ACTION AND FUNCTION OF MMP-20 DURING ENAMEL FORMATION

描述(申请人提供)：这项建议的目的是促进我们对基质金属蛋白酶-20(釉质溶素)在釉质生物矿化过程中的作用和功能的理解。最近关于转基因釉质溶素缺陷小鼠的釉质形成异常的发现强调了釉质溶素在釉质发育过程中的关键作用。牙釉质细胞外基质成分的逐步加工研究对于理解牙釉质形成的病理性是必不可少的，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，基质金属蛋白酶-20裂解特定的结构域：a)在釉原蛋白内，改变其蛋白分解产物的组装及其与磷灰石晶体的相互作用，以及细胞外骨架的结构组织；b)在釉质蛋白和成釉蛋白内，产生具有明确生理功能的多肽，如控制晶体成核和生长。为了验证上述假说，我们提出了以下具体目标：i)通过以下方式来检验基质金属蛋白酶-20的多肽键特异性：a)使用市售的多肽底物，b)使用基于混合物的定向多肽文库系统地测定基质金属蛋白酶-20在N端(P1-Pn)和C-末端(P1‘-Pn’)的裂解位点。研究基质金属蛋白酶-20作用对釉原蛋白纳米球在溶液中组装和拆解的影响。IV)利用原子力显微镜、扫描电子显微镜和动态光散射法研究基质金属蛋白酶-20作用对“凝胶”状态下釉原蛋白基质结构组成的影响。V)测定基质金属蛋白酶-20在溶液中对重组成釉蛋白的切割位置，以及在分离的釉质晶体上的吸附情况。Vi)研究基质金属蛋白酶-20对合成多肽的作用，这些多肽来源于釉蛋白上潜在的切割位点。综上所述：当应用基质金属蛋白酶-20、成釉蛋白、成釉蛋白和成釉蛋白缺失和转基因小鼠策略时，建议的体外研究将是对当前成釉发生的研究的补充。我们从拟议的实验中获得的知识将为体内动物模型研究的解释提供坚实的基础。从体外研究中获得的科学的化学原理将对釉质生物矿化、基质金属蛋白酶、牙齿发育以及我们对釉质形成病理性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，这些材料未来可能在临床牙科以及其他生物医学和生物材料技术领域应用。

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.