Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Heterophilic NCAM-Mediated Cell Adhesion to Proteoglycans from Chick Embryonic Brain Membranes

The vertebrate neural cell adhesion molecule NCAM mediates adhesion by both homophilic and heterophilic mechanisms, with heparan sulfate proteoglycans (HSPGs) being likely heterophilic ligands. In this study, transfected chicken NCAM polypeptides expressed on mouse L cells mediated the adhesion of these cells to several different heparan sulfate proteoglycans in nonionic detergent extracts of Embryonic Day 10 chicken brain membranes. In addition, adhesion inhibition experiments suggested a hitherto-undetected role for chondroitin sulfate proteoglycans in the stimulation of NCAM-mediated adhesion to some, but not all, of the HSPG ligands. Our experiments support the view that NCAM is a multivalent adhesive molecule whose function is affected by interactions with extracellular matrix and cell surface molecules.     

The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.     

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.     

Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

Modulation of the homophilic interaction between the first and second Ig modules of neural cell adhesion molecule by heparin

The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate-binding sites (HBS and CBS, respectively) in IgII coincide, and that this site overlaps with the homophilic binding site. Using NMR and surface plasmon resonance (SPR) analyses we demonstrate that interaction between IgII and heparin indeed interferes with the homophilic interaction between IgI and IgII. Accordingly, we show that treatment of cerebellar granule neurons (CGNs) with heparin inhibits NCAM-mediated outgrowth. In contrast, treatment with heparinase III or chondroitinase ABC abrogates NCAM-mediated neurite outgrowth in CGNs emphasizing the importance of the presence of heparan/chondroitin sulfates for proper NCAM function. Finally, a peptide encompassing HBS in IgII, termed the heparin-binding peptide (HBP), is shown to promote neurite outgrowth in CGNs. These observations indicate that neuronal differentiation induced by homophilic NCAM interaction is modulated by interactions with heparan/chondroitin sulfates.     

Molecular cloning of a dendritic cell-associated transmembrane protein, DC-HIL, that promotes RGD-dependent adhesion of endothelial cells through recognition of heparan sulfate proteoglycans

We isolated a novel molecule (DC-HIL) expressed abundantly by the XS52 dendritic cell (DC) line and epidermal Langerhans cells, but minimally by other cell lines. DC-HIL is a type I transmembrane protein that contains a heparin-binding motif and an integrin-recognition motif, RGD, in its extracellular domain (ECD). A soluble fusion protein (DC-HIL-Fc) of the ECD and an immunoglobulin Fc bound to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated polysaccharides (e.g. heparin and fucoidan) inhibited binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an integrin inhibitor (RGDS tetramer) had no effect on binding to SVEC, but prevented adhesion of SVEC. This differential RGD requirement was confirmed by the finding that DC-HIL-Fc mutant lacking the RGD motif can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore, DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing heparan sulfate proteoglycans on endothelial cells, thereby inducing adhesion of SVEC in an RGD-dependent manner. We propose that DC-HIL serves as a DC-associated, heparan sulfate proteoglycan-dependent integrin ligand, which may be involved in transendothelial migration of DC.     

Cathepsin X binds to cell surface heparan sulfate proteoglycans

Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.     

NCAM-Mediated Adhesion of Transfected Cells to Agrin

The vertebrate neural cell adhesion molecule NCAM mediates heterophilic adhesion to heparan sulfate proteoglycans in embryonic chick brain membranes. In this study, mouse L cells transfected with chicken NCAM were used to identify two of these ligands as agrin and the target of the 6C4 monoclonal antibody. A third heparan sulfate proteoglycan, perlecan, appeared not to support NCAM-mediated adhesion. Enzymatic degradation of chon-droitin sulfates decreased adhesion in agrin-containing membrane fractions but increased adhesion if the agrin had previously been removed by immunoprecipitation, suggesting that interactions between heparan sulfate and chondroitin sulfate proteoglycans have important influences on adhesion. Our experiments support the view that NCAM can interact with multiple, but not with all, heparan sulfate and chondroitin sulfate proteoglycans in chick brain membranes in both positive and negative ways to influence cell adhesion.     

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.     

Identification of heparin affin regulatory peptide domains with potential role on angiogenesis

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Heparan sulfate and chondroitin sulfate proteoglycans inhibit E-selectin binding to endothelial cells

E-selectin is a cell adhesion molecule involved in the initial rolling and adhesion of leukocytes to the endothelium during inflammation. In addition, in vitro studies have suggested that an interaction between E-selectin and binding sites such as sialyl Lewis X-containing oligosaccharides on endothelial cells may be important for angiogenesis. In order to investigate the binding of E-selectin to endothelial cells, we developed an ELISA assay using chimeric E-selectin-Ig molecules and endothelial cells fixed on poly-L-lysine coated plates. Our results indicate that E-selectin-Ig binds to both bovine capillary endothelial cells and human dermal microvascular endothelial cells in a calcium-dependent and saturable manner. The binding is inhibited markedly by heparin and by syndecan-1 ectodomain, and moderately by chondroitin sulfate, but not by sialyl Lewis X-containing oligosaccharides. These results suggest that heparan sulfate and chondroitin sulfate proteoglycans on endothelial cells are potential ligands for E-selectin.     

Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation

Heparin sulfate and the less sulfated glycosaminoglycan heparan sulfate enhance human plasminogen (Pg) conversion to plasmin by tissue-type plasminogen activator (t-PA). Kinetic studies indicate that both heparin and heparan increase the kcat of t-PA-mediated Pg activation by 25- and 3.5-fold, respectively. The Km of plasmin formation is unaltered by the presence of either heparin or heparan. Both heparin and heparan stimulate the activity of t-PA by interacting with the finger domain of t-PA, with association constants of 1 microM and 200 nM, respectively. Additionally, the lipoproteins lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) inhibit the heparin enhancement of Pg activation. Lp(a) is a competitive inhibitor and LDL is a mixed inhibitor of t-PA-mediated Pg activation, with inhibition constants of 30 and 70 nM, respectively. The inhibition constants correspond to physiologic concentrations of these lipoproteins. These data suggest that heparin, heparan, and lipoproteins may play an important in vivo role in regulating cell surface associated activation of the fibrinolytic system.     

Identification of cell-binding site of angiomodulin (AGM/TAF/Mac25) that interacts with heparan sulfates on cell surface.

Angiomodulin (AGM/TAF/mac25) is a 30-kDa glycoprotein that was identified as an integrin-independent cell adhesion protein secreted by human bladder carcinoma cells. AGM is highly accumulated in small blood vessels of tumor tissues. In the present study, we attempted to identify the cell surface receptor and the cell-binding site of AGM using ECV-304 human vascular endothelial cells and BALB/c3T3 mouse fibroblasts. Heparin, heparan sulfate, and dextran sulfate, but not chondroitin sulfate, inhibited both adhesion of the two cell lines to AGM-coated plates and binding of AGM to these cells. Treatment of cells with heparinase, but not chondroitinase, inhibited both cell adhesion to AGM and AGM binding to cells. These results strongly suggested that heparan sulfates are the major receptor for AGM. Furthermore, we determined a 20-amino acid sequence within AGM molecule as its major cell-binding site. The synthetic peptide for the cell-binding sequence showed cell adhesion activity comparable to that of AGM, and the activity was inhibited by heparin and heparan sulfate. The peptide competitively inhibited cell adhesion to AGM and the binding of AGM to cells. These results indicated that AGM binds to cells through interaction of the identified cell-binding sequence with heparan sulfates on cell surface. It was also found that the heparan sulfate-binding peptide inhibited the formation of capillary tube-like structures of vascular endothelial cells in culture.     

Regulation of platelet heparanase during inflammation: Role of pH and proteinases

Heparan sulfate is rapidly degraded by an endoglycosidase (heparanase) secreted by activated platelets. Since the cleavage and release of heparan sulfate would profoundly alter the local physiology of the endothelium, platelet heparanase activity should be tightly regulated. Consistent with this hypothesis, platelet heparanase was found to degrade endothelial cell heparan sulfate at pH 6.0 but not at pH 7.4, even though 25% of maximum activity was detected at pH 7.4. Loss of heparanase activity occurred rapidly (t_1/2 ≅ 20 min) and reversibly at physiologic pH but did not occur at acidic pH (<7.0). Inactivation of heparanase at pH 7.4 did not affect heparin binding and was reversed by 0.5 M NaCl or by heparan sulfate but not by chondroitin sulfate, suggesting inactive heparanase could be tethered on cell surfaces and the function regulated by heparan sulfate. Heparanase was gradually inactivated by trypsin and urokinase (t_1/2 = 5 h) but resisted cleavage by leukocyte cathepsin G, leukocyte elastase, plasmin, and thrombin. These findings are consistent with a model in which platelet heparanase is active at the low pH of inflammation but inactive under physiologic conditions preventing inadvertent cleavage of heparan sulfate and loss of physiologic functions of endothelial cells. J. Cell. Physiol. 175:255–267, 1998. © 1998 Wiley-Liss, Inc.     

Thrombin enhances degradation of heparan sulfate in the extracellular matrix by tumor cell heparanase.

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.     

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.     

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.     

Chondroitinases release acetylcholinesterase from chick skeletal muscle

Bacterial chondroitinases (both ABC and AC types) release asymmetric and globular forms of AChE from chick skeletal muscle samples. Heparinases, however, including heparitinase I, fail to do so under different incubation conditions. These results do not support the direct implication of the heparin/heparan sulfate family of GAGs in the interaction of the different AChE molecular forms with the muscle ECM. GAGs of the chondroitin/dermatan sulfate group could however be involved, either directly or indirectly, in the attachment of the AChE collagen-like tail to the muscle basal lamina.