Fibronectin Fibrillogenesis Involves the Heparin II Binding Domain of Fibronectin*

Hermann Bultmann, Amy J. Santas, and Donna M. Pesciotta PetersDagger

From the Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, Wisconsin 53706

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fibronectin matrix assembly is thought to involve binding interactions between the amino-terminal I1-5 repeats and the first type III repeat (III1). Here we report that a third site, located within the III12-14 repeats of the carboxyl-terminal heparin II domain of fibronectin, is also involved in fibrillogenesis. Heparin II fragments inhibited fibril formation and binding of 125I-labeled fibronectin and/or 70-kDa fragments to the cell surface, deoxycholate-insoluble matrix, and adsorbed 160-kDa cell adhesion fragments of fibronectin. The inhibitory effects of heparin II fragments were as large or up to 20 times larger than those of a 44-kDa fibronectin fragment containing the III1 repeat. Under physiological conditions, amino-terminal fragments of fibronectin containing the I1-5 repeats interacted preferentially with proteolytically derived heparin II fragments and a recombinant III12-14 peptide both in solution and in solid phase, indicating that matrix assembly may involve direct interactions between I1-5 and III12-14 repeats. Interactions between the I1-5 repeats and 160-kDa fragments containing the III12-14 and III1 repeats could be inhibited by >=  90% by either an anti-III13-14 monoclonal antibody (mAb) (IST-2) or an anti-III1 mAb (9D2), suggesting that cooperative interactions between III12-14 and III1 repeats may also promote binding of the I1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in combination, inhibited binding of 125I-labeled 70-kDa fragments to cycloheximide-treated cells plated on the 160-kDa substrate, suggesting that additional I1-5 binding sites, independent of the III1 and III12-14 repeats, may be involved in fibrillogenesis.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fibronectin is required for normal growth and development (1) and plays an important role in regulating cell attachment and movement, wound healing, and tumorigenesis (for review, see Refs. 2 and 3). It is a 500-kDa disulfide-bonded dimer consisting of similar subunits and is found as a soluble glycoprotein in blood and other body fluids and as an insoluble fibrous matrix component in tissues. Each subunit of fibronectin consists of three different types of repeating sequences, called types I, II, and III, which are arranged into discrete structural and functional modules.

Assembly of dimeric fibronectin into the extracellular matrix involves multiple consecutive binding interactions with integrin receptors, with itself, and with matrix components such as type I collagen (for review, see Refs. 4-6). Although the alpha 5beta 1 integrin appears to be the primary fibronectin receptor involved in matrix assembly (7-12), at least two other integrins, alpha IIbbeta 3 and alpha vbeta 3, can also support fibronectin fibrillogenesis (13, 14). High affinity binding interactions between these integrins and the RGD site in the 10th type III repeat (III10) of fibronectin are thought to promote fibrillogenesis by exposing appropriate self-assembly sites in fibronectin. Such sites may become exposed through local integrin-induced conformational changes in III10 repeats (15) or through integrin-mediated stretching (reversible unfolding) of one or a whole array of type III repeats in fibronectin in response to cell movements (13, 16).

Self-assembly of fibronectin dimers into fibrils is currently thought to involve primarily interactions between the first five type I repeats (I1-5) and the first type III (III1) repeat (17, 18). The I1-5 repeats are critical for matrix assembly, i.e. peptides including these repeats block assembly of fibronectin into fibrils, and fibronectin dimers lacking these repeats will not be incorporated into fibrils (19-22). The III1 repeats are also important for fibril formation, and either anti-III1 monoclonal antibodies or peptides derived from III1 repeats can block assembly of fibronectin into matrix (23, 24). The mechanism, however, by which I1-5-III1 interactions affect matrix assembly remains controversial. For example, Hocking et al. (25) have shown that III1 repeats will interact not only with I1-5 repeats but also with heat-denatured III10 repeats. They have proposed that the latter interaction activates the III1 repeat thereby allowing it to function as a receptor for the amino termini of a second fibronectin dimer. In contrast, Sechler et al. (26) have shown that fibronectin dimers lacking III1-7 repeats are readily polymerized into fibrils. In fact, the mutated dimers are rendered deoxycholate-insoluble more rapidly than intact dimers, suggesting that III1 repeats do not promote, but rather inhibit matrix assembly by keeping fibronectin in a compact form through intramolecular interactions with I1-5 repeats.

Earlier studies by Homandberg and Erickson (27) had indicated that another complementary binding site of the I1-5 repeats is located within the III12-14 repeats of the carboxyl-terminal heparin II binding domain of fibronectin, but the functional significance of this site was never investigated. Here we report that the heparin II domain plays an important role in matrix assembly, and we demonstrate that peptides containing the III12-14 repeats specifically block binding of fibronectin and its 70-kDa amino-terminal fragments to cells and their matrix. We also present evidence indicating that the inhibitory effects of the heparin II domain may involve direct interactions between the I1-5 and the III12-14 repeats and that binding of the I1-5 repeats may depend on cooperative interactions between the III12-14 and III1 repeats. Finally, the data presented here confirm our earlier report (12) and show that the amino terminus participates in additional binding interactions that are independent of either the III1 or the III12-14 repeats.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture and Binding Assays-- Neonatal human skin fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Binding assays in confluent cultures of matrix-deprived cells were performed as described before (12). Briefly, cells were harvested with trypsin and EDTA, washed in the presence of soybean trypsin inhibitor, and kept in suspension for 1 h at 21 °C in binding buffer (Dulbecco's modified Eagle's medium containing 25 µg/ml cycloheximide, 2 mg/ml BSA,1 100 units/ml penicillin G, 5 µM streptomycin sulfate, and 25 mM Hepes, pH 7.4). Cells were then plated in 96-well polystyrene microtiter wells precoated with 40-120 µg/ml of the 160-kDa or 5 µg/ml of the 75-kDa cell adhesion fragments of fibronectin. These coating concentrations had been shown previously to be optimal for cell spreading and binding of 70-kDa fragments (12). Plates were blocked overnight with 2 mg/ml heat-denatured BSA (5 min at 80 °C). In some experiments, antibodies were added to the cell suspension just before plating. After 3 h at 37 °C, cells were incubated with 125I-labeled fibronectin (1 × 107 cpm/pmol) or 70-kDa fragments (1.5-3 × 106 cpm/pmol) for 1 or 2 h at 37 °C, and wells were washed, separated, and counted.

Binding interactions between ligand and substrate which occur in the absence of cells were also measured in microtiter wells as described previously (12). For some of these experiments (see Figs. 6 and 7), the pH of the binding buffer was adjusted to 6.6 with HCl. Nonspecific binding was measured either in wells that had been coated only with the blocking agent (2 mg/ml heat-denatured BSA) or in the presence of a >100-fold excess of unlabeled ligand. Nonspecific binding measured in the presence of excess competitor represented 10-20% and 20-50% of total binding in the presence and absence of cells, respectively, and was independent of the addition of any of the antibodies.

Matrix Assembly Assay-- Cells were plated at confluence and maintained in the presence of 10% fetal bovine serum for 3 days. Cells plated in 35-mm dishes were then incubated with 0.1 nM 125I-fibronectin (1 × 106 cpm/ml) in 0.25 ml of binding buffer, pH 7.4, for 2 h at 37 °C with gentle rocking. Cells plated in 60-mm dishes were incubated with 0.75 nM 125I-labeled 70-kDa fragments (2 × 106 cpm/ml) in 1 ml of binding buffer, pH 7.4, for 4 h at 37 °C with gentle rocking. The cells were washed five times with binding buffer and lysed at 4 °C with 1% deoxycholate in hypotonic buffer (1.5 ml/dish 50 mM Tris-HCl, pH 8.3, containing 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin) and scraped from the dishes. The deoxycholate-soluble cellular fraction (pool I) and insoluble matrix fraction (pool II) were separated by centrifugation at 35,000 × g as described (28). The deoxycholate-insoluble material was washed with 1% deoxycholate in hypotonic buffer and recentrifuged. All operations were done at 4 °C.

Isolation of Adherent Matrices-- Adherent matrices were prepared from fibroblast cultures that had been plated at confluence in 60-mm dishes and maintained in the presence of 10% fetal bovine serum for 3 days. Cells were lysed with 1% deoxycholate in hypotonic buffer for 5 min (28). The lysates in the plates were treated with DNase I (50 units/ml; Sigma), MgCl2, and CaCl2 (5 mg/ml each) in hypotonic buffer for 15 min, and the plates were rinsed with hypotonic buffer followed by binding buffer. The fibrous matrices, prepared in this way at 21 °C, remained firmly attached to the dishes in subsequent binding assays. Binding assays using isolated matrices were done as described above for intact cell layers.

Binding Interactions in Solution-- Binding reactions in solution were carried out at 21 °C by mixing 0.3-1.8 pmol of the 125I-ligand (3-16 × 105 cpm/pmol) with 25 pmol of biotinylated 70-kDa fragments in 240 µl of 50 mM sodium phosphate, pH 7.4, containing 0.08 M NaCl (PBS) in the absence or presence of 1 mg/ml bis(sulfosuccinimidyl) suberate (BS3; Pierce). After 30 min, the cross-linker was inactivated by adding 27 µl of 1.5 M glycine, pH 7.4. Twenty min later 11 µl of 50 mM BSA was added. From each reaction vial, 250 µl was transferred to a new vial containing 6 µl of packed streptavidin-conjugated agarose beads (1.2 mg streptavidin/ml; Sigma) and 40 µl of packed Sepharose CL-4B (Pharmacia Biotech Inc.). The agarose beads and Sepharose were preblocked with 2 mg/ml BSA in PBS to reduce nonspecific binding. Binding to the beads was carried out at 21 °C for 1 h on an end-over-end rotator. The beads were then sedimented, washed three times with 1 ml of PBS containing 0.05% Tween 20 and counted in a gamma  counter. In each case, BS3-dependent as well as BS3-independent binding increased linearly with the concentration of the ligand (data not shown).

Proteolytic Fragments of Fibronectin and Antibodies-- Fig. 1 illustrates the fragments used in this study. Human plasma fibronectin and its 29-kDa (type I1-5 repeats), 40-kDa (type I6-I9 repeats), 70-kDa (type I1-I9 repeats), and 160-kDa (type III1-III14 repeats) proteolytic fragments were prepared as described before (29-31).


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Fig. 1.   Schematic diagram of fibronectin indicating the fibronectin fragments and antibodies used. The diagram represents a monomer of plasma fibronectin consisting of type I (rectangles), type II (ovals), type III (numbered squares) repeats, and the variable connecting sequence (CS). The two main heparin binding domains (Hep I, Hep II) and the binding site of the alpha 5beta 1 integrin (RGD) are also indicated. The location of the epitopes recognized by the monoclonal antibodies used in this study are indicated above. The location of the proteolytic fragments used in this study are indicated below together with their molecular mass. The proteolytically derived heparin II-binding fragments consisting of five peptides and their weighed average molecular mass (50 kDa) are indicated by a solid line. Some of these fragments may extend further as indicated by the dotted line.

The carboxyl-terminal heparin II-binding fragments were isolated from a catheptic D digest of plasma fibronectin, from which gelatin-binding fragments (notably the 70-kDa fragment) had been removed by first passing the digest over on a gelatin-Sepharose column. The flow-through from the gelatin-Sepharose column was then passed through a heparin-agarose column, and the bound material was eluted with increasing concentrations of NaCl (70-600 mM in 10 mM Tris-HCl, pH 7.4). SDS-polyacrylamide electrophoresis and Western blots indicated that the carboxyl-terminal heparin-binding fragments were included in the last peak eluted from the column. This peak contained five fragments (65, 46, 43, 41, and 39 kDa). None of these fragments contained the COOH-terminal disulfide bond. Calculations of molar concentrations are based on the weighed average molecular mass of these fragments (50 kDa).

The 44- and 75-kDa fragments were obtained by digesting reduced and alkylated plasma fibronectin with 10 µg of tosylphenylalanyl chloromethyl ketone-treated trypsin/mg of fibronectin for 30 min at 37 °C and separating the resulting fragments on a Sephacryl S-300 column (Pharmarcia) equilibrated with 20 mM Tris-HCl, 0.15 M NaCl, pH 7.4. The 44-kDa fragments were purified further on a heparin-agarose column to remove traces of contaminating material reacting with mAb IST-2.

