Heparan Sulfate Domains Research Articles

Fluorescent-tagged heparan sulfate precursor oligosaccharides to probe the enzymatic action of heparitinase I

Heparitinase I, a key lyase enzyme essential for structural analysis of heparan sulfate (HS), degrades HS domains that are undersulfated at glucuronyl residues through an elimination mechanism. Earlier studies employed viscosimetric measurements and electrophoresis to deduce the mechanism of action of heparitinase I and two other related lyases, heparitinase II and heparitinase III. However, these findings lack molecular evidence for the intermediates formed and could not distinguish whether the cleavage occurred from the reducing end or the nonreducing end. In the current study, 2-aminoacridone (2-AMAC)-labeled HS precursor oligosaccharides of various sizes were prepared to investigate the mechanism of heparitinase I-mediated depolymerization using sensitive and quantitative methodologies. Furthermore, fluorescent (2-AMAC) tagging of HS precursor oligosaccharides allowed us to distinguish fragments that result from cleavage of the substrates at various time intervals and sites farther away from the reducing and nonreducing ends of oligosaccharide substrates. This study provides the first direct molecular evidence for a predominantly random endolytic mechanism of cleavage of HS precursor oligosaccharides by heparitinase I. This robust strategy can be adapted to deduce the mechanism of action of other heparitinases and also to deduce structural information of complex HS oligosaccharides of biological importance.

Heparan Sulfate Regulates Fibrillin-1 N- and C-terminal Interactions

Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized within the domain encoded by exon 7 after the first hybrid domain. Rodent embryonic fibroblasts adhered to PF1 and deletion fragments, and, when cells were plated on fibrillin-1 or fibronectin Arg-Gly-Asp cell-binding fragments, cells showed heparin-dependent spreading and focal contact formation in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions.

Reduction of anionic sites in the glomerular basement membrane by heparanase does not lead to proteinuria

Heparan sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases contributes to proteinuria.

Heparan sulfate domains on cultured activated glomerular endothelial cells mediate leukocyte trafficking

Heparan sulfate (HS) proteoglycans by playing key roles in the leukocyte-endothelial interactions are thought to mediate inflammatory cell influx in proliferative glomerulonephritis. Here, we evaluated the specific features within glomerular endothelial HS that promote leukocyte adhesion. Mouse and human glomerular endothelial cells activated by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta increased expression of inflammatory N- and 6-O-sulfated HS domains. In addition, altered expression of HS-modifying enzymes occurred, a feature also found in mouse kidneys with anti-glomerular basement membrane disease or lupus nephritis. Inhibition of the nuclear factor (NF)-kappaB pathway repressed cytokine-induced alterations in HS and gene expression of modifying enzymes. Firm adhesion of leukocytes to activated mouse glomerular endothelial cells decreased after removal of endothelial HS or addition of sulfated heparinoids. Specific antibodies that block N- and 6-O-sulfated HS domains on activated mouse endothelial cells reduced the number of rolling and firmly adhering leukocytes under dynamic flow conditions, while they increased the average leukocyte-rolling velocity. Our study shows that N- and 6-O-sulfated domains in HS on activated glomerular endothelium are crucial for leukocyte trafficking and are possible therapeutic targets.

Syndecan-1 deficiency aggravates anti-glomerular basement membrane nephritis

During the heterologous phase of experimental anti-glomerular basement membrane (anti-GBM) nephritis, leukocyte influx peaks within hours, whereas albuminuria occurs within 1 day. In the subsequent autologous phase, endogenous anti-GBM IgG develops and albuminuria persists. Heparan sulfate (HS) proteoglycans like syndecan-1 play multiple roles during inflammation and we evaluate its role in experimental anti-GBM disease using syndecan-1 knockout (sdc-1-/-) mice. During the heterologous phase, glomerular leukocyte/macrophage influx was significantly higher in the sdc-1-/- mice and this was associated with higher glomerular endothelial expression of specific HS domains. In the autologous phase, glomerular influx of CD4+/CD8+ T cells was higher in the sdc-1-/- mice and these mice had persistently higher albuminuria and serum creatinine levels than wild-type mice. This resulted in a more sever glomerular injury and increased expression of extracellular matrix proteins. The sdc-1-/- mice developed higher plasma levels and glomerular deposits of total mouse Ig and IgG1 anti-rabbit IgG, whereas the levels of mouse IgG2a anti-rabbit IgG were lower. Furthermore, decreased Th1 and higher Th2 renal cytokine/chemokine expression were found in the sdc-1-/- mice. Our studies show that syndecan-1 deficiency exacerbates anti-GBM nephritis shifting the Th1/Th2 balance towards a Th2 response.

