receptor superfamily, shows that the structure of the individual domains is very similar but that the relative domain-domain orientation differs greatly. Even though the WSXWS box characteristic of the class I cytokine receptors is not present in tissue factor, the analogous residues have the identical polyproline helical conformation. Mapping of residues important for biological activity on the structure shows that all these are located on Beta-strands in a small number of distinct clusters, on the opposite side of the molecule compared to the ligand binding determinants of the growth hormone receptor.

• Bateman A, Jouet M, MacFarlane J, Du JS, Kenwrick S, Chothia C
• Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders.
• EMBO J. 1996; 15: 6050-9
• Display abstract

• The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties.

• Nagai T
• [Fibronectin--synergy cell recognition site]
• Rinsho Byori. 1996; 44: 1119-24
• Display abstract

• Fibronectin is a major and multifunctional cell adhesive extracellular matrix protein. Many studies have been carried out to reveal its structures and functions. One of the most important question was the cell adhesive structures on the molecule and how to associate with cell surface receptors. It was striking that very short amino acid sequence Arg-Gly-Asp (RGD) in the central cell binding domain of fibronectin was determined as minimum cell adhesive sequence. However, other data indicate that additional subregions besides the RGD motif are required for its cell adhesive activity mediated by alpha 5 beta 1 fibronectin receptor. This putative synergistic site, second site, was analyzed by site directed mutagenesis studies, homology scanning using intramolecular chimeras and epitope mapping using inhibitory monoclonal antibodies. Those studies cleared that the functional sequence, Pro-His-Ser-Arg-Asn (PHSRN), synergistically enhances the cell-adhesive activity of RGD motif depends on the activation state of the integrin.

• Potts JR, Campbell ID
• Structure and function of fibronectin modules.
• Matrix Biol. 1996; 15: 313-20
• Display abstract

• Fibronectin is an important component of the extracellular matrix and is involved in a diverse range of physiological processes. It is a mosaic protein composed almost entirely of three types of module, F1, F2 and F3. Although the structures of single F1, F2 and F3 modules have been available for a number of years, in many cases the key to understanding the structure-function relationships in fibronectin and other proteins containing these modules lies in studies of module pairs and larger domains. This review focuses on recent advances in the understanding of the structure and function of fibronectin modules.

• Mohri H, Katoh K, Motomura S, Okubo T
• Novel synthetic peptides from the C-terminal heparin binding domain of fibronectin with heparin binding activity.
• Peptides. 1996; 17: 1079-81
• Display abstract

• To identify a minimal peptide ligand that participates in recognition of heparin, we synthesized peptides extending a 29-kDa fragment in the C-terminal heparin binding domain of fibronectin. We obtained evidence that two peptides (designated D5 and D1) exhibit inhibitory activity on binding of the 29-kDa fragment to heparin-Sepharose CL 6B. It dose inhibitory studies, peptide D5 showed the potent inhibitory activity with IC50 of 210 +/- 37 microM. A new heparin binding site in the C-terminal heparin binding domain of fibronectin is demonstrated and provides a rationale for understanding the mechanism of cell adhesion and spreading.

• Plaxco KW, Spitzfaden C, Campbell ID, Dobson CM
• Rapid refolding of a proline-rich all-beta-sheet fibronectin type III module.
• Proc Natl Acad Sci U S A. 1996; 93: 10703-6
• Display abstract

• Fibronectin type III modules contain approximately 90 residues and are an extremely common building block of animal proteins. Despite containing a complex all-beta-sheet topology and eight prolines, the refolding of the 10th type III module of human fibronectin has been found to be very rapid, with native core packing, amide hydrogen bonding, and backbone conformation all recovered within 1 s at 5 degrees C. These observations indicate that this domain can overcome many structural characteristics often thought to slow the folding process.

• Alfandari D, Ramos J, Clavilier L, DeSimone DW, Darribere T
• The RGD-dependent and the Hep II binding domains of fibronectin govern the adhesive behaviors of amphibian embryonic cells.
• Mech Dev. 1996; 56: 83-92
• Display abstract

• In early amphibian development, interactions between fibronectin and both ectoderm and mesoderm cells are critical in the progression of gastrulation movements. In the Pleurodeles waltl embryo, it has been established that ectoderm cells of the animal hemisphere organize a fibrillar-extracellular matrix containing fibronectin. Mesoderm cells migrate along the blastocoel roof using these fibronectin fibrils as substratum. Fibronectin is an adhesive glycoprotein which possesses multiple cell-binding domains. From previous studies, it is clear that amphibian ectoderm and mesoderm cells interact with fibronectin in an RGD-dependent manner, whereas the contributions of RGD-independent domains in the adhesive behaviors of gastrula cells has not been defined. To study this question, we have used bacterially expressed Pleurodeles waltl fibronectin-fusion proteins. The approach consisted of in vitro adhesion assays with either isolated cells or tissue fragments of embryos dissected at the onset of gastrulation. Tissues were obtained from regions of the embryo which represent presumptive ectoderm cells or from the dorsal-marginal zone which contains cells of the presumptive cephalic, chordal and somitic mesoderm. The results show that both the RGD-dependent and the Hep II domains of fibronectin mediate attachment and spreading of isolated cells. Both regions cooperate to control the proper expansion of a sheet of dorsal mesoderm cells. The Hep II domain promotes the migration of cells ahead of the mesoderm-cell sheet.

• Carnemolla B, Leprini A, Querze G, Urbini S, Zardi L
• Novel self-association fibronectin sites.
• Biochem Cell Biol. 1996; 74: 745-8
• Display abstract

• In this study, we report a strong interaction between two contiguous proteolytic fragments of fibronectin, each having a mass of about 16 kDa. This interaction was stable in 4 M NaCl and 4 M urea and dissociation of the two fragments required buffers containing 0.5% sodium dodecyl sulphate. After purification, these peptides maintained their ability to interact when mixed. One fragment was made up of type III repeat 4 and part of 5, the other by repeat 6 and part of 5. Such strong interaction between two fibronectin regions may play a role in fibronectin conformation as well as during fibronectin fibril formation.

• Fischer D, Chiquet-Ehrismann R, Bernasconi C, Chiquet M
• A single heparin binding region within the fibrinogen-like domain is functional in chick tenascin-C.
• J Biol Chem. 1995; 270: 3378-84
• Display abstract

• Tenascin-C binds to cell surface and matrix proteoglycans and to heparin. Two heparin binding regions have recently been localized per tenascin-C monomer, one in the C-terminal fibrinogen-like domain and the other in fibronectin type III repeats 3-5. Here we show that a single region in each subunit is necessary and sufficient for heparin binding by whole tenascin-C at physiological ionic strength. First, native tenascin-C was bound to heparin-agarose and digested with Pronase. A 29-kDa fragment retained on the heparin column was recognized by a monoclonal antibody against the fibrinogen-like domain. In contrast, small fragments labeled by an antibody against fibronectin type III repeats 2-5 were released. Second, mild tryptic digestion of tenascin-C yielded two related fragments of 180 and 170 kDa. The latter missed part of the fibrinogen domain and had lost affinity for heparin, in contrast to the former. Finally, chick tenascin-C constructs were recombinantly expressed in human cells. Whereas the complete protein and a mutant lacking fibronectin type III repeats 1-5 bound to heparin-agarose, recombinant tenascin-C missing the C-terminal fibrinogen-like globe did not. Thus, whole chick tenascin-C contains one essential heparin binding region per subunit, located in the fibrinogen-like domain within 10 kDa from the C terminus.

