Yamazaki Hajime, Beniash Elia, Yamakoshi Yasuo, Simmer James P, Margolis Henry C
Center for Biomineralization, The Forsyth InstituteCambridge, MA, United States.
Department of Developmental Biology, Harvard School of Dental MedicineBoston, MA, United States.
Front Physiol. 2017 Jun 29;8:450. doi: 10.3389/fphys.2017.00450. eCollection 2017.
Previously, we have shown that serine-16 phosphorylation in native full-length porcine amelogenin (P173) and the Leucine-Rich Amelogenin Peptide (LRAP(+P)), an alternative amelogenin splice product, affects protein assembly and mineralization . Notably, P173 and LRAP(+P) stabilize amorphous calcium phosphate (ACP) and inhibit hydroxyapatite (HA) formation, while non-phosphorylated counterparts (rP172, LRAP(-P)) guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases. To test this hypothesis, we have utilized Fourier transform infrared spectroscopy (FTIR) to determine the secondary structure of LRAP(-P) and LRAP(+P) in the absence/presence of calcium and selected mineral phases relevant to amelogenesis; i.e., hydroxyapatite (HA: an enamel crystal prototype) and (ACP: an enamel crystal precursor phase). Aqueous solutions of LRAP(-P) or LRAP(+P) were prepared with or without 7.5 mM of CaCl at pH 7.4. FTIR spectra of each solution were obtained using attenuated total reflectance, and amide-I peaks were analyzed to provide secondary structure information. Secondary structures of LRAP(+P) and LRAP(-P) were similarly assessed following incubation with suspensions of HA and pyrophosphate-stabilized ACP. Amide I spectra of LRAP(-P) and LRAP(+P) were found to be distinct from each other in all cases. Spectra analyses showed that LRAP(-P) is comprised mostly of random coil and β-sheet, while LRAP(+P) exhibits more β-sheet and α-helix with little random coil. With added Ca, the random coil content increased in LRAP(-P), while LRAP(+P) exhibited a decrease in α-helix components. Incubation of LRAP(-P) with HA or ACP resulted in comparable increases in β-sheet structure. Notably, however, LRAP(+P) secondary structure was more affected by ACP, primarily showing an increase in β-sheet structure, compared to that observed with added HA. These collective findings indicate that phosphorylation induces unique secondary structural changes that may enhance the functional capacity of native phosphorylated amelogenins like LRAP to stabilize an ACP precursor phase during early stages of enamel mineral formation.
此前，我们已经表明，天然全长猪牙釉蛋白（P173）和富含亮氨酸的牙釉蛋白肽（LRAP(+P)，一种牙釉蛋白的替代剪接产物）中的丝氨酸-16磷酸化会影响蛋白质组装和矿化。值得注意的是，P173和LRAP(+P)能稳定无定形磷酸钙（ACP）并抑制羟基磷灰石（HA）形成，而非磷酸化的对应物（rP172，LRAP(-P)）则引导有序的HA晶体束生长。基于这些发现，我们推测全长牙釉蛋白和LRAP的磷酸化会诱导构象变化，这对其与正在形成的磷酸钙矿相相互作用的能力产生关键影响。为了验证这一假设，我们利用傅里叶变换红外光谱（FTIR）来确定LRAP(-P)和LRAP(+P)在有无钙以及与牙釉质形成相关的选定矿相（即羟基磷灰石（HA：牙釉质晶体原型）和ACP（牙釉质晶体前体相））存在时的二级结构。在pH 7.4条件下，制备含有或不含7.5 mM CaCl₂的LRAP(-P)或LRAP(+P)水溶液。使用衰减全反射获得每种溶液的FTIR光谱，并分析酰胺-I峰以提供二级结构信息。在与HA和焦磷酸盐稳定的ACP悬浮液孵育后，类似地评估LRAP(+P)和LRAP(-P)的二级结构。发现在所有情况下，LRAP(-P)和LRAP(+P)的酰胺I光谱彼此不同。光谱分析表明，LRAP(-P)主要由无规卷曲和β-折叠组成，而LRAP(+P)表现出更多的β-折叠和α-螺旋，几乎没有无规卷曲。加入钙后，LRAP(-P)中的无规卷曲含量增加，而LRAP(+P)中的α-螺旋成分减少。LRAP(-P)与HA或ACP孵育导致β-折叠结构有相当程度的增加。然而，值得注意的是，与添加HA时观察到的情况相比，LRAP(+P)的二级结构受ACP的影响更大，主要表现为β-折叠结构增加。这些共同发现表明，磷酸化会诱导独特的二级结构变化，这可能会增强天然磷酸化牙釉蛋白（如LRAP）在牙釉质矿化早期稳定ACP前体相的功能能力。
