3-O-Sulfated Oligosaccharide Structures Are Recognized by Anti-heparan Sulfate Antibody HS4C3

Abstract

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 ...

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

“…Fig. 4D demonstrates basal surface staining for HS4C3 in co-cultures of primary rat hepatocytes (26). Finally, the small leucine-rich proteoglycan, decorin, which was previously shown to be important in the long-term maintenance of hepatocyte function can be also detected in oxygenated cocultures (29).…”

“…4C demonstrates that human hepatocytes seeded in 95% oxygen form functional bile canaliculi at the onset of culture. Another aspect of hepatocyte polarity is the presence of 3-O-sulfated heparan sulfate (HS4C3) on the basal surfaces of the cells (26). Heparan sulfate plays a critical role in the clearance of lipoproteins which are thought to …”

A Developmentally Regulated Heparan Sulfate Epitope Defines a Subpopulation with Increased Blood Potential During Mesodermal Differentiation

“…Although HS4C3 binds weakly to any N-, 2-O-, and/or 6-O-sulfated octasaccharide fragment, the addition of an extra 3-O-sulfated glucosamine residue results in strong binding [23]. Thus the two binding populations seen are likely to represent a change in epitope presentation by the cell from a less-preferred to a more-preferred site, rather than an increase in number of epitopes presented (model presented in Fig.…”

“…1I, 1J). Antibody HS4C3 has been shown in vitro to require both Nand 6-O-sulfate groups for HS binding, with considerable redundancy between sequences containing different modification patterns [23]. However, all these sequences bind HS4C3 relatively weakly compared with the same parent saccharide containing an additional single 3-O-sulfate group; this addition appears critical for the presentation of high-affinity binding sequences [23].…”

“…Antibody HS4C3 has been shown in vitro to require both Nand 6-O-sulfate groups for HS binding, with considerable redundancy between sequences containing different modification patterns [23]. However, all these sequences bind HS4C3 relatively weakly compared with the same parent saccharide containing an additional single 3-O-sulfate group; this addition appears critical for the presentation of high-affinity binding sequences [23]. It is therefore possible that the HS4C3 high population may represent cells that express HS chains containing unique 3-O-sulfated groups, providing a new distinct epitope with increased binding affinity, rather than just an increase in the number of HS4C3 epitopes expressed.…”