A Refined Model for the TSG-6 LINK Module in Complex with Hyaluronan: use of Defined Oligosaccharides to Probe Structure and Function [Glycobiology and Extracellular Matrices]

January 8th, 2014 by Higman, V. A., Briggs, D. C., Mahoney, D. J., Blundell, C. D., Sattelle, B. M., Dyer, D. P., Green, D. E., DeAngelis, P. L., Almond, A., Milner, C. M., Day, A. J.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interact with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide, based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of ten distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a GlcUA ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.
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