Abstract
The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K(D) determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.
Highlights
Curonic acid (GlcA) [1, 2]
TSG-6, an HA-binding protein associated with inflammation [15, 16], is another member of the Link module superfamily containing a single Link module, which binds to HA, but in contrast to other members it binds to chondroitin 4-sulfate (C4S) (see [17])
As the binding of Link_TSG6 to HA in this experiment would increase by only 2% the mass of the HA-Fluorescein Isothiocyanate (FITC)-Link_TSG6 complex, this was unlikely to have any significant effect on the diffusion coefficient of HA
Summary
Materials—Unless otherwise stated, all reagents, chemicals, and consumables were obtained from Sigma. Fractions (250 l) collected containing the FITC-labeled protein were quantitated by a bicinchoninic acid protein assay kit (Sigma) For both the G1 and Link_TSG6 the labeling was ϳ2–3 FITC mol/mol, based on the fluorescence of a known concentration of protein in solution and free FITC standard. This level of FITC labeling was sufficient to obtain a strong signal for confocal FRAP bleach recovery experiments at the protein concentrations used and over the pH range investigated. The affinity of G1-HA binding was determined at different pH using mixtures of FITC-G1 (100 g/ml) and HA (800 kDa, 100 g/ml), prepared in citrate buffers (containing 100 mM Naϩ ion) at pH values ranging from pH 3.5 to 8.0. Based on experimental data from this study, a 10-mer binding site was assumed for FITC-Link_TSG6, whereas a 12-mer binding site was assumed for FITC-G1
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