Thesis presented October 20, 2023

Abstract:
Glycosaminoglycans (GAGs) are involved in various biological functions, regulating tissue homeostasis and cellular differentiation in the human body. These long polysaccharide chains interact with a wide range of proteins that modulate the cellular behavior, including growth factors, chemokines, cytokines and morphogens. The interactions of GAGs with proteins mainly rely on negatively charged sulfate groups distributed along the polysaccharide chain, which bind to positively charged regions of proteins. Through these sulfation-dependent interactions, GAGs regulate the bioactivity and spatial distribution of proteins, thereby modulating their induced cellular responses. Among GAGs, heparan sulfate (HS) was reported to have a crucial role in regulating the bioactivity of bone morphogenetic proteins (BMPs), and in particular BMP2. As suggested by their name, BMPs are involved in the regulation of bone development. Interestingly, the deficiency of HS polymerization enzymes has been associated to an elevated BMP signaling and to a human disorder characterized by the formation of osteochondroma. In contrast to HS, the role of other GAGs in the regulation of BMP signaling has not been determined.
In this PhD thesis, we aimed at deciphering the role of the different GAGs in BMP signaling, including HS, chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA). To this end, we conducted molecular interaction studies and cellular studies, aiming to provide a comprehensive understanding of the interactions between GAGs and BMP2, and of their role in modulating the signaling. The binding affinities and kinetic properties of the GAG-BMP2 molecular interactions were investigated with biolayer interferometry (BLI). Our findings revealed a much stronger affinity of BMP2 for HS compared to the other GAGs, and differences could be observed between different types of CS. Alongside, we investigated the effect of the interactions between GAGs and BMP2 on the cellular response using previously developed biomaterials, called biomimetic streptavidin platforms. Methodological developments were achieved to allow a platform construction on glass substrates and the automation of the process permitting their functionalization in 96-well plates. Using these platforms, we explored the role of distinct GAGs on BMP signaling, but also of their localization (cell surface or extracellular), an aspect which has not been fully understood. Our results highlighted a distinct role of cell surface and extracellular GAGs, and distinct roles of HS and CS. Specifically, cell surface HS appeared to inhibit BMP2 signaling while cell surface CS promoted it. On the other hand, extracellular HS promoted the signaling, while CS had no observable effect.
The sulfation patterns of GAGs is key factor regulating the binding and bioactivity of proteins. In the literature, the extensively studied interactions of HS with antithrombin III and FGF1 are striking examples of how specific sulfation motifs and sequences are crucial for the bioactivity of proteins. We aimed to explore whether in a similar way the interaction of BMP2 with HS is dependent to specific sulfation motifs. To address this, we prepared a library of HS-oligosaccharides and we investigated the molecular interactions of the synthesized compounds with BMP2 using BLI technique. Our findings revealed a HS trisulfated disaccharide sequence (IdoA(2S)-GlcNS(6S)) that appeared to mediate high-affinity binding with BMP2. However, this sequence was not unique in mediating high-affinity binding and our data suggest that BMP2 exhibits some structural plasticity that allows its binding to various HS sulfation patterns. The multiple findings of this thesis constitute important bricks in the understanding of the complex interplay between GAGs and growth factors.

Keywords:
Glycosaminoglycans, BMP, Biomimetism, Interactions, Signaling