SMART MODE:

NORMAL GENOMIC

• Muller KM, Arndt KM, Alber T
• Protein fusions to coiled-coil domains.
• Methods Enzymol. 2000; 328: 261-82

• Smith SP, Hashimoto Y, Pickford AR, Campbell ID, Werner JM
• Interface characterization of the type II module pair from fibronectin.
• Biochemistry. 2000; 39: 8374-81
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• The lone (1)F2(2)F2 modular pair of fibronectin is found in the collagen-binding region. This exclusive localization suggests the (1)F2(2)F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the (1)F2(2)F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the (1)F2(2)F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. (15)N NMR relaxation data identify significant motion occurring in the linker region of the (1)F2(2)F2 pair, and analyses of the anisotropic diffusion properties of the (1)F2(2)F2 pair are consistent with the modules in the F2 pair tumbling independent of one another.

• Hashimoto Y, Smith SP, Pickford AR, Bocquier AA, Campbell ID, Werner JM
• The relative orientation of the fibronectin 6F1(1)F2 module pair: a 15N NMR relaxation study.
• J Biomol NMR. 2000; 17: 203-14
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• The structure of a pair of modules (6F1(1)F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from 1H-1H NOE and 3J(HalphaHN) data [Bocquier et al. (1999) Structure, 7, 1451-1460] and a weak module-module interface, comprising Leu19 and Leu28, in 6F1, and Tyr68 in 2F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of 15N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and 3J(HalphaHN) data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where 15N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443-449].

• Schnepel J, Tschesche H
• The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E. coli expression.
• J Protein Chem. 2000; 19: 685-92
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• Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain. The enzyme could be generated and activated in the presence of Ca2+ from the purified 70-kDa pFN fragment produced by cathepsin D digestion. In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, alpha- and beta-casein, insulin b-chain, and a synthetic Mca-peptide. In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates. Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.

• Park JS, Shin HS, Doi RH
• Fusion proteins containing cellulose-binding domains.
• Methods Enzymol. 2000; 326: 418-29

• Clayton D, Brereton IM, Kroon PA, Smith R
• Three-dimensional NMR structure of the sixth ligand-binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities.
• FEBS Lett. 2000; 479: 118-22
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• The sixth ligand-binding module of the low-density lipoprotein receptor contributes to the binding of apolipoprotein B100-containing lipoproteins. 1H NMR spectroscopy, DYANA and X-PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E-containing lipoproteins in addition to apolipoprotein B100-containing lipoproteins.

• Briknarova K, Grishaev A, Banyai L, Tordai H, Patthy L, Llinas M
• The second type II module from human matrix metalloproteinase 2: structure, function and dynamics.
• Structure Fold Des. 1999; 7: 1235-45
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• BACKGROUND: Matrix metalloproteinase 2 (MMP-2, gelatinase A, 72 kDa type IV collagenase) has an important role in extracellular matrix degradation during cell migration and tissue remodeling. It is involved in development, inflammation, wound healing, tumor invasion, metastasis and other physiological and pathological processes. The enzyme cleaves several types of collagen, elastin, fibronectin and laminin. Binding to collagen is mediated by three repeats homologous to fibronectin type II modules, which are inserted in the catalytic domain in proximity to the active site. RESULTS: We have determined the NMR solution structure of the second type II module from human MMP-2 (col-2). The module exhibits a typical type II fold with two short double-stranded antiparallel beta sheets and three large loops packed around a cluster of conserved aromatic residues. Backbone amide dynamics, derived from (15)N relaxation experiments, correlate well with solvent accessibility and intramolecular hydrogen bonding. A synthetic peptide with the collagen consensus sequence, (Pro-Pro-Gly)(6), is shown to interact with the module. CONCLUSIONS: Spectral perturbations induced by (Pro-Pro-Gly)(6) binding reveal the region involved in the interaction of col-2 with collagen. The binding surface comprises exposed aromatic residues Phe21, Tyr38, Trp40, Tyr47, Tyr53 and Phe55, and the neighboring Gly33-Gly37 segment.

• Lockless SW, Ranganathan R
• Evolutionarily conserved pathways of energetic connectivity in protein families.
• Science. 1999; 286: 295-9
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• For mapping energetic interactions in proteins, a technique was developed that uses evolutionary data for a protein family to measure statistical interactions between amino acid positions. For the PDZ domain family, this analysis predicted a set of energetically coupled positions for a binding site residue that includes unexpected long-range interactions. Mutational studies confirm these predictions, demonstrating that the statistical energy function is a good indicator of thermodynamic coupling in proteins. Sets of interacting residues form connected pathways through the protein fold that may be the basis for efficient energy conduction within proteins.

• Tordai H, Patthy L
• The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2.
• Eur J Biochem. 1999; 259: 513-8
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• We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.

• Bloom L, Ingham KC, Hynes RO
• Fibronectin regulates assembly of actin filaments and focal contacts in cultured cells via the heparin-binding site in repeat III13.
• Mol Biol Cell. 1999; 10: 1521-36
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• Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central alpha5beta1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29-amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7-11 (which contains the integrin alpha5beta1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.

• Corchero JL, Villaverde A
• Tolerance of Escherichia coli beta-galactosidase C-terminus to different-sized fusions.
• Biotechnol Bioeng. 1999; 64: 644-9
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• The tolerance of the beta-galactosidase C-terminus to foreign protein fusions has been explored by using different-sized derivatives of the chimeric protein LACVP1. While the molecular mass of the partner domain shows a minor influence on protein toxicity for the producing E. coli cells, it dramatically affects the proteolytic susceptibility of the whole fusion. Surprisingly, the observed structural modulation of proteolysis is not an all-or-nothing process, but it exhibits a continuous effect concomitantly with the length of the fusion. The conformational effects caused by increasingly sized partners seem to progressively expose cryptic protease target sites, initiating a proteolytic cascade that dramatically reduces the yield of the recombinant protein.

• Calvete JJ, Campanero-Rhodes MA, Raida M, Sanz L
• Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109.
• FEBS Lett. 1999; 444: 260-4
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• PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around beta-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer.

• Bultmann H, Santas AJ, Peters DM
• Fibronectin fibrillogenesis involves the heparin II binding domain of fibronectin.
• J Biol Chem. 1998; 273: 2601-9
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• Fibronectin matrix assembly is thought to involve binding interactions between the amino-terminal I1-5 repeats and the first type III repeat (III1). Here we report that a third site, located within the III12-14 repeats of the carboxyl-terminal heparin II domain of fibronectin, is also involved in fibrillogenesis. Heparin II fragments inhibited fibril formation and binding of 125I-labeled fibronectin and/or 70-kDa fragments to the cell surface, deoxycholate-insoluble matrix, and adsorbed 160-kDa cell adhesion fragments of fibronectin. The inhibitory effects of heparin II fragments were as large or up to 20 times larger than those of a 44-kDa fibronectin fragment containing the III1 repeat. Under physiological conditions, amino-terminal fragments of fibronectin containing the I1-5 repeats interacted preferentially with proteolytically derived heparin II fragments and a recombinant III12-14 peptide both in solution and in solid phase, indicating that matrix assembly may involve direct interactions between I1-5 and III12-14 repeats. Interactions between the I1-5 repeats and 160-kDa fragments containing the III12-14 and III1 repeats could be inhibited by >/= 90% by either an anti-III13-14 monoclonal antibody (mAb) (IST-2) or an anti-III1 mAb (9D2), suggesting that cooperative interactions between III12-14 and III1 repeats may also promote binding of the I1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in combination, inhibited binding of 125I-labeled 70-kDa fragments to cycloheximide-treated cells plated on the 160-kDa substrate, suggesting that additional I1-5 binding sites, independent of the III1 and III12-14 repeats, may be involved in fibrillogenesis.

• Yan S, Rodrigues RG, Roberts DD
• Hemoglobin-induced binding of Candida albicans to the cell-binding domain of fibronectin is independent of the Arg-Gly-Asp sequence.
• Infect Immun. 1998; 66: 1904-9
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• Hemoglobin specifically induces fibronectin (FN) binding to the pathogenic yeast Candida albicans. When grown in the complex medium Sabouraud broth, C. albicans expresses receptors that bind to several domains of FN. Growth in defined medium supplemented with 0.1% hemoglobin, however, enhanced the binding of FN to a single class of receptors, with a Kd = 4.6 x 10(-8) M. Competitive binding assays using recombinant and proteolytic fragments of FN revealed that the cell-binding domain mediated this interaction. A recombinant 40-kDa fragment of FN consisting of type III repeats 9 to 13 had an inhibitory activity similar to that of the entire 120-kDa cell-binding domain, indicating that the C-terminal portion of the cell-binding domain contains the binding site. A recombinant 33-kDa fragment of the cell-binding domain and a 33-kDa fragment with the RGD sequence deleted had the same inhibitory activities, demonstrating that the RGD sequence recognized by some mammalian integrins is not required. The addition of hemoglobin to the culture medium also enhanced Candida cell adhesion to immobilized FN and to 120- and 40-kDa fragments of FN but not to the collagen-binding or fibrin I domains. Using ligand protection, we identified a surface protein from C. albicans with an apparent molecular mass of 55 kDa that was protected by both FN and the 40-kDa fragment derived from the cell-binding domain. Therefore, hemoglobin both induces FN binding and changes the relative affinities of C. albicans for the cell- and collagen-binding domains of FN.

• Sampoli Benitez BA, Hunter MJ, Meininger DP, Komives EA
• Structure of the fifth EGF-like domain of thrombomodulin: An EGF-like domain with a novel disulfide-bonding pattern.
• J Mol Biol. 1997; 273: 913-26
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• The structure of the fifth EGF-like domain (residues Q387 to E426) of thrombomodulin (TMEGF5) has been determined by two-dimensional NMR. TMEGF5 binds to thrombin with a Ki of 1.9 &mgr;M and has been shown to have a novel disulfide bonding pattern in a fully active fragment of TM. In EGF, the disulfide bonding pattern is (1-3,2-4, 5-6), while TMEGF5 has an uncrossed (1-2,3-4,5-6) pattern. The structure of this novel domain, determined from 483 NOE-derived distance restraints, appears to have diverged from the common EGF-like structure. Superposition of the 14 lowest-energy structures of TMEGF5 gives an overall r.m.s.d. of 1.09 A for the backbone atoms. The central two-stranded beta-sheet common to all EGF-like domains is not present in TMEGF5. The A loop, residues C390 to C395, is twisted away from interacting with the B loop, residues C399 to C407, as in EGF, and is close to the C loop, residues C409 to C421. This twist causes the N and C termini to be closer together in TMEGF5 than in EGF. Most of the residues that are important for activity lie on one face of the molecule, which is likely to be the thrombin-binding surface of the domain. The structure of the C loop within the domain, which is a beta-hairpin similar to EGF, is similar to the structure of a synthetic version of the loop bound to thrombin as determined by transferred NOE experiments. Despite the similarity in the structures of the loops, the residues immediately following C421 are in different positions in the two structures suggesting that these "tail" residues may change conformation upon thrombin binding.Copyright 1997 Academic Press Limited

• Ehnis T, Dieterich W, Bauer M, Kresse H, Schuppan D
• Localization of a binding site for the proteoglycan decorin on collagen XIV (undulin).
• J Biol Chem. 1997; 272: 20414-9
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• Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth. In this study we investigated the known binding of decorin to human collagen XIV. This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase. Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase. Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV. In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.

• Shimizu M, Minakuchi K, Moon M, Koga J
• Difference in interaction of fibronectin with type I collagen and type IV collagen.
• Biochim Biophys Acta. 1997; 1339: 53-61
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• In our studies on fibronectin, difference in binding to type I collagen and type IV collagen was observed and analysed. Four different fragments, which consist of I6-II1-II2-I7-I8-I9, I6-II1-II2-I7, I6-II1-II2, and I8-I9 within the collagen binding domain, have been isolated from proteolytic digests of fibronectin. The N-terminal fragments of the collagen binding domain showed the binding affinity to both of type I and type IV collagens. On the other hand, the C-terminal portion of the domain, I8-I9, only bound to type I collagen. The newly developed monoclonal antibody FN 40, which recognizes type I9 homology repeat of the collagen binding domain, inhibited the binding of fibronectin to type I collagen in a dose-dependent manner. Three overlapping fragments, I6-II1-II2-I7, I6-II1-II2-I7-I8, and I7-I8-I9, which have been expressed in Escherichia coli, showed the similar binding affinity to type IV collagen, whereas the fragment containing type I9 repeat (I7-I8-I9) showed the significantly stronger binding activity to type I collagen. In addition, the expressed fragment lacking I9 competitively inhibited the binding of fibronectin to type IV collagen. In contrast, the interference of this fragment to type I collagen binding activity of fibronectin was not significant. These data indicate that the collagen binding domain contains at least 2 sites for interaction with each type of collagen and that type I9 repeat is important for type I collagen binding, whereas I7 is for type IV collagen binding. Chemical modification studies revealed that the three-dimensional structure of fibronectin is more important for type I collagen binding.

• Sorensen MD et al.
• Solution structure and backbone dynamics of the human alpha3-chain type VI collagen C-terminal Kunitz domain,.
• Biochemistry. 1997; 36: 10439-50
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• The solution structure and backbone dynamics of the 58-residue C-terminal Kunitz domain fragment [alpha3(VI)] of human alpha3-chain type VI collagen has been studied by two-dimensional 1H-1H and 1H-15N nuclear magnetic resonance spectroscopy at 303 K. The solution structure is represented by an ensemble of 20 structures calculated with X-PLOR using 612 distance and 47 dihedral angle restraints. The distance restraints were obtained by a complete relaxation matrix analysis using MARDIGRAS. The root mean squared (rms) deviation is 0.91 A for the backbone atoms of the residues Thr2(8)-Gly12(18), Arg15(21)-Tyr35(41), and Gly40(46)-Pro57(63). The central beta-sheet [residues Ile18(24)-Tyr35(41)] and the C-terminal alpha-helix [residues Gln48(54)-Cys55(61)] are better defined with a backbone rms deviation of 0.46 A. The solution structure of alpha3(VI) is virtually identical to the crystal structure of alpha3(VI) and to the solution structure of bovine pancreatic trypsin inhibitor (BPTI). The 15N spin-lattice and spin-spin relaxation rates and the 1H-15N heteronuclear nuclear Overhauser enhancement (NOE) were analyzed using both the "model-free" formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping procedure [Farrow, N. A., Szabo, A., Torchia, D. A., & Kay, L. E. (1995) J. Biomol.NMR 6, 153-162]. The results obtained from the "model-free" analysis include an overall correlation time tauc of 3. 00 ns and backbone order parameters S2 in the range from 0.28 to 0. 93. The necessity of including an exchange term in the analysis of the relaxation data from 14 residues indicated that these residues are involved in motions on the micro- to millisecond time scale. The majority of the 14 residues are located in the vicinity of the Cys14(20)-Cys38(44) disulfide bond, suggesting the presence of a disulfide bond isomerization similar to the one observed in BPTI [Otting, G., Liepinsh, E., & Wuthrich, K. (1993) Biochemistry 32, 3571-3582]. It is suggested that this disulfide bond isomerization is the main reason for the surprisingly small effect on trypsin inhibition observed when Thr13(19) of alpha3(VI) is substituted with Pro.

• Spitzfaden C, Grant RP, Mardon HJ, Campbell ID
• Module-module interactions in the cell binding region of fibronectin: stability, flexibility and specificity.
• J Mol Biol. 1997; 265: 565-79
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• The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-dependent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein-protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

• Fukai F et al.
• Identification of the anti-adhesive site buried within the heparin-binding domain of fibronectin.
• J Biochem (Tokyo). 1997; 121: 189-92
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• We recently found that heparin-binding domain 2 (Hep 2) of fibronectin (FN) exhibits cryptic anti-adhesive activity. In order to locate the anti-adhesive site, a number of synthetic peptides which represents the primary structure of the Hep 2 domain were characterized as to their ability to decrease the adhesion of A375SM melanoma cells to FN substrate. Only one peptide (T-E-A-T-I-T-G-L-E-P-G-T-E-Y-T-I-Y-V-I-A-L, residues 1835-1855) (peptide III14-2), which is situated between the previously identified adhesive sites, FN-C/H-I and II, decreased the cell adhesion to FN. Assaying of the anti-adhesive activities of sub-peptides showed that the hydrophobic moiety of peptide III14-2 (underlined sequence) seems to be indispensable for the anti-adhesive activity. These results suggest that anti-adhesive activity is closely associated with the sequence, Y-T-I-V-I-A-L, that is usually buried within the Hep 2 domain structure because of its hydrophobic nature.

• Connolly PJ, Stern AS, Hoch JC
• Solution structure of LSIII, a long neurotoxin from the venom of Laticauda semifasciata.
• Biochemistry. 1996; 35: 418-26
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• We report the sequence-specific proton assignments and solution structure of the long neurotoxin LSIII from the venom of Laticauda semifasciata determined by two- and three-dimensional 1H NMR. Input for structure calculations consisted of 497 NOE-derived distance restraints and 45 dihedral angle restraints obtained from J couplings. A two-particle-per-residue representation of protein structure was used to generate 200 initial structures which were then subjected to all-atom refinement by simulated annealing. Twenty-three final structures consistent with the experimental restraints were obtained; the average atomic RMS difference between the individual structures and the mean structure was 0.82 A for the backbone heavy atoms and 1.3 A for all heavy atoms (residues 1-26, 37-60). The main elements of regular secondary structure are a three-stranded antiparallel beta-sheet and three finger-like loops protruding from a globular core, consistent with previously reported structures of long neurotoxins. The end of the prominent loop II, which is involved in binding to acetylcholine receptor, is disordered relative to the rest of the molecule. A novel finding of this study is that the loop has a well defined local structure; this and other observations suggest this region moves as a rigid body. We propose that this motion is a heretofore unrecognized general feature of long neurotoxins, with specific consequences for binding to the acetylcholine receptor.

• Sonnichsen FD, DeLuca CI, Davies PL, Sykes BD
• Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.
• Structure. 1996; 4: 1325-37
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• BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (?10&1macr;0?) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

• Hocking DC, Smith RK, McKeown-Longo PJ
• A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.
• J Cell Biol. 1996; 133: 431-44
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• Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

• Martins JC et al.
• H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus.
• J Mol Biol. 1996; 258: 322-33
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• The conformation in water of antimicrobial protein 2 from Amaranthus caudatus (Ac-AMP2) was determined using 1H NMR, DIANA and restrained molecular modeling. Ac-AMP2 is a 30 amino acid residue, lectin-like protein that specifically binds to chitin, a polymer of beta-1,4-N-acetyl-D-glucosamine. After sequence specific resonance assignments, a total of 198 distance restraints were collected from 2D NOESY buildup spectra at 500 MHz at pH 2, supplemented by a 2D NOESY spectrum at 600 MHz. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of Ac-AMP2 is presented as a set of 26 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol in the AMBER force field, with a backbone r.m.s.d. for the well defined Glu3-Cys28 segment of 0.69(+/-0.12) angstroms. The main structural element is an antiparallel beta-sheet from Met13 to Lys23 including a betaI-turn over Gln17-Phel8 with a beta bulge at Gly19. In addition, a beta'I turn over Arg6-Gly7, a beta'III turn over Ser11-Gly12 and a helical turn from Gly24 to Cys28 are identified. This structure is very similar to the equivalent regions of the X-ray structure of wheat germ agglutinin and the NMR structure of hevein.

• Gorlach J, Schmid J
• Introducing StuI sites improves vectors for the expression of fusion proteins with factor Xa cleavage sites.
• Gene. 1996; 170: 145-6
• Display abstract

• The Arg-encoding triplet (AGG) in the recognition sequence Ile-Glu-Gly-Arg for factor Xa can be used to generate a StuI restriction site (AGGCCT) which greatly facilitates the construction of DNA fragments encoding fusion proteins. Following proteolytic cleavage with factor Xa, a protein with the desired N terminus can be obtained.

• Iankovskii OI et al.
• [Complex-formation between myeloperoxidase and fibronectin and its modulation by fibronectin ligands]
• Vopr Med Khim. 1996; 42: 137-40
• Display abstract

• Human blood plasma fibronectin immobilised on agarose in physiological interacts with soluble myeloperoxidase (Kd = 2.43 mM). Interaction of myeloperoxidase with fibronectin adsorbed on immobilised gelatin a, natural fibronectin ligand, resulted in formation more stable complex (Kd = 0.94 mM). The presence of thermoaggregated (but not native) IgG in the liquid phase increased a stability of the complex (0.06 mM). It is suggested that myeloperoxidase could represent a component of complex super molecular structure, real immune complex.

• Sorimachi K, Jacks AJ, Le Gal-Coeffet MF, Williamson G, Archer DB, Williamson MP
• Solution structure of the granular starch binding domain of glucoamylase from Aspergillus niger by nuclear magnetic resonance spectroscopy.
• J Mol Biol. 1996; 259: 970-87
• Display abstract

• The solution structure of the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger has been determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy and simulated annealing. A total of 1092 nuclear Overhauser enhancement-derived 1H-1H distance constraints, 137 dihedral constraints and 86 hydrogen bond constraints were incorporated into an X-PLOR simulated annealing and refinement protocol. The family of calculated structures shows a well defined beta-sheet structure consisting of one parallel and six antiparallel pairs of beta-strands which forms an open-sided beta-barrel. The root-mean-square deviation (rmsd) of 53 individual structures to the calculated average structure for the backbone atoms of residues excluding the N terminus and two mobile loops is 0.57(+/-0.10) A while the rmsd for backbone atoms in beta-strands is 0.45(+/-0.08) A. Structural features of the SBD in solution are compared to the X-ray crystal structure of a homologous domain of cyclodextrin glycosyltransferase (CGTase) in the free and bound forms. Titration studies with two ligands, maltoheptaose and beta-cyclodextrin, show the existence of two binding sites. Examination of the tertiary structures shows these two sites to be at one end of the molecule on opposite faces. The majority of residues showing the largest 1H and 15N chemical shift changes are located in loop regions. Many residues implicated in binding, based on these changes, are similar in location to previously identified binding site residues in the crystal structures of CGTase. Overall, the shift changes are small indicating that the SBD does not undergo large conformational changes upon ligand binding.

• Xu GY et al.
• Solution structure of a cellulose-binding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy.
• Biochemistry. 1995; 34: 6993-7009
• Display abstract

• Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.

• Akiyama SK, Aota S, Yamada KM
• Function and receptor specificity of a minimal 20 kilodalton cell adhesive fragment of fibronectin.
• Cell Adhes Commun. 1995; 3: 13-25
• Display abstract

• Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti-fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin.

• Penc SF, Blumenstock FA, Kaplan JE
• A role for a 70-kDa amino-terminal fibronectin fragment in supporting soluble gelatin interactions with rat peritoneal macrophages.
• J Leukoc Biol. 1995; 58: 501-9
• Display abstract

• Seventy-kilodalton amino-terminal and 180-kDa cell-binding fibronectin fragments were used to determine which fibronectin domains support soluble gelatin interactions with macrophages. At each time measured, intact and 180-kDa fibronectin supported significantly larger quantities of cell-associated gelatin than control levels (P < 0.05). Throughout the time course fibronectin supported more binding than 180 kDa. Seventy kilodalton did not augment gelatin binding until 2 h, but by 6 h 70 kDa supported more binding than intact fibronectin (P < 0.01). This appeared to result from a cellular response initiated by 70-kDa-gelatin interactions with the macrophages. Within 4 h the majority of gelatin associated with cells under control conditions, and in the presence of fibronectin or 180 kDa, was internalized. Seventy-kilodalton-mediated binding remained localized primarily to the cell surfaces at all times. The macrophages partially degraded the internalized and external gelatin fractions. These results demonstrate that intact fibronectin and specific fibronectin fragments support soluble gelatin interactions with macrophages.

• Ingham KC, Brew SA, Novokhatny VV
• Influence of carbohydrate on structure, stability, and function of gelatin-binding fragments of fibronectin.
• Arch Biochem Biophys. 1995; 316: 235-40
• Display abstract

• The gelatin-binding region of fibronectin contains three Asn-linked carbohydrate moieties, one on the second type II module and two on the eighth type I "finger" module. Carbohydrate groups were enzymatically removed from two nonoverlapping gelatin-binding fragments (GBFs), 21-kDa GBF (modular composition I8-I9) and, with much greater difficulty, 30-kDa GBF (modular composition I6-II1-II2-I7). The gelatin-binding properties of these fragments were affected only slightly or not at all. Fluorescence and calorimetric analyses indicated that module I8 was strongly destabilized by deglycosylation such that the apo form melts near physiological temperature. A similar effect was caused by decreasing the pH of the holo form to 6.0, suggesting that one or more histidines are important for stability of module I8. The 21-kDa fragment exhibited an acid-induced change in fluorescence that occurred at higher pH in the deglycosylated derivative, providing further evidence of a stabilizing role for one or both carbohydrate moieties. By contrast, the stability of module II2 was unaffected by removal of its single carbohydrate. The effects of carbohydrate on the stability of module I8 may be important in efforts to express it or fragments containing it in bacteria.

• Sojar HT, Lee JY, Genco RJ
• Fibronectin binding domain of P. gingivalis fimbriae.
• Biochem Biophys Res Commun. 1995; 216: 785-92
• Display abstract

• P. gingivalis fimbriae play an important role in attachment of bacteria to various salivary components as well as to host cells and matrix proteins including fibronectin. In the present study, we investigated the binding domain of P. gingivalis fimbriae to fibronectin using synthetic peptides. A series of 20 mer fimbrillin peptides were used. Binding of fibronectin to purified fimbriae and synthetic peptides was assayed using polyclonal fibronectin antibodies as well as iodinated fibronectin. Purified fimbriae and peptide 126-146 (RMAFTEIKVQMSAAYDNIYTF) showed high levels of binding to fibronectin, while peptide 318-337(HLNVQCTVAEWVLVGQNATW) showed low but statistically significant binding. Our results suggest V-Q-X-X-X-A or V-X-X-X A common domain/domains present in both peptides might be involved in protein-protein interaction between P. gingivalis fimbriae and fibronectin.

• Gmeiner B, Leibl H, Zerlauth G, Seelos C
• Affinity binding of distinct functional fibronectin domains to immobilized metal chelates.
• Arch Biochem Biophys. 1995; 321: 40-2
• Display abstract

• Fibronectin has been found to bind metal ions. Using metal chelate chromatography and limited proteolysis to generate the distinct functional domains of fibronectin we set out to address the metal binding sites to well-defined regions of fibronectin. The results show that the affinity binding of fibronectin to Co2+ is mediated exclusively via the collagen binding domain of the molecule, whereas fibronectin will bind to Zn2+, Ni2+, and Cu2+ by metal binding sites located in two, three, and four well-defined regions of fibronectin, respectively. Fe2+ and Mn2+ chelates did not bind any of the isolated fibronectin domains. Combined metal chelate affinity chromatography opens a possibility to isolate particular fibronectin domains.

• Steffensen B, Wallon UM, Overall CM
• Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. High affinity binding to native type I collagen but not native type IV collagen.
• J Biol Chem. 1995; 270: 11555-66
• Display abstract

• 72-kDa gelatinase/type IV collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type II-like modules, was expressed in Escherichia coli. Similar to full-length 72-kDa gelatinase and the type II modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types IV and V collagens, but neither native types IV and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-kDa gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD123 to the alpha 1(I) collagen cyanogen bromide fragment CB2 from the NH2-terminal telopeptide. rCBD123 also bound alpha 1(I)-CB7, which encompasses the fibronectin-binding site, and to alpha 1(I)-CB8, a fragment not bound by fibronectin. Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may facilitate enzyme localization in connective tissue matrices.

• Fujita H, Mohri H, Kanamori H, Iwamatsu A, Okubo T
• Binding site in human plasma fibronectin to HL-60 cells localizes in the C-terminal heparin-binding region independently of RGD and CS1.
• Exp Cell Res. 1995; 217: 484-9
• Display abstract

• A 29-kDa monomeric dispase-digestive fragment of human plasma fibronectin has been purified by heparin affinity chromatography. The NH2-terminal sequence was determined as Ala1687-Val-Thr-Thr-Ile-Pro-Ala-Pro. By mass spectrometry the molecular weight was determined to be 30,241.9 with standard deviation of 3.9 amu. Therefore, we defined the C-terminal sequence of the 29-kDa fragment as Arg1957-Lys-Lys-Thr-Gly-Gln-Glu. This indicates that the fragment is composed of 277 amino acids. 125I-fibronectin and the 125I-labeled 29-kDa fragment bound to HL-60 (human acute promyelocytic leukemia) cells in a time-dependent, saturable, and reversible manner. Approximately 120 min was required to reach maximal binding. There were no differences in quantity or rate of binding of labeled fibronectin and 29-kDa fragment at temperatures of 4 degrees, 22 degrees, and 37 degrees C. The number of binding sites per HL-60 cell of fibronectin and the 29-kDa fragment were 140,000 with a Kd of 133 nM and 108,000 with a Kd of 250 nM, respectively. The binding of fibronectin to HL-60 cells was completely inhibited by this fragment, and by the peptides of RGDS and CS1 with IC50s of 3.6, 840, and 670 microM, respectively. Native fibronectin inhibited the direct binding of the 29-kDa fragment to HL-60 cells; however, RGDS peptide, peptide CS1, or two melanoma cell adhesion-promoting domain peptides in this 29-kDa fragment (peptide I; Tyr1906-Val1924, peptide II; Asp1946-Thr1960) did not block this binding. Neither heparitinase nor chondroitinase treatment of cells had any effect on these bindings. These results indicate that the C-terminal cell- and heparin-binding domain of fibronectin mediates HL-60 cell binding by direct interaction independently of RGD, CS1, and melanoma cell adhesion domains in this fragment.

• Denizli A, Piskin E, Dixit V, Arthur M, Gitnick G
• Collagen and fibronectin immobilization on PHEMA microcarriers for hepatocyte attachment.
• Int J Artif Organs. 1995; 18: 90-5
• Display abstract

• Polyhydroxyethylmethacrylate (PHEMA) microcarriers in a size range of 150-250 microns were prepared by a suspension polymerization in an aqueous phase containing magnesium oxide. The hydroxyl groups on the PHEMA microcarriers were activated by cyanogen bromide. In order to improve cell attachment, cell-adhesive proteins, namely, collagen and fibronectin were immobilized onto PHEMA microcarriers. The nonspecific adsorption values for collagen and fibronectin were 0.10 mg collagen/g PHEMA and 0.044 mg fibronectin/g PHEMA, respectively. Collagen and fibronectin immobilization on PHEMA microcarriers were studied at different pH by using single protein solutions containing different amounts of proteins, at a constant temperature of 20 degrees C. The maximum immobilizations were 0.85 mg collagen/g PHEMA (at pH: 9.5) and 0.52 mg fibronectin/g PHEMA (at pH: 7.4). Hepatocyte attachment onto these biologically modified PHEMA microcarriers was studied. Hydrophilic PHEMA microcarriers did not support cell attachment. High hepatocyte attachment yields (up to 75% surface coverage) were observed on collagen and fibronectin immobilized PHEMA microcarriers.

• Aguirre KM, McCormick RJ, Schwarzbauer JE
• Fibronectin self-association is mediated by complementary sites within the amino-terminal one-third of the molecule.
• J Biol Chem. 1994; 269: 27863-8
• Display abstract

• The formation of a fibrillar fibronectin (FN) extracellular matrix requires self-association of FN dimers. In this report, we show that the major sites for self-association are the amino-terminal repeats I1-5 and the first type III repeats. Recombinant FNs and fragments were generated by baculovirus expression of cysteine-rich domains and by bacterial expression of type III repeats as fusion proteins with maltose binding protein. When recombinant polypeptides were immobilized on microtiter wells, FN bound to 70-kDa amino-terminal fragment and to fusion proteins containing repeats III1-2 and III1-6 but not to other type III repeats. Similar results were obtained with a gel overlay assay. Binding was concentration-dependent and saturable. The amino-terminal binding site for III1-2 was further localized to repeats I1-5. Therefore, at least two different sites for FN-FN interaction reside near the amino terminus of the molecule. A model for the regulation of FN matrix assembly is proposed based on intramolecular interactions between these amino-terminal sites.

• Hocking DC, Sottile J, McKeown-Longo PJ
• Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin.
• J Biol Chem. 1994; 269: 19183-7
• Display abstract

• Cultured fibroblasts express binding sites for the amino-terminal region of fibronectin on their cell surface that mediate the assembly of soluble fibronectin into disulfide-stabilized fibrils. These binding sites have been termed matrix assembly sites and have been studied in binding assays using a 125I-labeled 70-kDa fragment derived from the amino terminus of fibronectin. In an attempt to isolate the protein(s) responsible for binding the 70-kDa fragment, cell surface proteins were cleaved from fibroblast monolayers by mild trypsinization. Trypsinization of monolayers generated a series of fibronectin fragments that bound the 125I-labeled 70-kDa fragment by ligand blot assay and affinity chromatography. All of the fibronectin fragments that bound the 70-kDa fragment contained the III-1 module. In solid phase binding assays, the 125I-labeled 70-kDa fragment bound preferentially to reduced fibronectin as compared with unreduced fibronectin fragments. Binding of the 125I-labeled 70-kDa fragment to reduced fibronectin was inhibited by a monoclonal antibody directed against the III-1 domain. Isolated III-1, however, did not bind the 125I-labeled 70-kDa fragment when adsorbed to plastic tissue culture wells. Heat denaturation of III-1 prior to adsorption conferred 70-kDa fragment binding properties on the isolated module. The 125I-labeled 70-kDa fragment did not bind to heat-denatured III-2, suggesting that 70-kDa fragment binding was a property of the III-1 module and not a general characteristic of all type III modules. The binding of 125I-labeled 70-kDa fragment to III-1 was of high affinity (KD = 1.8 x 10(-8) M). These results indicate that a binding site for the 70-kDa amino terminus of fibronectin is contained within a cryptic site found in the first type III module of fibronectin. Unfolding of the III-1 module on the cell surface may control matrix assembly site expression and represent an important step in the initiation of cell-dependent fibronectin polymerization.

• Lapiere JC, Chen JD, Iwasaki T, Hu L, Uitto J, Woodley DT
• Type VII collagen specifically binds fibronectin via a unique subdomain within the collagenous triple helix.
• J Invest Dermatol. 1994; 103: 637-41
• Display abstract

• Type VII collagen is the major component of anchoring fibrils, structures within basement membranes beneath stratified squamous epithelium thought to mediate the adherence of the epidermis to the dermis of human skin. Type VII collagen has affinity for fibronectin. The interaction between type VII collagen and fibronectin is mediated through the collagen-binding domain on the amino terminus of fibronectin. Heretofore, the domain on the type VII collagen molecule that binds to fibronectin was not known. In this study, we mapped the binding site of fibronectin to a specific subdomain of the triple helical collagenous region of type VII collagen, immediately adjacent to the small carboxyl terminal non-collagenous domain. This fibronectin-binding site within the type VII collagen molecule lies between nucleotide residues 615 and 1161.

• Ingham KC, Brew SA, Migliorini M
• An unusual heparin-binding peptide from the carboxy-terminal hep-2 region of fibronectin.
• Arch Biochem Biophys. 1994; 314: 242-6
• Display abstract

• A synthetic 22 residue peptide, N22W, with sequence NVSPPRRARVTDATETTITISW, derived from the amino terminus of type III module 13 in the carboxy-terminal hep-2 domain of fibronectin, was found to exhibit unusual heparin binding properties. Titration of fluoresceinamine-labeled heparin (FA-heparin) with N22W at 25 degrees C and pH 7.4 in 0.02 M Tris buffer containing 0.15 M NaCl (TBS) produced a cooperative sigmoidal increase in fluorescence polarization anisotropy with half-saturation near 2.5 microM. The increase in anisotropy was even greater than that produced by the much larger 30-kDa hep-2 fragment of fibronectin and saturation was achieved at lower concentration. Simply deleting the C-terminal Trp from the peptide abolished its heparin-binding activity as did deletion of residues TETTITIS or mutation of the RR doublet to SS. Further analysis suggested that peptide-peptide interactions mediated by the carboxy-terminal region of N22W play an important role in its binding to heparin. A branched tetrameric peptide containing four copies of N21S caused a nearly hyperbolic increase in anisotropy of FA-heparin with an apparent Kd of 0.3 microM in TBS, 10-fold lower than that of the monomer or of the parent domain from which the peptide was derived. The results illustrate that peptide-peptide interactions can lead to stronger binding by allowing multiple points of contact with the negatively charged polysaccharide.

• Li X, Bokman AM, Llinas M, Smith RA, Dobson CM
• Solution structure of the kringle domain from urokinase-type plasminogen activator.
• J Mol Biol. 1994; 235: 1548-59
• Display abstract

• The solution structure of the kringle domain from urokinase-type plasminogen activator (u-PA) has been determined using 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. A total of 35 structures, 20 generated using a distance geometry method prior to simulated annealing and 15 generated using initial random phi, psi values, have been calculated based on 946 experimental nuclear Overhauser effect distance constraints and 48 dihedral angle constraints. Excluding the N- and C-terminal residues (-1 to 12, 77 to 82) and a number of surface residues (M18, G19, S42, D55 to R60, G67) that are disordered or flexible, the root mean square deviation values from the mean structure are 0.49(+/- 0.14) A and 0.65(+/- 0.16) A for the backbone atoms, and 1.03(+/- 0.21) A and 1.39(+/- 0.24) A for all heavy atoms, for the two sets of structures, respectively. An extended binding site for anionic polysaccharides such as heparin has been located on a relatively flat facet of the molecule, involving three consecutive arginines, R57, R58 and R60 (there is a deletion at site 59 of the consensus sequence), which form a cationic triad facing the solvent, and two histidines, H37 and H40, at the opposite end of the molecule. Comparison between the u-PA kringle structure and the crystal and NMR solution structures of tissue-type plasminogen activator kringle 2 has shown that the two proteins have similar global folds but demonstrate a number of local differences.

• Sakata N, Jakab E, Wadstrom T
• Human plasma fibronectin possesses second binding site(s) to Staphylococcus aureus on its C-terminal region.
• J Biochem (Tokyo). 1994; 115: 843-8
• Display abstract

• The existence of a second binding site to Staphylococcus aureus on fibronectin was explored by using a recombinant protein, alb-FnBPA, which was obtained from an Escherichia coli clone containing a part of the gene for fibronectin-binding protein of S. aureus. Limited digestion with trypsin produced a 215K fragment from the B chain of fibronectin by releasing an N-terminal 32 kDa region. This C-terminal large fragment captured alb-FnBPA in spite of lacking the N-terminal binding region to S. aureus. This treatment also produced a 185K fragment from A chain by releasing N-terminal 32 kDa and C-terminal 37 kDa regions, and the 185K fragment possessed no binding ability. Thermolysin digestion was also performed to investigate the effect on the binding of releasing the C-terminal region from both of the chains and to locate the binding region to alb-FnBPA on the tryptic 215K fragment. But this digestion produced no fragment binding to alb-FnBPA except for the N-terminal 24K and its transient precursory fragments. These results indicate that both the N-terminal and the C-terminal regions of fibronectin bind to S. aureus, and suggest that the binding appears to involve cooperation between type III repeats and C-terminal type I repeats.

• Negre E, Vogel T, Levanon A, Guy R, Walsh TJ, Roberts DD
• The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans.
• J Biol Chem. 1994; 269: 22039-45
• Display abstract

• A 30-kDa proteolytic fragment from the gelatin/collagen-binding domain of fibronectin is a potent inhibitor of fibronectin binding to Candida albicans, with a molar inhibition constant equal to that of intact fibronectin. Recombinant and proteolytic fragments from the cell-, the fibrin I-, and the heparin II-binding domains also inhibit fibronectin binding, but are 13-1000-fold less active. In suspension, binding of fibronectin to C. albicans is regulated by growth conditions and is specific, saturable, time-dependent, reversible, and divalent cation-independent. Scatchard plot analyses indicate the presence of high affinity (Kd = 1.3 x 10(-9) M) and low affinity (Kd = 1.2 x 10(-7) M) receptors. Recombinant or proteolytic fragments from four binding domains of fibronectin promote adhesion of C. albicans. A recombinant fragment corresponding to the cell-binding domain but with the sequence Arg-Gly-Asp-Ser deleted promotes C. albicans adhesion and inhibits fibronectin binding to C. albicans with the same activity as the natural sequence. Furthermore, four peptides containing the Arg-Gly-Asp-Val sequence and the peptides CS-1 and Arg-Glu-Asp-Val did not block the binding of fibronectin to C. albicans. Thus, in contrast to the specific binding of soluble fibronectin, recognition of immobilized fibronectin by C. albicans is mediated by several domains of the protein. Interactions with the cell-binding domain are not mediated by the Arg-Gly-Asp or other known recognition sequences as it has been suggested. Binding of fibronectin also did not correlate with C3d binding to the avirulent clones of C. albicans strain H12 or with iC3b binding to variants of the strain 4918.

• Hsieh JC, Wu C, Chung AE
• The binding of fibronectin to entactin is mediated through the 29 kDa amino terminal fragment of fibronectin and the G2 domain of entactin.
• Biochem Biophys Res Commun. 1994; 199: 1509-17
• Display abstract

• Previous work has shown that fibronectin and entactin, an ubiquitous basement membrane glycoprotein, co-localize in the extracellular matrix of the embryonal carcinoma-derived 4CQ cell. Glutathione-S-transferase (GST) fusion proteins containing different domains of entactin have been obtained in the pGEX3X expression vector. These fusion proteins, GST-G1, GST-G2, GST-E and GST-G3, were purified with a glutathione affinity column. By using a solid phase binding assay, it was shown that the 125I-labeled 29 kDa amino terminal fragment of bovine fibronectin bound specifically to the immobilized GST-G2 fusion protein but not to GST-G1, GST-E, and GST-G3. Half saturation for binding of the 29 kDa fibronectin fragment to the immobilized GST-G2 fusion protein was obtained at a concentration of approximately 5 nM. It is suggested that the strong association between GST-G2, which contains the second globular domain of entactin, and the 29 kDa amino terminal fragment of fibronectin may be involved in the assembly of certain types of extracellular matrices.

• Kar L, Lai CS, Wolff CE, Nettesheim D, Sherman S, Johnson ME
• 1H NMR-based determination of the three-dimensional structure of the human plasma fibronectin fragment containing inter-chain disulfide bonds.
• J Biol Chem. 1993; 268: 8580-9
• Display abstract

• Human plasma fibronectin is a plasma glycoprotein that plays an important role in many biological processes. It consists of two identical 230-250-kDa subunits that are joined by two disulfide bonds near their carboxyl termini. Each subunit contains various binding domains composed of three types of homologous repeats. Recent work has determined the three-dimensional structures of various repeat fragments, but little is known about the three-dimensional structure of the carboxyl-terminal region. A recent NMR study of a plasmin-digested carboxyl-terminal inter-chain disulfide-linked heptapeptide dimer has proposed that the two subunits are arranged in an antiparallel fashion (An et al. (1992) Biochemistry 31, 9927-9933). We have now determined the three-dimensional structure for a substantial portion of a trypsin-digested interchain disulfide-linked 52-residue (6 kDa) fragment of the carboxyl-terminal of human plasma fibronectin (which includes the above-mentioned heptapeptide dimer) using two-dimensional NMR methods and a new strategy for NMR-based protein structure determination. The NMR data requires that the two chains in the dimer be linked in a symmetric, antiparallel arrangement. The resulting monomer conformation consists of two twisted or coiled segments, Thr3-Asn6 and Ile9-Phe12, connected by the Cys7-Pro8 residues in extended conformations, with the two monomer chains cross-linked at residues Cys7 and Cys11. The conformation of the heptapeptide dimer region differs substantially from the conformation proposed by An et al.

• Shibata K et al.
• An attempt to substitute the cell binding domain of human fibronectin in lambda phage J protein: computer design and expression.
• Biochimie. 1993; 75: 459-65
• Display abstract

• We superimposed hydropathic indexes of the human fibronectin cell binding domain (CBD) on the lambda phage J protein by computer, and substituted 22 amino acids from the fibronectin CBD for a part of the lambda phage J protein. The fibronectin cell binding domain -Arg-Gly-Asp-Ser- (-RGDS-) localizes at the junction of hydrophobicity and hydrophilicity. We selected a similar hydrophobic-to-hydrophilic junction in the J protein region for substitution. This junction corresponds to 150 bp of the PstI fragment of J protein cDNA. We synthesized 150 bp of the relevant PstI fragment that includes the cell binding domain region. The region was then constructed by serial cloning as an expression vector, pJCBD. The vector pJCBD expressed the fused protein named JCBD (M(r) 32 kDa) in E coli XL1-BLUE. The expressed JCBD protein was identified by Western blot analysis in the extract of the pJCBD carrying bacterial lysate using both rabbit anti-lambda phage antiserum and anti-CBD of fibronectin antibody. The JCBD protein appeared to recognize retinoblast cell membrane RGDS-directed receptors, detected by enzyme-linked immuno-sorbent assay and also by binding competition assay with synthetic pentapeptides, Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES). The former competitor inhibited completely fibronectin CBD-dependent binding activity of JCBD, the latter had no inhibitory activity. These results suggest that certain functional proteins engineered by computer design between human fibronectin cell binding domain and lambda phage J protein can be produced.

• Reece RJ, Rickles RJ, Ptashne M
• Overproduction and single-step purification of GAL4 fusion proteins from Escherichia coli.
• Gene. 1993; 126: 105-7
• Display abstract

• A DNA fragment encoding the yeast GAL4 transcriptional activator DNA-binding domain (amino acids 1-93) was cloned into an Escherichia coli expression vector such that the overproduced protein is tagged with six histidine residues and a factor Xa protease cleavage site. The vector also contains unique restriction sites at the 3' end of the gene to allow the construction of fusion proteins. These fusion proteins can easily be purified to homogeneity and their activity tested in vitro.

• Dzamba BJ, Wu H, Jaenisch R, Peters DM
• Fibronectin binding site in type I collagen regulates fibronectin fibril formation.
• J Cell Biol. 1993; 121: 1165-72
• Display abstract

• Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.

• Drake SL, Varnum J, Mayo KH, Letourneau PC, Furcht LT, McCarthy JB
• Structural features of fibronectin synthetic peptide FN-C/H II, responsible for cell adhesion, neurite extension, and heparan sulfate binding.
• J Biol Chem. 1993; 268: 15859-67
• Display abstract

• FN-C/H II (KNNQKSEPLIGRKKT), a heparin-binding peptide derived from the COOH-terminal heparin-binding domain of fibronectin, mediates cell adhesion for a variety of cell types and promotes neurite outgrowth. By systematic amino acid substitution of synthetic peptide analogues of FN-C/H II, the basic structural features necessary for activity have been identified in the COOH-terminal residues LIGRKK. This biologically "active" sequence has been located in several other heparin/heparan sulfate-binding proteins and may represent a potential binding motif for sulfated polyanions. NMR structural studies indicate that the COOH-terminal segment of FN-C/H II displays significant multiple-turn or helix-like character suggesting that the RKK sequence may lie on the same surface of the protein, as opposed to alternating in an extended chain motif.

• Miles AM, Smith RL
• Functional methionines in the collagen/gelatin binding domain of plasma fibronectin: effects of chemical modification by chloramine T.
• Biochemistry. 1993; 32: 8168-78
• Display abstract

• Chemical modification of plasma fibronectin (pFn) or its 40-kDa collagen/gelatin binding (CGB) domain by low concentrations of chloramine T (CT), a methionine-specific oxidant, caused decreased binding affinity between pFn or the isolated CGB domain and Sepharose-immobilized denatured collagen or a Texas Red-labeled CNBr fragment CB7 from the alpha 1 chain of type I collagen. Kds obtained by fluid-phase fluorescence polarization binding assays increased upon oxidation about 17-fold for pFn and by 4-fold for the CGB domain. Comparison of CT-oxidized and native CGB domains by endogenous tryptophan fluorescence and CD spectra gave no indication of conformational changes. delta GH2O, the free energy of unfolding at infinite denaturant dilution, derived from guanidinium chloride denaturation curves, differed by less than 0.7 kcal/mol for the oxidized and native CGB domains, indicating essentially equivalent conformational stabilities. We show here that methionyl residues found at positions 412, 432, and 446 are the sites of the oxidative modification. Modification protection experiments carried out in the presence of gelatin demonstrated specific protection of otherwise oxidizable methionyl residues and preservation of high-affinity binding. These results implicate methionyl residues as functional in contributing to high-affinity binding interaction between fibronectin and gelatin.

• Wilke MS, Vespa J, Skubitz AP, Furcht LT, McCarthy JB
• Human keratinocytes adhere to and spread on synthetic peptide FN-C/H-V derived from fibronectin.
• J Invest Dermatol. 1993; 101: 43-8
• Display abstract

• In a previous study, we reported that two synthetic peptides derived from the 33-kD carboxyl terminal cell/heparin-binding fragment of fibronectin A chain promoted keratinocyte adhesion but not spreading. Because keratinocytes are capable of spreading on the 33/66-kD fragments, we focused on identifying additional chemically synthesized peptides from the cell/heparin-binding fragments of fibronectin that might promote cell spreading. When plastic substrata were coated with peptide FN-C/H-V (WQPPRARI), which is derived from the carboxyl-terminal heparin-binding domain of all plasma fibronectin isoforms, keratinocytes adhered and displayed a spread morphology. In solution, soluble peptide FN-C/H-V inhibited cell spreading on intact fibronectin and on the 33/66-kD fragments. Furthermore, polyclonal antibodies raised against peptide FN-C/H-V also inhibited keratinocyte spreading on fibronectin and the 33/66-kD fragments. These data support the hypothesis that keratinocyte cell adhesion and cell spreading on fibronectin are mediated by multiple distinct domains and different regulatory processes.

• Garcia-Pardo A, Gold LI
• Further characterization of the binding of fibronectin to gelatin reveals the presence of different binding interactions.
• Arch Biochem Biophys. 1993; 304: 181-8
• Display abstract

• The specific interaction between fibronectin and collagen has permitted the isolation of fibronectin from plasma using gelatin-Sepharose affinity matrices. In this study we obtained evidence suggesting the presence of an additional high affinity binding interaction between fibronectin and collagen. The following information supports this idea: (i) Successive isolations of fibronectin using the same regenerated gelatin matrix resulted in progressively increased yields of fibronectin, thus indicating the progressive occupation of high affinity fibronectin-binding sites within collagen. (ii) This tightly bound fibronectin could not be eluted from gelatin-Sepharose matrices with strong denaturing agents, such as 8.0 M urea or 6.0 M guanidium chloride. (iii) The fibronectin-occupied matrices supported fibroblast adhesion and spreading, indicating that the amount of tightly bound fibronectin was quantitatively sufficient for this function, and that the fibronectin that bound with high affinity retained its biologically active conformation. (iv) Two fibronectin-derived fragments, CB52kDa and T55 kDa, selectively bound to fresh gelatin-Sepharose but not to gelatin-Sepharose previously employed to purify fibronectin, suggesting that these fragments recognize only the high affinity binding sites in gelatin. N-terminal amino acid sequence analysis of T55 kDa showed that it contained the previously described collagen-binding domain and included the repeat III-1 of fibronectin. These results support the existence of multiple fibronectin-binding sites in collagen and implicate a high affinity site(s) with a propensity for tenacious binding.

• Lai CS et al.
• Solution structure of human plasma fibronectin under different solvent conditions. Fluorescence energy transfer, circular dichroism and light-scattering studies.
• J Mol Biol. 1993; 230: 625-40
• Display abstract

• Human plasma fibronectin is a high molecular weight (530,000), multi-domain, modular glycoprotein, consisting of two nearly identical subunits disulfide-bridged close to their C-terminal ends. Three sites that can be differentially labeled with fluorescent probes are present on each fibronectin subunit, the transglutaminase-sensitive Gln3 residue and the two free sulfhydryl residues, Cys1201 and Cys2196. These sites are located, respectively, in the N-terminal heparin/fibrin-binding domain, between the central DNA and cell-binding domains, and just before the C-terminal fibrin-binding domain. To map the relative spatial arrangement of these domains, steady-state and lifetime fluorescence energy transfer techniques were employed. Our results show that the minimal intramolecular distances between the labeled Gln3-Cys1201 and Gln3-Cys2196 pairs are 5.5(+/- 0.6) nm and 5.7(+/- 0.7) nm, respectively, as measured by steady-state methods. Lifetime methods gave somewhat higher distances of 8.1(+/- 0.2) nm and 7.6(+/- 0.2) nm, respectively, between these sites. The binding of heparin or subjection to high ionic strength had only a minor effect, while in the presence of 50% (w/v) glycerol, an increase of about 25% in the intramolecular distances between these sites was observed. A similar effect was induced by binding of fibronectin to the surface of Cytodex beads, an event which was previously shown instead to markedly increase the intersubunit distances between the Gln3-Gln3 and Cys1201-Cys1201 pairs. The solution structure of fibronectin was further investigated by elastic light-scattering and circular dichroism measurements. By elastic light-scattering, the radius of gyration of fibronectin was found to be 15.3(+/- 0.8) nm in the presence of 30% (w/v) glycerol, in contrast to a value of 8.6(+/- 0.3) nm under physiological conditions. Far and near ultraviolet circular dichroism spectra showed that only minor changes in the secondary structure of fibronectin take place on increasing the glycerol content of the solvent up to 34% (w/v). Our results complement previously available information on the solution structure of fibronectin and on its transition from the native compact conformation to a more expanded form on increasing ionic strength or glycerol content. In either situation, fibronectin seems to retain a basic structural core, in which the N-terminal, the central and the C-terminal regions of the two subunits strongly interact with each other. A major role of hydrophobic forces, in stabilizing the fibronectin conformations under these conditions, is therefore postulated. The transition to the extended forms seen in many electron micrographs can instead be explained by disruption of the proposed structural core upon adsorption to surfaces.

• Sottile J, Mosher DF
• Assembly of fibronectin molecules with mutations or deletions of the carboxyl-terminal type I modules.
• Biochemistry. 1993; 32: 1641-7
• Display abstract

• Fibronectin is a large modular protein that is assembled into fibrils in a stepwise process that involves the binding of soluble fibronectin to the cell surface and formation of fibronectin multimers that are stabilized by disulfides. Fibronectin contains two types of disulfide-containing repeat modules, types I and II. The type I modules form units that mediate binding to assembly sites (I-1 through I-5), mediate binding to gelatin (I-6 through I-9 plus the type II modules), or have no known function other than fibrin binding (I-10 through I-12). All type I modules contain four cysteines that are disulfide-linked in a 1-3, 2-4 arrangement, except for I-12 that contains six cysteines disulfide-bonded in an unknown arrangement. I-12 contains the consensus sequence Cys-Xaa-Yaa-Cys found in a number of proteins involved in disulfide exchange reactions [Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237; Boniface, J. J., & Reichert, L. E., Jr. (1990) Science 247, 61]. We explored the role of I-12 and adjacent type I modules of fibronectin in matrix assembly. We generated mutant fibronectins in which the second and sixth or fifth and sixth cysteine residues in I-12 were changed to serines (CS mutants) or that contained deletions of the 12th (delta 12) or 10th through 12th (delta 10-12) type I modules. Expression of I-12 as a fusion protein with the gelatin binding part of fibronectin indicated that this module folds independently and that the most likely disulfide pairing is 1-4, 2-6, 3-5.(ABSTRACT TRUNCATED AT 250 WORDS)

• Dardel F, Davis AL, Laue ED, Perham RN
• Three-dimensional structure of the lipoyl domain from Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.
• J Mol Biol. 1993; 229: 1037-48
• Display abstract

• The structure of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of nuclear magnetic resonance spectroscopy. A total of 452 nuclear Overhauser effect distance constraints and 76 dihedral angle restraints were employed as the input for the structure calculations, which were performed using a hybrid distance geometry-simulated annealing strategy and the programs DISGEO and X-PLOR. The overall structure of the lipoyl domain (residues 1 to 79 of the dihydrolipoamide acetyltransferase polypeptide chain) is that of a flattened eight-stranded beta-barrel folded around a core of well-defined hydrophobic residues. The lipoylation site, lysine 42, is located in the middle of a beta-turn, and the N and C-terminal residues of the domain are close together in adjacent beta-strands at the opposite end of the molecule. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the C alpha atoms of residues 15 to 39 and 52 to 76 being almost superimposable on those of residues 52 to 76 and 15 to 39, respectively (root-mean-square deviation = 1.48 A). The amino acid residues at key positions in the structure are conserved among all the reported primary structures of lipoyl domains, suggesting that the domains all fold in a similar way.

• Sipes JM, Guo N, Negre E, Vogel T, Krutzsch HC, Roberts DD
• Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin.
• J Cell Biol. 1993; 121: 469-77
• Display abstract

• The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin-binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.

• Peake P, Gooley A, Britton WJ
• Mechanism of interaction of the 85B secreted protein of Mycobacterium bovis with fibronectin.
• Infect Immun. 1993; 61: 4828-34
• Display abstract

• The 85B protein of Mycobacterium bovis is a member of the secreted antigen 85 complex, which has been identified in a number of pathogenic mycobacteria. The 85 complex contains three components with molecular masses of 30 to 32 kDa which share the property of binding to fibronectin, a large glycoprotein present in plasma. To investigate this activity we have expressed the M. bovis 85B antigen as a recombinant protein and studied its interaction with human fibronectin. Fibronectin bound to the immobilized 85B protein in a solid-phase enzyme-linked immunosorbent assay (ELISA) and in the fluid phase in a radioimmunoassay using 125I-labelled 85B protein. In addition, fibronectin reacted with immobilized 85B in immunoblots and vice versa. Fibronectin also bound to three fragments of a cyanogen bromide digest of 85B which were subsequently identified by N-terminal sequencing. These fragments contained fibronectin-reactive peptides identified in ELISAs utilizing a set of 28 overlapping 20-mer peptides encompassing the 85B sequence. Further studies showed that the 85B protein reacted with a 32-kDa polypeptide from a limited tryptic digest of fibronectin which was identified as the collagen-binding domain. This region was confirmed as the 85B binding site by the fact that gelatin but not heparin inhibited the binding of fibronectin to 85B. These data indicate that the 85B-fibronectin interaction involves the binding of multiple regions of the 85B protein to the collagen-binding domain of fibronectin.

• Kusnadi AR, Ford C, Nikolov ZL
• Functional starch-binding domain of Aspergillus glucoamylase I in Escherichia coli.
• Gene. 1993; 127: 193-7
• Display abstract

• We have fused three starch-binding domains (SBD) encoding gene fragments (residues 511-616, 495-616 and 481-616) of glucoamylase I (GAI) to the 3' end of the Escherichia coli malE gene encoding maltose-binding protein (MBP). The fusion proteins were produced in E. coli and were purified by chromatography on cross-linked amylose. Factor Xa digestion of the fusion proteins resulted in the release of functional SBD fragments which were separated from MBP on the basis of their differential binding to cross-linked amylose. The amino acid (aa) composition of the purified SBD fragments agreed with the respective amino acid compositions of GAI. The sizes of the SBD fragments were 11.9, 13.8 and 15.6 kDa, respectively.

• Talay SR, Valentin-Weigand P, Jerlstrom PG, Timmis KN, Chhatwal GS
• Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.
• Infect Immun. 1992; 60: 3837-44
• Display abstract

• The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to epithelial cells.

• Klyashchitsky BA, Mitina VK, Frantsuzova NA, Krasnopolsky YuM, Shvets VI
• Use of biospecific interactions of collagen, fibronectin and their fragments in affinity chromatography.
• J Chromatogr. 1992; 577: 267-73
• Display abstract

• Various aspects of the application of fibronectin-collagen biospecific interactions in affinity chromatography are described. A new biospecific method for one-stage isolation of collagen peptides containing fibronectin-binding sites is proposed. The alpha 1 CB7-peptide of type-I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45,000 relative molecular mass) of fibronectin. The method gives highly purified preparations of alpha 1 CB7-peptide. This peptide, as well as some other collagen molecular fragments (alpha-chains, beta-components, alpha 1 CB8-peptide), were immobilized on Sepharose, and the properties of such affinity adsorbents obtained were studied. Adsorbents with immobilized alpha-chains and alpha 1 CB7-peptide had a fibronectin-binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Large-scale production of highly purified fibronectin from human plasma, using affinity chromatography on immobilized individual alpha-chains of collagen, was developed.

• Litvinovich SV, Novokhatny VV, Brew SA, Ingham KC
• Reversible unfolding of an isolated heparin and DNA binding fragment, the first type III module from fibronectin.
• Biochim Biophys Acta. 1992; 1119: 57-62
• Display abstract

• Several fragments containing all or part of the first type III homology unit of fibronectin were isolated and their folding properties examined by fluorescence spectroscopy and differential scanning calorimetry. Each fragment exhibits a reversible unfolding transition when heated or titrated with guanidinium chloride. This indicates that an isolated type III module can fold independently in the absence of neighboring modules. A comparison of the specific enthalpies of unfolding of these fragments with those of well-studied globular proteins suggests that this type III unit is composed of a stable core flanked by less compact or unstructured regions. Comparison of the heparin-binding properties of these fragments revealed that removal of 12 amino acids from the amino terminus of the largest one (Ile-585 to Val-675) increased its affinity for immobilized heparin such that it now binds at physiological ionic strength.

• Gold LI, Rostagno A, Frangione B, Passalaris T
• Localization of the cleavage sites on fibronectin following digestion by urokinase.
• J Cell Biochem. 1992; 50: 441-52
• Display abstract

• Urokinase (u-PA) proteolytically cleaves both human plasma (pFn) and cellular (cFn) dimeric fibronectin (M(r) 440,000) into four major polypeptides of approximately M(r) 210,000, 200,000, 25,000, and 6,000. Amino acid sequence analysis of the polypeptide fragments indicated that the enzymatic cleavage of Fn occurs at two sites: 1) between an arginine/alanine peptide bond located C-terminal to residue 259; this cleavage liberates the N-terminal M(r) 25,000 fragment and the M(r) 210,000 and M(r) 200,000 polypeptides derived from the A and B chains of Fn, respectively; and 2) between an arginine/threonine peptide bond located C-terminal to residue 2,299, thereby yielding an M(r) 6,000 dimeric fragment containing the C-terminal interchain disulfide bonds. Predigestion of Fn with u-PA increased the molecule's vulnerability to further attack by the enzymes plasmin and cathepsin D. These data provide further biochemical evidence for the proteolytic cleavage of fibronectin by plasminogen activators and substantiate that u-PA digestion of Fn may be an initial event in the local degradation of the extracellular matrix by malignant cells, possessing elevated levels of these enzymes.

• Zafar RS, Zeng Z, Walz DA
• Localization of two binding domains for thrombospondin within fibronectin.
• Arch Biochem Biophys. 1992; 297: 271-6
• Display abstract

• Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.

• Ingham KC, Brew SA
• Integrity of refolded and reoxidized gelatin-binding fragments of fibronectin.
• Proteins. 1992; 12: 180-7
• Display abstract

• The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.

• Bozzini S, Visai L, Pignatti P, Petersen TE, Speziale P
• Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.
• Eur J Biochem. 1992; 207: 327-33
• Display abstract

• The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.

• Hatai M, Takahara K, Hashi H, Kato I, Yaoi Y
• Effects of fibronectin-type V collagen recombinant fusion protein on cell adhesion and cell proliferation.
• Cell Struct Funct. 1992; 17: 293-300
• Display abstract

• An expression vector pTF7520-Col-V-In, which encodes a fusion protein of the cell-binding domain of fibronectin (C277) and the insulin- and heparin-binding domain of the alpha 1 chain of human type V collagen, was constructed. E. coli transfected with this plasmid synthesized a 50-kDa fusion protein. This fusion protein, C277-V, was purified from the crude extract by a single step heparin HPLC. Similar amounts of insulin bound to purified C277-V and to the alpha 1 chain of type V collagen as judged by the binding of peroxidase-conjugated insulin. Cell-adhesive activity of C277-V was lower than that of the original fibronectin fragment C274, but similar numbers of cells adhered to both protein substrates when the culture dishes were coated with 1 mM of each protein. Insulin bound to the C277-V substratum stimulated the growth of mouse mammary tumor MTD cells in serum-free culture medium.

• Carnemolla B, Leprini A, Allemanni G, Saginati M, Zardi L
• The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.
• J Biol Chem. 1992; 267: 24689-92
• Display abstract

• We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.

• Collier IE, Krasnov PA, Strongin AY, Birkedal-Hansen H, Goldberg GI
• Alanine scanning mutagenesis and functional analysis of the fibronectin-like collagen-binding domain from human 92-kDa type IV collagenase.
• J Biol Chem. 1992; 267: 6776-81
• Display abstract

• The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases contain a domain consisting of three contiguous copies of the fibronectin (FN)-derived type II homology unit (T2HU), T2HU-1, T2HU-2, and T2HU-3. To investigate the functional role of this domain, we have constructed plasmids expressing beta-galactosidase fusion proteins with one or more of the CLG4B-derived T2HU. The gelatin binding assays demonstrate that a single copy of T2HU-2 renders beta-galactosidase capable of binding gelatin. The three repeats, however, differ dramatically in their capacity to bind gelatin, with T2HU-1 and T2HU-3 having significantly less binding activity than T2HU-2. Using alanine scanning mutagenesis we have defined the amino acid residues (Arg307, Asp309, Asn319, Tyr320, Asp323) that are critical for gelatin binding of T2HU-2. The low gelatin binding of T2HU-1 compared to T2HU-2 was traced to the non-conserved residues Ala228-Ala and Leu253-Pro. The results suggest that the gelatin binding of the type IV collagenase proenzyme is mediated by the FN-like domain, although the presence of another gelatin-binding site cannot be excluded. The FN domain-mediated binding, however, is not a rate-limiting step in the hydrolysis of gelatin by the enzyme.

• Kimizuka F et al.
• Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.
• J Biochem (Tokyo). 1991; 110: 284-91
• Display abstract

• An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.

• Westerlund B et al.
• Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and fsoF influence fimbrial binding to renal tubuli and immobilized fibronectin.
• Mol Microbiol. 1991; 5: 2965-75
• Display abstract

• P fimbriae of the F7(1) serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Gal alpha(1-4)Gal-specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Gal alpha(1-4)Gal-containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin-coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F7(2), F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue-binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.

• Byeon IJ, Llinas M
• Solution structure of the tissue-type plasminogen activator kringle 2 domain complexed to 6-aminohexanoic acid an antifibrinolytic drug.
• J Mol Biol. 1991; 222: 1035-51
• Display abstract

• The solution structure of a recombinant tissue-type plasminogen activator kringle 2 domain, complexed with the antifibrinolytic drug 6-aminohexanoic acid (6-AHA) was determined via 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. The structure determination is based on 610 intramolecular kringle 2 and 14 intermolecular kringle 2-6-AHA interproton distance restraints, as well as on 82 torsion angle restraints. Three sets of simulated annealing structures were computed from three different classes of starting structures: (1) random conformations devoid of disulfide bridges; (2) random conformations that contain correct disulfide bonds; and (3) a folded conformation modeled after the homologous prothrombin kringle 1 X-ray crystallographic structure. All three sets of structures are well defined, with averaged atomic root-mean-square deviations between individual structures and mean set structures of 0.77, 0.99 and 0.70 A for backbone atoms, and 1.36, 1.55 and 1.41 A for all atoms, respectively. Kringle 2 is an oblate ellipsoid with overall dimensions of approximately 34 A x 30 A x 17 A. It exhibits a compact globular conformation characterized by a number of turns and loop elements as well as by one right-handed alpha-helix and five (1 extended and 4 rudimentary) antiparallel beta-sheets. The extended beta-sheet exhibits a right-handed twist. Close van der Waals' contacts between the Cys22-Cys63 and Cys51-Cys75 disulfide bridges and the central hydrophobic core composed of the Trp25, Leu46, His48a and Trp62 side-chains are among the distinguishing features of the kringle 2 fold. The binding site for 6-AHA appears as a rather exposed cleft with a negatively charged locus defined by the Asp55 and Asp57 side-chains, and with an aromatic pocket structured by the Tyr36, Trp62, His64 and Trp72 side-chains. The Trp62 and His64 rings line the back surface of the pocket, while the Tyr36 and Trp72 rings confine it from two sides. The Trp62 and Trp72 indole rings conform a V-shaped groove. The methyl groups of Val35 also contribute lipophilic character to the ligand-interacting surface. It is suggested that the positively charged side-chains of Lys34 and, potentially, Arg69 may favor interactions with the carboxylate group of the ligand. The Trp25 and Tyr74 aromatic rings, although conserved elements of the binding site structure, seem not to undergo direct contacts with the ligand.

• Lambert Vidmar S, Lottspeich F, Emod I, Imhoff JM, Keil-Dlouha V
• Collagen-binding domain of human plasma fibronectin contains a latent type-IV collagenase.
• Eur J Biochem. 1991; 201: 79-84
• Display abstract

• Cleavage of the 45-kDa gelatin-binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40-kDa FN-type-IV collagenase A degrades heat-denatured type-I collagen, laminin and also native collagen type IV. The 27-kDa FN-type-IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N-terminal sequence VYQPQPH- (residues 262-268 of fibronectin) but, contrary to the FN-type-IV collagenase A, the FN-type-IV collagenase B has lost the C-terminal region of type I repeats, where the major gelatin-binding determinants of fibronectin are located. The FN-type-IV collagenases A and B are sequentially similar to the middle domain (domain II) of collagenase type IV, secreted by H-ras-transformed human bronchial epithelial cells. Substrate and inhibition specificity of FN-type-IV collagenase A and B are different from those of FN-gelatinase and FN-laminase, isolated previously from the central and C-terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix-degrading metalloproteinases.

• Chernousov MA, Fogerty FJ, Koteliansky VE, Mosher DF
• Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.
• J Biol Chem. 1991; 266: 10851-8
• Display abstract

• Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.

• Hayashi-Nagai A, Kitagaki-Ogawa H, Matsumoto I, Hayashi M, Seno N
• Hydrophobic properties of porcine fibronectin and its functional domains.
• J Biochem (Tokyo). 1991; 109: 83-8
• Display abstract

• The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.

• Constantine KL, Ramesh V, Banyai L, Trexler M, Patthy L, Llinas M
• Sequence-specific 1H NMR assignments and structural characterization of bovine seminal fluid protein PDC-109 domain b.
• Biochemistry. 1991; 30: 1663-72
• Display abstract

• Sequence-specific resonance assignments for the isolated second or b domain of the bovine seminal fluid protein PDC-109 have been obtained from analysis of two-dimensional 1H NMR experiments recorded at 500 MHz. These assignments include the identification of all aromatic and most aliphatic amino acid resonances. Stereospecific assignment of resonances stemming from the Val2 CH3 gamma,gamma' groups and from seven CH beta,beta' geminal pairs has been accomplished by analysis of 3J alpha beta coupling constants in conjunction with patterns of cross-peak intensities observed in two-dimensional nuclear Overhauser effect (NOESY) spectra. Analysis of NOESY and 3J alpha NH data reveals a small antiparallel beta-sheet involving stretches containing residues 25-28 and 39-42, a cis-proline residue (Pro4), antiparallel strands consisting of residues 1-3, 5-7, and 10-13, and an aromatic cluster composed of Tyr7, Trp26, and Tyr33. The results of distance geometry and restrained molecular dynamics calculations indicate that the global fold of the PDC-109 b domain, a type II module related to those found in fibronectin, is somewhat different from that predicted by modeling the structure on the basis of homology between type II and kringle units. A shallow depression in the molecular surface which presents a solvent-exposed hydrophobic area--a potential ligand-binding site-is identified in the NMR-based models.

• Lambert Vidmar S, Lottspeich F, Emod I, Planchenault T, Keil-Dlouha V
• Latent fibronectin-degrading serine proteinase activity in N-terminal heparin-binding domain of human plasma fibronectin.
• Eur J Biochem. 1991; 201: 71-7
• Display abstract

• The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262-265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond K85-D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca(2+)-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.

• Borsi L, Balza E, Leprini A, Ponassi M, Zardi L
• Procedure for the purification of the fibronectin proteolytic fragments containing the ED-B oncofetal domain.
• Anal Biochem. 1991; 192: 372-9
• Display abstract

• Fibronectin (FN) is the blend of structurally different molecules (isoforms) whose makeup varies depending on the FN sources. Fibronectin polymorphism is caused by three sequences (called ED-A, ED-B, and IIICS) which may be included or excluded from the FN molecule depending on the alternative splicing patterns of a single primary transcript. The sequence ED-B, which is a complete type III repeat of 91 amino acids, presents some interesting peculiarities: it is the most conserved FN region and, in normal adult tissues, the ED-B-containing FN has an extremely restricted distribution while having a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989, J. Cell Biol. 108, 1139-1148), suggesting that the ED-B sequence may confer to the FN molecules specific biological activities required during ontogenesis and oncogenetic processes. Here we describe a detailed procedure to purify fibronectin fragments containing the ED-B sequence. These purified fragments are useful reagents in the study of the biological function(s) of the ED-B-containing FN molecules.

• Chen LJ, Ford C, Nikolov Z
• Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase.
• Gene. 1991; 99: 121-6
• Display abstract

• We have constructed and purified by affinity chromatography three beta-galactosidase (beta Gal) fusion proteins (BSB133, BSBCD8, and BGA134) containing amino acid (aa) sequences from Aspergillus glucoamylase (GA). BSB133, containing the C-terminal 133 aa of GA (aa 484-616), adhered to native starch granules with a much higher affinity (Kad = 18 ml/g starch) than a beta Gal control (Kad = 0.9 ml/g starch). Two other fusion proteins, BSBCD8 and BGA134, similar in size to BSB133, adhered to starch with a relatively low affinity (Kad = 7 ml/g starch, and Kad = 4 ml/g starch, respectively). BSBCD8 differs from BSB133 by a truncation of 8 aa at the C terminus. BGA134 contains 134 aa from an overlapping region of GA (aa 380-513). These results confirm the presence of a strong starch-binding region (SBR) included in the C-terminal 133 aa of GA and indicate that the SBR can confer starch-binding activity on a fusion protein produced in Escherichia coli. In the presence of crude soluble cell extracts, the fusion proteins adsorbed by native starch granules with an affinity similar to that of the purified enzymes. BSB133 that had been adsorbed by starch from crude extracts could be eluted at a high level of purity, similar to that achieved by affinity chromatography. These results suggest that it may be feasible to use native starch as an adsorbent for the recovery and purification of recombinant fusion proteins containing the SBR. Starch has many favorable qualities for this application: it is inexpensive, stable, nontoxic, and easy to recover by centrifugation.

• Visai L, Bozzini S, Petersen TE, Speciale L, Speziale P
• Binding sites in fibronectin for an enterotoxigenic strain of E. coli B342289c.
• FEBS Lett. 1991; 290: 111-4
• Display abstract

• The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N-terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N-terminal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N-terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N-terminal end of fibronectin, may be structurally related.

• Mosher DF, Fogerty FJ, Chernousov MA, Barry EL
• Assembly of fibronectin into extracellular matrix.
• Ann N Y Acad Sci. 1991; 614: 167-80
• Display abstract

• The results summarized above suggest that assembly of fibronectin is a fundamental biological process and that knowledge of the process of assembly may reveal new ways by which cells interact with extracellular molecules. Deposition of a fibronectin matrix seems to be regulated as tightly as synthesis of fibronectin or expression of adhesion receptors for fibronectin and is influenced profoundly by two products of blood coagulation--TGF-beta released from platelets and factor XIII activated by thrombin. Fibronectin assembly may be important in all sorts of physiological and pathophysiological processes. Cell A--for instance, a stromal cell--can influence the behavior of cell B--for instance, a lymphocyte--by assembling fibronectin made by cell C--for instance, a hepatocyte. We hope that the testable models of assembly presented in this paper will lead to new understanding of the process of assembly and suggest new modalities for treatment of diseases that result in fibrosis, damaged tissues, and neoplastic growth.

• Gonzalez EA et al.
• Virulent Escherichia coli strains for chicks bind fibronectin and type II collagen.
• Microbios. 1990; 62: 113-27
• Display abstract

• 125I-fibronectin and 125I-collagen (type II) binding was detected in Escherichia coli strains isolated from chickens and poults. High fibronectin binding-strains also bind the 29 kD aminoterminal fragment of fibronectin. Binding properties in strain CK28 were partially characterized. The highest binding of 125I-fibronectin and 125I-collagen for strain CK28 was obtained with bacteria grown at 33 degrees C. Binding of 125I-fibronectin, its 125I-29 kD fragment, and 125I-collagen, was very rapid, reaching a maximum in 5 min. Binding of 125I-fibronectin and 125I-collagen was considerably inhibited by preincubation of bacteria with unlabelled fibronectin and unlabelled type I collagen respectively, but not inhibited with human immunoglobulin G or bovine serum albumin. Inhibition experiments showed that the reversibility of 125I-fibronectin binding was estimated at approximately 50%, while reversibility for 125I-collagen binding was higher than 90%. Receptors for fibronectin, its 29 kD fragment, and collagen were released from the bacterial surface by treatment at different temperatures, and surface material released at 100 degrees C inhibited binding. There was cross-inhibition for both fibronectin and collagen binding when unlabelled fibronectin and unlabelled collagen were used as inhibitors, suggesting that binding receptors for both proteins may be closely located.

• Emod I, Lafaye P, Planchenault T, Lambert Vidmar S, Imhoff JM, Keil-Dlouha V
• Potential proteolytic activity of fibronectin: fibronectin laminase and its substrate specificity.
• Biol Chem Hoppe Seyler. 1990; 371: 129-35
• Display abstract

• The purified 190-kDa fibronectin fragment produced by cathepsin D can be spontaneously activated in the presence of CaCl2. This activation generates new proteolytic activities and also results in the formation of several subfragments. One of them exhibits the activity of FN-gelatinase that preferentially splits type I denatured collagen and fibronectin (see preceding paper). In this work we describe the purification and characterization of another fragment (25 kDa), issued from the same autodigest. This fragment may be activated to yield another proteinase, that splits preferentially laminin and denatured collagen type I. This enzyme will be referred as FN-laminase. Purified FN-laminase specifically reacted with antibodies against fibronectin. The specificity of bond cleavage by FN-laminase was studied with various synthetic peptides analogous to collagen repeats. FN-laminase cleaves the Ala-Gly bond in the sequence GPAGPR; the arginine residue in position P3' is important for this cleavage. The enzyme is inhibited by pepstatin A and phenylmethanesulfonyl fluoride, like retroviral aspartic proteinases. It is also inhibited by EDTA. No inhibition was obtained with 1,10-phenanthroline or 4-chloromercuribenzoate, inhibitors of Zn-metalloproteinases or cysteine proteinases, respectively.

• Tatunashvili LV et al.
• Co-operative domains in fibronectin.
• J Mol Biol. 1990; 211: 161-9
• Display abstract

• The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.

• Lohr KM, Kurth CA, Xie DL, Seyer JM, Homandberg GA
• The amino-terminal 29- and 72-Kd fragments of fibronectin mediate selective monocyte recruitment.
• Blood. 1990; 76: 2117-24
• Display abstract

• Proteolytic fragments of fibronectin (Fn) can possess properties not inherent to intact Fn. Previously, only mixtures of low molecular weight Fn fragments, and the 120-Kd fibroblastic cell-binding segment, but not intact Fn, were shown to be selectively chemotactic for human monocytes (MOs). In order to determine if other structural domains of Fn were responsible, we tested six Fn fragments. The amino-terminal 72-Kd fragment at 1.5 microns was about 75% as potent as zymosan-activated serum (ZAS). Its amino-terminal 29-Kd degradation product at 1.0 micron was about one third as potent as ZAS. Checkerboard analysis confirmed chemotaxis. Complexing gelatin to 72-Kd fragments reduced MO chemotaxis by 28% to 30%. Reducing disulfide bonds in 29- and 72-Kd segments had no effect. A synthetic peptide containing the thrombin cleavage site between the 29- and 50-Kd segments of the 72-Kd fragment was chemotactic. The 50-, 190/170-, 35-, and 160/150/120-Kd fragments, and intact Fn were not chemotactic for MOs. The data suggest that the 72-Kd fragment and its 29-Kd subfragment are additional Fn fragments that mediate selective MO chemotaxis. We speculate that proteinases present at inflammatory sites can liberate such fragments that selectively recruit MOs.

• Sottile J, Selegue J, Mosher DF
• Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria.
• Protein Expr Purif. 1990; 1: 104-10
• Display abstract

• Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.

• Imhoff JM, Blondeau X, Planchenault T, Emod I, Keil-Dlouha V
• Collagenase activation of latent matrix-degrading proteinases from human plasma fibronectin.
• Biol Chem Hoppe Seyler. 1990; 371: 137-44
• Display abstract

• Fibronectin contains two latent gelatinolytic enzymes, FN-gelatinase and FN-laminase that can be activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. The results of this work show that Achromobacter collagenase cleaves fibronectin and generates an active FN-gelatinase. In contrast to the cathepsin D digest, the collagenase digest directly exhibits gelatinolytic activity without additional activation. The gelatinolytic activity of the total collagenase digest can be inhibited by phenylmethanesulfonyl fluoride, a serine proteinase inhibitor and by pepstatin A, an aspartic-acid proteinase inhibitor. FN-laminase activity, when assayed with its synthetic substrate GPAGPR and also with laminin was revealed after separation of the collagenase digest of fibronectin on heparin Ultrogel. FN-gelatinase and FN-laminase activities were found in heparin unretained and heparin strongly retained fractions. These results have demonstrated that in contrast to cathepsin D, Achromobacter collagenase activates two matrix-degrading proteinases from fibronectin, FN-Gelatinase und FN-Laminase.

• Khan MY, Medow MS, Newman SA
• Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin.
• Biochem J. 1990; 270: 33-8
• Display abstract

• Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.

• Holland IB, Kenny B, Steipe B, Pluckthun A
• Secretion of heterologous proteins in Escherichia coli.
• Methods Enzymol. 1990; 182: 132-43

• Zhu BC, Laine RA, Barkley MD
• Intrinsic tryptophan fluorescence measurements suggest that polylactosaminyl glycosylation affects the protein conformation of the gelatin-binding domain from human placental fibronectin.
• Eur J Biochem. 1990; 189: 509-16
• Display abstract

• Glycosylation can affect the physical and biochemical properties of the polypeptide chain in glycoproteins. Asparagine-N-linked polylactosaminyl glycosylation of the chymotryptic 44-kDa gelatin-binding domain from human placental fibronectin confers protease resistance [Zhu, B. C. R., Fisher, S. F., Panda, H., Calaycay, J., Shively, J. E. & Laine, R. A. (1984) J. Biol. Chem. 259, 3962-3970] and weaken the binding to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041-4045]. Intrinsic tryptophan fluorescence of the gelatin-binding domain was used to probe glycosylation-dependent protein conformation changes. In gelatin-binding fragments containing incrementally smaller polylactosamine oligosaccharides, the fluorescence intensity progressively decreased and the emission spectrum shifted about 7 nm to the blue. Removal of the polylactosamine chains from a highly glycosylated fragment with endo-beta-galactosidase from Escherichia freundii also quenched the protein fluorescence. The fluorescence lifetimes did not appear to be affected by the extent of glycosylation, suggesting static quenching of the tryptophan emission in the low glycosylated fragments. Acrylamide quenching studies showed that the accessibility of the tryptophans to small solutes was not altered by glycosylation. The steady-state emission anisotropy increased with decreasing polylactosamine chain length. The results indicate that the polylactosamine chains alter the tryptophan environments in the gelatin-binding domain, probably by changing the polypeptide conformation. These putative protein conformation changes may be partially responsible for the altered gelatin binding, protease resistance, and cell adhesion functions of fetal tissue fibronectin.

• Nilsson B, Abrahmsen L
• Fusions to staphylococcal protein A.
• Methods Enzymol. 1990; 185: 144-61

• Planchenault T et al.
• Potential proteolytic activity of human plasma fibronectin: fibronectin gelatinase.
• Biol Chem Hoppe Seyler. 1990; 371: 117-28
• Display abstract

• Human plasma fibronectin contains a latent proteinase that after activation cleaves gelatin and fibronectin. The autoactivation propensity of the two purified cathepsin D-produced fragments of fibronectin (190 and 120 kDa) was compared. Both polypeptides were spontaneously activated in the presence of Ca2+. This activation was inhibited by EDTA. The active gelatinase was isolated from the autodigest of the 190-kDa fragment. Among various protein substrates, including laminin and native type I and IV collagens, the purified enzyme degraded only gelatin and fibronectin. We have named this proteinase FN-gelatinase. FN-gelatinase is inhibited by phenylmethanesulfonyl fluoride and also by pepstatin A like retroviral aspartic proteinases. The amino-acid composition of the purified enzyme (35 kDa) was compared with the entire fibronectin sequence using the computer programme FIT. The optimal fit indicated that the 35-kDa fragment corresponds to the stretch # 1043-1404. This sequence contains a 93-residue segment (# 1140-1233) analogous to retroviral aspartic proteinases, comprising the sequence DTG of their putative active site.

• Guidry C, Miller EJ, Hook M
• A second fibronectin-binding region is present in collagen alpha chains.
• J Biol Chem. 1990; 265: 19230-6
• Display abstract

• The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.

• Tomkinson B, Zetterqvist O
• Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin.
• Biochem J. 1990; 267: 149-54
• Display abstract

• Tripeptidyl peptidase II (TPP II) is a large intracellular exopeptidase with an active site of the subtilisin type. Affinity-purified hen antibodies against human erythrocyte TPP II cross-reacted with fibronectin in an immunoblot analysis. Furthermore, antibodies against human fibronectin cross-reacted with TPP II. Antibodies against a 65 kDa cell-binding fragment of fibronectin specifically reacted with TPP II, whereas antibodies against the collagen-binding domain, the main heparin-binding domain or the N-terminal fibrin-binding domain did not react. Moreover, the affinity-purified antibodies against TPP II reacted with a 105 kDa cell-binding fragment of fibronectin but not with the fibrin-binding domain or the collagen-binding domain. When native TPP II was dissociated into smaller units through dialysis against a dilute Tris buffer, it could be digested by chymotrypsin into three stable fragments of 70 kDa, 42 kDa and 20 kDa. It could be demonstrated that the 42 kDa fragment was specifically recognized by antibodies against the 65 kDa cell-binding fragment of fibronectin. Furthermore, labelling with di-[3H]isopropyl phosphorofluoridate and N-terminal sequence determination showed that the 70 kDa fragment contained the active-site serine residue. In conclusion, our findings suggest that one domain of the TPP II molecule bears structural resemblance to a cell-binding fragment of fibronectin.

• Kumar A et al.
• Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain.
• J Biol Chem. 1990; 265: 17094-100
• Display abstract

• A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.

• Visai L, Speziale P, Bozzini S
• Binding of collagens to an enterotoxigenic strain of Escherichia coli.
• Infect Immun. 1990; 58: 449-55
• Display abstract

• An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity (G. Froman, L. M. Switalski, A. Faris, T. Wadstrom, and M. Hook, J. Biol. Chem. 259:14899-14905, 1984). We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides [alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4] were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.

• Yaoi Y, Hashimoto K, Koitabashi H, Takahara K, Ito M, Kato I
• Primary structure of the heparin-binding site of type V collagen.
• Biochim Biophys Acta. 1990; 1035: 139-45
• Display abstract

• The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.

• Westerlund B, Kuusela P, Vartio T, van Die I, Korhonen TK
• A novel lectin-independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin.
• FEBS Lett. 1989; 243: 199-204
• Display abstract

• Binding of P fimbriae of uropathogenic Escherichia coli to purified human fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin. Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by alpha-D-Gal-(1-4)-beta-D-Gal-1-O-Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I-labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.

• Bychkova VV, Berman AE
• [Isolation and purification of collagen proteins using biospecific chromatography on a sorbent with collagen-binding fragment of fibronectin]
• Vopr Med Khim. 1989; 35: 129-33
• Display abstract

• A new procedure is described for isolation of collagens from various preparations containing small amounts of these proteins. The procedure is based on specific affinity of collagens of various types to one of peptide fragments of fibronectin--collagen-binding peptide (CBP). Collagens were isolated after single-step chromatography of tissue homogenate on the sorbent containing immobilized CBP. High specificity and reproducibility of the procedure was shown when various preparations were used: chicken embryo proteins with total radioactive label. Proteins synthesized in fibroblast culture; polypeptides synthesized in cell-free system of protein synthesis.

• Ingham KC, Brew SA, Migliorini MM
• Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules.
• J Biol Chem. 1989; 264: 16977-80
• Display abstract

• Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.

• Barry EL, Mosher DF
• Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments.
• J Biol Chem. 1989; 264: 4179-85
• Display abstract

• Factor XIIIa cross-links plasma fibronectin as it is being assembled into the extracellular matrix of cultured human skin fibroblasts (Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 262, 10464-10469). We have further characterized this process. Fibroblasts were metabolically labeled with proline in the presence or absence of ascorbate and Factor XIIIa. Endogenous fibronectin in the extracellular matrix was cross-linked by Factor XIIIa. There was no evidence for cross-linking of collagenous proteins. Fibro-blast cell layers were incubated with iodinated 27-kDa heparin-binding or 70-kDa collagen- and heparin-binding amino-terminal fibronectin fragments. Factor XIIa cross-linked the fragments into high molecular weight aggregates. The amounts of cross-linked fragments reaches a steady state after 1 to 2 h, whereas intact fibronectin continues to be cross-linked for 24 h. When fibroblast cell layers were pulsed with iodinated fibronectin or amino-terminal fragments and Factor XIIIa was included in the chase media, the high molecular weight aggregates were formed in a step-wise manner. The smallest cross-linking steps were to high molecular weight extracellular matrix molecules forming approximately 270-, 300-, and 440-kDa complexes for the 27-kDa fragment, 70-kDa fragment, and intact fibronectin, respectively. When iodinated fibronectin was bound to fibroblast cell layers and chased into the matrix pool in the absence of Factor XIIIa, it could also be cross-linked into high molecular weight complexes when Factor XIIIa was added to the media. These results, therefore, indicate that both cellular and plasma fibronectin and amino-terminal fragments are cross-linked specifically by Factor XIIIa, that the cross-linking is probably to other fibronectin molecules rather than to collagenous proteins, and that both assembling and assembled fibronectin are substrates for Factor XIIIa.

• Delannoy P, Montreuil J
• Deglycosylation of the chymotryptic collagen-binding fragment of human plasma fibronectin does not modify its affinity to denatured collagen.
• FEBS Lett. 1989; 247: 25-7
• Display abstract

• N-Glycanase deglycosylation of purified 44 kDa chymotryptic collagen-binding domain from human plasma fibronectin does not significantly modify its behavior on gelatin affinity chromatography. This indicates that carbohydrates do not play any role in the binding affinity of fibronectin to collagen. The influence of changes in glycosylation on the biological functions of fibronectin is discussed.

• Lewandowska K, Kaetzel CS, Zardi L, Culp LA
• Binding characteristics of complementary fibronectin fragments on artificial substrata.
• FEBS Lett. 1988; 237: 35-9
• Display abstract

• Various properties have been evaluated for the binding to tissue culture substrata of proteolytic fragments of human plasma or cellular fibronectins containing complementary sequences from the individual and alternatively spliced chains, since related fragments are known to yield differing adhesive responses from cells. These studies utilize ELISA methods and a polyclonal antiserum directed to human pFN for direct measurement or an occupancy test utilizing anti-albumin. Very related fragments (with or without an extra type III homology unit or extra domaina or b) have significantly different properties in substratum binding and such differences provide a partial explanation for alteration of cellular adhesive responses on such fragments.

• Ingham KC, Brew SA, Isaacs BS
• Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen.
• J Biol Chem. 1988; 263: 4624-8
• Display abstract

• Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.

• Karasaki Y, Gotoh S, Kubomura S, Higashi K, Hirano H
• Nucleotide sequence analysis of the DNA binding region of the chicken fibronectin gene.
• J UOEH. 1988; 10: 357-63
• Display abstract

• We have determined the nucleotide sequence of 2.0 kb EcoRI segment from the clone lambda FC32 of the genomic chicken fibronectin gene, which is called DNA binding domain. This segment overlapped another clone lambda FC36 and contained three exons which were 16, 17 and 18. They were classified as Type III repeat as originally shown in bovine plasma fibronectin. The average homologies of these three exons among the chicken, rat and human fibronectins in amino acid level are very high (87-98%) compared with that (79-88%) of the exons in the cell binding domain, indicating that this region is highly conservative during the evolution.

• Khan MY, Jaikaria NS, Frenz DA, Villanueva G, Newman SA
• Structural changes in the NH2-terminal domain of fibronectin upon interaction with heparin. Relationship to matrix-driven translocation.
• J Biol Chem. 1988; 263: 11314-8
• Display abstract

• The effects of heparin and various related polysaccharides on the circular dichroic spectra of fibronectin and its 31-kDa NH2-terminal tryptic fragment were studied. These effects were evaluated with respect to (i) spectral features of the native proteins that are sensitive to pH denaturation and breaking of disulfide bonds, (ii) sensitivity of spectral changes to Ca2+, and (iii) the fibronectin-dependent interfacial interaction known as "matrix-driven translocation." We found that native heparin causes an attenuation of the positive CD peak at 228 nm with both the intact protein and the fragment, and causes a small but reproducible red shift in the spectrum of the fragment. All of these changes are analogous to spectral changes seen with denaturation or reduction of the proteins. In contrast to the situation with the intact protein, the heparin-induced spectral changes in the fragment were abolished in the presence of 10 mM Ca2+. Desulfation of heparin lessened or destroyed its ability to induce these changes, and carboxymethylated heparin and dextran sulfate induced different kinds of spectral alterations. Fibronectin and heparin determinants required for the induction of the characteristic spectral shift of the NH2-terminal domain corresponded to those required for matrix-driven translocation, suggesting that the associated conformational change in fibronectin plays a role in this biophysical effect.

• Clark RA, Wikner NE, Doherty DE, Norris DA
• Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.
• J Biol Chem. 1988; 263: 12115-23
• Display abstract

• Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.

• Obara M, Kang MS, Yamada KM
• Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function.
• Cell. 1988; 53: 649-57
• Display abstract

• Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.

• Molla A, Tanase S, Hong YM, Maeda H
• Interdomain cleavage of plasma fibronectin by zinc-metalloproteinase from Serratia marcescens.
• Biochim Biophys Acta. 1988; 955: 77-85
• Display abstract

• Limited proteolysis of porcine plasma fibronectin by the 56 kDa proteinase (56K proteinase) (EC 3.4.24.4) from Serratia marcescens released six polypeptides: a 27 kDa peptide, the heparin-binding domain which comprises the NH2-terminal end; a 50 kDa peptide, a mid-molecule that mediates binding to gelatin or collagen; a 160 kDa peptide, that contained the heparin-binding domain with cell-spreading activity; and a 140 and a 20 kDa peptide which released from the 160 kDa peptide. Each fragment was purified and characterized by its chemical and biological properties, and it was found that they were respectively different domains. Both the 160 and the 140 kDa peptide contained one cysteine per mole of peptide. The 160 kDa peptides were connected by a 6 kDa peptide, which was present at the COOH-terminal end of the molecule and was biologically inactive. Only 6 kDa peptide contained a disulfide bond and produced 3 kDa peptide after reduction, whereas other fragments did not change with or without reduction on SDS-polyacrylamide gel electrophoresis. NH2-terminal sequence analyses of the released peptides showed that the 56K proteinase cleaved the fibronectin between the Arg-Thr (located at two different sites), Leu-Ser and Gln-Glu bonds. Out of 118 Arg residues, there are nine sequences containing Arg-Thr, and two of them near or at an interdomain location (at Arg 259 and 2239) were cleaved. Out of 124 Leu residues, there are 11 Leu-Ser sequences and only one, at 687, was cleaved. The above fragments with functional domain activity could be aligned according to the previously reported amino-acid sequence of human or bovine plasma fibronectin. The treatment of fibroblast cells by the 56K proteinase resulted in loss of morphological integrity and extracellular matrix.

• Easter DW, Hoyt DB, Ozkan AN
• Immunosuppression by a peptide from the gelatin binding domain of human fibronectin.
• J Surg Res. 1988; 45: 370-5
• Display abstract

• Circulating immunosuppressive peptides are found in conjunction with elevated protease activity in injured patients' serum. Previous work suggests that fibronectin may be a source of these peptides. Elastase-generated fibronectin degradation products were chromatographically separated and assessed for immunosuppressive capacity in the neutrophil chemotaxis and mixed lymphocyte reaction bioassays. The suppressive fibronectin degradation products fraction 22 inhibited chemotaxis by 53% (P less than 0.01) and mixed lymphocyte reaction by 41% (P less than 0.001). Incubation of the suppressive fraction 22 with gelatin, neuraminidase, or anti-SAP (suppressor active peptide) antibody reverses the chemotaxis inhibition to control values. These results indicate that an elastase-generated fragment of fibronectin, which potently inhibits neutrophil and lymphocyte activity, is located within the sialated gelatin binding portion of the molecule. Immunosuppression reversal by anti-SAP antibody, and the requirement for sialic acid, suggests similarity between fraction 22 and SAP, although other explanations are plausible. This peptide fragment from fibronectin may impact on the clinical immunosuppression seen in patients following severe trauma.

• Tressel T, Shively JE, Pande H
• Human placental fibronectin: demonstration of structural differences between the A and B chains in the extra domain-A region.
• Arch Biochem Biophys. 1988; 266: 639-43
• Display abstract

• Fibronectins are a class of cell adhesion proteins produced from a single gene. Soluble plasma fibronectin plays a role in wound healing and the insoluble cellular fibronectin form anchors cells to the substrata. The proteins possess multiple macromolecular binding domains including collagen, fibrin, and heparin. Alternative RNA splicing in at least three regions (ED-A, ED-B, and III CS) is responsible for this fibronectin polymorphism. We have been studying this polymorphism at the protein level in placental fibronectin, a poorly soluble form of cellular fibronectin. Cathepsin D-digested placental fibronectin applied to a heparin-agarose column and eluted with a NaCl stepwise gradient (0.1, 0.3, 0.5 M) gave two polypeptides (80-100 and 65 kDa) in the 0.3 M NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the carboxy-terminal heparin-binding domain) and IST-9 (specific for the ED-A portion of fibronectin) suggest that both peptides contain the carboxy-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the ED-A segment. The two peptides were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to Polybrene-coated polyvinyl difluoride membranes, and characterized by microsequence analysis. This analysis confirmed that both fragments start with the same amino acid sequence, 17 amino acids before the start of ED-A. These results demonstrate that placental fibronectin is a heterodimer, structurally distinct from plasma fibronectin due to the presence of a unique domain modification that is not seen in the plasma form.

• Brentani RR, Ribeiro SF, Potocnjak P, Pasqualini R, Lopes JD, Nakaie CR
• Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach.
• Proc Natl Acad Sci U S A. 1988; 85: 364-7
• Display abstract

• It has been shown that a significant correlation is seen when the hydropathy scores of amino acids encoded by the coding strand of double-helical DNA are plotted against those of the noncoding strand. Thus, peptides encoded by complementary DNA strands might form amphiphilic structures and bind one another. We have used this approach to study the interaction between fibronectin (FN) and its cell receptor. Taking into consideration the nucleotide sequence from published rat cDNA clones that corresponds to the cell binding site (Arg-Gly-Asp-Ser) in the FN molecule, the deduced amino acid sequence found for the putative receptor binding site was Trp-Thr-Val-Pro-Thr-Ala. This peptide was chemically synthesized and coupled to an AH-Sepharose column. FN bound appreciably to this column and was eluted much more efficiently by a solution of Arg-Gly-Asp-Ser-containing peptide than by a solution of related but inactive Arg-Gly-Glu-Ser-containing peptide. Binding of labeled FN to receptor-rich MG63 human osteosarcoma cells was inhibited by the hexapeptide. The hexapeptide Gly-Ala-Val-Ser-Thr-Ala predicted similarly from the nucleotide sequence of human FN was equally efficient in such inhibition. Antibodies produced against Trp-Thr-Val-Pro-Thr-Ala recognized with equal efficiency Gly-Ala-Val-Ser-Thr-Ala in an ELISA assay. Furthermore, they were able to recognize a single 140-kDa band in whole-cell extracts from Chinese hamster ovary cells, attesting to their specificity. Identification of the recognized protein was provided by showing that this antibody was also able to bind to affinity-purified FN receptor from human osteosarcoma MG63 cells.

• Quade BJ, McDonald JA
• Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats.
• J Biol Chem. 1988; 263: 19602-9
• Display abstract

• Fibronectin is organized into disulfide cross-linked, insoluble pericellular matrix fibrils by fibroblasts in vitro. Two sites, the Arg-Gly-Asp-Ser-containing cell attachment domain and a site located in the first 70 kDa of fibronectin, are required for matrix assembly. The first 70 kDa of fibronectin contain two structural motifs termed type I and type II homologies, which are repeated nine and two times, respectively. Previous work has implicated the amino-terminal region and the carboxyl terminus containing three type I repeats in matrix assembly, suggesting that type I repeats possess binding activity essential for fibronectin matrix assembly. To test this hypothesis, we developed a sensitive capture immunoassay to quantify insoluble matrix fibronectin and tested a panel of fibronectin fragments, containing all of the type I repeats found in the intact protein, for their ability to inhibit matrix assembly. Only fragments containing the first five type I repeats inhibited fibronectin matrix assembly, although sequences carboxyl-terminal to this domain enhanced this activity. Additional evidence for the specific recognition of the amino-terminal type I repeats by matrix assembling cells was found when the reversible, detergent-sensitive binding of a 125I-labeled fragment containing the first five type I repeats (29 kDa) to cell monolayers was studied. Only monolayers of cell lines that incorporate fibronectin into a fibrillar matrix specifically bound 125I-labeled 29 kDa. Binding of the radiolabeled amino-terminal fragment to matrix-forming cells was inhibited by unlabeled fragments containing the first five type I repeats but not by unlabeled fragments containing the remaining seven type I repeats. Matrix assembly is therefore not a generalized property of type I repeats. Rather, a critical site is located within the first 29 kDa of fibronectin.

• Duffaud GD, March PE, Inouye M
• Expression and secretion of foreign proteins in Escherichia coli.
• Methods Enzymol. 1987; 153: 492-507

• Obara M, Kang MS, Rocher-Dufour S, Kornblihtt A, Thiery JP, Yamada KM
• Expression of the cell-binding domain of human fibronectin in E. coli. Identification of sequences promoting full to minimal adhesive function.
• FEBS Lett. 1987; 213: 261-4
• Display abstract

• Two cDNA subfragments containing the cell-attachment site of human fibronectin (FN) were expressed as beta-galactosidase fusion proteins in E. coli. The products were purified to homogeneity by monoclonal antibody affinity chromatography and assayed for activity in a standard cell-adhesion assay. A fusion protein containing an 80 kDa fragment of human FN appeared functionally equivalent to intact FN purified from human plasma, whereas a truncated fusion protein of 33 kDa still containing a previously postulated cell-attachment site was approx. 50-fold less active. Our study establishes a system for analyzing adhesive protein function by DNA manipulation, rules out any major role for eukaryotic post-translational modifications in FN adhesive function, and localizes additional functional activity to a 1.3 kb region.

• Seidah NG, Manjunath P, Rochemont J, Sairam MR, Chretien M
• Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin.
• Biochem J. 1987; 243: 195-203
• Display abstract

• Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109.

• Newman SA, Frenz DA, Hasegawa E, Akiyama SK
• Matrix-driven translocation: dependence on interaction of amino-terminal domain of fibronectin with heparin-like surface components of cells or particles.
• Proc Natl Acad Sci U S A. 1987; 84: 4791-5
• Display abstract

• During the process of matrix-driven translocation, certain types of cells or polystyrene latex beads are transported between compositionally different regions of a collagen matrix. Under appropriate conditions this translocation depends on an interaction between the cell or particle surface and fibronectin. We now show that this interaction takes place at a site located within the first 31 kDa of the amino-terminal end of the fibronectin molecule. Using defined fibronectin fragments and monoclonal antibodies directed against specific fibronectin domains, this site is established as both necessary and sufficient for the promotion of matrix-driven translocation. Competition experiments using heparin, heparan sulfate, and other sulfated polysaccharides show that this fibronectin site interacts with heparin-like cell or particle surface components in promoting matrix-driven translocation. Treatment of cells with heparinase renders them unresponsive to the translocational effect. An antibody directed against the amino-terminal domain of fibronectin completely inhibits matrix-driven translocation without interfering with heparin binding, suggesting that a post-binding conformational change in fibronectin may be required for promotion of the effect.

• McDonald JA et al.
• Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix.
• J Biol Chem. 1987; 262: 2957-67
• Display abstract

• Fibroblasts organize the modular cell-adhesive glycoprotein fibronectin into a highly structured pericellular matrix by poorly understood mechanisms. Previous studies implicated an amino-terminal domain in matrix assembly and suggested that fibronectin's cell-adhesive domain and the corresponding fibroblast receptor were not involved in this process. To further elucidate the fibronectin region(s) involved in matrix assembly, we mapped a library of proteolytic fragments and antibodies to various fibronectin domains. The fragments and antibodies were used to probe the role of fibronectin's amino-terminal and cell-adhesive domains in a fibroblast matrix assembly assay. We found that fibronectin fragments including the first 25-kDa sequence of fibronectin and antibodies to amino-terminal domains inhibited pericellular matrix assembly. Polyclonal antibodies to the 40-kDa collagen binding domain following the 25-kDa amino-terminal domain also inhibited matrix assembly. However, collagen binding is not required for matrix assembly as neither monoclonals blocking collagen binding nor purified collagen binding domains themselves inhibited matrix assembly. Therefore, the amino-terminal region of fibronectin contains a site important in matrix assembly, and most activity is present in the first 25-kDa of fibronectin. Fibronectin's cell-adhesive domain and the fibroblast receptor binding to this domain also play an important role in fibronectin matrix assembly. Apart from a monoclonal antibody to the amino-terminal domain, only monoclonal antibodies binding to fibronectin's cell-adhesive domain and inhibiting cell adhesion also inhibited matrix assembly. In addition a 105-kDa fragment containing the cell-adhesive domain inhibited matrix assembly. We conclude that at least two discrete and widely separated sites in fibronectin with different binding properties--the carboxyl-terminal fibroblast cell-adhesive domain and an amino-terminal matrix assembly domain localized primarily within the first 25 kDa--are required for fibronectin pericellular matrix assembly by fibroblasts. Fibronectin's cell-adhesive domain and its cell surface-receptor complex appear to be involved in the matrix assembly process prior to a step involving the amino-terminal domain. We believe that this step is likely to be the initiation of cell-associated fibronectin fibril formation by the fibronectin-adhesive-receptor complex.

• Pande H et al.
• Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain.
• Eur J Biochem. 1987; 162: 403-11
• Display abstract

• Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.

• Chhatwal GS, Blobel H
• Heterogeneity of fibronectin reactivity among streptococci as revealed by binding of fibronectin fragments.
• Comp Immunol Microbiol Infect Dis. 1987; 10: 99-108
• Display abstract

• Streptococci belonging to serological groups A, B, C, G, L and U were studied for their interaction with 125I-labelled fibronectin and its fragments. Fibronectin purified from humans plasma by affinity chromatography on gelatin-agarose and heparin-agarose was cleaved by thrombin into a 29,000 Dalton and a 210,000 Dalton fragments. Terminal analysis of purified fragments indicated that 29,000 fragment was from amino-terminal and 210,000 fragment from carboxyl-terminal domain of fibronectin. Binding of fibronectin was observed in all streptococci except those of group B. Streptococci of groups A, G, L, U and S. equisimilis reacted only with 29,000 Dalton fragment whereas S. dysgalactiae, S. zooepidermicus and S. equi reacted only with 210,000 Dalton fragment. The streptococcal binding sites for these two fragments were distinct from each other. Fibrinogen blocked the binding of 210,000 Dalton fragment but not of 29,000 Dalton fragment. Trypsinization of streptococci did not affect their binding sites for 210,000 Dalton fragment but destroyed those for 29,000 Dalton fragment. The results indicate that the streptococci of group A and G as well as S. equisimilis which are mainly pathogenic in humans bind amino-terminal fragment of fibronectin. This may facilitate the adherence of these pathogens. On the other hand, the streptococci isolated from animal infections had different binding sites recognized only by carboxyl-terminal part of fibronectin.

• Homandberg GA, Kramer-Bjerke J
• Thrombospondin binds to amino-terminal fragments of plasma fibronectin.
• Thromb Res. 1987; 48: 329-35
• Display abstract

• Thrombospondin is a 420-kD trimeric glycoprotein that can bind type V collagen, heparin, fibrinogen and certain cells and may be one of the lectins responsible for platelet aggregation. Thrombospondin binds another glycoprotein, fibronectin, that is also released during platelet aggregation and also binds similar ligands. This work shows that the amino-terminal 29-kD segment of fibronectin binds thrombospondin, the interaction occurs within minutes, and one 29-kD molecule binds per thrombospondin subunit. The interaction was not inhibited by fibrinogen, type V collagen, or heparin. Two subfragments of the 29-kD fragment, an amino-terminal 20-kD and a carboxyl-terminal 8-kD subfragment, the latter containing a single disulfide-rigidified type I loop of the five homologous loops in the 29-kD fragment, reacted with thrombospondin while the reduced counterparts did not.

• Papp B, Kovacs T, Lerant I, Nagy Z, Machovich R
• Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin.
• Biochim Biophys Acta. 1987; 925: 241-7
• Display abstract

• The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.

• Homandberg GA
• Characterization of the interactions of an amino-terminal fibronectin fragment with the native molecule: implications for polymerization of fibronectin.
• Biopolymers. 1987; 26: 2087-98

• Homandberg GA
• Inaccessibility to ligands of the amino-terminal region of plasma fibronectin.
• Thromb Res. 1987; 48: 321-7
• Display abstract

• Fragments of plasma fibronectin can display properties not expressed by fibronectin. This work shows that the relative affinities of smaller fragments for gelatin or heparin increases with decreasing fragment size. When heparin or gelatin were added to fragments or to fibronectin, the affinities for the alternate ligand increased. The UV and CD spectra of a mixture of amino-terminal 29-kD and 50-kD fragments differed from that of the precursor, the intact 72-kD fragment, suggesting differences in secondary structure. Therefore, biologic activities of the 29-kD and 50-kD segments in fibronectin may be suppressed by structural constraints but may be expressed more fully by interaction with ligands.

• Charonis AS, Tsilibary EC, Saku T, Furthmayr H
• Inhibition of laminin self-assembly and interaction with type IV collagen by antibodies to the terminal domain of the long arm.
• J Cell Biol. 1986; 103: 1689-97
• Display abstract

• Laminin is a major glycoprotein of the basement membrane. Although its precise localization and orientation within this structure is unknown, it is presumably anchored to other macromolecules such as type IV collagen or proteoheparan sulfate. In vitro, laminin has the ability to self-assemble and to bind to type IV collagen molecules at distinct sites. To identify more precisely the domains of the complex, cross-shaped laminin molecule that are involved in these interactions, images of laminin-laminin dimers and laminin-type IV collagen complexes obtained by the rotary shadowing method were analyzed. We observed that the complex domain at the end of the long arm of laminin is predominantly involved in these interactions. By using Fab fragments of antibodies specific for a peptide fragment derived from this complex domain, it is shown that laminin self-assembly is inhibited in their presence, as measured by turbidity and by electron microscopy. In addition, these antibodies inhibit the specific interaction of laminin with type IV collagen. These data suggest that the complex domain at the end of the long arm of laminin contains binding sites of potential importance for the assembly of basement membranes.

• Sato TN, Hayashi M
• On the origin of species specificity of fibronectin. Immunological identification of a species-specific domain of human fibronectin.
• J Biochem (Tokyo). 1986; 99: 653-61
• Display abstract

• We have investigated whether species-specific epitopes of human fibronectin are localized at a specific domain of fibronectin using rabbit polyclonal antibodies. Tryptic fragments of human fibronectin were tested for reactivity with anti-human fibronectin antibody, which had been previously absorbed with other animal fibronectin to establish species specificity. Human-specific epitopes were found to be present on 75,000, 65,000, and 42,000 dalton fragments. The 42,000-dalton fragment shares almost all the epitopes with the 75,000 and 65,000 dalton fragments. It does not promote BHK cell spreading, whereas the 75,000 and 65,000 dalton fragments do. The amino acid sequence from the amino terminus of the 42,000-dalton fragment is Asp/Gly-Gln/Val-?-Ile-Val-, which is almost identical to the sequence Asp-Gln-Cys-Ile-Val- located in the carboxyl terminal 1/3 of the collagen-binding domain of human fibronectin (Kornblihtt et al. (1985) EMBO J. 4, 1755-1759). These results suggest that human fibronectin bears human-specific epitopes mainly on the amino-terminal half of domain 4 (Hayashi & Yamada (1983) J. Biol. Chem. 258, 3332-3340) located between the collagen and cell binding domains almost at the center of the fibronectin polypeptide. The domain specific for human fibronectin may be a general species-specific domain of animal fibronectins.

• Thompson LK, Horowitz PM, Bentley KL, Thomas DD, Alderete JF, Klebe RJ
• Localization of the ganglioside-binding site of fibronectin.
• J Biol Chem. 1986; 261: 5209-14
• Display abstract

• It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule.

• Griffin CA, Calaycay J, Shively JE, Smith RL
• Two plasma fibronectin fragments with different gelatin-binding properties.
• Thromb Res. 1986; 43: 469-77
• Display abstract

• Two subtilisin-generated fragments of human plasma fibronectin exhibit significantly different gelatin affinities. One fragment of 60 kdaltons elutes from gelatin-Sepharose, after intact fibronectin, with pH 5.5, 50 mM citrate, 0.1 M NaCl. A 40 kdalton fragment, which can be derived from the 60 kdalton fragment, remains bound to gelatin under the same conditions. Furthermore, upon urea gradient elution, the 60 kdalton fragment dissociates from gelatin before the smaller fragment. Of the two fragments, the 60 kdalton fragment also demonstrates affinity for heparin. Identical amino-terminal sequences for the two fragments indicate that the heparin-binding activity resides in the difference region at the carboxyl-terminal of the 60 kdalton fragment.

• Keller KM, Keller JM, Kuhn K
• The C-terminus of type I collagen is a major binding site for heparin.
• Biochim Biophys Acta. 1986; 882: 1-5
• Display abstract

• The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH 6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15-1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TCB, was shown to have greater binding affinity for heparin than the N-terminal TCA fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind.

• Savill CM, Ayad SR
• The interaction of fibronectin fragments with cultured cells and isolated nuclei.
• Anticancer Res. 1986; 6: 315-9
• Display abstract

• 125I-fibronectin was partially digested by trypsin and the interaction of the labelled fragments produced with cultured cells and isolated nuclei was studied. Fragments not only became associated with the surface of cultured cells but a significant amount of radioactivity remained associated with the cell pellet following trypsin treatment, indicating that certain fragments may have been internalized. In addition to binding at the cell surface, fibronectin fragments also showed the ability to bind to isolated nuclei in a dose-dependent nonsaturable manner. Considerably reduced binding was observed at 4 degrees C compared to that at 25 degrees C. Cells allowed to grow and proliferate in the presence of 125I-fibronectin also incorporated the label onto their cell surface. A significant proportion of radioactivity again remained associated with the cell pellet after trypsin treatment and on subsequent fractionation approximately one-half of this radioactivity was found to co-purify with the isolated nuclear pellet in the form of low molecular weight peptides Mr 20,000 to 50,000.

• Savill CM, Ayad SR
• The mitogenic activity of a heparin-binding fibronectin fragment (Mr 35,000) produced by cathepsin D digestion.
• Anticancer Res. 1986; 6: 321-6
• Display abstract

• Fibronectin was partially digested using the lysosomal proteinase cathepsin D and the fragments produced tested for their effect on the rate of DNA synthesis in quiescent Chinese Hamster Fibroblasts (CH23). A mitogenically active digest was produced after digestion of fibronectin with cathepsin D for 2 to 3h at a 1:200 (w/w) ratio of enzyme to substrate at 37 degrees C. Affinity chromatography of the active digest on both gelatin and heparin 4BCl columns demonstrated that the active fragment was gelatin-nonbinding but heparin-binding, and therefore derived from one of the two or three heparin binding domains of intact fibronectin. The active fragment was found to have a Mr35,000 by gel filtration on sephacryl S200. A dose-response curve of the mitogenic fragment purified by gel filtration against DNA synthesis showed it to be maximally active at a concentration of approximately 10 micrograms/ml.

• Homandberg GA, Erickson JW
• Model of fibronectin tertiary structure based on studies of interactions between fragments.
• Biochemistry. 1986; 25: 6917-25
• Display abstract

• Human plasma fibronectin aggregates in solution and is thought to form fibrils on cell surfaces, perhaps by self-associating and by interacting with other components such as proteoglycans. We have localized the self-association domains by testing the ability of various fragments of fibronectin to interact with each other. Complexation between fluorescamine-labeled fragments and unlabeled fragments or whole molecules was assessed by gel filtration high-performance liquid chromatography. The fragments studied included nonoverlapping fragments that are situated on the fibronectin polypeptide chain in the following order, beginning from the amino terminus: the 29-, 50-, 120-, 35-, and 25-kDa fragments, as well as multiple-domain fragments of 72 kDa containing the 29- and 50-kDa segments, a fragment of 150 kDa containing the 120- and 35-kDa segment, a fragment of 190 kDa containing the 120- and 35-kDa segments, a fragment of 190 kDa containing the 50-, 150-, and 25-kDa segments, and a 45-kDa fragment containing the 35-kDa segment. The amino-terminal 29-kDa fragment bound to the carboxyl-terminal heparin-binding (Hep II) 35-kDa fragment as well as the 150- and 190-kDa fragments that contain the 35-kDa segment. On the other hand, carboxyl-terminal 35- and 45-kDa Hep II containing fragments bound to each other as well as to amino-terminal 29- and 72-kDa fragments and to the 190-kDa fragment. Further, the 25-kDa carboxyl-terminal fibrin-binding fragment bound the 190-kDa fragment, the only fragment containing the 25-kDa segment.(ABSTRACT TRUNCATED AT 250 WORDS)

• Sekiguchi K, Klos AM, Hirohashi S, Hakomori S
• Human tissue fibronectin: expression of different isotypes in the adult and fetal tissues.
• Biochem Biophys Res Commun. 1986; 141: 1012-7
• Display abstract

• The isotype of fibronectin present in human adult and fetal lung tissues was studied by limited proteolysis by cathepsin D followed by immunoblot analysis with domain-specific antibodies. The results indicate that the fibronectin in the adult lung tissue is predominantly the plasma-type whereas the fibronectin in the fetal lung tissue is more related to the cellular-type than to the plasma-type. Thus, it appears that the fibronectin isotype in tissue switches from the cellular-type to the plasma-type during ontogenesis.

• Siri A, Balza E, Carnemolla B, Castellani P, Borsi L, Zardi L
• DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin.
• Eur J Biochem. 1986; 154: 533-8
• Display abstract

• We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.

• Castellani P, Siri A, Rosellini C, Infusini E, Borsi L, Zardi L
• Transformed human cells release different fibronectin variants than do normal cells.
• J Cell Biol. 1986; 103: 1671-7
• Display abstract

• Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.

• Aycock RS, Raghow R, Stricklin GP, Seyer JM, Kang AH
• Post-transcriptional inhibition of collagen and fibronectin synthesis by a synthetic homolog of a portion of the carboxyl-terminal propeptide of human type I collagen.
• J Biol Chem. 1986; 261: 14355-60
• Display abstract

• We evaluated the effects of a synthetic copy of a highly conserved portion (residues 225-246) of the COOH-propeptide of human pro-alpha 2(I) procollagen on collagen, fibronectin, and total protein synthesis by human fibroblasts. Incubation of COOH-propeptide 225-246 with fibroblasts resulted in a concentration-dependent inhibition of both type I procollagen and fibronectin when compared with controls; a 50% inhibition of both fibronectin and type I collagen was observed at a concentration of 45 microM. Since the overall cellular protein synthesis was only minimally affected, COOH-propeptide appeared to specifically inhibit collagen and fibronectin synthesis. The peptide was nontoxic to cells and the inhibition was completely reversible upon removal of the peptide. We measured the steady-state levels of mRNAs coding for procollagen, fibronectin, and beta-actin by hybridization to specific recombinant cDNA probes; there was no significant change in the steady-state level of mRNAs of the three proteins. These results strongly suggest that the biosynthesis of procollagen and fibronectin in COOH-propeptide-treated cells is inhibited at a post-transcriptional level. These data establish a link between collagen and fibronectin synthesis and further define the important interaction of these molecules in the formation of the extracellular matrix.

• Keil-Dlouha V, Planchenault T
• Potential proteolytic activity of human plasma fibronectin.
• Proc Natl Acad Sci U S A. 1986; 83: 5377-81
• Display abstract

• Evidence is presented that fibronectin (FN) polypeptide chain contains a latent proteinase. Human plasma FN was cleaved with cathepsin D into three main fragments: 140-kDa and 70-kDa single-chain and 140-kDa double-chain polypeptides. Their separation was achieved according to their affinity for heparin-Sepharose. A single-chain 140-kDa fragment (H-1) was eluted in the first peak. This peptide corresponds to the already described fragment that originates from the central part of FN; it contains a low-affinity heparin-binding site, one free SH group, and a cell-binding site. After reduction and further purification by preparative polyacrylamide gel electrophoresis, this fragment revealed a spontaneous decomposition, which could be attributed to proteolytic degradation. The subfragments, ranging from 25 to 95 kDa, yielded the same proteolytically active doublet of 28-30 kDa when tested by NaDodSO4/polyacrylamide gel electrophoresis in a gel containing copolymerized gelatin or fibrinogen. The proteolytic activity was inhibited by specific SH proteinase inhibitors. The proteinase forms a labeled complex after its incubation with 125I-labeled cystatin. Neither FN, cathepsin D, nor any products from previous purification steps were proteolytically active under the conditions of the assay. It was suggested that the same fragment may also yield an inhibitor, since structural analogies were found between the cell-binding region of FN and SH proteinase inhibitors.

• Skorstengaard K, Jensen MS, Petersen TE, Magnusson S
• Purification and complete primary structures of the heparin-, cell-, and DNA-binding domains of bovine plasma fibronectin.
• Eur J Biochem. 1986; 154: 15-29
• Display abstract

• The complete amino acid sequences of the heparin-, cell- and DNA-binding domains of bovine plasma fibronectin have been determined. The fragments were generated from the 170-kDa central plasmic fragment by extensive digestion with chymotrypsin, and they contain 268, 300 and 269 amino acid residues, respectively. No half-cystines or cysteines were found in these sequences. A glucosamine-based oligosaccharide group is attached to Asn-108 in the sequence of the DNA-binding domain. Only one of the three types of internal homology found in fibronectin [Petersen et al. (1983) Proc. Natl Acad. Sci. USA 80, 137-141], namely the type III homology, occurs in these three fragments, and each of them consists of approximately three stretches of this type III homology. Part of the arrangement of peptides was derived by comparison with the partial cDNA sequence for human fibronectin recently reported [Kornblihtt et al. (1984) Nucleic Acids Res. 12, 5853-5868].

• Chernousov MA, Metsis ML, Koteliansky VE
• Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments.
• FEBS Lett. 1985; 183: 365-9
• Display abstract

• Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.

• Pande H, Calaycay J, Hawke D, Ben-Avram CM, Shively JE
• Primary structure of a glycosylated DNA-binding domain in human plasma fibronectin.
• J Biol Chem. 1985; 260: 2301-6
• Display abstract

• The complete amino acid sequence of a DNA-binding domain isolated from human plasma fibronectin after limited trypsin digestion has been obtained. It contains 132 amino acids and one biantennary glycosyl unit at residue 104, for an estimated Mr of 16,931. The fragment can be purified by a two-step procedure consisting of DNA-affinity chromatography and reverse-phase high performance liquid chromatography. It can also be purified by heparin-affinity chromatography. The domain is unusual in its susceptibility to tryptic-like cleavages even by neutral or aromatic residue-specific proteases. It has no cysteine residues and is predicted to favor a beta-sheet structure by Chou and Fasman analysis. Based on this analysis we have proposed a model which exhibits a clustering of aromatic and basic residues, consistent with similar involvement of basic and aromatic residues in other DNA-binding proteins. The net charge of the domain at neutral pH (+1, without sialic acid) argues against a nonspecific charge interaction with polyanionic macromolecules such as DNA and heparin. Internal sequence repeats occur at intervals of 30, 60, and 90 residues, thus suggesting a maximum size for a repetitive building block which gave rise to this domain.

• Forastieri H, Ingham KC
• Interaction of gelatin with a fluorescein-labeled 42-kDa chymotryptic fragment of fibronectin.
• J Biol Chem. 1985; 260: 10546-50
• Display abstract

• A 42-kDa gelatin binding fragment of human plasma fibronectin was labeled with fluorescein and its fluid-phase interaction with gelatin was investigated. At 25 degrees C in 0.1 M Tris, 0.15 M NaCl, pH 7.3, a dissociation constant, Kd = 0.6 microM, was obtained from the dependence of fluorescence polarization on gelatin concentration. An identical value was obtained for the unlabeled fragment by competition. Binding was unaffected by higher concentrations of NaCl up to 1.0 M, but increased as much as 20-fold at low ionic strength. The dependence of Kd on temperature revealed that dissociation of the complex is accompanied by an increase in entropy. Thus, the interaction is not dominated by either hydrophobic or electrostatic forces; an important role for hydrogen binding is proposed.

• Hormann H, Richter H, Jelinic V
• Fibrinmonomer binding to macrophages mediated by fibrin-binding fibronectin fragments.
• Thromb Res. 1985; 39: 183-94
• Display abstract

• Binding of 125-I-fibrinmonomer to peritoneal macrophages was investigated in dependence of plasma fibronectin and of its thrombin- or plasmin-derived fragments. Plasma fibronectin failed to enhance cell binding of 125-I-fibrinmonomer. In contrast, 30kD-fragments derived from the N-termini of the fibronectin subunits improved binding considerably. The association with the cell surface was completely inhibited by EDTA, 2-5 mM putrescine and to about 40 per cent by 0.1 mM dansylcadaverine suggesting that a transamidase-catalyzed cross-linking reaction was involved. Thrombin-derived 200kD-remnants of the fibronectin subunit chains failed to mediate cell binding of 125-I-fibrinmonomer provided they had been deprived of residual thrombin activity. Otherwise they were active and their activity was inhibited by the thrombin inhibitor hirudin. Plasmin-derived 200 kD-fragments were inactive as well.

• Calaycay J et al.
• Primary structure of a DNA- and heparin-binding domain (Domain III) in human plasma fibronectin.
• J Biol Chem. 1985; 260: 12136-41
• Display abstract

• The complete amino acid sequence of a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin has been obtained. The domain contains 90 amino acids with a calculated molecular weight of 10,225. The apparent molecular mass of this domain is 14 kDa when analyzed by sodium dodecyl sulfate-gel electrophoresis. The anomalously high molecular size estimation may be due to the inaccuracy of this method in the low range. The structure was established from microsequence analysis of the chymotryptic, tryptic, and Staphylococcus aureus protease peptides. The molecular ion of each of the chymotryptic peptides was obtained by fast atom bombardment mass spectrometry. The domain has a preponderance of basic residues with a net charge of +5 at neutral pH. The basic nature of the domain may account for its affinity for the polyanions, DNA and heparin. The predicted secondary structure is beta-sheet, in common with all of the type III internal sequence homology structures obtained for fibronectin so far. The location of the domain in fibronectin was made possible by limited thermolysin digestion and identification of the fragments and by comparison of the sequence of the 14-kDa fragment with the partial structure of bovine plasma fibronectin. The domain comprises residues 585-675 and defines a region immediately adjacent to the collagen-binding domain. Numbering domains beginning at the amino terminus, this domain is Domain III after the fibrin/heparin/actin/S. aureus binding Domain I and the collagen-binding Domain II. The domain was obtained from a larger precursor (56 kDa) which bound heparin, DNA, and gelatin. Further digestion of the 56-kDa fragment gave rise to a 40-kDa fragment which only bound gelatin, and a 14-kDa fragment which only bound heparin or DNA. The 14-kDa fragment (Domain III) marks the beginning of the type III homology region in fibronectin, for there may be up to 15 repeats of 90 amino acids. The size of this domain corresponds to one repeat of 90 amino acids and it has some sequence homology to the other type III sequences found thus far in fibronectin.

• Czop JK, Kadish JL, Zepf DM, Austen KF
• Characterization of the opsonic and monocyte adherence functions of the specific fibronectin fragment that enhances phagocytosis of particulate activators.
• J Immunol. 1985; 134: 1844-50
• Display abstract

• The functional opsonic and monocyte adherence domains within the 180,000 m.w. opsonic fibronectin fragment (180K-opFnf) that selectively augments human monocyte phagocytosis of particulate activators of the alternative complement pathway were analyzed with Fab fragments of monoclonal anti-fibronectin antibodies BC7, CE9, BD4, AB3, and CPG1, and with fragments of intact human plasma fibronectin derived by cathepsin cleavage and isolated by affinity chromatography. Monoclonals AB3 and CPG1, which recognize epitopes within 40,000 daltons of the carboxy terminus of intact fibronectin, and the cathepsin D-derived, disulfide-linked fragments that contain these epitopes each inhibited the opsonic function of 180K-opFnf. Monoclonals AB3 and CPG1 inhibited monocyte ingestion of rabbit erythrocytes (Er) by 60 and 50%, respectively, when 180K-opFnf was pretreated with 20 micrograms of these monoclonals, but neither monoclonal affected the enhanced monocyte ingestion of Er pretreated with the fibronectin fragment. The pretreatment of Er with 5 micrograms and 40 micrograms of the disulfide-linked, cathepsin D derivatives isolated from high and low affinity heparin fractions, respectively, inhibited the proportion of ingesting monocytes by 60%, but these types of fragments had little effect when concurrently incubated with the opsonic fragment and Er. Monoclonals CE9 and BD4, which recognize epitopes located adjacent to or within the cell-adhesive domain of intact fibronectin, respectively, inhibited the monocyte adherence function of 180K-opFnf, as evidence by their comparable inhibitory effects when present before or after Er were opsonized with 180K-opFnf. When 20 micrograms of monoclonals CE9 and BD4 were each introduced before and after Er were opsonized with 180K-opFnf, monocyte ingestion was inhibited by 60 and 65% and by 51 and 60%, respectively. At 42 micrograms, cathepsin D-derived, non-gelatin-binding, low affinity heparin fragments that contained both BD4 and CE9 determinants or only the BD4 determinant inhibited monocyte ingestion by 53 and 74%, respectively, when concurrently incubated with 180K-opFnf and target Er, but were without effect when used to pretreat Er before the addition of 180K-opFnf. Thus, the inhibitory effects produced by monoclonals AB3 and CPG1 and by cathepsin D-derived, disulfide-linked fragments containing their corresponding epitopes demonstrated that the opsonic domain within 180K-opFnf is immunologically similar to regions within the carboxy terminus of intact plasma fibronectin.(ABSTRACT TRUNCATED AT 400 WORDS)

• Bronner-Fraser M
• Effects of different fragments of the fibronectin molecule on latex bead translocation along neural crest migratory pathways.
• Dev Biol. 1985; 108: 131-45
• Display abstract

• Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.

• Garcia-Pardo A, Pearlstein E, Frangione B
• Primary structure of human plasma fibronectin. Characterization of a 31,000-dalton fragment from the COOH-terminal region containing a free sulfhydryl group and a fibrin-binding site.
• J Biol Chem. 1985; 260: 10320-5
• Display abstract

• The 31-kDa domain of human plasma fibronectin has been completely characterized. This fragment is located at the COOH-terminal end of the molecule immediately preceding the 3-kDa interchain disulfide-containing peptide. The 31-kDa domain was obtained after trypsin digestion of fibronectin and purified by affinity chromatography on gelatin- and heparin-Sepharose columns. The fragment eluted in the heparin-unbound fraction and was further purified by DEAE-cellulose and high performance liquid chromatography. The 31-kDa fragment contained a fibrin-binding site (fibrin II site) which was only active at physiological NaCl concentrations and therefore differed from that located in the NH2-terminal domain which also bound at lower NaCl concentrations. The 31-kDa domain bound to thiopropyl-Sepharose and was shown to contain a free sulfhydryl group located at position 35 in the sequence. To determine the complete amino acid sequence of this fragment, a trypsin digestion was performed on the reduced and alkylated 31-kDa domain, and the 17 resulting peptides were isolated by high performance liquid chromatography; their amino acid compositions and amino acid sequences have been determined, and the arrangement of peptides was achieved by comparison with the sequences deduced from human and rat cDNA clones and with a related plasmic fragment from bovine fibronectin. Comparison of these three sequences showed 23 amino acid differences between human and rat fibronectin and 16 between human and bovine fibronectin. This represents a 91 and 94% homology, respectively. An interesting finding is that the 31-kDa fragment contains a deletion of 31 residues when compared to the rat cDNA sequence. This deletion appears to represent a species difference since it is due to a shorter mRNA in the case of human fibronectin.

• Akiyama SK, Hasegawa E, Hasegawa T, Yamada KM
• The interaction of fibronectin fragments with fibroblastic cells.
• J Biol Chem. 1985; 260: 13256-60
• Display abstract

• We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed.

• Schwarzbauer JE, Paul JI, Hynes RO
• On the origin of species of fibronectin.
• Proc Natl Acad Sci U S A. 1985; 82: 1424-8
• Display abstract

• Multiple different subunits of fibronectin are known to occur and their origin has been unclear. Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing suggested an explanation for some of this diversity of fibronectin subunits. Because the alternative splicing events occur within the coding region, the mRNAs differ in coding potential. We have prepared recombinant phage containing a rat fibronectin cDNA segment that is present in some fibronectin mRNAs and not in others. This segment was inserted in the beta-galatosidase gene of lambda gt11, and fusion protein produced by lysogens of the recombinant phage was purified and used as immunogen. The resulting antisera recognized some subunits of rat and hamster fibronectins but not others, indicating that inclusion or removal of this segment gives rise to mRNAs that encode different fibronectin subunits. In particular, presence or absence of a 95 amino acid segment appears to account for differences in size among the subunits of plasma fibronectin, whose origin is therefore explained by alternative patterns of RNA splicing.

• Click EM, Balian G
• Domain structure of human plasma and cellular fibronectin. Use of a monoclonal antibody and heparin affinity to identify three different subunit chains.
• Biochemistry. 1985; 24: 6685-96
• Display abstract

• The domain structure of human plasma fibronectin was investigated by using heparin-binding and antibody reactivity of fibronectin and its proteolytically derived fragments. Digestion of human plasma fibronectin with a combination of trypsin and cathepsin D produced six major fragments. Affinity chromatography showed that one fragment (Mr 45 000) binds to gelatin and three fragments (Mr 31 000, 36 000, and 61 000) bind to heparin. The 31K fragment corresponds to NH2-terminal fragments isolated from other species. The 36K and 61K fragments are derived from a region near the C-terminus of the molecule and appear to be structurally related as demonstrated by two-dimensional peptide maps. A protease-sensitive fragment (Mr 137 000), which binds neither gelatin nor heparin but which has been shown previously to be chemotactic for cells [Postlethwaite, A. E., Keski-Oja, J., Balian, G., & Kang, A. H. (1981) J. Exp. Med. 153, 494-499], separates the NH2-terminal heparin- and gelatin-binding fragments from the C-terminal 36K and 61K heparin-binding fragments. A monoclonal antibody to fibronectin that recognized the 61K heparin-binding fragment was used to isolate a sixth fragment (Mr 34 000) that did not bind to heparin or gelatin and that represents a difference between the 61K and 36K heparin-binding fragments. Cathepsin D digestion produced an 83K heparin-binding, monoclonal antibody reactive fragment that contains the interchain disulfide bond(s) linking the two fibronectin chains at their C-termini. The data indicate that plasma fibronectin is a heterodimeric molecule consisting of two very similar but not identical chains (A and B). In contrast, enzymatic digestion of cellular fibronectin produced a 50K heparin-binding fragment lacking monoclonal antibody reactivity which suggests that the cellular fibronectin subunit is similar to the plasma A chain in enzyme susceptibility but contains a larger heparin-binding domain. A model relating the differences in the three fibronectin polypeptides to differences in published cDNA sequences is presented.

• Nagai K, Thogersen HC
• Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli.
• Nature. 1984; 309: 810-2
• Display abstract

• High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences. To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E. coli genes. However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage. Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E. coli. We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain. As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.

• Reid KB, Edmondson J
• Location of the binding site in subcomponent C1q for plasma fibronectin.
• Acta Pathol Microbiol Immunol Scand Suppl. 1984; 284: 11-7
• Display abstract

• Previous studies on the interaction of fibronectin with C1q have yielded apparently conflicting results since both the globular head regions (produced by collagenase digestion) and the collagen-like domains (produced by limited pepsin digestion) have been reported to bind to fibronectin. In this study, the binding of 125I-labelled fibronectin to either intact C1q, or the collagenase or pepsin digestion products, immobilised on plastic microtitre plates was examined. Inhibition of the C1q-fibronectin interaction by the C1q digestion products was also examined. The results confirmed that both globular 'head' region preparations and collagen-like region preparations, can interact with fibronectin. Since the fragments used in these studies share a section of common amino acid sequence from the C1q molecule it can be concluded that the binding site, on C1q for fibronectin, is located in a region formed from the residues 81-97 of each of the three chains of the C1q molecule.

• Zhu BC, Fisher SF, Pande H, Calaycay J, Shively JE, Laine RA
• Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. Presence of polylactosamine glycopeptides and properties of a 44-kilodalton chymotryptic collagen-binding domain: difference from human plasma fibronectin.
• J Biol Chem. 1984; 259: 3962-70
• Display abstract

• Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins.

• Garcia-Pardo A, Pearlstein E, Frangione B
• Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges.
• Biochem Biophys Res Commun. 1984; 120: 1015-21
• Display abstract

• The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.

• Ali IU
• Structural analysis of fibronectin and its collagen-binding fragment from several cell lines.
• Proc Natl Acad Sci U S A. 1984; 81: 28-32
• Display abstract

• Tryptic peptide mapping of fibronectin from various species indicates that (i) there exists a remarkable similarity between methionine-labeled tryptic peptides of fibronectins isolated from cells of widely separated species such as human, chicken, mouse, hamster, and rat; (ii) fibronectins from normal and transformed or tumor cells of the same species are very similar, if not identical; and (iii) collagen-binding regions of fibronectin from different species share identical major peptides.

• Suzuki S, Pierschbacher MD, Hayman EG, Nguyen K, Ohgren Y, Ruoslahti E
• Domain structure of vitronectin. Alignment of active sites.
• J Biol Chem. 1984; 259: 15307-14
• Display abstract

• The structure of vitronectin, an adhesive protein isolated from human plasma, was studied by chemical fragmentation. Partial cleavage of vitronectin with cyanogen bromide in 70% formic acid generated four main fragments with masses of 53,000, 43,000, 35,000, and 12,000 daltons arising from both the 75- and 65-kDa vitronectin polypeptides and a 10-kDa fragment arising only from the 75-kDa polypeptide. By varying the reaction conditions, four BrCN cleavage sites and one acid cleavage site could be identified. The latter site gave rise to 40-, 32-, and 26-kDa fragments. The order of these fragments within the vitronectin polypeptides was determined by comparison of the NH2-terminal sequences of the polypeptides and their fragments, by further cleavage of the largest fragments with BrCN or 70% formic acid, and by assaying for heparin-binding and cell-attachment activities. The NH2-terminal sequences of the intact vitronectin polypeptides are the same and identical to a 44-amino acid serum peptide called somatomedin B, indicating that vitronectin may be the precursor of somatomedin B. The cell-attachment site appears to be located within approximately 5 kDa of the NH2 terminus, but it is distinct from the somatomedin B domain. The heparin-binding site is contained in the 12-kDa fragment near the COOH terminus. This fragment was shown to bind to a chondroitin sulfate proteoglycan in addition to heparin. The NH2-terminal amino acid sequence of this glycosaminoglycan-binding fragment is remarkably rich in basic amino acids. The NH2-terminal sequences of this and the other vitronectin fragments showed no homology with the amino acid sequence of the heparin-binding domain of fibronectin or other known sequences from fibronectin. These results show that the biological activities of vitronectin are located in distinct parts of both of the vitronectin polypeptides, which appear to be identical except for the presence of an additional 10-kDa fragment near or at the COOH terminus of the 75-kDa polypeptide.

• Kuusela P, Vartio T, Vuento M, Myhre EB
• Binding sites for streptococci and staphylococci in fibronectin.
• Infect Immun. 1984; 45: 433-6
• Display abstract

• Purified cathepsin G fragments of fibronectin were used to locate the binding sites for streptococci and staphylococci in the fibronectin molecule. The iodinated, NH2-terminal, 30-kilodalton (kd) fragment bound to group A and G streptococci and to Staphylococcus aureus. The 125I-labeled, COOH-terminal, 120- to 140-kd fragment bound weakly to group A streptococcus strain and to S. aureus when tested in a buffer of low ionic strength. The 30- and 120- to 140-kd fragments inhibited the binding of iodinated fragments to bacteria. The two fragments were, on a molar basis, equally effective, and they were more potent inhibitors than intact fibronectin. The gelatin-binding 40-kd fragment neither bound to any of the bacterial strains nor inhibited the binding of 125I-labeled 30-kd or 125I-labeled 120- to 140-kd fragments to bacteria. The results indicate that fibronectin has at least two separate binding sites for streptococci and staphylococci, one in the NH2-terminal region and another in the COOH-terminal region of the molecule, both capable of specific interaction with a complementary structure exposed on streptococcal and staphylococcal cell surfaces.

• Rourke FJ, Blumenstock FA, Kaplan JE
• Effect of fibronectin fragments on macrophage phagocytosis of gelatinized particles.
• J Immunol. 1984; 132: 1931-6
• Display abstract

• Rat plasma fibronectin enhances the binding and ingestion of gelatin-coated, formalin-fixed, or tanned sheep erythrocytes by elicited rat peritoneal macrophages. Fibronectin binding to the gelatinized erythrocytes is required for this enhancement, because macrophages preferentially recognize the surface bound molecule. This enhancement of particle uptake by fibronectin required the presence of a renewable, trypsin-sensitive component(s) on the macrophage surface (fibronectin receptor). When subjected to plasminolysis for 3 hr, fibronectin was degraded into gelatin-binding fragments (170 to 210 kd) and a 25-kd nongelatin binding fragment. The 170 to 210 kd gelatin binding fragments retained uptake-enhancing activity but were less active on a weight and molar basis than intact, dimeric fibronectin. The nongelatin binding 25 kd fragment alone did not enhance uptake. These results indicate that the sites for interaction with both the gelatinized erythrocyte surface and macrophages are retained on 170 to 210 kd fragments. However, the fibronectin dimeric structure is required for maximal expression of opsonic activity.

• Yamada KM, Kennedy DW
• Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.
• J Cell Biol. 1984; 99: 29-36
• Display abstract

• Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5-10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor.

• Vartio T et al.
• Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them.
• Eur J Biochem. 1983; 135: 203-7
• Display abstract

• Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.

• Vuento M, Sekiguchi K, Korkolainen M
• Identification of fibronectin fragments that bind to carboxy-group-modified proteins.
• Biochem J. 1983; 215: 613-6
• Display abstract

• Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.

• Cierniewski CS, Babinska A, Niewiarowska J, Augustyniak W
• Alteration of the antigenic structure of human fibronectin caused by complexing with collagen.
• Hoppe Seylers Z Physiol Chem. 1983; 364: 515-8
• Display abstract

• The complex formation between fibronectin and denatured collagen is associated with a significant decrease of antigenic expression of the fibronectin epitopes. This conclusion is evidenced by critical changes in the value of competitive inhibition parameters such as CImax. Generally, an alteration of the fibronectin antigenic structure is similar to that observed after thermic denaturation of the molecule.

• Molnar J, Lai MZ, Siefring GE Jr, Lorand L
• Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity.
• Biochemistry. 1983; 22: 5704-9
• Display abstract

• Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.

• Albini A, Richter H, Pontz BF
• Localization of the chemotactic domain in fibronectin.
• FEBS Lett. 1983; 156: 222-6
• Display abstract

• Fragments derived from human plasma fibronectin by enzymatic degradation were tested in the Boyden chamber for chemotactic activity towards various fibroblast strains. The results provide clear evidence that the chemotactic activity is restricted to a defined region of the fibronectin molecule which is the same for various fibroblast strains. The active domain is localized between the collagen binding site and the major heparin binding site, about 170 kDa apart from the N-terminal and about 70 kDa from the C-terminal ends of the two subunit peptide chains.

• Hayashi M, Yamada KM
• Domain structure of the carboxyl-terminal half of human plasma fibronectin.
• J Biol Chem. 1983; 258: 3332-40

• Gold LI, Frangione B, Pearlstein E
• Biochemical and immunological characterization of three binding sites on human plasma fibronectin with different affinities for heparin.
• Biochemistry. 1983; 22: 4113-9

• Sekiguchi K, Hakomori S
• Domain structure of human plasma fibronectin. Differences and similarities between human and hamster fibronectins.
• J Biol Chem. 1983; 258: 3967-73

• Garcia-Pardo A, Pearlstein E, Frangione B
• Primary structure of human plasma fibronectin. The 29,000-dalton NH2-terminal domain.
• J Biol Chem. 1983; 258: 12670-4
• Display abstract

• The complete amino acid sequence of the NH2-terminal domain obtained after trypsin digestion of human plasma fibronectin has been determined. It contains residues 1-259 and has a Mr of 29,000. The 29-kDa fragment was isolated from the other major tryptic cleavage products of 200, 180, and 31 kDa by affinity chromatography on gelatin- and heparin-Sepharose columns. The two high Mr fragments bound to gelatin and were easily removed; the 31-kDa fragment failed to bind to heparin while the 29-kDa fragment did and was eluted with 0.15 M NaCl. The 29-kDa domain has a blocked NH2-terminal (pyrrolidone carboxylic acid) which was removed by digestion with pyroglutamate aminopeptidase, and the amino acid sequence of the first 36 residues was obtained. The sequence showed a glutamine residue at position 3 which is probably the acceptor site for transglutaminase as reported for bovine fibronectin. Extensive trypsin digestion of the completely reduced and alkylated 29-kDa fragment yielded twenty-four peptides which were separated and purified by high performance liquid chromatography; their amino acid composition and amino acid sequence has been determined and the arrangement of peptides was achieved by comparison with the sequence recently reported for bovine fibronectin. The sequences in human and bovine fibronectin were nearly identical with only nine amino acid differences which can all be explained by single base substitutions. Apparently this domain is highly conserved in the two species studied thus far.

• Amphlett GW, Hrinda ME
• The binding of calcium to human fibronectin.
• Biochem Biophys Res Commun. 1983; 111: 1045-53
• Display abstract

• The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25 degrees in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr congruent to 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.

• Skorstengaard K, Thogersen HC, Vibe-Pedersen K, Petersen TE, Magnusson S
• Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them. Overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus.
• Eur J Biochem. 1982; 128: 605-23
• Display abstract

• Twelve cyanogen bromide fragments (CB1-12) from bovine plasma fibronectin have been isolated and eight of these completely sequenced. Altogether they account for 502 of the total expected 1880 residues in each of the two chains of fibronectin. Four of these fragments (CB1-4) constitute residues 1-289 in fibronectin with CB4 overlapping the N-terminal 29-kDa plasmic fragment to the second plasmic fragment, of 170-kDa in fibronectin. Fragments CB 5-9 are all contained within a 45-kDa gelatin-binding region, which is N-terminal in the 170-kDa fragment. The sequence of two of these five fragments in the 45-kDa fragment (CB7-8) contains two mutually homologous stretches with 57% sequence identity. Another two fragments (CB10-11) are derived from the heparin-binding region of the 170-kDa fragment. CB12 constitutes the C-terminal 13-residue stretch in fibronectin and contains a partly phosphorylated serine residue in the C-terminal sequence: -Arg-Glu-Asp-Ser(P)-Arg-Glu.

• Hayashi M, Yamada KM
• Divalent cation modulation of fibronectin binding to heparin and to DNA.
• J Biol Chem. 1982; 257: 5263-7
• Display abstract

• Fibronectin is an adhesive glycoprotein that binds to heparin and to DNA. The binding of tryptic fragments of human plasma fibronectin to these ligands is found to be highly dependent on the concentration of divalent cations. We have identified 3 types of binding to heparin. 1) Calcium-sensitive binding is inhibited by CaCl2, but not by MgCl2 or by MnCl2. The NH2-terminal 31,000-dalton fragment (fragment 23) has this type of binding, which is half-maximally inhibited by 3 to 4 mM CaCl2. 2) Divalent cation-sensitive binding is exhibited by a 75,000-dalton fragment (fragment 13); its binding is inhibited by all 3 divalent cations. 3) Divalent cation-insensitive binding is characteristic of a 95,000-dalton fragment (fragment 10) and larger fragments. These 3 fragments (fragments 10, 13, and 23) are not disulfide-bonded to other fragments. Specific tryptic fragments of fibronectin also bind readily to native DNA in the presence of EDTA, but the binding of all fragments is abolished by the presence of 10 mM CaCl2 or MgCl2. Our results indicate that the binding of specific domains of fibronectin to heparin or to DNA can be modulated by divalent cations.

• Pande H, Shively JE
• NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin.
• Arch Biochem Biophys. 1982; 213: 258-65

• Vibe-Pedersen K, Sahl P, Skorstengaard K, Petersen TE
• Amino acid sequence of a peptide from bovine plasma fibronectin containing a free sulfhydryl group (cysteine).
• FEBS Lett. 1982; 142: 27-30

• Hoch SO
• DNA-binding domains of fibronectin probed using Western blots.
• Biochem Biophys Res Commun. 1982; 106: 1353-8

• Smith DE, Furcht LT
• Localization of two unique heparin binding domains of human plasma fibronectin with monoclonal antibodies.
• J Biol Chem. 1982; 257: 6518-23
• Display abstract

• Monoclonal antibodies to human plasma fibronectin were used to study the topological arrangement of several biologically active sites on the 220,000-dalton fibronectin subunit. Plasma fibronectin was cleaved into a number of biologically active fragments by trypsin and cathepsin D. Fragments that bind gelatin and heparin bind to both gelatin and heparin were isolated by affinity chromatography. These fragments were further characterized by their ability to bind to two different monoclonal antibodies: monoclonal 2-8 and monoclonal 180-8. Using this approach, we have established the positions of two unique heparin-, a gelatin-, and two monoclonal antibody-binding sites on the fibronectin subunit.

• Vartio T
• Characterization of the binding domains in the fragments cleaved by cathepsin G from human plasma fibronectin.
• Eur J Biochem. 1982; 123: 223-33

• Richter H, Hormann H
• Early and late cathepsin D-derived fragments of fibronectin containing the C-terminal interchain disulfide cross-link.
• Hoppe Seylers Z Physiol Chem. 1982; 363: 351-64
• Display abstract

• Fibronectin consists of two very similar subunits connected by disulfide bonds close to their C-terminal ends. After short digestion with cathepsin D a fragment containing an intact subunit linked to a C-terminal piece of the other chain was isolated. Both possible combinations were realized. The remnant chain, removed by reduction, had a relative molecular mass (Mr) of 65000 or 75000, respectively, whether it originated from the shorter or the longer fibronectin subunit. After prolonged cathepsin D digestion another disulfide-linked fragment of Mr 90000 containing peptide chains of Mr 65000 and Mr 36000 was found. The same fragment also formed during prolonged digestion of a previously described cathepsin D-derived heparin-binding piece of Mr 140000 which consisted of disulfide-linked chains of Mr 65000 and Mr 75000. The cut-off material emerged as a mixture of two similar heparin binding peptides of Mr 35000 and Mr 37000. The residual disulfide-linked peptide of Mr 90000 was retained by heparin-Sepharose, too, indicating that the non-degraded chain of Mr 65000 also contained a heparin-binding site. Plasminolysis of the Mr 140000 fragment first liberated the two individual chains and subsequently cleaved the longer one into two domains of nearly equal size. A rather basic one (pI 8.4-8.8) with low cystine content bound to heparin-Sepharose, while the other cystine-rich and more acidic domain (pI 5.0-5.8) was not retained. The shorter peptide chain, also showing affinity to heparin, was attacked by plasmin rather slowly. It, however, also appeared to be composed of two differently charged regions as it had an isoelectric point in the neutral range (pI 6.4-6.8) in spite of its heparin-binding properties. In the longer chain the heparin-binding domain was located in a distal position to the C-terminal disulfide link. The same is assumed for the shorter chain by reason of homology.

• Ledger PW, Tanzer ML
• The phosphate content of human fibronectin.
• J Biol Chem. 1982; 257: 3890-5
• Display abstract

• The nature and distribution of the covalently bound phosphate of human fibronectin have been investigated. Phosphate was detected only as phosphoserine and is not present on asparagine-linked oligosaccharides. Fibroblast fibronectin labeled with 3H-amino acids and [32P]orthophosphate was subjected to limited tryptic digestion. Initial cleavages which reduced the molecule to nondisulfide-bonded polypeptides of Mr approximately 210,000-220,000 did not entail the loss of phosphate. Subsequent cleavage products included intermediate-sized 32P-phosphopeptides (43,000 and 37,000), but all 32P label became increasingly associated with lower molecular weight material (13,000 and 9,000) eventually terminating in a phosphopeptide (or peptides) of Mr approximately 6,000. The phosphorylated region(s) of human fibronectin are apparently not located at the extreme COOH-terminal region containing the interchain disulfide bond, but seem to be within a 40,000-50,000 segment near one end of the molecule. Cyanogen bromide cleavage of 32P-labeled fibronectin isolated from fibroblast culture medium produced one major phosphopeptide (Mr approximately 5,000) which co-purified through two chromatographic procedures with a CNBr phosphopeptide derived from plasma fibronectin. The phosphorylation of fibronectin is evidently restricted to a small region of the protein molecule, implying a very site-specific event. The phosphorylated region is present on human fibronectins obtained from plasma, fibroblast surfaces, and fibroblast culture medium, indicating that the role of the phosphate may be important to all forms of fibronectin.

• Bell ML, Engvall E
• The specific detection of collagenous proteins after electrophoresis using enzyme-conjugated collagen-binding fibronectin fragments.
• Anal Biochem. 1982; 123: 329-35

• Price TM, Rudee ML, Pierschbacher M, Ruoslahti E
• Structure of fibronectin and its fragments in electron microscopy.
• Eur J Biochem. 1982; 129: 359-63
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• Human plasma fibronectin and a series of its large proteolytic fragments were analyzed by electron microscopy using tungsten shadowing on carbon and polystyrene films. On carbon, intact fibronectin appeared as a randomly coiled strand, while on polystyrene it appeared as an elongated structure. Two fragments of fibronectin, Mr = 205000 and 190000, which lack the NH2-terminal domain of fibronectin and retain the collagen-binding, cell-attachment and heparin-binding functions, and a Mr = 170000 fragment, which retains the collagen-binding and cell-attachment functions, were seen as rods with varying degrees of nodularity while a Mr = 100000 fragment, which only binds to collagen, had two clear-cut domains. These results support the existing biochemical evidence that the segregation of the functional activities in the fibronectin molecule is based on distinct structural domains and provides evidence for the existence of an additional structural domain not revealed by biochemical and functional studies.

• Williams EC, Janmey PA, Ferry JD, Mosher DF
• Conformational states of fibronectin. Effects of pH, ionic strength, and collagen binding.
• J Biol Chem. 1982; 257: 14973-8
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• Human plasma fibronectin was enzymatically labeled with dansylcadaverine using plasma Factor XIIa. Fluorescence polarization studies of dansylcadaverine-labeled fibronectin indicate that fibronectin has a significant degree of chain flexibility in physiologic solution and that there is an increase in chain flexibility at high pH or ionic strength. Binding of a collagen peptide to dansylcadaverine-fibronectin results in a decrease in fluorescence polarization, suggesting that such binding causes a conformational change which also results in increased chain flexibility.l Quasielastic light scattering and intrinsic viscosity measurements of fibronectin were performed under physiologic conditions and at high pH and ionic strength. Shape calculations based on these data indicate that fibronectin is in an elongated configuration under physiologic conditions and further unfolds at high pH or ionic strength into a very flexible, strand-like configuration. Light scattering studies of fibronectin after binding of a collagen fragment indicate that such binding results in a decrease in the diffusion coefficient, suggesting that collagen binding also results in a partial unfolding of fibronectin. These results suggest that published electron micrographs of fibronectin showing a long, strand-like molecule do not reflect the conformation of plasma fibronectin under physiologic conditions; fibronectin, however, may assume an unfolded conformation upon binding to collagen in the tissue matrix.

• Pierschbacher MD, Ruoslahti E, Sundelin J, Lind P, Peterson PA
• The cell attachment domain of fibronectin. Determination of the primary structure.
• J Biol Chem. 1982; 257: 9593-7
• Display abstract

• The complete amino acid sequence of the cell attachment domain of human plasma fibronectin (Pierschbacher, M. D., Hayman, E. G., and Ruoslahti, E. (1981) Cell 26, 259-267) has been determined by automated sequential degradation of a peptic fragment comprising this region and of peptides derived from this fragment by digestion with thermolysin, staphylococcal V8 protease, cyanogen bromide cleavage, and partial acid hydrolysis. The fragment contains 108 residues with isoleucine and methionine as the NH2- and carboxyl-terminal amino acids, respectively. No cysteines are present. The calculated molecular weight of the cell attachment fragment, based on the amino acid sequence, is 11,482, which is in good agreement with the molecular weight estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and ultracentrifugation. There are no homologies in this fragment with other published sequences. The implications of the structure of the cell attachment fragment to the molecular mechanism of cell-fibronectin interaction are discussed.

• Richter H, Seidl M, Hormann H
• Location of heparin-binding sites of fibronectin. Detection of a hitherto unrecognized transamidase sensitive site.
• Hoppe Seylers Z Physiol Chem. 1981; 362: 399-408
• Display abstract

• Resolution of a cathepsin D digest of plasma fibronectin on heparin-Sepharose yielded, in addition to non-bound and weakly retained material (Fraction I and II), various fragments which were not eluted until 0.25M NaCl (Fraction III) and 0.5M NaCl (Fraction IV) was applied. Fraction III contained predominantly a peptide of Mr 70 000 originating from the N-terminus of the fibronectin subunits as well as high molecular weight precursors yielding the Mr 70 000 peptide by further digestion. All those peptides were retained by immobilized denatured collagen, type I, indicating the presence of the known gelatin-binding domain. In addition, they contained a transamidase-sensitive site as revealed from a digest of fibronectin previously labelled with [14C]putrescine by a transamidase-mediated reaction. Plasminolysis of the fragment of Mr 70 000 resulted in two peptides of Mr 30 000 and 40 000, only the former being retained by heparin-Sepharose. Fraction IV contained a fragment of Mr 140 000 which, after reduction, dissociated into two peptides of Mr 75 000 and 65 000. Apparently, it included the disulfide bond(s) connecting the two fibronectin subunits close to their C-terminal ends. Partial digestion of the two electrophoretically separated peptide chains with protease of Staphylococcus aureus V8 yielded for each chain a number of peptides with equal electrophoretic migration rate. In addition, however, some peptides were different in the two digests. The results were consistent with an identical or homologous structure of the two peptide chains with an additional sequence in the longer chain. The latter (Mr 75 000) uniquely contained a transamidase susceptible site as demonstrated by processing of [14C]putrescine-labelled fibronectin.

• Pierschbacher MD, Hayman EG, Ruoslahti E
• Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule.
• Cell. 1981; 26: 259-67
• Display abstract

• Proteolytic fragments of human plasma fibronectin were used to identify monoclonal antibodies reacting with the various domains of fibronectin. One of these antibodies, which reacts with cell-attachment-promoting fragments of fibronectin, inhibits attachment of cells to fibronectin-coated surfaces. A cell-attachment-promoting, chymotryptic, 120 kilodalton fragment was cleaved further with pepsin into three main fragments. The smallest, 15 kilodalton fragment was purified by affinity chromatography on the cell-attachment-inhibiting antibody insolubilized on Sepharose. This fragment is active in promoting cell attachment but lacks the other known binding activities of fibronectin. It can be localized between the collagen-binding and heparin-binding domains, about 127 to 197 kilodaltons from the NH2 terminus of the polypeptide. These results show that the interaction of fibronectin with cells is restricted to a defined portion of the molecule and is independent of the direct involvement of the known affinities toward other macromolecules.

• Akiyama SK, Yamada KM, Hayashi M
• The structure of fibronectin and its role in cellular adhesion.
• J Supramol Struct Cell Biochem. 1981; 16: 345-8
• Display abstract

• Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin had been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypeptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protease-resistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutaminase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low serum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.

• Engvall E, Bell ML, Ruoslahti E
• Affinity chromatography of collagen on collagen-binding fragments of fibronectin.
• Coll Relat Res. 1981; 1: 505-16
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• The affinity of fibronectin for collagen was exploited in biospecific affinity chromatography of collagen. Matrices with excellent capacity and stability were obtained by coupling collagen-binding fragments of fibronectin to Sepharose. Collagen-binding tryptic (30,000 daltons) and chymotryptic (45,000 daltons) fragments, lacking the binding sites of intact fibronectin for various other substances, were coupled to Sepharose and used to chromatograph gelatin and type I collagen. Gelatin was rapidly and quantitatively bound to these matrices at 4 degrees C and 37 degrees C, while binding of type I collagen took place more slowly and was temperature dependent. The collagen did not bind at 4 degrees C but bound quantitatively at 37 degrees C if preincubated at this temperature. These results suggest that a temperature-dependent perturbation of the triple helical structure of the collagen uncovers the binding site for fibronectin, allowing the collagen to bind to the insolubilized collagen-binding fibronectin fragments. Enzyme affinity assays showed that the conformational change in the fibronectin-binding region of collagen was irreversible. Affinity chromatography on collagen-binding fragments of fibronectin could provide a method for the study of structure and function of collagens and may prove useful for the isolation of minor collagens.

• Atherton BT, Taylor DM, Hynes RO
• Structural analysis of fibronectin with monoclonal antibodies.
• J Supramol Struct Cell Biochem. 1981; 17: 153-61
• Display abstract

• The reactivity of six monoclonal antibodies with fragments of fibronectin produced with trypsin, chymotrypsin, and Staphylococcus aureaus V8 protease is described. All these antibodies reacted with fragments derived from the C-terminal one-third of fibronectin. This region probably contains sites for the binding of fibronectin to cells, and to heparin and may also contain active sites for the reattachment, spreading, and alignment of transformed cells. Analysis of the reactivities of different sets of proteolytic fragments with the antibodies and with other ligands (eg. heparin) allows one to determine overlaps between the fragments and to locate the positions of the different binding sites for antibodies and ligands. One of the antibodies has allowed us to identify a site of structural difference between cellular and plasma fibronectins from hamsters. The site recognized by this antibody is located near to, but not at, the C-terminal end and does not involve carbohydrate groups. Because of its internal location in fibronectin, this difference suggests that there are probably different genes for cellular and plasma fibronectin. These monoclonal antibodies should be useful for further probing the functions present in the C-terminal regions of fibronectin and for determining their locations.

• Kleinman HK, Wilkes CM, Martin GR
• Interaction of fibronectin with collagen fibrils.
• Biochemistry. 1981; 20: 2325-30
• Display abstract

• The interaction of fibronectin with native collagen during collagen fibril formation was investigated. Fibronectin prepared from serum, or from the cell surface, bound to the forming collagen fibrils while less fibronectin bound to preformed fibers. Denatured collagen competed with native collagen in binding fibronectin. Fibronectin delayed the precipitation of collagen fibrils but did not alter the total amount of fibrils formed. Fibronectin which was heated to 30 degrees C for 30 min did not promote cell adhesion but still bound to native collagen and delayed fiber formation. The collagen-binding fragment of fibronectin produced by digestion either with chymotrypsin or with neutrophil elastase had a similar effect in delaying fibril formation, but the cell-binding fragment was not active. These studies indicate that fibronectin can bind to aggregating collagen fibers probably at the same site shown previously to bind to denatured collagen. Since fibronectin inhibits the rate of collagen fibrillogenesis, it may regulate the size of collagen fibers.

• Aggeler J, Engvall E, Werb Z
• An irreversible tissue inhibitor of collagenase in human amniotic fluid: characterization and separation from fibronectin.
• Biochem Biophys Res Commun. 1981; 100: 1195-201

• Ehrismann R, Chiquet M, Turner DC
• Mode of action of fibronectin in promoting chicken myoblast attachment. Mr = 60,000 gelatin-binding fragment binds native fibronectin.
• J Biol Chem. 1981; 256: 4056-62

• Keski-Oja J, Yamada KM
• Isolation of an actin-binding fragment of fibronectin.
• Biochem J. 1981; 193: 615-20
• Display abstract

• We have identified a specific actin-binding site in the adhesive glycoprotein fibronectin, isolated from chicken fibroblasts. Affinity chromatography of fragments, released from fibronectin by limited proteolysis with trypsin, chymotrypsin and subtilisin, on actin-Sepharose and other protein-Sepharose columns was used to locate the binding site. A 27 000-mol.wt. subtilisin-digest fragment bound efficiently to actin. The results suggest that the actin-binding site is close to, but not identical with, the reported collagen-binding site.

• Haim S
• [Fibronectin]
• Harefuah. 1981; 100: 584-5

• Ruoslahti E, Hayman EG, Engvall E, Cothran WC, Butler WT
• Alignment of biologically active domains in the fibronectin molecule.
• J Biol Chem. 1981; 256: 7277-81
• Display abstract

• Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.

• De Petro G, Barlati S, Vartio T, Vaheri A
• Transformation-enhancing activity of gelatin-binding fragments of fibronectin.
• Proc Natl Acad Sci U S A. 1981; 78: 4965-9
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• Studies have established that cryoprecipitates of the plasma of tumor patients contain a biological activity enhancing morphological cell transformation (transformation-enhancing factor; TEF) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We report here that similar TEF activity is effected by defined fragments of human plasma fibronectin obtained by limited digestion with major humoral or tissue proteinases. TEF activity was obtained from plasminolytic fragments of fibronectin and from cathepsin G-treated fibronectin. No activity was recorded from intact dimeric fibronectin or its reduced and alkylated subunits, from fibrinogen or its plasminolytic fragments, or from plasmin (EC 3.4.21.7) or cathepsin G (EC 3.4.21.20) treated or untreated with proteinase inhibitors. All of the TEF activity of the proteolytic fragments of fibronectin was located on the gelatin-binding peptides. The minimum effective doses in the TEF assay were 750 ng/ml of plasmin-treated fibronectin, 100 ng/ml of gelatin-binding plasminolytic fibronectin (enriched in Mr 180,000--190,000 polypeptides), and 100 ng/ml of gelatin-binding fragments of cathepsin G-treated fibronectin (enriched in a Mr 30,000 fragment). TEF activity of proteinase-treated fibronectin was inhibited by gelatin and by intact dimeric fibronectin. The potent TEF activity of proteolytic fragments of fibronectin raises the possibility that they may have a role in malignant transformation.

• Sekiguchi K, Fukuda M, Hakomori S
• Domain structure of hamster plasma fibronectin. Isolation and characterization of four functionally distinct domains and their unequal distribution between two subunit polypeptides.
• J Biol Chem. 1981; 256: 6452-62

• McDonald JA, Broekelmann TJ, Kelley DG, Villiger B
• Gelatin-binding domain-specific anti-human plasma fibronectin Fab' inhibits fibronectin-mediated gelatin binding but not cell spreading.
• J Biol Chem. 1981; 256: 5583-7
• Display abstract

• Antisera against a Mr = 60,000 peptide containing the gelatin-binding domain of human plasma fibronectin (McDonald, J. A., and Kelley, D. G. (1980) J. Biol. Chem. 255, 8848-8858) bound the Mr = 60,000 peptide and intact fibronectin but not three other fragments released by leukocyte elastase proteinolysis (the Mr = 25,000 amino-terminal sequence, Mr = 140,000 sequence containing cell adhesive activity, and a Mr = 31,000 fragment). Affinity-purified Fab' blocked Mr = 60,000 peptide binding to gelatin and inhibited plasma and cellular fibronectin gelatin binding without affecting fibronectin-mediated cell spreading. In contrast, antifibronectin Fab' absorbed with the gelatin-binding fragment completely blocked fibronectin-mediated cell spreading. These data indicate that the gelatin-binding domain of fibronectin is immunogenic, and antisera against this domain recognize cellular fibronectin gelatin-binding sites. Inhibition of gelatin binding but not cell spreading by anti-gelatin binding domain Fab' confirms the hypothesis that fibronectin has separate sites mediating these activities. Selective inhibition of fibronectin-collagen binding by domain-specific antisera may help elucidate the role of fibronectin in organization of the extracellular matrix.

• Postlethwaite AE, Keski-Oja J, Balian G, Kang AH
• Induction of fibroblast chemotaxis by fibronectin. Localization of the chemotactic region to a 140,000-molecular weight non-gelatin-binding fragment.
• J Exp Med. 1981; 153: 494-9
• Display abstract

• Plasma and cell-derived fibronectin are potent chemoattractants for human dermal fibroblasts in vitro. The chemotactic property of fibronectin resides in a major 140,000-mol wt non-gelatin-binding fragment of the native molecule. Human monocytes and neutrophils do not recognize fibronectin as a chemotactic stimulus. These findings suggest that fibronectin and perhaps certain fragments of fibronectin may function in vivo as a specific chemoattractant for fibroblasts and could, therefore, induce directional migration of fibroblasts to sites of tissue injury, remodeling or morphogenesis.

• Ruoslahti E, Hayman EG, Engvall E
• Molecular interactions of fibronectin.
• Prog Clin Biol Res. 1980; 41: 821-8
• Display abstract

• Fibronectin mediates attachment of cells to surfaces in vitro, and its abundance in basement membrane structures suggests that it serves a similar function in vivo. The cell attachment promoting activity of fibronectin depends on its capacity to interact with a number of other macromolecules. These include collagen, fibrin(ogen), glycosaminoglycans, and as yet unidentified receptors on the surfaces of various mammalian and avian cells and on the surfaces of Staphylococci. Denatured collagens bind more avidly to fibronectin than the native forms. Of the latter, type III is the most active one. The collagen-binding is inhibited by fibrinogen and vice versa, indicating that the same (or overlapping) binding site are responsible for the binding of fibronectin to collagen and fibrinogen. The binding site for collagen in fibronectin can be localized in a homogeneous fragment with a molecular weight of 30,000. This binding site is distinct from the one interacting with cell surfaces, since the cell attachment promoting activity is found associated with collagen-nonbinding fragments of fibronectin. These same fragments also bind to Staphylococci, suggesting that the cell surface receptor in these bacteria may be similar to that of eukaryotic cells. Glycosaminoglycans bind to collagen-fibronectin complexes resulting in a complex more stable to disruption with urea than complexes of any two of the components. This phenomenon may be important in the formation of the fibronectin-containing, adhesive, extracellular matrix, and its significance to the apparent disturbances that many malignant cells have in forming such a matrix in particular, warrants further study.

• Hayashi M, Schlesinger DH, Kennedy DW, Yamada KM
• Isolation and characterization of a heparin-binding domain of cellular fibronectin.
• J Biol Chem. 1980; 255: 10017-20
• Display abstract

• A protease-resistant functional polypeptide domain of fibronectin that contains a heparin-binding site has been purified from chick cellular fibronectin following pronase digestion, heparin-Sepharose affinity chromatography, and molecular sieve chromatography on Sephacryl S-200. This polypeptide migrates as a single band of apparent Mr = 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction by dithiothreitol. It has a Stokes radius of 35 to 37 A and a calculated frictional ratio of 1.4 to 1.5. Its amino acid composition is unusual in that cysteine and methionine constitute only 1 and 2 mol/mol of the polypeptide, respectively. The NH2-terminal amino acid sequence is Glu-Asp-Asp. In contrast to intact fibronectin, the heparin-binding polypeptide contains virtually no neutral sugars. This 50,000-dalton domain is inactive in four bioassays for intact fibronectin, including hemagglutination, spreading of cells on tissue culture plastic, restoration of alignment of tumor cells, and attachment of cells to collagen. Moreover, excess amounts of this polypeptide do not inhibit the activity of intact fibronectin in all of these assays; these results suggest that the heparin-binding domain does not contain a cell surface-binding site. This domain of fibronectin appears to be a unique region free of carbohydrate that is a globular, protease-resistant functional site.

• Lineback-Zins J, Brew K
• Preparation and characterization of an NH2-terminal fragment of human serum transferrin containing a single iron-binding site.
• J Biol Chem. 1980; 255: 708-13

• Hormann H, Jilek F
• Interaction of fibrinogen/fibrin and fibronectin with collagen.
• Artery. 1980; 8: 482-6
• Display abstract

• Interaction of fibrinogen, soluble fibrin and fibronection with native collagen, type I and III, was investigated by means of affinity chromatography and radio binding assay. Among collagens type III was most active. It associated stronger with fibronectin than with fibrinogen and fibrin suggesting that fibronectin might form the nuclei for deposition of plasma proteins on exposed collagen structures.

• Furie MB, Rifkin DB
• Proteolytically derived fragments of human plasma fibronectin and their localization within the intact molecule.
• J Biol Chem. 1980; 255: 3134-40

• Reiser KM, Last JA
• Quantitation of specific collagen types from lungs of small mammals.
• Anal Biochem. 1980; 104: 87-98

• Hormann H, Jelinic V
• Fibronectin, VII. Binding of cold-insoluble globulin and of denatured collagen by macrophages.
• Hoppe Seylers Z Physiol Chem. 1980; 361: 379-87

• Ruoslahti E, Hayman EG
• Two active sites with different characteristics in fibronectin.
• FEBS Lett. 1979; 97: 221-4

• Balian G, Click EM, Crouch E, Davidson JM, Bornstein P
• Isolation of a collagen-binding fragment from fibronectin and cold-insoluble globulin.
• J Biol Chem. 1979; 254: 1429-32
• Display abstract

• Limited proteolytic cleavage of fibronectin and plasma cold-insoluble globulin with cathepsin D produced two major fragments. The smaller, Mr = 72,000 fragment bound to collagen and contained most of the cysteine in the molecule. This region contains intrachain disulfide bonds which maintain a conformation that is necessary for interaction with collagen. Cleavage of the intact protein and the 72,000-dalton fragment with plasmin localized the collagen-binding region in cold-insoluble globulin to a sequence of about 42,000 daltons. This region is located approximately two-thirds of the linear distance from the NH2 terminus of each chain in the dimeric molecule.

• Hahn LH, Yamada KM
• Identification and isolation of a collagen-binding fragment of the adhesive glycoprotein fibronectin.
• Proc Natl Acad Sci U S A. 1979; 76: 1160-3
• Display abstract

• We have identified and purified a polypeptide region containing the collagen-binding site of the adhesive glycoprotein fibronectin. Chicken cellular fibronectin isolated from cultured embryonic fibroblasts was permitted to bind to gelatin coupled to agarose beads and was then digested extensively with chymotrypsin. A prominent 40,000-dalton fragment of fibronectin consisting of a single polypeptide chain was detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of material remaining bound to the gelatin-agarose. This fragment appeared within 10 min after the digestion was initiated and persisted for more than 20 hr. This proteolytic fragment was isolated in electrophoretically pure form and retained its affinity for collagen. Plasma fibronectins from chicken and human blood also contained collagen-binding proteolytic fragments of similar size. This finding suggest that the collagen-binding sites of cellular and plasma fibronectins are homologous.

• Gold LI, Pearlstein E
• Stability of fibronectin biological activity following chemical modification.
• Biochim Biophys Acta. 1979; 581: 237-51
• Display abstract

• Fibronectin isolated from human plasma functions in vitro as a mediator of adhesion and spreading of trypsinized fibroblasts on native or denatured collagen. As a means of elucidating structural characteristics which might contribute to fibronectin's biological activity, we have modified and digested the protein with several chemicals. Following various treatments, the protein was utilized to mediate cell adhesion and spreading on collagen to determine which alteration disrupted its activity. Fibronectin remained functionally intact after partial or complete reduction and alkylation, oxidation of 59% of the carbohydrates with sodium periodate, citraconylation, carbodiimide-catalyzed amide formation, and oxidation of 35.2 residues of tryptophan/molecule with N-bromosuccinimide. Dinitrofluorobenzene treatment, which phenylated ten residues/molecule of fibronectin, successfully inactivated fibronectin's in vitro biological function. Effective modification of the protein was determined by appropriate analytical procedures. Since fibronectin retained its biological function after several treatments that presumably affected its molecular conformation, we concluded that its secondary or tertiary structure appears not to be essential for its in vitro activity, or alternatively that the protein possesses a biologically active domain relatively resistant to chemical modification.

• Gold LI, Garcia-Pardo A, Frangione B, Franklin EC, Pearlstein E
• Subtilisin and cyanogen bromide cleavage products of fibronectin that retain gelatin-binding activity.
• Proc Natl Acad Sci U S A. 1979; 76: 4803-7
• Display abstract

• The gelatin-binding region of fibronectin has been obtained by subtilisin digestion and cyanogen bromide cleavage of the molecule. Enzymatic digestion yielded two fragments of molecular weights 50,000 (S50K) and 30,000 (S30K) which were isolated by elution from gelatin-Sepharose affinity columns. Because the S50K fragment also mediated the adhesion of fibroblasts to collagen, it contains both the collagen and cell binding sites on the fibronectin molecule. Both fragments had valine as the NH2-terminal residue, were enriched in half-cystine and methionine residues compared to the whole molecule, and were identical by immunodiffusion. The S50K fragment begins with the sequence Val-Tyr-Gln-Pro-Gln-Pro-His-Pro-Gln-Pro-(Pro)-(Gly)-Tyr-Gly-His-( )-Val, a region with an extended conformation which is susceptible to proteolysis and connects this domain to the remainder of the fibronectin molecule. The S50K fragment appears to be located in the COOH-terminal one-third of the fibronectin molecule but does not contain the interchain disulfide bridge(s); the S30K fragment is probably derived from the NH2-terminal region of S50K.

• Ruoslahti E, Hayman EG, Kuusela P, Shively JE, Engvall E
• Isolation of a tryptic fragment containing the collagen-binding site of plasma fibronectin.
• J Biol Chem. 1979; 254: 6054-9

• Engvall E, Ruoslahti E, Miller EJ
• Affinity of fibronectin to collagens of different genetic types and to fibrinogen.
• J Exp Med. 1978; 147: 1584-95
• Display abstract

• Fibronectin, a fibroblast surface protein, was purified from human and chicken plasma and extracts of cultured chicken fibroblasts with affinity chromatography on gelatin coupled to Sepharose particles. A fibronectin-like protein was also isolated from the plasma of Torpedo fish. The collagen binding properties of fibronectin were studied with several genetically distinct collagens. Heat denatured types I, II, and III collagens were equal in their binding capacity and more active than the native collagens or A and B chains. Native type III collagen was more active than the other native collagens. Human and chicken fibronectins showed approximately the same pattern of specificity. Identical specificities were shown by the plasma and fibroblast forms of chicken fibronectin. Two cyanogen bromide peptides of the collagen alpha1 (II) chain, CB8 and CB12, derived from different parts of the chain, were active in fibronectin binding. A polymer of the tripeptide pro-gly-pro, and polyproline were inactive. Fibronectin also binds to fibrinogen and fibrin. Comparison of this binding to collagen binding showed that fibrinogen inhibited binding of fibronectin to collagen, but was less active than native collagen. Two other fibrous proteins, tropoelastin and keratin, did not bind fibronectin. The binding of fibronectin to fibrinogen was inhibited by collagen and incorporation of fibronectin into blood clot in the cold was inhibited by gelatin. These results suggest that the binding of fibronectin to collagen and fibrinogen depends on the same binding site in the fibronectin molecule. It is proposed that cell surface fibronectin mediates attachment of cells to the collagenous extracellular matrix and to a temporary fibrin matrix in a wound.

• Weickmann JL, Himmel ME, Smith DW, Fahrney DE
• Arginine deiminase: demonstration of two active sites and possible half-of-the-sites reactivity.
• Biochem Biophys Res Commun. 1978; 83: 107-13

• Cannon DJ, Davison PF
• Crosslinking in type III collagen of fetal tissue.
• Biochem Biophys Res Commun. 1978; 85: 1373-8

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