Trypanosoma cruzi heparin-binding proteins and the nature of the host cell heparan sulfate-binding domain

Trypanosoma cruzi invasion is mediated by receptor–ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)-binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S 35]methionine labeled proteins eluted from heparin–sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi–cardiomyocyte interaction.

The Heparin/Heparan Sulfate Sequence That Interacts with Cyclophilin B Contains a 3-O-Sulfated N-Unsubstituted Glucosamine Residue

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.

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.

Novel ‘phage display antibodies identify distinct heparan sulfate domains in developing mammalian lung

Heparan sulfate proteoglycans (HSPGs) are essential to respiratory morphogenesis in species as diverse as Drosophila and mice; they play a role in the regulation of numerous HS-binding growth factors, e.g. fibroblast growth factors. Moreover, an HS analogue, heparin, modulates lung growth in vitro. However, it has been difficult to assess the roles of specific HS structures in lung development due to technical barriers to their spatial localisation. Lungs from Sprague-Dawley rats were harvested between E15.5 and E19.5 and immediately fixed in 4 % (w/v) paraformaldehyde (in 0.1 M phosphate-buffered saline (PBS), pH 7.4). Lungs were washed in PBS, cryoprotected with 20% (w/v) sucrose (in PBS), gelatin embedded [7.5% (w/v) gelatin, 15% (w/v) sucrose in PBS], before being covered in Cryo-M-Bed (Bright, Huntingdon, UK) and snap frozen at -40 degrees C. Cryosections were cut at 8 microm and stained with the HSPG core protein specific antibody 3G10 and a HS 'phage display antibody, EW4G2V. 3G10 and EW4G2V immunohistochemistry highlighted the presence of specific HS structures in lungs at all gestational ages examined. 3G10 strongly labelled airway basement membranes and the surrounding mesenchyme and showed weak staining of airway epithelial cells. EW4G2V, however, was far more selective, labelling the airway basement membranes only. Mesenchymal and epithelial cells did not appear to possess the HS epitope recognised by EW4G2V at these gestational ages. Novel 'phage display antibodies allow the spatial distribution of tissue HS to be analysed, and demonstrate in situ that distinct cellular compartments of a tissue possess different HS structures, possibly on the same proteoglycan core protein. These probes offer a new opportunity to determine the role of HS in the pathogenesis of congenital defects such as congenital diaphragmatic hernia (CDH), where lung development is aberrant, and the resulting pulmonary hypoplasia and hypertension are a primary cause of mortality.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Interactions between heparan sulfate and proteins: the concept of specificity

Proteoglycan (PG) coreceptors carry heparan sulfate (HS) chains that mediate interactions with growth factors, morphogens, and receptors. Thus, PGs modulate fundamental processes such as cell survival, division, adhesion, migration, and differentiation. This review summarizes recent biochemical and genetic information that sheds new light on the nature of HS–protein binding. Unexpectedly, many interactions appear to depend more on the overall organization of HS domains than on their fine structure.

The Morphogenic Properties of Oligomeric Endostatin Are Dependent on Cell Surface Heparan Sulfate

Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with N- and 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface.

Overexpression of Heparan Sulfate 6-O-Sulfotransferases in Human Embryonic Kidney 293 Cells Results in Increased N-Acetylglucosaminyl 6-O-Sulfation

Heparan sulfate (HS) interacts with a variety of proteins and thus mediates numerous complex biological processes. These interactions critically depend on the patterns of O-sulfate groups within the HS chains that determine binding sites for proteins. In particular the distribution of 6-O-sulfated glucosamine residues influences binding and activity of HS-dependent signaling molecules. The protein binding domains of HS show large structural variability, potentially because of differential expression patterns of HS biosynthetic enzymes along with differences in substrate specificity. To investigate whether different isoforms of HS glucosaminyl 6-O-sulfotransferase (6-OST) give rise to differently sulfated domains, we have introduced mouse 6-OST1, 6-OST2, and 6-OST3 in human embryonic kidney 293 cells and compared the effects of overexpression on HS structure. High expression of any one of the 6-OST enzymes resulted in appreciably increased 6-O-sulfation of N-sulfated as well as N-acetylated glucosamine units. The increased 6-O-sulfation was accompanied by a decrease in nonsulfated as well as in iduronic acid 2-O-sulfated disaccharide structures. Furthermore, overexpression led to an altered HS domain structure, the most striking effect was the formation of extended 6-O-sulfated predominantly N-acetylated HS domains. Although the effect was most noticeable in 6-OST3-expressing cells, these results were largely independent of the particular 6-OST isoform expressed and mainly influenced by the level of overexpression.

