Module Of Fibronectin Research Articles

Rapid refolding of a proline-rich all-beta-sheet fibronectin type III module.

Fibronectin type III modules contain approximately 90 residues and are an extremely common building block of animal proteins. Despite containing a complex all-beta-sheet topology and eight prolines, the refolding of the 10th type III module of human fibronectin has been found to be very rapid, with native core packing, amide hydrogen bonding, and backbone conformation all recovered within 1 s at 5 degrees C. These observations indicate that this domain can overcome many structural characteristics often thought to slow the folding process.

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.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

High-resolution structural studies of the factor Xllla crosslinking site and the first type 1 module of fibronectin

The N-terminal domain of fibronectin undergoes factor XIIIa-catalysed crosslinking to fibrin, bacteria and collagen. The reactive glutamine residue is in an extended, random coil 'tail' of about 18 residues that would be accessible for crosslinking.

Protein ligands of the human adenovirus type 2 outer capsid identified by biopanning of a phage-displayed peptide library on separate domains of wild-type and mutant penton capsomers.

A filamentous phage-displayed random hexapeptide library was screened on the adenovirus type 2 (Ad2) penton capsomer and its separate domains, penton base, full-length fiber, fiber shaft and fiber knob. Affinity supports were designed to immobilize the penton ligate with a preferred orientation, via immuno-adsorption to pre-coated antibody. Three classes of phagotopes were distinguished in the eluates from the penton and fiber domains. (i) The first class represented peptide sequences identified in certain Ad2 capsid proteins, protein IIIa, protein pVIII, penton base and penton fiber. Data from specific ligand elution of phages bound to fiber and penton base wild-types and mutants suggested that the region overlapping the RLSNLLG motif at residues 254-260 in the penton base and the FNPVYP motif at residues 11-16 in the fiber tail formed mutual interacting sites in the penton capsomer. (ii) The second class consisted of phagotopes homologous to peptide sequences found in host cell membrane proteins involved in receptor or adhesion functions. One of the most abundant species corresponded to a conserved motif present in the beta-strand B of type III modules of human fibronectin. In addition, phages which were screened for their failure to bind to penton base RGD mutants were found to carry consensus motifs to peptide sequences present in the RGD recognition site of human integrin beta subunits. (iii) The third class comprised peptide motifs common to both viral and cellular proteins, suggesting that a mechanism of ligand exchange could occur during virus entry and uncoating, and virus assembly and release.

Heparin Binding by Fibronectin Module III-13 Involves Six Discontinuous Basic Residues Brought Together to Form a Cationic Cradle

The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding consensus sequence, Arg6-Arg7-Ala8-Arg9 (residues 1697-1700 in plasma fibronectin). Barkalow and Schwarzbauer (Barkalow, F.J., and Schwarzbauer, J.E. (1991) J. Biol. Chem. 266, 7812-7818) showed that mutation of Arg6-Arg7 in domain III-13 of recombinant truncated fibronectins abolished their ability to bind heparin-Sepharose. However, synthetic peptides containing this sequence have negligible affinity for heparin (Ingham, K.C., Brew, S.A., Migliorini, M. M., and Busby, T.F. (1993) Biochemistry 32, 12548-12553). We generated a three-dimensional model of fibronectin type III-13 based on the structure of a homologous domain from tenascin. The model places Arg23, Lys25, and Arg54 parallel to and in close proximity to the Arg6-Arg7-Ala8-Arg9 motif, suggesting that these residues may also contribute to the heparin-binding site. Domain III-13 and six single-site mutants containing Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli. All of the purified mutant domains melted reversibly with a Tm near that of the wild type indicating that they were correctly folded. When fluorescein-labeled heparin was titrated at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a Kd of 5-7 microM, close to that of the natural domain obtained by proteolysis of fibronectin. The R54S mutant bound 3-fold weaker and the remaining mutants bound at least 10-fold weaker than wild type. The results point out that the Arg6-Arg7-Ala8-Arg9 consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in the context of a folded domain. Thus, the heparin-binding site in fibronectin is more complex than previously realized. It is formed by a cluster of 6 positively charged residues that are remote in the sequence but brought together on one side of domain III-13 to form a "cationic cradle" into which the anionic heparin molecule could fit.

