Nonhelical,fibronectin-binding basement-membrane collagen from endodermal cell culture

A novel method of affinity chromatography on insolubilized collagen-binding fragments of fibronectin was utilized to isolate a random-coil collagenous protein from culture media of mouse teratocarcinoma-derived endodermal cells. These cells also produced another collagenous protein, which did not bind to fibronectin but could be isolated by differential salt precipitation. The affinity-purified collagen differs from its conventionally isolated counterpart in that it is not triple-helical in structure, its polypeptides are not disulfide-crosslinked and it has affinity for fibronectin in its native state. Both collagens resemble previously characterized type IV basement-membrane collagens with respect to their amino acid composition, cyanogen bromide peptides, chain size, immunological reactivity and tissue localization. The random-coil collagen is directly active in promoting the attachment of some lines of cells, but for attachment of the endodermal cells addition of fibronectin is required. This suggests that the presence of nonhelical, fibronectinbinding collagen may have biological significance in the interaction of cells with the extracellular matrix.     

Lectins as probes for assessing the accessibility of N‐linked glycans in relation to the conformational changes of fibronectin

Fibronectin, a ≈450‐kDa protein with 4–9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal‐induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose‐binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal‐induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. Copyright © 2015 John Wiley & Sons, Ltd.     

Stability of fibronectin biological activity following chemical modification

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.     

Fibronectin quantification without antibodies: a bioassay for the detection of the gelatin-captured macromolecule

We have developed a new cell-adhesion-bioassay (CAA) for the quantitative determination of fibronectin in biological fluids. The assay is based on two particular properties of fibronectin: it specifically binds to gelatin with high affinity and simultaneously it can anchor to different surface molecules of a cell. First fibronectin, derived from very different biological fluids, is purified in situ, within the wells of the microtiter plates applied for the assay, using solid surface bound gelatin. After capturing the macromolecule, it is quantified based on its cell adhesive properties. In contrast to ELISA the CAA does not require specific antibodies, and as the Jurkat cells used as indicator cells, seem to recognize fibronectin from different species equally; species specificity of the reagent plays smaller, perhaps negligible, role in the determination of the amount of the macromolecule. The CAA method may not replace fibronectin specific ELISA-s, but using its principle, improved applications, for example a capture EIA for determining fibronectin can easily be envisioned and CAA may serve as a viable alternative for EIA-s when specific antibodies are not available or when relative measurement of not only the soluble but cell surface associated fibronectin is necessary.     

GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through enhanced integrin alpha5beta1-fibronectin interaction

Carcinoembryonic antigen (CEA) and CEA family member CEACAM6 are glycophosphatidyl inositol (GPI)-anchored, intercellular adhesion molecules that are up-regulated in a wide variety of human cancers, including colon, breast, and lung. When over-expressed in a number of cellular systems, these molecules are capable of inhibiting cellular differentiation and anoikis, as well as disrupting cell polarization and tissue architecture, thus increasing tumorigenicity. The present study shows that perturbation of the major fibronectin receptor, integrin alpha5beta1, underlies some of these biological effects. Using confocal microscopy and specific antibodies, CEA and CEACAM6 were demonstrated to co-cluster with integrin alpha5beta1 on the cell surface. The presence of CEA and CEACAM6 was shown to lead to an increase in the binding of the integrin alpha5beta1 receptor to its ligand fibronectin, without changing its cell surface levels, resulting in increased adhesion of CEA/CEACAM6-expressing cells to fibronectin. More tenacious binding of free fibronectin to cells led to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. Disruption of this process with specific monoclonal antibodies against either fibronectin or integrin alpha5beta1 led to the restoration of cellular differentiation and anoikis in CEA/CEACAM6 producing cells.     

Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.     

Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen

A proteoglycan isolated from a rat yolk sac tumor and characterized as a chondroitin sulfate proteoglycan with a smaller amount of dermatan sulfate was studied with respect to complex formation with collagen and fibronectin. The proteoglycan co-precipitated with native collagen from neutral salt solutions at 6 degrees C and 37 degrees C. Addition of fibronectin in such precipitation mixtures resulted in incorporation of fibronectin to the precipitate. Treatment of the proteoglycan with alkali to separate the glycosaminoglycan chains from the protein part and digestion of the protein part with papain greatly reduced the capacity of the proteoglycan to precipitate collagen and fibronectin. A defined extracellular matrix as represented by the complexes of collagen, proteoglycan, and fibronectin constructed here may be useful for studies on the biological effects of extracellular matrices. The multiple interactions of matrix macromolecules exemplified by these results may play a role in the formation of extracellular matrices and in the maintenance of their integrity.     

