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PubMed1998

Fibronectin-binding peptides. I. Isolation and characterization of two unique fibronectin-binding peptides from gelatin.

Gelatin binds to fibronectin with a high affinity although the fibronectin-binding components have not been located. Fibronectin plays an important role in tumor cell metastasis and gelatin may have a profound effect on the metastatic process. In this study, fractionated acid-washed gelatin was cleaved with trypsin and resultant peptides fractionated by fibronectin-Sepharose affinity chromatography. After further purification using size exclusion HPLC and then reverse-phase HPLC, two unique peptides were obtained and sequenced. The binding affinities of these two peptides to fibronectin were evaluated by an ELISA method developed during this study and compared with the gelatin. Both possessed significantly higher binding affinities to fibronectin than gelatin alone.

Amino Acid Sequence↗

PubMed1995

Antibody specific for extra domain B of fibronectin demonstrates elevated levels of both extra domain B(+) and B(-) fibronectin in osteoarthritic canine cartilage.

A fusion protein containing the alternately spliced Extra Domain B [ED-B(+)] sequence of canine fibronectin was expressed in E. coli and the purified protein was used to produce an antibody specific for the ED-B(+) segment. This antibody recognized canine cartilage fibronectin but not canine plasma or synovial fluid fibronectin. Using this antibody, it was determined that ED-B(+) fibronectin increased proportionally with the total fibronectin in osteoarthritic cartilage. From this result, it was concluded that the chondrocyte is the source of much of the elevated fibronectin that appears in osteoarthritic cartilage.

Amidohydrolases↗

PubMed2003

Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin.

In this study, we have investigated the interactions of a Staphylococcal recombinant fibronectin-binding protein A (rFnbA) with fibronectin, fibrinogen, and fibrin. Using analytical size-exclusion chromatography, we evaluated the stoichiometry of reversible binding of FnbA to fibronectin and demonstrated that, in solution, it can accommodate at least two molecules of fibronectin. Results of ELISA experiments demonstrated that rFnbA binds with equally high affinity to both immobilized fibrinogen and fibrin. When included into a thrombin-induced fibrin polymerization reaction, rFnbA strongly inhibited fibrin assembly in a dose-dependent manner. In this study, we have shown that rFnbA can act as a substrate for coagulation factor XIIIa. Factor XIIIa catalyzes the incorporation of amine donor (dansylacadaverine) and amine acceptor (peptide patterned on the N-terminal sequence of fibronectin) synthetic probes into rFnbA, suggesting that it serves as a bifunctional substrate containing reactive glutamine and lysine residues. We have demonstrated that the reversible complex formed by rFnbA and fibronectin or rFnbA and fibrin is covalently stabilized by the transglutaminase action of factor XIIIa. Incubation of rFnbA in the presence of either of its ligands and factor XIIIa results in the introduction of intermolecular epsilon-(gamma-glutamyl)lysine isopeptide bond(s) and the formation of high molecular mass heteropolymers. These findings suggest a novel mechanism by which pathogenic Staphylococcus aureus may utilize the transglutaminase activity of factor XIIIa for attachment to soluble proteins, cell surfaces, and matrixes.

Adhesins, Bacterial↗

PubMed1998

Fibronectin fibrillogenesis involves the heparin II binding domain of fibronectin.

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.

Antibodies↗

PubMed2005

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes.

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.

Adhesins, Bacterial↗

PubMed1987

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin.

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.

Animals↗

PubMed1987

[Plasma fibronectin in acute leukemia. Effects of the cytostatic therapy on plasma fibronectin level].

