Direct association of fibronectin and actin molecules in vitro

Affinity chromatography with actin-Sepharose conjugates of purified human fibronectin, normal human plasma, or serum-free culture fluid from human fibroblasts showed that fibronectin molecules can directly bind to actin. A quantitative recovery of soluble human fibronectin was accomplished by chromatography on actin immobilized on Sepharose beads. Human fibronectin molecules bound to actin-Sepharose were eluted with 0.25--0.35 M potassium bromide, and these molecules competed in a species-specific radioimmunoassay for human fibronectin. The subunits of fibronectin isolated by actin-Sepharose chromatography comigrated in SDS polyacrylamide gel electrophoresis with those of electrophoretically homogeneous fibronectin purified by conventional procedures. The efficient direct binding of fibronectin to actin suggests that interactions between these proteins might also take place in vivo but further studies are needed to elucidate the biological significance of this affinity.     

Expression of mouse and human fibronectin in mouse-human somatic cell hybrids

Using antisera to the human and mouse fibronectin (Large External Transformation Sensitive (LETS) protein), we studied the expression of mouse and human fibronectin in mouse-human somatic cell hybrids segregating either human or mouse chromosomes. The results of this study show that, in such somatic cell hybrids, mouse and human fibronectin are coexpressed. In addition, activation of the synthesis of mouse fibronectin was observed in somatic cell hybrids between mouse peritoneal macrophages, which do not produce detectable amounts of fibronectin, and either SV40-transformed human cells or cells derived from a human fibrosarcoma.     

Assignment of a fibronection gene to human chromosome 2 using monoclonal antibodies

The locus coding for the presumed structural gene for fibronectin has been mapped to human chromosome 2 using human-mouse somatic cell hybrids. The assignment of fibronectin has been made by testing man-mouse somatic cell hybrids with two anti-human fibronectin monoclonal antibodies which recognize different antigenic determinants of human, but not mouse, fibronectin, Both monoclonal antibodies demonstrate a highly concordant association between the presence of two different human fibronectin antigens and human chromosome 2.     

Species-specific monoclonal antibodies in the assignment of the gene for human fibronectin to chromosome 2.

Using three different species-specific monoclonal antibodies we have studied, in human-mouse and human-hamster somatic cell hybrids, the correlation between the presence of different human chromosomes and the ability to release human fibronectin into the tissue culture medium. Presence of human fibronectin was determined by an affinity-radioimmunoassay. In addition, tissue culture media of the different hybrids were separated on SDS-polyacrylamide gels, the proteins were blotted onto a nitrocellulose sheet and human fibronectin visualized by an immunoenzymatic technique. Karyology and determination of isoenzyme markers of specific human chromosomes show that the ability to produce human fibronectin segregated with the presence of human chromosome 2.     

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.     

The influence of exogenous fibronectin on blood granulocyte adherence to vascular endothelium in vitro

Fibronectin is a major cell surface and extracellular matrix glycoprotein. It binds to a variety of substrata and supports the attachment and spreading of a number of cell types. We have found that purified human plasma fibronectin can also support blood granulocyte adhesion to cultured human umbilical vein endothelial cells. This activity is protected by treatment of the fibronectin with a sulphhydryl-containing agent. The effect of granulocyte attachment was observed at fibronectin concentration of 100 ng/ml with maximum effect at a concentration of 10 μg/ml. The attached granulocytes retained a rounded appearance, compared with the flattening that occurs on attachment to plastic. Granulocytes attached poorly to cultured human vascular smooth muscle cells and no enhancement occurred when fibronectin was added. Immunofluorescence microscopy using monospecific rabbit anti-human fibronectin demonstrated that the sulphhydryl-treated fibronectin accumulated on the endothelial cell surface, forming aggregates on the apical surface by 3 h of continued incubation. Washed, cultured endothelial cells not exposed to fibronectin or exposed to untreated purified plasma fibronectin did not demonstrate an aggregation of cell-surface fibronectin.     

On the origin of species specificity of fibronectin. Immunological identification of a species-specific domain of human fibronectin

We have investigated whether species-specific epitopes of human fibronectin are localized at a specific domain of fibronectin using rabbit polyclonal antibodies. Tryptic fragments of human fibronectin were tested for reactivity with anti-human fibronectin antibody, which had been previously absorbed with other animal fibronectin to establish species specificity. Human-specific epitopes were found to be present on 75,000, 65,000, and 42,000 dalton fragments. The 42,000-dalton fragment shares almost all the epitopes with the 75,000 and 65,000 dalton fragments. It does not promote BHK cell spreading, whereas the 75,000 and 65,000 dalton fragments do. The amino acid sequence from the amino terminus of the 42,000-dalton fragment is Asp/Gly-Gln/Val-?-Ile-Val-, which is almost identical to the sequence Asp-Gln-Cys-Ile-Val- located in the carboxyl terminal 1/3 of the collagen-binding domain of human fibronectin (Kornblihtt et al. (1985) EMBO J. 4, 1755-1759). These results suggest that human fibronectin bears human-specific epitopes mainly on the amino-terminal half of domain 4 (Hayashi & Yamada (1983) J. Biol. Chem. 258, 3332-3340) located between the collagen and cell binding domains almost at the center of the fibronectin polypeptide. The domain specific for human fibronectin may be a general species-specific domain of animal fibronectins.     

