Insight from NMR observations on glycosaminoglycan interactions with the Link module of TSG-6
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Tumor necrosis factor stimulated gene-6 (TSG-6) is a glycosaminoglycan (GAG) binding protein expressed in inflammatory diseases and inflammatory-like processes. The Link module, one domain of TSG-6, is well known for its interactions with hyaluronan (HA), which play important roles in protecting against cartilage matrix destruction during inflammation and stabilizing the HA-rich extracellular matrix (ECM) during ovulation. Interactions with HA, and to some extent heparin/heparan sulfate (Hp/HS), have been structurally studied, indicating that HA and Hp/HS interact with the Link module at totally different binding sites. However, its interactions with chondroitin sulfate (CS) have yet to be adequately described. In this thesis, we isolated a well-defined homogeneous hexasaccharides of CS using enzymatic digestions followed by chromatographic separation and determined their structures using Nuclear Magnetic Resonance (NMR) methods. A spin-labeled analog of a CS hexasaccharide was synthesized as well. These, in turn, were used in further binding studies with the Link module. The interactions between the Link module and CSs were monitored by NMR chemical shift perturbation (CSP), suggesting possible CS binding sites and multiple binding modes for the Link module. The production of a Link dimer during the binding process was confirmed by measuring relaxation rates of 15N sites in the Link module as well as by dynamic light scattering (DLS) measurements. Paramagnetic relaxation enhancement (PRE) data from 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the Link module in the absence and presence of the spin labeled analog of CS added to binding site definition. A solution structure of the induced dimer of the Link module was produced, using triple resonance assignment methods for NMR resonance assignments and Nuclear Overhause Effect (NOE) and Residual Dipolar Coupling (RDC) data for structural constraints. Finally, a dimer model of Link module with the 4,4,4 sulfated, TEMPO adduct of a CS hexasaccharide bound was generated using CSP, PRE, and RDC restraints in docking program (HADDOCK). This provides insights into the cross-linking of CS in the ECM that can play an additional role in the remodeling and stabilization of the ECM during inflammation.