Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Heparanase induces endothelial cell migration via protein kinase B/Akt activation

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor.     

A peptide derived from tenascin-C induces beta1 integrin activation through syndecan-4

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor G?6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.     

A unique sequence of the laminin alpha 3 G domain binds to heparin and promotes cell adhesion through syndecan-2 and -4

Laminin-5, consisting of the alpha 3, beta 3, and gamma 2 chains, is localized in the skin basement membrane and supports the structural stability of the epidermo-dermal linkage and regulates various cellular functions. The alpha chains of laminins have been shown to have various biological activities. In this study, we identified a sequence of the alpha 3 chain C-terminal globular domain (LG1-LG5 modules) required for both heparin binding and cell adhesion using recombinant proteins and synthetic peptides. We found that the LG3 and LG4 modules have activity for heparin binding and that LG4 has activity for cell adhesion. Studies with synthetic peptides delineated the A3G75aR sequence (NSFMALYLSKGR, residues 1412--1423) within LG4 as a major site for both heparin and cell binding. Substitution mutations in LG4 and A3G75aR identified the Lys and Arg of the A3G75aR sequence as critical for these activities. Cell adhesion to LG4 and A3G75aR was inhibited by heparitinase I treatment of cells, suggesting that cell binding to the A3G75aR site was mediated by cell surface heparan sulfate proteoglycans. We showed by affinity chromatography that syndecan-2 from fibroblasts bound to LG4. Solid-phase assays confirmed that syndecan-2 interacted with the A3G75aR peptide sequence. Stably transfected 293T cells with expression vectors for syndecan-2 and -4, but not glypican-1, specifically adhered to LG4 and A3G75aR. These results indicate that the A3G75aR sequence within the laminin alpha 3 LG4 module is responsible for cell adhesion and suggest that syndecan-2 and -4 mediate this activity.     

PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate-matrix interactions

PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P < 0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P < 0.01) and heparan sulfate (HS) (P < 0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P < 0.05) and a reduction in cell speed (P < 0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Furthermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes.     

Heparan sulfate regulates targeting of syndecan-1 to a functional domain on the cell surface

In polarized B lymphoid cells, syndecan-1 is targeted specifically to a discrete membrane domain termed the uropod that is located at the cell's trailing edge. Within this functional domain, syndecan-1 promotes cell-cell adhesion and concentration of heparin binding growth factors. The present study reveals the surprising finding that targeting of syndecan-1 to uropods is mediated by its heparan sulfate chains and that targeting is regulated by cell surface events rather than solely by intracellular mechanisms. The addition of exogenous heparin or the treatment of polarized cells with heparitinase initiates a rapid and dramatic redistribution of uropod syndecan-1 over the entire cell surface, and a mutated syndecan-1 lacking heparan sulfate chains fails to concentrate within uropods. Interestingly, the heparan sulfate-bearing proteoglycans glypican-1 and beta glycan fail to concentrate in uropods, indicating that targeting may require heparan sulfate structural motifs unique to syndecan-1 or that the core protein of syndecan-1 participates in specific interactions that promote heparan sulfate-mediated targeting. These findings suggest functional specificity for syndecan-1 within uropods and, in addition, reveal a novel mechanism for the targeting of molecules to discrete membrane subcellular domains via heparan sulfate.     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties

Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain.     

Heparanase enhances syndecan-1 shedding: a novel mechanism for stimulation of tumor growth and metastasis

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.     

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated β1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.     

Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.     

A Liquid Chromatography-Mass Spectrometry-based Approach to Characterize the Substrate Specificity of Mammalian Heparanase

Extracellular heparanase activity releases growth factors and angiogenic factors from heparan sulfate (HS) storage sites and alters the integrity of the extracellular matrix. These activities lead to a loss of normal cell matrix adherent junctions and correlate with invasive cellular phenotypes. Elevated expression of heparanase is associated with several human cancers and with vascular remodeling. Heparanase cleaves only a limited fraction of glucuronidic linkages in HS. There have been few investigations of the functional consequences of heparanase activity, largely due to the heterogeneity and complexity of HS. Here, we report a liquid chromatography-mass spectrometry (LC-MS)-based approach to profile the terminal structures created by heparanase digestion and reconstruct the heparanase cleavage sites from the products. Using this method, we demonstrate that heparanase cleaves at the non-reducing side of highly sulfated HS domains, exposing cryptic growth factor binding sites. This cleavage pattern is observed in HS from several tissue sources, regardless of overall sulfation degree, indicating a common recognition pattern. We further demonstrate that heparanase cleavage of HS chains leads to increased ability to support FGF2-dependent cell proliferation. These results suggest a new mechanism to explain how heparanase might potentiate the uncontrolled cell proliferation associated with cancer through its ability to activate nascent growth factor-promoting domains within HS.     

