Abstract
Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.
Highlights
Ameloblastin domain redistribution during development serves to pattern mineral microstructure
Nucleotide sequencing revealed that each of the seven candidates was preserved in the same reading frame as the GAL4 activating domain (AD), supporting the notion that the encoded protein could interact with ameloblastin
On tangential sections (Fig. 2, g–k) that favor imaging en face visualization of the Tomes’ processes (Fig. 1), immunostaining revealed ameloblastin distribution in a decussating pattern, corresponding to the positional pattern of Tomes’ processes and most visualized in three-dimensional reconstruction of a confocal Z-stack of images (Fig. 2, j and k). This decussating staining pattern corresponds precisely to the arrangement of enamel rods observed in mature mouse enamel, suggesting the ameloblastin was localized to the secretory surface of a rod with little ameloblastin appearing in the interrod matrix shared among adjacent ameloblasts
Summary
Ameloblastin domain redistribution during development serves to pattern mineral microstructure. In the truncated ameloblastin animal, rescue of the enamel rod microstructure abnormalities has been achieved with expression of a full-length ameloblastin transgene [10] These observations suggest that the distributions of ameloblastin domains within the forming enamel matrix play important roles in establishing the enamel microstructure comprising the rod-interrod pattern of organization and in producing the favorable material properties found in mature enamel. Based on these observations, we hypothesized that the N-terminal ameloblastin domain undergoes redistribution to the ameloblast cell periphery, serving to segregate the forming enamel matrix into individual units (rods) of enamel microstructure. We performed in vitro proteasome digestion assays to investigate the potential functional significance of the ameloblastin-Psma interaction
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