• Weber P, Zimmermann DR, Winterhalter KH, Vaughan L
• Tenascin-C binds heparin by its fibronectin type III domain five.
• J Biol Chem. 1995; 270: 4619-23
• Display abstract

• Two sites on tenascin mediate interactions with glycosaminoglycan chains of proteoglycans. One is situated on the fibrinogen-like domain, whereas the other lies within the fibronectin type III homology region (Aukhil, I., Joshi, P., Yan, Y.Z., and Erickson, H.P. (1993) J. Biol. Chem. 268, 2542-2553.). We now characterize the latter binding site more closely by means of recombinant protein fragments derived from the type III homology region of tenascin. Using a heparin-Sepharose column, we localize the second heparin binding site to the fifth fibronectin type III domain. This is confirmed in solid phase assays by incubation of fusion proteins with biotin-labeled heparin. In addition, we demonstrate the binding of heparan sulfate and dermatan sulfate to domain five. Molecular modelling of this domain reveals a conserved heparin-binding motif that we propose as the putative binding site. The fact, that different glycosaminoglycans may bind to this domain, implies that different classes of proteoglycans may in vivo compete for the same site.

• Potts JR, Phan I, Williams MJ, Campbell ID
• High-resolution structural studies of the factor XIIIa crosslinking site and the first type 1 module of fibronectin.
• Nat Struct Biol. 1995; 2: 946-50
• Display abstract

• The N-terminal domain of fibronectin undergoes factor XIIIa-catalysed crosslinking to fibrin, bacteria and collagen. The reactive glutamine residue is in an extended, random coil 'tail' of about 18 residues that would be accessible for crosslinking.

• Aota S, Yamada KM
• Fibronectin and cell adhesion: specificity of integrin-ligand interaction.
• Adv Enzymol Relat Areas Mol Biol. 1995; 70: 1-21

• Ely KR, Kunicki TJ, Kodandapani R
• Common molecular scaffold for two unrelated RGD molecules.
• Protein Eng. 1995; 8: 823-7
• Display abstract

• The sequence arginine-glycine-aspartic acid (RGD) is important for recognition of cell adhesion proteins by cell surface receptors (integrins). This tripeptide sequence is present in a number of proteins including fibronectin, vitronectin, von Willebrand factor and fibrinogen. Specific and selective binding of the RGD sequence by different receptors suggests that the conformational orientation of the tripeptide is critical for stereochemical recognition. The crystal structures of two proteins that contain the RGD signal were determined: (i) the cell-binding type III module of fibronectin (FNIII10) and (ii) an anti-receptor antibody fragment (OPG2) that is a functional RGD ligand mimic with an RYD recognition site in the variable (VH) domain. Both of these modules are folded into beta-barrels with two layers of antiparallel beta-sheets enclosing a hydrophobic core. Since these molecules each contain the RGD (RYD) sequence, there is a unique opportunity for direct structural comparison. The comparison has defined a common molecular scaffold in these two unrelated molecules. Within this framework, the RGD (RYD) sites are located in structurally related loops in the two modules, i.e. at one end of the scaffold in a long loop connecting the last two strands in one of the beta-sheets. This shared scaffold is used for the stereochemical presentation of the RGD site for receptor recognition.

• Ugarova TP et al.
• Conformational transitions in the cell binding domain of fibronectin.
• Biochemistry. 1995; 34: 4457-66
• Display abstract

• Plasma fibronectin readily changes shape in response to environmental conditions which may, in turn, lead to differential expression of its multiple functional sites. To test this possibility, the expression of two of the type III modules within cell binding domain of fibronectin was assessed with monoclonal antibodies (mAb). Utilizing proteolytic and recombinant fragments of plasma fibronectin, the epitopes recognized by mAbIII-9 and mAbIII-10 were localized to the ninth and tenth (RGD-containing) type III repeats of fibronectin, respectively. Both mAb inhibited the adhesion of platelets to immobilized fibronectin, suggesting that the recognized epitopes resided in close spatial proximity to the cell binding sites. Radioimmunoassay and Scatchard analyses showed that, in solution, each dimeric fibronectin molecule bound two mAbIII-9 but only one mAbIII-10 molecule (ionic strength 0.15, pH 7.4). The binding of a single mAbIII-10 per fibronectin molecule was verified by electron microscopy. Heparin, heparan sulfate, gangliosides (but not chondroitin sulfates A and B and hyaluronic acid), and self-association increased the apparent affinity of mAbIII-10 for soluble fibronectin. Adsorption of fibronectin onto a polystyrene surface resulted in the appearance of an additional binding site for mAbIII-10. MAbIII-9 binding also was altered by fibronectin immobilization. These results suggest that the deposition of fibronectin and its interaction with components of the extracellular matrix can modulate the expression of the cell binding domains including the RGDS-containing type III repeat. Exposure of the second tenth type III repeat within the fibronectin dimer, as a result of unfolding on a surface, could contribute to the enhanced adhesiveness of adsorbed fibronectin.

• Miles AM, Brew SA, Ingham KC, Smith RL
• Photoinduced structural changes in the collagen/gelatin binding domain of fibronectin.
• Biochemistry. 1995; 34: 6941-6
• Display abstract

• Prolonged exposure of human plasma fibronectin (pFn) and its 40- and 21-kDa collagen/gelatin binding fragments (GBFs) to 280-nm irradiation decreased their affinity for gelatin and for TR-CB7, a fluorescently labeled CNBr fragment of the alpha-1 chain of type I collagen. Fluorescence polarization binding assays of TR-CB7 with pFn and the 40-kDa GBF yielded progressively higher Kd's with increased time of exposure to 280-nm light at 25 degrees C. Binding of nonirradiated and irradiated pFn and fragments to gelatin-Sepharose correlated with the polarization data, confirming diminished gelatin binding following exposure to 280-nm light. Fluorescence spectra of intrinsic tryptophans in the 21- and 40-kDa GBFs exhibited changes indicative of photoinduced conformational changes; the maximum fluorescence wavelength red-shifted from between 340 and 350 nm to 360 nm, with concomitant increases in fluorescence intensity. Exposure of 21- and 40-kDa GBFs and pFn to 280-nm light also generated approximately two, four, and six free sulfhydryl groups per molecule, respectively. No sulfhydryl release was observed in other Trp- and disulfide-containing proteins under the same conditions. We propose that the fluorescence changes as well as the changes in affinity for gelatin or the collagen fragment result from structural changes secondary to the breakage of disulfide bonds, as a consequence of energy transfer from nearby tryptophans in one or more of the Fn type I repeats in the gelatin binding region of fibronectin.

• Danen EH, Ruiter DJ, Van Muijen GN
• Mechanisms of melanoma cell adhesion to fibronectin.
• Biochem Soc Trans. 1995; 23: 403-403

• Leahy DJ, Erickson HP, Aukhil I, Joshi P, Hendrickson WA
• Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.
• Proteins. 1994; 19: 48-54
• Display abstract

• Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.

• Yokosaki Y et al.
• The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.
• J Biol Chem. 1994; 269: 26691-6
• Display abstract

• We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.

