DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.     

Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.     

Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin.

Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.     

Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature, raising a question as to the physiological significance of the cryptic anti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragment), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.     

Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.     

Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin.

The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a Kd of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of > 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.     

Interaction of fibronectin with heparin in model extracellular matrices: role of arginine residues and sulfate groups

The interaction of heparin with the NH2-terminal domain of human plasma fibronectin was studied by using matrix-driven translocation, an assay for the adhesion of extracellular macromolecules with cell or particle surfaces within artificial collagen matrices. Partial desulfation of heparin rendered it ineffective in competitively inhibiting the interaction of the fibronectin NH2-terminal domain with heparin-coated particles, suggesting a role for sulfate groups of heparin in the interaction. Analysis of the fibronectin domain in terms of its primary structure, its proposed organization into "type I modules", and its hydrophilic and flexible segments led to the identification of several arginine-containing sites of potential interaction with the sulfate groups of heparin. Modification of increasing numbers of arginine side chains with 1,2-cyclohexanedione under mild conditions eventually led to decreases in translocation-promoting activity, and of heparin binding capacity as measured in a gel-shift assay, but the major portions of these functions were retained even when the four most accessible arginines (attributed to sites in and adjacent to the large loops of the type I modules) were modified. With the modification of additional arginines (attributed to sites in the small loops), both functions were lost. The peptide Gly-Arg-Gly, corresponding to a repeated determinant at the tips of two small loops, inhibited translocation, but arginine alone did not. Cleavage of the large loops by CNBr also led to loss of translocation-promoting activity. The correspondence between the molecular determinants of matrix-driven translocation and those previously found for mesenchymal morphogenesis indicates the utility of this system in the analysis of adhesive interactions of biological importance.     

Co-operative domains in fibronectin

The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.     

DNA-binding domains of human fibronectin

Here we report the use of the technique of transferring proteins from polyacrylamide gels to nitrocellulose sheets to identify the DNA-binding domains of human plasma fibronectin. After separation of fibronectin and its tryptic digest on a sodium dodecylsulfate-polyacrylamide gel, the polypeptides were transferred to nitrocellulose sheets and the DNA-binding polypeptides were identified by incubation of the nitrocellul$\text{o}$ se sheets with ³²P-labeled human DNA followed by autoradiography.The following results were obtained: a) only two fibronectin trypsin resistant peptides (50 and 60 kd) showed DNA-binding capacity; b) in competition experiments a 300 fold excess of E. coli DNA did not inhibit the human DNA-human fibronectin binding, indicating the specificity of the interaction.     

Reversible unfolding of an isolated heparin and DNA binding fragment, the first type III module from fibronectin.

Several fragments containing all or part of the first type III homology unit of fibronectin were isolated and their folding properties examined by fluorescence spectroscopy and differential scanning calorimetry. Each fragment exhibits a reversible unfolding transition when heated or titrated with guanidinium chloride. This indicates that an isolated type III module can fold independently in the absence of neighboring modules. A comparison of the specific enthalpies of unfolding of these fragments with those of well-studied globular proteins suggests that this type III unit is composed of a stable core flanked by less compact or unstructured regions. Comparison of the heparin-binding properties of these fragments revealed that removal of 12 amino acids from the amino terminus of the largest one (Ile-585 to Val-675) increased its affinity for immobilized heparin such that it now binds at physiological ionic strength.     

Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca2+ and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca2+ dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a Kd of approximately 21 nM for the interaction of annexin V with end-chain immobilized heparin and a Kd of approximately 49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The Kd of a heparin octasaccharide binding to annexin V was approximately 1 microM with a binding stoichiometry of 1:1.     

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.     

Interaction of the sex-lethal RNA binding domains with RNA.

Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo.     

Interaction of fibronectin with its receptor on platelets

We report that the 12,000 dalton domain of fibronectin that interacts with fibroblast cell surfaces also binds specifically to thrombin-inducible, saturable receptors on platelets. Furthermore, we have used chemical cross-linking and monoclonal antibodies to show that the 12,000 dalton domain of fibronectin interacts directly with glycoprotein IIIa at the platelet cell surface. Both binding and cross-linking of this domain to platelets are competed by a hexapeptide previously shown to block fibroblast adhesion to fibronectin. Finally, we show that a complex of the platelet glycoproteins IIIa and IIb binds to affinity columns of a cell-attachment fragment of fibronectin. These results localize a major fibronectin-platelet interaction to a specific domain of fibronectin and to a specific platelet glycoprotein.     

Interaction of enzymatically modified low-density lipoproteins with fibronectin

Interaction of human low-density lipoproteins (LDL) with homologous fibronectin fixed on collagen-Sepharose was studied. LDL were digested with pepsin, the degree of hydrolysis amounting to 10%. Upon passing modified LDL through a fibronectin-collagen-Sepharose column the desorption of fibronectin occurred. Addition of the increasing amount of fibronectin to the pepsin-treated LDL solution in the presence of Ca2+ ions led to the formation of LDL-fibronectin insoluble complexes. Interaction of native LDL with fibronectin was not observed. The data suggest that enzymatic modification of LDL increasing interaction of modified LDL with fibronectin, a component of extracellular matrix, could promote the accumulation of such LDL in arterial walls.     

Interaction of fibronectin with polymorphonuclear leukocytes normally and in anaphylaxis

Fibronectin is known as an opsonic glycoprotein possessing a wide range of specificity. Fibronectin interaction with neutrophils (normal ones and those obtained at peak of anaphylactic shock) was studied. Neutrophils were brought in contact with purified homologous fibronectin previously bound to gelatin-Sepharose granules. The presence of fibronectin-binding proteins on the surface of human and rabbit neutrophils was demonstrated, the binding being strongly dependent on Ca2+ and especially Mg2+ ions. "Anaphylactic" neutrophils, in contrast to normal ones, did not interact with fibronectin, which may be important for the pathogenesis of the immediate type hypersensitivity reactions.     

Aggregation and fibril formation of plasma fibronectin by heparin

The precipitation of plasma fibronectin by heparin in dependence on various parameters was investigated. Rising heparin concentration augmented the precipitates up to a maximum beyond which precipitation decreased. Yields close to 80% were obtained at low temperatures, but some precipitation was observed at 37 degrees C as well. Insolubilization was considerably dependent on the ionic strength, indicating that electrostatic forces play a major role in the aggregation of fibronectin. Calcium already prevented precipitation by heparin at low concentrations. If precipitation was performed on hydrophobized glass cover slides, the formation of fibrils visible by phase-contrast microscopy was observed. On hydrophilic surfaces amorphous precipitates were generally obtained, most likely due to trapping of aggregates by adsorption prior to their arrangement to fibrils. The results are discussed on the basis of a model assuming that heparin induces a conformational rearrangement of plasma fibronectin so that masked binding sites responsible for self-association become exposed.