Abbarin Nastaran, San Miguel Symone, Holcroft James, Iwasaki Kengo, Ganss Bernhard
Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Canada.
J Bone Miner Res. 2015 May;30(5):775-85. doi: 10.1002/jbmr.2411.
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 ((P)S(P) SEEL), 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.
釉成熟蛋白（AMTN）是一种最近发现的蛋白质，在牙釉质形成的成熟阶段特异性表达。它定位于釉质表面和成釉细胞顶端表面之间的界面处。AMTN基因敲除小鼠的釉质矿化不足，而过度表达AMTN的转基因小鼠的釉质羟基磷灰石（HA）微结构致密但紊乱，这表明AMTN可能直接参与调节HA矿化。在本研究中，基于光散射测量，我们证明溶解在亚稳缓冲系统中的重组人（rh）AMTN可促进HA沉淀。通过扫描电子显微镜、透射电子显微镜和电子衍射对矿物沉淀进行了表征。对矿物沉积物中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（形成釉质基质的主要蛋白质）相当。在小鼠颅骨细胞中过表达AMTN也增加了培养基中钙沉积物的形成。在体内釉质形成的分泌阶段过表达AMTN导致釉质矿化迅速且不受控制。潜在的丝氨酸磷酸化基序SSEEL的位点特异性诱变将体外矿物沉淀减少至不到25%，表明该基序对该蛋白质的HA矿化功能很重要。含有SSEEL基序的合成短肽仅在其磷酸化形式（(P)S(P)SEEL）下能够促进矿化，这表明该基序对于AMTN的矿化特性是必要的但不是充分的。这些发现表明AMTN通过促进HA矿化对生物矿化有直接影响，并表明AMTN在釉质形成成熟阶段致密的无棱柱釉质表层形成中起关键作用。