Twice a week plasma (Pl.)-fibronectin was determined quantitatively in the course of disease with immunoelectrophoresis according to Laurell in 12 patients suffered from acute non-lymphoblastic leukemia (ANLL) and in 12 patients affected with acute lymphoblastic leukemia (ALL). At diagnosis Pl.-fibronectin concentration was found to be significantly lowered only in those patients affected with ANLL. During the induction therapy Pl.-Fibronectin could be observed to decline significantly in all patients: in acute non-lymphoblastic leukemia from mean 270 micrograms/ml, s 93 micrograms/ml, to mean 185 micrograms/ml, s 89 micrograms/ml (p less than 0.01), and in acute lymphoblastic leukemia from mean 290 micrograms/ml, s 98 micrograms/ml, to mean 180 micrograms/ml, s 94 micrograms/ml (p less than 0.01). After administering L-asparaginase there is a strong decline of Pl.-fibronectin. Pl.-fibronectin concentration could be observed to be significantly lower in patients without remission in comparison to those with remission. A correlation between Pl.-fibronectin concentration and tumour mass could not be identified.

Acute Disease↗

PubMed1989

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin.

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.

Actin Cytoskeleton↗

PubMedDate not supplied

[The use of immobilized antibodies against fibronectin for isolation of macromolecular complexes of fibronectin with other secreted proteins from human fibroblasts].

Interaction of labelled proteins from human fibroblast culture and the antibodies to fibronectin, which were used as soluble and Sepharose-immobilized preparations, was studied. Electrophoretic mobilities of the proteins bound to antibodies were compared. Soluble antibodies bound only fibronectin, while immobilized antibodies bound, except for fibronectin, other low molecular proteins. After treatment of the cultural proteins with bacterial collagenase two main components, besides fibronectin, were eliminated, thus suggesting their collagen-like nature. Two dimers of fibronectin per one trimer of procollagen were present in the material bound to immobilized antibodies to fibronectin. Control studies demonstrated that the data obtained were not an artefact occurring due to sorption of proteins on Sepharose or to unspecific actions of antibodies.

Antibodies↗

PubMed1985

Polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin.

Gelatin-binding chymotryptic fragments from placental fibronectin contain polylactosamine carbohydrates (Zhu, B.C.R., Fisher, S.R., Pande, H., Calaycay, J. Shively, J.E., and Laine, R.A. (1984) J. Biol. Chem. 259, 3962-3970). We have separated polylactosamine-containing gelatin-binding fragments of placental fibronectin from their counterparts containing smaller "complex" N-linked saccharides using Sephadex G-200 gel permeation chromatography. The peptide portions of both fragments have similar amino acid composition and N-terminal sequence (see reference above). The strength of binding of these two glycosylation types of chymotryptic fragments to gelatin differs as shown by the following experiments. 1) Upon urea gradient elution of affinity-bound fibronectin fragments from gelatin-Sepharose chromatography, the apex of the elution peak for polylactosamine-containing fragments occurs at 2.0 M urea while the peak for complex N-linked carbohydrate-containing fragments maximized at 2.5 M urea indicating a tighter binding. Removal of polylactosamine sequences from the former glycopeptide by endo-beta-galactosidase digestion caused the elution peak for this fraction to change from 2.0 to 2.5 M, the same as for the complex N-linked carbohydrate-containing glycopeptide. 2) Competitive displacement experiments give an apparent dissociation constant of polylactosamine-containing fragments at 3 X 10(-9) M whereas this constant for complex carbohydrate-containing fragments is 1 X 10(-9) M. These results indicate that the binding of placental fibronectin to gelatin is weakened by the presence of high molecular weight polylactosamine carbohydrate. To our knowledge this is the first report that the type and extent of glycosylation of a glycoprotein can affect its binding affinity to a proteinacious ligand. Thus, fetal placental fibronectin may have different biological properties than fibronectins containing only the smaller N-linked complex carbohydrate.

Amino Sugars↗

PubMedDate not supplied

Fibronectin in leprosy lesions: observations using monoclonal antibodies to human fibronectin.

