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J Sottile

Publications and source records attributed to J Sottile.

At least 19 recordsLinked to original sources
PubMed2001

Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin.

Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.

Adhesins, Bacterial↗
PubMed2000

Stimulation of integrin-mediated cell contractility by fibronectin polymerization.

Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.

Amino Acid Sequence↗
PubMed2000

Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms.

Many aspects of cell behavior are regulated by cell-extracellular matrix interactions, including cell migration and cell growth. We previously showed that the addition of soluble fibronectin to collagen-adherent fibronectin-null cells enhances cell growth. This growth-promoting effect of fibronectin depended upon the deposition of fibronectin into the extracellular matrix; occupancy and clustering of fibronectin-binding integrins was not sufficient to trigger enhanced cell growth. To determine whether the binding of integrins to fibronectin's RGD site is required for fibronectin-enhanced cell growth, the ability of fibronectin lacking the integrin-binding RGD site (FN(Delta)RGD) to promote cell growth was tested. FN(Delta)RGD promoted cell growth when used as an adhesive substrate or when added in solution to collagen-adherent fibronectin-null cells. Addition of FN(Delta)RGD to collagen-adherent fibronectin-null cells resulted in a 1.6-1.8x increase in cell growth in comparison with cells grown in the absence of fibronectin. The growth-promoting effects of FN(Delta)RGD and wild-type fibronectin were blocked by inhibitors of fibronectin polymerization, including the anti-fibronectin antibody, L8. In addition, FN(Delta)RGD-induced cell growth was completely inhibited by the addition of heparin, and was partially blocked by either heparitinase-treatment or by addition of recombinant fibronectin heparin-binding domain. Heparin and heparitinase-treatment also partially blocked the growth-promoting effects of wild-type fibronectin, as well as the deposition of wild-type fibronectin into the extracellular matrix. These data suggest that cell surface heparan-sulfate proteoglycans contribute to the growth-promoting effects of FN(Delta)RGD and wild-type fibronectin. Addition of heparin, treatment with heparitinase, or incubation with monoclonal antibody L8 all inhibited the formation of short linear FN(Delta)RGD fibrils on the cell surface. Inhibitory (beta)1 integrin antibodies had no effect on FN(Delta)RGD fibril formation, FN(Delta)RGD-induced cell growth, or cell adhesion on FN(Delta)RGD-coated substrates. These data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans.

Animals↗
PubMed1999

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin.

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin's heparin-binding domain inhibited both beta(1) and non-beta(1) integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins alpha-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.

Animals↗
PubMed1999

Identification of protein-disulfide isomerase activity in fibronectin.

Assembly and degradation of fibronectin-containing extracellular matrices are dynamic processes that are up-regulated during wound healing, embryogenesis, and metastasis. Although several of the early steps leading to fibronectin deposition have been identified, the mechanisms leading to the accumulation of fibronectin in disulfide-stabilized multimers are largely unknown. Disulfide-stabilized fibronectin multimers are thought to arise through intra- or intermolecular disulfide exchange. Several proteins involved in disulfide exchange reactions contain the sequence Cys-X-X-Cys in their active sites, including thioredoxin and protein-disulfide isomerase. The twelfth type I module of fibronectin (I12) contains a Cys-X-X-Cys motif, suggesting that fibronectin may have the intrinsic ability to catalyze disulfide bond rearrangement. Using an established protein refolding assay, we demonstrate here that fibronectin has protein-disulfide isomerase activity and that this activity is localized to the carboxyl-terminal type I module I12. I12 was as active on an equal molar basis as intact fibronectin, indicating that most of the protein-disulfide isomerase activity of fibronectin is localized to I12. Moreover, the protein-disulfide isomerase activity of fibronectin appears to be partially cryptic since limited proteolysis of I10-I12 increased its isomerase activity and dramatically enhanced the rate of RNase refolding. This is the first demonstration that fibronectin contains protein-disulfide isomerase activity and suggests that cross-linking of fibronectin in the extracellular matrix may be catalyzed by a disulfide isomerase activity contained within the fibronectin molecule.

Base Sequence↗
PubMed1998

Activation of distinct alpha5beta1-mediated signaling pathways by fibronectin's cell adhesion and matrix assembly domains.

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the alpha5beta1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed "matrix assembly sites," which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the alpha5beta1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the alpha5beta1 integrin, suggesting that these two domains of fibronectin cannot bind to the alpha5beta1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the alpha5beta1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

Amino Acid Sequence↗
PubMed1998

Opposing effects of low and high molecular weight kininogens on cell adhesion.

