Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion

To define regions within fibronectin (Fn) recognized by platelet binding sites, inhibition of Fn binding by an Fn fragment and synthetic peptides has been analyzed. A highly purified 120-kDa chymotryptic fragment, which has cell attachment activity but did not bind to insolubilized heparin or gelatin, inhibited Fn binding to platelets with an ID50 approximately 3 microM. Previous work indicates that fibroblasts attach to an 11.5-kDa subfragment of this 120-kDa fragment, and that one of four 30-residue synthetic peptides containing sequences from this region supports cell attachment. Only the peptide containing the COOH terminus of the 11.5-kDa fragment inhibited Fn binding to platelets, with an ID50 approximately 10 microM and is the peptide which supports fibroblast attachment. Of the smaller peptides studied from this sequence, all peptides containing the Arg-Gly-Asp-Ser sequence, including the tetrapeptide itself, were active in inhibiting Fn binding to platelets (ID50 values approximately 10-20 microM). The same peptides support fibroblast attachment. Those which lacked this sequence including Gly-Asp-Ser-Pro and Thr-Gly-Arg-Gly (immediately adjacent tetrapeptides) lacked both activities. Further evidence for specificity of inhibition was provided by structurally modified peptides in which substitution of a Glu for Asp abolished inhibitory activity and substitution of Lys for Arg or Ala for Gly reduced activity 6- and 8-fold, respectively. In addition, Arg-Gly-Asp-Ser-containing peptides inhibited the rate and extent of thrombin-induced platelet aggregation. These data suggest that the Arg-Gly-Asp-Ser tetrapeptide contains a recognition specificity involved in the binding of Fn to platelets and that platelets share features of this recognition specificity with fibroblasts.     

A fibronectin-related synthetic peptide, Pro-Ala-Ser-Ser, inhibits fibronectin binding to the cell surface, fibronectin-promoted cell migration in vitro, and cell migration in vivo

The biological activity of the amino acid sequence consisting of the immediate carboxyl terminus side of the Arg-Gly-Asp-Ser (RGDS) amino acid sequence in the cell-binding domain of intact fibronectin (FN) molecules was examined using synthetic peptides [RGDS, Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro (RGDSPASSKP), Pro-Ala (PA), Pro-Ala-Ser (PAS), Pro-Ala-Ser-Ser (PASS), and Pro-Ala-Ser-Ser-Lys (PASSK)]. These peptides were applied to the primary mesenchyme cells (PMCs) of the sea urchin, Clypeaster japonicus. In vitro immunohistochemistry indicated that the binding of exogenous FN to the PMC surface was inhibited by the peptides RGDSPASSKP and PASS, but not by RGDS, GRGDSP, PA, or PAS. PASS and RGDS introduced into the blastocoel also inhibited PMC migration in vivo. FN-promoted PMC migration in vitro was also inhibited by PASS and RGDS. The present results indicate that the PASS peptide inhibits FN binding to the PMC surface and promotes PMC migration, suggesting that the FN molecule uses the PASS amino acid sequence to bind to the PMC surface and to promote PMC migration in the blastocoel.     

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.     

Biologically active synthetic peptides as probes of embryonic development: a competitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos

We describe a new method for analyzing embryonic events dependent on a specific peptide recognition signal. A short, specific amino acid sequence in fibronectin has been implicated as a recognition site in fibronectin-mediated interactions. Fibroblast adhesion to fibronectin is competitively inhibited by certain synthetic peptides, including the decapeptide Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, which appears to contain the cell recognition sequence. We found that this peptide inhibited both amphibian gastrulation and avian neural crest cell migration in vivo, as well as the attachment and migration of neural crest cells in vitro. These processes are major cell migratory events previously suggested to involve fibronectin. Negative controls included another conserved fibronectin peptide from the collagen-binding region containing the sequence Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr and another peptide. Our results demonstrate the feasibility of using synthetic peptides directed at recognition sites in extracellular proteins as probes of morphogenetic processes, and they provide further support for the hypothesis that fibronectin is involved in gastrulation and neural crest cell migration.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Fluorescence-activated cell-sorting analysis of fibronectin peptides binding to Trypanosoma cruzi trypomastigotes

