Abstract
The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.
Highlights
The superficial layer of a tooth is enamel, the hardest tissue known in vertebrates
We focus on how the adsorption of amelogenin to HAP crystals affects its degradation by enamel proteinases, Matrix metalloproteinase 20 (MMP20) and KLK4
Because UV detection showed only a trace amount of MMP20 and KLK4 bound to HAP during digestion process, their effects on the measurement of amelogenin amount were not included from our analysis
Summary
The unique morphological structure and outstanding mechanical properties of the enamel make it different from other mineralized tissues in the body, such as bone, dentin, and cementum. The unique morphology and organization of enamel crystals determine its excellent mechanical characteristics while raise a persistent question of how these enamel crystals form in such a special shape. Solving this puzzle will advance our knowledge of the basic principle of amelogenin-mediated mineralization during enamel development, help us to better understand the fundamental mechanism of an enamel disease, amelogenesis imperfecta, and provide useful information for future acellular tooth enamel regeneration
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