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J. Biol. Chem., Vol. 283, Issue 47, 32294-32301, November 21, 2008

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Hyaluronan Binding to Link Module of TSG-6 and to G1 Domain of Aggrecan Is Differently Regulated by pH^*

Boon Chin Heng, Philip M. Gribbon^¶, Anthony J. Day¹, and Timothy E. Hardingham²

From the Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom, Abbott Vascular Inc., Santa Clara, California 95054, and ^¶GlaxoSmithKline Research and Development Ltd., Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, United Kingdom

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 HA₈ and HA₁₀ 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.

Received for publication, May 30, 2008 , and in revised form, September 15, 2008.

^* This work was supported by the Seikagaku Corporation (Tokyo, Japan), Arthritis Research Campaign Grants D0540 and 16539 (to A. J. D.), and the Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence may be addressed. Tel.: 44-161-275-1495; E-mail: anthony.day{at}manchester.ac.uk. ²To whom correspondence may be addressed. Tel.: 44-161-275-5511; E-mail: timothy.e.hardingham{at}manchester.ac.uk.

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