Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium
Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium
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DOI：
10.1042/bj20060662
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发表时间：
2006-10-01
影响因子：
4.1
通讯作者：
Nanci, Antonio
中科院分区：
文献类型：
--
作者：
Moffatt, Pierre;Smith, Charles E.;Nanci, Antonio
牙釉质的形成是一个非常复杂的过程，其中由成釉细胞分泌的一组特定蛋白质发挥着原始作用。作为鉴定大鼠门牙 EO（釉质器官）细胞分泌的新蛋白质的筛选程序的一部分，我们分离了部分 cDNA 片段 (EO-017)，它是最近描述的小鼠 Amtn (amelotin) 基因的同源物 [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gau, Wendel 和 Ganss (2005) J. Dent。资源。 84、1127-1132]。本文介绍了大鼠和猪全长 cDNA 及其推导的蛋白质序列的克隆。通过 Northern 印迹分析和 RT（逆转录酶）-PCR 对大鼠和小鼠组织进行的详细表达谱显示，下颌骨中表达最高，更具体地说，在 EO 的成熟阶段。在所有测试的组织中，仅在牙周膜、肺、胸腺和牙龈中检测到低表达。计算机分析显示，Amtn 基因在其他七种哺乳动物中高度保守，但在鱼类、鸟类和两栖动物中不存在。 Amtn 蛋白富含脯氨酸、亮氨酸、谷氨酰胺和苏氨酸（占总量的 52%），并含有完美保守的蛋白激酶 CK2 磷酸化位点。 HEK-293 细胞（人胚胎肾细胞）中的瞬时转染实验表明，分泌的 Amtn 可能通过苏氨酸残基上的 O-连接寡糖进行翻译后修饰。与其主要表达位点一致，大鼠和小鼠下颌骨内的免疫荧光定位显示Amtn定位于门牙和未萌出磨牙的成熟阶段成釉细胞的基底层。在磨牙交界上皮的内基底层中也检测到了强烈的 Amtn 蛋白表达。 Amtn 独特的细胞定位表明其在细胞粘附中发挥作用。
Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.