Analysis of CD44-hyaluronan interactions in an artificial membrane system: insights into the distinct binding properties of high and low molecular weight hyaluronan.

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

On the adsorption behavior of biotin-binding proteins on gold and silica.

Streptavidin (SAv), avidin (Av), and neutravidin (NAv) have become widely used molecular tools in biotechnology thanks to their remarkable affinity for biotin. Their tetravalency renders these molecules particularly interesting for the functionalization of solid-liquid interfaces. Using the quartz crystal microbalance with dissipation monitoring, we systematically investigate the deposition of biotin-binding proteins to two surfaces that are popular in biotechnology: gold and silica. We find that simple physisorption of biotin-binding proteins is a viable method to confer biotin-binding functionality to gold surfaces. Both SAv and Av form dense, stable protein monolayers that retain biotin-binding activity and are largely inert to the unspecific binding of bovine serum albumin. Furthermore, we report that SAv resists adsorption to silica over a wide range of pH and ionic strength. The contrast in the binding behavior of SAv on silica and on gold suggests a simple strategy for the selective biofunctionalization of nano- or microstructured surfaces.