Functional Roles of Prolines at Amelogenin C Terminal during Tooth Enamel Formation
Functional Roles of Prolines at Amelogenin C Terminal during Tooth Enamel Formation
复制标题
牙釉质形成过程中釉原蛋白 C 末端脯氨酸的功能作用
作者：
Li Zhu;K. Tanimoto;T. Le;P. DenBesten;Wu Li
釉原蛋白是釉基质中的主要蛋白质，在基质金属蛋白酶-20（MMP-20）的作用下发生降解，促进晶体生长。脯氨酸是釉原蛋白中最丰富的残基，位于釉原蛋白序列中所有MMP-20切割位点的上游。Pro41对釉原蛋白N-末端加工至关重要，而脯氨酸在釉原蛋白C末端的作用尚未确定。本研究旨在阐明C-末端脯氨酸对磷灰石结合和MMP-20水解的影响。为了比较磷灰石亲和力，将重组全长人釉原蛋白（rh 174）和突变变体（P156 T和P164 T）与羟基磷灰石（HAP）一起孵育，并通过Bradford测定定量未结合的蛋白质。rh 174和突变体，以及3个寡肽，包括野生型肽和含有2个突变的肽，与MMP-20在溶液中和HAP上孵育。酶解产物经SDS-PAGE、反相高效液相色谱和质谱分析。与野生型相比，突变的釉原蛋白对HAP的亲和力显著降低（P156 T <P164 T <rH 174）。氨基酸位置164处的脯氨酸突变显著降低了溶液中的釉原蛋白或HAP上结合的蛋白质的初始水解，这通过釉原蛋白寡肽测定证实。结果表明，脯氨酸在釉原蛋白C末端是必不可少的初始加工的釉原蛋白和釉原蛋白矿物质的相互作用。
Amelogenins, the chief proteins in enamel matrix, undergo progressive degradation by matrix metalloproteinase-20 (MMP-20) to facilitate crystal growth. Proline is the most abundant residue in amelogenin and is located upstream to all MMP-20 cleavage sites in the amelogenin sequence. Pro41 is critical for amelogenin N-terminal processing, while the role of prolines at the amelogenin C terminus have not been determined. This study sought to elucidate the effect of the C-terminal prolines on apatite binding and MMP-20 hydrolysis. To compare apatite affinity, recombinant full-length human amelogenin (rh174) and mutated variants (P156T and P164T) were incubated with hydroxyapatite (HAP), and the unbound proteins were quantified by the Bradford assay. rh174 and mutants, as well as 3 oligopeptides, including wild-type peptide and peptides containing 2 mutations, were incubated with MMP-20 both in solution and on HAP. The digested products were analyzed by SDS-PAGE, reverse-phase high-performance liquid chromatography and mass spectrometry. Mutated amelogenins displayed a significantly lower affinity to HAP than the wild type (P156T < P164T < rH174). The proline mutation at amino acid location 164 significantly reduced the initial hydrolysis of either the amelogenins in solution or the proteins bound on HAP, which was confirmed by amelogenin oligopeptide assays. It was concluded that prolines at the amelogenin C terminus are essential for the initial processing of amelogenin and amelogenin-mineral interactions.
影响因子：
6.9
作者：
Moradian-Oldak, J;Bouropoulos, N;Gharakhanian, N
通讯作者：
Gharakhanian, N
影响因子：
2.9
作者：
Moradian-Oldak, J;Jimenez, I;Fincham, AG
通讯作者：
Fincham, AG