STRUCTURE/FUNCTION OF BONE GLYCOPROTEINS

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Bone is a composite of diverse cell types, extracellular matrix and mineral. Normal function is dependent upon the co-ordinate effort of several cell types, but it is the osteoblast under the influence of local and systemic factors that is primarily responsible for the synthesis, organization and deposition of an extracellular matrix that becomes mineralized during bone formation. When the balance between resorption and deposition of the bone matrix is disrupted, a variety of metabolic bone diseases such as osteoporosis, Paget's disease, and osteopetrosis can result. The goal of these studies is to define the role of the extracellular matrix deposition in bone formation. These studies will lead to a greater understanding of bone homeostasis and thereby have broad application to the spectrum of metabolic bone diseases and the pathology of connective tissues. The specific aim of this project is to apply cutting edge molecular and cell biology techniques to define the role of biglycan and bone sialoprotein in the formation of the bone extracellular matrix. Many of the proteins of the bone matrix undergo extensive post-translational modifications including glycosylation, phosphorylation and sulfation. Biological assays have been hampered by the requirement for denaturing solvents during purification and inherently poor yields from tissues. To produce post-translationally processed recombinant glycoproteins in chemical amounts, we will develop a novel system for vaccinia virus-driven expression in osteoblast-like cells. This important advance in the study of matrix biology will enable the role of the complex post-translational modifications of these molecules to be defined. The ultimate goal of this research is to determine the function of these molecules and define their interaction with the extracellular matrix, the resident cells, and growth factors. This will be achieved by (a) transient, short-term expression and (b) stable, long-term expression of wild-type and mutant extracellular matrix glycoproteins. Intermolecular interactions will be studied (a) by in cellulo expression in osteoblast cultures that deposit mature collagenous extracellular matrices and (b)by in vitro binding studies, primarily based on the technique of surface plasmon resonance available in the BIAcore System (Pharmacia).