The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.     

Heparin-mediated conformational changes in fibronectin expose vascular endothelial growth factor binding sites

Regulation of angiogenesis involves interactions between vascular endothelial growth factor (VEGF) and components of the extracellular matrix, including fibronectin and heparan sulfate. In the present study, we identified two classes of VEGF binding sites on fibronectin. One was constitutively available whereas the availability of the other was modulated by the conformational state of fibronectin. Atomic force microscopy studies revealed that heparin and hydrophilic substrates promoted the extended conformation of fibronectin, leading to increased VEGF binding. The ability of heparin to enhance VEGF binding to fibronectin was dependent on the chemical composition and chain length of heparin, since long (>22 saccharides) heparin chains with sulfation on the 6-O and N positions of glucosamine units were required for full activity. Treatment of the complex endothelial extracellular matrix with heparin also increased VEGF binding, suggesting that heparin/heparan sulfate might regulate VEGF interactions within the extracellular matrix by controlling the structure and organization of fibronectin matrices.     

Glycosaminoglycans differentially bind HARP and modulate its biological activity

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.     

Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix

The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane- intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin- resistant molecule that binds cells to the sequence RGD and a trypsin- labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.     

Potential role for heparan sulfate proteoglycans in regulation of transforming growth factor-beta (TGF-beta) by modulating assembly of latent TGF-beta-binding protein-1

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-beta in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-beta into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-beta into the ECM, with increased amounts of soluble latent TGF-beta. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by beta-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-beta availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.     

Heparin and heparan sulfate binding sites on B-16 melanoma cells

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.     

Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro

The involvement of embryonic cell surface proteoglycans in the attachment and outgrowth of cultured mouse embryos has been investigated. Several lines of evidence indicate that periimplantation stage blastocysts express heparin/heparan sulfate proteoglycans on their cell surfaces that can mediate embryo attachment and trophoblast outgrowth on a variety of matrices. First, in the presence of soluble heparin, the rate at which embryos attach and outgrow on laminin, fibronectin, or monolayers of uterine epithelial cells is reduced considerably. In the case of fibronectin, the rate of outgrowth in the presence of the heparin is slower than in the presence of the Arg-Gly-Asp-Ser-containing peptide that is recognized by a fibronectin receptor. Embryos also attach and exhibit a limited ability to outgrow on platelet factor IV, a heparin binding protein that does not possess the additional binding domains of laminin or fibronectin. Attachment on platelet factor IV is inhibited by heparin. Second, cell surface digestion of attachment-component embryos with heparinase, but not chondroitinase ABC, slows the rate of outgrowth on tissue culture plates in the presence of serum. Third, selective staining for sulfated molecules on the trophectoderm surface of periimplantation stage embryos indicates that such molecules are abundant and uniformly distributed on these cell surfaces. Last, heparin/heparan sulfate proteoglycans are detected as major cell surface components of embryos using vectorial labeling with lactoperoxidase and Na125I. Collectively, these data indicate that heparin/heparan sulfate-bearing molecules have a direct role in attachment and outgrowth of implantation stage blastocysts.     

Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa.

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.     

Identification and characterization of heparan sulfate-binding proteins from human lung carcinoma cells

The heparan sulfate proteoglycan/heparin-binding proteins of the human lung carcinoma cell line LX-1 have been identified, partially purified, and characterized. Analysis of the binding of [3H]heparin to membranes isolated from LX-1 cells indicated the presence of two classes of binding sites, with Kd values of approximately 2 x 10(-10) and 4 x 10(-8) M and corresponding Bmax values of 1 x 10(5) and 2 x 10(7) binding sites/cell. Binding was also observed with isolated heparan sulfate chains and with intact heparan sulfate proteoglycan isolated from two different cell types. With each ligand, binding was inhibited by addition of unlabeled heparin. The binding proteins were extracted from LX-1 cell membranes in detergent solution, and two size classes of binding proteins were identified by overlaying transblots of electrophoretically separated proteins with radioactive ligands. These two classes of binding proteins were shown to contain doublets with estimated molecular masses of approximately 16 kDa (HSBP1A and HSBP1B) and approximately 32 kDa (HSBP2A and HSBP2B). The proteins were partially purified by heparin-Sepharose chromatography and shown to bind heparin and heparan sulfate proteoglycan. By amino acid composition, N-terminal amino acid sequence, and reactivity with antibody, HSBP1A was shown to be very similar to histone 2B; HSBP1B may also be related to histone 2A. HSBP2A and HSBP2B, however, did not react with antibodies to the major histones and had compositions different from one another and from HSBP1.     

