Novel model systems for the investigation of polyvalent protein-hyaluronan interactions

The gel-like hyaluronan (HA) rich coat that surrounds many cells has been linked to a variety of vital cellular functions, but the regulatory mechanisms at the cell-HA matrix interface remain poorly understood. For the thorough investigation of specific interactions between the cell surface and HA as well as the structural properties of this supra-molecular matrix, it is desirable to switch from the complex cellular environment to simplified systems. This work aims to find routes towards the development of new in-vitro model systems of HA-rich coats. One approach relies on the immobilization of ectodomains of HA's main cell surface receptor, CD44, on supported lipid membranes (SLBs). Model surfaces with tunable receptor density are exploited to investigate the polyvalent interaction between HA and CD44 in a biologically relevant arrangement. On surfaces that provide a high density of receptors, HA binding increases in a sigmoidal fashion with molecular weight, and becomes reversible below 30 kDa. The physico-chemical properties of these HA films reveal many similarities between the binding behavior of HA chains and flexible polymer chains adsorbing to a homogeneously attractive surface. On surfaces with low receptor surface density binding of HA of sufficiently high molecular weight is irreversible for all, and proportional to the amount of receptors. Quantifying the number of receptors that are available per HA chain provides insight how polyvalent interactions stabilize HA binding on receptor covered surfaces. HA binding is though regulated by both polyvalency and the intrinsic affinity of individual receptors, and we outline a method to disentangle the contributions of both effects. Another approach envisages the immobilization of HA via a terminal biotin moiety at controlled anchor separation. The binding and activity of biotin-receptors on gold and silica is systematically examined with the objective to selectively functionalize gold nanostructured surfaces. We find that streptavidin adsorbs in a functional conformation on gold while resisting binding to silica. First attempts to exploit this selectivity on gold nanostructures are presented. The confinement of the model systems to solid supports enables qualitative and quantitative characterization by complementary surface sensitive techniques. Here, quartz crystal microbalance with dissipation monitoring and ellipsometry - alone or in combination - as well as microinterferometry provide detailed insight into the formation, stability and morphology of the model surfaces and the HA films, and involved molecular interactions. The developed model systems with tunable properties can serve as a well-controlled experimental platform for the investigation of the interactions between proteins and HA in a supra-molecular context, and between cells and HA-matrices