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. 2021 Aug 1;39(4):419-424.
doi: 10.7518/hxkq.2021.04.007.

Self-assembly and mineralization of full-length human amelogenin and its functional fragments in vitro

[Article in English, Chinese]
Xiu Zhong  1   2 , Ting-Ting Lai  1 , Liang Chen  3 , Kun Tian  1   2
Affiliations

Self-assembly and mineralization of full-length human amelogenin and its functional fragments in vitro

[Article in English, Chinese]
Xiu Zhong et al. Hua Xi Kou Qiang Yi Xue Za Zhi. .

Abstract
in English, Chinese

Objectives: To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) in vitro and its role in hydroxyapatite (HA) crystal formation.

Methods: The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified in vitro. The protein solution of 100 µg‧mL-1, pH=8, was prepared in Tris-HCl and incubated at room temperature for 1-15 min. Their self-assembly behaviors were observed and compared under a transmission electron microscope (TEM). The full-length AM was added to artificial saliva and incubated for 3 days. A scanning electron microscope (SEM) was used in observing the morphology of the induced new crystals. Then, TARP and LRAP were added. The resulting solution was incubated for 3 days and then observed again.

Results: When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor.

Conclusions: The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals in vivo, providing a theoretical basis for the role of the fragments in the growth and maturation of HA crystals.

目的: 探讨人釉原蛋白（AM）全长及其N端酪氨酸富集段（TRAP）、C端亮氨酸富集段（LRAP）体外自组装的动态过程及其在羟磷灰石（HA）晶体形成中的作用。方法: 体外重组、纯化人AM全长及其功能片段TRAP、LRAP，在三氨基甲烷（Tris-HCl）中配制成100 μg·mL-1、pH=8的蛋白溶液，室温孵育1~15 min，透射电镜（TEM）下观察比较AM全长、TARP和LRAP的自组装行为；在人工唾液中加入AM全长孵育3 d，扫描电镜（SEM）观察诱导形成的新生晶体形貌，继续加入TARP和LRAP孵育3 d后再次观察。结果: pH为8时，人AM全长及TRAP自发启动组装，15 min后均可形成“纳米球”结构，其中TRAP形成的“纳米球”孤立存在，大小均匀，没有明显内部结构；而AM全长分级组装，形成“纳米球”后进一步各向延伸趋势，形成链状结构，随后聚集成网；LRAP的自组装行为不明显，蛋白多以单体形式存在，无“纳米球”生成，仅可见少量低聚物。AM全长诱导3 d后形成棒状HA晶体，加入TRAP和LRAP蛋白继续诱导3 d后晶体在c轴明显伸长，而a、b平面生长欠佳。结论: 人AM全长、TRAP和LRAP的自组装和矿化行为与体内HA晶体定向生长的机制契合，为它们在HA晶体生长、成熟过程中的作用提供了理论依据。.

Keywords: human amelogenin; leucine-rich amelogenin peptide; mineralization in vitro; self-assembly; tyrosine-rich amelogenin peptide.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

图 1
图 1. AM全长及LRAP、TRAP基因的重组
Fig 1 Recombination of AM, LRAP and TRAP genes 左：PCR扩增的AM基因，M为Marker，1为AM基因；右：PCR扩增的LRAP、TRAP基因，M为Marker，1为TRAP基因，2为LRAP基因。
图 2
图 2. AM全长及LRAP、TRAP的SDS-PAGE图谱
Fig 2 SDS-PAGE of AM, LRAP and TRAP M为Marker，1~3依次为LRAP、TRAP和AM目的蛋白。箭头所示为蛋白目的片段。
图 3
图 3. AM全长自组装的观察结果
Fig 3 Micrographs of full-length AM self-assembly 上：分别为AM自组装1、15 min TEM ×500 000；下：AM自组装中间结构特写 TEM ×1 200 000。
图 4
图 4. LRAP自组装的观察结果 TEM × 500 000
Fig 4 Micrographs of LRAP self-assembly TEM × 500 000 左：LRAP自组装1 min；右：LRAP自组装15 min。
图 5
图 5. TRAP自组装的观察结果
Fig 5 Micrographs of TRAP self-assembly 上：分别为TRAP自组装1、15 min TEM × 350 000；下：TRAP纳米球特写 TEM × 1 750 000。
图 6
图 6. 不同时间点加入相应蛋白诱导生成的晶体
Fig 6 The crystals induced by the corresponding proteins were added at different time 左：AM在玻片上矿化3 d SEM × 80 000；右：加入LRAP、TRAP继续矿化3 d SEM × 40 000。
图 7
图 7. 新生晶体的X射线衍射图
Fig 7 XRD pattern of the newborn crystal
See this image and copyright information in PMC

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