• Linnala A, von Koskull H, Virtanen I
• Isoforms of cellular fibronectin and tenascin in amniotic fluid.
• FEBS Lett. 1994; 337: 167-70
• Display abstract

• Amniotic fluid (AF) obtained from second trimester pregnancies presented extradomain (ED) A, B and an oncofetal (onc-f) domain containing isoforms of cellular fibronectin (cFn) in Western blotting of gelatin-bound polypeptides and directly of AF. Western blotting after sequential immunoprecipitation suggested at least three Fn molecules: one containing EDA and the onc-f domain and another minor component distinctly containing all the domains, and a third one only containing EDA. The immunoblotting results for EDA-cFn and onc-f-cFn were closely similar to that for total Fn, whereas in plasma samples of normal and pregnant women only traces of EDA-cFn and onc-f-cFn, but no EDB-cFn, were found. Western blotting of AF also indicated the presence of three isoforms of tenascin (Tn), M(r) 190,000 and 280,000 polypeptides earlier found in many cells, and a M(r) 200,000 polypeptide, novel for AF and not present in plasma. The results suggest a novel extracellular matrix polypeptide composition for AF.

• Schenk S, Chiquet-Ehrismann R
• Tenascins.
• Methods Enzymol. 1994; 245: 52-61

• Chiquet-Ehrismann R, Hagios C, Matsumoto K
• The tenascin gene family.
• Perspect Dev Neurobiol. 1994; 2: 3-7
• Display abstract

• Tenascin is believed to be an important extracellular matrix protein involved in regulating numerous developmental processes, such as morphogenetic cell migration and organogenesis. This function was implied by its tissue distribution and regulated expression during embryogenesis. However, such an important function was questioned recently, since mice lacking a functional tenascin gene apparently developed normally. To us, this is not conclusive evidence that tenascin has no function, for we believe that the loss of tenascin could be compensated for. Since in several other cases compensation occurs by other members of a gene family, we started to investigate the family of tenascin-like genes and we review in this article the structures of the original tenascin/cytotactin (tenascin-C), restrictin/J-160/180 (tenascin-R), and the tenascin-like gene present in the major histocompatibility complex class III locus (tenascin-X). Furthermore, we present evidence for the likely existence of even more members of this tenascin family.

• Schachner M, Taylor J, Bartsch U, Pesheva P
• The perplexing multifunctionality of janusin, a tenascin-related molecule.
• Perspect Dev Neurobiol. 1994; 2: 33-41
• Display abstract

• The extracellular matrix glycoprotein janusin, closely related to tenascin in its repeated motifs of epidermal growth factor, fibronectin type III, and fibrinogen-like domains, displays in vitro a broad spectrum of functional diversity. Synthesized by oligodendrocytes and subpopulations of neurons at late developmental stages in the rodent central nervous system, it can be adhesive or antiadhesive, depending on the neural cell type that interacts with it. It promotes neurite outgrowth of some neural cell types, when offered as a uniform culture substrate, but inhibits neurite outgrowth of other neuronal populations. When offered as a sharp substrate boundary in congruence with a permissive substrate, it acts as a barrier for neurite outgrowth. Like tenascin, it can modify the adhesive substrate properties of another extracellular matrix glycoprotein, fibronectin, whereby the smaller, 160 kD component of janusin exerts its effects by interaction with fibronectin and the 180 kD janusin component functionally modifies the fibronectin receptor via a disialoganglioside receptor. In neurons, the antiadhesive and neurite outgrowth inhibiting signal is mediated by the F3/11 immunoglobulin superfamily recognition molecule. In oligodendrocytes, yet another receptor for janusin mediates adhesion and process formation. A prerequisite for any intracellular response to occur is a transient lock-and-key recognition manifesting itself in short-term binding between the interacting partners. As for tenascin, the different functions exerted by janusin are likely to be encoded in the different domains of the janusin molecule, which can act on different receptors, whereby the receiving cell is able to interpret the cell surface trigger in different ways, depending on the particular cell type involved.(ABSTRACT TRUNCATED AT 250 WORDS)

• Erickson HP
• Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin.
• Proc Natl Acad Sci U S A. 1994; 91: 10114-8
• Display abstract

• The elastic protein titin comprises a tandem array of fibronectin type III and immunoglobulin domains, which are structurally similar 7-strand beta-sandwiches. A proposed mechanism for stretching titin, by sequential denaturation of individual fibronectin type III-immunoglobulin domains in response to applied tension, is analyzed here quantitatively. The folded domain is approximately 4 nm long, and the unraveled polypeptide can extend to 29 nm, providing a 7-fold stretch over the relaxed length. Elastic recoil is achieved by refolding of the denatured domains when the force is released. The critical force required to denature a domain is calculated to be 3.5-5 pN, based on a net free energy for denaturation of 7-14 kcal/mol, plus 5 kcal/mol to extend the polypeptide (1 cal = 4.184 J). This force is comparable to the 2- to 7-pN force generated by single myosin or kinesin molecules. The force needed to pull apart a noncovalent protein-protein interface is estimated here to be 10-30 pN, implying that titin will stretch internally before the molecule is pulled from its attachment at the Z band. Many extracellular matrix and cell adhesion molecules, such as fibronectin, contain tandem arrays of fibronectin type III domains. Both single molecules and matrix fibers should have elastic properties similar to titin.

• Erickson HP
• Evolution of the tenascin family--implications for function of the C-terminal fibrinogen-like domain.
• Perspect Dev Neurobiol. 1994; 2: 9-19
• Display abstract

• The three members of the tenascin (TN) family, TN-C, TN-R, and TN-X, are apparently conserved in all vertebrates and therefore must have functions that contribute to survival. One specific domain of tenascins, the fibrinogen-like terminal knob, can be argued to have an essential function. Its position at the C-terminus makes it most vulnerable to loss through mutation or deletion, and it should have been eliminated in evolution if there were no selective pressure to maintain it. The epidermal growth factor and fibronectin III domains probably play an important role as spacers, placing the terminal knob at the end of the tribrachion or hexabrachion arms. In addition to functioning as spacers, at least some of these domains may have additional functional interactions. The conservation of these domains in evolution is comparable to that of some growth factors, consistent with this possibility. A phylogenetic tree of all known fibrinogen-related domains, including those in tenascins, is presented.

• Morla A, Zhang Z, Ruoslahti E
• Superfibronectin is a functionally distinct form of fibronectin.
• Nature. 1994; 367: 193-6
• Display abstract

• Fibronectin is an extracellular matrix protein that is important in development, wound healing and tumorigenesis. In the blood it is dimeric, but in tissues forms disulphide crosslinked fibrils. Here we show that a fragment from the first type-III repeat of fibronectin binds to fibronectin and induces spontaneous disulphide crosslinking of the molecule into multimers of high relative molecular mass which resemble matrix fibrils. Treatment of fibronectin with this inducing fragment also converts fibronectin into a form that has greatly enhanced adhesive properties (hence the term superfibronectin) and which suppresses cell migration. Whereas cells attach to fibronectin through integrins, cell attachment to superfibronectin is mediated both by integrins and by receptors with properties distinct from those of integrins. Superfibronectin may be closely related to the natural matrix form of fibronectin.

• Dickinson CD et al.
• Crystal structure of the tenth type III cell adhesion module of human fibronectin.
• J Mol Biol. 1994; 236: 1079-92
• Display abstract

• The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data. The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands. The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands. The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

• Diamond MS, Springer TA
• The dynamic regulation of integrin adhesiveness.
• Curr Biol. 1994; 4: 506-17
• Display abstract

• The integrins are a family of transmembrane heterodimeric adhesion molecules that play important roles in wound healing, immune system function and organ development. Recent studies indicate that adhesion of integrins to their ligands is not constitutive but is dynamically regulated by intracellular signal transduction pathways.

