Common epitopes of mammalian amelogenins at the C-terminus and possible functional roles of the corresponding domain in enamel mineralization.
Common epitopes of mammalian amelogenins at the C-terminus and possible functional roles of the corresponding domain in enamel mineralization.
复制标题
哺乳动物牙釉蛋白 C 末端的常见表位以及相应结构域在牙釉质矿化中的可能功能作用。
DOI：
10.1007/bf00296224
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发表时间：
1992
影响因子：
4.2
通讯作者：
Inage,T
中科院分区：
文献类型：
--
作者：
Aoba,T;Shimoda,S;Shimokawa,H;Inage,T
本研究旨在探讨哺乳动物釉原蛋白C端的共同表位的存在及其保守的C端结构域在哺乳动物釉质形成过程中的矿化作用。从猪、牛、大鼠和兔切牙的分泌性釉质中分离出釉质蛋白，包括完整的釉原蛋白及其降解多肽。兔和大鼠抗肽血清，以及大鼠抗25 kD和20 kD猪釉原蛋白血清，用于确定釉原蛋白之间的分离的基质蛋白的每种动物物种。先前（Aoba等人[19]）使用两种合成肽C13和C25作为免疫原开发了抗肽血清，这两种合成肽分别对应于猪完整釉原蛋白的最后12个（加上Cys用于KLH结合）和25个氨基酸残基。通过Western印迹分析检测釉质蛋白与每种抗血清的反应性。免疫印迹结果显示，抗C13血清可识别每种哺乳动物中的一些釉基质蛋白，特别是25 kD的猪釉原蛋白（以及27、22和18 kD的痕量组分），28 kD的牛釉原蛋白（26、22、19和14 kD的痕量组分）、28和26 kD的大鼠釉原蛋白（和20 kD的痕量组分）和24和21 kD的兔釉原蛋白（和13 kD的痕量组分）。抗C25血清还与23 kD的猪釉原蛋白、27 kD的牛釉原蛋白（主要基质成分）和19 kD的兔蛋白发生反应。抗猪20 kD釉原蛋白（缺乏最后25个氨基酸残基的C-末端）血清与大量的猪，牛，和大鼠釉原蛋白，但有趣的是，没有兔釉蛋白反应。用Maclura pomiferalectin对大鼠釉蛋白的分析表明，大鼠釉蛋白的糖基化具有异质性，特别是在28和26 kD完整的釉蛋白之间。在平行吸附研究中，从每个物种中分离的部分釉质蛋白样品被用作吸附物，以研究牙釉原蛋白在羟基磷灰石上的选择性吸附。吸附到晶体上的蛋白质的免疫印迹分析显示，在C-末端具有共同表位的哺乳动物釉原蛋白，一般来说，优先吸附到羟基磷灰石上。降解的釉原蛋白的吸附亲和力显着下降，对抗C13血清的反应性的损失。整体结果支持的论点，完整的哺乳动物釉原蛋白，包括大鼠和兔釉原蛋白，共享共同的抗原决定簇在C-末端和保守的C-末端结构域起着重要的作用，设置完整的釉原蛋白的分子结构，以促进蛋白质-釉矿物质的相互作用。
The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD. The anti-pig 20 kD amelogenin (lacking the last 25 amino acid residues at the C-terminus) serum reacted with a large number of pig, cow, and rat amelogenins but, interestingly, with none of the rabbit enamel proteins. Probing of rat enamel proteins withMaclura pomiferalectin showed the heterogeneity of glycosylation of rat amelogenins, particularly between the 28 and 26 kD intact amelogenins. In parallel adsorption studies, part of the enamel protein samples isolated from each of the species was used as adsorbates to investigate the selective adsorption of amelogenins onto hydroxyapatite. Immunoblot analysis of the proteins adsorbed onto the crystals revealed that the mammalian amelogenins having the common epitopes at the C-terminus, in general, adsorb preferentially onto hydroxyapatite. The adsorption affinity of the degraded amelogenins decreased significantly with the loss of reactivity toward the anti-C13 serum. The overall results support the contention that the intact mammalian amelogenins, including rat and rabbit amelogenins, share common epitopes at the C-terminus and that the conserved C-terminal domain plays an important role in setting the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction.