Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Quantitative study of cell interaction with collagen and fibronectin

The interaction of BHK-fibroblasts with collagen or fibronectin-collagen complex was investigated quantitatively. For that purpose an improved method for production of defined cell substrata was developed. The method permitted reproducible coupling of different ligands to glass via an amino or carboxyl group. BHK-cells grown on collagen required a minimum density of 15-20 ng collagen/cm2 for spreading. When grown on fibronectin adsorbed on collagen the cells were found to remove fibronectin from the substratum at a rate of 0.15 pg/(cell X h).     

Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.     

Characterisation of the immune response to type I collagen in scleroderma

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSE^low). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4⁺, activated (CD25⁺), memory (CD45RO⁺) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.     

Fibronectin binding site in type I collagen regulates fibronectin fibril formation

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin- sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.     

The mammalian chitinase-like lectin, YKL-40, binds specifically to type I collagen and modulates the rate of type I collagen fibril formation

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 x 10(-3) to 4.50 x 10(-3) s(-1)). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.     

Altered rabbit endothelial cell phenotypic expression in response to human fibronectin

When rabbit corneal endothelial cells were cultured in excess concentrations of human fibronectin an altered phenotypic expression was observed. Cell appearance was changed radically and collagen synthesis was specifically inhibited in a dose-response fashion. This study provides further evidence that fibronectin may be one of the developmental signals which act a molecular level and is capable of interspecies activity.     

Flow perfusion rate modulates cell deposition onto scaffold substrate during cell seeding

The combination of perfusion bioreactors with porous scaffolds is beneficial for the transport of cells during cell seeding. Nonetheless, the fact that cells penetrate into the scaffold pores does not necessarily imply the interception of cells with scaffold substrate and cell attachment. An in vitro perfusion system was built to relate the selected flow rate with seeding efficiency. However, the in vitro model does not elucidate how the flow rate affects the transport and deposition of cells onto the scaffold. Thus, a computational model was developed mimicking in vitro conditions to identify the mechanisms that bring cells to the scaffold from suspension flow. Static and dynamic cell seeding configurations were investigated. In static seeding, cells sediment due to gravity until they encounter the first obstacle. In dynamic seeding, 12, 120 and 600 \(\upmu \hbox {l/min}\) flow rates were explored under the presence or the absence of gravity. Gravity and secondary flow were found to be key factors for cell deposition. In vitro and in silico seeding efficiencies are in the same order of magnitude and follow the same trend with the effect of fluid flow; static seeding results in higher efficiency than dynamic perfusion although irregular spatial distribution of cells was found. In dynamic seeding, 120 \(\upmu \hbox {l/min}\) provided the best seeding results. Nevertheless, the perfusion approach reports low efficiencies for the scaffold used in this study which leads to cell waste and low density of cells inside the scaffold. This study suggests gravity and secondary flow as the driving mechanisms for cell-scaffold deposition. In addition, the present in silico model can help to optimize hydrodynamic-based seeding strategies prior to experiments and enhance cell seeding efficiency.     

Heparin enhances the rate of binding of fibronectin to collagen.

125I-labelled fibronectin is shown to bind to both native and denatured collagen immobilized on Sephadex beads in reactions that exhibit different kinetics. The rates of both reactions were enhanced by the presence of heparin or highly sulphated dextran sulphate but not by other glycosaminoglycans or dextran sulphates having low sulphate contents.     

Monoclonal antibodies to the collagen binding domain of human plasma fibronectin

Five independent hybrids producing monoclonal antibodies to human plasma fibronectin have been obtained by fusing P3/X63-Ag8 myeloma cells with immune mouse splenocytes. The specificity of these monoclonal antibodies (MABs) for fibronectin was demonstrated by three independent tests: binding to the purified soluble molecule, immunofluorescence staining of insoluble extracellular matrices produced by endothelial cells in vitro, immunostaining of fibronectin tryptic peptides after separation on SDS-PAGE and transfer to nitrocellulose sheets. Two antibodies (MAB 29 and 52) recognized selectively human fibronectin while the others (MAB 5, 30 and 59) reacted also with plasma fibronectin from calf, hamster and chicken. Four distinct epitopes were recognized by the MABs studied. MAB 5, 30, 52 and 59 reacted with distinct antigenic sites, while MAB 29 and 52 bind to the same site. Antigenic fragments were identified by immunostaining of fibronectin tryptic peptides. MAB 5 reacted with a collagen binding fragment with a molecular weight of 120 K. In addition, each of the MAB 29, 30, 52 and 59 reacted with peptides with a molecular weight of 40 K that bind to gelatin. Since these antibodies do not inhibit fibronectin-collagen interaction, it is concluded that their corresponding epitopes are clustered in a region close, but not coincident, to the collagen binding site of fibronectin.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.     

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.     

Role of collagen and fibronectin in neural crest cell adhesion and migration

Cultured neural crest cells which are freshly trypsinized require serum or purified fibronectin to attach to collagen substrates of types I–V. Furthermore, neural crest cells migrate in a Boyden chamber in response to fibronectin, and a “checkerboard” analysis demonstrates that fibronectin is both chemotactic and chemokinetic for these cells. It is proposed that collagen serves as a substrate for neural crest cells as they migrate in the early embryo. By mediating the cells' attachment to collagen, fibronectin may influence the movement of the cells. Local differences in fibronectin concentration or availability in the matrix could affect the degree of attachment of the cells to the collagen substrate and could also direct their migration by serving as a chemoattractant.     

Adhesion to fibronectin or collagen I gel induces rapid,extensive, biosynthetic alterations in epithelial cells

Extracellular matrix influences many cellular events. In this study, we demonstrate that adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by β₁ integrins, results in substantial alterations in protein and RNA expression profiles. The large numbers of changes in expression suggest that simply changing the adhesive substrate has basic effects on the regulation of cellular biosynthesis. Two-dimensional electrophoresis of [³⁵S]methionine-labeled HSG cell proteins identified significant differences in the patterns of protein expression by cells cultured on nonprecoated substrates, collagen I gels or fibronectin. Thirty-two differentially expressed cDNA clones, which included both novel and previously sequenced genes, were up-regulated within 6 hr by culturing HSG cells on fibronectin or collagen I gels. Therefore, adhesion to collagen I or fibronectin resulted in rapid, widespread changes in cellular biosynthetic control. Expression of some genes was induced by ligation of β₁ integrins with antifunctional antibodies, whereas the expression of other genes was not induced. Most of the differentially expressed genes were up-regulated by adhesion to both fibronectin- and collagen I gel-coated substrates, but a few genes were selectively up-regulated on only one substrate. Furthermore, the up-regulated expression of some genes was detected within 3 hr, whereas changes in others required 6 hr. Discrete adhesive substrates and integrin molecules differentially affected the expression of a significant number of genes, suggesting that the cellular responses to adhesion were triggered through several signaling pathways. J. Cell. Physiol. 175:163–173, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells

Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is though to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of ³H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.     

Fluid shear stress modulates endothelial cell invasion into three-dimensional collagen matrices

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of approximately 5 dyn/cm2, whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm2 shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells.

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.     

Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.