The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets

Fibrinogen binding to receptors on activated platelets is a prerequisite for platelet aggregation. However, the regions of fibrinogen interacting with these receptors have not been completely characterized. Fibronectin also binds to platelet fibrinogen receptors. Moreover, the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin, is located near the carboxyl-terminal region of the alpha-chain of fibrinogen. We have examined the ability of this tetrapeptide to inhibit platelet aggregation and fibrinogen binding to activated platelets. Arg-Gly-Asp-Ser, but not the peptide Arg-Gly-Tyr-Ser-Leu-Gly, inhibited platelet aggregation stimulated by ADP, collagen, and gamma-thrombin without inhibiting platelet shape change or secretion. At a concentration of 60-80 microM, Arg-Gly-Asp-Ser inhibited the aggregation of ADP-stimulated gel-filtered platelets approximately equal to 50%. Arg-Gly-Asp-Ser, but not Arg-Gly-Tyr-Ser-Leu-Gly, also inhibited fibrinogen binding to ADP-stimulated platelets. This inhibition was competitive with a Ki of approximately equal to 25 microM but was incomplete even at higher tetrapeptide concentrations, indicating that Arg-Gly-Asp-Ser is a partial competitive inhibitor of fibrinogen binding. These data suggest that a region near the carboxyl-terminus of the alpha-chain of fibrinogen interacts with the fibrinogen receptor on activated platelets. The data also support the concept that the sequence Arg-Gly-Asp-Ser has been conserved for use in a variety of cellular adhesive processes.     

Fluorescence-Activated Cell-Sorting Analysis of Fibronectin Peptides Binding to Trypanosoma cruzi Trypomastigotes1

ABSTRACT. The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells.     

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.     

Synthetic transit peptides inhibit import and processing of mitochondrial precursor proteins

We have demonstrated that a synthetic peptide corresponding to the rat mitochondrial malate dehydrogenase (mMDH) transit peptide (TP-28) inhibits the binding of pre-mMDH to isolated mitochondria. Synthetic peptides derived from chloroplast transit peptide sequences, which have a similar net charge, did not inhibit import. In addition, this peptide (TP-28) inhibits import of ornithine transcarbamylase, another mitochondrial matrix protein, thus suggesting that common import pathways exist for both mMDH and ornithine transcarbamylase. A smaller synthetic peptide corresponding to residues 1-20 of the mMDH transit peptide (TP-20) also inhibits binding. However, several substitutions for leucine-13 in the smaller peptide relieve import inhibition, thus providing evidence that this neutral residue plays a crucial role in transit peptide binding to the mitochondrial surface. Proteolytic processing of pre-mMDH by a mitochondrial matrix fraction to both the mature and intermediate forms of mMDH was also inhibited by TP-28. The ability of synthetic peptides to inhibit distinct steps in the import of mitochondrial precursor proteins corresponds precisely to their ability to interact with the same components used by transit peptides on intact precursors. Furthermore, inhibition at multiple points along the import pathway reflects the functions of several independent structures contained within transit peptides.     

Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: unique and shared features

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly-Arg-Gly-Asp-Ser was considerably more active than Arg-Gly-Asp-Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser-Asp-Gly-Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly-Arg-Gly-Asp-Ser was only weakly active, but the inverted peptide Ser-Asp-Gly-Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser-Asp-Gly-Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

Plasminogen Activator Inhibitor-1 Represses Integrin- and Vitronectin-Mediated Cell Migration Independently of Its Function as an Inhibitor of Plasminogen Activation

Cell migration involves the integrins, their extracellular matrix ligands, and pericellular proteolytic enzyme systems. We have studied the role of plasminogen activator inhibitor-1 (PAI-1) in cell migration, using human amnion WISH cells and human epidermoid carcinoma HEp-2 cells in an assay measuring migration from microcarrier beads and a modified Boyden-chamber assay. Active, but not latent or reactive center-cleaved, PAI-1 inhibited migration. A PAI-1 mutant without ability to inhibit plasminogen activation was as active as wild-type PAI-1 as a migration inhibitor, showing that inhibition of plasminogen activation was not involved. PAI-1 specifically interfered with integrin- and vitronectin-mediated migration: Migration onto vitronectin-coated but not onto fibronectin-coated surfaces was inhibited by PAI-1, a cyclic RGD peptide inhibited migration, and both cell lines expressed vitronectin-binding α_v-integrins. In addition, active PAI-1, but not latent or reactive center-cleaved PAI-1, inhibited vitronectin binding to integrins in anin vitrobinding assay, without affecting binding of fibronectin. Monoclonal antibodies against the urokinase receptor, another vitronectin binding protein, did not affect cell migration in the beads assay, while some inhibitory effect was observed in the Boyden-chamber assay. We conclude that PAI-1, independently of its role as a proteinase inhibitor, inhibits cell migration by competing for vitronectin binding to integrins, while the interference of PAI-1 with binding of vitronectin to the urokinase receptor may play a secondary role. These data define a novel function for the serpin PAI-1, enabling it to regulate cell migration over vitronectin-rich extracellular matrix in the body.     

