Hyaluronan binding to link module of TSG-6 and to G1 domain of aggrecan is differently regulated by pH.
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.
Heng
Boon Chin
BC
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.
Gribbon
Philip M
PM
Day
Anthony J
AJ
Hardingham
Timothy E
TE
D053674:Aggrecans / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002645:Chickens; D005786:Gene Expression Regulation; D018960:Hyaluronan Receptors / Q000737:chemistry* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007249:Inflammation; D007700:Kinetics; D018613:Microscopy, Confocal; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D053674:Aggrecans; D015815:Cell Adhesion Molecules; D018960:Hyaluronan Receptors; D009844:Oligosaccharides; D006820:Hyaluronic Acid