High molecular weight kininogen (HK) blocks cell spreading but not cell attachment to surfaces coated with vitronectin and other ligands of beta3 integrins. We sought to learn the structural basis of this phenomenon. Monoclonal antibodies against the histidine-rich D5 domain in the light chain of 2-chain HK abolished the inhibitory effect of 2-chain HK on spreading of MG-63 osteosarcoma cells on vitronectin-coated tissue-culture plastic. The antibodies were effective only if incubated with 2-chain HK in solution and did not abolish the anti-cell-spreading effect of 2-chain HK that was pre-adsorbed to tissue-culture plastic. Exposure of an epitope in the histidine-rich domain was less when HK was adsorbed to tissue-culture plastic (oxidized polystyrene) than when it was adsorbed to ELISA plastic (untreated polystyrene). Loss of the epitope correlated with increased anti-cell-spreading activity of HK on tissue-culture plastic. The light chain of 2-chain HK containing D5 and that containing recombinant D5 both had anti-cell-spreading activity, but only when present in solution during adhesion assays. Pre-adsorption of recombinant D5 to tissue-culture plastic resulted in a surface on which adsorbed 2-chain HK had little anti-cell-spreading activity. Binding study revealed that HKa bound to immobilized vitronectin. The histidine-rich D5 domain of light chain of HK was identified as one of the binding sites of vitronectin, suggesting that the masking of the RGD cell-binding site of immobilized vitronectin is the molecular mechanism of anti-cell-spreading effect of HKa. In contrast, low molecular weight kininogen (LK), which lacks D5, augmented cell spreading on vitronectin-coated tissue-culture plastic. Thus, HK and LK have opposing effects on VN-dependent cell adhesion. The augmenting effect of LK was greater if LK was preincubated with cells or adsorbed to the surface at pH>7.0. Analysis of fragments of LK and antibody inhibition studies localized the cell-adhesion activity to the D3 domain that is common to LK and HK. These findings indicate that the D5 domain mediates the adsorption of HK or 2-chain HK to vitronectin substratum in anti-adhesive conformations, i.e., masking of the RGD cell-binding site of vitronectin. Such conformers inhibit cell spreading on vitronectin even though a cell-adhesion site is present in D3.

Antibodies, Monoclonal↗
PubMed1998

Fibronectin matrix assembly enhances adhesion-dependent cell growth.

Cell growth control in non-transformed cells depends, in part, on adhesive interactions with the extracellular matrix. Following injury, excess or altered fibronectin deposition into the extracellular matrix may contribute to the pathogenesis of fibrosis and atherosclerosis by triggering changes in specific cell functions associated with wound repair, including cell proliferation and migration. To assess the role of fibronectin polymerization on cell growth, we isolated mouse embryonic cells that lack endogenous fibronectin (fibronectin-null cells) and established them in culture under serum-free conditions. These fibronectin-null cells do not produce any detectable fibronectin, but are capable of assembling a fibronectin matrix when cultured in the presence of exogenously added fibronectin. Our data indicate that adhesion-dependent growth in fibronectin-null cells is dramatically increased (>2-5x) by culturing cells in the presence of fibronectin. This fibronectin-induced cell growth was blocked by inhibiting fibronectin matrix assembly. Arg-Gly-Asp peptides or fragments of fibronectin that contain the Arg-Gly-Asp cell binding site promoted clustering of the (&agr ;)5beta1 integrin in focal adhesions, but did not enhance cell growth. These data indicate that the polymerization of fibronectin into the extracellular matrix positively regulates cell growth, and that occupancy and clustering of fibronectin-binding integrins alone are not sufficient to trigger increased cell growth.

Animals↗
PubMed1997

N-terminal type I modules required for fibronectin binding to fibroblasts and to fibronectin's III1 module.

Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fiftBiomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706U.S.A. Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conh as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

Animals↗
PubMed1996

Binding and degradation of thrombospondin-1 mediated through heparan sulphate proteoglycans and low-density-lipoprotein receptor-related protein: localization of the functional activity to the trimeric N-terminal heparin-binding region of thrombospondin-1.

Thrombospondin-1 (TSP-1) is a multimodular trimeric protein involved in cell adhesion, motility and growth. TSP-1 binds to cells and is internalized and degraded in a process that requires the presence of heparan sulphate proteoglycan; the process is inhibited by heparin or receptor-associated protein (RAP), an antagonist of the low-density-lipoprotein receptor (LDLR) family. We characterized the attributes of TSP-1 that mediate the process. TSP277, which is truncated at Gln-277 of TSP-1 and contains the heparin-binding domain and the heptad repeat region that mediates trimerization, bound to and was degraded by a variety of cells with kinetics similar to those of the binding and degradation of intact TSP-1. Degradation of TSP277 was inhibited by heparin or RAP with dose responses similar to those for inhibition of degradation of TSP-1. Binding and degradation of TSP277 were decreased in Chinese hamster ovary cells lacking heparan sulphate. These results indicate that the N-terminal heparin-binding domain in a trivalent configuration is sufficient to mediate binding and degradation of TSP-1 via the proteoglycan-LDLR family pathway.

Animals↗
PubMed1996

Substratum-dependent stimulation of fibroblast migration by the gelatin-binding domain of fibronectin.

Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibroblast migration in the transmembrane (or 'Boyden chamber') assay; in contrast, the gelatin-binding domain (GBD) of fibronectin has consistently been reported to be devoid of migration-stimulating activity in this assay. We have examined the effects of fibronectin and several of its purified functional domains on the migration of human skin fibroblasts in what is presumably a more physiologically relevant assay involving the movement of cells into a 3-D matrix of native type I collagen fibrils. We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin, as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femtomolar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 microgram/ml). The correct assignment of migration-stimulating activity to GBD, rather than to a contaminant, was confirmed by: (a) the use of several fibronectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed against epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of native fibronectin; and (d) obtaining an identical dose-response curve with a genetically engineered GBD peptide. The cryptic migration-stimulating activity of GBD was not affected by the presence of serum or native fibronectin, but was inhibited by TGF-beta 1. Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migration-stimulating activity in this assay when membranes coated with gelatin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen. Cells preincubated with GBD for 24 hours whilst growing on plastic tissue culture dishes and then plated onto native collagen matrices in the absence of further GBD also displayed an elevated migration compared to controls. Taken together, these observations suggest that: (a) the interaction of GBD with a putative cell surface receptor (and not the collagen substratum) initiates a persistent alteration in cell phenotype which is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a hitherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.

Cell Movement↗
PubMed1994

Properties of recombinant mouse thrombospondin 2 expressed in Spodoptera cells.

A baculovirus system was used to express full-length recombinant mouse thrombospondin 2 (rTSP2) as a disulfide-bonded homotrimer with an NH2 terminus beginning with Asp20.rTSP2, like TSP1, was more sensitive to trypsin digestion if depleted of calcium ion. The trypsin digestion pattern of rTSP2 and TSP1 differed in that trypsin cut between the first and second type 1 modules of rTSP2. For bovine aortic endothelial cells adhering to TSP-coated polystyrene plates, reduction after coating caused both TSPs to be much more adhesive; these adhesions were blocked completely by RGDS peptide or antibody to alpha v beta 3 integrin.rTSP2 and TSP1 also mediated the adhesion of HT-29 human colon adenocarcinoma cells that carry alpha v beta 5 but not alpha v beta 3 integrin. Antibody to alpha v beta 5 did not inhibit adhesion of HT-29 cells to TSP1 or rTSP2. Rather, adhesion of HT-29 cells was decreased by treatment of TSPs with EDTA, abolished by reduction of the TSPs, and, in the case of rTSP2, blocked by heparin. Adhesion of MG63 cells to both TSPs was complex. Treatment with EDTA enhanced the adhesive activity of rTSP2 but decreased the adhesive activity of TSP1. These results show that TSP2 can be processed and secreted when overexpressed using baculovirus, TSP1 and rTSP2 differ in protease susceptibility in the type 1 module region, and TSP1 and rTSP2 mediate cell adhesion by complex and similar but not identical mechanisms.

Amino Acid Sequence↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Assembly of amino-terminal fibronectin dimers into the extracellular matrix.

Fibronectin is a dimeric adhesion molecule that consists of three types of repeating modules. Adherent cells bind soluble fibronectin and incorporate it into insoluble fibrils in the extracellular matrix. The amino-terminal 70-kDa portion of fibronectin mediates binding to the cell surface, but amino-terminal fragments do not accumulate in the extracellular matrix. The ninth type I and first type III modules, the cell adhesion region, and the cysteines that form the interchain disulfide bonds have also been implicated in matrix assembly. To further define which regions of fibronectin are essential for matrix assembly, we generated a dimeric protein (d70 kDa) in which the 70-kDa amino terminus is directly linked to the last 51 amino acids of fibronectin, which contain the cysteines involved in interchain disulfide bonding. d70 kDa bound to cells and accumulated in the extracellular matrix. Incorporation of d70 kDa into the extracellular matrix was dependent upon protein synthesis; in cycloheximide-treated cultures that lacked a pre-existing matrix, d70 kDa accumulated in the extracellular matrix only in the presence of intact fibronectin. Monomeric 70-kDa protein was not incorporated into the matrix in the presence or absence of cycloheximide. These data indicate that fibronectin molecules containing only the amino-terminal 70-kDa region and the carboxyl-terminal 51 amino acids can become assembled into the extracellular matrix.

Animals↗
PubMed1994

Baculovirus-mediated expression of the epidermal growth factor-like modules of human factor IX fused to the factor XIIIa transamidation site in fibronectin. Evidence for a direct interaction between the NH2-terminal epidermal growth factor-like module of factor IXa beta and factor X.

Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.

Amino Acid Sequence↗
PubMed1993

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)

Amino Acid Sequence↗
PubMed1992

Assembly of extracellular matrix.

A great challenge in understanding how different extracellular matrices assemble is to sort through the vast number of possible interactions between and among matrix molecules. The most profound insights are likely to come from patients with defined defects of matrix molecules and the use of transgenic mice or other experimental technologies that mimic the complexity of the human system.

Amino Acid Sequence↗