Interactions Of Fibronectin Research Articles

A second fibronectin-binding region is present in collagen alpha chains.

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.

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation

Abstract Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinyli-dene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.

Interactions of Fibronectin with Streptococci: The Role of Fibronectin as a Receptor for Streptococcus pyogenes

Current evidence suggests that lipoteichoic acid (LTA) on the surface of Streptococcus pyogenes is centrally involved in the adherence of these bacteria to the oral mucosa. The major receptor on the surface of buccal mucosal cells for S. pyogenes is fibronectin. Fibronectin is a complex glycoprotein found in blood, extracellular matrices, and saliva. Many species of streptococci bind to fibronectin, although the characteristics of these interactions are different. Furthermore, whereas LTA is an efficient inhibitor of the binding of fibronectin to group A streptococci, it has little ability to inhibit the binding of fibronectin to Streptococcus pneumoniae or Staphylococcus aureus. Studies conducted with fibronectin show that different bacteria bind to different sites. The ability of various bacteria to interact with different domains on the fibronectin molecule may play an important role in bacterial adherence and tissue tropism.

Antibody-independent interactions of fibronectin, C1q, and human neutrophils with Treponema pallidum.

Although recent evidence suggests that fibronectin may be involved in the attachment of treponemes to mammalian cells, its possible role in promoting phagocytosis of Treponema pallidum has not been investigated. In the present study, we examined the antibody-independent interactions of fibronectin, C1q, and human polymorphonuclear leukocytes with T. pallidum. Binding of [125I]fibronectin was specific and saturable with an affinity constant of approximately 2 X 10(7) M-1. The number of binding sites per treponeme at 37 degrees C, irrespective of the mammalian source of fibronectin, was between 2,500 and 7,500, with a mean of approximately 4,700. Binding of [125I]C1q to T. pallidum, in the absence of antibodies to the organism, also was saturable and specific. Pretreatment of treponemes with C1q enhanced binding of soluble [125I]fibronectin two- to threefold and also increased attachment of 125I-surface-labeled treponemes to fibronectin-coated surfaces. Treatment of 125I-labeled T. pallidum with fibronectin alone, or together with C1q, however, did not enhance surface phagocytosis by neutrophils.

Characterization of a membrane‐associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.

Preferential in vitro interaction of fibronectin with histone H2A + H3

The interactions of fibronectin with unfractionated histone and histone subfractions (H1, H2A + H3, H2B and H4) were studied under physiological conditions by laser nephelometry. It was found that fibronectin interacted preferentially with histone H2A + H3. Due to self-aggregation, fibronectin showed a very low relative light scattering.

Tyrosine optical activity as a probe of the conformation and interactions of fibronectin.

AbstractA very intense negative band is observed at ∼ 183 nm in the CD spectrum of fibronectin from bovine plasma. This transition has not previously been reported, probably because it occurs in a spectral region that has not been readily accessible in earlier studies. At longer wavelength, the observed CD is very similar to spectra reported for human and chick material, having positive bands at ∼230 and ∼200 nm, and a negative band at ∼215nm. The low molar ellipticity of the negative band ([θ] ≈ −2.5 × 103 deg cm2 dmol−1) suggests little α‐helix or β‐sheet structure. The new transition, and the two positive bands at higher wavelength, do not correspond to known transitions of the peptide backbone, but all three are present in the CD of N‐acetyltyrosineamide. It is therefore suggested that the observed CD behavior of fibronectin arises predominantly from the optical activity of tyrosine side chains. The contribution of this side‐chain optical activity to the CD of other proteins is discussed.On raising pH to ionize tyrosine residues, the positive CD band at ∼230 nm is lost in both N‐acetyltyrosineamide and in fibronectin. The spectral change is fully reversible in the model compound, but only partially reversible in fibronectin. From this evidence, and the magnitude of the 183‐nm band, it is suggested that some or all of the tyrosine residues in fibronectin may be present within ordered domains. The possible role of SS bonds in maintaining tertiary structure is discussed. The interaction of fibronectin with heparin is accompanied by a large increase in the 183‐nm band and by slight enhancement of the negative band at 215 nm, consistent with some limited formation of β‐sheet.Present results indicate that CD may be of considerable value in characterization of the molecular organization and biologically relevant interactions of fibronectins and of related glycoproteins of the extracellular matrix.

Molecular and Biological Interactions of Fibronectin

Fibronectin is a high molecular weight glycoprotein present at cell surfaces and in various body fluids. It is involved in cellular adhesion. The frequent absence of fibronectin from the surface of malignant cells may contribute to the invasive growth of tumors. To develop a better understanding of the functions of fibronectin, we have studied its structure by making use of proteolytic fragmentation and monoclonal antibodies. Fragments of plasma fibronectin which retain one or several of the affinities of intact fibronectin have been purified using affinity chromatography on collagen and heparin, and on monoclonal antibodies. These studies reveal a structure in which the different functions are located in separate protease-resistant molecular domains. The NH2-terminal domain contains the binding sites for fibrin, actin, and Staphylococci; this is followed by a collagen-binding domain, the cell attachment domain, and at the COOH-terminal end, the heparin-binding domain. These binding sites allow fibronectin to participate in multiple interactions with collagens and proteoglycans. The insoluble complexes formed by these 3 components that can be generated in vitro may represent a model for the basic structure of extracellular matrices. The role of fibronectin in such a matrix may be to contribute to the stability of the complex and to provide adhesion sites for cells. Disturbances in the interactions involving fibronectin could be potentially important to understanding diseases of connective tissues and in malignancy.

