Enzymatic Digestion of Amelogenin Protein Bound to Apatite is Hindered

During enamel maturation proteases eliminate most of the extracellular matrix proteins and facilitate the formation of a highly mineralized hard tissue with a unique and remarkable microstructure. Objective: The purpose of this study was to determine whether amelogenin proteolysis by MMP-20 or trypsin is altered when the assembled amelogenin protein is bound to apatite mineral. Method: Recombinant human full-length amelogenin (0.4 mg/ml) was immobilized onto a fluoroapatite (FAP) glass-ceramic substrate and subsequently digested by MMP-20 (0.03µg/l) or trypsin (0.5 g/l) for up to 120 h at 37ºC and pH 7.5. Four samples were prepared for each group. The substrate surface with the immobilized protein was imaged by atomic force microscopy after 0, 2, 4, 6, 12, 24, 48, 72 and 120 h of enzyme application. Results: Tapping-mode AFM-imaging revealed that amelogenin nanospheres with a diameter of 20 to 25 nm adhered homogeneously to both glass matrix and FAP crystals of the substrate. Amelogenin nanospheres attached to the glass matrix decreased gradually in number and size over time when exposed to MMP-20 or trypsin and completely disappeared at 6 or 24 h, respectively. In contrast, amelogenin nanospheres attached to FAP crystals remained even after 120 h of enzyme application. The size of the nanospheres increased slightly (to about 30 nm) when MMP-20 was applied, while larger diameters of up to 100 nm were observed for trypsin-treated samples. Nanospheres, however, could be removed by NaOCl or using contact-mode AFM indicating their proteinacious nature. Conclusions: Apatite binding hinders digestion of assembled amelogenin proteins by MMP-20 and tryspin. We suggest that the steric structure of amelogenin changes when bound to apatite, altering exposed domains which results in a delayed proteolysis. Support: NIH/NIDCR P01DE09859 and R21DE015416