Amyloid Fibers Guide Mineralization in Enamel Protein Matrices

Objectives: The mechanism guiding enamel mineralization has not been fully described, in part because the organic matrix is completely degraded during tissue maturation. Here we investigate the self-assembly of amelogenin, the main protein present in developing enamel, into an amyloid in porcine enamel extract and in recombinant human amelogenins.
Methods: Bioinformatics fibrillation prediction methods were used to identify regions within amelogenin prone to aggregation. Self-assembly of amelogenin, C-terminus modified amelogenin and amelogenin-derived peptides was characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), fluorescence microscopy and spectroscopy, dot blot and circular dichroism.
Results: Porcine enamel extract tested positive for amyloids with dot blot and Thioflavin T analyses; microscopy images showed that it is composed of nanoribbons of similar dimension and morphology as those described in vitro for recombinant amelogenin (rH174). Sequence analyses by seven different algorithms predicted a region near the N-terminus of human amelogenin, 14P2, to have a high β-aggregation score. Experimentally, 14P2 self-assembled into nanoribbons similar to the full-length protein. When 14P2 was deleted from amelogenin, ribbons no longer formed. 14P2 and amelogenin nanoribbons fluoresced in the presence of Thioflavin T, indicating the presence of amyloids in vitro. With increasing solution pH acicular growth of nanocrystals occurred in association with amelogenin ribbons.
Conclusions: This study describes for the first time the presence of an amyloid involved in the control of extracellular tissue mineralization. We identified a specific region in the human amelogenin responsible for β-sheet aggregation and nanoribbon development. Our results suggest that amelogenin amyloids act as scaffolds for uniaxial growth of apatite nanofibers in enamel and are the supramolecular structures that govern the mineralization process in developing human teeth.