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（搪瓷酶）在牙釉质生物矿化过程中的作用机制和功能的理解。近年来对转基因釉质酶缺陷小鼠的异常牙釉质形成的研究表明，釉质酶在牙釉质发育中的重要作用。牙釉质细胞外基质成分的分步加工研究是理解牙釉质病理形成的基础，也是开发仿生牙釉质材料的关键步骤之一。我们的一般假设是，MMP-20切割特定区域：a)在淀粉原蛋白内，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)在釉素和成釉蛋白内，产生具有特定生理功能的多肽，如控制晶体成核和生长。为了验证上述假设，我们提出了以下具体目标：I)通过以下方法来检测MMP-20的肽键特异性：a)使用市买多肽底物，b)使用基于混合物的定向肽库系统地确定MMP-20在n端（P1-Pn）和c端（P1'-Pn'）的裂解位点基序。Il)利用MMP-20测定重组淀粉原蛋白rp172和rp148在溶液中的裂解位点以及在离体釉质晶体上的吸附位点。ii)研究MMP-20作用对溶液中淀粉原纳米球组装和拆卸的影响。IV)利用原子力显微镜、扫描电镜、动态光散射等方法研究MMP-20作用对凝胶状淀粉原基质结构组织的影响。V)利用MMP-20测定重组成釉蛋白在溶液中的裂解位点以及吸附在分离釉质晶体上的裂解位点。6)研究MMP-20对从瓷釉蛋白潜在切割位点衍生的合成肽的作用。综上所述，当采用MMP-20、成釉原蛋白、成釉细胞素和釉素无效和转基因小鼠策略时，所提出的体外研究将对目前的成釉发生研究形成补充。从我们提出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具有潜在未来应用于临床牙科和其他生物医学和生物材料技术领域的新型生物材料所需的基础知识。

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.