Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions

Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions.     

Heparan sulfate proteoglycans from mouse mammary epithelial cells: localization on the cell surface with a monoclonal antibody

Mouse mammary epithelial cells, of the normal murine mammary gland (NMuMG) cell line, bear a heparan sulfate-rich proteoglycan (HSPG) on their surfaces. A hybridoma (281-2) secreting a monoclonal antibody that recognizes this HSPG was produced by fusion of SP-2/0 myeloma cells with spleen cells from rats immunized with NMuMG cells. The 281-2 monoclonal antibody is directed against the core protein of the cell surface HSPG, as demonstrated by (a) recognition of the isolated proteoglycan but not its glycosaminoglycan chains, (b) co-localization of 281-2-specific antigen and radioactive cell surface HSPG on gradient polyacrylamide gel electrophoresis and on isopycnic centrifugation, and (c) abolition of immunofluorescent staining of the NMuMG cell surface by the intact, but not the protease-digested ectodomain of the cell surface HSPG. The antibody is specific for cell surface HSPG and does not recognize the HSPG that accumulates extracellularly beneath the basal cell surface. Therefore, the 281-2 antibody may be used to isolate the cell surface HSPG and to explore its distribution in tissues.     

Heparan sulfate at the surface of HeLa cells.

HeLa cells, labeled with Na235SO4, release into the culture medium 35SO4 bound to plasma membrane vesicles next to 35SO4-glycoproteins and free 35SO4. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain 35SO4 and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.     

Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens

A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells apparently intercalated into their plasma membranes. Mild treatment of the cells with trypsin releases the GAG-bearing region (ectodomain) of this molecule as a discrete proteoglycan which is readily purified. At physiological pH and ionic strength, the ectodomain binds collagen types I, III, and V but not types II, IV, or denatured type I. The proteoglycan binds to a single class of high affinity saturable sites on type I collagen fibrils, sites which are selective for heparin-like glycosaminoglycans. The binding of NMuMG cells to type I collagen duplicates that of their cell surface proteoglycan; cells bind to native but not denatured collagen, and binding is inhibited by heparin but not by other glycosaminoglycans. These binding properties suggest that cell surface heparan sulfate proteoglycans could act as receptors for interstitial collagens and mediate changes in cell behavior induced by collagenous matrices.     

Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin

We have reported previously that Noggin is a heparin-binding protein and associates with the cell surface through heparan sulfate proteoglycans, where it remains functional for the binding of bone morphogenetic proteins (BMPs). Here we report that the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are required for the interaction. Noggin binds most efficiently to heparin sequences composed of 10 or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1 selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.     

Cell-surface heparan sulfate proteoglycans potentiate chordin antagonism of bone morphogenetic protein signaling and are necessary for cellular uptake of chordin

Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cell-surface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed.     

Breast and ovarian cancers: a survey and possible roles for the cell surface heparan sulfate proteoglycans

Tumor markers are widely used in pathology not only for diagnostic purposes but also to assess the prognosis and to predict the treatment of the tumor. Because tumor marker levels may change over time, it is important to get a better understanding of the molecular changes during tumor progression. Occurrence of breast and ovarian cancer is high in older women. Common known risk factors of developing these cancers in addition to age are not having children or having children at a later age, the use of hormone replacement therapy, and mutations in certain genes. In addition, women with a history of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression.     

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.     

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.     

Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans.

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, we identified a sixth member of the glypican family. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (identity of 63%). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily comprising so far glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might in part be regulated by the presence of glypican-6 on the cell surface.     

Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.

Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). To detect binding between MGP and BMP-2, we performed immunoprecipitation using MGP and BMP-2 tagged with FLAG and c-Myc. The results showed co-precipitation of BMP-2 with MGP. To quantify the effect of MGP on BMP-2 activity, we assayed for alkaline phosphatase activity and showed a dose-dependent effect. Low levels of MGP relative to BMP-2 (<1-fold excess) resulted in mild enhancement of osteoinduction, whereas intermediate levels (1-15-fold excess) resulted in strong inhibition. High levels of MGP (>15-fold excess), however, resulted in pronounced enhancement of the osteoinductive effect of BMP-2. Cross-linking studies showed that inhibitory levels of MGP abolished BMP-2 receptor binding. Immunoblotting showed a corresponding decrease in activation of Smad1, part of the BMP signaling system. Enhancing levels of MGP resulted in increased Smad1 activation. To determine the cellular localization of BMP-2 in the presence of MGP, binding assays were performed on whole cells and cell-synthesized matrix. Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein.     

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.     

Specific interaction between cartilage proteoglycans and hyaluronic acid at the chondrocyte cell surface.

Binding of exogenous [35S]sulphate-labelled cartilage proteoglycans to cells was studied with suspension cultures of calf articular-cartilage chondrocytes. Proteoglycans interact with hyaluronic acid at the cell surface via their hyaluronic acid-binding region. The binding is time-dependent and saturable, but does not appear to be freely reversible. The bound 35S-labelled proteoglycans are located at the cell surface, and only small proportions of the proteoglycans are internalized.     

Upregulation of acetylcholine synthesis by bone morphogenetic protein 9 in a murine septal cell line.

Previous studies showed that bone morphogenetic protein 9 (BMP-9) induces the expression of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter, and upregulates ACh synthesis in cultured primary neurons from embryonic mouse septum [I. López-Coviella, B. Berse, R. Krauss, R.S. Thies, J.K. Blusztajn, Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. Science 289 (2000) 313-316]. In the present studies we investigated the effects of BMP-9 on ACh synthesis in the cholinergic mouse SN56T17 septal cell line. BMP-9 increased ACh synthesis in these cells up to 2.5-fold in a time- and dose-dependent, saturable manner. The maximal effect of BMP-9 was observed after a 3-day treatment and the median effective concentration of BMP-9 was 0.5 ng/ml. These data show that SN56T17 cells are a useful model for studies of the effects of BMPs on the cholinergic phenotype.     

USAG-1: a bone morphogenetic protein antagonist abundantly expressed in the kidney

Bone morphogenetic proteins (BMPs) play critical roles in cellular proliferation, differentiation, and programmed cell death in multiple tissues. An increasing body of recent evidence has suggested that classes of molecules collectively termed BMP antagonists play important roles for the local regulation of BMP actions by binding BMPs and neutralizing their activities. Uterine sensitization-associated gene-1 (USAG-1) was previously reported as a gene of unknown function, preferentially expressed in sensitized endometrium of the rat uterus. Here, we show that USAG-1 is abundantly expressed in the kidney and functions as a BMP antagonist. Recombinant USAG-1 binds directly to BMPs and antagonizes the BMP-mediated induction of alkaline phosphatase in C2C12 cells. USAG-1 also induces formation of secondary axis and/or hyperdorsalization when its mRNA is injected to Xenopus embryos. In the early stage of mouse embryogenesis, USAG-1 is expressed in the first and second branchial arches and in metanephros, while in later stages the expression is confined to renal tubules and ameloblasts of teeth. Postnatally, the expression is further restricted to distal tubules of kidney, in a pattern similar to the localization of BMP-7, which has been shown to be important in the development of kidney and preservation of adult renal functions under pathological stresses. Collectively, we suggest that USAG-1 is a BMP antagonist that interacts with BMP-7 in the developing and adult kidney.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans.

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

Presence of a laminin-binding chondroitin sulfate proteoglycan at the cell surface of a human melanoma cell Mel-85

Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with -mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. The molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [³⁵S]-sulfate and chemical -elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin.