Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell-binding domain using site-directed mutagenesis

Previous studies of adhesion mediated by the central cell-binding domain of fibronectin suggest that additional polypeptide information besides the Arg-Gly-Asp sequence is required for full activity. We analyzed this putative second, synergistic region of fibronectin more extensively by deletion analysis and oligonucleotide-based site-directed mutagenesis. Resulting mutated fusion proteins expressed using lambda gt11 were assayed for baby hamster kidney fibroblast cell spreading activity. Deletion mutants truncating from the amino terminus showed a decrease of activity in two apparently discrete steps. Complementary studies using a series of overlapped internal deletions designed to retain the repetitive fibronectin structure also indicated that two distinct peptide regions besides the RGD sequence were necessary for full activity. Removal of the carboxyl-terminal region resulted in the greatest loss of activity (greater than or equal to 20- versus 3-5-fold). Very similar results were obtained with HT-1080 cells dependent on the alpha 5 beta 1 integrin receptor for adhesion to fibronectin. An anti-fibronectin monoclonal antibody that inhibits cell adhesion was found to bind to the carboxyl-terminal functional region, and a point mutation caused specific loss of its epitope. These studies reveal unexpected complexity in the organization of these functional regions, which contrasts with adhesion models based only on simple, short peptide recognition sequences.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Effects of different fragments of the fibronectin molecule on latex bead translocation along neural crest migratory pathways

Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.     

Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of type III homology repeats in cell adhesive function within the cell-binding domain of fibronectin

Recombinant fibronectin (FN) fragments and their mutant proteins were produced to elucidate the role of type III homology repeats in cell adhesive activity within the cell-binding domain of FN. Cell adhesive activity of the 11.5-kDa fragment, the cell attachment site of the cell-binding domain, was less than 0.1% that of native FN despite the presence of the Arg-Gly-Asp-Ser sequence. The activity increased as type III homology repeats were added to the N terminus of the 11.5-kDa fragment, and a 52-kDa fragment with four additional type III repeats had almost the same activity of native FN. Deletion of Arg-Gly-Asp from the fully active fragments completely abolished the cell adhesive activity. Deletion of one or two repeats from the 52-kDa fragment affected the extent of the cell adhesive activity, the degree of the effect being inversely correlated with the distance of the deletion from the type III repeat containing Arg-Gly-Asp-Ser. Rearrangement of type III repeats caused much loss of activity. These results suggest that the number and kinds of type III repeats and their correct alignment rather than the putative synergistic site decide the extent of the specific cell adhesive activity.     

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates. At the same time, heparin or two monoclonal antibodies against the heparin-binding domain were able to inhibit the haptotactic migration to CH-271 or fibronectin, though not to C-274 or a mixture of C-274 and H-271. This suggests that although heparin did not affect tumor cell adhesion to the cell-binding domain near the heparin-binding domain in CH-271 or fibronectin, it did lead to a modulation of cell motility. It seems likely that the regulatory mechanism may depend on interaction between heparin-like molecules on the cell surface and the heparin-binding domain in fibronectin, rather than on simple steric hindrance or on the masking of the cell-binding domain caused by the binding of heparin to heparin-binding domain.     

Interaction between cell-binding domain and extracellular matrix-binding domain of fibronectin determined by fluorescence depolarization

Interaction of domains in fibronectin was observed by photometry of fluorescence polarization of three kinds of dye; [N-(1-anilinonaphthyl-4)]maleimide (ANM tau = 5 ns), [N-(3-fluoranthyl)]maleimide (FAM tau = 20 ns), and [N-(3-pyrene)]maleimide (PRM tau = 100 ns). Each dye was labeled at a free sulfhydryl group in the cell-binding domain. Neither fluorescence of ANM with short fluorescent lifetime, FAM with long lifetime, nor PRM with longer fluorescent lifetime on fibronectin depolarized as much as the free dye. It was found that each dye was firmly fixed in the cell-binding domain. When heparin or gelatin was added in the solution of PRM-fibronectin complex, the fluorescence polarization tended to increase principally by combining heparin or gelatin to fibronectin. It was found that the rotation of whole or partial fibronectin containing the cell-binding domain through fluorescent lifetime of 100 ns was suppressed by combining of heparin or gelatin to fibronectin. When heparin or gelatin was added in the solution of ANM- or FAM-fibronectin complex, on the contrary, the fluorescence polarization tended to decrease, that is, slightly depolarize through the fluorescent lifetime of 5 or 20 ns, respectively. It was found that the rotation of the cell-binding domain, or of part of the fibronectin molecule containing the domain, was slightly promoted by combining heparin or gelatin to its domain. These results indicate that an interaction of the heparin- or gelatin-binding domain with the cell-binding domain was induced by the combining of heparin or gelatin to the respective domains.     

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.     

Mechanism of human keratinocyte migration on fibronectin: unique roles of RGD site and integrins.

The migration of human keratinocytes over the wound bed plays an important role in the re-epithelialization of cutaneous wounds. Fibronectin, a large glycoprotein matrix component that is abundant within cutaneous wound beds, promotes keratinocyte migration. However, the mechanisms by which keratinocytes migrate over fibronectin are unknown. In this study, we sought to identify specific sites within the fibronectin molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. The data show that the domain within the fibronectin molecule that induces human keratinocyte migration is the 120 kD cell-binding domain close to the carboxyl terminus. The 40 kD heparin-binding domain near the carboxyl terminus and the 45 kD gelatin-binding domain near the amino terminus did not promote keratinocyte migration. In addition, keratinocyte migration on both fibronectin and the 120 kD cell-binding domain was completely inhibited by the presence of GRGDSP peptide, suggesting that keratinocyte migration on fibronectin is mediated by recognizing the RGD sequence located within the cell-binding domain of fibronectin. Furthermore, keratinocytes were able to migrate directly on immobilized RGD substratum. Cell migration on fibronectin is mediated by the alpha 5 beta 1 integrin since antibodies blocking the alpha 5 and the beta 1 subunits completely inhibited keratinocyte migration on fibronectin. In addition, we demonstrate that human keratinocytes express alpha 5 beta 1 integrin in culture by flow cytometry.     

Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and "synergy" sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin alpha5beta1.     

Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments.

Fibronectin has been shown previously to promote complete cell adhesion in the absence of other serum components or de novo protein synthesis. Recently a sequence of four amino acids from the cell-binding domain of fibronectin has been termed the 'cell recognition site' of this multidomain molecule since it mediates cell attachment and inhibits cell adhesion to intact fibronectin. We show here, however, that substrata coated with an isolated cell-binding domain of fibronectin are not sufficient for complete cell adhesion; cells attach and spread but, unlike those adhering to intact fibronectin, they do not form stress fibres terminating in focal adhesions. An additional external stimulus is needed for this cytoskeletal reorganisation and may be provided by one of two heparin-binding fragments of fibronectin. The two 'signals' required for complete adhesion need not be provided simultaneously since focal adhesion formation can be promoted by stimulating cells pre-spread on a cell-binding fragment of fibronectin with a soluble heparin-binding fragment. This second stimulation may involve cell membrane heparan sulphate proteoglycans.     

Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin.

Tripeptidyl peptidase II (TPP II) is a large intracellular exopeptidase with an active site of the subtilisin type. Affinity-purified hen antibodies against human erythrocyte TPP II cross-reacted with fibronectin in an immunoblot analysis. Furthermore, antibodies against human fibronectin cross-reacted with TPP II. Antibodies against a 65 kDa cell-binding fragment of fibronectin specifically reacted with TPP II, whereas antibodies against the collagen-binding domain, the main heparin-binding domain or the N-terminal fibrin-binding domain did not react. Moreover, the affinity-purified antibodies against TPP II reacted with a 105 kDa cell-binding fragment of fibronectin but not with the fibrin-binding domain or the collagen-binding domain. When native TPP II was dissociated into smaller units through dialysis against a dilute Tris buffer, it could be digested by chymotrypsin into three stable fragments of 70 kDa, 42 kDa and 20 kDa. It could be demonstrated that the 42 kDa fragment was specifically recognized by antibodies against the 65 kDa cell-binding fragment of fibronectin. Furthermore, labelling with di-[3H]isopropyl phosphorofluoridate and N-terminal sequence determination showed that the 70 kDa fragment contained the active-site serine residue. In conclusion, our findings suggest that one domain of the TPP II molecule bears structural resemblance to a cell-binding fragment of fibronectin.     

Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions

Two domains of fibronectin deliver two different but cooperative signals required for focal adhesion formation. The signal from the cell-binding domain is mediated by integrins, whereas the signal from the heparin-binding domain is recognized by heparan sulfate proteoglycans, of which syndecan-4 has been hypothesized to be involved in focal adhesion formation. We generated mice deficient in syndecan-4 to study its role directly. Even in fibroblasts from syndecan-4-deficient mice, focal adhesions were formed, and actin fibers terminated normally at focal adhesions when they were cultured on coverslips coated with fibronectin or with a mixture of its cell-binding and heparin-binding fragments. However, when the cells were cultured on the cell-binding fragment and the heparin-binding fragment was added to the medium, focal adhesion formation was impaired in the syndecan-4 null fibroblasts as compared with that in wild-type cells. Therefore, syndecan-4 is essential for promoting focal adhesion formation only when the signal of the heparin-binding domain of fibronectin is delivered as a soluble form, most probably from the apical surface. When the signal is delivered as a substratum-bound form, other molecule(s) also participate(s) in the signal reception.     

Binding of fibronectin by the acute phase reactant C-reactive protein

Following tissue injury, the concentration of C-reactive protein (CRP) is known to increase in plasma rapidly, while that of fibronectin often decreases. We now report that CRP immobilized onto polystyrene surfaces binds soluble plasma fibronectin (Kd = 1.5 X 10(-8) M). The binding of fibronectin by CRP was relatively sensitive to ionic conditions, being maximal at physiological NaCl concentrations. A decrease of pH from neutral to 5-6 greatly enhanced the binding of fibronectin by CRP. Ca2+ ions at greater than 1 mM inhibited binding. No binding was observed between fibronectin and CRP in soluble phase. CRP was found also to bind fibrinogen, which competed with fibronectin for CRP-binding sites. This was shown to explain why fibronectin was effectively bound from serum but not from plasma by immobilized CRP. The amount of CRP immobilized was critical in binding fibronectin; a too dense molecular layer of CRP inhibited the binding, as did the postsaturation of free surfaces with albumin, which itself was not bound by CRP. Soluble fibronectin agglutinated CRP-coated latex particles. Most or all of the CRP-binding activity in the fibronectin molecule was localized to the 120-140-kilodalton fragment, which also contains cell-binding and heparin-binding domains of fibronectin. The results provide a link between acute phase response and tissue repair.     

Isolation and characterization of two monoclonal antibodies that recognize remote epitopes on the cell-binding domain of human fibronectin

Two monoclonal anti-fibronectin antibodies that inhibit fibronectin-mediated cell adhesion have been established and characterized. One antibody, FN12-8, inhibited attachment of rat kidney fibroblasts on the fibronectin-coated substrate in a concentration-dependent manner, attaining a maximal inhibition of greater than 85% at 850 micrograms/ml. Another antibody, FN30-8, caused about 70% inhibition at a concentration as low as 0.85 microgram/ml, although further increase of the antibody concentration did not significantly augment the inhibitory effect. Immunoblot analysis with defined proteolytic fragments revealed that both antibodies are directed to the cell-binding domain of fibronectin. The epitopes for these antibodies were further narrowed down using recombinant cell-binding fragments expressed in Escherichia coli. FN12-8 recognized the 11.5-kDa cell-binding fragment previously characterized by Pierschbacher et al. (1981, Cell 26, 259-267), suggesting that FN12-8 blocks the Arg-Gly-Asp (RGD) cell adhesion signal. FN30-8 could not bind this fragment but did recognize a longer cell-binding fragment containing additional greater than 111 amino acid residues upstream of the 11.5-kDa fragment. Since the RGD-dependent cell adhesion seems to require another signal located at a region 50-160 residues upstream of the 11.5-kDa fragment for full activity, FN30-8 may exert its inhibitory effect by blocking the latter signal.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

Localization of the major heparin-binding site in fibronectin

We have identified the major site required for the interaction of fibronectin (FN) with heparin. Affinity chromatography was used to test the binding ability of a library of truncated, monomeric forms of fibronectin (deminectins) containing deletions or two point mutations in the heparin-binding domain. This domain consists of type III repeats 12, 13, and 14. Deletions of individual repeats showed that both III13 and III14 are required for complete binding. Small deletions within these repeats localized a major site of heparin interaction to the amino-terminal half of III13. Site-directed mutagenesis of adjacent arginines within this sequence to uncharged residues reduced heparin binding by 98%, identifying these positively charged amino acids as essential for the interaction. A significant role for the flanking alternatively spliced regions and for repeat III12 was not found. We conclude that, while both repeats III13 and III14 participate in heparin binding, there is a major site of interaction in repeat III13 that accounts for nearly all of the activity. The significance of multiple heparin-binding sites within this domain is discussed and a model is proposed to account for how these sites may function in vivo.     

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.     

Transglutaminase-sensitive glutamine residues of human plasma fibronectin revealed by studying its proteolytic fragments

The sites of transglutamination of fibronectin and fibronectin fragments, by coagulation factor XIIIa and tissue transglutaminase, were studied. It was shown that the intact fibronectin molecule has two sites sensitive to coagulation factor XIIIa and four sites sensitive to tissue transglutaminase: 180--190-kDa gelatin/heparin-binding fragments, 2 and 5--6 sites; 29-kDa heparin-I/fibrin-I-binding N-terminal fragments, 1 and 2 sites; 70-kDa gelatin-binding fragments, 0 and 1 site; 60-kDa cell-binding central fragments, 1 and 3--4 sites; 60-kDa, 45-kDa, 30-kDa heparin-II-binding C-terminal fragments, 1 and 2 sites. Thus, we have found a new coagulation-factor-XIIIa-sensitive site localized in the cell-binding central fragment, inaccessible to enzyme in the intact fibronectin molecule. Tissue transglutaminase appeared to interact with all of the three coagulation-factor-XIIIa-sensitive sites and, in addition, some others which are either available on the intact molecule or can be revealed only in proteolytic fragments of the fibronectin. We suggest that interdomain and intersubunit interactions in the intact fibronectin molecule account for the masking of glutamine residues potentially accessible to transglutaminases.     

Influence of decorin on fibroblast adhesion to fibronectin.

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