Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine

Fibronectin contains at least two major domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific adhesion site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell adhesion site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.     

The alternatively spliced type III connecting segment of fibronectin is a zinc-binding module.

Fibronectin (FN) is a prototypic adhesive glycoprotein that is widely expressed in extracellular matrices and body fluids. The fibronectin molecule is dimeric, and composed of a series of repeating polypeptide modules. A recombinant fragment of FN incorporating type III repeats 12-15, and including the alternatively-spliced type three connecting segment (IIICS), was found to bind Ni(2+), Cu(2+) and Zn(2+) divalent cations, whereas a similar fragment lacking the IIICS did not. Mutation of two pairs of histidine residues in separate spliced regions of the IIICS reduced cation binding to near the level of the variant lacking the IIICS, suggesting a zinc finger-like mode of cation coordination. Analysis of native FNs purified from plasma or amniotic fluid revealed significant levels of zinc associated with those isoforms that contain the complete IIICS. Taken together, these data demonstrate that the IIICS region of FN is a novel zinc-binding module.     

Extra domain A and type III connecting segment of fibronectin in assembly and cleavage

To determine the role of the extra domain A (EDA) and type III connecting segment (IIICS) of fibronectin in fiber assembly, topographical distribution and proteolytic cleavage, eight full-length human fibronectin cDNA variants (aa0, aa64, aa89, and aa120 variations in the IIICS with or without the EDA) tagged with the V5 epitope were cloned from human endothelial cells and were expressed in CHO-K1 cells. All eight variants were assembled on cell surfaces. However, only the EDA(+) variants, regardless of the type of the IIICS domain, formed extensive fibrous networks. In contrast, the EDA(-)/aa64 and EDA(-)/aa89 variants were present predominantly as a soluble form. Western analysis of both soluble and cell-associated fibronectin/V5 variants showed that aa64, aa89, and aa120 variants with or without the EDA domain produced the major 50- to 62-kDa C-terminal fragments, whereas the aa0 variants did not, suggesting that the IIICS domain provides proteolytic cleavage sites.     

The role of the ninth and tenth type III domains of human fibronectin in cell adhesion

Fibronectins (FN) contain sites, in addition to the cell recognition site RGD in the tenth type III domain (FIII10), that are required for adhesive activity. The role of FIII10 and the adjacent FIII9 was analysed in functional cell adhesion assays recombinant FIII domains in which the domain boundaries were strictly conserved. FIII9 had no adhesive activity. FIII10, and FIII9 plus FIII10 had less activity than FN, whereas the activity of FIII9-10 was similar to FN. We conclude that FIII9 acts synergistically with FIII10 in cell adhesion, and that this synergy is dependent upon the structural integrity of the FIII9-10 pair of domains.     

Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment

In this report we have studied the mechanism of human B lymphocyte adhesion to fibronectin and to proteolytic fragments of this protein. B cells adhered to fibronectin and to a 38-kDa fragment, derived from the A chain, containing the Hep II domain and most of the type III connecting segment, IIICS, of fibronectin. Cells did not bind to an 80-kDa fragment containing the RGD adhesive sequence of fibronectin. Attachment to fibronectin or to the 38-kDa fragment was not affected by the 80-kDa fragment, the GRGDSPC synthetic peptide, or by a mAb specific for the alpha chain of the RGD-dependent fibronectin receptor, alpha 5 beta 1. However, B cell adhesion to fibronectin was inhibited by the synthetic peptides CS-1, comprising the first 25 amino acids of IIICS and B12, containing the sequence LHGPEILDVPST of CS-1 (residues 14-25). Moreover, this sequence was shown to be sufficient to induce stable cell adhesion when coated on plastic surfaces. A mAb specific for the alpha-subunit of the alpha 4 beta 1 integrin, completely inhibited B cell attachment to B12, CS-1, 38 kDa, and fibronectin coated substrata. These data clearly indicate that adhesion of B lymphocytes to fibronectin is exclusively mediated by the interaction of alpha 4 beta 1 with residues 14-25 of the IIICS region in fibronectin. Therefore this interaction constitutes an alternative pathway of adhesion to fibronectin, independent of RGD and alpha 5 beta 1.     

