Kimberly E Forsten and Matthew A Nugent 1 Introduction

A large number of proteins (>100) have been demonstrated to bind to heparan sulfate proteoglycans, and in many instances these interactions have important biological consequences (1). The best-studied example is the fibroblast growth factor (FGF) family of proteins. The FGFs regulate a wide range of cellular functions, including proliferation, differentiation, and migration. The best-characterized FGF family member, basic fibroblast growth factor (bFGF) or FGF-2, has been shown to require interaction with heparan sulfate on cell surfaces in order to induce maximal activity (2,3). However, interaction of bFGF with heparan sulfate within the extracellular matrix can limit bFGF diffusion and access to cell surfaces (4,5). Thus the interaction of bFGF with heparan sulfate has been targeted as a site for regulation of both endogenous and exog-enously administered bFGF. For example, bFGF and many other heparin-binding proteins have been demonstrated to play pivotal roles in the growth of the new blood vessels (angiogenesis), a process that is essential for both efficient wound healing and the development of malignant tumors. Indeed, inhibition of endogenous bFGF activity has been suggested as a possible treatment to prevent tumor growth and metastasis, whereas enhancement of angiogenesis by pharmacological bFGF has been proposed to stimulate repair of damaged tissue (i.e., ischemic heart muscle) (6). In both instances, effective treatments might make use of small compounds, which inhibit bFGF binding to heparan sulfate proteoglycans. These compounds might have applications as inhibitors of bFGF binding and activity at cell surfaces, or in turn they might enhance the transport of added bFGF through connective tissue and allow cell stimulation distant from the administration site. Compounds that bind either to the growth factor or to heparan sulfate could block bFGF binding to heparan sulfate, yet might have very different effects biologically. To screen various potential inhibitors of growth factor/heparan sulfate binding effectively, a simple, rapid, and semi-quantitative assay is required.

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

In this chapter we report a simple assay to determine equilibrium binding constants for inhibitors of bFGF/heparan sulfate proteoglycan binding that is based on the semiquantitative retention of sulfated proteoglycans on cationic nylon filters (7). Using a modification of previously published conditions, samples containing proteoglycan and growth factor are subject to filtration through cationic nylon. While the large negatively charged proteoglycans are retained on the filter, only a small fraction of growth factor is retained unless bound to the proteoglycan (see Fig. 1). Thus, using standard preparations of bFGF and heparan sulfate proteoglycan (isolated from bovine aortic endothelial cell conditioned media [mostly perlecan]), baseline measurements were made to determine the bFGF/heparan sulfate proteoglycan binding affinity. A range of concentrations of potential inhibitors are then included in the reaction, and decreased bFGF retention on the cationic membrane is measured. These data are then analyzed by fitting to a generalized equation for binding using a commercially available software package (Mathematica, version 3.0, Wolfram Research) to determine the binding constant for each inhibitor (binding to either bFGF or heparan sulfate). This assay has been established with bFGF and has been shown to be effective with inhibitors that bind either bFGF or heparan sulfate proteoglycans. Minor modifications of the base assay should allow it to be transferable for the analysis of a large number of heparin-binding proteins.

2. Materials

1. Human recombinant 125I-bFGF is prepared by a modification of the Bolton-Hunter procedure (8) and frozen in single-use aliquots to avoid freeze-thaw cycles.

2. Proteoglycans (E-HS) are purified from bovine aortic endothelial cell conditioned medium as described previously (4) (see Note 1). Briefly, confluent bovine endothelial cells (isolated from freah calf aorta or purchased from Coriell Cell Repositories) are established in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf serum. The medium is then replaced by DMEM without any additives and the cells incubated for 1 h at 37°C as a wash, followed by a 24 h incubation in fresh DMEM. Conditioned medium is collected and centrifuged at 3000g for 30 min at 4°C to remove cell debris. The conditioned media is equilibrated with urea (1 M final) for at least 30 min prior to being applied to an anion-exchange column (Q-sepharose, Pharmacia-LKB) in Tris buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 M urea). The column is washed extensively with 50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 M urea, and the proteoglycan eluted in the same buffer containing 1.5 M NaCl. Proteoglycan fractions are identified using the dimethylmethylene blue (DMMB) dye binding assay (9) and dialyzed extensively in 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl. Proteoglycan concentration is based on the mass of glycosaminoglycan using the DMMB assay (9) with a bovine kidney heparan sulfate standard (Sigma).

3. Incubation buffer: 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 2 mg/mL bovine serum albumin. BSA, biotech grade (Fisher), worked well.

4. Cationic membrane (Zeta-Probe membrane) and Dot-blot apparatus (Bio-Dot Microfiltration Apparatus) (Bio-Rad)

5. Test compound, we have used glycosaminoglycans (Sigma), protamine sulfate (TCI America), and sucrose octasulfate.

|>~-(i) 125I-bFGF

A

Heparin-binding Inhibitor

E-HS

bFGF Binding

Inhibitor

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