The main objective of this proposal is to understand the molecular mechanisms involved in the mineralization of the extracellular matrix of bone and dentin, and more generally, the mechanism of construction of all biogenic mineralized tissues. Our basis hypothesis is that acidic macromolecules of the matrix direct the deposition of the mineral cations, with these macromolecules in turn being localized by their interaction with the structural component of the matrix. In dentin we believe the important acidic macromolecule is phosphophoryn (PP) and the structural component is the collagen fiber network. To examine this hypothesis in detail we need to know: 1) the structure and sequence of the PP; 2) the nature of the interaction between PP and calcium ion; 3) the nature of the interaction between PP and collagen; 4) the calcium binding and apatite nucleation properties of the PP-collagen complex; and 5) the pathway by which PP enters into the extracellular matrix to reach the collagen in dentin. Three specific approaches are to be used to gain this knowledge: 1) A cDNA library has been prepared for rat odontoblasts, and a few clones apparently related to PP located in screening experiments. These clones will be used to sequence rat incisor PP. Peptide sequencing techniques will also be used for confirmation studies. 2) Anti-rat incisor PP antibody has been prepared and will be used at the light and electron microscopic levels to immunohistochemically localize the PP and determine its route of secretion into the mineralization front. These studies will be correlated with the processing of the pro-PP molecule. 3) Chemical and physicochemical studies of interactions between PP, collagen and the calcium ion binding process will be continued and extended. These approaches will all be carried out within the broader context of bone and other mineralized tissues so that any mechanistic insights gained could be applied to those systems. Mineralization is a crucial biological process, we expect this work to lead to an enlarged understanding of this event.
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