Structural and Functional Characterization of Noncollagenous Bone Matrix Proteins

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Mineralization in bones and teeth involves noncollagenous matrix proteins (NCPs) that regulate the formation of mineral. Evolutionary analysis has shown that genes for these NCPs derive from a common ancestor and are clustered together. NCPs in collagen-based calcified tissues form the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family and share a number of biochemical properties and functions. The SIBLINGs are characterized by motifs and domains that determine their interaction with cells, matrix and mineral. Understanding the role of these motifs and domains is thus of fundamental importance for defining how SIBLINGs regulate mineralization. We have focused on one of the SIBLINGs - bone sialoprotein (BSP) - because it is associated with initial mineralization events. BSP has a motif that increases osteoblast-related gene expression and matrix mineralization. It also contains two acidic domains that are believed to be partially responsible for the nucleating mineral and binding to it, although there is only circumstantial evidence for this in vivo. We have shown that mutating both domains has no effect on in vitro osteogenesis. However, unexpectedly, removal of the second motif results in a major inhibition of mineral formation, a novel finding that has important functional implications. As all SIBLINGs, BSP is believed to contain a motif that confers both mineral inhibition and promotion capacity, yet little is known about this motif in BSP. Our objective is to obtain a comprehensive understanding of the functional domains of the BSP molecule by exploiting integrative experimental approaches. We will: 1.carry out cell culture and animal studies to test the activity of the functional domains. These studies will uniquely take into account the physiology of the body. 2.examine functional changes induced by relevant modifications of the BSP structure. 3.investigate the binding efficiency of normal and mutated BSP molecules – so far only studied in non-physiological assays - using methods our group has pioneered for studies in animals. Taking advantage of the knowledge acquired for BSP, in the longer term we intend to extend our studies to other members of the SIBLINGs in order to obtain a comprehensive understanding of how this family of proteins regulates both physiologic and pathological mineralization. HQPs in the proposed research program will be exposed to a unique training environment that is only available in a limited number of laboratories throughout the world. Their training is also expected to be in high demand industry and research institutions. We are requesting annual support for 2 MSc and 1 PhD students. MSc students will be primarily implicated in vitro procedures and studies. The PhD student will assume advanced molecular analyses and studies in animals. The proposed research will provide fundamental information on the function of NCPs necessary for understanding the basic biology of hard tissue formation and how changes in protein structure can lead to disease.