title: A refined model for the TSG-6 link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
abstract: 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 interacts 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 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 10 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 (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 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.
authors.affiliation: From the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Higman
authors.forename: David C
authors.initials: DC
authors.lastname: Briggs
authors.forename: David J
authors.initials: DJ
authors.lastname: Mahoney
authors.forename: Charles D
authors.initials: CD
authors.lastname: Blundell
authors.forename: Benedict M
authors.initials: BM
authors.lastname: Sattelle
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Dyer
authors.forename: Dixy E
authors.initials: DE
authors.lastname: Green
authors.forename: Paul L
authors.initials: PL
authors.lastname: DeAngelis
authors.forename: Andrew
authors.initials: A
authors.lastname: Almond
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Milner
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
mesh_terms: D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010060:Ovulation / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: C497483:CD44 protein, human; D015815:Cell Adhesion Molecules; D018960:Hyaluronan Receptors; D009844:Oligosaccharides; C072273:TNFAIP6 protein, human; D006820:Hyaluronic Acid
keywords: Chondroitin; Defined Sugars; Hyaluronate; Isothermal Titration Calorimetry; Molecular Modeling; NMR; TSG-6