Functional and structural characterization of Bone Morphogenetic Protein 2 and the antagonist Noggin

The bone morphogenetic protein-2 (BMP-2) is a multifunctional growth factor involved in numerous functions in the development and maintenance of tissues. For instance, during chondrogenesis, BMP-2 is known to induce the differentiation of mesenchymal stem cells into mature chondrocytes, which are responsible for cartilage synthesis. But BMP-2 also promotes the differentiation of mature chondrocytes into hypertrophic chondrocytes, which are involved in cartilage mineralization. This biological activity confers a great clinical potential to BMP-2, notably for the regeneration of osteo-articular tissues. However, BMP-2 induces its biological functions through signalling pathways that are tightly regulated by an intricate combination of factors, e.g. receptors and antagonists. The lack of knowledge of this signalling leads to the apparition of several side effects. In this context, our project aims a better understanding of the interaction between BMP-2 and its antagonists Noggin and Gremlin-1. This knowledge will further allow us to modify the affinity of the BMP for its two antagonists, aiming to enhance the biological functions of the growth factor. Towards this task, we first produced BMP-2, Noggin and Gremlin-1. Thus, these three proteins were expressed as inclusion bodies in E.coli and subsequently refolded and purified. The native structures of BMP-2 and Noggin were confirmed using a combination of biochemical methods, while their biological activities were measured using in vitro cell-based assays. Furthermore, we solved the X-ray crystal structure of the BMP-2:Noggin complex, which revealed details of the protein-protein interface and allowed us to design relevant BMP-2 mutants. Afterwards, to better study the biological activity of BMP-2 and its variants, we developed new in vitro cell-based assays to visualize the kinetics of the chondrogenic/hypertrophic differentiation. Thus, we combined Incucyte S3, a Live-cell imaging system, with chondrogenic markers that are specific to mature or hypertrophic chondrocytes (i.e. mature chondrocytes differentiation was assessed using Col2a immunohistochemistry, while the hypertrophic phenotype was followed by measuring tissue mineralization with Calcein green staining). Finally, four BMP-2 mutants were produced, studied and compared to their wild-type counterpart using the biochemical and biological methods developed in this study. Two of them (i.e. BMP-2 P36K and BMP-2 L100K) showed promising biological activities. Thus BMP-2 L100K exhibits increased biological activity compared to wild-type, while BMP-2 P36K, combined with Noggin, is able to trap the majority of cells in a mature chondrocyte state