Modulation of fibronectin and tenascin production in human bronchial epithelial cells by inflammatory cytokines in vitro.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix glycoproteins participating in tissue morphogenesis and repair. The regulation of their synthesis and deposition during airway inflammation and their possible contribution in asthma are poorly understood. In this study, modulation of Fn and Tn production was investigated in transformed human bronchial epithelial cells in culture. The cells were treated with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), a combination of these cytokines, interleukins 3 and 6 (IL-3 and IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), and a combination of IL-3 and IL-6 for 48 h. Immunofluorescence and immunoblotting methods with monoclonal antibodies to Fn and Tn antibodies suggested the production of some Fn and Tn in the untreated cells. Fn was minimally induced in response to IFN-gamma and TNF-alpha, when compared with the untreated cells, whereas TNF-alpha and especially the IFN-gamma plus TNF-alpha combination resulted in a prominent Tn induction. Interleukins and GM-CSF did not induce Fn or Tn in any case. These results show that human bronchial epithelial cells are capable of producing Fn and Tn. The modulation of Fn and Tn may have an important impact on the pathology of epithelial cells during airway inflammation in vivo.

Analysis of the linkage between fibronectin alternative spliced sites during ageing in rat tissues

The modulation of fibronectin (FN) functions in cell-cell and cell-substrate interactions in a variety of physiological situations is achieved by the selective expression of different isoforms, which in the rat are generated by alternatively splicing of at least three regions of the molecule: EIIIA, EIIIB and V. Extensive information has been collected on the regulation of the differential processing of the single alternatively spliced regions but up to now there was scant knowledge about a possible coordinated expression of the three regions in the same transcript. Using a long range RT-PCR system we have shown that most of the splicing regulation is autonomous for each individual region but we have also observed a preferential expression of the EIIIA + form linked to the EIIIB — variant that is age independent. Furthermore the analysis of the single regions showed interesting variations occurring in brain during the ageing. There is a decrease in the V120 form between the 6-and the 24-month-old rat brain (from 57% to 39%), whereas despite a constant prevalence of the EIIIA — form in the young rats (65%) there is a random individual variation of this form in the old animals. Noteworthy no variations have been detected in the EIIIB region (90% EIIIB -). These data suggest a role for the V and EIIIA regions, but not for the EIIIB, during the ageing process at least in brain, since no variations were detected in kidney between the 6-and 24-month-old animals.

Modulation of fibronectin and thymic stromal cell-dependent thymocyte maturation by retinoic acid.

Retinoic acid (RA) controls the differentiation of a variety of cell types, although its role in influencing T cell development and the mechanisms potentially involved have not been thoroughly investigated. To study the ability of RA to modulate T cell development, we established a thymic stromal cell line (TC-1S) that supports the phenotypic maturation of CD4-8- double negative (DN) or CD3-4-8- triple negative (TN) thymocyte precursors. Cocultures of either DN or TN thymocytes on a monolayer of TC-1S cells resulted in the appearance of thymocytes with a more mature phenotype (CD4+8+ double positive, CD4+ or CD8+ single positive, and CD3(low) cells). Double negative T cell contact with TC-1S cells also increased the production of fibronectin (FN) by the thymic stroma and the expression of the VLA-4 FN receptor on the DN cells. Ab-mediated inhibition of the interaction between FN and its receptors significantly reduced the level of induced T cell maturation. Addition of RA either to TC-1S cells alone or to the coculture with DN cells decreased stromal cell FN expression, antagonized DN cell-induced increase in stromal cell FN production and significantly inhibited in vitro thymocyte maturation. The effects of RA were likely mediated by RA acid receptors alpha and gamma expressed both in DN thymocytes and TC-1S cells. Together these data suggest that FN/VLA-4 interaction may be an important component of stromal cell-dependent thymocyte phenotypic differentiation and that this interaction can be one of the targets for the influence of RA in T cell development.

Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin.

Cultured fibroblasts express binding sites for the amino-terminal region of fibronectin on their cell surface that mediate the assembly of soluble fibronectin into disulfide-stabilized fibrils. These binding sites have been termed matrix assembly sites and have been studied in binding assays using a 125I-labeled 70-kDa fragment derived from the amino terminus of fibronectin. In an attempt to isolate the protein(s) responsible for binding the 70-kDa fragment, cell surface proteins were cleaved from fibroblast monolayers by mild trypsinization. Trypsinization of monolayers generated a series of fibronectin fragments that bound the 125I-labeled 70-kDa fragment by ligand blot assay and affinity chromatography. All of the fibronectin fragments that bound the 70-kDa fragment contained the III-1 module. In solid phase binding assays, the 125I-labeled 70-kDa fragment bound preferentially to reduced fibronectin as compared with unreduced fibronectin fragments. Binding of the 125I-labeled 70-kDa fragment to reduced fibronectin was inhibited by a monoclonal antibody directed against the III-1 domain. Isolated III-1, however, did not bind the 125I-labeled 70-kDa fragment when adsorbed to plastic tissue culture wells. Heat denaturation of III-1 prior to adsorption conferred 70-kDa fragment binding properties on the isolated module. The 125I-labeled 70-kDa fragment did not bind to heat-denatured III-2, suggesting that 70-kDa fragment binding was a property of the III-1 module and not a general characteristic of all type III modules. The binding of 125I-labeled 70-kDa fragment to III-1 was of high affinity (KD = 1.8 x 10(-8) M). These results indicate that a binding site for the 70-kDa amino terminus of fibronectin is contained within a cryptic site found in the first type III module of fibronectin. Unfolding of the III-1 module on the cell surface may control matrix assembly site expression and represent an important step in the initiation of cell-dependent fibronectin polymerization.

Crystals of the Cell-binding Module of Fibronectin Obtained from a Series of Recombinant Fragments Differing in Length

A recombinant fragment corresponding to the cell adhesion module (FNIII 10) of human fibronectin has been crystallized at pH 8·6 from solutions containing polyethylene glycol as precipitant. The crystals formed in the space group P2 1 with a = 30·76 Å, b = 35·07 Å, c = 37·66 Å, β = 106·9°. There is one molecule per asymmetric unit and the crystals diffract beyond 1·75 Å resolution. To improve the prospects for successful crystallization of the FNIII 10 module, a series of recombinant fragments was produced with minor differences in the length of N or C-terminal segments. Only one of these variants crystallized. Interestingly, the C-terminal residue of this variant formed stable intermolecular contacts with a symmetry-related molecule in the crystal lattice.

The NH2-terminal fibrin-binding site of fibronectin is formed by interacting fourth and fifth finger domains. Studies with recombinant finger fragments expressed in Escherichia coli

The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five finger modules of fibronectin (F1-5) binds reversibly to fibrin and facilitates cross-linking by Factor XIII. To narrow down the fibrin-binding site within this region, we have used recombinant technology to express a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and their pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli. These recombinant fragments were separated from the carrier maltose-binding protein by digestion with human factor Xa or other proteases, and their structural integrity was confirmed by spectroscopic and calorimetric methods. The recombinant F1 and F4-5 exhibited fluorescence-detected melting transitions of the same magnitude and with the same midpoint (Tm) as their natural analogues prepared from Fib-1 by proteolysis. Differential scanning calorimetry experiments further demonstrated that these fragments are properly folded and have compact structures identical to the natural ones. Isolated rF4 melts at a much lower temperature than rF5 or the bimodular fragment rF4-5, indicating the loss of a stabilizing interaction between fingers 4 and 5. Comparison of fluorescence spectra of individual rF4 and rF5 with that of rF4-5 was also consistent with an interaction that affects the environment of Trp residue(s). rF2 also melts at a lower temperature than rF3 or rF2-3, suggesting a stabilizing interaction between the second and third fingers as well. When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind. All other fragments, including individual fingers 4 and 5, failed to bind. Thus, fibrin binding is not a common property of all fingers. The results indicate that a recognition site for fibrin is located within fingers 4 and 5. The interaction between these neighboring domains may play an important role in proper orientation of the residues forming this site.

Crystal structure of the tenth type III cell adhesion module of human fibronectin

The crystal structure of the cell adhesion module of fibronectin (FNIII 10)_has been determined at 1·8 Å resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2 1 with a = 30·, b = 35·1 and c = 37·7 A ̊ and β = 107°. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factpr for the final model of the 91 amino acid module plus 56 solvent atoms is 0·18 for 10 to 1·8 Å data. The module consists of two layers of β-sheet, one with three antiparallel strands and the other with four antiparallel strands. The β-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two β-strands. The tertiary structure of the FNIII 10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

In Vivo Prevention of Corticosteroid-Induced Skin Atrophy by Tretinoin in the Hairless Mouse Is Accompanied by Modulation of Collagen, Glycosaminoglycans, and Fibronectin

In an earlier study we showed that tretinoin could prevent corticosteroid-induced skin atrophy in hairless mice. In this study, we examined the histochemical, biochemical, and immunochemical changes that accompanied the atrophy and its prevention. Mice were treated dorsally for 3 weeks in the morning and afternoon (AM:PM) as follows: 1) vehicle:vehicle, 2) steroid:vehicle, 3) steroid:tretinoin. Tretinoin concentration was 0.05% in an ethanol:propylene glycol vehicle. The steroid was clobetasol propionate (0.05%). The normally sparse dermal glycosaminoglycans were further reduced by steroid:vehicle treatment and increased to greater than vehicle:vehicle amounts by steroid:retinoid. Mast cells were similarly affected. Biochemical quantification of glycosaminoglycans confirmed the histochemical findings. Collagen, non-collagenous protein, and total protein content were reduced by the steroid. The latter two were returned to more normal levels by tretinoin whereas with collagen there was only a trend toward normal levels. Fibronectin, which was increased by the steroid:vehicle treatment, was reduced to more normal levels by steroid:tretinoin. We conclude that tretinoin has the ability to prevent the major steroid-induced biomechanical changes in hairless mouse dermal connective tissue that contribute to atrophy.