All fragments were characterized by SDS-polyacrylamide gel electrophoresis, Western blots, and direct or competitive enzyme-linked immunoassays. The large 160-kDa cell adhesion fragment was recognized by mAbs 9D2 (anti-III1), C6F10 (anti-III8-11), and IST-2 (anti-III13-14) but not by IST-7 (anti-III15), whereas the smaller 75-kDa cell adhesion fragment was recognized by C6F10 only. The 44-kDa fragment was recognized by 9D2 but not by L8 (anti-I9-III1) or C6F10. None of the four antibodies recognized the amino-terminal 29-, 40-, and 70-kDa fragments. All of the COOH-terminal heparin-binding fragments, except for the 43-kDa fragment, were recognized by mAbs IST-2 and IST-7. None of these COOH-terminal fragments was recognized by the mAbs C6F10 and 9D2. All fragments were iodinated with carrier-free Na125I by the chloramine-T method (32). These radioiodinated probes were also used to measured adsorption and desorption of fibronectin fragments in microtiter wells. Biotinylated 70-kDa fragments were prepared using Enzotin (NZM Biochem, New York) as described by the manufacturer.

The mAbs L8, 9D2, and C6F10 were provided by Dr. Deane Mosher, University of Wisconsin. The mAbs IST-2 and IST-7 were obtained from Dr. Luciano Zardi, Instituto Nationale per La Ricera Sul Cancro. The antibodies were concentrated by precipitation with 30% ammonium sulfate and/or solvent extraction with flakes of polyethylene glycol followed by dialysis against 20 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl. IgG concentrations were determined by using a mouse IgG kit (Boehringer Mannheim).

Preparation of Recombinant Type III12-14 Peptide-- Human full-length fibronectin cDNA, pFH100 (33), was used as a template for polymerase chain reaction amplification (34) of a DNA sequence (bases 5072-5923) encoding the III12-14 repeats of fibronectin (amino acid Glu1687 through Pro1970). The DNA was generously provided by Dr. Deane Mosher (who received it from Dr. Jean Paul Thiery, CNRS URA, Paris). The bases are numbered starting with the G before the first codon of the first amino acid of the mature protein (GenBankTM accession no. X02761), and the amino acids are numbered starting with the pyroglutamic acid (35). This region spans the entire length of the III12-14 repeats and also includes 3 amino acids upstream of the III12 repeat (QST) as well as 10 amino acids downstream of the III14 repeat (DELPQLVTLP). The sense primer, 5'-GAATTCCAGTCCACAGCTATTCCTG, generated an EcoRI site (in boldface), whereas the antisense primer, 5'-CTCGAGCTATGGAAGGGTTACCAGTTG, generated an XhoI site (in boldface) and also introduced a stop codon (underlined). The polymerase chain reaction-amplified DNA was purified using the WizardTM PCR Preps DNA Purification System (Promega, Madison, WI) and subsequently ligated into the pGEM-T vector (Promega) according to the manufacturer's instructions. The pGEM-T vector containing the insert was transfected through electroporation (ElectroCell Manipulator 600 BTX Electroporation System) into JM109 bacteria. The pGEM-T vectors containing the appropriate sized insert were digested with EcoRI and XhoI, and the cDNA fragment encoding the III12-14 repeats was gel purified using the WizardTM PCR Preps DNA Purification System. This purified DNA fragment was then cloned in-frame into the bacterial expression vector pGEX-4T1 (Pharmacia) and electroporated as described above. Expression of the glutathione S-transferase fusion peptide (GST-III12-14 repeats) was confirmed using Western blot analysis with mAb IST-2.

The GST-III12-14 fusion peptide was purified using modifications of the procedure described previously (36). The modifications included adding 1 mM Pefabloc®SC (Boehringer Mannheim) and lysing the cells with a French press followed by sonication (three cycles of 10 s each). The fusion peptide was adsorbed to glutathione-Sepharose 6B beads (Pharmacia) and digested overnight at 4 °C with thrombin (10 NIH units/500 ml of original culture). The beads were removed by centrifugation at 250 × g for 5 min at 4 °C. The supernatant containing the cleaved peptide was incubated with p-aminobenzamidine Sepharose 6B beads (Sigma) for 20 min at 4 °C to remove thrombin. The beads were removed by centrifugation at 250 × g for 5 min, and the recombinant III12-14 peptide in the final supernatant was concentrated on flakes of polyethylene glycol and dialyzed into PBS. SDS-polyacrylamide gel electrophoresis and Western blots showed that the recombinant III12-14 peptide migrated as a doublet (~29-kDa) and was recognized by the mAb IST-2 (data not shown). Western blot analysis using a goat anti-GST serum (gift from Dr. James Tracy, University of Wisconsin, Madison) showed that the recombinant peptide did not contain any uncleaved fusion peptide or GST (data not shown).

Immunofluorescence Microscopy-- Teflon® coated 12-well slides (Polysciences, Warrington, PA) were precoated for 2 h at 21 °C with 20 µg/ml of the 160-kDa fragment of fibronectin in Hanks' balanced salt solution. Fibroblasts suspended in Dulbecco's modified Eagle's medium containing 7.5% fetal bovine serum, 100 units/ml penicillin G, and 5 µM streptomycin sulfate were plated at a density of 3 × 105 cells/well and incubated at 37 °C to allow the cells to attach. After 1 h, the medium was replaced with serum-free medium (Dulbecco's modified Eagle's medium, 2 mg/ml BSA, 25 µg/ml cycloheximide, 100 units/ml penicillin G, and 5 µM streptomycin sulfate), and the cells were allowed to spread for 3 h at 37 °C. The medium was removed again and replaced with fresh serum-free medium containing 1 µg/ml human plasma fibronectin. In some wells, either the 70-kDa (93 µg/ml) or heparin II-containing fragments (200 µg/ml) were added as inhibitors. Fibroblasts were kept overnight at 37 °C, washed with Hanks' balanced salt solution, and fixed with 4% paraformaldehyde, 0.1 M sodium phosphate buffer at pH 7.4 for 30 min at 21 °C. Fibronectin fibrils were labeled with a polyclonal anti-fibronectin serum as described previously (37) and viewed by epifluorescence with a Nikon Optiphot microscope. Images were digitized using a Photometrics Image Point®CCD camera and Image Pro Plus® program.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Heparin II-containing Fragments of Fibronectin Inhibit Assembly of Fibronectin into Matrix-- Earlier studies by Homandberg and Erickson (27) had indicated that the heparin II binding domain of fibronectin may contain an amino-terminal binding site. Because an amino-terminal binding site in the III1 repeat of fibronectin had been shown to play an important role in the assembly of fibronectin into fibrils (17, 18, 25, 26), we wanted to know whether the heparin II binding domain of fibronectin also participates in fibrillogenesis. To examine this question, cycloheximide-treated fibroblasts capable of assembling exogenous plasma fibronectin into fibrils (38) were tested for their ability to assemble plasma fibronectin into fibrils in the presence, or absence, of proteolytically derived fragments of fibronectin. In the absence of any fibronectin fragments, numerous fibronectin fibrils were observed in these cultures (arrowheads, Fig. 2a). If, however, fragments of fibronectin containing the heparin II domain were added, fibril formation was inhibited completely (Fig. 2c), indicating that the heparin II domain may indeed play a role in fibrillogenesis. The 70-kDa amino-terminal fragment of fibronectin, as expected (19, 39), also blocked incorporation of exogenous fibronectin into matrices (Fig. 2b).


