ENAMEL WITHOUT ENAMELIN

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DESCRIPTION (provided by applicant): Enamel Without Enamelin: Our long-term objective is to understand the molecular mechanisms of normal and diseased dental enamel formation. Mutations in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. In this investigation, we study enamelin's functions during amelogenesis. The two Specific Aims propose to investigate enamelin function by: SA 1: characterizing enamel formation in the absence of mouse enamelin expression. SA 2: characterizing enamelin protein structural and functional properties. In Specific Aim 1 we generate a knock-in mouse that replaces the enamelin gene with the coding region for bacterial 13-galactosidase fused to a mouse nuclear localization signal (NLS-lacZ). We then compare enamel formed in the absence of enamelin expression to enamel formed in the wild-type mice. Expression of the enamelin-NLS-lacZ reporter gene will be characterized by X-gal staining in frozen sections and whole mounts. Biochemical analyses will include SDS-PAGE, Western blotting, and amino acid analysis. Mineral analyses will include Light Transmission Microscopy (LTM), Scanning Electron Microscopy (SEM) to look for pitting, chipping or other surface defects, Transmission Electron Microscopy (TEM) to determine crystal morphology, X-ray microanalysis to determine elemental composition, Selected-Area Electron Diffraction (SAED) to determine mineral type, Dual-Energy X-ray Absorption (DEXA) to measure mineral density, histomorphometric measurement and Faxitron radiology for high-resolution X-ray imaging to determine enamel thickness, and Micro-computed tomography for 3-D reconstruction for mineral area and volume. In Specific Aim 2 enamelin proteolytic cleavage products are isolated from the pig enamel matrix and characterized to reveal their posttranslational modifications. How these enamelins influence crystal growth in vitro is analyzed using pH stat and constant composition assays of hydroxyapatite formation, seeded growth assays for inhibition of hydroxyapatite growth, and by analysis of hydroxyapatite growth habit by X-ray diffractometry This study will determine if enamelin catalyzes enamel crystal nucleation and crystal elongation, or controls crystal habit. The findings will establish an appropriate knowledge base for framing future hypotheses concerning the molecular mechanisms by which enamelin controls enamel biomineralization.