Assembly of fibronectin molecules with mutations or deletions of the carboxyl-terminal type I modules.

Fibronectin is a large modular protein that is assembled into fibrils in a stepwise process that involves the binding of soluble fibronectin to the cell surface and formation of fibronectin multimers that are stabilized by disulfides. Fibronectin contains two types of disulfide-containing repeat modules, types I and II. The type I modules form units that mediate binding to assembly sites (I-1 through I-5), mediate binding to gelatin (I-6 through I-9 plus the type II modules), or have no known function other than fibrin binding (I-10 through I-12). All type I modules contain four cysteines that are disulfide-linked in a 1-3, 2-4 arrangement, except for I-12 that contains six cysteines disulfide-bonded in an unknown arrangement. I-12 contains the consensus sequence Cys-Xaa-Yaa-Cys found in a number of proteins involved in disulfide exchange reactions [Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237; Boniface, J. J., & Reichert, L. E., Jr. (1990) Science 247, 61]. We explored the role of I-12 and adjacent type I modules of fibronectin in matrix assembly. We generated mutant fibronectins in which the second and sixth or fifth and sixth cysteine residues in I-12 were changed to serines (CS mutants) or that contained deletions of the 12th (delta 12) or 10th through 12th (delta 10-12) type I modules. Expression of I-12 as a fusion protein with the gelatin binding part of fibronectin indicated that this module folds independently and that the most likely disulfide pairing is 1-4, 2-6, 3-5.(ABSTRACT TRUNCATED AT 250 WORDS)

The three-dimensional structure of the tenth type III module of fibronectin: An insight into RGD-mediated interactions

The solution structure of the tenth type III module of fibronectin has been determined using nuclear magetic resonance techniques. The molecule has a fold similar to that of immunoglobulin domains, with seven β strands forming two antiparallel β sheets, which pack against each other. Both β sheets contribute conserved hydrophobic residues to a compact core. The topology is more similar to that of domain 2 of CD4, PapD, and the extracellular domain of the human growth hormone receptor than to that of immunoglobulin C domains. The module contains an Arg-Gly-Asp sequence known to be involved in cell adhesion. This tripeptide is solvent exposed and lies on a conformationally mobile loop between strands F and G, consistent with its cell adhesion function.

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

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

The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin.

Trypanosoma cruzi expresses a developmentally regulated neuraminidase (TCNA) implicated in parasite invasion of cells. We isolated full-length DNA clones encoding TCNA. Sequence analysis demonstrated an open reading frame coding for a polypeptide of 1,162 amino acids. In the N-terminus there is a cysteine-rich domain containing a stretch of 332 amino acids nearly 30% identical to the Clostridium perfringens neuraminidase, three repeat motifs highly conserved in bacterial and viral neuraminidases, and two segments with similarity to the YWTD repeats found in the low density lipoprotein (LDL) receptor and in other vertebrate and invertebrate proteins. This domain is connected by a structure characteristic of type III modules of fibronectin to a long terminal repeat (LTR) consisting of 44 full length copies of twelve amino acids rich (75%) in serine, threonine, and proline. LTR is unusual in that it contains at least 117 potential phosphorylation sites. At the extreme C-terminus is a hydrophobic segment of 35 amino acids, which could mediate anchorage of TCNA to membranes via a glycosylphosphatidylinositol linkage. This is the first time a protozoan protein has been found to contain a YWTD repeat and a fibronectin type III module. The domain structure of TCNA suggests that the enzyme may have functions additional to its catalytic activity such as in protein-protein interaction, which could play a role in T. cruzi binding to host cells.

Five type I modules of fibronectin form a functional unit that binds to fibroblasts and Staphylococcus aureus

Fibronectin is a cell-adhesive protein comprised of three types of repeating homologous sequences, I, II, and III (Petersen, T.E., Thøgersen, H.C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L., and Magnusson, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 137-141). The amino-terminal portion of fibronectin is comprised of five type I modules and mediates assembly of dimeric soluble fibronectin into insoluble fibrils by cultured fibroblasts, binding and cross-linking of fibronectin to Staphylococcus aureus, and binding and cross-linking of fibronectin to fibrin. It is not known whether these binding activities require individual type I modules, several modules, or all five modules. To answer this question, we generated recombinant truncated fibronectin molecules with deletions of or mutations in the amino-terminal type I modules. Binding to cellular fibronectin assembly sites and S. aureus required all five type I modules. In contrast, proteins with deletions of type I modules interacted well with fibrin.

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.