Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (V H 3 Heparan sulfate (HS) 1 represents a heterogeneous class of molecules within the group of glycosaminoglycans. It has been implicated in many basic cellular phenomena, such as cell growth, migration, and differentiation (1-4). HS binds and modulates various proteins, including growth factors and cytokines, enzymes, protease inhibitors, and extracellular matrix proteins. Studies involving specific enzymatic or chemical cleavage and subsequent analysis of the resulting oligosaccharides indicate the existence of many HS species and the presence of domain structures within the HS molecule (5-15). There are clues that specific monosaccharide sequences within the molecule dictate the specific features of a given species, e.g. a pentasaccharide for the binding of HS/heparin to anti-thrombin III and a preferential sequence for the binding of HS to bFGF (4, 16 -19). The appreciation of the structural diversity of HS species and its role in pathological conditions is strongly hampered by the lack of appropriate methodologies. Sequence strategies are not at hand, and specific antibodies, obvious tools for studying diversity, are difficult to raise. HS, and glycosaminoglycans in general, are almost nonimmunogenic, and consequently, only a few specific antibodies have been described (20, 21). To circumvent this, we adopted antibody phage display technology because this system allows one to generate antibodies against "self" antigens. We report here on the generation and application of three specific antibodies against HS species using this technique. We compared these antibodies with two described mouse monoclonal antibodies, with regard to immunostaining on sections of rat kidney, immunoreactivity toward various HS preparations, and reactivity with bFGF sites on HS. EXPERIMENTAL PROCEDURES MaterialsA "single pot" human semisynthetic phage library (22) (now officially named synthetic scFv library 1) was generously provided by Dr G. Winter, Cambridge University, Cambridge, United Kingdom. This library contains 50 different V H genes with synthetic random complementarity-determining region 3 segments, which are 4 -12 amino acid residues in length. The heavy chains are combined with a single light chain gene (DPL 16). The library contains Ͼ 10 8 different clones.Two Escherichia coli strains were used: the suppressor strain TG1 (K12, D(lac-pro), supE, thi, hsdD5/FЈtraD36, proA ϩ B ϩ , laqI q , lac-ZDM15), and the nonsuppressor strain HB2151 (K12, ara, D(lac-p...

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...

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