Proteinases are present in forming enamel and are known to cleave both amelogenins and enamelins. Although there is general agreement that the degradation of enamel proteins is an important step in enamel formation, determining the actual function of the proteinases is difficult because of the exceedingly low concentrations of the enzymes in the tissue. Thus, it is very difficult to directly isolate these enzymes using biochemical procedures. In the proposed studies, molecular biology strategies will be used to address this problem. Proteinases will be identified based on their expression in the enamel organ (Aim 1). This will be done by performing RT-PCR on mRNA isolated from the enamel organ. Degenerate primers designed for cDNA which codes for conserved regions of the serine or metalloproteinase gene families will be used in PCR to generate the corresponding cDNAs. Results from in situ hybridization experiments will determine if the ameloblasts produce an identified proteinase (Aim 2). To confirm that a proteinase is actually exported into the forming enamel, the proteinase will be cloned from a cDNA library and antisera will be generated for immunolocalization studies. This antisera will also be used in conjunction with Northern analysis to assess the regulation of individual proteinases at different stages of tooth development (Aim 3). Recombinant proteinases will be produced in a mammalian expression system and substrate specificity will be determined using recombinant amelogenin or extracted enamel proteins (Aim 4). Taken together, these studies will provide key information regarding the functional role of proteinases during amelogenesis.