Adhesive substrates for fibronectin

In order to promote cell attachment, fibronectin must first undergo activation by a suitable substrate. In this study, 52 materials have been surveyed for their ability (a) to bind fibronectin, (b) to activate the cell-adhesive property of fibronectin, and (c) to support the growth of cells. Many plastics, polysaccharides, metals, and ceramics were found to support cell growth as well as the fibronectin-dependent attachment of cells. Several other substrates have been identified that were inactive in promoting either cell attachment or growth. Hydrophobic substrates were found to be active in fibronectin activation, whereas hydrophilic substrates were found to be inactive. Since fibronectin binds to substrata of extremely varied chemical composition, it is clear that the binding of fibronectin to such substrata is nonspecific in nature. Since protein pretreatment of all substrata, except collagen and poly(L-lysine), abolished the physical binding of fibronectin, the binding of fibronectin to artificial substrata is probably ascribable to a nonspecific hydrophobic protein-substratum interaction. In contrast, several lines of evidence indicate that the interaction between fibronectin and collagen displays biological specificity. Poly(hydroxyethylmethacrylate)(poly(HEMA)), which has previously been shown to be nonadhesive for cells, is demonstrated here to be unique in its inability to bind fibronectin. Addition of one part per million of an adhesive polymer to poly(HEMA) permits fibronectin binding to occur.     

Synthetic peptides competitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin

The 75,000-dalton cell-binding domain of fibronectin (f75k) and synthetic peptides derived from its sequence have been used to examine the cell-surface fibronectin receptor. The spreading of baby hamster kidney fibrolasts on f75k-coated substrates and the direct binding of [3H]f75k to these cells were both competitively inhibited by a synthetic peptide of fibronectin with the sequence Gly-Arg-Gly-Asp-Ser, indicating that the peptide and f75k interact with the same cell-surface sites. Related peptides, including the inverted sequence Ser-Asp-Gly-Arg, also competed for f75k binding. Our results provide the first evidence that fibroblastic baby hamster kidney cells recognize the same fibronectin amino acid sequence in direct binding of soluble ligand and in indirect inhibitory biological assays, thus correlating our direct fibronectin-receptor binding assay with biological functional assays for the fibronectin receptor in fibroblasts.     

Deglycosylation of the chymotryptic collagen-binding fragment of human plasma fibronectin does not modify its affinity to denatured collagen

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.     

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii , we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria–fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.     

Specificity of miR-378a-5p targeting rodent fibronectin

One criterion for microRNA identification is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3′UTR. Because most miRNA target sites identified are highly conserved across different species, it is not clear whether miRNA targeting is species-specific. To predict miRNA targeting, we aligned all available fibronectin 3′UTRs and observed significant conservation of all 20 species. Twelve miRNAs were predicted to target most fibronectin 3′UTRs, but rodent fibronectin showed potential binding sites specific for five different miRNAs. One of them, the miR-378a-5p, contained a complete matching seed-region for all rodent fibronectin, which could not be found in any other species. We designed experiments to test whether the species-specific targeting possessed biological function and found that expression of miR-378a-5p decreased cancer cell proliferation, migration, and invasion, resulting in inhibition of tumor growth. Silencing fibronectin expression produced similar effects as miR-378a-5p, while transfection with a construct targeting miR-378-5p produced opposite results. Tumor formation assay showed that enhanced expression of fibronectin in the stromal tissues as a background environment suppressed tumor growth, while increased fibronectin expression inside the tumor cells promoted tumor growth. This was likely due to the different signaling direction, either inside-out or outside-in signal. Our results demonstrated that species-specific targeting by miRNA could also exert functional effects. Thus, one layer of regulation has been added to the complex network of miRNA signaling.     

A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin

FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.     

Trigramin, an RGD-containing peptide from snake venom, inhibits cell-substratum adhesion of human melanoma cells

Trigramin, a cysteine-rich, RGD-containing peptide isolated from the venom of the Trimeresurus gramineus snake, inhibited the adhesion of human melanoma cells to fibronectin and fibrinogen. Compared on a molar basis to GRGDSP, trigramin was approximately 500 times more potent than the hexapeptide at inhibiting cell adhesion to fibronectin. The activity of trigramin was abolished by chemical reduction of the molecule, indicating that the secondary structure is important to the biological activity. Trigramin presents an example of an effective inhibitor of cell adhesion that has developed in nature and may prove to be a useful probe in studying the cell surface receptors involved in cell adhesion.     

