Conformational Changes Of Fibronectin Research Articles

VEGF and VEGFR2 bind to similar pH-sensitive sites on fibronectin, exposed by heparin-mediated conformational changes

Physical interactions between vascular endothelial growth factor (VEGF), a central player in blood endothelial cell biology, and fibronectin, a major fibrillar protein of the extracellular matrix, are important determinants of angiogenic activity in health and disease. Conditions signaling the need for new blood vessel growth, such as hypoxia and low extracellular pH, increase VEGF–fibronectin interactions. These interactions can be further fine-tuned through changes in the availability of the VEGF-binding sites on fibronectin, regulated by conformational changes induced by heparin and heparan sulfate chains within the extracellular matrix. These interactions may alter VEGF bioavailability, generate gradients, or alter the way VEGF is recognized by and activates its cell-surface receptors. Here, using equilibrium and kinetic studies, we discovered that fibronectin can also interact with the extracellular domain of the VEGF receptor 2 (VEGFR2). The VEGFR2-binding sites on fibronectin show great similarity to the VEGF-binding sites, as they were also exposed upon heparin-induced conformational changes in fibronectin, and the interaction was enhanced at acidic pH. Kinetic parameters and affinities for VEGF and VEGFR2 binding to fibronectin were determined by surface plasmon resonance measurements, revealing two populations of fibronectin-binding sites for each molecule. Our data also suggest that a VEGF/VEGFR2/fibronectin triple complex may be formed by VEGF or VEGFR2 first binding to fibronectin and subsequently recruiting the third binding partner. The formation of such a complex may lead to the activation of distinct angiogenic signaling pathways, offering new possibilities for clinical applications that target angiogenesis.

Enhanced osteogenic activity of anatase TiO2 film: Surface hydroxyl groups induce conformational changes in fibronectin

In this study, with an attempt to identify the effects of TiO2 crystalline phase compositions on the osteogenic properties, the anatase and rutile TiO2 thin films with similar film thickness, surface topography and hydrophilicity were prepared on Si (100) substrates by atomic layer deposition (ALD), subsequent thermal annealing and ultraviolet irradiation. The films were studied with XRD, XPS, FE-SEM, AFM, FTIR and contact angle measurements. In vitro cellular assays showed that the anatase phase led to better osteoblast compatibility in terms of adhesion, proliferation, differentiation, mineralization as well as osteogenesis-related gene expression when compared with the rutile phase. We investigated the difference between the anatase and rutile TiO2 films at the biomolecular level to explain the enhanced osteogenic activity of the anatase film. It was found that the presence of more TiOH groups on anatase surface induced more cell-binding sites of fibronectin (FN) exposed on its surface, causing a more active conformation of the adsorbed FN for subsequent osteoblast behaviors.

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.

Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

Shear-Induced Fibrillar-like Supramolecules of Plasma Fibronectin: A New Form of Fibronectin with Enhanced Activity in Platelet Adhesion and Aggregation