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Fig. 2.   Inhibition of fibronectin fibrillogenesis in the presence of the 70-kDa or heparin II-containing fragments. Cycloheximide-treated fibroblasts plated on the 160-kDa fragment of fibronectin were incubated overnight with plasma fibronectin (panel a), plasma fibronectin and the 70-kDa amino-terminal fragment (panel b), or plasma fibronectin and heparin II-containing fragments (panel c) as described under "Materials and Methods." At the concentrations used, 70-kDa (1.3 µM) and heparin II fragments (4.4 µM) inhibited binding of 125I-fibronectin by 50 and 40%, respectively (see Fig. 4A). A phase image of the cell layer is shown in panel d. All cultures were fixed and labeled with an anti-fibronectin serum followed by an anti-rabbit IgG conjugated to rhodamine as described under "Materials and Methods." Fibronectin fibrils (arrowheads) can be found only in cultures incubated with plasma fibronectin alone. Cultures incubated with either fibronectin fragment did not form fibrils. Bar = 50 µm.

The inhibitory effect of the heparin II domain on fibrillogenesis was confirmed biochemically using the matrix assembly assay described by McKeown-Longo and Mosher (19, 28). In this assay, incorporation of 125I-fibronectin into the deoxycholate-insoluble matrix (pool II) was inhibited with increasing concentrations of unlabeled 70-kDa fragments (Fig. 3). The IC50 of the inhibition was 1.0 µM. At 10-fold higher concentrations, fragments containing the III12-14 repeats were equally effective inhibitors (IC50 = 10 µM). The 44-kDa fragment containing the III1-4 repeats was also inhibitory at these higher concentrations, however it never blocked incorporation of 125I-fibronectin by more than 40%. In contrast, 75-kDa fragments containing the III5-10 repeats or BSA had little (20%) or no inhibitory effects.


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Fig. 3.   The incorporation of fibronectin into deoxycholate-insoluble matrix is inhibited by various fibronectin fragments. The incorporation of 125I-fibronectin into matrix in confluent cell layers was measured as described under "Materials and Methods." Increasing concentrations of unlabeled amino-terminal 70-kDa fragments (bullet ), heparin II-binding fragments (open circle ), 44-kDa fragments containing the III1 repeat (black-triangle), 75-kDa cell adhesion fragments (square ), or BSA (triangle ) were added to cell layers in the presence of 125I-fibronectin. Incorporation of fibronectin is expressed as percent of controls. The controls had incorporated 0.5 ± 0.02 fmol/dish 125I-fibronectin over the 2-h labeling period. The data are means of two or three measurements; bars indicate the S.E. Curves are second order regression lines.

Fragments containing the heparin II binding domain also inhibited binding of 125I-fibronectin to the cell surface in confluent cell layers of matrix-deprived cycloheximide-treated fibroblasts plated on the 75-kDa cell adhesion fragment of fibronectin (12). As shown in Fig. 4A, the 70-kDa fragment was again the most effective inhibitor (IC50 = 0.1 µM) followed by the heparin II (IC50 = 3 µM) and the 44-kDa (IC50 approx  20 µM) fragments. The 75-kDa fragment and BSA had little or no inhibitory effect. Thus, under physiological conditions, III12-14-containing fragments inhibited binding of 125I-fibronectin to cell layers at least 10 times more efficiently than either the III1-4- or III5-10-containing fragments. These studies clearly establish that besides the amino-terminal I1-5 repeats, the type III repeats contained in the carboxyl-terminal heparin II-binding fragments are the most effective inhibitor of fibronectin matrix assembly and that they are capable of inhibiting the initial binding of fibronectin to the cell surface as well as its subsequent incorporation into fibrils.


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Fig. 4.   Inhibition of the binding of 125I-labeled fibronectin or 70-kDa fragments to matrix-deprived fibroblasts by various fibronectin fragments. Cycloheximide-treated fibroblasts (3 × 104 cells/well) were plated for 3 h at 37 °C in microtiter wells coated with 0.4 pmol of the 75-kDa fragment of fibronectin. Cultures were incubated for 2 h with either 0.9 nM (1 × 107 cpm/well) 125I-fibronectin (panel A) or 2.8 nM (5 × 106 cpm/well) 125I-labeled 70-kDa fragments of fibronectin (panel B) in the presence or absence unlabeled 70-kDa fragments (bullet ), heparin II-containing fragments (open circle ), 44-kDa fragments containing type III1 repeats (black-triangle), 75-kDa cell adhesion fragment (square ), or BSA (triangle ). None of the added peptides had any discernible effects on the appearance of the cells or the cell density. All data represent means of triplicate measurements. Bars indicate the S.E. Data were normalized to the amount of ligand binding seen in the absence of any additions (for 125I-fibronectin (panel A): 0.26 ± 0.01 fmol/well, n = 15; for 125I-labeled 70-kDa fragments (panel B): 0.63 ± 0.02 fmol/well, n = 12). All curves are third order regression lines.

Heparin II-containing Fragments Inhibit Binding of 125I-Labeled 70-kDa Fragments to Cell Layers, 160-kDa Fibronectin Fragments, and Matrix Fibrils-- Because the assembly of fibronectin into fibrils is thought to be mediated by the amino-terminal I1-5 repeats (19, 20), we also examined whether the heparin II-binding fragments could interfere directly with binding of 125I-labeled 70-kDa fragments to cell layers. As shown in Fig. 4B, unlabeled 70-kDa (IC50 = 0.006 µM), heparin II (IC50 = 2 µM), and 44-kDa (IC50 = 20 µM) fragments inhibited binding of 125I-labeled 70-kDa fragments to cells in almost exactly the same manner as they inhibited binding of 125I-fibronectin (see Fig. 4A). Thus, all of the binding interactions of fibronectin in cell layers which can be specifically inhibited by these fibronectin fragments appear to be I1-5-dependent binding interactions. In control experiments, high concentrations of BSA elevated the binding of 125I-labeled 70-kDa fragments slightly.

To test whether the inhibitory effects of heparin II fragments involve direct fibronectin-fibronectin binding interactions independent of cells, we measured the ability of these fragments to inhibit direct binding of 125I-labeled 70-kDa fragments to 160-kDa cell adhesion fragments of fibronectin adsorbed to plastic wells (12). This reaction was specifically inhibited by unlabeled 70-kDa (IC50 = 0.02 µM), heparin II (IC50 = 0.1 µM), and 44-kDa fragments (IC50 = 2 µM), whereas the 75- and 160-kDa cell adhesion fragments had the same nonspecific effect as high concentrations of BSA (Fig. 5). Both the heparin II fragments and the 44-kDa fragments thus inhibited binding of the 125I-labeled 70-kDa ligand to the 160-kDa substrate 10 times more efficiently than they inhibited binding of 70-kDa fragments to the cell surface. This suggests that the III12-14 repeats, like the III1 repeats, may preferentially affect I1-5-dependent fibronectin-fibronectin interactions in the extracellular matrix.


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Fig. 5.   Inhibition of the binding of 125I-labeled 70-kDa fragments to adsorbed 160-kDa substrate by fibronectin fragments containing the III12-14 or the III1 repeats. Microtiter wells, which had adsorbed 2.9 pmol of 160-kDa fragments, were labeled with 2.8 nM 125I-labeled 70-kDa fragments (5 × 105 cpm/well) in 60 µl of binding buffer, pH 7.4, for 2 h at 37 °C in the absence or presence of unlabeled 70-kDa fragments (bullet ), fragments containing the III12-14 repeats (open circle ), or 44-kDa fragments containing the III1 repeat (black-triangle) at the indicated concentrations. Nonspecific effects caused by increasing the protein concentration were measured by supplementing the binding buffer with additional BSA (triangle ). All data represent means of triplicate measurements (bars indicate the S.E.) normalized to the binding of 125I-labeled 70-kDa fragments seen in the absence of any additions (0.90 ± 0.07 fmol/well; n = 9). Curves are third order regression lines.

To examine this possibility, we compared the ability of heparin II fragments to block the binding of 125I-labeled 70-kDa fragments to the cell surface (the deoxycholate-soluble pool I) and to the matrix (deoxycholate-insoluble pool II) in confluent cell layers. Earlier experiments had shown that compared with intact fibronectin, 70-kDa fragments were poorly incorporated into pool II (19). We found, however, that over a 4-h period, as much as 44% of the total 125I-labeled 70-kDa fragments binding to confluent normal cell layers could be recovered in pool II (Table I); under the same conditions, ~60% of 125I-fibronectin entered pool II (data not shown). We also found that both fibronectin and its 70-kDa fragment could bind directly to isolated matrices, provided that the deoxycholate-insoluble extracted cell layers were first treated with DNase to remove any DNA that was released during cell lysis and had coated the adherent matrices. Direct binding to such matrices over a 4-h period amounted to nearly 70% of the binding of 125I-labeled 70-kDa fragments (Table I) or 125I-fibronectin (data not shown) in intact cell layers. Incorporation of 125I-labeled 70-kDa fragments into pool I, pool II, and isolated matrices was equally inhibited with unlabeled 70-kDa fragments (~35% inhibition with 0.8 µM). The incorporation of 125I-labeled 70-kDa fragments to pool I, pool II, and isolated matrices was also significantly (p > 0.05) inhibited by the addition of unlabeled heparin II-binding fragments. At the concentration chosen (4 µM), heparin II fragments inhibited 15, 25, and 29% of the total binding or 42, 69, and 88% of the specific binding of 125I-labeled 70-kDa fragments into pool I, pool II, and isolated matrices, respectively. We conclude, therefore, that the amino terminus of fibronectin can interact directly with the matrix in cell layers and that the heparin II-binding fragment can interfere with such binding significantly. Presumably, the ability of the heparin II fragments to inhibit the binding interaction between the 70-kDa ligand and the adsorbed 160-kDa substrate (Fig. 5) is physiologically significant in that it reflects this aspect of fibronectin matrix assembly.

                              
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Table I
Inhibition of amino-terminal binding to cell layers and isolated matrices by fragments containing the III12-14 repeats
Deoxycholate soluble (pool I) and insoluble fractions (pool II) and isolated fibronectin matrices were prepared as described under "Materials and Methods." Percent inhibition of specific binding refers to the percent inhibition by the heparin II containing fragments (hep II) relative to the percent inhibition by the 70-kDa fragment (0.8 µM). All data represent the means of four measurements ± S.E. The differences between controls and dishes treated with the inhibitor were significant at the 95% confidence level in each of the seven experiments (t test; comparison of means).