Croystat sections of dermal lesions from 33 untreated patients with leprosy were studied by indirect immunofluorescence using monoclonal antibodies to human fibronectin. Macrophages in borderline (BL) and lepromatous (LL) leprosy showed intense staining with antifibronectin antibodies. In the tuberculoid lesions cells of the mononuclear phagocyte series in an epithelioid cell granuloma stained for fibronectin. The lymphocytes surrounding these granulomas also showed the presence of fibronectin. These results suggest that the granuloma of leprosy consists of macrophages expressing fibronectin and the lymphocytes in the lesion also appear to express this protein. In six borderline cases, the sub epidermal collagen band showed intense staining with antifibronectin antibodies. In addition, the distribution of Ia-like antigens and fibronectin was studied on the plastic adherent cells obtained from peripheral blood of 11 untreated patients with leprosy. Results indicate that higher percentage of adherent cells from lepromatous patients express fibronectin in comparison to adherent cells from tuberculoid patients or controls. However, no difference was observed in the expression of the Ia-like antigens by the adherent cells, from these patients.

Antibodies, Monoclonal↗

PubMed1982

Effect of bilateral nephrectomy on plasma fibronectin levels: its influence on acute fibronectin response to trauma and RE blockade.

Plasma fibronectin is important to reticuloendothelial (RE) function and resistance to experimental shock. However, the factors that regulate its level have remained undefined. The present study evaluated the concept that the kidneys may play a role in the modulation of plasma fibronectin levels. Immunoreactive and opsonically active fibronectin in blood over a 28-hour period following bilateral nephrectomy was assessed in male rats (250-350 gm) by electroimmunoassay, peritoneal macrophage monolayer assay, and liver slice bioassay. Controls consisted of sham-operated rats. An acute 25%-30% fibronectin deficiency was detected within the initial 1-2 hours posttrauma in both experimental and control rats. Thereafter, a significant (P less than 0.05) increase in immunoreactive fibronectin developed by 24 hours which was much greater after bilateral nephrectomy as compared to controls (P less than 0.05). With the peritoneal macrophage assay or with the liver slice assay, the plasma or serum from bilateral nephrectomized rats has greater opsonic activity as compared to controls. This corresponded to increased hepatic RE phagocytic function in vivo after nephrectomy. In the 24-hour postnephrectomized rats, fibronectin decreased acutely after RE blockade or traumatic shock (NCD) indicating it was biologically active. These data suggest that the kidneys may participate in regulating the blood level of this protein, which has a role in reticuloendothelial system function and shock resistance.

Animals↗

PubMed1995

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation of Rana pipiens.

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frog Rana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373-1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124-131 of beta 1 subunit of a variety of integrins including alpha 5 beta 1. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).

Amino Acid Sequence↗

PubMed1982

[Fibronectin and fibronectin receptors on the surfaces of polymorphonuclear leukocytes].

Interaction between human neutrophils and fibronectin bound to gelatin-Sepharose granules was studied. It was found that fibronectin following cell-induced dissociation from gelatin-Sepharose caused neutrophil aggregation which was inhibited by EDTA or pretrypsinization of the cells. Besides, polymorphonuclear lymphocytes were found to be capable to adhere to and to spread on gelatin-Sepharose granules. This process was enhanced in the presence of heparin and inhibited by neutrophil pretrypsinization. The conclusion has been drawn on simultaneous existence of fibronectin and fibronectin receptors on the surface of neutrophils. A hypothesis is advanced of the existence in the fibronectin molecule of two independent binding sites, one being responsible for cell attachment and the other for gelatin binding.

Fibronectins↗

PubMed1996

Glomerulopathy associated with predominant fibronectin deposits: exclusion of the genes for fibronectin, villin and desmin as causative genes.

