Kwak Seo-Young, Wiedemann-Bidlack Felicitas B, Beniash Elia, Yamakoshi Yasuo, Simmer James P, Litman Amy, Margolis Henry C
Department of Biomineralization, The Forsyth Institute, Boston, Massachusetts 02115, USA.
J Biol Chem. 2009 Jul 10;284(28):18972-9. doi: 10.1074/jbc.M109.020370. Epub 2009 May 14.
The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.
通过体外矿化研究阐明了釉原蛋白磷酸化在釉质形成中的潜在作用。研究聚焦于天然的20 kDa猪釉原蛋白蛋白水解裂解产物P148，其在发育中的釉质中含量显著。实验条件支持磷酸钙自发沉淀，并初步形成无定形磷酸钙（ACP）。在没有蛋白质的情况下，发现ACP会相对快速地转变为随机取向的板状磷灰石晶体。在存在非磷酸化重组全长釉原蛋白rP172的情况下，观察到诱导期更长，在此期间相对较小的ACP纳米颗粒被短暂稳定。在存在rP172的情况下，发现这些纳米颗粒排列形成线性针状颗粒，随后转变并组织成磷灰石针状晶体的平行阵列。与这些发现形成鲜明对比的是，在丝氨酸16上带有单个磷酸基团的P148被发现可抑制磷酸钙沉淀并稳定ACP形成超过1天。使用非磷酸化重组体（rP147）和P148的部分去磷酸化形式（去磷酸-P148）的进一步研究表明，P148中的单个磷酸基团是其对体外矿化产生深远影响的原因。本研究首次提供了证据，表明天然蛋白水解裂解产物P148可能在釉质形成过程中调节矿化方面具有重要功能作用，即通过在釉质发生的分泌阶段防止釉质基质内不必要的矿物质形成。所获得的结果还为全长釉原蛋白中高度保守的亲水性C末端的功能作用提供了新的见解。
