The Enamel Protein Amelotin Is a Promoter of Hydroxyapatite Mineralization
The Enamel Protein Amelotin Is a Promoter of Hydroxyapatite Mineralization
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DOI：
10.1002/jbmr.2411
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发表时间：
2015-05-01
影响因子：
6.2
通讯作者：
Ganss, Bernhard
中科院分区：
文献类型：
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作者：
Abbarin, Nastaran;San Miguel, Symone;Ganss, Bernhard
Amelotin (AMTN) 是最近发现的一种蛋白质，在牙釉质形成的成熟阶段特异性表达。它位于牙釉质表面和成釉细胞顶面之间的界面处。 AMTN 敲除小鼠的牙釉质矿化不足，而过表达 AMTN 的转基因小鼠则具有致密但无组织的牙釉质羟基磷灰石 (HA) 微观结构，表明 AMTN 可能直接参与调节 HA 矿化。在本研究中，我们根据光散射测量结果证明，重组人 (rh) AMTN 溶解在亚稳态缓冲系统中可促进 HA 沉淀。通过扫描和透射电子显微镜以及电子衍射对矿物沉淀物进行了表征。矿床中 AMTN 的胶体金免疫标记显示蛋白质分子与 HA 晶体相关。发现 rh-AMTN 与 HA 的结合亲和力与釉原蛋白（形成牙釉质基质的主要蛋白质）的结合亲和力相当。 AMTN 在小鼠颅盖细胞中的过度表达也增加了培养基中钙沉积物的形成。 AMTN 在体内牙釉质形成的分泌阶段过度表达导致快速且不受控制的牙釉质矿化。潜在丝氨酸磷酸化基序 SSEEL 的位点特异性诱变将体外矿物质沉淀降低至 25% 以下，表明该基序对于蛋白质的 HA 矿化功能非常重要。含有 SSEEL 基序的合成短肽只能以磷酸化形式 ((SSEEL)-S-P-S-P) 促进矿化，表明该基序对于 AMTN 的矿化特性是必要的，但还不够。这些发现表明，AMTN 通过促进 HA 矿化对生物矿化产生直接影响，并表明 AMTN 在釉质形成成熟阶段的致密棱柱形牙釉质表面层的形成中发挥着关键作用。 (c) 2015 年美国骨与矿物质研究学会。
Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock-out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh-AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site-specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form ((SSEEL)-S-P-S-P), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation stage of amelogenesis. (c) 2015 American Society for Bone and Mineral Research.