CHARACTERIZATION DMP1 A DENTIN PHOSPHOPROTEIN

点击翻译按钮获取中文摘要

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.