Regulation of cell motility, morphology, and growth by sulfated glycosaminoglycans

Due to the recent observation that heparin binds to several growth factors and cell adhesion molecules, the effect of heparin on biological processes governed by growth factors and cell adhesion molecules was investigated. Pharmacological doses of heparin were found to alter cell growth rate, cellular morphology, and cell motility. Concentrations (microgram/ml) of heparin or dextran sulfate decreased cell growth rate, but not the final cell density attained in plateau phase. The effect of heparin on cell growth rate was most pronounced when cells were cultured in low concentrations of serum. A heparin-induced decrease in cell growth rate could be reversed by addition of platelet-derived growth factor (PDGF), a heparin-binding growth factor. Heparin altered the morphology of all cell lines studied to various degrees. The effect of heparin on cell morphology was quantitated by measuring the heparin-induced change in cell surface area. HT-1080 and HeLa cells nearly doubled in surface area upon exposure to 10 micrograms/ml heparin. Since several heparin-binding cell adhesion proteins mediate both cell spreading and cell migration, the influence of heparin on cell migration was investigated with an improved version of the phagokinetic track technique. Low concentrations of heparin and dextran sulfate were found to increase the rate of cell migration in a dose-dependent fashion. Since the quantitative effect of heparin on cell growth rate, morphology, and migration depends on the cell line studied, it is suggested that three separate phenomena may be involved. The results presented indicate a central role for sulfated glycosaminoglycans in the control of both cell growth and cell-cell interactions.     

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.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.     

A novel synthetic peptide from the B1 chain of laminin with heparin- binding and cell adhesion-promoting activities

Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]heparin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.     

Heparin-functionalized thermoresponsive surface

Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.     

Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor''s intracellular SH2-binding motif.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell-cell adhesion. PECAM-1 has been shown to mediate cell-cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.     

Modulation of endothelial cell proliferation,adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin

Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence -Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependet and reversible. Thus, the antiproliferative activities of the thrombospondin peptides and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.     

Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells

Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN(347-361) (the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (tau = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (tau = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN(347-361), demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions.     

The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans

The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. In the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them, heparin presented the highest inhibitory activity (K_i = 2.5 ± 0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. In permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. In addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking.     

Design of peptides with high affinities for heparin and endothelial cell proteoglycans

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.     

FGFRL1 affects chemoresistance of small‐cell lung cancer by modulating the PI3K/Akt pathway via ENO1

Fibroblast growth factor receptor‐like 1 (FGFRL1), a member of the FGFR family, has been demonstrated to play important roles in various cancers. However, the role of FGFRL1 in small‐cell lung cancer (SCLC) remains unclear. Our study aimed to investigate the role of FGFRL1 in chemoresistance of SCLC and elucidate the possible molecular mechanism. We found that FGFRL1 levels are significantly up‐regulated in multidrug‐resistant SCLC cells (H69AR and H446DDP) compared with the sensitive parental cells (H69 and H446). In addition, clinical samples showed that FGFRL1 was overexpressed in SCLC tissues, and high FGFRL1 expression was associated with the clinical stage, chemotherapy response and survival time of SCLC patients. Knockdown of FGFRL1 in chemoresistant SCLC cells increased chemosensitivity by increasing cell apoptosis and cell cycle arrest, whereas overexpression of FGFRL1 in chemosensitive SCLC cells produced the opposite results. Mechanistic investigations showed that FGFRL1 interacts with ENO1, and FGFRL1 was found to regulate the expression of ENO1 and its downstream signalling pathway (the PI3K/Akt pathway) in SCLC cells. In brief, our study demonstrated that FGFRL1 modulates chemoresistance of SCLC by regulating the ENO1‐PI3K/Akt pathway. FGFRL1 may be a predictor and a potential therapeutic target for chemoresistance in SCLC.     

Effect of extension of the heparin binding pocket on the structure,stability, and cell proliferation activity of the human acidic fibroblast growth factor

Acidic human fibroblast growth factor (hFGF1) plays a key role in cell growth and proliferation. Activation of the cell surface FGF receptor is believed to involve the glycosaminoglycan, heparin. However, the exact role of heparin is a subject of considerable debate. In this context, in this study, the correlation between heparin binding affinity and cell proliferation activity of hFGF1 is examined by extending the heparin binding pocket through selective engineering via charge reversal mutations (D82R, D84R and D82R/D84R). Results of biophysical experiments such as intrinsic tryptophan fluorescence and far UV circular dichroism spectroscopy suggest that the gross native structure of hFGF1 is not significantly perturbed by the engineered mutations. However, results of limited trypsin digestion and ANS binding experiments show that the backbone structure of the D82R variant is more flexible than that of the wild type hFGF1. Results of the temperature and urea-induced equilibrium unfolding experiments suggest that the stability of the charge-reversal mutations increases in the presence of heparin. Isothermal titration calorimetry (ITC) data reveal that the heparin binding affinity is significantly increased when the charge on D82 is reversed but not when the negative charge is reversed at both positions D82 and D84 (D82R/D84R). However, despite the increased affinity of D82R for heparin, the cell proliferation activity of the D82R variant is observed to be reduced compared to the wild type hFGF1. The results of this study clearly demonstrate that heparin binding affinity of hFGF1 is not strongly correlated to its cell proliferation activity.     