Localization of the human fibronectin (FN) gene on chromosome 8 by a specific enzyme immunoassay

The fibronectin produced by clonal murine-human hybrid cell lines containing various complements of human chromosomes was measured. Human and murine fibronectins were assayed by specific immunoassay, and the production of human fibronectin was correlated with karyology and isozyme markers for specific human chromosomes. The data show a 100% concordance between the expression of human fibronectin and glutathione reductase, a marker for human chromosome 8, indicating that chromosome 8 codes for the fibronectin polypeptide.     

Augmentation of phagocytosis by a specific fibronectin fragment that links particulate activators to the fibronectin adherence receptor of human monocytes

Intact human plasma fibronectin of 44,000 m.w. and a fibronectin fragment of 180,000 m.w. promote dose-dependent adherence of gelatin-coated particles to human monocytes without phagocytosis. Both of these proteins, however, augment monocyte ingestion of gelatin-coated targets that are particulate activators of the alternative complement pathway or of nonactivators bearing IgG. Unlike intact fibronectin, the 180,000 m.w. fragment also binds directly to particulate activators that lack gelatin to augment their phagocytosis by human monocytes. Prior attachment to monocytes of gelatin-coated sheep erythrocytes bearing increasing concentrations of intact fibronectin decreases in a dose-dependent fashion the capacity of these monocytes to engage in augmented phagocytosis of particulate activators opsonized with the 180,000 m.w. fibronectin. Occupation of the monocyte fibronectin receptors with particle-bound, intact fibronectin does not decrease monocyte phagocytosis of plain particulate activators or of IgG-coated particles. Thus, the 180,000 m.w. fibronectin fragment both directly opsonizes particulate activators and interacts with monocyte fibronectin receptors to promote particle adherence, thereby enhancing phagocytosis through a concerted action with the distinct receptors for particulate activators.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

Local production of fibronectin by ectopic human retinal cells

Summary The distribution of fibronectin mRNA and fibronectin in adult human retina and epiretinal membranes was investigated by in situ hybridisation and immunohistochemical techniques. The cells in normal adult retina contained little or no fibronectin mRNA and the retina only showed fibronectin immunoreactivity in retinal vessels. The cells in detached neuroretina did not contain fibronectin message but the vitreoretinal interface of the detached retina exhibited variable fibronectin immunoreactivity. Retinal glia, retinal pigment epithelium and fibroblast-like cells in membranes at the vitreoretinal juncture (epiretinal membranes) showed variable labelling with the fibronectin mRNA probe and all the membranes immunostained for fibronectin. No difference could be detected between membrane cell types in the intensity of labelling with the mRNA probe or for fibronectin immunoreactivity. The results indicate that cells in situ in attached and detached adult human retina do not produce fibronectin. Although fibronectin at the vitreoretinal juncture in retinal detachment is probably partly derived from plasma fibronectin resulting from breakdown of the blood-retinal barrier, ectopic retinal cells produce fibronectin and contribute to the glycoprotein in epiretinal membranes.     

Hydrophobic properties of porcine fibronectin and its functional domains

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.     

Visualization of purified fibronectin-transglutaminase complexes.

It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.     

Human plasma gelsolin binds to fibronectin

Human plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering, sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites.     

Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.     

A family of fibronectin mRNAs in human normal and transformed cells

Previously, two fibronectin mRNAs, generated by alternative splicing of the extra domain (ED) and type III connecting segments (IIICS) sequences, have been described in a human transformed cell line and in human liver, respectively. We now report on a family of fibronectin mRNAs identified by Northern blotting analysis in two normal human fibroblast strains (HEL 299 and Flow 7000) and five transformed cell lines (8387 and HT-1080, fibrosarcomas; G-361, melanoma; JEG-3, choriocarcinoma; and RD, rhabdomyosarcoma). Seven different fibronectin mRNA forms with electrophoretic mobilities ranging between 8.6 and 7.7 kb were identified. Each cell line contains three (HEL 299, Flow 7000 and 8387) or two (HT-1080, G-361, JEG-3 and RD) fibronectin mRNAs species with characteristic size. In all cell lines we detected one fibronectin mRNA form which lacks the ED sequence (ED- fibronectin mRNA) and one or two fibronectin mRNAs containing it (ED+ fibronectin mRNA). These data show that the presence of ED+ and ED- fibronectin mRNAs is a general feature of all cells tested. Moreover, the fibronectin mRNA pattern is characteristic of the cell type analyzed, suggesting the occurrence of specifically programmed splicing mechanisms in each cell line.     

Study of the structural and functional properties of fibronectin--a high molecular weight plasma glycoproteins--by using monoclonal antibodies

Functional and structural properties of fibronectin--high molecular weight glyco-protein from human plasma--were studied by monoclonal antibodies against fibronectin. It was shown that monoclonal antibodies against human plasma fibronectin exhibit a certain species specificity. Antigenic determinant for our monoclonal antibody is located in the central part of the protein polypeptide chain--in the structural domain. The monoclonal antibodies studied do not inhibit any tested functions of fibronectin. In contrast, polyclonal antibodies are not species specific and inhibit all fibronectin functions.     

A novel lectin-independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Binding of P fimbriae of uropathogenic Escherichia coli to purified human fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin. Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by alpha-D-Gal-(1-4)-beta-D-Gal-1-O-Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I-labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.     

Exogenous fibronectin is not required for organogenesis in vitro

The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50 micrograms/ml) was a strong mitogen for kidney cells, whereas the addition of soluble fibronectin (50 to 250 micrograms/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. However, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 micrograms/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250 micrograms/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro.     

Transformed human cells release different fibronectin variants than do normal cells

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.