M180 Amelogenin Processed by MMP20 is Sufficient for Decussating Murine Enamel

Abstract

Amelogenin (AMELX) and matrix metalloproteinase-20 (MMP20) are essential for proper enamel development. Amelx and Mmp20 mutations cause amelogenesis imperfecta. MMP20, a protease secreted by ameloblasts, is responsible for processing enamel proteins, including AMELX, during the secretory stage of enamel formation. Of at least 16 different amelogenin splice products, the most abundant isoform found in murine ameloblasts and developing enamel is the M180 protein. To understand the role of MMP20 processing of M180 AMELX, we generated AmelxKO/Mmp20KO (DKO) mice with an amelogenin (M180Tg) transgene. We analyzed the enamel phenotype by SEM to determine enamel structure and thickness, µCT, and by nanoindentation to quantify enamel mechanical properties. M180Tg/DKO mouse enamel had 37% of the hardness of M180Tg/AmelxKO teeth and demonstrated a complete lack of normal prismatic architecture. Although molar enamel of M180Tg/AmelxKO mice was thinner than WT, it had similar mechanical properties and decussating enamel prisms, which were abolished by the loss of MMP20 in the M180Tg/DKO mice. Retention of the C-terminus or complete lack of this domain is unable to rescue amelogenin null enamel. We conclude that among amelogenins, M180 alone is sufficient for normal enamel mechanical properties and prism patterns, but that additional amelogenin splice products are required to restore enamel thickness.

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus

“…In vitro studies have further shown that these mutations or deletion of the N-terminal conserved sequences lead to misfolding and uncontrolled aggregation of amelogenin [17,25,26]. The observation that the thin enamel seen in amelogenin knockout mice lacks prismatic-inter-prismatic architecture supports the notion that matrix-cell binding may be disturbed in the absence of amelogenin [27,28,29]. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta with mutations at the amelogenin N-terminal may be the result of defective amelogenin-cell interactions.…”

Truncated amelogenin and LRAP transgenes improve Amelx null mouse enamel

“…μCT was used to compare mineral densities of incisor and molar enamel in specific regions in CTRNCTg/LRAPTg/ Amelx −/− mice and controls. Adult mouse incisor and molar enamel were assessed for mineralization levels [29]. Hemimandibles with soft tissues removed were scanned in a μCT-40 (Scanco, Brüttisellen, Switzerland) at 70 kV, 114 mA, and 6 μm resolution.…”

“…Hemi-mandibles were embedded in plastic and polished longitudinally for scanning electron microscopy (SEM) analysis for enamel prism organization and enamel thickness [29]. Mandibles from 6 week-old male and female mice and controls were dissected, and erupted portions of mandibular incisors and molars were washed and dehydrated with graded alcohol and acetone, and embedded in Embed812 resin (Electron Microscopy Sciences, Hatfield, PA), ground in a sagittal plane, and polished to 0.25 μm with diamond suspensions (Electron Microscopy Sciences), etched with 0.1M phosphoric acid for 15 s, gold coated and imaged with a Zeiss Evo LS 10 SEM.…”

Mouse Genetic Background Influences the Dental Phenotype

“…Mandibles were fixed overnight in 4% paraformaldehyde and prepared for analysis as described (Gibson et al, 2011) except that scans were performed on a microtomograph imaging system (μCT 40, Scanco Medical AG, Brüttisellen, Switzerland) with 16 μm resolution at 70 kVp (Pugach et al, 2013). The images were processed by three-dimensional reconstruction software (μCT Evaluation Program v6.0, Scanco Medical) and analyzed for enamel and dentin density and volume.…”