Introduction: Plasma fibronectin (FN) is synthesized by hepatocytes and secreted into the circulation in a soluble, compact and non-fibrillar form. Plasma FN is assembled by cells or adherent platelets into functional fibrils. Reports have indicated that the process to incorporate FN into multimer fibrils can also occur in cell-free models in vitro by incubation with denaturants, reducing agents, or anastellin (FN peptidic fragment). Here, we report on (1) the formation of insoluble fibrillar-like supramolecules of plasma FN (FN fibrils) by exposing the molecules to increasing shear rates and (2) the functional characterization in platelet adhesion and aggregation.Methods: To induce the formation of FN fibrils, 1 ml of plasma FN solution (100 μg/ml) was added to plates pre-coated with FN (100 μg/ml). Subsequently, the FN solutions were exposed to shear (50 to 5000 s-1 within 5 min and subsequently 5000 to 50 s-1 within 5 min) generated by a cone-plate rheometer (Haaka Rheostress 1; Thermo Scientific). Viscosities of FN solutions were recorded. To quantify the formation of FN fibrils, FN solutions after exposure to shear were collected and incubated with 2% deoxycholate (DOC). The DOC-insoluble pellets containing FN fibrils were isolated by centrifugation at 19,000 g for 20 min at 4°C and resuspended in 1% SDS buffer for Western blot analyses. For adhesion experiments, washed platelets (107/ml) in HEPES Tyrode’s buffer were labeled with 10 μM 5-chloromethylfluorescein diacetate and placed on 96-well plates pre-coated with FN or FN fibrils (25 µg/ml) for 30 min at 37°C. In parallel experiments, platelets resuspended in FN-depleted plasma (107/ml) were placed onto immobilized collagen, fibrinogen, FN (10 µg/ml) in the presence of FN (300 µg/ml) or FN fibrils (10 µg/ml). For aggregation experiments, FN (5, 10, 300 µg/ml) or FN fibrils (5, 10 µg/ml) was added to platelet-rich plasma (PRP) or platelets resuspended in FN-depleted plasma (2.5 × 108/ml). Aggregation was induced by 400 nM PMA (Phorbol 12-myristate 13-acetate), or 10 µg/ml collagen.Results: The initial viscosities (mPa’s) of FN solutions were 7.62 ± 0.98. Upon exposure to dynamic shear for 10 min, viscosities increased to 10.98 ± 1.81 (p = 0.02, n = 4), suggesting conformational changes of FN. Western blot analyses of DOC-insoluble fractions revealed bands of FN in the range of 220 – 250 kDa (reducing condition), indicative of the formation of insoluble fibrils in FN solutions after exposure to shear. Platelet adhesion and aggregation experiments were performed to compare the activity of FN fibrils with normal plasma FN. For adhesion experiments, washed platelets in HEPES Tyrode’s buffer were placed onto immobilized FN or FN fibrils (25 µg/ml). The adhesion rates (mean fluorescence signal ± SD) of washed platelets were higher onto surfaces coated with FN fibrils (0.5 ± 0.06) than onto surfaces coated with plasma FN (0.4 ± 0.01) (p = 0.04, n = 3). In parallel adhesion experiments using platelets resuspended in FN-depleted plasma, addition of plasma FN (300 µg/ml) increased platelet adhesion rates onto immobilized collagen (from 0.14 ± 0.005 to 0.2 ± 0.01, p = 0.0007), fibrinogen (from 0.16 ± 0.03 to 0.22 ± 0.01, p = 0.03), and FN (from 0.14 ± 0.01 to 0.18 ± 0.02, p = 0.04) (n = 3). Addition of FN fibrils at low concentration of 10 µg/ml had a similar supportive effect. FN showed an inhibitory effect in platelet aggregation. Activation by 400 nM PMA induced aggregation of PRP by 81% (amplitude). In the presence of plasma FN at 5, 10, 300 µg/ml, platelet aggregation was reduced to 50 %, 41 %, and 29.5 %, respectively. A stronger inhibition on platelet aggregation was seen when FN fibrils were added. PRP aggregated by 35.4 % and 17 % in the presence of 5 and 10 µg/ml FN fibrils, respectively. The same phenomenon was observed in aggregation assays using platelets resuspended in FN-depleted plasma and collagen (10 µg/ml) as activating agonist.Conclusion: Our study shows that dynamic shear rates induce the formation of insoluble fibrillar-like form of plasma FN in cell-free model in vitro. Fibril formation of FN can be monitored by measuring viscosities of FN solutions during exposure to shear and quantified by Western blot. Shear-induced formed FN fibrils have an explicitly stronger activity in supporting platelet adhesion and inhibiting platelet aggregation than normal plasma FN. This finding emphasizes the importance of FN assembly on its activity in platelet functions. DisclosuresNo relevant conflicts of interest to declare.

