A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation.

A heparin-binding protein was isolated from bovine uteri and purified to homogeneity. This protein appears as a double band of approx. 78 kDa in SDS/polyacrylamide-gel electrophoresis and has an isoelectric point of 5.2. The binding of heparin to this protein is saturable. No other glycosaminoglycan from mammalian tissue, such as hyaluronic acid, chondroitin sulphate, dermatan sulphate or keratan sulphate, binds to the 78 kDa protein. Dextran sulphate binds in a non-saturable fashion. Certain heparan sulphate polysaccharide structures are required for binding to the 78 kDa protein. Some proteoheparan sulphates, such as endothelial cell-surface proteoheparan sulphate, show only weak interaction with the 78 kDa protein in contrast with a basement-membrane proteoheparan sulphate from HR-9 cells. Antibodies against the 78 kDa protein inhibit binding of proteoheparan [35S]sulphate from basement membranes to smooth-muscle cells. Conventional antibodies, Fab fragments and some monoclonal antibodies, inhibit smooth-muscle cell proliferation in a similar range as that observed for heparin. The protein was detected in a variety of tissues and cells but not in blood cells. A possible role of this protein as a receptor for heparin or heparan sulphate and its function in the control of the arterial wall structure are discussed.     

Release of diamine oxidase into plasma by glycosaminoglycans in rats

Plasma diamine oxidase (DAO) values are enhanced by intravenous injection of heparin which releases the enzyme, synthesized in small bowel enterocytes, from binding sites located on endothelial cells of the intestinal microvasculature. Intestinal DAO, in analogy with lipoprotein lipase (another heparin-released enzyme), is believed to be electrostatically linked to endothelial binding sites composed of a glycosaminoglycan (GAG) which is presumably heparan sulphate, but the complete mechanism of enzyme release is not known. In this study we assayed in rats the DAO-releasing capability of heparan sulphate, dermatan sulphate, chondroitin sulphate A and hyaluronic acid, all heparin related compounds. Heparan sulphate, a compound with the same hexosamine as heparin but with a lower concentration of sulphated iduronic acid, induced a very high release of DAO (3-fold less than heparin), while the other tested GAGs, composed of higher proportions of non sulphated uronic acid and with galactosamine instead of glucosamine, induced a significantly lower release. In rats treated with 60 mg heparan sulphate the significant decrease in ileal mucosal DAO activity indicates that, in analogy with heparin, the high plasma enzymatic activity induced is of enterocytic origin. It is suggested that the high charge density of the compounds tested, due to the degree of sulphatation, is the decisive factor in promoting the release of intestinal DAO.     

Heparin interferes with translocation of Yop proteins into HeLa cells and binds to LcrG, a regulatory component of the Yersinia Yop apparatus

Yersiniae are equipped with the Yop virulon, an apparatus that allows extracellular bacteria to deliver toxic Yop proteins inside the host cell cytosol in order to sabotage the communication networks of the host cell or even to cause cell death. LcrG is a component of the Yop virulon involved in the regulation of secretion of the Yops. In this paper, we show that LcrG can bind HeLa cells, and we analyse the role of proteoglycans in this phenomenon. Treatment of the HeLa cells with heparinase I, but not chondroitinase ABC, led to inhibition of binding. Competition assays indicated that heparin and dextran sulphate strongly inhibited binding, but that other glycosaminoglycans did not. This demonstrated that binding of HeLa cells to purified LcrG is caused by heparan sulphate proteoglycans. LcrG could bind directly to heparin-agarose beads and, in agreement with these results, analysis of the protein sequence of Yersinia enterocolitica LcrG revealed heparin-binding motifs. In vitro production and secretion by Y . enterocolitica of the Yops was unaffected by the addition of heparin. However, the addition of exogenous heparin decreased the level of YopE–Cya translocation into HeLa cells. A similar decrease was seen with dextran sulphate, whereas the other glycosaminoglycans tested had no significant effect. Translocation was also decreased by treatment of HeLa cells with heparinitase, but not with chondroitinase. Thus, heparan sulphate proteoglycans have an important role to play in translocation. The interaction between LcrG and heparan sulphate anchored at the surface of HeLa cells could be a signal triggering deployment of the Yop translocation machinery. This is the first report of a eukaryotic receptor interacting with the type III secretion and associated translocation machinery of Yersinia or of other bacteria.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

