BMPER interactions in BMP signalling and disease

The extracellular matrix (ECM) is comprised of multiple agonists and antagonists responsible for regulating Bone Morphogenetic Protein (BMP) signalling. Bone Morphogenetic Protein Endothelial Cell Precursor-Derived Regulator (BMPER) can either be an agonist or antagonist depending on its context and concentration relative to BMP and interactions with other proteins. BMPER is known to regulate BMP through its interactions with BMP, other BMP-binding regulators such as Twisted Gastrulation (Tsg) and Chordin, and BMP receptor type I (BMPR-I). The BMPER domains that interact with BMP and Chordin, respectively, had been previously mapped to inform how BMP signalling was regulated through a series of synergistic or competitive mechanisms among BMP regulators. However, the BMPER domains required to bind Tsg and BMP receptors are still unknown. In this thesis, four overlapping von Willebrand Factor C (vWFC) domain doublet pairs that represent the N-terminal half of BMPER (N-BMPER) were generated and used with N-BMPER in binding studies to find the binding sites for BMPR-IA/B, BMP receptor type II (BMPR-II) and Tsg. N-BMPER bound BMPR-II with low affinity where a fast off-rate of N-BMPER may be required to free BMPR-II for signalling. Tsg bound N-BMPER but not the vWFC doublet domains, suggesting the Tsg epitope spans more than two domains. BMPER/BMPR-I/BMP has been modelled as a transient ternary complex involved in the regulation of BMP for signalling. Here, the formation of an N-BMPER/BMPR-II/BMP ternary complex was reported but its functional significance is still unknown. To study the reported pro-BMP role of BMPER mediated by Chordin, the role of autocatalytic cleavage product of BMPER (N-BMPER) and Tolloid-mediated partial cleavage product of Chordin (Chordin without vWFC1, Delta N-Chordin) in BMP regulation was investigated. N-BMPER was a more potent BMP inhibitor compared to Delta N-Chordin. Together, N-BMPER and Delta N-Chordin were able to co-operatively inhibit BMP and N-BMPER formed a ternary complex with Delta N-Chordin and BMP. These data suggest that the more diffusible N-BMPER may work cooperatively with Chordin or its cleavage product. The role of a BMPER missense mutation (P370L) at the GDPH autocatalytic cleavage site which contributes to the rare skeletal disorders Diaphanospondylodysostosis (DSD) and Ischiospinal dysostosis (ISD) was investigated. The mutant protein was secreted but the mutation prevented intracellular cleavage at the GDPH site. However, BMPER-P370L was extracellularly cleaved at a downstream site that was independent of matrix metalloproteinase 9 (MMP-9) and furin. BMPER and BMPER-P370L were equally potent BMP inhibitors and exhibited similar high-affinity binding with Delta N-Chordin. Taken together, this thesis provided further insights on how different higher-order complexes involving BMPER could be involved in BMP regulation and broadened our understanding on BMPER function in the ECM with Chordin, and at close proximity to the cell surface with the BMPR-II receptor.