Generation and Application of Type-specific Anti-Heparan Sulfate Antibodies Using Phage Display Technology

Abstract

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...

The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer

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Critical comparative analyses of anti–α‐actinin and glomerulus‐bound antibodies in human and murine lupus nephritis

“…An enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies to nucleosomes, H1, dsDNA, DNase I, type IV collagen, heparan sulfate, and ␣-actinin, as described previously (27)(28)(29)(30). Essentially, microtiter plates (MaxiSorp; Nunc, Copenhagen, Denmark) were coated with nucleosomes (10 g/ml as DNA), dsDNA (10 g/ml), H1 (2 g/ml), heparan sulfate (10 g/ml), ␣-actinin (5 g/ml), DNase I (5 g/ml), and type IV collagen (5 g/ml).…”

Heparanase induces a differential loss of heparan sulphate domains in overt diabetic nephropathy

“…Cryosections were then incubated for 1 h in blocking buffer with the phage display-derived VSV-tagged anti-heparan sulphate antibodies HS4C3 and EW3D10 [20,21], both of which recognise sulphated heparan sulphate domains. Bound antibodies were detected by incubation with mouse IgG anti-VSV tag antibody P5D4 and rabbit IgG anti-VSV tag antibody 9E10 (both 1:10; Boehringer Mannheim, Mannheim, Germany), followed by Alexa 488-conjugated goat antimouse IgG and Alexa 594-conjugated goat anti-rabbit IgG JM403, JM72, anti-K5 antibody and anti-heparanase antibody Frozen kidney sections were air-dried and fixed in 100% (v/v) acetone for 10 min at 4°C.…”