Several different investigations have reported the presence of enamel resident serine and metalloproteinases and there is a general agreement that proteinase activity is necessary to form healthy enamel. However, the enamel proteinases are present in minute quantities and are extremely difficult to purify from the forming enamel. Thus, until recently, no proteinase had been cloned from the enamel matrix. This application seeks to replicate the successful molecular biology techniques that were used to clone the enamel resident proteinases name enamelysin and enamel matrix serine proteinase-1. Specifically, PCR- based homology cloning will be used to isolated cDNA fragments of proteinases and/or their inhibitors that are expressed within the enamel organ (Aim 1). Four candidate cDNA fragments and one full-length proteinase cDNA (MMP-14) have already been cloned using this methodology. Since most, if not all, of the proteins secreted into the developing enamel are secreted by ameloblasts, each isolated cDNA fragment will be used for in situ hybridization experiments designed to verify that the ameloblasts produce the proteinase or proteinase inhibitor. Norther blot analysis will also be used to quantify the levels of gene expression (Aim 2). If the ameloblasts do express the gene of interest, then peptide-specific antiserum will be generated for use in Western blot analysis and Immunohistochemical experiments designed to determine if the proteinase or proteinase inhibitor is secreted into the developing enamel. A developmental analysis of expression patterns will also be performed in order to gain insight into the overall proteinase or proteinase inhibitor function during enamel formation (Aim 3). Those proteinase or proteinase inhibitors that are present within the forming enamel will be manufactured as recombinant protein can be assessed (Aim 4). The completion of this experimental protocol will lead to a better understanding of how proteinase, proteinase substrates, and proteinase-inhibitors interact with one another to form healthy dental enamel.
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