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Studies on Molecular Structure of Amelogenins and Their Functional Roles in Enamel Formation

釉原蛋白的分子结构及其在牙釉质形成中的功能作用研究

基本信息

批准号：
07457434
负责人：
AOBA Takaaki
金额：
$ 4.35万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1. In the current work, we conducted studies on the molecular structure of the amelogenins by combining multiple techniques in order to gain more concrete information about the structure/function relationship of the major matrix proteins involveld in enamel formation. Techniques used entailed proton nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), small angle x-ray scattering analysis (SAXS), low angle laser light scattering analysis (LALLS), atomic force microscopy (AFM), and immunohistochemistry with epitope-specific antisera. The intact amelogenin protein and its degraded products were isolated and purified from the secretory enamel of slaughtered piglets of 5 to 6 months old. In addition, the 25-residue peptide corresponding to the segment at tha C-terminus was synthesized. The results lead to the following conclusion that : (1) the molecular structure of the intact porcine amelogenin consists of discrete foldin … More g units, i.e., the N-terminal domain containing many aromatic residues, the central domain containing many His and characteristic (QPX)_n repeats, and the hydrophilic domain at the C-terminus : (2) these units have little, if any, interaction with each other. The 5 kD segment at the N-terminus probably has a folded structure, tentatively assigned as beta-sheet structures from CD spectrum, and is more reactive with the cations, particularly calcium. The assumed beta-sheet conformation in the N-terminal region may mediate the intermolecular association of the cleaved fragments of the amelogenin proteins as commonly found at neural pH.The spectral characteristics of the C-terminal domain are similar to those of a random coil conformation. Also the conserved C-terminal domain may direct the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction. The plausible conformation of the central domain was characterized by a strong negative ellipticity at 203 nm, similar to that of collagen, although its nature remains to be defined.Based on the information gained in the current work, we propose a tentative structural model of the 20 kD porcine amelogenins, which adopts an elongated bundle structure consisting of one part of beta-sheet and two parts of polyglycine II,interspersed with beta-turn of "loop". It is of interest that the folding sites correspond to the cleavages of the amelogenin in situ, supporting the view that the programd extracellular processing is directly mediated by the conserved primary structure and the resulting folded structure in solution. Less

1. 在目前的工作中，我们结合多种技术对淀粉原蛋白的分子结构进行了研究，以期更具体地了解参与牙釉质形成的主要基质蛋白的结构/功能关系。使用的技术包括质子核磁共振（NMR）光谱、傅里叶变换红外光谱（FTIR）、圆二色（CD）、小角度x射线散射分析（SAXS）、低角度激光散射分析（LALLS）、原子力显微镜（AFM）和抗原表位特异性抗血清的免疫组织化学。从5 ~ 6月龄屠宰仔猪的分泌牙釉质中分离纯化完整的淀粉原蛋白及其降解产物。此外，还合成了与该c端片段对应的25个残基肽。结果表明：(1)完整猪淀粉原蛋白的分子结构是由离散的折叠蛋白组成的，含有较多的g单元，即n端结构域含有较多的芳香残基，中心结构域含有较多的His和特征（QPX）_n重复序列，以及c端亲水性结构域；(2)这些单元之间相互作用很小（如果有的话）。在n端5kd片段可能具有折叠结构，从CD谱中暂定为β -片结构，并且与阳离子，特别是钙具有更强的活性。假设在n端区域的β -片构象可以介导在神经ph中常见的淀粉原蛋白的裂解片段的分子间结合。c端结构域的光谱特征与随机线圈构象相似。此外，保守的c端结构域可能指导完整的淀粉原蛋白的分子结构，从而促进蛋白质与珐琅质矿物的相互作用。中心结构域的合理构象在203 nm处具有很强的负椭圆性，与胶原蛋白相似，尽管其性质仍有待确定。在此基础上，我们提出了一种20 kD猪淀粉原蛋白的初步结构模型，该模型采用由一部分β -sheet和两部分聚甘氨酸II组成的细长束结构，其间穿插β -turn“loop”。有趣的是，这些折叠位点对应于原淀粉原的原位切割，支持了程序化的细胞外加工是由保守的一级结构和溶液中产生的折叠结构直接介导的观点。少