This new application focuses on the determination of the mechanism by which the predominant extracelluar enamel matrix protein, amelogenin, regulates initial enamel mineral formation and tissue organization. Based on extensive prior findings, our overall hypothesis is that phosphorylation of serine-16 modulates the structure and hierarchical assembly of native (phosphorylated) full-length amelogenin, resulting in its greatly enhanced capacity to stabilize amorphous calcium phosphate (ACP) nanoparticles. We further hypothesize that subsequent proteolytic modification of native full-length amelogenin is required to promote the alignment and transformation of ACP to ordered bundles of apatitic crystals, as seen in the secretory stage of enamel development. We propose that enamelysin plays a critical role in this transformation process. The proposed studies are designed to provide fundamental insight into how matrix proteins, like amelogenin, control mineralization and structure in mineralized tissues. As a long-term goal, our findings should aid in the development of novel approaches for the regeneration and repair of diseased or damaged dental enamel. Given the high prevalence of dental caries, there is a tremendous need for restorative procedures that are superior to those presently available. Hypotheses will be tested in vitro, primarily using native amelogenins that contain a single phosphorylated site, and in vivo, using enamelysin null mice.
Four specific aims will be carried out using multiple complementary approaches, including: dynamic light scattering, transmission electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy, Raman microspectroscopy, small angle x-ray scattering, and cryomicroscopy. Specifically:
Aim 1. To determine the effect of phosphorylation on the step-wise hierarchical assembly of native full-length amelogenin, to test the hypothesis that phosphorylation affects the formation and structure of amelogenin oligomers and their subsequent higher-order assembly;
Aim 2. To determine the mechanism by which phosphorylated native full-length amelogenin effectively stabilize nanoparticles of ACP, to test the hypothesis that phosphorylation of the single serine-16 site affects protein conformation and structural changes that uniquely enhance the capacity of the full-length amelogenin to interact with forming ACP nanoparticles;
Aim 3. To conduct in vitro studies to test the hypothesis that the alignment and subsequent transformation of ACP nanoparticles seen in vivo to ordered bundles of hydroxyapatite (HA) crystals is induced by specific proteolytic modifications of native full-length amelogenin;
and Aim 4. To verify in vivo that enamelysin plays an essential role in regulating the transformation of aligned ACP particles to ordered bundles of HA crystals, by testing the hypothesis that disordered ACP-like minerals will persist in vivo, in the absence of enamelysin, in contrast to what is seen in wild type mice.

Public Health Relevance

The proposed studies are designed to determine the mechanism by which native enamel matrix proteins regulate initial enamel mineral formation and structural organization. The successful completion of this work will provide new insights into how all hard tissues form and aid in the development of improved methods for regeneration of tooth enamel. Given the high prevalence of dental caries, there is much need for new and more effective restorative procedures.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Skeletal Biology Development and Disease Study Section (SBDD)
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Wan, Jason
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Forsyth Institute
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Yamazaki, Hajime; Beniash, Elia; Yamakoshi, Yasuo et al. (2017) Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide. Front Physiol 8:450
Connelly, Christopher; Cicuto, Thomas; Leavitt, Jason et al. (2016) Dynamic interactions of amelogenin with hydroxyapatite surfaces are dependent on protein phosphorylation and solution pH. Colloids Surf B Biointerfaces 148:377-384
Kwak, S Y; Yamakoshi, Y; Simmer, J P et al. (2016) MMP20 Proteolysis of Native Amelogenin Regulates Mineralization In Vitro. J Dent Res 95:1511-1517
Kwak, Seo-Young; Kim, Sonia; Yamakoshi, Yasuo et al. (2014) Regulation of calcium phosphate formation by native amelogenins in vitro. Connect Tissue Res 55 Suppl 1:21-4
Margolis, Henry C; Kwak, Seo-Young; Yamazaki, Hajime (2014) Role of mineralization inhibitors in the regulation of hard tissue biomineralization: relevance to initial enamel formation and maturation. Front Physiol 5:339
Fang, Ping-An; Margolis, Henry C; Conway, James F et al. (2013) CryoTEM study of effects of phosphorylation on the hierarchical assembly of porcine amelogenin and its regulation of mineralization in vitro. J Struct Biol 183:250-7