The catabolism of intravenously injected heparan N-[35S]sulphate in the rat

The metabolic fate of heparan N-[(35)S]sulphate was studied in rats. Heparan sulphate was obtained from either bovine aorta or lung and labelled with (35)S by desulphation and subsequent resulphation in vitro. Experiments in which heparan N-[(35)S]sulphate was administered intravenously to either free-range or wholly anaesthetized rats with ureter cannulae established that substantial desulphation occurs in vivo, with elimination of inorganic [(35)S]sulphate in urine. Oligosaccharides labelled with (35)S, possible intermediates in heparan sulphate degradation, could not be detected in urine or blood. The general distribution of radioactivity after administration of heparan N-[(35)S]sulphate, as demonstrated by whole-body radioautography, suggested that desulphation was not restricted to one organ in particular. Support for this view was obtained in experiments in which heparan N-[(35)S]sulphate was administered to animals after the removal of kidneys, liver, spleen, pancreas or gastrointestinal tract. In all cases inorganic [(35)S]sulphate was still produced. The ability of rats of desulphate heparan N-[(35)S]sulphate was progressively impaired by increasing concentrations of heparin administered simultaneously. It was concluded that heparan sulphate is metabolized at a number of sites in the body by a sequence of degradative events leading to the formation of inorganic sulphate. It is also concluded that at least some of these events are common to heparan sulphate and heparin.     

Structure and metabolism of rat liver heparan sulphate

Rat liver cells grown in primary cultures in the presence of [³⁵S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO₂ treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In ³H-labelled heparan sulphate, isolated after incubation of the cells with [³H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [³⁵S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [³⁵S]sulphate by rat liver cells incubated in the presence of [³⁵S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.     

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

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

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with (3)H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.     

Binding of heparan sulphate and heparin to control and virus-transformed cells

A cloned embryonic mouse cell line contained specific cell-surface receptors for heparin and both the number and affinity appeared to be unchanged in a simian-virus-40-transformed subclone. In competitive binding assays heparan sulphate from the control clone was bound preferentially compared to that from the transformed subclone, indicating that the altered sulphation of heparan sulphate from transformed cells results in a lowered affinity for cell-surface receptors. Evidence was obtained suggesting that endogenous proteoglycans were not held at the cell surface by binding to these receptors alone. However the possibility that proteoglycans embedded in the plasma membrane may interact with the receptor has not been ruled out.     

Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.     

Binding of the asymmetric forms of acetylcholinesterase to heparin.

The interaction between acetylcholinesterase (EC 3.1.1.7) and heparin, a sulphated glycosaminoglycan, was studied by affinity chromatography. A specific binding of the asymmetric acetylcholinesterase to an agarose gel containing covalently bound heparin was demonstrated. This interaction required an intact collagenous tail, shown by the fact that the binding is abolished by pretreatment with collagenase. The globular forms did not bind to the column. Both total and intracellular asymmetric acetylcholinesterase forms isolated from the endplate region of the rat diaphragm muscle showed higher affinity for the heparin than did the enzyme from the non-endplate region. The binding to the resin was destabilized with 0.55 M-NaCl, and, among the various glycosaminoglycans tested, only heparin was able to displace the acetylcholinesterase bound to the column. Our results added further support to the concept that the asymmetric acetylcholinesterase forms are immobilized on the synaptic basal lamina via interactions with heparin-like molecules, probably related to heparan sulphate proteoglycans.     

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

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

Interaction between glial-cell-line-derived neurotrophic factor (GDNF) and 2-O-sulphated heparin-related glycosaminoglycans

Glial cell line-derived growth factor (GDNF) is a cytokine of the transforming growth factor (TGF)-beta family with potent neuroprotective activity. Clinical trials of recombinant GDNF in advanced Parkinson's disease are currently under way. It is known that mice homozygous for disruption of the gene encoding heparan sulphate 2-O-sulphotransferase die perinatally, due to the complete absence of kidneys. Similar phenotypes arise from targeted disruption of the genes encoding either GDNF, or its receptors, GFRalpha1 and c-Ret. It may therefore be proposed that GDNF normally binds to 2-O-sulphate-rich heparan sulphate within kidney progenitor tissues, and that this interaction is essential for its activity in kidney development. In support of this hypothesis we have shown in ELISA studies that GNDF binds to heparin and heparan sulphate. This binding is unusually sensitive to the chemical 2-O-desulphation, and promotes the binding of GNDF to GFRalpha1.     

