TNAP-mediated hydrolysis of phosphorylated enamel matrix proteins drives local phosphate generation and enamel mineralization

Dental enamel is the most highly mineralized tissue in the body, and its formation requires a precisely regulated supply of Pi. Although phosphate transport in ameloblasts has been studied, how phosphate is generated within the enamel remains unclear. Tissue-nonspecific alkaline phosphatase (TNAP) is a key enzyme in skeletal mineralization, but its functional contribution to enamel formation has not been fully established. Herein, we examined the presence, activity, and catalytic potential of TNAP in porcine enamel matrix. To determine the localization and functional contribution of TNAP within the enamel matrix during the secretory and maturation stages, we performed histochemical staining, gene expression analysis, and immunohistochemistry. TNAP activity, low-molecular-weight phosphate-related compounds, and levamisole-sensitive Pi production were quantified in enamel matrix extracts and ex vivo reactions. Purified enamel proteins were incubated with recombinant human TNAP (rhTNAP) to assess phosphate release. TNAP was detected and showed catalytic activity. PPi and ATP were present; Pi generation was significantly suppressed by levamisole, indicating TNAP dependence. rhTNAP directly hydrolyzed phosphorylated enamel proteins, including amelogenins and enamelin, as well as ATP, and TNAP-dependent hydrolysis of PPi was observed. Proteolytic processing of amelogenins enhanced TNAP-dependent phosphate release. Stage-dependent differences in enzyme distribution and substrate availability suggested regulation during enamel formation. These findings demonstrate that the enamel matrix is not merely a passive site of mineral deposition, but a functionally active compartment of phosphate metabolism, and identify TNAP as a central integrator that coordinates phosphate generation with protease-dependent processing of enamel proteins to support proper enamel mineralization.