The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.

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-α 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.

Organic electronics for precise delivery of neurotransmitters to modulate mammalian sensory function.

Significant advances have been made in the understanding of the pathophysiology, molecular targets and therapies for the treatment of a variety of nervous-system disorders. Particular therapies involve electrical sensing and stimulation of neural activity, and significant effort has therefore been devoted to the refinement of neural electrodes. However, direct electrical interfacing suffers from some inherent problems, such as the inability to discriminate amongst cell types. Thus, there is a need for novel devices to specifically interface nerve cells. Here, we demonstrate an organic electronic device capable of precisely delivering neurotransmitters in vitro and in vivo. In converting electronic addressing into delivery of neurotransmitters, the device mimics the nerve synapse. Using the peripheral auditory system, we show that out of a diverse population of cells, the device can selectively stimulate nerve cells responding to a specific neurotransmitter. This is achieved by precise electronic control of electrophoretic migration through a polymer film. This mechanism provides several sought-after features for regulation of cell signalling: exact dosage determination through electrochemical relationships, minimally disruptive delivery due to lack of fluid flow, and on-off switching. This technology has great potential as a therapeutic platform and could help accelerate the development of therapeutic strategies for nervous-system disorders.

Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4.

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 ubiquitouys, 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.

Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development.

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 beta (PDGFRbeta) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.

3-O-sulfated oligosaccharide structures are recognized by anti-heparan sulfate antibody HS4C3.

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 anti-thrombin binding pentasaccharide sequence.