The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.     

Identification of a novel recognition sequence for fibronectin within the NH2-terminal beta-sandwich domain of tissue transglutaminase

Tissue transglutaminase belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. Unlike other transglutaminases, it is involved in cell-matrix interactions and serves as an adhesion co-receptor for fibronectin. Previous work established that the fibronectin-binding motif(s) is located within the NH2-terminal proteolytic fragment of the protein consisting of residues 1-272. Here we identify a novel fibronectin recognition site within this sequence of tissue transglutaminase. Substitution of individual domains of tissue transglutaminase with those from homologous factor XIIIA showed that the major fibronectin-binding site is present within the first beta-sandwich domain of the protein. Experiments with deletion mutants of the first domain revealed that amino acids 81-140 of tissue transglutaminase are involved in fibronectin binding. Using synthetic peptides encompassing this region, we found that the peptide 88WTATVVDQQDCTLSLQLTT106 inhibited the interaction of tissue transglutaminase with fibronectin and decreased transglutaminase-dependent cell adhesion and spreading. In the three-dimensional structure of the first domain, amino acids 88-106 comprise an extended hairpin formed by antiparallel beta strands 5 and 6. Mutations of Asp94 and Asp97 within the beta5/beta6 hairpin to Ala significantly reduced the affinity of tissue transglutaminase for fibronectin, indicating that these residues are critical for fibronectin binding. Identification of the fibronectin-binding site on tissue transglutaminase will help to dissect the role of this protein in cell-matrix interactions.     

Direct determination of the interleukin-6 binding epitope of the interleukin-6 receptor by NMR spectroscopy

All cytokines belonging to the interleukin-6 (IL-6)-type family of cytokines utilize receptors that have a modular build of several immunoglobulin-like and fibronectin type III-like domains. Characteristic of these receptors is a cytokine receptor homology region consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 first binds to its specific receptor and subsequently to a homodimer of the signal transducer protein gp130. The interleukin-6 receptor consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane proximal fibronectin-like domain accounts for more than 90% of the binding energy to IL-6. Here, the key residues of this fibronectin-like domain involved in the interaction with IL-6 are described. Chemical shift mapping data with 15N-labeled IL-6R-D3 and unlabeled IL-6 coupled with recent structural data clearly reveal the epitope within the IL-6R-D3 responsible for mediating the high affinity interaction with its cognate cytokine.     

Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein.

We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.     

Influence of decorin on fibroblast adhesion to fibronectin.

Decorin is a ubiquitous small dermatan sulfate proteoglycan carrying a single glycosaminoglycan chain. It is known for its ability to bind, via its core protein, to interstitial collagens. Decorin was purified from the secretions of cultured human skin fibroblasts under non-denaturing conditions. The intact proteoglycan and its glycosaminoglycan-free core protein were tested for their interference with fibroblast adhesion to a fibronectin substrate. Concentrations of 40 nmoles or more of hexuronic acid/ml of decorin or equivalent amounts of core protein inhibited cell adhesion. Inhibition was caused by an interaction of core protein with fibronectin and not by masking of the fibronectin receptor. When cell-binding fragments of fibronectin were used as substrates, a similar inhibition of cell adhesion by decorin core protein was found, and in vitro assays demonstrated an interaction of core protein with the cell-binding domain of fibronectin. Decorin core protein also inhibited the low degree of cell adhesion to heparin-binding fragments on the N-terminus and near the C-terminus of the fibronectin molecules.     

Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.

The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.     

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion.     

Secretion and regulation Hrp mutants of Erwinia amylovora trigger different responses in apple

The interaction between two Hrp mutants of Erwinia amylovora, the parental virulent strain, and a host plant (Malus sp.) was studied with apple seedlings and apple flowers, using inoculation procedures which reduce the possibility of injury. We showed that one hrp regulatory mutant protected apple tissue against the virulent strain to a greater degree than a secretion mutant. The dynamics of bacterial growth in this interaction indicated that the protection was associated with the inhibition of the multiplication of the virulent strain. The activity of two enzymes involved in plant defence responses, phenylalanine-ammonia lyase and guaiacol peroxidase, increased in leaves treated with the regulatory mutant, but not in the leaves treated with the secretion mutant. The role of regulatory genes of the hrp cluster in this interaction is discussed.     

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.     

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.     

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes

Fibronectin-binding surface proteins are found in many bacterial species. Most strains of Streptococcus pyogenes, a major human pathogen, express the fibronectin-binding protein F1, which promotes bacterial adherence to and entry into human cells. In this study, the role of fibronectin in S. pyogenes virulence was investigated by introducing the protein F1 gene in an S. pyogenes strain lacking this gene. Furthermore, transgenic mice lacking plasma fibronectin were used to examine the relative contribution of plasma and cellular fibronectin to S. pyogenes virulence. Unexpectedly, protein F1-expressing bacteria were less virulent to normal mice, and virulence was partly restored when these bacteria were used to infect mice lacking plasma fibronectin. Dissemination to the spleen of infected mice was less efficient for fibronectin-binding bacteria. These bacteria also disseminated more efficiently in mice lacking plasma fibronectin, demonstrating that plasma fibronectin bound to the bacterial surface downregulates S. pyogenes virulence by limiting bacterial spread. From an evolutionary point of view, these results suggest that reducing virulence by binding fibronectin adds selective advantages to the bacterium.     

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.     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.     

Evidence that an RGD-dependent receptor mediates the binding of oligodendrocytes to a novel ligand in a glial-derived matrix

A simple adhesion assay was used to measure the interaction between rat oligodendrocytes and various substrata, including a matrix secreted by glial cells. Oligodendrocytes bound to surfaces coated with fibronectin, vitronectin and a protein component of the glial matrix. The binding of cells to all of these substrates was inhibited by a synthetic peptide (GRGDSP) modeled after the cell-binding domain of fibronectin. The component of the glial matrix responsible for the oligodendrocyte interaction is a protein which is either secreted by the glial cells or removed from serum by products of these cultures; serum alone does not promote adhesion to the same extent as the glial-derived matrix. The interaction of cells with this glial-derived matrix requires divalent cations and is not mediated by several known RGD-containing extracellular proteins, including fibronectin, vitronectin, thrombospondin, type I and type IV collagen, and tenascin.