Heparanase and syndecan-1 interplay orchestrates fibroblast growth factor-2-induced epithelial-mesenchymal transition in renal tubular cells

The epithelial-mesenchymal transition (EMT) of proximal tubular epithelial cells (PTECs) into myofibroblasts contributes to the establishment of fibrosis that leads to end stage renal disease. FGF-2 induces EMT in PTECs. Because the interaction between FGF-2 and its receptor is mediated by heparan sulfate (HS) and syndecans, we speculated that a deranged HS/syndecans regulation impairs FGF-2 activity. Heparanase is crucial for the correct turnover of HS/syndecans. The aim of the present study was to assess the role of heparanase on epithelial-mesenchymal transition induced by FGF-2 in renal tubular cells. In human kidney 2 (HK2) PTEC cultures, although FGF-2 induces EMT in the wild-type clone, it is ineffective in heparanase-silenced cells. The FGF-2 induced EMT is through a stable activation of PI3K/AKT which is only transient in heparanase-silenced cells. In PTECs, FGF-2 induces an autocrine loop which sustains its signal through multiple mechanisms (reduction in syndecan-1, increase in heparanase, and matrix metalloproteinase 9). Thus, heparanase is necessary for FGF-2 to produce EMT in PTECs and to sustain FGF-2 intracellular signaling. Heparanase contributes to a synergistic loop for handling syndecan-1, facilitating FGF-2 induced-EMT. In conclusion, heparanase plays a role in the tubular-interstitial compartment favoring the FGF-2-dependent EMT of tubular cells. Hence, heparanase is an interesting pharmacological target for the prevention of renal fibrosis.     

Heparanase plays a dual role in driving hepatocyte growth factor (HGF) signaling by enhancing HGF expression and activity

Hepatocyte growth factor (HGF) is a heparin-binding cytokine that enhances growth, motility, and angiogenesis of many tumor types, including multiple myeloma where it is often highly expressed. However, little is known regarding what controls HGF level and activity in these tumors. Evaluation of bone marrow biopsies from myeloma patients revealed a strong positive correlation between the levels of HGF and heparanase, an endoglucuronidase known to promote aggressive tumor behavior. In vitro, addition of recombinant heparanase to myeloma cells or transfection of myeloma cell lines with the cDNA for heparanase significantly increased tumor cell expression and secretion of biologically active HGF. Shed syndecan-1, whose levels in myeloma are also enhanced by heparanase expression, binds to secreted HGF. This syndecan-1-HGF complex is active as shown by its ability to stimulate paracrine signaling via c-Met, the cell surface receptor for HGF. Surprisingly, heparanase enzyme activity was not required for up-regulation of HGF expression by the tumor cells. This is in contrast to the heparanase-mediated enhanced syndecan-1 shedding, which does require activity of the enzyme. This suggests that two different functional domains within the heparanase enzyme (the enzyme active site and a separate site) contribute to events leading to enhanced HGF signaling. These findings demonstrate a novel mechanism driving the HGF pathway whereby heparanase stimulates an increase in both HGF expression and syndecan-1 shedding to enhance HGF signaling. This work also provides further mechanistic insight into the dynamic role of heparanase in driving aggressive tumor progression.     

Heparanase-enhanced Shedding of Syndecan-1 and Its Role in Driving Disease Pathogenesis and Progression

Both heparanase and syndecan-1 are known to be present and active in disease pathobiology. An important feature of syndecan-1 related to its role in pathologies is that it can be shed from the surface of cells as an intact ectodomain composed of the extracellular core protein and attached heparan sulfate and chondroitin sulfate chains. Shed syndecan-1 remains functional and impacts cell behavior both locally and distally from its cell of origin. Shedding of syndecan-1 is initiated by a variety of stimuli and accomplished predominantly by the action of matrix metalloproteinases. The accessibility of these proteases to the core protein of syndecan-1 is enhanced, and shedding facilitated, when the heparan sulfate chains of syndecan-1 have been shortened by the enzymatic activity of heparanase. Interestingly, heparanase also enhances shedding by upregulating the expression of matrix metalloproteinases. Recent studies have revealed that heparanase-induced syndecan-1 shedding contributes to the pathogenesis and progression of cancer and viral infection, as well as other septic and non-septic inflammatory states. This review discusses the heparanase/shed syndecan-1 axis in disease pathogenesis and progression, the potential of targeting this axis therapeutically, and the possibility that this axis is widespread and of influence in many diseases.     

Identification and characterization of a novel heparin‐binding peptide for promoting osteoblast adhesion and proliferation by screening an Escherichia coli cell surface display peptide library

Heparin/heparan sulfate (HS) plays a key role in cellular adhesion. In this study, we utilized a 12‐mer random Escherichia coli cell surface display library to identify the sequence, which binds to heparin. Isolated insert analysis revealed a novel heparin‐binding peptide sequence, VRRSKHGARKDR, designated as HBP12. Our analysis of the sequence alignment of heparin‐binding motifs known as the Cardin–Weintraub consensus (BBXB, where B is a basic residue) indicates that the HBP12 peptide sequence contains two consecutive heparin‐binding motifs (i.e. RRSK and RKDR). SPR‐based BIAcore technology demonstrated that the HBP12 peptide binds to heparin with high affinity (K_D = 191 nM ). The HBP12 peptide is found to bind the cell surface HS expressed by osteoblastic MC3T3 cells and promote HS‐dependent cell adhesion. Moreover, the surface‐immobilized HBP12 peptide on titanium substrates shows significant increases in the osteoblastic MC3T3‐E1 cell adhesion and proliferation. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and {alpha}5{beta}1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and β1 integrin co-signaling pathway. By using α5 null cells, β1 integrin functional blocking antibody, and a α5β1 integrin targeting peptide A5-1, we demonstrate that the α5 and β1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.     

Transglutaminase-2 interaction with heparin: identification of a heparin binding site that regulates cell adhesion to fibronectin-transglutaminase-2 matrix

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg(19) and Arg(28) also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.