Potentiation and inhibition of bFGF binding by heparin: a model for regulation of cellular response

Basic fibroblast growth factor (bFGF) binds to cell surface tyrosine kinase receptor proteins and to heparan sulfate proteoglycans. The interaction of bFGF with heparan sulfate on the cell surface has been demonstrated to impact receptor binding and biological activity. bFGF receptor binding affinity is reduced on cells that do not express heparan sulfate. The addition of soluble heparin or heparan sulfate has been demonstrated to rescue the bFGF receptor binding affinity on heparan sulfate deficient cells yet has also been shown to inhibit binding under some conditions. While the chemical requirements of the heparin-bFGF-receptor interactions have been studied in detail, the possibility that heparin enhances bFGF binding in part by physically associating with the cell surface has not been fully evaluated. In the study presented here, we have investigated the possibility that heparin binding to the cell surface might play a role in modulating bFGF receptor binding and activity. Balb/c3T3 cells were treated with various concentrations of sodium chlorate, so as to express a range of endogenous heparan sulfate sites, and [(125)I]bFGF binding was assessed in the presence of a range of heparin concentrations. Low concentrations of heparin (0.1-30 nM) enhanced bFGF receptor binding to an extent that was inversely proportional to the amount of endogenous heparan sulfate sites present. At high concentrations (10 microM), heparin inhibited bFGF receptor binding in cells under all conditions. The ability of heparin to stimulate and inhibit bFGF-receptor binding correlated with altered bFGF-stimulated tyrosine kinase activity and cell proliferation. Under control and chlorate-treated conditions, [(125) I]heparin was observed to bind with a high affinity to a large number of binding sites on the cells (K(d) = 57 and 50 nM with 3.5 x 10(6) and 3.6 x 10(6) sites/cell for control and chlorate-treated cells, respectively). A mathematical model of this process revealed that the dual functions of heparin in bFGF binding were accurately represented by heparin cell binding-mediated stimulation and soluble heparin-mediated inhibition of bFGF receptor binding.     

Perlecan inhibits smooth muscle cell adhesion to fibronectin: role of heparan sulfate

Smooth muscle cell migration, proliferation, and deposition of extracellular matrix are key events in atherogenesis and restenosis development. To explore the mechanisms that regulate smooth muscle cell function, we have investigated whether perlecan, a basement membrane heparan sulfate proteoglycan, modulates interaction between smooth muscle cells and other matrix components. A combined substrate of fibronectin and perlecan showed a reduced adhesion of rat aortic smooth muscle cells by 70-90% in comparison to fibronectin alone. In contrast, perlecan did not interfere with cell adhesion to laminin. Heparinase treated perlecan lost 60% of its anti-adhesive effect. Furthermore, heparan sulfate as well as heparin reduced smooth muscle cell adhesion when combined with fibronectin whereas neither hyaluronan nor chondroitin sulfate had any anti-adhesive effects. Addition of heparin as a second coating to a preformed fibronectin matrix did not affect cell adhesion. Cell adhesion to the 105- and 120 kDa cell-binding fragments of fibronectin, lacking the main heparin-binding domains, was also inhibited by heparin. In addition, co-coating of fibronectin and (3)H-heparin showed that heparin was not even incorporated in the substrate. Morphologically, smooth muscle cells adhering to a substrate prepared by co-coating of fibronectin and perlecan or heparin were small, rounded, lacked focal contacts, and showed poorly developed stress fibers of actin. The results show that the heparan sulfate chains of perlecan lead to altered interactions between smooth muscle cells and fibronectin, possibly due to conformational changes in the fibronectin molecule. Such interactions may influence smooth muscle cell function in atherogenesis and vascular repair processes.     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

Complexing of fibronectin glycosaminoglycans and collagen

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

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.     

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.     

Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Initial interaction of herpes simplex virus with cells is binding to heparan sulfate.

We have shown that cell surface heparan sulfate serves as the initial receptor for both serotypes of herpes simplex virus (HSV). We found that virions could bind to heparin, a related glycosaminoglycan, and that heparin blocked virus adsorption. Agents known to bind to cell surface heparan sulfate blocked viral adsorption and infection. Enzymatic digestion of cell surface heparan sulfate but not of dermatan sulfate or chondroitin sulfate concomitantly reduced the binding of virus to the cells and rendered the cells resistant to infection. Although cell surface heparan sulfate was required for infection by HSV types 1 and 2, the two serotypes may bind to heparan sulfate with different affinities or may recognize different structural features of heparan sulfate. Consistent with their broad host ranges, the two HSV serotypes use as primary receptors ubiquitous cell surface components known to participate in interactions with the extracellular matrix and with other cell surfaces.     

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.