• Aukhil I, Joshi P, Yan Y, Erickson HP
• Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins.
• J Biol Chem. 1993; 268: 2542-53
• Display abstract

• We have produced a set of bacterial expression proteins corresponding to 10 segments of tenascin and two of fibronectin and tested them for heparin binding and cell adhesion. We used polymerase chain reaction cloning to terminate the segments precisely at domain boundaries. Heparin binding activity was mapped to two different tenascin segments: one comprising the fourth and fifth fibronectin type III domains, and to TNfbg, the fibrinogen-like terminal knob. TNfbg, but none of the other tanascin segments, also supported adhesion of primary rat embryo skin fibroblasts. The fibroblasts did not spread on TNfbg but remained rounded. Cell binding to TNfbg occurred in the presence or absence of divalent cations and was not inhibited by RGD peptides, suggesting that integrins are not involved. Fibroblast binding to TNfbg was strongly inhibited by soluble heparin, by treating the cells with heparitinase, or by culture conditions that cause undersulfation of proteoglycans. These observations suggest that cell attachment to TNfbg is mediated by cell surface proteoglycans. We have also made full-length cDNA constructs for the largest and smallest splice variants of human tenascin, as well as one truncated after the 14th epidermal growth factor-like domain, in the pNUT mammalian cell expression vector. Stably transfected baby hamster kidney cell lines secreted large quantities of tenascin, and this was assembled into normal hexabrachions, the arm length corresponding to the construct.

• Hedin U, Holm J, Hansson GK
• Expression of tenascin in arterial tissue.
• Am J Pathol. 1993; 142: 659-659

• Erickson HP
• Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions.
• Curr Opin Cell Biol. 1993; 5: 869-76
• Display abstract

• "Mice develop normally without tenascin", was a shock to biologists studying the extracellular matrix. Could tenascin be a useless protein? This seems most improbable, as it is conserved in every vertebrate species. Moreover, two new proteins have been discovered in the last year that are closely related to the original tenascin, providing evidence for a tenascin family. Speculations on functions are ripe for re-evaluation.

• Bristow J, Tee MK, Gitelman SE, Mellon SH, Miller WL
• Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B.
• J Cell Biol. 1993; 122: 265-78
• Display abstract

• A human gene termed XB overlaps the P450c21B gene encoding steroid 21-hydroxylase and encodes a protein that closely resembles extracellular matrix proteins. Sequencing of phage and cosmid clones and of cDNA fragments shows that the XB gene spans 65 kb of DNA, consisting of 39 exons that encode a 12-kb mRNA. The predicted protein of over 400 kD consists of five distinct domains: a signal peptide, a hydrophobic domain containing three heptad repeats, a series of 18.5 EGF-like repeats, 29 fibronectin type III repeats, and a carboxy-terminal fibrinogen-like domain. Because the structure of the protein encoded by the XB gene closely resembles tenascin, we term this protein tenascin-X (TN-X), and propose a simplified nomenclature system for the family of tenascins. RNase protection experiments show that the TN-X transcript is expressed ubiquitously in human fetal tissues, with the greatest expression in the fetal testis and in fetal skeletal, cardiac, and smooth muscle. Two adrenal-specific transcripts, P450c21B (steroid 21-hydroxylase) and Y (an untranslated transcript) overlap the XB gene on the complementary strand of DNA, yielding a unique array of overlapping transcripts: a "polygene." In situ hybridization histochemistry experiments show that the TN-X transcript and the P450c21 and Y transcripts encoded on the complementary DNA strand are all expressed in the same cells of the human adrenal cortex. Genetic data suggest that TN-X may be essential for life.

• Johnson WC Jr, Pagano TG, Basson CT, Madri JA, Gooley P, Armitage IM
• Biologically active Arg-Gly-Asp oligopeptides assume a type II beta-turn in solution.
• Biochemistry. 1993; 32: 268-73
• Display abstract

• The sequence Arg-Gly-Asp (RGD) has been found to be the consensus sequence of matrix proteins for binding cell surface receptors (integrins). Studies with synthetic peptides containing the RGD sequence show that the biological activity of these oligopeptides is removed upon a conservative substitution of Glu for Asp in the RGD sequence. Two-dimensional 1H NMR methods were used to investigate the secondary structures in aqueous solution for two such oligopeptides of differing biological activity. The sequence Tyr-Gly-Arg-Gly-Asp-Ser-Pro, which binds to selected integrins, is found to assume a type II beta-turn at both pH 4 and 7. In contrast, the sequence Tyr-Gly-Arg-Gly-Glu-Ser-Pro, which does not interfere with integrin-mediated cell attachment, is found to assume a type I or III beta-turn at both pH 4 and 7. This comparison confirms not only that oligopeptides can assume a secondary structure in aqueous solution, but also that these structures may be important to biological functions.

• Edelman GM
• A golden age for adhesion.
• Cell Adhes Commun. 1993; 1: 1-7

• Appel F, Holm J, Conscience JF, Schachner M
• Several extracellular domains of the neural cell adhesion molecule L1 are involved in neurite outgrowth and cell body adhesion.
• J Neurosci. 1993; 13: 4764-75
• Display abstract

• The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth. To analyze structure-function relationships of L1 in neurite outgrowth and cell body adhesion, we have expressed and purified a set of different fragments of the extracellular part of this glycoprotein in CHO cells and in Escherichia coli. When neurite outgrowth from small cerebellar neurons was measured on substrate-coated L1 or L1 fragments, neurite outgrowth was promoted by the immunoglobulin-like domains I-II, III-IV, and V-VI, and by the fibronectin type III homologous repeats 1-2, while the fibronectin type III homologous repeats 3-5 were ineffective. In contrast, cell bodies of small cerebellar neurons adhered mostly to the immunoglobulin-like domains I-II and V-VI, and to the fibronectin type III homologous repeats 3-5, but less to the immunoglobulin-like domains III-IV and fibronectin type III homologous repeats 1-2. In both assays, the neuronal cell surface receptor for all active protein fragments was identified as L1. No significant differences in functional activities were found between fragments with and without carbohydrate structures. These findings indicate that L1 uses several domains for homophilic interactions overlapping for the two functions analyzed here, but also showing some regional specialization. Furthermore, we show that a homophilic molecule uses several domains in one function, with neurite outgrowth requiring more domains than adhesion for maximal activity.

• Sherman SA, Johnson ME
• Derivation of locally accurate spatial protein structure from NMR data.
• Prog Biophys Mol Biol. 1993; 59: 285-339

• Chiquet-Ehrismann R
• Tenascin and other adhesion-modulating proteins in cancer.
• Semin Cancer Biol. 1993; 4: 301-10
• Display abstract

• A loss in cell-cell adhesion and an increase in cell motility are clearly prerequisites for the development of invasive tumors and metastasis. Since disseminating tumor cells have to interact with and migrate through extracellular matrices it is of interest to compare the composition of extracellular matrix proteins in normal and tumor tissues, in order to determine whether the extracellular matrix could be critically involved in affecting tumor cell adhesion and migration. The question will be addressed whether tumor extracellular matrix contains increased amounts of potentially antiadhesive extracellular matrix components and whether cells behave differently depending on the extracellular matrix they encounter. The results reviewed support the hypothesis that changes in the extracellular matrix and the cellular receptors are indeed important in the process of tumorigenesis.