Identification of two distinct regulatory regions adjacent to the human beta-interferon gene

To study the regulation of the human beta-interferon (beta-IFN) gene by poly(I)-poly(C), we analyzed the expression of deletion mutants of the cloned gene introduced into mouse cells on a new bovine papilloma virus (BPV) vector. In stable cell lines transformed by a BPV-IFN plasmid containing the beta-IFN structural gene with 210 bp of DNA to the 5' side of its mRNA cap site (denoted -210), human beta-IFN mRNA is induced approximately 400-fold by poly(I)-poly(C), and reproducible levels of expression are observed for independent cell lines. Our studies indicate that there are two distinct regulatory regions adjacent to the gene, located between -77 and -19, and between -210 and -107. The -77 to -19 region is required for constitutive and induced IFN gene expression, and both are drastically reduced by deletion to -73. When sequences between -210 and -107 are deleted, the constitutive level of IFN gene expression is increased 5- to 10-fold, while induced expression is essentially unaffected. Deletion of the -210 to -107 region also alters the kinetics of induction of the gene.     

PEGylated human plasma fibronectin is proteolytically stable,supports cell adhesion,cell migration,focal adhesion assembly,and fibronectin fibrillogenesis

Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS‐PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 493–504, 2013     

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

1H NMR assignment and secondary structure of the cell adhesion type III module of fibronectin.

The secondary structure of the tenth type III module from human fibronectin has been determined using NMR. This type of module appears many times in a wide variety of proteins. The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion. The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry. 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum. The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates. The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family [Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938]. The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G.     

Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.     

Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X

Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.     

1H and 15N resonance assignment of the second fibronectin type III module of the neural cell adhesion molecule

We report here the NMR assignment of the second fibronectin type III module of the neural cell adhesion molecule (NCAM). This module has previously been shown to interact with the fibroblast growth factor receptor (FGFR), and the FGFR-binding site was mapped by NMR to the FG-loop region of the module. The FG-loop region also contains a putative nucleotide-binding motif, which was shown by NMR to interact with ATP. Furthermore, ATP was demonstrated to inhibit binding of the second F3 module of NCAM to FGFR.     

Identification and characterization of two distinct ligand binding regions of cubilin

Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin B(12); Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110-115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca(2+) dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60-65%. However, a combination of CUB domains 7 and 8 or 6-8 was needed to demonstrate significant Ca(2+) dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds.     

Identification of two distinct heparin cofactors in human plasma: Separation and partial purification

Two distinct components having potent heparin cofactor activity have been separated from human blood plasma and partially purified by means of precipitation with polyethylene glycol, adsorption on Al(OH)₃ gel and ion exchange chromatography on DEAE-cellulose and hydroxyapatite. The first component, cofactor A, is distinguished from the second, cofactor B, by its very low activity in the progressive antithrombin assay. Ion exchange rechromatography experiments indicate that cofactors A and B are not interconvertible, and that neither is an artifact of the polyethylene glycol precipitation step. Molecular weights, estimated by gel filtration were found to be 105,000 and 87,000 daltons for cofactors A and B respectively. Except for the molecular weight, the bulk of evidence suggests that cofactor B and the antithrombin III purified by Abildgaard (3) are identical.     

Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule

Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant > 100 μM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.     

A novel alpha-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.     

Identification of two distinct regions within the binding sites for fibrinogen and fibronectin on the IIb-IIIa human platelet membrane glycoprotein complex by monoclonal antibodies P2 and P4

The effect of two monoclonal antibodies P2 (LyP 2) or P4 (LyP 4), specific for the platelet membrane glycoprotein IIb/IIIa complex, on binding of 125I-labelled fibrinogen or 125I-labelled fibronectin to thrombin-stimulated platelets was studied. These monoclonal antibodies are directed against different determinants on the IIb-IIIa complex and react only with the complex and not with the individual glycoproteins. Fibrinogen binding to thrombin-stimulated platelets was significantly inhibited by P2 but not by P4. Fibronectin binding to thrombin-stimulated platelets was significantly inhibited by P4 but only poorly by P2. These results indicate the presence of specific regions on the glycoprotein IIb-IIIa complex which act as binding sites for fibrinogen or fibronectin. Other authors [Haverstick et al. (1985) Blood 66, 946-952; Ginsberg et al. (1985) J. Biol. Chem. 260, 4133-4138] have shown that a tetrapeptide, Arg-Gly-Asp-Ser, inhibited the binding of fibrinogen, fibronectin, and von Willebrand factor (vWf) to stimulated platelets and that fibrinogen competes with vWf and fibronectin for binding. These findings, together with previous studies, therefore indicate the presence of specific regions as well as a common region in the binding sites for fibrinogen and fibronectin on the IIb-IIIa complex.     

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.