Macromolecular crowding by hyaluronan: implications for binding reactions in the pericellular matrix and at the cell surface

In common with many glycosaminoglycan binding growth factors, the interactions of fibroblast growth factor-2 (FGF-2) with heparin/heparan sulfate regulate its biological activity. Consequently, much effort has been expended in characterising growth factor-glycoaminoglycan interactions. Many experiments are performed in vitro in dilute solution. However, the cell surface is an extremely crowded environment. We have, therefore, examined the parameters of FGF-2 binding to a heparin-derived tetradecasaccharide in the presence of two different crowding agents, dextran, a commonly used crowding agent, as well as hyaluronan (HA), which is normally present at the cell surface. The heparin tetradecassaccaride was immobilised through its reducing end onto a biosensor surface and the crowding agents were used in solution. The results show that the observed on-rate rate of association (kon) decreased over two-fold with increasing concentrations of both crowding agents. The slope of initial rate of association similarly decreased, indicating that as the solution became more crowded the diffusion of FGF-2 becomes increasingly limited. One thousand kDa HA was shown to be the most effective crowding agent, as diffusion became limited at concentrations above 0.5 mg/ml, compared to 1 mg/ml and 10 mg/ml for 100kDa HA and dextran, respectively. Such concentrations of hyaluronan are readily found extracellularly. The results, therefore, suggest that the effect of crowding at the cell surface and in the extracellular matrix may play an important role in governing the interactions of at least FGF-2 with its heparan sulfate co-receptor.