• Minguell JJ, Hardy CL, Tavassoli M
• Adhesive interaction of hemopoietic progenitor cell membrane with the RGD domain of fibronectin.
• Biochim Biophys Acta. 1993; 1151: 120-6
• Display abstract

• The binding of two cloned hemopoietic progenitor cell lines, B6Sut (multipotential) and FDCP-1 (bipotential) to dishes coated with fibronectin or its chymotryptic fragments was studied by labeling the cells with 51Cr or [35S]methionine. Intact fibronectin molecule and its 120 kDa fragment, containing the Arg-Gly-Asp (RGD) sequence motif, as well as a synthetic RGD-containing peptide Peptite 2000 all bound progenitor cells. However, the 40 or 45 kDa fragments, containing the heparin-binding and CS-1 domains, failed to bind the cells in a comparable magnitude. The binding of intact fibronectin and its 120 kDa fragment was inhibited in a dose-dependent fashion with increasing concentration of RGD-containing Gly-Arg-Gly-Asp-Ser peptide, but not with Gly-Arg-Gly-Glu-Ser control peptide that does not contain the RGD sequence motif. To explore the nature of the receptor for this fragment of fibronectin, membrane proteins were labeled with 125I and subjected to affinity chromatography using a matrix to which the 120 kDa fragment of fibronectin was covalently bound. Specific competitive elution with RGD yielded two bands with molecular masses of 160 and 110 kDa, corresponding, respectively, to those of alpha 5 and beta 1 chains of integrin molecule. Western blotting of whole-cell-lysate proteins with a monospecific, polyclonal serum specific for vertebrate beta 1 integrins identified a beta 1 integrin in these cells. Thus, it appears that an interaction involving alpha 5 beta 1 integrin with 120 kDa fragment of fibronectin may be involved between hemopoietic progenitor cells and the fibronectin component of extracellular matrix.

• Vrucinic-Filipi N, Chiquet-Ehrismann R
• Tenascin function and regulation of expression.
• Symp Soc Exp Biol. 1993; 47: 155-62
• Display abstract

• Tenascin is a large hexameric extracellular matrix protein. Each subunit consists of a linear array of the following structural domains: an N-terminal cysteine-rich region and heptad repeats both involved in the oligomerization, followed by EGF-like repeats, fibronectin type III domains and a C-terminal globular domain homologous to fibrinogens. Depending on the species, the number of EGF-like repeats as well as of the fibronectin type III repeats differs. A model proposing a new nomenclature for the numbering of the fibronectin type III repeats is presented. It enables the assignment of corresponding repeats from different species and shows that for human tenascin several more alternatively spliced fibronectin type III repeats have been described than for chicken or mouse tenascin. Tenascin is generally classified as an anti-adhesive protein, since many cells do not adhere to tenascin or if they adhere they do not spread. The addition of tenascin can also lead to the loss of focal contacts in well spread cells. On the other hand growth cones adhere well to tenascin and a tenascin substrate can support neurite extension. Using recombinant fragments of tenascin we could identify an adhesive as well as an anti-adhesive region within one tenascin subunit. The major interest in tenascin was prompted by its interesting tissue distribution. Tenascin is often transiently expressed during organogenesis, tissue remodeling, cell migration and the development of the nervous system. We thus investigated possibilities to regulate tenascin expression in primary chick embryo fibroblast cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

• Fuss B, Wintergerst ES, Bartsch U, Schachner M
• Molecular characterization and in situ mRNA localization of the neural recognition molecule J1-160/180: a modular structure similar to tenascin.
• J Cell Biol. 1993; 120: 1237-49
• Display abstract

• The oligodendrocyte-derived extracellular matrix glycoprotein J1-160/180 is a recognition molecule expressed exclusively in the central nervous system. J1-160/180 has been shown to be adhesive for astrocytes and repellent towards neurons and growth cones. We report here the complete nucleotide sequence of J1-160/180 in the rat. The predicted amino acid sequence showed a structural architecture very similar to tenascin: a cysteine-rich amino terminal region is followed by 4.5 epidermal growth factor-like repeats, 9 fibronectin type III homologous repeats and a domain homologous to fibrinogen. Sequence comparison analysis revealed highest homology of rat J1-160/180 to mouse tenascin and chicken restrictin with a similarity of 66% and 85%, respectively. The J1-160/180-coding mRNA is derived from a single copy gene. Using the polymerase chain reaction we could show that two J1-160/180 isoforms are generated by alternative splicing of the sixth fibronectin type III homologous repeat. Localization of J1-160/180 mRNA by in situ hybridization in the cerebellum, hippocampus and olfactory bulb confirmed the expression of J1-160/180 by oligodendrocytes with a peak of transcription at 7-14 d after birth, indicating a functional role during myelination. In addition, J1-160/180-specific RNA was found in a small subset of neurons in all three structures of the CNS analyzed. These neurons continue to express J1-160/180 in the adult.

• Ingham KC, Brew SA, Migliorini MM, Busby TF
• Binding of heparin by type III domains and peptides from the carboxy terminal hep-2 region of fibronectin.
• Biochemistry. 1993; 32: 12548-53
• Display abstract

• The major sites of heparin binding by fibronectin are located in fragments of 30 or 40 kDa that contain type III modules 12 through 14 or 15. Various proteolytic or recombinant subfragments and several synthetic peptides derived from this region have been compared with respect to their binding to fluorescein-labeled heparin in solution. Binding was monitored by the change in fluorescence anisotropy at 25 degrees C and pH 7.4 in 0.02 M Tris buffer, alone (TB) or with 0.15M NaCl (TBS). A 23-kDa fragment containing III13 and III14 but lacking III12 had Kd values of 0.3 and 1.8 microM in TB, and TBS, respectively, indistinguishable from the 30-kDa parent. Fragments containing only module III13 bound 2-3-fold weaker than the parent while those containing only III14 bound 6-50-fold weaker depending on the ionic strength. Fragments containing only III12 or III15 failed to bind at all in TBS. A cationic peptide derived from the amino terminus of III13 and containing the Arg-Arg-Ala-Arg consensus sequence, whose integrity was shown by Barkalow and Schwarzbauer [Barkalow, F. J., & Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818] to be critical, failed to bind in TBS but bound weakly in TB. Two additional cationic peptides derived from the middle and C-terminal regions of III14 showed similar behavior. Thus while the major determinant(s) of heparin binding are located in III13, those determinants are only active when part of a properly folded structure. Furthermore, module III13 when isolated had a slightly lower affinity than fragments containing both III13 and III14.(ABSTRACT TRUNCATED AT 250 WORDS)

• Prieto AL, Andersson-Fisone C, Crossin KL
• Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin.
• J Cell Biol. 1992; 119: 663-78
• Display abstract

• The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGF-like repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.

