NORMAL GENOMIC

• Wemmer D
• Solid-state nuclear magnetic resonance of protein deposits.
• Methods Enzymol. 1999; 309: 536-59

• Corbett SA, Lee L, Wilson CL, Schwarzbauer JE
• Covalent cross-linking of fibronectin to fibrin is required for maximal cell adhesion to a fibronectin-fibrin matrix.
• J Biol Chem. 1997; 272: 24999-5005
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• In a blood clot, fibrin and plasma fibronectin (pFN) are covalently cross-linked by activated factor XIII (factor XIIIa) to form pFN-fibrin multimers. To determine the functional significance of covalent pFN-fibrin interactions, we have developed an in vitro model which allows the incorporation of recombinant FN (recFN) molecules into a covalently cross-linked recFN-fibrin matrix. Using the baculovirus expression system, we have expressed recFN monomers composed of the amino-terminal 70-kDa region and the first 11 type III repeats (WT) with mutations in the glutamines at positions 3 and 4 (Q2) or at 3, 4, and 16 (Q3). Examination of the covalent incorporation of these recFNs into fibrin clots confirms that glutamines 3 and 4 are major participants in FN-fibrin cross-linking as the mutation of these sites reduces cross-linking efficiency by 65%. Additional mutation of the glutamine at position 16, however, eliminates >99% of cross-linking suggesting that it also may be factor XIIIa reactive. When the Q3 recFN-fibrin clots were used as substrates for cell adhesion, there was a decrease in both cell attachment and spreading when compared with the WT recFN-fibrin clots. These data demonstrate that for maximal cell attachment to a FN-fibrin clot, FN must be cross-linked to fibrin by factor XIIIa.

• Matsuka YV, Migliorini MM, Ingham KC
• Cross-linking of fibronectin to C-terminal fragments of the fibrinogen alpha-chain by factor XIIIa.
• J Protein Chem. 1997; 16: 739-45
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• Fibronectin binds specifically to fibrin and is covalently cross-linked to the fibrin alpha chain by activated factor XIII (XIIIa). This reaction is important for wound healing. Here we investigate XIIIa-catalyzed cross-linking of fibronectin and some of its fragments to a recombinant fragment representing the COOH-terminal 30 kDa of the fibrin alpha chain (alpha C30K:His 368-Val 610). Only fibronectin and those fragments containing an intact NH2-terminus were able to form cross-linked complexes. As many as 10 of the 17 lysines in alpha C30K can serve as amine donors in this reaction. Analysis of the rate of XIIIa-catalyzed cross-linking of fibronectin NH2-terminal peptides and fragments with alpha C30K revealed that the presence of the first type I "finger" module accelerates the cross-linking reaction; addition of fingers 2-5 had no further effect.

• Matsuka YV, Medved LV, Migliorini MM, Ingham KC
• Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen.
• Biochemistry. 1996; 35: 5810-6
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• Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

• Willbold D et al.
• Structural studies of the equine infectious anemia virus trans-activator protein.
• Eur J Biochem. 1996; 240: 45-52
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• Trans-activator (tat) proteins are necessary components for the completion of the T replication cycle of lentiviruses. The three-dimensional structure of the equine infectious anemia virus (EIAV) tat protein (e-tat) was studied with CD spectroscopy, NMR spectroscopy, and restrained molecular-dynamics calculations. No stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix-forming tendency, e-tat exhibits a flexible tertiary structure, and only the amino acids comprising the core sequence region form a well-defined tertiary fold. The three-dimensional structure allows discussion of biochemical data as well as data from molecular biological investigations of lentiviral tat proteins.

• House-Pompeo K, Xu Y, Joh D, Speziale P, Hook M
• Conformational changes in the fibronectin binding MSCRAMMs are induced by ligand binding.
• J Biol Chem. 1996; 271: 1379-84
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• Bacterial adherence to host tissue involves specific microbial surface adhesins of which a subfamily termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) specifically recognize extracellular matrix components. We now report on the biophysical characterization of recombinant fibronectin binding MSCRAMMs originating from several different species of Gram-positive bacteria. The far-UV CD spectra (190-250 nm) of recombinant forms of the ligand binding domain of the MSCRAMMs, in a phosphate-buffered saline solution at neutral pH, were characteristic of a protein containing little or no regular secondary structure. The intrinsic viscosity of this domain was found to be the same in the presence or absence of 6 M guanidine hydrochloride, indicating that the native and denatured conformations are indistinguishable. On addition of fibronectin NH2 terminus as ligand to the recombinant adhesin there is a large change in the resulting far-UV CD difference spectra. At a 4.9 M excess of the NH2 terminus the difference spectra shifted to what was predominately a beta-sheet conformation, as judged by comparison with model far-UV CD spectra. The fibronectin NH2-terminal domain undergoes a minute but reproducible blue-shift of its intrinsic tryptophan fluorescence on addition of rFNBD-A, which contains no tryptophan residues. Since this result indicates that there is no large change in the environment of the tryptophan residues of the NH2 terminus on binding, the large shift in secondary structure observed by CD analysis is attributed to induction of a predominately beta-sheet secondary structure in the adhesin on binding to fibronectin NH2 terminus.

• Atta MS, Powell RJ, Todd I
• Re: Characterization of a novel human antibody xenoreactive with fibronectin.
• Clin Immunol Immunopathol. 1996; 80: 336-7

• Huff S, Matsuka YV, McGavin MJ, Ingham KC
• Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy.
• J Biol Chem. 1994; 269: 15563-70
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• The N-terminal 29-kDa fragment of fibronectin (Fn29K) contains five type I "finger" modules. It binds to heparin, fibrin, and bacteria and is involved in fibronectin (Fn) matrix assembly. Binding to Staphylococcus aureus involves a cell wall-associated protein that contains approximately three repeats of a 38-residue D motif (Signas, C., Raucci, G., Jonsson, K., Lindgren, P.-E., Anantharamaiah, G.M., Hook, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). Synthetic peptides representing D1, D2, and D3, when labeled with fluorescein isothiocyanate (FITC), exhibited increases in fluorescence anisotropy upon addition of Fn29K but not other Fn fragments. The response could be reversed by titration with unlabeled peptides to yield inhibition constants that agreed with the dissociation constants obtained by fitting the initial response. Values of Kd ranged between 2 and 12 microM, with D3 having the highest affinity. Specificity of D3 for Fn29K was further illustrated by the fact that its C-terminal half (D3b, Lys801 to Lys821), when immobilized, selectively adsorbed Fn29K from a thermolysin digest of fibronectin. The binding site in Fn was further localized within Fn29K by analyzing smaller proteolytic or recombinant subfragments. Those containing fingers, F3-5 and F4-5, were purified on D3b-Sepharose and bound FITC-D3b with Kd values of 4-6 microM. Subfragments containing pairs of fingers 1-2, 2-3, or single fingers 1, 4, or 5 were inactive. Whole D1-3, expressed in Escherichia coli and labeled with fluorescein, bound 1.9 mol/mol of Fn29K with Kd = 1.5 nM. F4-5 and F2-3 bound with respective Kd values of 0.35 and 4.4 microM. These and other results indicate that binding of the individual D region peptides is mediated through their C-terminal halves, primarily to fingers 4 and 5 of fibronectin. The possible basis of the much higher affinity of D1-3 is discussed.

• Smith BO, Downing AK, Dudgeon TJ, Cunningham M, Driscoll PC, Campbell ID
• Secondary structure of fibronectin type 1 and epidermal growth factor modules from tissue-type plasminogen activator by nuclear magnetic resonance.
• Biochemistry. 1994; 33: 2422-9
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• A segment of human tissue-type plasminogen activator (t-PA) corresponding to the fibronectin type 1 (F1) and epidermal growth factor-like (G) pair of modules, residues 1-91, has been produced as a recombinant protein in Saccharomyces cerevisiae, with a single conservative Cys to Ser substitution. The sequence-specific assignment of the 1H and 15N nuclear magnetic resonances from the pair of modules has been completed using 2D 1H nuclear magnetic resonance (NMR) spectra in conjunction with 3D, 15N-edited, 1H and 2D 15N-1H NMR spectra. Slowly exchanging amide protons have been identified, and estimates of a number of backbone 3JNH-C alpha H coupling constants were obtained by line-shape-fitting. The secondary structure of the F1 module in the pair closely matches that previously determined for the isolated F1 module from t-PA, and that of the G module conforms to the "consensus" G module structure determined previously from several isolated G modules. In the module pair, the residues linking the two modules appear to form an extended beta-strand, the carboxy-terminal end of which makes up a third strand of the major beta-sheet of the G module. The intermodule interface is defined by NOEs between residues in the ranges 22-24 in the F1 module and 65-72 in the G module. The NMR data indicate that there is little or no reorientation of the two modules with respect to one another but rather that they combine with a fixed hydrophobic contact dominated by the side chain of leucine-22.