Determination of the putative binding site for fibronectin on platelet glycoprotein IIb-IIIa complex through a hydropathic complementarity approach

We have applied the principle of complementary hydropathy to the prediction of the binding site for fibronectin (FN) and for the alpha-chain of fibrinogen in the platelet receptor complex glycoprotein (GP) IIb-IIIa. Since both ligands bind to it through their respective RGDS (Arg-Gly-Asp-Ser) domains and since both have been cloned, we were able to deduce the amino acid sequence of the binding site from the nucleotide sequence coding for RGDS in both proteins. The deduced peptides were very similar. Antibodies raised against a synthetic peptide WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) deduced from the cloned rat FN RGDS domain block ADP-mediated platelet aggregation; this block can be overcome by additional fibrinogen. In Western blots of whole cell platelet extracts run under reducing conditions, this antibody binds to a 108-kDa band. It also binds to affinity-purified GP IIIa. Furthermore, it reacts strongly with GP IIIa immunoprecipitated by a commercially available anti-GP IIb-IIIa monoclonal antibody. Binding of affinity-purified GP IIb-IIIa complex to fibronectin is inhibited by the 110-kDa FN fragment. Similar inhibitions can be effected by WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) and GAVSTA (Gly-Ala-Val-Ser-Thr-Ala) predicted from the rat and human fibronectin nucleotide sequences, respectively. GAGSTA (Gly-Ala-Gly-Ser-Thr-Ala) and GARSTA (Gly-Ala-Arg-Ser-Thr-Ala) related to the human peptide but with discrepant hydropathies are noninhibitory.     

The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation

The plasma membrane of murine erythro-leukemia (MEL) cells contains a 140-kD protein that binds specifically to fibronectin. A 125I-labeled 140-kD protein from surface-labeled uninduced MEL cells was specifically bound by an affinity matrix that contained the 115-kD cell binding fragment of fibronectin, and specifically eluted by a synthetic peptide that has cell attachment-promoting activity. The loss of this protein during erythroid differentiation was correlated with loss of cellular adhesion to fibronectin. Both MEL cells and reticulocytes attached to the same site on fibronectin as do fibroblasts since adhesion of erythroid cells to fibronectin was specifically blocked by a monoclonal antibody directed against the cell-binding fragment of fibronectin and by a synthetic peptide containing the Arg-Gly-Asp-Ser sequence found in the cell-binding fragment of fibronectin. Erythroid cells attached specifically to surfaces coated either with the 115-kD cell-binding fragment of fibronectin or with the synthetic peptide-albumin complex. Thus, the erythroid 140-kD protein exhibits several properties in common with those described for the fibronectin receptor of fibroblasts. We propose that loss or modification of this protein at the cell surface is responsible for the loss of cellular adhesion to fibronectin during erythroid differentiation.     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins

Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*- peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

Synthetic peptides competitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin

The 75,000-dalton cell-binding domain of fibronectin (f75k) and synthetic peptides derived from its sequence have been used to examine the cell-surface fibronectin receptor. The spreading of baby hamster kidney fibrolasts on f75k-coated substrates and the direct binding of [3H]f75k to these cells were both competitively inhibited by a synthetic peptide of fibronectin with the sequence Gly-Arg-Gly-Asp-Ser, indicating that the peptide and f75k interact with the same cell-surface sites. Related peptides, including the inverted sequence Ser-Asp-Gly-Arg, also competed for f75k binding. Our results provide the first evidence that fibroblastic baby hamster kidney cells recognize the same fibronectin amino acid sequence in direct binding of soluble ligand and in indirect inhibitory biological assays, thus correlating our direct fibronectin-receptor binding assay with biological functional assays for the fibronectin receptor in fibroblasts.