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

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

The immunoelectronmicroscopic localization of human platelet factor 4 in tissue mast cells

Initial immunohistochemical localization of human platelet factor 4 (PF4) in tissue mast cells suggested that the protein was present in the mast cell granule. It was proposed that this could reflect binding of PF4 to heparin or heparan sulphate, known granule constituents. We report here the confirmation of granule localization by an immunoelectron microscopical method. The possible role of such binding is unknown, but the potential for cationic proteins of platelet origin interacting with vessel wall constituents is discussed.     

Structural features in heparin which modulate specific biological activities mediated by basic fibroblast growth factor

The biological activity of basic fibroblast growth factor (bFGF)is influenced greatly by direct binding to heparin and heparansulphate (HS). Heparin-derived oligosaccharides have been utilizedto determine the structural requirements present in the polymerthat account for bind ing to bFGF. We had previously demonstratedthat fragments >6 mer can inhibit the interaction betweencell surface heparan sulphate proteoglycan (HSPG) and bFGF,and bFGF-induced proliferation of adrenocortical endothelial(ACE) cells. In contrast, oligosaccharides > 10 mer can enhancethe binding of bFGF to its high-affinity receptor or supportbFGF-induced mitogenesis in ACE cells (Ishihara et al., J. Biol.Chem., 268, 4675–4683, 1993). We have extended these studiesto size- and structure-defined oligosaccharides from heparin,2-O-desulphated (2-O-DS-) heparin, 6-O-desulphated (6-O-DS-)heparin, carboxyreduced (CR-) heparin and carboxy-amidomethylsulphonated(AMS-) heparin. Oligosaccharides from these polymers were fractionatedon a bFGF-affinity column and were assessed as inhibitors orenhancers of specific bFGF-derived biological activities. Theresults of these studies indicate that both 2-O-sulphate andthe negative charge of the carboxy group [L-iduronic acid (IdoA)residues] are required for specific interactions of heparin-derivedoligosaccharides with bFGF and for modulation of bFGF mitogenicactivity. In addition, the charge of the carboxy groups in uronicacids can be replaced by other functional groups with a negativecharge, such as the amidomethyl sulphonate moiety describedhere. basic fibroblast growth factor heparan sulphate heparin oligosaccharides     

The binding of the circumsporozoite protein to cell surface heparan sulfate proteoglycans is required for plasmodium sporozoite attachment to target cells

The major surface protein of malaria sporozoites, the circumsporozoite protein, binds to heparan sulfate proteoglycans on the surface of hepatocytes. It has been proposed that this binding event is responsible for the rapid and specific localization of sporozoites to the liver after their injection into the skin by an infected anopheline mosquito. Previous in vitro studies performed under static conditions have failed to demonstrate a significant role for heparan sulfate proteoglycans during sporozoite invasion of cells. We performed sporozoite attachment and invasion assays under more dynamic conditions and found a dramatic decrease in sporozoite attachment to cells in the presence of heparin. In contrast to its effect on attachment, heparin does not appear to have an effect on sporozoite invasion of cells. When substituted heparins were used as competitive inhibitors of sporozoite attachment, we found that sulfation of the glycosaminoglycan chains at both the N- and O-positions was important for sporozoite adhesion to cells. We conclude that the binding of the circumsporozoite protein to hepatic heparan sulfate proteoglycans is likely to function during sporozoite attachment in the liver and that this adhesion event depends on the sulfated glycosaminoglycan chains of the proteoglycans.     

Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7)

Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound. The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane. This is established by extraction experiments and confocal microscopy. The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment. The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion. Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle. The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; the K(D) is 5-10 nM. Zymographic measurement of MMP-7 activity is greatly enhanced by heparin. Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands. Evidence is also found for the binding of MMP-2, -9, and -13. Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels. Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins. Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis.