Molecular mechanism of abnormal dentin calcification in dentinogenesis imperfecta

牙本质发育不全II型(DGI-TypeII)是一种常染色体显性遗传病，患病率为每6,000-8,000人中有1人。先前的连锁研究已经将DGI定位到染色体4Q13-21，在该染色体上也定位了几个编码牙齿细胞外基质蛋白(成釉蛋白，牙本质基质蛋白1)的候选基因。我们聚焦了其中的两个基因(成釉蛋白和牙本质基质蛋白1，牙本质涎磷蛋白)，并从分子水平对其进行了分析。人类成釉蛋白(AMBN)基因目前还没有测序。因此，我们克隆了人AMBN的全长cDNA.序列分析显示，AMBN的开放阅读框为1341bp，编码一个447个氨基酸的蛋白质.与猪、牛、大鼠和小鼠的AMBN序列比较显示，AMBN的氨基酸序列具有很高的相似性，并通过内部序列重复得到了一个新的78bp(26个氨基酸)的插入片段。对一个人类基因组克隆的DNA分析表明，AMBN基因由13个外显子和一个新的78bp片段组成，该片段由两个小外显子组成。牙本质基质蛋白1(Dentin Matrix Protein 1，DMP1)是一种在牙齿、器官和骨骼中表达的酸性磷酸蛋白。我们通过Northern印迹分析证明，在不能形成牙齿的鸡中，DMP1mRNA在骨骼中高表达。为了阐明DMP1在骨骼中表达的意义，我们检测了DMP1mRNA及其蛋白在鸡和大鼠体内的表达。在鸡和大鼠中，DMP1mRNA仅在骨组织中检测到，在骨细胞中定位，而在成骨细胞中不表达。制备抗大鼠DMP1多肽的抗血清，免疫组织化学方法检测DMP1的定位。这些数据表明，DMP1是一种在骨细胞中特异表达的骨基质蛋白。

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.