• Schaefer EM, Erickson HP, Federwisch M, Wollmer A, Ellis L
• Structural organization of the human insulin receptor ectodomain.
• J Biol Chem. 1992; 267: 23393-402
• Display abstract

• To provide an experimental system amenable to a detailed biochemical and structural investigation of the extracellular (ligand binding) domain of the insulin receptor, we developed a mammalian heterologous cell expression system from which tens of milligrams of the soluble secreted ectodomain (the IR921 protein) can be routinely purified using methods that do not require harsh elution conditions. The purified IR921 protein has a Stokes radius of 6.8 nm and a sedimentation coefficient of 9.8 S, from which we calculate a hydro-dynamic mass of 281 kDa. Electron microscopic images, using both rotary shadowing and negative staining techniques, demonstrate a characteristic substructure for the IR921 protein consisting of two elongated arms, with a globular domain at each end, connected to each other at a point somewhat off-center to form a Y structure. Analysis using circular dichroism and fluorescence spectroscopy illustrate that insulin binding results in conformational changes in the ectodomain. Furthermore, fluorescence anisotropy decay data reveal segmental mobility within the IR921 protein that is successively frozen as a result of insulin binding, in contrast to results obtained in a previous study of the epidermal growth factor receptor ectodomain. This result suggests a divergence in hormone-induced signaling mechanisms used by the insulin and epidermal growth factor receptors.

• Norenberg U, Wille H, Wolff JM, Frank R, Rathjen FG
• The chicken neural extracellular matrix molecule restrictin: similarity with EGF-, fibronectin type III-, and fibrinogen-like motifs.
• Neuron. 1992; 8: 849-63
• Display abstract

• Restrictin is a chick neural extracellular matrix protein implicated in neural cell attachment and found to be associated with the cell surface recognition protein F11. Here we show by cDNA cloning that restrictin is a large multidomain protein composed of 4 structural motifs. At the N-terminus restrictin contains a cysteine-rich segment of about 140 aa that might link restrictin monomers into oligomers. This region is followed by 4.5 epidermal growth factor-like repeats and then by 9 consecutive motifs that are similar to fibronectin type III motifs. At the C-terminus restriction is related to the beta and gamma chains of fibrinogen, including similarity to a calcium-binding segment. Restrictin shows substantial sequence similarity with tenascin (cytotactin) throughout the polypeptide, and like tenascin, it forms oligomeric structures, as revealed by electron microscopy of immunoaffinity-purified restriction. The cell attachment site of restrictin is mapped to the C-terminal region by antibody perturbation experiments.

• Darribere T et al.
• Distinct regions of human fibronectin are essential for fibril assembly in an in vivo developing system.
• Dev Dyn. 1992; 194: 63-70
• Display abstract

• In early vertebrate development, the proper assembly of fibronectin into fibrils is crucial for embryonic cells to adhere and to migrate on the extracellular matrix. The molecular mechanisms by which such a process occurs in vivo are poorly understood. In the amphibian embryo Pleurodeles waltl fibronectin fibrils appear first at the blastula stage. They form a fibrillar matrix on the basal surface of animal cells facing the blastocoel. Using competition and perturbation experiments with purified proteolytic fragments and domain-specific monoclonal antibodies, we demonstrate that at least three fibronectin sites are essential for assembly of fibronectin fibrils in the blastula of Pleurodeles waltl. Two sites, the RGDS sequence and the synergistic domain in the 10th type III repeat, are both involved in receptor recognition. A third site that spans the 9th type I and 1st type III homology sequences is also likely to participate in fibronectin-fibronectin interactions.

• Hendrickson WA
• Structural biology of cell surface interactions.
• Harvey Lect. 1992; 88: 49-65

• Jones EY, Davis SJ, Williams AF, Harlos K, Stuart DI
• Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2.
• Nature. 1992; 360: 232-9
• Display abstract

• The crystal structure of a soluble form of the T lymphocyte antigen CD2 provides the first complete view of the extracellular region of a cell adhesion molecule. The topology of the molecule, which comprises two immunoglobulin-like domains, is the same as that of the first two domains of CD4 but the relative domain orientation is altered by a fairly flexible linker region. The putative ligand-binding beta-sheet forms a flat surface towards the top of the molecule. Crystal contacts between these surfaces suggest a plausible model for the adhesive interaction.

• Matsumoto K, Arai M, Ishihara N, Ando A, Inoko H, Ikemura T
• Cluster of fibronectin type III repeats found in the human major histocompatibility complex class III region shows the highest homology with the repeats in an extracellular matrix protein, tenascin.
• Genomics. 1992; 12: 485-91
• Display abstract

• Walking and sequencing a genome portion centromeric of CYP21B in the human MHC class III region disclosed a cluster of fibronectin type III repeats in an approximately 50-kb DNA segment. Fibronectin type III repeats are known to consist of ca. 90 amino acid residues and exist in a wide range of protein species. Homology searches in protein databases showed that the repeats found had the highest homology with the repeats of human tenascin, an extracellular matrix protein. One cDNA sequence located immediately centromeric of CYP21B, the 3' portion of which is transcribed by the opposite strand of CYP21B, was found also to have six type III repeats followed by a fibrinogen domain. Pairwise homology comparison of these repeats in the MHC locus with those of human tenascin showed a general parallelism in their gene organization, indicating that the newly found repeats are elements of certain tenascin-like genea.

• Weller A, Beck S, Ekblom P
• Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis.
• J Cell Biol. 1991; 112: 355-62
• Display abstract

• We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).

• Jaikaria NS, Rosenfeld L, Khan MY, Danishefsky I, Newman SA
• Interaction of fibronectin with heparin in model extracellular matrices: role of arginine residues and sulfate groups.
• Biochemistry. 1991; 30: 1538-44
• Display abstract

• The interaction of heparin with the NH2-terminal domain of human plasma fibronectin was studied by using matrix-driven translocation, an assay for the adhesion of extracellular macromolecules with cell or particle surfaces within artificial collagen matrices. Partial desulfation of heparin rendered it ineffective in competitively inhibiting the interaction of the fibronectin NH2-terminal domain with heparin-coated particles, suggesting a role for sulfate groups of heparin in the interaction. Analysis of the fibronectin domain in terms of its primary structure, its proposed organization into "type I modules", and its hydrophilic and flexible segments led to the identification of several arginine-containing sites of potential interaction with the sulfate groups of heparin. Modification of increasing numbers of arginine side chains with 1,2-cyclohexanedione under mild conditions eventually led to decreases in translocation-promoting activity, and of heparin binding capacity as measured in a gel-shift assay, but the major portions of these functions were retained even when the four most accessible arginines (attributed to sites in and adjacent to the large loops of the type I modules) were modified. With the modification of additional arginines (attributed to sites in the small loops), both functions were lost. The peptide Gly-Arg-Gly, corresponding to a repeated determinant at the tips of two small loops, inhibited translocation, but arginine alone did not. Cleavage of the large loops by CNBr also led to loss of translocation-promoting activity. The correspondence between the molecular determinants of matrix-driven translocation and those previously found for mesenchymal morphogenesis indicates the utility of this system in the analysis of adhesive interactions of biological importance.

• Litvinovich SV, Strickland DK, Medved LV, Ingham KC
• Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded.
• J Mol Biol. 1991; 217: 563-75
• Display abstract

• The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.

• Schwarzbauer JE
• Identification of the fibronectin sequences required for assembly of a fibrillar matrix.
• J Cell Biol. 1991; 113: 1463-73
• Display abstract

• During extracellular matrix assembly, fibronectin (FN) binds to cell surface receptors and initiates fibrillogenesis. As described in this report, matrix assembly has been dissected using recombinant FN polypeptides (recFNs) expressed in mammalian cells via retroviral vectors. RecFNs were assayed for incorporation into the detergent-insoluble cell matrix fraction and for formation of fibrils at the cell surface as detected by indirect immunofluorescence. Biochemical and immunocytochemical data are presented defining the minimum domain requirements for FN fibrillogenesis. The smallest functional recFN is half the size of native FN and contains intact amino- and carboxy-terminal regions with a large internal deletion spanning the collagen binding domain and the first seven type III repeats. Five type I repeats at the amino terminus are required for assembly and have FN binding activity. The dimer structure mediated by the carboxy-terminal interchain disulfide bonds is also essential. Surprisingly, recFNs lacking the RGDS cell binding site formed a significant fibrillar matrix. Therefore, FN-FN interactions and dimeric structure appear to be the major determinants of fibrillogenesis.

