Comparative immunohistochemical analyses of the localization of amelogenin domains during a tooth enamel development inlowervertebrates

为了解低等脊椎动物釉原蛋白的系统发育特征，测定了爬行动物(鬣蜥)和两栖动物(Newt)的釉原蛋白基因序列，并对其N-端、C-端和中心结构域的多肽进行了免疫组织化学和免疫印迹分析。根据鬣蜥和鬣蜥中釉原蛋白的识别部分，将这些抗体命名为：In(鬣蜥N-末端)、IM(鬣蜥中央结构域)、IC(鬣蜥C-末端)、NN(Newt N-末端)、NM(Newt中央结构域)和NC(Newt C-末端)。ELISA法分析表明，每一种抗体对相应的多肽都有很高的特异性。用蛋白A-GOLD法对鬣蜥进行IN、IM和IC免疫组织化学分析，用NN、NM和NC对鬣蜥进行免疫组织化学分析。因此，除NC外，所有抗体均在鬣蜥成釉细胞的釉质基质和成釉细胞的分泌颗粒上显示出强烈而均匀的免疫反应。在Newt中，只有NC在釉质基质上显示出不均匀的反应性分布。与NC的强烈反应集中在釉质基质的表层，向中层向深层逐渐减少。内田等人也报道了类似的现象。等(1991)通过使用猪釉原蛋白C末端的抗体免疫组织化学显示猪牙釉质形成中的釉原蛋白。因此，本研究可能表明，与哺乳动物的成釉作用类似，该物种具有釉原蛋白的降解机制。然而，Western印迹分析表明，蝾螈和鬣蜥中都存在22 kDa的釉原蛋白，但在20 kDa以下的釉原蛋白中没有低分子量的釉原蛋白。这些数据表明，与哺乳动物相比，爬行动物和两栖动物的釉质发生对釉原蛋白的降解是不完整的。

In order to understand the phylogenetic characterization of amelogenin protein in lower vertebrates, cDNA sequences of amelogenin in reptile (iguana) and amphibia (newt) were determined, then immunohistochemical and western blot analyses were performed in them, using antibodies against the peptides consisting the N-terminus, C-terminus and central domain of their amelogenin. These antibodies were termed as follows by the recognizing portions of amelogenin in iguana and newt : IN (iguana N-terminus), IM (iguana central domain), IC (iguana C-terminus), NN (newt N-terminus), NM (newt central domain) and NC (newt C-terminus). ELISA analysis indicated that each antibody had a high specificity for the corresponding peptide. Immunohistochemical analysis was examined by the protein-A-gold method, for the iguana using IN,IM and IC, and for the newt using NN, NM and NC. Consequently, all antibodies, except NC, showed intense and uniform immunoreactivities over the enamel matrix and the secretory granules of the ameloblasts in iguana and newt, respectively. Only NC showed ununiform distribution of reactivities over the enamel matrix in newt. The intense reactions with NC were concentrated on the surface layer of an enamel matrix and decreased gradually toward the middle layer to deep layer. Similar phenomenon was reported by Uchida, et. al.(1991) by immunohistochemical demonstration of amelogenin in the porcine tooth enamel formation using the antibody against the C-terminus of the porcine amelogenin. The present study, therefore, may indicates that the newt has the degraded mechanism of the amelogenin similar to mammalian amelogenesis. Western blot analysis, however, showed the presence of 22 kDa amelogenin in both newt and iguana but the absence of low molecular weight of amelogenins under 20 kDa in them. These data suggest that the amelogenesis in reptile and amphibia were incomplete for the degradation of amelogenin proteins compared with that in mammals.