ACTION AND FUNCTION OF MMP-20 DURING ENAMEL FORMATION

描述（由申请人提供）：本提案的目的是促进我们对基质金属蛋白酶-20（溶釉素）在釉质生物矿化过程中的作用机制和功能的理解。最近的研究发现，在转基因的釉质溶解素缺陷小鼠的异常釉质形成突出了釉质溶解素在釉质发育过程中的关键功能。釉质细胞外基质成分的分步加工研究对于理解病理性牙釉质形成至关重要，也是开发釉质仿生材料的关键步骤之一。我们的一般假设是MMP-20切割特定的结构域：a）在釉原蛋白内以改变其蛋白水解产物的组装及其与磷灰石晶体的相互作用以及细胞外框架的结构组织，B）在釉蛋白和成釉蛋白内以产生具有确定的生理功能如控制晶体成核和生长的多肽。提出了以下具体目标来检验上述假设：I）通过以下方法检验MMP-20的肽键特异性：a）使用市售多肽底物，B）使用基于混合物的定向肽文库系统地测定N-末端（P1-Pn）和C-末端（P1 ′-Pn ′）的MMP-20切割位点基序。目的：研究MMP-20对重组釉原蛋白rp 172和rp 148的切割位点。III）研究MMP-20作用对溶液中釉原蛋白纳米球的组装和分解的影响。IV）使用原子力显微镜、SEM和动态光散射研究MMP-20作用对“凝胶样”状态下的釉原蛋白基质的结构组织的影响。V）确定MMP-20在溶液中以及吸附到分离的釉质晶体上对重组成釉蛋白的切割位点。VI）检查MMP-20对衍生自釉蛋白上的潜在切割位点的合成肽的作用。总的来说：当MMP-20、釉原蛋白、成釉蛋白、釉蛋白缺失和转基因小鼠策略被应用时，拟议的体外研究将是对当前关于釉质形成的研究的补充。从我们提出的实验中获得的知识将为解释体内动物模型研究提供坚实的基础。从体外研究中获得的科学化学原理将对牙釉质生物矿化、基质金属蛋白酶、牙齿发育以及我们对病理性牙釉质形成的理解产生重大影响。此外，这些研究将有助于设计和开发新型生物材料所需的基础知识，这些生物材料在临床牙科和其他生物医学和生物材料技术领域具有潜在的未来应用。

DESCRIPTION (provided by applicant): The goal of this proposal is to advance our understanding of the mechanism of action and function of the matrix metalloproteinase-20 (enamelysin) during enamel biomineralization. Recent findings on the abnormal enamel formation in transgenic enamelysin-deficient mice have highlighted the critical function of enamelysin during enamel development. The study on the stepwise processing of the enamel extracellular matrix components is essential for the understanding of pathological dental enamel formation and is one of the critical steps towards the development of enamel-inspired biomimetic materials. Our general hypothesis is that MMP-20 cleaves specific domains: a) within amelogenin to alter the assembly of its proteolytic products and their interactions with apatite crystals as well as the structural organization of the extracellular framework, b) within enamelin and ameloblastin to generate polypeptides with defined physiological function such as control of crystal nucleation and growth. The following specific aims are proposed to examine the above hypothesis: I) To examine peptide bond specificity of MMP-20 by: a) using commercially available polypeptide substrates, b) systematically determining MMP-20 cleavage site motifs at both the N-terminal (P1-Pn) and C-terminal (P1'-Pn') using mixture-based oriented peptide libraries.Il) To determine cleavage sites on recombinant amelogenin rp172 and rp148 by MMP-20 in solution as well as adsorbed on isolated enamel crystals. Ill) To investigate the effect of MMP-20 action on the assembly and disassembly of amelogenin nanospheres in solution. IV) To investigate the effect of MMP-20 action on the structural organization of the amelogenin matrix in a "gel-like" state using atomic force microscopy, SEM, and dynamic light scattering. V) To determine cleavage sites on recombinant ameloblastin by MMP-20 in solution as well as adsorbed onto isolated enamel crystals. VI) To examine the action of MMP-20 on synthetic peptides derived from potential cleavage sites on enamelin proteins. In summary: The proposed in vitro studies will be complementary to current research on amelogenesis when MMP-20, amelogenin, ameloblastin, and enamelin null and transgenic mice strategies are applied. The knowledge gained from our proposed experiments will provide a solid base for interpretation of the in vivo animal model studies. The scientific chemical principles gained from the proposed in vitro studies will have a great impact on the field of enamel biomineralization, matrix metalloproteinases, tooth development, and our understanding of pathological enamel formation. In addition, these studies will contribute to the basic knowledge required for the design and development of novel biomaterials with potential future application in clinical dentistry and other areas of biomedical and biomaterial technology.