Electrochemical modulation of plasma fibronectin surface conformation enables filament formation and control of endothelial cell–surface interactions

Electrochemical modulation of a gold surface charge induces conformational changes in fibronectin when immobilized on the surface. A negatively-charged surface yields an open and filamentous fibronectin which significantly improves endothelial cell adhesion.

Titanium alloy surface oxide modulates the conformation of adsorbed fibronectin to enhance its binding to α5β1 integrins in osteoblasts

Our laboratory has previously demonstrated that heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of a titanium alloy (Ti6Al4V) increased the net negative charge of the alloy's surface oxide and the attachment of osteoblastic cells to adsorbed fibronectin. The purpose of the current study was to investigate the biological mechanism by which these surface pretreatments enhance the capacity of fibronectin to stimulate osteoblastic cell attachment. Each pretreatment was found to increase the binding (measured by ELISA) of a monoclonal anti-fibronectin Ig to the central integrin-binding domain of adsorbed fibronectin, and to increase the antibody's inhibition of osteogenic cell attachment (measured by hexosaminidase assay). Pretreatments also increased the binding (measured by ELISA) of anti-integrin IgG's to the α(5) and β(1) integrin subunits that became attached to fibronectin during cell incubation. These findings suggest that negatively charged surface oxides of Ti6Al4V cause conformational changes in fibronectin that increase the availability of its integrin-binding domain to α(5) β(1) integrins.

Block copolymer arrangement and composition effects on protein conformation using atomic force microscope-based antigen-antibody adhesion

The conformational changes of fibronectin (FN) deposited on various block copolymers where one block is composed of poly(methyl methacrylate) (PMMA) and the other block is either poly(acrylic acid) (PAA) or poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated using a functionalized atomic force microscope (AFM) tip. The tip was modified with an antibody sensitive to the exposure of the arginine-glycine-aspartic acid (RGD) groups in FN. By studying the adhesive interactions between the antibody and the proteins adsorbed on the block copolymer surface and phase imaging, it was found that the triblock copolymers PAA-b-PMMA-b-PAA and PMMA-b-PHEMA-b-PMMA, which both have large domain sizes, are conducive to the exposure of the FN RGD groups on the surface. On the basis of these results, it is concluded that the surface chemistry as well as the nanomorphology dictated by the block copolymer arrangement could both tune protein conformation and orientation and optimize cell adhesion to the biomaterial surface.

Antibody sensed protein surface conformation

An antibody-modified atomic force microscope (AFM) tip was used to detect conformational changes of fibronectin deposited on a poly(methyl methacrylate)/poly(acrylic acid) block copolymer compared to PMMA and a random poly(methyl methacrylate)/poly(acrylic acid) copolymer with an identical chemical composition. Based on the antibody-protein adhesive force maps and phase imaging, it was found that the nanomorphology of the triblock copolymer induces the desired conformation of fibronectin. This finding demonstrates that block copolymer nanomorphology can be used to regulate protein conformation and potentially cellular response.

Protein conformation changes on block copolymer surfaces detected by antibody‐functionalized atomic force microscope tips

Conformational changes of fibronectin (Fn) deposited on poly(methyl methacrylate) and poly(acrylic acid) block copolymers with identical chemical compositions were detected using an antibody-functionalized atomic force microscope (AFM) tip. Based on the antibody-protein adhesive force maps and phase imaging, it was found that the nanomorphology of the triblock copolymer is conducive to the exposure of the arginine-glycine-aspartic acid (RGD) groups in Fn. For the first time, X-ray photoelectron spectroscopy was used to elucidate surface chemical composition and confirm AFM results. The findings demonstrate that block copolymer nanomorphology can be used to regulate protein conformation and potentially cellular response.

