Large, Tissue-regulated Domain Diversity of Heparan Sulfates Demonstrated by Phage Display Antibodies

Abstract

Heparan sulfates (HS) are long, linear polysaccharides with a high degree of variability. They bind to a vast number of proteins such as growth factors and cytokines, and these interactions are likely to be mediated by specific HS domains. To investigate the structural diversity and topological distribution of HS domains in tissues, we selected a panel of 10 unique anti-HS antibodies using phage display technology. All 10 antibodies recognize a specific HS epitope as demonstrated by enzyme-linked immunosorbent assay using defined synthetic HS oligosaccharides, modified HS/ heparin molecules, and HS isolated from a variety of organs. The chemical groups involved in the epitopes could be indicated and the position of sulfate groups is of major importance. All HS epitopes have a defined tissue distribution as shown by immunohistochemistry using rat organs. Taken together, the data show that in vivo, a large number of defined HS epitopes exist that do not occur randomly but are tightly, topologically regulated.Heparan sulfates (HS), 1 a class of glycosaminoglycans, are long linear complex polysaccharides covalently bound to a protein core. They have a ubiquitous distribution in the extracellular matrix and on cell surfaces and have been implicated in many basic biological phenomena such as cell migration, adhesion, and differentiation. They play a role in such diverse processes as growth factor/cytokine handling, enzyme regulation, lipid metabolism, blood coagulation, and viral entry (1-4). This involvement is mediated by the interactions of HS with a vast number of proteins such as growth factors/cytokines, enzymes, protease inhibitors, extracellular matrix molecules, and viral coat proteins (5, 6). The large number of interactions suggests an extensive structural variation within HS. Chemical analysis of HS-derived disaccharides indeed indicates a large structural diversity (7-9), which is brought about by specific chain modifications during HS biosynthesis. The importance of defined monosaccharide sequences for specific interactions with proteins has been demonstrated for the binding and activation of antithrombin III by HS/heparin (10, 11). In addition, specific structural requirements for binding to basic fibroblast growth factor and hepatocyte growth factor have been defined (12, 13). These observations indicate that HS modifications do not occur randomly but that a controlled expression of specific domains/sequences in HS exists. To investigate whether a large number of different HS domains indeed occur in tissues, we selected a panel of epitope-specific anti-HS antibodies using phage display technology. Using these antibodies, we chemically and topologically characterized the HS epitopes involved. We chose antibody phage display because it allows for the generation of antibodies against poorly immunogenic molecules such as HS. EXPERIMENTAL PROCEDURES MaterialsA human semisynthetic antibody phage display library (14, now officially named synthetic scFv Library No. 1) was generously provided by Dr G. Winter, C...

Detection of 2-O-Sulfated Iduronate and N-Acetylglucosamine Units in Heparan Sulfate by an Antibody Selected against Acharan Sulfate (IdoA2S-GlcNAc)

“…Both the N-acetyl and 2-O-sulfate groups were found essential for antibody recognition. In general, anti-GAG single chain antibodies react well with oligosaccharides consisting of five or more monosaccharides (6), and therefore the most compatible epitope in AS may be (IdoA2S-GlcNAc) 3 .…”

“…These antibodies were selected against HS from various sources and stained differentially in tissues sections from various organs. Defined HS oligosaccharides were used to reveal details of the epitopes recognized by the antibodies (6). Although some chemical groups in HS essential for antibody reactivity could be determined, the oligosaccharide sequence recognized by these antibodies remains largely unknown.…”

Heparan Sulfate 3-O-Sulfotransferase Isoform 5 Generates Both an Antithrombin-binding Site and an Entry Receptor for Herpes Simplex Virus, Type 1

“…It is very interesting to note that a unique subset of HS was identified in human skeletal muscle tissues by a set of antibodies that bind to HS (37). An elegant study was recently published (38) to determine the binding sequences of these antibodies. However, it still remains to be investigated whether or not the unique subset of HS found in the skeletal muscle tissue is associated with 3-OST-5 modification.…”

Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion

“…1 e and f ) of midgut microvilli from blood-fed mosquitoes suggests that these anionic polysaccharides are analogous to CS. Midgut sections from blood-fed mosquitoes stained with scFv RB4EA12 (10) anti-HS antibodies showed basal lamina localization of HScharide units (GlcA␤1-3GalNAc) (13). MAb CS-56 recognizes the monosulfated GlcA-GalNAc (4S) disaccharide ''A-unit'' and GlcA-GalNAc (6S) ''C-unit,'' the disulfated GlcA (2S)-GalNAc(6S) disaccharide ''D-unit'', and octasaccharides that contained an internal trisulfated tetrasaccharide ''A-D core unit'' composed of GlcA-GalNAc(4S)-GlcA(2S)-GalNAc(6S) in CS chains (Fig.…”