Molecular and biological interactions of fibronectin.

Fibronectin is a high molecular weight glycoprotein present at cell surfaces and in various body fluids. It is involved in cellular adhesion. The frequent absence of fibronectin from the surface of malignant cells may contribute to the invasive growth of tumors. To develop a better understanding of the functions of fibronectin, we have studied its structure by making use of proteolytic fragmentation and monoclonal antibodies. Fragments of plasma fibronectin which retain one or several of the affinities of intact fibronectin have been purified using affinity chromatography on collagen and heparin, and on monoclonal antibodies. These studies reveal a structure in which the different functions are located in separate protease-resistant molecular domains. The NH2-terminal domain contains the binding sites for fibrin, actin, and Staphylococci; this is followed by a collagen-binding domain, the cell attachment domain, and at the COOH-terminal end, the heparin-binding domain. These binding sites allow fibronectin to participate in multiple interactions with collagens and proteoglycans. The insoluble complexes formed by these 3 components that can be generated in vitro may represent a model for the basic structure of extracellular matrices. The role of fibronectin in such a matrix may be to contribute to the stability of the complex and to provide adhesion sites for cells. Disturbances in the interactions involving fibronectin could be potentially important to understanding diseases of connective tissues and in malignancy.

Masking of extracellular collagen and the co-distribution of collagen and fibronectin during matrix formation by cultured embryonic fibroblasts.

Embryonic chicken heart fibroblasts (CHF) were examined by double immunofluorescence in order to study the interactions of collagen and fibronectin. Double-labelling studies of permeabilized whole mounts of CHF, cultured for 15 min to 2 days after plating, detected extracellular collagen at very early times (15 min to 1 h) but not at times later than 3 h. Abundant fibronectin was seen at all times. These observations suggested that the collagen/procollagen antigen was being "masked' by matrix components at the later times. This possibility was tested in two ways: (1) immunolabelling of frozen sections of multilayered 4-day CHF cultures, which showed extensive extracellular collagen superimposable with fibronectin. (2) Limited trypsinization of 4-day CHF cultures, which allowed collagen to be immunolabelled concomitantly with a partial decrease in the degree of fibronectin labelling. The results are discussed in terms of the secretion and assembly of collagen and fibronectin in cultures of embryonic fibroblasts.

Effects of hyaluronate and heparan sulphate on collagen-fibronectin interactions

Interactions of fibronectin and glycosaminoglycans and the involvement of heparan sulphate and hyaluronate in fibronectin-collagen interactions have been studied by affinity chromatography. Partially periodate-oxidized glycosaminoglycans were coupled to adipic acid dihydrazide-substituted agarose. The elution of fibronectin was performed by using increasing concentrations of NaCl. Of the copolymeric glycosaminoglycans, heparin and self-associating heparan sulphates display the highest affinity towards fibronectin while hyaluronic acid and chondroitin 6-sulphate do not bind fibronectin. Competitive release experiments suggest the existence of common binding sites for copolymeric glycosaminoglycans on the fibronectin backbone. Heparan sulphate favours the formation of collagen-fibronectin complexes at low molarity, while hyaluronate is ineffective at low concentrations and prevents the formation of complexes when present at concentrations > 1 mg ml −1. It is suggested that heparan sulphate promotes the formation of complexes which bind with fibronectin thus producing steric changes that increase the affinity for collagen, while hyaluronate prevents the binding of fibronectin to collagen by a steric exclusion mechanism.

Binding of fibronectin to actin is inhibited by gelatin

An enzyme-linked immunosorbent assay was developed to study the ability of fibronectin to bind to actin. Plastic microtiter wells were coated with actin and the binding of fibronectin was detected using purified fibronectin antibodies conjugated to alkaline phosphatase. The binding was dependent on the concentration of actin used for coating and on the amount of fibronectin that was subsequently permitted to bind. The binding could be inhibited by actin and gelatin, but not by heparin or bovine serum albumin. No major inhibition was observed by amines known to interfere with some of the other interactions of fibronectin. The ability of gelatin to inhibit the binding suggests that actin and collagen cannot bind to fibronectin simultaneously, and that the cell-binding and actin-binding sites of fibronectin are separate since cells attach to collagen-bound fibronectin.

Binding and factor XIIIa-mediated cross-linking of a 27-kilodalton fragment of fibronectin to Staphylococcus aureus.

A 27-kilodalton tryptic fragment, derived from the amino terminus of the 200-kilodalton fibronectin subunit, inhibited binding of intact fibronectin to Staphylococcus aureus and could be cross-linked to Staphylococcus aureus by blood coagulation Factor XIIIa. Interactions of fibronectin with Staphylococcus aureus via this fragment may be important for bacterial opsonization and attachment.

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [ 35S]heparin. Fibronectin attached directly to Sepharose also bound [ 35S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [ 35S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit. The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads. It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.