• Esposito G, Lesk AM, Molinari H, Motta A, Niccolai N, Pastore A
• Probing protein structure by solvent perturbation of NMR spectra. II. Determination of surface and buried residues in homologous proteins.
• Biopolymers. 1993; 33: 839-46
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• The experimental assignment of most residues in a protein to the surface or interior is in principle possible without prior solution of a complete three-dimensional structure. The method described is based on nmr measurements that determine the amino acid composition of the surface of a protein [A. Petros, L. Mueller, and K.D. Kopple (1990) Biochemistry, Vol. 29, pp. 10041-10048; G. Esposito, A. M. Lesk, H. Molinari, A. Motta, N. Niccolai, and A. Pastore (1992) Journal of Molecular Biology, Vol. 224, pp. 659-670]. If these measurements are carried out on several homologous proteins of known sequence, it is possible to combine the results to determine, in most cases, which positions in the sequence contain exposed residues.

• Williams MJ, Phan I, Baron M, Driscoll PC, Campbell ID
• Secondary structure of a pair of fibronectin type 1 modules by two-dimensional nuclear magnetic resonance.
• Biochemistry. 1993; 32: 7388-95
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• The fourth and fifth type 1 module pair, corresponding to residues 151-244 from the amino terminus of human fibronectin, has been produced as a recombinant protein using a yeast expression system and studied by two-dimensional homonuclear 1H nuclear magnetic resonance (NMR) spectroscopy. The sequence-specific resonance assignment of the 1H NMR spectrum has been completed using a combination of 2D 1H nuclear Overhauser effect (NOE) spectroscopy, homonuclear Hartmann-Hahn, and correlated spectroscopy spectra recorded under a variety of pH and temperature conditions. Slow exchanging amide protons have been identified and estimates of many backbone 3JNH-C alpha H coupling constants were obtained by line shape fitting. The secondary structures of each module conform closely to the "consensus" fibronectin type 1 module structure determined previously for two other single type 1 modules. In the module pair described here, the two modules are linked by a short five-residue linker which appears to form a turn. The intermodule interface is defined by NOEs observed between a hydrophobic three-residue sequence from the fourth type 1 module and residues in the first double-stranded beta-sheet of the fifth type 1 module. The interaction is dominated by a tryptophan residue (unconserved in other type 1 sequences) within the fourth module, which causes large upfield ring current shifts for several proton resonances from the beta-sheet of the fifth module. The NMR data indicate that there is little or no relative reorientation of the two modules about the linker region but rather that the two modules combine with a fixed and intimate hydrophobic contact.

• Blystone SD, Kaplan JE
• The role of fibronectin in macrophage fibrin binding: a potential mechanism for high affinity, high capacity clearance of circulating fibrin.
• Blood Coagul Fibrinolysis. 1993; 4: 769-81
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• Fibrin generation occurs as the result of a wide variety of pathological conditions. Extraneous deposition of fibrin outside a wound or inflammatory locus can lead to severe circulatory and respiratory complications. Fibrin within the circulation is removed by the hepatic macrophage (Kupffer cell). While many mechanisms for macrophage fibrin binding have been delineated in vitro, the complete pathway for in vivo fibrin clearance has not been determined. This article reviews these varied mechanisms and describes in detail a novel potential fibronectin-dependent pathway for hepatic fibrin clearance.

• Baron M, Main AL, Driscoll PC, Mardon HJ, Boyd J, Campbell ID
• 1H NMR assignment and secondary structure of the cell adhesion type III module of fibronectin.
• Biochemistry. 1992; 31: 2068-73
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• The secondary structure of the tenth type III module from human fibronectin has been determined using NMR. This type of module appears many times in a wide variety of proteins. The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion. The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry. 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum. The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates. The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family [Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938]. The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G.

