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PubMed1994

The binding of fibronectin to entactin is mediated through the 29 kDa amino terminal fragment of fibronectin and the G2 domain of entactin.

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.

Animals↗
PubMed1999

Analysis of splice variants of the fibronectin gene in thyroid carcinomas by reverse transcription-polymerase chain reaction: increased expression of oncofetal fibronectin mRNA in papillary carcinomas is not caused by the alternation in splicing.

The expression levels of each splice variant of the fibronectin gene in the normal thyroid and in thyroid tumors were examined by reverse transcription-polymerase chain reaction (RT-PCR). In thyroid papillary carcinomas, insertion of a variant exon in the ED-A and ED-B domains, and three of five types of splice variants in the IIICS domain were observed. In spite of the marked increase in the expression of oncofetal fibronectin mRNA with the IIICS sequence in papillary and anaplastic carcinomas in the previous reports, the relative expression levels of each splice variant with or without the IIICS sequence showed no difference among all the tumor types. Therefore, the much increased expression of oncofetal fibronectin mRNA in these carcinomas is not caused by the alternation in splicing, but may be caused by an increase in promoter activity or stability of mRNA of the fibronectin gene.

Adenocarcinoma, Follicular↗
PubMedDate not supplied

Effect of cyclic Arg-Gly-Asp-containing peptide on fibronectin binding to activated platelets; role of fibronectin on platelet aggregation.

The interaction of fibronectin with glycoprotein IIb/IIIa (GPIIb/IIIa) was studied. Fibronectin bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with 98,000 molecules/platelet (Kd = 2.76 x 10(-7) M). The binding was inhibited by other adhesive proteins with IC50S of 0.076-0.105 microM and the anti-GPIIb/IIIa monoclonal antibody (LJ-CP8). The binding was also inhibited by the cyclic GRGDSPA peptide and the linear GRGDSPA peptide with IC50S of 0.223 microM and 3.75 microM, respectively. Native fibronectin, the cyclic peptide, and the linear peptide blocked thrombin-induced aggregation with IC50 of 0.95 microM, 32 microM, and 190 microM, respectively. These observations imply that the conformation of RGD-containing sequence may play an important role for an increasing affinity of the binding, and fibronectin may prevent thrombus formation by interfering with the interaction between fibrinogen and activated GPIIb/IIIa.

Amino Acid Sequence↗
PubMed2000

Identification of residues involved in the interaction of Staphylococcus aureus fibronectin-binding protein with the (4)F1(5)F1 module pair of human fibronectin using heteronuclear NMR spectroscopy.

Many pathogenic Gram-positive bacteria express cell surface proteins that bind to components of the extracellular matrix. This paper describes studies of the interaction between ligand binding repeats (D3 and D1-D4) of a fibronectin-binding protein from Staphylococcus aureus with a module pair ((4)F1(5)F1) from the N-terminal region of fibronectin. When D3 was added to isotope-labeled (4)F1(5)F1, (1)H, (15)N, and (13)C NMR chemical shift changes indicate that binding is primarily via residues in (4)F1, although a few residues in (5)F1 are also affected. Both hydrophobic and electrostatic interactions appear to be involved. The NMR data indicate that part of the D3 repeat converts from a disordered to a more ordered, extended conformation on binding to (4)F1(5)F1. In further NMR experiments, selective reduction of the intensity of D1-D4 resonances was observed on binding to (4)F1(5)F1, consistent with previous suggestions that in each of D1, D2, and D3 repeats, the main fibronectin binding site is in the C-terminal region of the repeat. In D1-D4, these regions also appear to go from a disordered to a more ordered conformation of fibronectin binding. Although the regions of the two proteins which interact had been previously identified, the findings presented here identify, for the first time, the specific residues in both proteins that are likely to be involved in the interaction.

Adhesins, Bacterial↗
PubMed1999

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1.

The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus, but not S. epidermidis, invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in noninvasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1-D4) completely blocked invasion. Integrin alpha5beta1 served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin alpha5beta1 is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.

Adhesins, Bacterial↗
PubMed1994

Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin.

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

Calorimetry↗
PubMed1996

Cross-linking of the NH2-terminal region of fibronectin to molecules of large apparent molecular mass. Characterization of fibronectin assembly sites induced by the treatment of fibroblasts with lysophosphatidic acid.

Cell surface molecules on adherent cells that bind 125I-labeled fibronectin or its 70-kDa N-terminal fragment were identified by cross-linking with factor XIIIa and by photoaffinity labeling. Such cross-linking caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, an enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecules that migrated in discontinuous sodium dodecyl sulfate-polyacrylamide gels at the top of the 3.3% stacking gel and near the top of the separating gel. Estimated sizes of these large apparent molecular mass molecules (LAMMs) were >>3 MDa and approximately 3 MDa. The label in 70-kDa fragment conjugated with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-1, 3'-dithiopropionate was associated with >>3-MDa LAMMs without reduction and with approximately 3-MDa LAMMs after reduction and transfer of the cleavable label. The LAMMs were expressed on monolayer cells shortly after adherence, required both 1% Triton X-100 and 2 M urea for efficient extraction, and were susceptible to digestion with trypsin but not to cathepsin D digestion. Complexes of 125I-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C protease digestion but were not cleaved by chondroitin lyase or heparitinase. Neither the uncleaved complexes nor the cleavage products were immunoprecipitated with anti-fibronectin antibodies directed toward epitopes outside the 70-kDa region. Thus, cell surface molecules that are either very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin.

Antineoplastic Agents↗
PubMed2000

Altered processing of fibronectin in mice lacking heparin. a role for heparin-dependent mast cell chymase in fibronectin degradation.

We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.

Amidohydrolases↗
PubMed1989

Activation of CD4 cells by fibronectin and anti-CD3 antibody. A synergistic effect mediated by the VLA-5 fibronectin receptor complex.

In this study, fibronectin synergized with anti-CD3 antibody to promote CD4 cell proliferation in a serum-free culture system. The cell-adhesive domain plus additional regions of the fibronectin molecule are involved in this synergy. Anti4B4(CDw29) antibody blocked the activation of CD4 cells in this system. Furthermore, it is the VLA-5 protein within the set of molecules recognized by anti-4B4 that serves as a fibronectin receptor on the CD4 lymphocytes. The VLA-5 fibronectin receptor was mainly expressed on CD4+ CD45R-CDw29+ cells and may in part contribute to the unique function of these cells.

Antibodies, Monoclonal↗
PubMed1996

Increased expression of the Ed-B-containing fibronectin (an embryonic isoform of fibronectin) in human osteoarthritic cartilage.

Fibronectin is non-collagenous protein which accumulates in osteoarthritic cartilage. The presence of fibronectin and its specific isoform containing the B sequence, Ed-B fibronectin (B.Fn), was studied in normal and osteoarthritic human cartilage using immunohistochemical and biochemical assays, with a specific monoclonal antibody. Results showed substantial amounts of B.Fn in osteoarthritic cartilage, especially in the superficial and middle layers. Western blot analysis confirmed the presence of B.Fn with a molecular mass of 220 and 55 kDa. In contrast, in normal cartilage, expression of B.Fn was extremely low. In conclusion, the expression of a specific isoform of fibronectin during the osteoarthritic process suggests that this isoform might have specific functions in extracellular matrix remodelling.

Aged↗
PubMed2006

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor.

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.

Adhesins, Bacterial↗
PubMed2002

Identification of CD9 extracellular domains important in regulation of CHO cell adhesion to fibronectin and fibronectin pericellular matrix assembly.

CD9, a 24-kDa member of the tetraspanin family, influences cellular growth and development, activation, adhesion, and motility. Our investigation focuses on the hypothesis that the CD9 second extracellular loop (EC2) is important in modulating cell adhesive events. Using a Chinese hamster ovary (CHO) cell expression system, we previously reported that CD9 expression inhibited cell adhesion to fibronectin and fibronectin matrix assembly. For the first time, a functional epitope on CD9 EC2 that regulates these processes is described. Binding of mAb7, an EC2-specific anti-CD9 monoclonal antibody, reversed the CD9 inhibitory activity on CHO cell adhesion and fibronectin matrix assembly. This reversal of cell phenotype also was observed in CHO cells expressing CD9 EC2 truncations. Furthermore, our data showed that the EC2 sequence (173)LETFTVKSCPDAIKEVFDNK(192) was largely responsible for the CD9-mediated CHO cell phenotype. Two peptides, (135)K-V(172) (peptide 5b) and (168)P-I(185) (peptide 6a), selectively blocked mAb7 binding to soluble CD9 and to CD9 on intact cells. These active peptides reversed the influence of CD9 expression on CHO cell adhesion to fibronectin. In addition, confocal microscopy revealed that CD9 colocalized with the integrin alpha(5)beta(1) and cytoskeletal F-actin in punctate clusters on the cell surface, particularly at the cell margins. Immunoprecipitation studies confirmed CD9 association with beta(1) integrin. The cellular distribution and colocalization of focal adhesion kinase and alpha-actinin with cytoskeletal actin was also influenced by CD9 expression. Thus, CD9 may exhibit its effect by modulating the composition of adhesive complexes important in facilitating cell adhesion and matrix assembly.

Actins↗
PubMed1980

Rapid determination of fibronectin by laser nephelometry. Fibronectin concentrations is plasma in human diseases, I.

Fibronectin (Synonyms: cold-insoluble globulin, antigelatin factor) is a cell surface glycoprotein. It has been shown that remarkably large amounts of fibronectin are present in human plasma. Owing to the difficult and expensive radioimmunoassays, only a few reports on the relation of fibronectin and human diseases have so far been published. In this communication a rapid and easily handled immunoassay, using the light scattering effect, is described. The first results on the measurement of fibronectin concentrations in the plasma of patients with chronic active liver diseases are reported.

Antibodies↗
PubMed1980

Serum levels of fibronectin and a fibronectin-like DNA-binding protein in patients with various diseases.

The levels of fibronectin and the fibronectin-like, malignant disease-associated DNA-binding protein (MAD-2) were determined in sera from patients with various diseases. The levels of both proteins were elevated in most serum samples from patients with various types of cancer and in some patients with nonmalignant diseases. The highest levels of these proteins were found in patients with carcinomas of the breast and lung. A subset of the lung cancer patients with small cell carcinoma had either normal or depressed marker levels. The levels of fibronectin and MAD-2 varied concordantly in the different patient populations studied, which suggests that the serum levels of the fragment are directly proportional to the total amount of fibronectin in the serum.

Breast Neoplasms↗
PubMed1979

Release of platelet fibronectin (cold-insoluble globulin) from alpha granules induced by thrombin or collagen; lack of requirement for plasma fibronectin in ADP-induced platelet aggregation.

Platelets lysed with Triton X-100 contain 3.44 +/- 1.27 (SD) microgram of fibronectin (cold-insoluble globulin) per 10(9) platelets. Fibronectin was partially released from washed whole platelets by collagen or thrombin, and its release by collagen was inhibited by aspirin. Analysis of subcellular fractions obtained by density-gradient centrifugation of disrupted platelets indicated that fibronectin was contained in the alpha granules. Fibrinogen depleted of fibronectin (less than 2 microgram/mg) supported ADP-induced aggregation as effectively as fibrinogen contaminated with this protein, thus reinforcing the generally held view that fibrinogen itself is the necessary protein cofactor in this reaction.

Adenosine Diphosphate↗
PubMed1992

Interaction of fibronectin and fibronectin binding protein (FnBP) of Staphylococcus aureus with murine phagocytes and lymphocytes.

In the present study we examined the in vitro and in vivo interactions of a cloned staphylococcal fibronectin binding protein (FnBP) and plasma fibronectin (Fn) with polymorphonuclear cells (PMNs) and macrophages, and how antibodies against FnBP affect the phagocytosis process in vitro. Moreover, the interaction of FnBP and Fn coupled on latex beads as 'artificial bacteria' and 'artificially opsonized bacteria' with murine spleen cells and peritoneal macrophages was tested. The major finding of the present study is that antibodies against the FnBP of Staphylococcus aureus (S. aureus) and low concentration of antibodies recognized two IgG-binding domains of protein A (SpA) are effective in the promotion of phagocytosis in vitro. It was also observed that FnBP has a chemoattractant activity and causes accumulation of PMNs and macrophages in the mouse peritoneal cavity when injected 24 h before irritation of peritoneal exudate. It seems likely that this activity is connected with binding to Fn molecules since formalin inactivation of FnBP (60%) abolished it. In the in vitro phagocytosis assay in the presence of FnBP (in a medium supplemented with serum depleted of Fn), ingestion of bacteria by phagocytes was identical to assay carried out in the presence of BSA. However, addition of plasma fibronectin caused an increased uptake of bacteria by macrophages and to a lesser degree by PMNs. We observed that in a population of normal splenocytes, those cells that effectively bound FnBP- and Fn-coated latex beads were mostly those cells exhibiting macrophage and dendritic morphology. In populations of spleen cells of animal infected with S. aureus, T lymphocytes were also found to bind FnBP- and Fn-coated latex beads. These data suggest that FnBP may have the ability to promote aggregation of immune cells, either directly or by interaction with plasma Fn, which can be helpful in certain cell-cell interactions taking place at the initial stages of specific immune response.

Animals↗
PubMed1988

A role for fibronectin in the migration of avian precardiac cells. I. Dose-dependent effects of fibronectin antibody.

An anterior-posterior concentration difference of fibronectin associated with the endoderm in early chick embryos has been implicated in the directional migration of precardiac mesoderm cells. We have examined the effect of increasing concentrations of an antibody to fibronectin (FN) to test the essentiality of FN to precardiac cell migration. For controls embryos were incubated in the presence of antibodies produced against several other extracellular components, such as laminin and anti-collagen types I and IV, as well as against integrin, a cell surface FN receptor. Embryos were also incubated in the presence of a high concentration of exogenous FN, as well as in the presence of an RGD-containing synthetic pentapeptide that is recognized by the FN receptor. After incubation of chick embryos in various concentrations of anti-FN (5 to 80 micrograms/ml), a dose-dependent effect of anti-fibronectin was observed, whereby heart development was arrested at high concentrations of anti-FN. Early developmental stages were more susceptible to lower antibody concentrations than later stages. Incubation in the presence of the RGD-containing synthetic peptide resulted in partial cardiabifida. None of the antibodies serving as controls affected cell migration or early heart development. These results support the hypothesis that FN is a major component in the migratory pathway and plays a role in the directional migration of precardiac cells to the embryonic midline.

Animals↗
PubMed1990

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria.

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.

Animals↗