Glomerulopathy with predominant fibronectin deposits (GFD) is a newly recognized autosomal dominant renal disease that leads to albuminuria, microscopic hematuria, hypertension, renal tubular acidosis type IV, and end-stage renal disease in the second to fourth decade of life. Light microscopy documents extensive deposits in the subendothelial space, which on electron microscopy consist of non-oriented 12 x 125 nm fibers. Deposits are strongly immunoreactive for antibodies to fibronectin. We examined the hypothesis that a genetic defect in the gene for fibronectin is responsible for the disease. In a 197 member pedigree, 13 relatives developed end-stage renal failure from the disease. In 99 individuals haplotype analysis was performed using 6 microsatellite markers spanning a > 56 cM interval in chromosome region 2q34, where fibronectin, villin, and desmin map in close proximity. Haplotype analysis resulted in exclusion of the whole range of 78 cM covered by the markers examined. This result excludes fibronectin, villin, and desmin from being the causative genes for GFD in this large kindred.

Calcium-Binding Proteins↗

PubMed1998

De novo expression of the alpha5beta1-fibronectin receptor in HT29 colon-cancer cells reduces activity of C-SRC. Increase of C-SRC activity by attachment on fibronectin.

Changes in integrin expression during malignant transformation have been observed in many tumors. Colon-carcinoma cells show reduced expression or even loss of the alpha5beta1 integrin compared to normal or adenoma cells. To determine the significance of absent alpha5beta1 integrin signaling, we transfected the cDNA coding for the alpha5 integrin sub-unit into the human colon-carcinoma cell line HT29, which constitutively lacks this subunit but does express the beta1 subunit. We show here that the newly expressed fibronectin receptor alpha5beta1 generates multiple signals, causing marked changes in cytoskeletal arrangements within a few minutes of adhesion to fibronectin. Cells expressing the alpha5beta1 integrin exhibit the formation of actin stress fibers and focal adhesions, as well as the induction of tyrosine phosphorylation of several proteins, within 10 min. We identified the focal adhesion kinase pp125FAK and the cytoskeletal protein paxillin as major phosphorylation substrates in these cells. These proteins remained hypophosphorylated when alpha5-negative control cells were plated on fibronectin. The tyrosine kinase pp60c-src, regarded as central in the regulation of cellular proliferation and constitutively over-expressed in HT29 and in colon-carcinoma cells, showed reduced intrinsic kinase activity in unstimulated HT29alpha5 cells. In contrast, fibronectin-induced signaling through alpha5beta1 increased pp60c-src activity. Moreover, immunoprecipitation of pp60c-src from extracts of HT29alpha5 cells cultivated on fibronectin for 20 min revealed complex formation of pp60c-src and tyrosine-phosphorylated pp125FAK. Our data suggest that de novo expression of the alpha5beta1 integrin in HT29 colon-cancer cells restores signaling via pp125FAK and pp60c-src. Thus, loss of this receptor during malignant transformation may contribute to tumor-cell autonomy, while reduced activity of pp60c-src in HT29alpha5-cells may participate directly in growth control.

Cell Adhesion↗

PubMed1992

Secretion of cell-adhesion-promoting factors, fibronectin, fibronectin fragments and a 53-kDa protein, by human rectal adenocarcinoma cells.

Production of cell-adhesion proteins was examined in 10 cell lines and 5 cultured human cancer cells at an early passage. Two-thirds of the tested cells produced and secreted into their culture medium variable amounts of material active in promoting cell attachment. One of the rectal carcinoma cell lines, CaR-I, grew well in serum-free medium and secreted a large amount of the active principle. The active principles produced by CaR-I cells were characterized after partial purification, and were found to be fibronectin and its fragments. The presence of fibronectin and its fragments was proved by the following facts: (1) reactivity to the monoclonal antibodies which recognize different epitopes of fibronectin, and (2) reactivity to RGD peptide which is the attachment sequence of fibronectin. In addition to fibronectin and its fragments, CaR-I cells were also shown to produce a 53-kDa attachment factor. Unexpectedly, the protein was proved to be most probably the p53 suppressor gene product.

Adenocarcinoma↗

PubMed1988

Fibronectin-like immunoreactivity in Helisoma buccal ganglia: evidence that an endogenous fibronectin-like molecule promotes neurite outgrowth.

We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.

Animals↗