Characterizing structure-function relationships for a homogeneous, highly 6-O-sulfated keratan sulfate from bovine cornea: collagen binding and anti-inflammatory potential.

A structurally homogeneous keratan sulfate (KS) was purified from bovine cornea using an optimized method integrating enzymatic digestion and ion-exchange chromatography. Comprehensive structural analyses, including 1D/2D NMR, FT-IR, and disaccharide profiling, demonstrated a highly sulfated, linear polymer composed of repeating [-3Galβ1-4GlcNAcβ1-] units, with predominant 6-O-sulfation on both galactose and N-acetylglucosamine residues, comprising nearly equal proportions of disulfated (Gal6S-GlcNAc6S, 48.63%) and monosulfated (GlcNAc6S, 51.11%) disaccharides, with only trace amounts of non-sulfated units (<0.3%). HPGPC confirmed a narrow molecular weight distribution centered at approximately 43 kDa. Fluorescence quenching spectroscopy demonstrated that KS interacts with collagen with moderate affinity, a 1:1 stoichiometric ratio, and an enthalpy-driven mechanism. Circular dichroism analysis further suggested that KS association induces partial destabilization of the collagen triple-helical structure. At the single-molecule level, fluorescence correlation spectroscopy confirmed the formation of the KS-collagen complex, with further mechanistic insights provided by molecular docking and molecular dynamics simulations. Functionally, KS attenuated LPS-induced pro-inflammatory cytokine expression in RAW 264.7 macrophages, with network pharmacology implicating multi-target regulation of the TLR4/NF-κB pathway. This study establishes a direct link between the fine structure of corneal KS and its biological functions, providing a quantitative framework for understanding stromal matrix organization and guiding KS-based biomaterial design.