Identification of L-selectin Binding Heparan Sulfates Attached to Collagen Type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.

Novel Heparan Sulfate Structures Revealed by Monoclonal Antibodies

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans. Because of the structural heterogeneity of HS, tissue-derived HS preparations represent a mixture of HS chains originating from different cell types and tissue loci. Monoclonal anti-HS antibodies have been employed to detect the localization of specific HS epitopes in tissues, but limited information has been available on the saccharide structures recognized by the antibodies. We have studied the saccharide epitope structures of four anti-HS antibodies, HepSS1, JM13, JM403, and 10E4, which all recognize distinct HS species as demonstrated by different patterns of immunoreactivity upon staining of embryonic rat and adult human tissues. The epitopes recognized by JM13 and HepSS1 were found almost exclusively in basement membrane HS, whereas JM403 and 10E4 reacted also with cell-associated HS species. The binding of HepSS1, JM403, and 10E4 to HS was dependent on the GlcN N-substitution of the polysaccharide rather than O-sulfation. HepSS1 thus interacted with N-sulfated HS domains, JM403 binding was critically dependent on N-unsubstituted GlcN residues, and 10E4 bound to "mixed" HS domains containing both N-acetylated and N-sulfated disaccharide units. By contrast, JM13 binding seemed to require the presence of 2-O-sulfated glucuronic acid residues.

Localization and Functional Characterization of Glycosaminoglycan Domains in the Normal Human Kidney as Revealed by Phage Display-Derived Single Chain Antibodies

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy.

Stem domains of heparan sulfate 6-O-sulfotransferase are required for Golgi localization, oligomer formation and enzyme activity.

Heparan sulfate O-sulfotransferases catalyze the O-sulfation of the glucosamine and uronic acid residues of heparan sulfate, thereby determining the binding sites for ligands necessary for important biological functions such as the formation of morphogen gradients and growth factor signaling. Here we investigated the localization of the three heparan sulfate 6-O-sulfotransferase (HS6ST) isoforms and the mechanism of their localization. All three GFP-tagged HS6STs localized in the Golgi apparatus. C-5 epimerase and HS2ST have been shown to form complexes that facilitate their localization in the Golgi but we found that the absence of HS2ST did not alter the localization of any of the HS6STs. Neither the forced expression of HS2ST in the rough endoplasmic reticulum (ER), the deletion of most of the lumenal domain nor increasing the length of the transmembrane domain had any effect on the localization of HS6STs. However, deletions in the stem region did affect the Golgi localization of the HS6STs and also reduced their sulfotransferase activity and oligomer formation. These findings suggest that the stem region of HS6ST plays an important role in normal functioning, including the transit of HS6ST to the Golgi apparatus and maintaining the active conformation essential for enzyme activity.

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.

Targeted Disruption of a Murine Glucuronyl C5-epimerase Gene Results in Heparan Sulfate Lacking l-Iduronic Acid and in Neonatal Lethality

The glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate signaling events in embryogenesis. All identified protein-binding HS epitopes contain l-iduronic acid (IdoA). We report that targeted disruption of the murine d-glucuronyl C5-epimerase gene results in a structurally altered HS lacking IdoA. The corresponding phenotype is lethal, with renal agenesis, lung defects, and skeletal malformations. Unexpectedly, major organ systems, including the brain, liver, gastrointestinal tract, skin, and heart, appeared normal. We find that IdoA units are essential for normal kidney, lung, and skeletal development, albeit with different requirement for 2-O-sulfation. By contrast, major early developmental events known to critically depend on heparan sulfate apparently proceed normally even in the absence of IdoA.

Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function

The anti-angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of >/=15 kDa bound quantitatively to ES whereas N-sulfated HS decasaccharides, with affinity for several fibroblast growth factor (FGF) species, failed to bind. Instead, ES-binding oligosaccharides composed of mixed N-sulfated and N-acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES-binding epitope was identified, with two N-sulfated regions separated by at least one N-acetylated glucosamine unit (SAS-domain). Cleavage at the N-acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial growth factor-induced endothelial cell migration was blocked by the ES-binding SAS-domains and by heparin oligosaccharides (12mers) similar in length to the ES-binding SAS-domains, but not by 6mers capable of FGF binding. We propose that SAS-domains modulate the biological activities of ES and other protein ligands with extended HS-binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.