CHARACTERIZATION OF DMP1--DENTIN PHOSPHOPROTEIN

骨骼和牙齿的适当矿化在 正常的人体生长发育和肌肉骨骼功能。这个 非胶原基质蛋白的作用及作用机制 (NCP)牙本质，特别是那些特别起源于 成牙本质细胞还没有被很好地理解。最重要的角色可能是 在牙本质形成和修复体形成过程中的表达 牙本质，并可能包括矿化过程的调节。在……里面 尽管经过了多年的研究，但非常复杂的主要牙本质NCP 没有得到详细的描述。不过，我们最近已完成 其中一种NCPs的DNA测序及其染色体定位，并通过 原位杂交，获得了其发育受调控的证据 在表达上。这种蛋白质最初是通过克隆命名来鉴定的， AG1是第一个被测序和定位的牙本质特异性蛋白 从染色体上看。本研究具有重要的临床应用价值。Dmp1地图 位于人类染色体上与牙齿形成基因相同的区域 不完全II型(DI-II)。在DI-II中牙本质的不完全矿化 是最重要的病理发现。基本假设是 像DMP1这样的非胶原蛋白与胶原基质在 特定的位点，成核并调节成矿过程。我们现在 建议从大鼠基因组文库中鉴定Dmp1基因并确定 基因5‘端的序列，从而识别启动子 这对于了解Dmp1的组织特异性表达是必要的。 为了了解蛋白质的功能，我们建议制作 大量的重组蛋白。实现这一目标的工作有两个目标： L)制备未经修饰的载脂蛋白，使其可用作 用于研究翻译后修饰的基础，如 磷酸化和糖基化；2)制备多克隆抗体。这 将使用抗Dmp1抗体来检测Dmp1在 修复性牙本质和套层牙本质以及Dmp1在牙周炎中的定位 发育过程中的细胞外基质。免疫电子显微镜 程序和金标记物DMP1抗体将用于 DMP1的定位及其分泌途径的确定 成牙本质细胞。这些实验将提供更多关于 Dmp1合成的分子机制 成牙本质细胞。长期目标是了解Dmp1在 牙本质形成。

The proper mineralization of bones and teeth has great importance in normal human growth and development and musculo-skeletal function. The roles and mechanisms of action of the non-collagenous matrix proteins (NCPs) of dentin and particularly those originating specifically in the odontoblasts are not well understood. The most important roles may be expressed during the periods of dentinogenesis and formation of reparative dentin, and may include regulation of the process of mineralization. In spite of many years of study the very complex principal dentin NCPs have not been characterized in detail. However we have recently completed the cDNA sequencing of one of these NCPs its chromosomal localization, and, by in situ hybridization, gained evidence that its developmentally regulated in expression. The protein, first identified by the cloning designation, AG1, is the first dentin-specific protein to be sequenced and localized chromosomally. This study has important clinical applications. Dmp1 maps to the same region on the human chromosome as the gene for dentenogenesis imperfecta type II (DI-II). In DI-II incomplete mineralization of dentin is the most important pathological finding. The basic hypothesis is that noncollagenous proteins like Dmp1 interacts with the collagen matrix at specific loci, nucleate and regulate the process of mineralization. We now propose to identify the Dmp1 gene from a rat genomic library and determine the sequences at the 5'end of the gene and thus identify the promoter which is necessary to understand the tissue specific expression of Dmp1. In order to understand the function of the protein we propose to make large amounts of recombinant protein. Work on this aim has two objectives: l) to prepare the unmodified apoprotein so that it can be used as a substrate for studying post-translational modifications like phosphorylation and glycosylation, 2) to prepare polyclonal antibody. This anti-Dmp1 antibody would be used to examine the presence of Dmp1 in reparative and mantle dentin as well as localization of Dmp1 in the extracellular matrix during development. Immunoelectron microscope procedures and gold-conjugated DMP1 antibody would be used for localization and to determine the pathway for secretion of DMP1 from the odontoblasts. These experiments will provide increased understanding of the molecular mechanisms involved in the synthesis of Dmp1 by the odontoblasts. The long -term goal is to understand the role of Dmp1 in dentinogenesis.