Deposition of fibronectin on material surfaces exposed to plasma: quantitative and biological studies

Experiments were carried out to characterize plasma fibronectin deposition onto material surfaces exposed to plasma solutions. Under nonclotting conditions, the amount of fibronectin adsorption on the surfaces, determined by an indirect radioactive antibody assay, was maximal at low plasma concentrations (0.1%). At higher concentrations of plasma, other plasma proteins appeared to compete with and inhibit adsorption of fibronectin. Biological activity (fibronectin-promoted cell spreading) was also greatest at low plasma concentrations and decreased as the plasma concentration was raised. When surfaces were exposed to plasma under clotting conditions (i.e., addition of Ca2+ and thrombin), fibronectin deposition on the surfaces and biological activity remained constant or increased as the plasma concentration was raised. Based on indirect immunofluorescent antibody assays, the fibronectin deposited from clotting plasma appeared to be in a punctate distribution over the entire material surface and occasionally was associated with discrete fibrillar structures. The increased deposition of fibronectin from clotting plasma compared to nonclotting plasma (approximately a 10-fold difference with 10% plasma) was partially a result of covalent crosslinking of fibronectin to fibrin based upon studies with putrescine added to inhibit crosslinking during clotting. On the other hand, the increase in biological activity that occurred if the surfaces were exposed to clotting plasma was completely inhibited by putrescine, indicating that fibronectin had to be crosslinked to fibrin to have biological activity under these conditions. Finally, fibronectin deposition also occurred on surfaces exposed to whole blood and was markedly enhanced when clotting occurred.     

In vitro denaturation-renaturation of fibronectin. Formation of multimers disulfide-linked and shuffling of intramolecular disulfide bonds

It is well established that fibronectin into extracellular matrix undergoes repeated tensions applied by cells, resulting into dramatic structural changes which reflect its elastic properties. However, there is currently no study reporting with precision the consequences of this elasticity on fibronectin structure and conformation. In the present work, we investigated fibronectin structural and conformational reorganization in vitro through a denaturation-renaturation approach. The similarities and differences between "refolded fibronectin" and "native fibronectin" were investigated using various spectroscopic methods, hydrodynamic characterization, molecular imaging and biochemical characterization. In the refolded form, secondary structure elements as well as local tyrosine and tryptophan environment are identical compared to the native form. Interestingly, some differences in global tertiary structure organization and molecular conformation were observed. These differences are due to the reactivity of the two free cysteines, which are buried in the native state but become accessible during the unfolding process. First, oxidation of these residues leading to the formation of intermolecular disulfide bonds results in formation of stabilized multimer. Second, some illegitimate intramolecular disulfide bonds are formed. The presence of iodoacetamide, the sulfhydryl alkylating agent, during the unfolding-refolding process prevents all these events. This study clearly demonstrates that, under near physiological conditions, competitive renaturation pathways occur, involving free cysteines in either multimer formation or intermolecular shuffling of disulfide bonds. These findings might have important implications for future studies and be helpful to develop a deeper understanding of fibronectin morphology.     

Amniotic fluid fibronectin. Characterization and synthesis by cells in culture

A glycoprotein immunologically related to plasma cold-insoluble globulin (CIG) and fetal skin fibroblast fibronectin has been purified from second-trimester human amniotic fluid. This protein (amniotic fluid fibronectin) migrated more slowly than CIG on sodium dodecyl sulfate gel electrophoresis and showed greater polydispersity which could result, at least in part, from heterogeneity in glycosylation. Cloned human amniotic fluid epithelioid and fibroblastic cells synthesized and secreted a protein with similar properties into the culture medium. Fibronectin was shown to be associated with the pericellular and extracellular matrix of cultured amniotic fluid cells by immunofluorescence, lactoperoxidase-catalyzed iodination, and labeling with ferritin-conjugated antibodies. The kinetics of secretion of the protein were consistent with its role as a matrix protein. We anticipate that amniotic fluid fibronectin will prove to be the same protein which elsewhere in the body is incorporated into connective tissues and basement membranes. Amniotic fluid could, therefore, serve as a convenient source of in vivo synthesized fibronectin for biological and structural studies.     

Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders.

The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties.     

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.