Direct Binding Interactions between I1-5 and III12-14 Repeats-- To determine whether direct interactions between I1-5 and III12-14 repeats could account for the inhibitory effects of heparin II-binding fragments in fibrillogenesis, 70-kDa fragments adsorbed to plastic wells were tested for their ability to bind proteolytically derived and recombinant peptides containing the III12-14 repeats. As shown in Fig. 6, 125I-labeled III12-14-containing peptides bound to absorbed 70-kDa fragments in a concentration-dependent manner. On a molar basis, the recombinant peptide was bound three to four times more efficiently than the proteolytic fragments. Binding of radioiodinated recombinant and proteolytic fragments was inhibited by ~50% in the presence of a 300-400-fold molar excess of unlabeled heparin II-binding fragments. No further inhibition could be achieved by increasing the concentration of the competitor (data not shown). Presumably, this was because of the self-association of the heparin II-binding fragments at the higher concentrations (27). Binding interactions between the heparin II-binding fragments and the 70-kDa fragments involve the amino-terminal I1-5 repeats. As shown in Fig. 7, binding of 125I-labeled amino-terminal fragments to increasing concentrations of adsorbed heparin II fragments is associated exclusively with the 29-kDa fragments containing the I1-5 repeats and not with the 40-kDa gelatin binding domain. In the experiments reported in Figs. 6 and 7, binding reactions were carried out at pH 6.6 because binding was considerably enhanced at this pH compared with the binding seen at pH 7.4 (data not shown).


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Fig. 6.   Binding of recombinant and proteolytically derived III12-14-containing peptides to adsorbed 70-kDa fragments. Microtiter wells that had been coated with the indicated amounts of the 70-kDa fragment were labeled for 4 h at pH 6.6 and 37 °C with 0.86 pmol/well 125I-labeled recombinant III12-14 peptide (2.7 × 105 cpm/well) (bullet ,open circle ) or 0.57 pmol/well 125I-heparin II-binding proteolytic fragments (4.6 × 105 cpm/well)(black-triangle,triangle ) in the absence (filled symbols) and in the presence of 240 pmol/well unlabeled 50-kDa fragments (open symbols). Data represent means of triplicate measurements. S.E. (data not shown) were all <0.8 fmol/well. Binding of the recombinant fragment was normalized to the input of the proteolytic fragment. Nonspecific binding to BSA-coated wells was subtracted. Curves are third order regression lines.


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Fig. 7.   Comparison of 29-, 40-, and 70-kDa amino-terminal fragments binding to adsorbed heparin II-binding fragments. 125I-Labeled 29-kDa (18.3 nM or 2.3 × 105 cpm/well) (open circle ), 70-kDa fragments (11.9 nM or 5.3 × 105 cpm/well) (bullet ), or 40-kDa gelatin-binding fragments (12.3 nM or 3.7 × 105 cpm/well) (triangle ) were incubated at pH 6.6 for 4 h at 37 °C in microtiter wells coated with increasing concentrations of heparin II-binding fragments. The data have been normalized to a ligand input of 10 nM, and they represent the means of triplicate measurements. Bars indicate the S.E. Curves are third order regression lines.

Adsorbed 70-kDa fragments clearly bound recombinant and proteolytically derived III12-14-containing peptides in preference to other fibronectin fragments such as the 44-, 75-, or 40-kDa fragments (Fig. 8A). Such preferential binding was not merely an artifact caused by denaturation of the adsorbed 70-kDa fragment, but could also be demonstrated in solution. As shown in Fig. 8B, soluble biotinylated 70-kDa fragments bound the recombinant and the proteolytically derived III12-14-containing peptides two or three times more efficiently than they bound the 44-, 75-, or 40-kDa fragments, both in the absence and the presence of a homobifunctional cross-linker (BS3). These studies leave little doubt that direct I1-5-III12-14 interactions contribute to the inhibitory effects of the heparin II-binding fragments. The modest preferential binding interactions between the heparin II and 70-kDa fragments seen in solution, however, suggest that direct binding interactions cannot readily account for the fact that the inhibitory effect of the heparin II fragments may be 20 times larger than those of the 44-kDa fragments (see Fig. 5). Thus, the III12-14 repeats may also affect binding interactions of the I1-5 repeats by other mechanisms.


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Fig. 8.   Binding interactions between biotinylated 70-kDa fragments and various other fragments of fibronectin. Binding reactions were done in a solid phase assay (panel A) or in solution (panel B). In panel A, the proteolytically derived heparin II fragment (7.4 × 105 cpm/pmol), the recombinant III12-14 peptide (3.3 × 105 cpm/pmol), the 44-kDa fragment containing the III1 repeat (4.8 × 105 cpm/pmol), the 75-kDa cell adhesion fragment (8.1 × 105 cpm/pmol), or the 40-kDa gelatin-binding fragment of fibronectin (8.2 × 105 cpm/pmol) was bound to biotinylated 70-kDa fragments (30 µg/ml) absorbed to microtiter wells. Binding was done for 4 h at 37 °C in PBS, pH 7.4, containing 2 mg/ml BSA. Binding to uncoated BSA-blocked wells was subtracted from the data. Nonbiotinylated 70-kDa fragments (adsorbed at 30 µg/ml) bound the various ligands in exactly the same way as the biotinylated substrate (data not shown). In panel B, soluble biotinylated 70-kDa fragments were incubated with the various 125I-fibronectin fragments as described under "Materials and Methods." Binding was measured in the presence (hatched bars) and the absence (open bars) of the cross-linker BS3. In both panels A and B, the data represent the means of triplicate measurements. Bars indicate the S.E.

mAbs IST-2 and 9D2 Inhibit Binding of Amino-terminal Fragments of Fibronectin to the 160-kDa Substrate but Not to Cells-- As shown in Figs. 3-5, maximal inhibition of the binding of 125I-fibronectin and 125I-labeled 70-kDa fragments by either the heparin II or the 44-kDa fragments does not plateau and may not be limited at all (note a possible exception in Fig. 3, black-triangle). This suggests that the inhibitory effects of the III12-14 and III1 repeats may not be independent of each other. To examine this possibility, we compared the roles of the III12-14 and III1 repeats in the binding of 125I-labeled 70-kDa fragments with the 160-kDa substrate using anti-III13-14 (IST-2) and anti-III1 (9D2) monoclonal antibodies.

