Essential role of amelogenin phosphorylation in tooth enamel formation

这项新的 R01 提案旨在阐明单个氨基酸磷酸化在 最丰富的牙釉质基质蛋白，釉原蛋白，在牙釉质形成的调节中发挥作用。提议 研究建立在我们广泛的新发现的基础上，这些新发现表明天然植物中单个丝氨酸位点 (S-16) 的磷酸化 釉原蛋白对于高度有序的牙釉质结构的形成至关重要。使用新型敲入 (KI) 小鼠 我们实验室开发的模型将 S16 替换为丙氨酸，可防止釉原蛋白磷酸化， 我们现在首次在体内证明牙釉蛋白磷酸化在 釉质发生的分泌阶段和成熟阶段。对发育中的牙釉质组织进行广泛分析 KI、杂合子 (HET) 和野生型 (WT) 同窝小鼠显示 KI 小鼠表现出不同的牙釉质表型， 包括，哺乳动物牙釉质标志性特征——牙釉质棒结构的丧失、众多表面缺陷、 牙釉质晶体较短、发育不全和钙化不足。特别值得注意的是，HET 珐琅被发现是马赛克的 自然界中也含有正常棱柱结构的区域，如 WT 牙釉质中所示。我们还发现 KI 成釉细胞缺乏 Tomes 突起，并表现出成釉细胞层组织的丧失，并且严重 通过分泌阶段逐渐建立的细胞病理学。这些发现以及最近的其他发现 来自我们实验室的证据，引导我们提出关于作用的新工作假设 釉原蛋白磷酸化在釉质矿化调节和维持成釉细胞完整性中的作用 和在釉质形成过程中的功能。拟议的矿化功能活动反映了 天然磷酸化全长牙釉蛋白及其主要磷酸化的能力增强 裂解产物以稳定矿物相前体，作为控制整个矿化的一种手段 釉质发生。我们进一步假设缺乏牙釉蛋白磷酸化会导致细胞破坏 基质相互作用和牙釉质基质蛋白的运输。提出了四 (4) 个具体目标： 确定釉原蛋白如何引导牙釉质晶体的线性对位生长和组织；到 确定 KI 突变体阶段特异性牙釉质发育异常的基础；确定是否为S-16 牙釉原蛋白磷酸化是牙釉蛋白与其他必需牙釉质基质蛋白相互作用所必需的 在牙釉质形成过程中；并阐明釉原蛋白磷酸化在维持 成釉细胞在整个成釉细胞形成过程中的完整性和功能。拟议的研究旨在提供 对磷酸化牙釉蛋白调节形成机制的基本了解 高度有序的牙釉质组织。从长远来看，我们的发现应该有助于我们对遗传的理解 牙釉质疾病和影响龋齿易感性的因素。本次工作的顺利完成 还将为开发改进牙釉质再生方法提供新的见解。 鉴于龋齿的高发病率，需要加深对这些领域的了解。

This new R01 proposal is designed to elucidate the essential role the phosphorylation of a single amino acid in the most abundant enamel matrix protein, amelogenin, plays in the regulation of enamel formation. Proposed studies build on our extensive new findings that show that phosphorylation of a single serine site (S-16) in native amelogenin is critical for the formation of the highly-ordered enamel structure. Using a novel knock-in (KI) mouse model developed in our laboratory with a S16 to alanine substitution that prevents amelogenin phosphorylation, we have now for the first time demonstrated in vivo that amelogenin phosphorylation plays an essential role in both the secretory and maturation stages of amelogenesis. Extensive analyses of developing enamel tissues from KI, heterozygous (HET) and wild-type (WT) littermates reveal that KI mice exhibit distinct enamel phenotypes, including, the loss of enamel rod structure, the hallmark feature of mammalian enamel, numerous surface defects, shorter enamel crystals, hypoplasia and hypocalcification. Of particular note, HET enamel was found to be mosaic in nature with regions that also contain normal prismatic structures as seen in WT enamel. We have also found that KI ameloblasts lack Tomes' processes and exhibit a loss of organization of the ameloblast layer and severe cell pathology that builds gradually through the secretory stage. These findings, along with other recent evidence from our laboratories, have lead us to develop new working hypotheses regarding the role of amelogenin phosphorylation in the regulation of enamel mineralization and in maintaining ameloblast integrity and function during amelogenesis. Proposed functional activities with respect to mineralization reflect the enhanced capacity of both native phosphorylated full-length amelogenin and its predominant phosphorylated cleavage products to stabilize mineral phase precursors, as a means to control mineralization throughout amelogenesis. We further hypothesize that lack of amelogenin phosphorylation leads to disruption of cell- matrix interactions and trafficking of enamel matrix proteins. Four (4) specific aims have been proposed: to determine how amelogenin guides the linear appositional growth and organization of enamel crystals; to determine the basis for stage-specific abnormal enamel development in the KI mutants; to determine if S-16 amelogenin phosphorylation is required for amelogenin interactions with other essential enamel matrix proteins during enamel formation; and to elucidate the importance of amelogenin phosphorylation in maintaining ameloblast integrity and function throughout amelogenesis. The proposed studies are designed to provide fundamental insight into the mechanism by which phosphorylated amelogenin serves to regulate the formation of the highly-ordered dental enamel tissue. Long-term, our findings should aid in our understanding of inherited enamel diseases and factors that influence dental caries susceptibility. The successful completion of this work will also provide new insights for the development of improved methods for the regeneration of tooth enamel. Given the high prevalence of dental caries, there is need for improved understanding in these noted areas.