Abstract
Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.
Highlights
Fibroblast growth factors (FGFs) are broad-range morphogens that have significant functional roles in early and late embryonic development
Fibroblast growth factor-2 (FGF2) purified with heparin-sepharose chromatography is likely to contain trace contamination of heparin stripped from the resin during the elution step, which is not the case for FGF2 purified by ionexchange chromatography
FGF2 mediates its biological activity by binding to specific cell-surface high-affinity tyrosine kinase receptors and heparin-like glycosaminoglycan [3,4]
Summary
Fibroblast growth factors (FGFs) are broad-range morphogens that have significant functional roles in early and late embryonic development. FGFs are thought to be implicated in renewal processes in the adult by promoting neuronal stem cell survival, neuron migration, and wound healing and tissue repair [1,2]. FGF2 is a potent angiogenic molecule that in vivo and in vitro stimulates smooth muscle cell growth, wound healing, and tissue repair [3,4]. It has been shown that FGF2 may stimulate haematopoiesis [5] and potentially plays an important role in the differentiation and/or function of the nervous system [6], the eye [7], and the skeleton [8]
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