Molecular mechanism of abnormal dentin calcification in dentinogenesis imperfecta

牙齿疾病“牙本质发生II型（DGI-II型）”是常染色体显性疾病，其患病率为6，000 - 8，000个体中的1个。先前的连锁研究将DGI定位于染色体4 q13 -21，其中编码牙齿细胞外基质蛋白（成釉蛋白，牙本质基质蛋白1）的几个候选基因也已被定位。我们对其中的两个基因（成釉蛋白和牙本质基质蛋白1、牙本质涎磷蛋白）进行了分子水平的分析，人成釉蛋白（AMBN）基因尚未测序。因此，我们克隆了人AMBN全长cDNA，序列分析表明其开放阅读框为1341 bp，编码447个氨基酸。与猪，牛，大鼠和小鼠AMBN序列的比较显示出较高的氨基酸序列相似性，并导致鉴定出一种新的78 bp（26个氨基酸）插入物，该插入物来自内部序列重复。通过对人类基因组克隆的DNA分析，AMBN基因由13个外显子和一个新的78 bp片段组成，该片段被证明包含两个小外显子。牙本质基质蛋白1（Dentin matrix protein 1，DMP 1）是一种酸性磷酸化蛋白，主要表达于牙体和骨组织中。通过北方印迹分析，我们证明了在不能形成牙齿的鸡中，DMP 1 mRNA在骨中高度表达。为了阐明骨组织中DMP 1表达的意义，检测了鸡和大鼠骨组织中DMP 1 mRNA及其蛋白的表达。在鸡和大鼠中，DMP 1 mRNA仅在骨组织中检测到，并且定位于骨细胞而不在成骨细胞中。抗血清从大鼠的DMP 1的肽，和DMP 1的定位进行了研究，通过免疫组织化学。这些数据表明，DMP 1是骨细胞中特异性表达的骨基质蛋白。

The dental disorder "dentinogenesis imperfecta type II (DGI-typeII)" is an autosomal dominant condition that has a prevalence of 1 in 6,000-8,000 individuals. Linkage studies had previously mapped DGI to chromosome 4q13-21, where several candidate genes that encode tooth extracellular matrix proteins (ameloblastin, dentin matrix protein 1, have also been mapped. We focused two genes (ameloblastin and dentin matrix protein 1, Dentin sialophosphoprotein) of them and analyzed them by molecular level.Human ameloblastin (AMBN) gene had not sequenced so far. Therefore, we isolated the full-length human AMBN cDNA.Sequence analysis of the AMBN cDNA revealed an open reading frame of 1341 bp encoding a 447-amino acid protein. Comparison with pig, cattle, rat, and mouse AMBN sequences showed high amino acid sequence similarity and led to the identification of a novel 78 bp (26-amino acid) insert resulting from internal sequence duplication. By DNA analysis of a human genomic clones, the AMBN gene was shown to consist of 13 exons and a novel 78 bp segment which proved to comprise two small exons. There were little polymorphisms of human AMBN gene in Asian populations.Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein expressed in tooth organ and bone. We demonstrated that in chicken, which is not capable of forming tooth, DMP1 mRNA is highly expressed in bone by Northern blot analysis. To clarify the significance of DMP1 expression in bone, the expression of DMP1 mRNA and its protein was examined in chicken and rat. In the chicken and rat, DMP1 mRNA was detected only in bone tissues and was localized in osteocytes but not in osteoblasts. Antiserum was raised against the peptide from rat DMP1, and the localization of DMP1 was examined by immunohistochemistry. These data demonstrate that DMP1 is a bone matrix protein specifically expressed in osteocytes.