Amyloids in Enamel Development

摘要 淀粉样蛋白是一类自发自组装成交叉β-折叠聚集体的蛋白质，其具有 与淀粉样变性和神经变性有关。然而，最近，无毒且 在哺乳动物生物合成途径中发挥功能性作用，这一发现对我们的研究提出了挑战。 目前认为淀粉样蛋白在哺乳动物中的唯一作用是细胞毒性。在这里，我们提出，釉原蛋白， 发育中的釉基质的主要蛋白质，适应交叉β折叠构型并发育成纤维状 淀粉样蛋白，以实现结构支持，引导体内和体外矿化。搪瓷，最硬， 人体中大多数矿化组织由独特的磷灰石纳米纤维组织组成， 直径约50纳米，长度几百微米。人们一致认为， 晶体形态和组织成杆和杆间釉质是蛋白质引导的单轴 磷灰石的生长过程，但目前还不清楚这种独特的微观和 纳米结构发展。虽然釉基质蛋白，特别是釉原蛋白， 已被广泛认为是控制釉质结构发育的关键因素， 有机超分子聚集体和牙釉质结构之间令人信服的关系，直到最近才 当我们发现重组人全长釉原蛋白（rH 174）形成 宽度为17 nm的带状物，其长度可增长至几微米。这种丝带具有自我- 排列并形成束，类似于釉质棒中排列的磷灰石微晶的外观。分析 釉原蛋白的一级结构揭示了几个高度倾向于形成β折叠的结构域，包括 形成淀粉样蛋白的可能性，并产生易于组装的14个残基的N-末端肽（14 P2 纳米带（6.7nm宽），可能具有淀粉样结构。 缺乏牙釉质特异性酶激肽释放酶4（KLK 4）的小鼠牙釉质中淀粉样蛋白染色呈阳性。两 釉组织和重组釉原蛋白纳米带的X射线衍射图显示， β折叠和淀粉样蛋白的特征。自组装釉原蛋白的酶促加工 精确切割成23 kDa和20 kDa片段，其抵抗MMP-20的进一步降解，因此 可能在分泌期成釉过程中为矿化提供稳定的有机模板，而 第二种釉质蛋白酶能够分解和降解釉原蛋白淀粉样蛋白。在此，我们将 进一步研究淀粉样蛋白在牙釉质中的存在，并提出淀粉样蛋白在牙釉质中起关键作用。 发展和组织的有机基质的釉质，并探讨这种结构是 对我们理解牙釉质的形成、疾病和修复至关重要。

ABSTRACT Amyloids are a class of proteins that spontaneously self-assemble into cross β-sheet aggregates which have been associated with amyloidosis and neurodegeneration. However, recently, amyloids that are non-toxic and rather play a functional role in a mammalian biosynthetic pathway have been discovered challenging our current views on the sole role of amyloids in mammals to be cytotoxic. Here we propose that amelogenin, the main protein of the developing enamel matrix, adapts cross-β sheet configuration and develops into fibrillar amyloids to achieve structural support to guide mineralization in vivo and in vitro. Enamel, the hardest and most mineralized tissue in the human body, is comprised of a unique organization of apatite nanofibers of about 50 nm in diameter and several hundreds of micrometers in length. There is agreement that the unique crystal morphology and organization into rod and interrod enamel is the result of a protein-guided uniaxial growth process of apatite, but it is unclear by which molecular mechanisms this unique micro- and nanostructure develops. While the role of self-assembly of enamel matrix proteins, in particular amelogenin, has widely been recognized as a crucial factor in controlling the structural development of enamel, a convincing relationship between organic supramolecular aggregates and enamel structure has only recently been observed, when we discovered that the recombinant human full-length amelogenin protein (rH174) forms ribbons of 17 nm in width, which grow to several micrometers in length. Such ribbons have the ability to self- align and form bundles resembling the appearance of aligned apatite crystallites in an enamel rod. Analysis of the primary structure of amelogenin revealed several domains with high propensity to form β-sheets, including the possibilty to form amyloids, and produced a 14 residue N-terminal peptide (14P2) that readily assembled into nanoribbons (6.7nm wide), with possible amyloid structure. Amyloid stains were positive in enamel from mice lacking the enamel-specific enzyme kallikrein 4 (KLK4). Both enamel tissue and recombinant amelogenin nanoribbons showed x-ray diffraction spacings at 4.7Å characteristic of β sheets and amyloids. Enzymatic processing of self-assembled amelogenin promoted precise cleavage into the 23 kDa and 20 kDa fragments which resisted further degradation by MMP-20, thus possibly providing a stable organic template for mineralization during secretory stage amelogenesis, whereas the second enamel protease is able to disassemble and to degrade amelogenin amyloids. Herein we will further investigate the presence of amyloids in enamel and propose that amyloids play a key role in the development and organization of the organic matrix of enamel and that an exploration of such structures is essential to our understanding of enamel formation, its diseases and repair.