During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development.We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/ N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N-and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor ␤ (PDGFR␤) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.[Keywords: PDGF-B; angiogenesis; heparan sulfate; pericyte; vascular development] Supplemental material is available at http://www.genesdev.org. Tissue morphogenesis depends on cell-cell interactions, controlling directed cell migration proliferation, differentiation, and cell survival. Specificity is often regulated at the level of selective ligand-receptor interaction. However, the spatial distribution and local concentration of the ligand determine the range of the signal, and, as exemplified by morphogens of the hedgehog, TGF␤, and Wnt family members, also the nature of the signal. Indeed, spatial restriction defines the activities of most peptide growth factors and many secreted neural guidance molecules. In vascular development, peptide growth factors of the VEGF and platelet-derived growth factor (PDGF) families regulate the migration and proliferation of endothelial cells and supporting mural cells; i.e., pericytes (PC) and vascular smooth muscle cells (vSMC). The longitudinal migration and proliferation of vSMC/PC depend on paracrine signaling of endothelial derived PDGF-B to PDGF receptor-␤ (PDGFR␤) expressed on vSMC/PC (Lindahl et al. 1997;Hellström et al. 1999). PDGF-B is secreted as a homodimer (PDGF-BB), which signals by mediating dimerization of its receptor. Conditional inactivation of Pdgf-b in the endothelium demonstrated that endothelial cells are the

Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitous, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin.Heparan sulfates (HS) 3 are linear, sulfated polysaccharides that occur covalently bound to core proteins in proteoglycan structures at cell surfaces and in extracellular matrices. Due to their negative charge, HS chains interact with a variety of proteins, and thus modulate important processes in embryogenesis and tissue homeostasis (1, 2). Biosynthesis of HS involves formation of a precursor polysaccharide composed of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) residues, which is subsequently modified through a series of enzymatic reactions. The modifications include N-deacetylation and N-sulfation of GlcNAc residues, C5-epimerization of GlcA to iduronic acid (IdoA) units, and finally O-sulfation at C2 of hexuronic acid and C6, more rarely C3, of glucosamine units. The reactions are generally incomplete, yielding products of extensive structural heterogeneity (3). The modification process is not under template control, yet tightly regulated, such that differences in structure of HS generated in different tissues are strikingly conserved (4, 5). Although aspects of specificity of HS-protein interactions remain unclear, these differences are presumably of functional significance (6). Elucidation of these problems is hampered by the lack of high-throughput tools for detailed structural characterization of HS, which remains a tedious task.Phage display antibodies have been developed to overcome the limited immunogenicity of HS, thereby making it possible to highlight tissue-specific HS structures and to follow changes in development and disease. Using such antibodies, the differential expression of HS motifs in various tissues has been demonstrated (7-9). Yet, few of these antibodies have so far been characterized for their epitope specificity, which hampers further use of these tools and interpretation of results. We have selected two of these phage display antibodies, AO4B08 and HS4E4, based on their tissue staining properties (8, 9) and their select...

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS NS4F5 ), which we have rigorously identified as (GlcNS6S-IdoA2S) 3 . HS NS4F5 exhibits a restricted expression in healthy adult tissues. Blocking HS NS4F5 on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS NS4F5 is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-␣ stimulated endothelial expression of HS NS4F5 , which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS NS4F5 was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS NS4F5 and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.Heparan sulfate proteoglycans (HSPGs), 3 major components of the cell surface and the extracellular matrix, are involved in a variety of biological phenomena, including cell adhesion, proliferation, differentiation, and inflammation as well as being associated with pathologic events such as atherosclerosis and amyloidosis. Because of their high negative charge, HS chains interact with a variety of proteins, including growth factors/ morphogens and their receptors, the amyloid precursor protein serum amyloid protein A (AA), chemokines, and extracellular matrix proteins. HS-protein interactions vary with regard to specificity and may depend on charge density in addition to strict sequence motifs of HS.The interaction of heparin and HS with FGFs and their receptors has been characterized in great detail. Specific HS structures are predominantly determined by the regulated positioning of N-, 2-, 6-, and 3-O-sulfate groups along HS chains (1). For example, FGF-2 requires both N-and 2-O-sulfate groups for binding to HS. The 6-O-sulfate group is not essential for binding to FGF-2 but is critical for activation of the FGF receptor (2). In contrast, binding of hepatocyte growth factor, platelet-derived growth factor, lipoprotein lipase, and herpes simplex virus glycoprotein C to HS is dependent on 6-O-sulfation (3). The activation of antithrombin III by HS/heparin is mediated by a specific pentasaccharide in which a 3-O-sulfate group is crucial (4). Thus, the biological functions of HSPGs are contro...

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O-and 6-O-sulfated hexa-to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mM. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mM salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the antithrombin binding pentasaccharide sequence. Heparan sulfate (HS)3 proteoglycans consist of a core protein with covalently linked HS side chains, and occur on cell surfaces and in the extracellular matrix. HS polysaccharides consist of up to ϳ200 repeating disaccharide units (glucosamine ␣1-4-glucuronic acid ␤1-4 and glucosamine ␣1-4-iduronic acid ␣1-4), which are variably modified by N-acetyl/N-sulfate and O-sulfate groups (1, 2). The HS chains have fundamental roles in embryonic development, homeostasis, and disease, by interaction with regulatory proteins (morphogens, growth factors, enzymes etc.), mediated by specific HS domains (3). HS-protein interactions are believed to be dictated not only by the overall charge of the HS chain but also by the distribution and positioning of the negatively charged carboxyl and sulfate groups within the HS chain (4). The structural diversity within the HS chain arises through the ordered action of sulfotransferases and an epimerase (1, 5, 6) during HS biosynthesis within the Golgi apparatus and may be further affected by the extracellular action of endosulfatases after biosynthesis (7). The biosynthetic HS modification reactions include N-deacetylation/N-sulfation of the glucosamine (GlcN) residues by N-deacetylase/N-sulfotransferases, C 5 epimerization of glucuronic ...

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HS (heparan sulphate) plays a key role in angiogenesis, by interacting with growth factors required in the process. It has been proposed that HS controls the diffusion, and thus the availability, of platelet-derived growth factor B that is needed for pericyte recruitment around newly formed capillaries. The present paper summarizes our studies on the importance of HS structure in this regulatory process.