A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan

Abstract

Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-6 during inflamma-tion and ovulation. Results: NMR spectroscopy on TSG-6 in the presence of defined sugars provided restraints that allowed modeling of a refined hyaluronan/TSG-6 complex. Conclusion: TSG-6 binding causes bending of hyaluronan that explains its condensation of this polysaccharide. Significance: This provides novel structural insights into protein-hyaluronan interactions. 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 inter-acts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid 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 confor-mation. 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 10 distinct HA oligosaccharides (from 4-to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligo-mers and an octasaccharide in which a single sugar ring was 13 C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interac-tions 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 provid-ing 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-␣-inhibitor onto HA, a process known to be essential for ovulation.