• Blystone SD, Weston LK, Kaplan JE
• Fibronectin dependent macrophage fibrin binding.
• Blood. 1991; 78: 2900-7
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• Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.

• Sottile J, Schwarzbauer J, Selegue J, Mosher DF
• Five type I modules of fibronectin form a functional unit that binds to fibroblasts and Staphylococcus aureus.
• J Biol Chem. 1991; 266: 12840-3
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• Fibronectin is a cell-adhesive protein comprised of three types of repeating homologous sequences, I, II, and III (Petersen, T.E., Thogersen, H.C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L., and Magnusson, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 137-141). The amino-terminal portion of fibronectin is comprised of five type I modules and mediates assembly of dimeric soluble fibronectin into insoluble fibrils by cultured fibroblasts, binding and cross-linking of fibronectin to Staphylococcus aureus, and binding and cross-linking of fibronectin to fibrin. It is not known whether these binding activities require individual type I modules, several modules, or all five modules. To answer this question, we generated recombinant truncated fibronectin molecules with deletions of or mutations in the amino-terminal type I modules. Binding to cellular fibronectin assembly sites and S. aureus required all five type I modules. In contrast, proteins with deletions of type I modules interacted well with fibrin.

• Hormann H, Richter H, Jelinic V
• The role of fibronectin fragments and cell-attached transamidase on the binding of soluble fibrin to macrophages.
• Thromb Res. 1987; 46: 39-50
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• Proteolytic fragments from the N-terminus of the fibronectin subunit chains were shown to mediate the binding of 125-I-fibrin to macrophages. With increasing molecular weight of the fragments, binding activity decreased and intact plasma fibronectin was inactive. Fibrin binding to macrophages was a time dependent reaction and proceeded considerably faster than binding of fibrinogen. The binding reaction was inhibited by putrescine suggesting the involvement of a transamidase. Pericellular transamidase was demonstrated on macrophages by incorporation of 14-C-putrescine into fibronectin 30 kD-fragment. Expression of this enzyme appeared to be rate-limiting for the binding reaction which was accelerated after loading the cells with placental transamidase.

• Liu MC, Suiko M
• Tyrosine sulfation site is located in the C-terminal fibrin-binding domain in secreted fibronectin from rat embryo fibroblasts, line 3Y1.
• Arch Biochem Biophys. 1987; 255: 162-7
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• Tryptic fragments of [35S]sulfate-labeled 3Y1 secreted fibronectin were fractionated by hydroxylapatite column chromatography and examined using sodium dodecyl sulfate gel electrophoresis, followed by autoradiography. Radioactive bands containing tyrosine-O-[35S]sulfate were detected at 17- and 40-kDa positions under reducing conditions. Under nonreducing conditions, the 17-kDa band was no longer present and new bands at 57- and 80-kDa positions appeared, indicating a disulfide linkage between the two smaller fragments in the native state. These fragments exhibited binding affinity toward fibrin and could be immunoprecipitated by the monoclonal antifibronectin Fib-2 domain antibody. These results suggested that the tyrosine sulfation site in 3Y1 secreted fibronectin is located in the C-terminal fibrin-binding (Fib-2) domain, being within 17 kDa of the C-terminus.

• Hynes R
• Molecular biology of fibronectin.
• Annu Rev Cell Biol. 1985; 1: 67-90

• Mosher DF, Johnson RB
• Specificity of fibronectin--fibrin cross-linking.
• Ann N Y Acad Sci. 1983; 408: 583-94
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• Our experiments indicate that (1) non-covalent binding of fibronectin to fibrin is mediated by sites in a 27 kd NH2-terminal region and a 31 kd COOH-terminal region of fibronectin; (2) the 31 kd region is probably present in both chains of the fibronectin dimer; and (3) covalent (factor XIIIa-mediated) cross-linking of fibronectin and fibrin is between a glutaminyl residue in the 27 kd region of fibronectin and a lysyl residue in the COOH-terminal two-thirds of the fibrin alpha chain.

• Richardson JS
• The anatomy and taxonomy of protein structure.
• Adv Protein Chem. 1981; 34: 167-339