As shown in Fig. 9, the mAb IST-2 inhibited binding of 70-kDa fragments in a concentration-dependent manner by at least 80% and in some experiments by >90% (Fig. 9; bullet ). In contrast, mAb C6F10, recognizing the III8-10 repeats, inhibited binding by no more than 20% over a wide range of concentrations (Fig. 9; open circle ). The difference is even more striking because at equal IgG concentrations mAb C6F10 binds to the 160-kDa fragment at least 100-fold more efficiently than mAb IST-2 (data not shown). Nonimmune mouse IgG is noninhibitory in this system (Fig. 9; black-triangle). Apparently, mAb IST-2 interferes with binding interactions of the type I1-5 repeats because it inhibits binding of 125I-labeled 29-kDa fragments as efficiently as it inhibits binding of 125I-labeled 70-kDa fragments (data not shown). If mAb 9D2 was used, binding of the 70-kDa ligand to the 160-kDa substrate was also blocked in a concentration-dependent manner by > 90% provided that the antibody concentrations were increased >=  10-fold over the effective IST-2 concentrations. Thus, either of the antibodies could substitute for the other (see below, Fig. 11, bullet ).


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Fig. 9.   Inhibition of binding interactions between 125I-labeled 70-kDa fragments and adsorbed 160-kDa substrate by mAb IST-2. Microtiter wells, which had been coated with 5.3 (bullet ,black-triangle) or 3.2 pmol/well (open circle ) of the 160-kDa cell adhesion fragment, were incubated for 4 h at 37 °C with mAbs IST-2 (bullet ), C6F10 (open circle ), or with nonimmune mouse IgG (black-triangle) at the indicated IgG concentrations. Labeling with the 125I-labeled 70-kDa fragment (1.8 × 105 cpm/well or 0.1 pmol/well) was done during the last hour. All data represent specific ligand binding normalized to specific binding measured in the absence of antibodies (0.8 fmol/well; nonspecific binding was 0.2 fmol/well). Points are means of five (bullet ,black-triangle) or two (open circle ) independent determinations. Bars represent the S.E. The smooth curve is a third order regression line.

When cycloheximide-treated cells were plated on the 160-kDa substrate, binding of 125I-labeled 70-kDa fragments in the absence of the antibodies was equal to the binding to the substrate alone (e.g. in Fig. 10, specific binding was 0.5 fmol/well in the presence as well as in the absence of cells), or it was enhanced up to 3-fold (12). Surprisingly, none of the binding of 125I-labeled 70-kDa fragments to these cell layers was inhibited by either the mAb IST-2 (Fig. 10), the mAb 9D2 (data not shown), or when both antibodies were used together (Fig. 11; open circle ), even though both antibodies could inhibit binding to the substrate (Fig. 11, bullet ). Instead, binding to cells was enhanced by 30-40% in the presence of either the mAb IST-2 (0.1-10 µg/ml) or the mAb 9D2 (data not shown). Binding to cell layers was unaffected by the mAb C6F10 or nonimmune IgG (Fig. 10). In all of these experiments, antibodies were present from the time cells were plated to the end of the labeling period. This procedure ensured that the antibodies had access to the 160-kDa substrate in the confluent cell layers (12). In conclusion, binding of 70-kDa fragments to cell surfaces appears to be independent of the type III12-14 and the type III1 repeats, whereas each of these domains proved to be critical for nearly all of the binding of the 70-kDa fragment to the 160-kDa substrate.


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Fig. 10.   Binding of 125I-labeled 70-kDa fragments to cell layers is not inhibited by mAb IST-2. Suspensions of cycloheximide-treated cells were mixed with mAbs IST-2 (open circle ), C6F10 (triangle ), or with nonimmune mouse IgG (black-triangle) at the indicated IgG concentrations. Cells were plated in microtiter wells that had been coated with 3.2 pmol/well of the 160-kDa cell adhesion fragment of fibronectin. Cell attachment and spreading were unaffected by any of the antibodies (not shown). After 3 h, the confluent monolayers (3 × 104 cell/well) were labeled for 1 h with the 125I-labeled 70-kDa fragment (2 × 105 cpm/well or 0.1 pmol/well). In the same experiment, cell-independent binding of the 70-kDa fragment to the 160-kDa substrate was measured in the presence of mAb IST-2 as described in Fig. 3 (bullet ). All data are means of triplicate measurements of specific binding normalized to specific binding seen in the absence of antibodies (0.4 fmol/well in the presence or absence of cells). Bars represent the S.E. Smooth curves are third order regression lines.


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Fig. 11.   Binding of 125I-75-kDa fragments to cell layers is not inhibited by the mAbs IST-2 and 9D2. Binding of the 125I-labeled 70-kDa fragments to cell layers (open circle ) and to substrate alone (bullet ) was measured at increasing concentrations of mAb 9D2 in the presence of a fixed amount of mAb IST-2 (1.6 µg/ml). Suspensions of cycloheximide-treated cells were mixed with mAb 9D2 and/or mAb IST-2 and plated in culture wells that had been coated with 3.2 pmol/well of the 160-kDa cell adhesion fragment. Cell attachment and spreading were unaffected by any of the antibody treatments (not shown). After 3 h, the confluent monolayers (3 × 104 cells/well) were labeled for 1 h with 125I-labeled 70-kDa fragments (2 × 105 cpm/well or 0.1 pmol/well). Binding to substrate alone was measured at the same time. Data represent means of triplicate measurements of specific binding normalized to specific binding seen in the absence of antibodies (0.5 fmol/well in the presence or absence of cells). Bars represent the S.E.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this paper we demonstrate that the carboxyl-terminal heparin II binding domain of fibronectin is involved in the assembly of fibronectin into fibrils. Using binding assays and immunofluorescence microscopy, we show that proteolytic fragments of fibronectin containing the III12-14 repeats inhibit binding of fibronectin, or its amino terminus, to cell layers and assembly of fibronectin into fibrils. Until now, the III1 repeat was the only type III repeat known to inhibit fibronectin fibril formation (23, 24). Under physiological conditions, the heparin II-binding fragments inhibited binding of fibronectin and amino-terminal 70-kDa fragments to cells >=  10 fold more efficiently than a 44-kDa fragment containing the III1 repeat. We conclude therefore that assembly of fibronectin fibrils involves at least two separate and distinct type III repeats.

The sequences within the heparin II binding domain which inhibit fibril formation are unknown. These sequences, like the inhibitory sequences within the III1 repeat, appear to be cryptic in fibronectin because a 160-kDa fragment of fibronectin containing the III12-14 and III1 repeats did not inhibit either the incorporation of fibronectin into the extracellular matrix or the binding of 70-kDa fragments to absorbed 160-kDa fragments.

