Amelogenesis imperfecta (AI) is an inherited developmental enamel defect affecting tooth masticatory function, esthetic appearance, and the well-being of patients. As one of the major enamel matrix proteins (EMPs), enamelin (ENAM) has three serines located in Ser-x-Glu (S-x-E) motifs, which are potential phosphorylation sites for the Golgi casein kinase FAM20C. Defects in FAM20C have similarly been associated with AI. In our previous study of Enam^Rgsc514 mice, the Glu⁵⁷ in the S⁵⁵-X⁵⁶-E⁵⁷ motif was mutated into Gly, which was expected to cause a phosphorylation failure of Ser⁵⁵ because Ser⁵⁵ cannot be recognized by FAM20C. The severe enamel defects in ENAM^Rgsc514 mice reminiscent of Enam-knockout mouse enamel suggested a potentially important role of Ser⁵⁵ phosphorylation in ENAM function. However, the enamel defects and ENAM dysfunction may also be attributed to distinct physicochemical differences between Glu⁵⁷ and Gly⁵⁷. To clarify the significance of Ser⁵⁵ phosphorylation to ENAM function, we generated two lines of Enam knock-in mice using CRISPR-Cas9 method to eliminate or mimic the phosphorylation state of Ser⁵⁵ by substituting it with Ala⁵⁵ or Asp⁵⁵ (designated as S55A or S55D), respectively. The teeth of 6-day or 4-week-old mice were subjected to histology, micro-CT, SEM, TEM, immunohistochemistry, and mass spectrometry analyses to characterize the morphological, microstructural and proteomic changes in ameloblasts, enamel matrix and enamel rods. Our results showed that the enamel formation and EMP expression in S55D heterozygotes (Het) were less disturbed than those in S55A heterozygotes, while both homozygotes (Homo) had no mature enamel formation. Proteomic analysis revealed alterations of enamel matrix biosynthetic and mineralization processes in S55A Hets. Our present findings indicate that Asp⁵⁵ substitution partially mimics the phosphorylation state of Ser⁵⁵ in ENAM. Ser⁵⁵ phosphorylation is essential for ENAM function during amelogenesis.

釉质生成不全 (AI) 是一种遗传性牙釉质发育缺陷，影响牙齿咀嚼功能、美观和患者的健康。作为主要的牙釉质基质蛋白 (EMP) 之一，牙釉质 (ENAM) 在 Ser-x-Glu (SxE) 基序中具有三个丝氨酸，这些基序是高尔基体酪蛋白激酶 FAM20C 的潜在磷酸化位点。FAM20C 的缺陷同样与人工智能相关。在我们之前对Enam ^Rgsc514小鼠的研究中， S ⁵⁵ -X ⁵⁶ -E ⁵⁷基序中的Glu ⁵⁷突变为 Gly，预计这会导致 Ser ⁵⁵磷酸化失败，因为 Ser ⁵⁵无法被 FAM20C 识别。^{ENAM Rgsc514}小鼠中严重的牙釉质缺陷让人想起Enam敲除小鼠的牙釉质，这表明 Ser ⁵⁵磷酸化在 ENAM 功能中具有潜在的重要作用。然而，牙釉质缺陷和 ENAM 功能障碍也可能归因于 Glu ⁵⁷和 Gly ⁵⁷之间明显的物理化学差异。^{为了阐明 Ser 55}磷酸化对 ENAM 功能的重要性，我们使用 CRISPR-Cas9 方法生成了两系Enam敲入小鼠，通过用 Ala ⁵⁵或 Asp ⁵⁵取代Ser ⁵⁵来消除或模拟 Ser 55 的磷酸化状态（命名为 S55A）或S55D），分别。对 6 天或 4 周龄小鼠的牙齿进行组织学、显微 CT、SEM、TEM、免疫组织化学和质谱分析，以表征成釉细胞、牙釉质基质和牙釉质棒的形态、微观结构和蛋白质组变化。我们的结果表明，S55D 杂合子 (Het) 中的牙釉质形成和 EMP 表达受到的干扰比 S55A 杂合子中的干扰要小，而两种纯合子 (Homo) 都没有成熟的牙釉质形成。蛋白质组学分析揭示了 S55A Hets 牙釉质基质生物合成和矿化过程的改变。我们目前的研究结果表明，Asp ^{55取代部分模拟了 ENAM 中 Ser 55}的磷酸化状态。Ser ⁵⁵磷酸化对于釉质形成过程中的 ENAM 功能至关重要。