Chemical modification of hyaluronan oligosaccharides to modulate interactions with hyaluronan-binding proteins

Abstract

Introduction: Hyaluronan binding proteins (HABPs) are a diverse family of proteins, united by their ability to interact with hyaluronan (HA) and thereby modulate the function of this ubiquitous glycosaminoglycan (GAG). In particular, the interactions of HA with the secreted anti-inflammatory protein TSG-6 and the major cell surface HA receptor CD44 have been studied in detail. Given the wealth of structural and biophysical information available for the recombinant HA-binding domains of TSG-6 (Link_TSG6; the Link module domain of human TSG-6) and CD44 (CD44_HABD; the Link module of human CD44 with flanking sequences), these represent a suitable model system within which to develop a series of protein-specific HA-based probes. The aim of this PhD was to design and synthesise a series of chemically modified HA oligosaccharides and characterise their differential binding to CD44_HABD and Link_TSG6 with the long-term goal of creating new HA-based probes for HA biology research. Methods: Various chemical groups were conjugated by reductive amination to the reducing terminus of HAnAN oligosaccharides (where A and N correspond to GlcA and GlcNAc at the non-reducing and reducing termini, respectively). Interactions of the modified species (and unmodified oligosaccharides of the same length) with Link_TSG6 and CD44_HABD were studied using biophysical techniques, including isothermal titration calorimetry (ITC), microscale thermophoresis (MST), and nuclear magnetic resonance (NMR) spectroscopy. The solution dynamic 3D structure of a modified oligosaccharide was determined by NMR in order to rationalise its improved binding affinity for Link_TSG6 and direct subsequent chemical modifications. An in vitro assay was used to investigate whether the modified HA oligosaccharides were substrates for the TSG-6-catalysed transfer of heavy chains (HC) from the serum protein inter-alpha-inhibitor. Results: In the first series of experiments, HA6AN oligosaccharides modified with 2-aminobenzoic acid (HA6-2AA), 3-aminobenzoic acid (HA6-3AA) and 2-amino-4-methoxybenzoic acid (HA6-2A4MBA) were found to have increased affinities for Link_TSG6 (~2-fold) compared to HA6AN. The same modifications did not improve the affinity of the oligosaccharide for CD44_HABD20-169, however modified tetrasaccharides (HA4-2AA, HA4-3AA, and HA4-4AA) did show increased affinities compared to HA4AN. The solution conformation of HA4-2AA was determined by NMR spectroscopy and used to inform modelling of a complex of HA6-2AA with either CD44_HABD or Link_TSG6; the latter indicated that the carboxyl moiety of 2AA is orientated towards residue R81 (which is thought to participate in a salt bridge with a GlcA carboxylate group of HA8AN) of Link_TSG6, whereas a similar interaction was not observed for HA6-2AA in the HA-binding groove of CD44_HABD. The aromatic moiety of 2AA was accommodated within a hydrophobic pocket of Link_TSG6 and this observation was targeted in a second series of chemical modifications of HAnAN with hydrophobic groups (including 3-aminopyrazole (3APY), 4-aminoindan (4AI), 5-aminoindan (5AI), and 6-aminoquinoline (6AQ)). Modification of HA8AN with either 3APY or 4AI most favoured binding to Link_TSG6 over CD44_HABD20-169, whereas modification with 6AQ (HA6-6AQ and HA8-6AQ) favoured binding to CD44_HABD20-169 over Link_TSG6. Several modified oligosaccharides were found to be substrates for HC-transfer, including HA4-4AA, HA4-2A4MBA, HA6-2AA, and HA6-3APY whereas the unmodified oligosaccharides, HA4AN and HA6AN were not. Discussion: These studies provide proof of concept that specific chemical modifications of HA oligosaccharides can differentially modulate binding to HABDs. While the modifications described have so far achieved 2-5-fold changes in affinities, the finding that some modifications alter the activity of HAnAN oligosaccharides as substrates in HC-transfer demonstrates the potential of chemically modified HA oligosaccharides as research tools and probe

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anthony Day (Supervisor) & Caroline Milner (Supervisor)