• Burgoon MP, Grumet M, Mauro V, Edelman GM, Cunningham BA
• Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily.
• J Cell Biol. 1991; 112: 1017-29
• Display abstract

• The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.

• Khan MY
• Structure and properties of fibronectin.
• Indian J Biochem Biophys. 1990; 27: 63-8

• Chiquet-Ehrismann R
• What distinguishes tenascin from fibronectin?
• FASEB J. 1990; 4: 2598-604
• Display abstract

• Tenascin and fibronectin are two major extracellular matrix glycoproteins. They both consist of large disulfide-linked subunits composed of multiple structural domains. More than half of each molecule consists of so-called fibronectin type III repeats, but the other domains differ. Fibronectin is a dimer, whereas tenascin is a hexamer. Often fibronectin and tenascin are colocalized in tissues, but the occurrence of tenascin is much more restricted when compared with fibronectin. Tenascin is transiently expressed in many developing organs such as connective tissues, the mesenchyme of epithelial organs, and also the central and peripheral nervous systems, and it reappears in the stroma of many tumors. The distinctive and highly regulated expression of tenascin has provoked interest in trying to identify possible functions of tenascin in cell-cell and cell-substratum adhesion, cell migration, growth, and cell differentiation during morphogenesis.

• Alon R, Bayer EA, Wilchek M
• Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin.
• Biochem Biophys Res Commun. 1990; 170: 1236-41
• Display abstract

• Streptavidin binds at low levels and high affinity to cell surfaces, the cause of which can be traced to the occurrence of a sequence containing RYD (Arg-Tyr-Asp) in the protein molecule. This binding is enhanced in the presence of biotin. Cell-bound streptavidin can be displaced by fibronectin, as well as by RGD- and RYD-containing peptides. In addition, streptavidin can displace fibronectin from cell surfaces. The RYD sequence of streptavidin thus mimics RGD (Arg-Gly-Asp), the universal recognition domain present in fibronectin and other adhesion-related molecules. The observed adhesion to cells has no relevance to biotin-binding since the RYD sequence is not part of the biotin-binding site of streptavidin. Since the use of streptavidin in avidin-biotin technology is based on its biotin-binding properties, researchers are hereby warned against its indiscriminate use in histochemical and cytochemical studies.

• Vuillard L, Roux B, Miller A
• The measurement of the rotational diffusion coefficient of bovine plasma fibronectin by electric birefringence technique.
• Eur J Biochem. 1990; 191: 333-6
• Display abstract

• Although the conformation of fibronectin has been widely investigated by various techniques, there has not yet been any determination of its rotational diffusion coefficient. We report here this determination by the transient electric birefringence study of solutions of bovine plasma fibronectin at physiological ionic strength. The solutions showed a positive birefringence. A linear relationship was observed between the intensity of the birefringence at equilibrium and the square of the electric field within the range of fields applied (up to 12.5 kV.cm-1). The field-independent decay of the induced birefringence was described by a single exponential with a relaxation time of 0.76 (+/- 0.08) microsecond at 23 degrees C. This establishes fibronectin in solution as a globally rigid structure with a rotational diffusion coefficient, at 20 degrees C, of 202,000 s-1. This result allows the first rigorous determination of the low-resolution structure of fibronectin. It is important to notice that the analysis combines only results obtained in physiological conditions on native molecules and follows a strict hydrodynamic interpretation. The conclusion of this work is that a hollow sphere of about 20 nm external diameter can be proposed as a model for the three-dimensional structure of the fibronectin molecule in solution. This new model suggests the fibronectin could have the structure of a carrier protein.

• Humphries MJ, Obara M, Olden K, Yamada KM
• Role of fibronectin in adhesion, migration, and metastasis.
• Cancer Invest. 1989; 7: 373-93

• Obara M
• [Molecular mechanisms for cell adhesion mediated by fibronectin molecules]
• Seikagaku. 1989; 61: 1462-6

• Ruoslahti E
• Versatile mechanisms of cell adhesion.
• Harvey Lect. 1988; 84: 1-17

• Rocco M, Infusini E, Daga MG, Gogioso L, Cuniberti C
• Models of fibronectin.
• EMBO J. 1987; 6: 2343-9
• Display abstract

• The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/- 0.8 nm and 10.7 +/- 0.9 nm respectively, for a constant mol. wt of 533,000 +/- 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end-to-end joined chains of 28 beads, was then constructed, and their calculated physico-chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino-terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy-terminal fibrin(ogen)-binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.

• Owens RJ, Baralle FE
• Exon structure of the collagen-binding domain of human fibronectin.
• FEBS Lett. 1986; 204: 318-22
• Display abstract

• Fibronectin consists of a series of three internal homology repeats (types I, II, and III [(1983) Proc. Natl. Acad. Sci. USA 80, 137-141]). The type III units are each coded for by two exons with the exception of the alternatively spliced extra domain type III [(1984) EMBO J. 3, 2511-2516]. To investigate the gene organisation of the type I and II repeats, genomic clones covering the collagen-binding domain of human fibronectin have been isolated and characterised. The results show that each of the type I and II homology units in this region corresponds to an exon. Taken together with the previous data concerning the type III units, the data lend support to a gene fusion model of fibronectin evolution.

• Pierschbacher MD, Hayman EG, Ruoslahti E
• The cell attachment determinant in fibronectin.
• J Cell Biochem. 1985; 28: 115-26
• Display abstract

• Fibronectin possesses a domain that interacts with cell surfaces. The ability of fibronectin to promote cell attachment can be duplicated with a short amino acid sequence, glycyl-L-arginyl-glycyl-L-aspartyl-L-serine, taken from that domain. The tripeptide Arg-Gly-Asp appears to be irreplaceable for maintenance of the activity of this peptide, whereas the serine residue can be replaced with some, but apparently not all, possible residues. This recognition sequence, or a closely related sequence, is present in a number of proteins other than fibronectin that interact with cells. These proteins include collagens, fibrinogen, thrombin, a bacterial surface protein, and two viral proteins, as well as discoidin-I, a protein implicated in the aggregation of Dictyostelium discoideum. A similar sequence is also repeated in some, but not all, fibronectin molecules, making it possible that some fibronectin molecules have more than a single cell attachment site. Synthetic peptides constructed from sequences taken from several of these other proteins have also been shown to promote cell attachment. The tripeptide sequence may, therefore, constitute an ancient cellular recognition mechanism common to many proteins.

