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Molecular mechanism of abnormal dentin calcification in dentinogenesis imperfecta

牙本质发育不全牙本质钙化异常的分子机制

基本信息

批准号：
11671800
负责人：
TOYOSAWA Satoru
金额：
$ 0.64万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671800/
关键词：
Ameloblastin Dentin matrix protein 1 Dentin sialophosphoprotein Dentinogenesis imperfecta Genome Polymorphism Osteocyte

项目摘要

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.

牙本质发育不全II型（dgi - II型）是一种常染色体显性遗传病，发病率为6000 - 8000人中有1人。连锁研究之前已经将DGI定位到染色体4q13-21上，其中一些编码牙齿细胞外基质蛋白（成釉细胞蛋白，牙本质基质蛋白1）的候选基因也被定位。我们对其中的两个基因（成釉细胞蛋白和牙本质基质蛋白1、牙本质唾液磷酸蛋白）进行了分子水平的分析。人成釉细胞素（AMBN）基因至今尚未测序。因此，我们分离了人AMBN全长cDNA。AMBN cDNA序列分析显示一个1341 bp的开放阅读框，编码447个氨基酸的蛋白。与猪、牛、大鼠和小鼠的AMBN序列比较显示出较高的氨基酸序列相似性，并鉴定出一个由内部序列重复引起的78 bp（26个氨基酸）的新插入。通过对人类基因组克隆的DNA分析，发现AMBN基因由13个外显子和一个新的78 bp片段组成，该片段由两个小外显子组成。人类AMBN基因在亚洲人群中几乎没有多态性。牙本质基质蛋白1 （DMP1）是一种在牙齿器官和骨骼中表达的酸性磷蛋白。我们通过Northern blot分析证明，在不能形成牙齿的鸡中，DMP1 mRNA在骨中高表达。为了阐明DMP1在骨中的表达，我们检测了鸡和大鼠DMP1 mRNA及其蛋白的表达。在鸡和大鼠中，DMP1 mRNA仅在骨组织中检测到，并且定位于骨细胞，而不定位于成骨细胞。采用免疫组化方法检测大鼠DMP1肽的定位。这些数据表明，DMP1是骨细胞特异性表达的骨基质蛋白。