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Abstract The glycosaminoglycan hyaluronan (HA) is a ubiquitous, non-sulphated polysaccharide with diverse biological roles mediated through its interactions with HA-binding proteins (HABPs). Most HABPs belong to the Link module superfamily, including the major HA receptor, CD44, and secreted protein TSG-6, which catalyzes the covalent transfer of Heavy Chains (HC) from inter-a-inhibitor (IaI) onto HA. The structures of the HA-binding domains (HABD) of CD44 (HABDCD44) and TSG-6 (LinkTSG6) have been determined and their interactions with HA extensively characterized. The mechanisms of binding are different, with LinkTSG6 interacting with HA primarily via ionic and CH−π interactions, whereas HABDCD44 binds solely via hydrogen bonds and van der Waals forces. Here we exploit these differences to generate HA oligosaccharides, chemically modified at their reducing ends, that bind specifically and differentially to these target HABPs. Hexasaccharides (HA 6 AN) modified with 2- or 3-aminobenzoic acid or 2-amino-4-methoxybenzoic acid (HA 6 -2AA, HA 6 -3AA, HA 6 -2A4MBA, respectively) had increased affinities for LinkTSG6 compared to unmodified HA 6 AN. These modifications did not increase the affinity for CD44HABD. A model of HA 6 -2AA (derived from the solution dynamic 3D structure of HA 4 -2AA) was docked into the LinkTSG6 structure, providing evidence that the 2AA-carboxyl forms a salt bridge with Arginine-81. These modeling results informed a 2 nd series of chemical modifications for HA oligosaccharides, which again showed differential binding to the two proteins. Several modifications to HA 4 and HA 6 were found to convert the oligosaccharide into substrates for HC-transfer, whereas unmodified HA 4 and HA 6 are not. This study has generated valuable research tools to further understand HA biology.
Dodd et al. (Thu,) studied this question.