Interactions of a laminin-binding peptide from a 33-kDa protein related to the 67-kDa laminin receptor with laminin and melanoma cells are heparin-dependent.

A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.     

The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains

Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH³⁵⁹, now shown to bind to heparin with a K_D of 13 μm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their β₁ integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X

Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.     

Glycosaminoglycan binding properties of the myxoma virus CC-chemokine inhibitor, M-T1

Poxviruses encode a number of secreted virulence factors that function to mitigate or modulate the host immune response. M-T1 is a secreted 43-kDa glycoprotein produced by the myxoma virus, a poxvirus pathogen of rabbits, that binds CC-chemokines with high affinity, blocks binding to their cognate G-protein coupled receptors, and thereby inhibits chemokine-induced leukocyte chemotaxis. The present study indicates that M-T1, but not the related vaccinia virus 35-kDa CC-chemokine-binding protein, can localize to cell surfaces through an interaction with glycosaminoglycan molecules. In addition to biochemically characterizing the nature of this interaction, we demonstrate that M-T1 can also simultaneously interact with CC-chemokines while bound to heparin, suggesting that the binding sites on M-T1 for chemokines and heparin are distinct. Furthermore, using recombinant baculovirus-expressed M-T1 truncation and internal deletion mutants, we localize the heparin-binding region of M-T1 to the C terminus of the protein, a region that contains a high abundance of basic residues and includes two clusters of basic amino acid residues that resemble Cardin and Weintraub heparin-binding consensus sequences. The ability of M-T1 to simultaneously interact with chemokines and glycosaminoglycans may enable M-T1 to tether to endothelial surfaces or extracellular matrix and capture host chemokines that are expressed close to sites of virus infection.     

Heparin localization and fine structure regulate Burkitt's lymphoma growth

Burkitt's lymphoma (BL) is a B-cell malignancy associated with the Epstein-Barr virus (EBV). Mounting evidence has implicated heparan sulfate proteoglycans and heparan sulfate-like glycosaminoglycans (HSGAGs) in the initiation, severity, and progression of the malignancy. The importance of HSGAGs in regulating BL cell growth was therefore examined. Extracellular exogenous heparin inhibited cell growth >30%, while heparin internalized with poly(beta-amino ester)s promoted proliferation up to 58%. The growth-modulating effects of heparin and internalized heparin were dependent on cell surface HSGAGs, PI3K, and Erk/Mek. Treatment of cells with protamine sulfate or with heparinases potently inhibited proliferation, with the greatest effects induced by heparinase I. Cell surface HSGAGs therefore play an important role in regulating BL proliferation and may offer a potential target for therapeutic intervention.     

Neuronal cell adhesion, mediated by the heparin-binding neuroregulatory factor midkine, is specifically inhibited by chondroitin sulfate E. Structural ans functional implications of the over-sulfated chondroitin sulfate

The heparin-binding neurotrophic factor midkine (MK) has been proposed to mediate neuronal cell adhesion and neurite outgrowth promotion by interacting with cell-surface heparan sulfate. We have observed that over-sulfated chondroitin sulfate (CS) D and CS-E show neurite outgrowth-promoting activity in embryonic day (E) 18 rat hippocampal neurons (Nadanaka, S., Clement, A., Masayama, K., Faissner, A., and Sugahara, K. (1998) J. Biol. Chem. 273, 3296-3307). In the present study, various CS isoforms were examined for their ability to inhibit the MK-mediated cell adhesion of cortical neuronal cells in comparison with heparin from porcine intestine and heparan sulfate from bovine kidney. E17-18 rat cortical neuronal cells were cultured on plates coated with recombinant MK in a grid pattern. The cells attached to and extended their neurites along the MK substratum. Cell adhesion was inhibited by squid cartilage over-sulfated CS-E as well as by heparin, but not by heparan sulfate or other CS isoforms. Direct interactions of MK with various glycosaminoglycans were then evaluated using surface plasmon resonance, showing that CS-E bound MK as strongly as heparin, followed by other over-sulfated CS isoforms, CS-H and CS-K. Furthermore, E18 rat brain extracts showed an E disaccharide unit, GlcUAbeta1-3GalNAc(4,6-O-disulfate). These findings indicate that CS chains containing the E unit as well as heparin-like glycosaminoglycans may be involved in the expression and/or modulation of the multiple neuroregulatory functions of MK such as neuronal adhesion and migration and promotion of neurite outgrowth.