• Yamada KM et al.
• Fibronectin and interactions at the cell surface.
• Prog Clin Biol Res. 1984; 151: 1-15

• Hynes RO, Schwarzbauer JE, Tamkun JW
• Fibronectin: a versatile gene for a versatile protein.
• Ciba Found Symp. 1984; 108: 75-92
• Display abstract

• We have isolated cDNA and genomic clones for rat fibronectins. A single gene gives rise to three different mRNA species by alternative splicing at a complex intron-exon boundary within the coding region. The fibronectins encoded by these three mRNAs differ by the insertion of different protein segments in the C-terminal heparin-binding domain. The amino acid sequences of the cell-, heparin- and fibrin-binding domains in the C-terminal third of fibronectin were deduced from the DNA sequences. The cell- and heparin-binding regions each consist of several similar repeating sequences known as type III homologies, while the fibrin-binding region comprises three repeats of a different type (type I homologies). The sequences suggest several hypotheses for the structure-function relationships of these domains of fibronectin; these hypotheses are now being tested. Determination of intron-exon boundaries within the fibronectin gene is beginning to reveal the modular structure of the gene and its relation to the repeating structure of fibronectin.

• Skorstengaard K, Thogersen HC, Petersen TE
• Complete primary structure of the collagen-binding domain of bovine fibronectin.
• Eur J Biochem. 1984; 140: 235-43
• Display abstract

• The complete amino acid sequence of the collagen-binding domain of bovine plasma fibronectin has been determined. The fragment, generated by digestion of fibronectin with plasmin and chymotrypsin, contains 340 residues (260-599 of fibronectin) with threonine and tryptophan as the amino-terminal and carboxyl-terminal amino acids, respectively. 24 half-cystines and no cysteines are present in the sequence. Three glucosamine-based oligosaccharide groups are attached to Asn-399, Asn-497 and to Asn-511, respectively. Two of the three types (I and II) [Petersen et al. (1983) Proc. Natl Acad. Sci. USA 80, 137-141] of internal homology occur in the fragment, namely four of the at least twelve stretches of type I sequence homology, 'fingers', and two stretches of type II homology. The type I homology is present in two other plasmic fragments from fibronectin, while the type II homology has been found in the collagen-binding domain only.

• Sekiguchi K, Siri A, Zardi L, Hakomori S
• Differences in domain structure between pericellular matrix and plasma fibronectins as revealed by domain-specific antibodies combined with limited proteolysis and S-cyanylation: a preliminary note.
• Biochem Biophys Res Commun. 1983; 116: 534-40
• Display abstract

• Differences in domain structure between human fibronectins obtained from pericellular matrix and plasma have been revealed by limited proteolysis and S-cyanylation, followed by identification of each domain with domain-specific antibodies. Although the overall domain structure is similar between pericellular and plasma fibronectins, the fragments derived from the COOH-terminal region of these fibronectins, which were defined by specific antibodies, exhibited clear differences in their molecular weights and protease susceptibility, suggesting that the structure near the COOH-terminal region is significantly different between these two proteins.

• Vartio T
• Fibronectin: multiple interactions assigned to structural domains.
• Med Biol. 1983; 61: 283-95
• Display abstract

• Characteristic features of fibronectin are its various molecular and biological interactions and a wide distribution in vivo. Structural studies of the protein have indicated that the functions correspond to specific interactive domains in the molecule and have revealed unique, internally homologous sequences in the primary structure. Fibronectin is easily degraded by proteinases and many of its biological effects are shared by the proteolytic fragments which also seem to have effects of their own.

• Ehrismann R, Roth DE, Eppenberger HM, Turner DC
• Arrangement of attachment-promoting, self-association, and heparin-binding sites in horse serum fibronectin.
• J Biol Chem. 1982; 257: 7381-7

• Yamada KM, Akiyama SK, Hayashi M
• Fibronectin structure and function, and its interactions with glycosaminoglycans.
• Biochem Soc Trans. 1981; 9: 506-8

• Ruoslahti E, Engvall E, Hayman EG
• Fibronectin: current concepts of its structure and functions.
• Coll Relat Res. 1981; 1: 95-128

• Adelmann-Grill BC
• Independence of reciprocal attachment sites on denatured collagen and fibronectin from antigenic sites, and binding of gelatin-based plasma substitutes to plasma fibronectin.
• Coll Relat Res. 1981; 1: 36775-36775
• Display abstract

• Fibronectin and denatured collagen possess attachment sites for each other and, in addition, antigenic sites reacting with the respective antibodies. Using an enzyme linked immunosorbent assay it was shown that attachment sites and antigenic sites on fibronectin and on collagen reacted independently of each other. Gelatin-based plasma substitutes blocked the binding of fibronectin to denatured collagen thus showing that attachment sites for fibronectin are preserved in the gelatin preparations used for manufacturing these plasma substitutes. Antigenic sites, in contrast, were destroyed during the manufacturing process as shown with antibodies to denatured collagen raised in guinea-pigs. It is concluded that complex formation of gelatin-based plasma substitutes with plasma fibronectin may occur upon infusion of such plasma substitutes but that formation of antigen/antibody complexes in vivo is unlikely.

• Sekiguchi K, Hakomori S
• Functional domain structure of fibronectin.
• Proc Natl Acad Sci U S A. 1980; 77: 2661-5
• Display abstract

• Structural domains of fibronectin (FN) and their ability to associate with cell surface components have been systematically investigated. Plasma FN was cleaved into three structural domains (Mr 150,000-140,000, 40,000, and 32,000) by sequential digestion with trypsin and thermolysin. A single digestion with thermolysin alone generated Mr 150,000-140,000, 40,000, and smaller fragments. With the inclusion of thermolysin, but not with other proteases, one can, with a high yield dissect FN simultaneously into three clearly distinctive functional domains. Of three major fragments only the Mr 40,000 fragment bound to a gelatin column; this fragment contained essentially all of the carbohydrates present in the original FN. In contrast, heparin-binding sites were localized on both the Mr 150,000-140,000 and 32,000 fragments but not on the Mr 40,000 fragment. Only the Mr 150,000-140,000 fragments and intact FN promoted cell spreading, whereas the Mr 40,000 and 32,000 fragments could induce cell attachment but failed to promote cell spreading. These results indicate that FN is composed of (at least) three structural domains that are functionally distinct from each other.

• Wagner DD, Hynes RO
• Topological arrangement of the major structural features of fibronectin.
• J Biol Chem. 1980; 255: 4304-12

• Yamada KM, Hahn LH, Olden K
• Structure and function of the fibronectins.
• Prog Clin Biol Res. 1980; 41: 797-819

• Sekiguchi K, Hakomori S
• Identification of two fibrin-binding domains in plasma fibronectin and unequal distribution of these domains in two different subunits: a preliminary note.
• Biochem Biophys Res Commun. 1980; 97: 709-15

• Johansson S, Hook M
• Heparin enhances the rate of binding of fibronectin to collagen.
• Biochem J. 1980; 187: 521-4
• Display abstract

• 125I-labelled fibronectin is shown to bind to both native and denatured collagen immobilized on Sephadex beads in reactions that exhibit different kinetics. The rates of both reactions were enhanced by the presence of heparin or highly sulphated dextran sulphate but not by other glycosaminoglycans or dextran sulphates having low sulphate contents.

• Furie MB, Frey AB, Rifkin DB
• Location of a gelatin-binding region of human plasma fibronectin.
• J Biol Chem. 1980; 255: 4391-4

• Mosesson MW, Amrani DL
• The structure and biologic activities of plasma fibronectin.
• Blood. 1980; 56: 145-58

• Mosher DF, Schad PE, Vann JM
• Cross-linking of collagen and fibronectin by factor XIIIa. Localization of participating glutaminyl residues to a tryptic fragment of fibronectin.
• J Biol Chem. 1980; 255: 1181-8

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