Inhibitory effect of porcine amelogenins on spontaneous mineralization

Prior studies in our laboratory suggest that full-length recombinant mouse amelogenin can provide an organized microstructure that regulates the formation of parallel arrays of apatitic crystals under conditions of spontaneous precipitation. The conserved hydrophilic C-terminus is believed to play a key role in these processes. Objective: The present study was carried out to determine the effect of a prominent native porcine amelogenin cleavage product P148, that lacks 25 C-terminal amino acids, on the spontaneous formation of calcium phosphates, in comparison to full-length recombinant porcine amelogenin rP172. Methods: Calcium and phosphate were sequentially added to ice-cold protein solutions (2mg/ml in water, pH<4) to yield final concentrations of 2.5mM Ca and 1.5mM P (vol: 0.06 – 1.8 mL). The solution was brought to 37^oC and the pH was quickly adjusted (KOH) to 7.4±0.1. The change in pH was monitored continuously, as an indication of mineralization progress. The formed minerals were analyzed using TEM and electron diffraction. Results: In the absence of protein, a sharp drop in pH was observed following a short induction period (~20 min), that corresponded to the formation of large amounts of randomly oriented plate-like apatite crystals. In the presence of rP172, a somewhat longer induction period (~60-90 min) was observed along with a smaller decrease in pH that corresponded to the formation of preferred oriented apatite crystals. However, using P148, relatively little pH change was observed for up to 24h and TEM analyses primarily showed the presence of nanoparticles of amorphous calcium phosphate (ACP). Conclusion: Results show that P148 can inhibit calcium phosphate crystallization by stabilizing an ACP precursor phase, while the full-length rP172 promotes the formation of ordered apatite crystals. These results suggest that P148 may have a functional role during enamel formation that is related to its unique structure and properties. Supported by NIDCR grant DE-16376.