The data presented in this paper suggest two alternative mechanisms by which the heparin II binding domain could inhibit the incorporation of fibronectin into fibrils. First, the III12-14 repeats could bind directly to the I1-5 repeats and inactivate the amino terminus. Second, the heparin II-binding fragments could block cooperative interactions between the III12-14 and III1 repeats which facilitate binding of the amino terminus.

Our studies confirm previous studies by Homandberg and Erickson (27) and show that I1-5 repeats bind preferentially to the III12-14 repeats, both in solid phase and in solution. This is consistent with the idea that direct I1-5-III12-14 interactions may contribute to fibronectin fibrillogenesis. It is also consistent with the idea that heparin II-containing fragments are inhibitory because they can bind to the I1-5 repeats in either fibronectin or the 70-kDa fragment and form a complex that is incapable of binding to cells or the 160-kDa cell adhesion substrate. It remains uncertain, however, to which extent direct I1-5-III12-14 interactions can account for the inhibitory effects of the heparin II-binding fragment because compared with the 44-kDa III1-containing fragment, III12-14-containing fragments bound soluble 70-kDa fragments only two to three times more efficiently whereas they could inhibit binding of 70-kDa fragments to cells or to the 160-kDa substrate >=  10 times better. This leaves the possibility that the III12-14 repeats may also facilitate binding of I1-5 repeats by alternative mechanisms.

One possible alternative mechanism suggested by our finding is that the IST-2 as well as the 9D2 antibody could inhibit the binding of the 70-kDa amino terminus to the 160-kDa substrate by >=  90% even though these antibodies recognize exclusive epitopes in the type III13-14 and type III1 repeats, respectively (23, 40). Because this finding seems to be incompatible with the simple notion that the heparin II domain and the III1 repeat represent two independent I1-5 binding sites, we suggest that binding of the I1-5 repeats to the 160-kDa substrate involves cooperative interactions between the III12-14 and the III1 repeats.

Conceivably, such cooperative interactions might depend on the III1 and III12-14 repeats coming into close contact with each other. Thus, binding of type I1-5 repeats would be inhibited by either mAb 9D2 or IST-2, whereas an antibody like mAb C6F10 (23) would have little or no effect because it recognizes an epitope in the III8-11 repeats. Binding of I1-5 repeats would also be expected to be completely and specifically inhibited by the heparin II or 44-kDa fragments because they could readily disrupt the apposition of III12-14 and III1 repeats in the 160-kDa substrate. Apposition of III1 and III12-14 repeats might be facilitated by the highly electronegative decapeptide in fibronectin (residues 722-731) in the type III2 repeat (41) because this decapeptide, like heparin, could bind to heparin binding sequences in the III12-14 repeats (42, 43). In intact fibronectin, apposition of the III12-14 and III1 repeats may also involve the binding interaction between the III1 and III15 repeats described recently by Ingham et al. (44).

Because the heparin II domain falls in between integrin binding sites, its activity may well be regulated through tensile forces created by integrin-mediated mechanochemical interactions between the cytoskeleton and fibronectin dimers (13). Such interactions are thought to promote reversible unfolding of one or more type III repeats (16) resulting in the exposure of self-association sites within fibronectin. The "activated" heparin II domain could then bind directly to the I1-5 repeats and/or facilitate I1-5 binding through cooperative interactions with another type III repeat, such as III1.

Our evidence for direct binding interactions between I1-5 and III12-14 repeats lends further support to the notion that the assembly of a complex fibronectin matrix requires multiple I1-5 binding sites in fibronectin. As predicted before (5, 45), single binding interactions of the I1-5 repeats can only give rise to uniform fibrils, whereas additional interactions would also promote fibril thickening and branching and thereby add considerable complexity and flexibility to a fibronectin matrix. The availability of multiple I1-5 binding sites in fibronectin could also reconcile conflicting views of the role of the III1 repeats in matrix assembly. For example, it now seems possible that the assembly of fibronectin dimers lacking the III1-7 repeats (26) into fibrils could proceed entirely via I1-5-III12-14 interactions.

Although our studies clearly attest to the importance of III12-14 as well as the III1 repeats in I1-5-dependent fibronectin-fibronectin binding interactions during fibrillogenesis, it is also evident that binding of the 70-kDa amino terminus to cell layers can occur independently of these two type III domains. As we have shown before (12), the 70-kDa fragments of fibronectin can bind to cycloheximide-treated cells in the absence of any III1 or III12-14 repeats. Such binding could not be attributed to any residual fibronectin left on the cell surface because the same cells failed to bind any 70-kDa fragments when they were plated on collagen or vitronectin. In addition, we have shown here that mAbs IST-2 and 9D2, even when used together, fail to inhibit binding of 70-kDa fragments to cycloheximide-treated cells. The inability of these antibodies to block binding of the amino terminus to cell layers cannot be attributed to an inaccessibility of their epitopes because these antibodies were added at the time cells were plated and thus had ample access to the substrate (12). Interestingly, the antibodies were not entirely ineffective in cell layers but actually enhanced binding of 70-kDa fragments by ~40%. Such enhancement is seen not only in our cycloheximide-treated cells, but also in normal fibroblast cultures treated with anti-III1 Fab' fragments (23). The fact that the antibodies fail to block binding of the amino terminus to cell layers may seem in conflict with our observation that fibronectin fragments can inhibit such binding effectively. In this case, however, the fragments, unlike the antibodies, could simply interact with the ligand in solution.

In conclusion, the heparin II domain in fibronectin, like the III1 repeat, plays an important role in fibronectin fibrillogenesis but does not appear to participate in all I1-5-dependent steps in matrix assembly. The III12-14 repeats may participate in fibrillogenesis by binding directly to the amino terminus and/or facilitate binding of the amino terminus through cooperative interactions with the III1 repeat.

    ACKNOWLEDGEMENTS

We thank Sara Brummel for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM47221 and EY0850 (to D. M. P. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be sent: Dept. of Pathology, Rm. 6590 MSC, 1300 University Ave., University of Wisconsin, Madison, WI 53706. Tel.: 608-262-4626; Fax: 608-265-3301.

1 The abbreviations used are: BSA, bovine serum albumin; PBS, phosphate-buffered saline; BS3, bis(sulfosuccinimidyl) suberate; mAb, monoclonal antibody; GST, glutathione S-transferase.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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