The functional transformation of a predominantly organic matrix into a 95 percent inorganic tissue of high structural organization and mechanical integrity (e.g., enamel, requires a spatial and timely accurate cleavage and degradation of matrix proteins). The metalloproteinase 20 (enamelysin) hydrolyses amelogenin proteins, the major protein component of the developing enamel matrix, at the C-terminal and N-terminal resulting in protein fragments including a 20 kDa amelogenin (Amg20), a tyrosine rich amelogenin peptide (TRAP) and a leucine rich amelogenin peptide (LRAP). Enamel maturation and the formation of the unique enamel microstructure must be a result of the involvement of these protein fragments by presumably controlling growth rate, orientation and morphology of apatite crystals. All MMP-20 cleavage sites in amelogenin contain an upstream proline residue that may be important for optimal enzyme function. A mutation of the proline near the TRAP cleavage site, resulted in reduced amelogenin hydrolysis in vitro, and has been linked to amelogenesis imperfecta in vivo. In this proposal, we aim to alter hydrolysis of amelogenin by MMP-20 at the N- and C-terminal hydrolysis sites in vivo and in vitro, to identify the role Amg20, TRAP and LRAP fragments in enamel biomineralization. We hypothesize that the proline residues nearby the MMP-20 cleavage sites in amelogenin, are critical for MMP-20 binding to amelogenin to form Amg20, TRAP and LRAP and that these cleavage products have unique functional roles in enamel biomineralization. The hypotheses will be tested by following specific aims: 1). To determine the role of proline residues near the MMP-20 proteinase cleavage sites, on the kinetics of amelogenin degradation by MMP-20 in vitro. 2). To determine the role of N- and C-terminal amelogenin hydrolysis by MMP-20 in enamel biomineralization in vivo by creating transgenic mouse models. 3). To determine the role of N- and C- terminal amelogenins created by MMP-20 and altered hydrolysis by mutations, on hydroxyapatite crystal growth in vitro. This proposal is designed to clarify mechanisms of enamel development, especially regarding the formation and function of Amg20, TRAP and LRAP during enamel maturation. This project will gain insights in the interactions between MMP-20 and amelogenin proteins at the molecular level as well as their interactions with forming apatite mineral. Furthermore the proposed studies will help us to gain knowledge in the pathogenesis of amelogenesis imperfecta.
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