Atomic force microscopy evidence for conformational changes of fibronectin adsorbed on unmodified and sulfonated polystyrene surfaces

The effect of polystyrene surface polarity on the conformation of adsorbed fibronectin (FN) has been studied with atomic force microscopy. We demonstrated that bare sulfonated and nonsulfonated polystyrene surfaces featured similar topographies. After the FN adsorption, direct comparison of both types of substrata revealed drastically different topographies, roughness values, and also cell-adhesive properties. This was interpreted in terms of FN conformational changes induced by the surface polarity. At high-solute FN concentrations the multilayer FN adsorption took place resulting, for the sulfonated substratum, in an increase of surface roughness, whereas for the nonsulfonated one the roughness was approximately stable. Conversely, the FN conformation characteristic for the first saturative layer tended to be conserved in the consecutive layers, as evidenced by height histograms. The height of individual FN molecules indicated, consonantly with the derived thickness of the adsorbed protein layer (the latter value being 1.4 nm and 0.6 nm, respectively, for an unmodified and sulfonated polystyrene surface), that molecules are flattened on polar surfaces and more compact on nonsulfonated ones. It was also demonstrated that the FN adsorption and conformation on polymeric substrata, and hence the resultant cell-adhesive properties, depended on the chemistry of the original surface rather than on its topography. Our results also demonstrated the ability of surface polarity to influence the protein conformation and its associated biological activity.

Semiquantitative evaluation of fibronectin adsorption on unmodified and sulfonated polystyrene, as related to cell adhesion

The process of human fibronectin (FN) adsorption on nonsulfonated and sulfonated polystyrene surfaces was studied in relation to mechanisms of L1210 cell adhesion. Radioisotope assays directed towards FN, as well as ELISA measurements of adsorbed FN and bovine serum albumin (BSA) were carried out. (125)I radioisotope assays led to linear FN adsorption isotherms. When combined to ELISA measurements for FN, they revealed the multilayer adsorption. Results indicated a large difference in the saturating first-layer surface density of FN adsorbed on sulfonated and nonsulfonated polystyrene surfaces: significantly (ca. factor of 5) less FN molecules are necessary to complete a monolayer on sulfonated than on nonsulfonated polystyrene. This suggests an unfolded conformation of FN on sulfonated polystyrene, and a more compact one on the nonsulfonated polymer. Significant conformational changes of FN are also indicated by the following: (1) early phase of cell adhesion to FN adsorbed on sulfonated polystyrene surfaces is significantly (ca. factor of 6) higher than to FN on nonsulfonated surfaces, and in the former case adhesion proceeds mostly via alpha(5)beta(1) integrins; (2) RGD, the crucial fragment within central cell binding domain, seems to be partially hidden in the protein structure adopted on nonsulfonated surfaces; (3) patterns of F-actin organization differ in cells adhering to FN on sulfonated and nonsulfonated surfaces. The ELISA study directed against BSA (this protein always present on the surface after the adsorption of FN), showed the importance of "free area," uncovered by both proteins, which influence the cell adhesion processes.

Kinetics of conformational changes of fibronectin adsorbed onto model surfaces

Fibronectin (FN), a large glycoprotein found in body fluids and in the extracellular matrix, plays a key role in numerous cellular behaviours. We investigate FN adsorption onto hydrophilic bare silica and hydrophobic polystyrene (PS) surfaces using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) in aqueous medium. Adsorption kinetics using different bulk concentrations of FN were followed for 2 h and the surface density of adsorbed FN and its time-dependent conformational changes were determined. When adsorption occurs onto the hydrophilic surface, FN molecules keep their native conformation independent of the adsorption conditions, but the amount of adsorbed FN increases with time and the bulk concentration. Although the protein surface density is the same on the hydrophobic PS surface, this has a strong impact on the average conformation of the adsorbed FN layer. Indeed, interfacial hydration changes induced by adsorption onto the hydrophobic surface lead to a decrease in unhydrated β-sheet content and cause an increase in hydrated β-strand and hydrated random domain content of adsorbed FN. This conformational change is mainly dependent on the bulk concentration. Indeed, at low bulk concentrations, the secondary structures of adsorbed FN molecules undergo strong unfolding, allowing an extended and hydrated conformation of the protein. At high bulk concentrations, the molecular packing reduces the unfolding of the stereoregular structures of the FN molecules, preventing stronger spreading of the protein.

Adsorption-Induced Conformational Changes in Fibronectin Due to Interactions with Well-Defined Surface Chemistries

Protein adsorption onto synthetic materials influences cell adhesion and signaling events that direct cell function in numerous biomedical applications. Adsorption of fibronectin (FN) to different surfaces alters protein structure and modulates α5β1 integrin binding, cell adhesion, cell spreading, and cell migration. In the present study, self-assembled monolayers of alkanethiols on Au were used to analyze the effects of surface chemistry (CH3, OH, NH2, and COOH) on the adsorption of a recombinant fragment of FN, FNIII7-10, that incorporates both the synergy and RGD cell binding motifs. Surface chemistry potentiated differential FNIII7-10 adsorption kinetics and adsorbed structure as determined by surface plasmon resonance spectroscopy and antibody binding assays. FNIII7-10 functional activity, determined by cell adhesion strength, was modulated in a fashion consistent with these structural changes (OH = NH2 > COOH > CH3). However, these changes in protein parameters did not correlate simply to differences in surface hydrophobicity, indicating that additional surface parameters influence protein adsorption. These results demonstrate that surface chemistry modulates adsorbed protein structure and activity and establish a relationship between surface-dependent changes in structural domains of FNIII7-10 and functional activity.

Dissolution/reprecipitation and protein adsorption studies of calcium phosphate coatings by FT‐IR/ATR techniques

The surfaces of bioactive Ca-P ceramics immediately change when exposed to proteinaceous solutions. The dissolution behavior and protein interactions of these bioactive materials at the bone/implant interface need to be investigated to understand their material-cellular interactions fully. In this study, FT-IR/ATR techniques were used to study the in situ phosphate release kinetics of Ca-P coatings. The net loss of phosphate molecules from coatings was slower in saline solutions compared with alpha-MEM solutions. Coatings exposed to alpha-MEM solutions containing fibronectin released phosphate molecules slower than coatings exposed to alpha-MEM solutions containing albumin. Conformational changes in fibronectin and albumin adsorbed onto Ca-P and uncoated germanium surfaces were also investigated using FT-IR/ATR spectroscopy. Analysis of changes in the amide I bands indicated that there was a greater loss of beta-sheet structure in adsorbed fibronectin on Ca-P coatings when compared with bare germanium surfaces. Although albumin did change its structure upon adsorption on both Ca-P and germanium, unlike fibronectin, adsorbed albumin structure was similar on Ca-P coatings and germanium. Furthermore, with time the conformation of adsorbed fibronectin and albumin appeared to be very stable on Ca-P coatings, whereas albumin adsorbed to germanium exhibited an increase in ratio of alpha-helix to beta-turn.

Conformational changes of fibronectin induced by polystyrene derivatives with a heparin-like function.

It was previously reported that polystyrene substituted with the sulfonate group, PSSO3, which has anticoagulant heparin-like properties, and then coated with fibronectin supports the growth of human umbilical vein endothelial cells. On the other hand, polystyrene substituted with the amino acid sulfamide group, PSSO2-Asp, which has a higher anticoagulant activity, and then coated with fibronectin no longer supported the growth of endothelial cells. We report here that, while the affinity of fibronectin to either polymer is of the same order of magnitude, fibronectin is adsorbed onto the PSSO2-Asp polymer in a different conformation compared to the PSSO3 polymer. This was shown by a higher binding of polyclonal antifibronectin antibodies to fibronectin-coated PSSO2-Asp polymer, and by a decreased susceptibility of the coated fibronectin to proteolysis by thermolysin. This study provides evidence that a solid phase substrate with a strong heparin-like function may influence the conformation and biological properties of fibronectin.

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.