人釉原蛋白全长及其功能片段的体外自组装和矿化行为的研究

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

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100μg·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。

Objective 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 100μg·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.Conclusion 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.