Immunocytochemical and immunochemical study of amelogenesis

用生化、免疫化学、免疫细胞化学和细胞生物学等方法研究了猪牙胚的成釉过程。主要研究结果如下：1.克隆了猪鞘蛋白的编码基因，并对其进行了鉴定。猪鞘蛋白是一种低相对分子质量的蛋白质，主要集中在未成熟釉质的棱柱鞘中。这些克隆的翻译产物可能是395和380个残基的分泌型蛋白。我们提出了翻译产物及其蛋白水解物的术语“鞘蛋白”。全长鞘蛋白与大鼠成釉蛋白的氨基酸序列有67%的同源性。2.鞘蛋白N端裂解的多肽约有100~150个氨基酸残基，定位于棱柱鞘中。C端裂解的多肽被鉴定为钙结合蛋白，分子量分别为29 kDa和27 kDa，均可被磷酸化和糖基化，定位于未成熟釉质的最外层。3.克隆了编码釉蛋白的基因。猪釉蛋白由1104个氨基酸残基组成。分泌的釉蛋白分子量约150 kDa，定位于靠近成釉细胞质膜的未成熟釉质中，与磷灰石晶体无亲和力。150 kDa釉质蛋白的N端切割蛋白为89 kDa，与磷灰石晶体有亲和力。32 kDa釉蛋白的氨基酸序列位于89 kDa釉蛋白的中间部分。4.综上所述，三类釉质蛋白在釉质形成中起着不同的作用。釉原蛋白主要形成釉质的形状，釉质蛋白控制初始钙化和磷灰石晶体的生长，鞘蛋白产生棱柱鞘，可能是釉质蛋白在釉质钙化过程中逃逸的一种方式。

Amelogenesis of the porcine tooth germ was investigated by means of biochemical, immunochemical, immunocytochemical and cell biological methods. The results are summarized as follows.1.The cDNAs encoding porcine sheath proteins, which are low molecular weight proteins concentrated in the prism sheath in immature enamel, were cloned and characterized. The translation products of these clones might be secreted proteins having 395 and 380 residues. We proposed the term "sheathlin" for the translation products and their proteolytic cleavage products. Full length sheathlin and rat ameloblastin shear 67% identity in their amino acid sequences.2.The N terminal cleaved peptides of sheathlin had about 100 to 150 amino acid residues and were specifically localized in the prism sheath. The C-terminal cleaved peptides were identified as calcium binding proteins having molecular weights of 29kDa and 27 kDa, both of which might be phosphorylated and glycosylated and were localized outer most layr of immature enamel.3.The cDNA encoding enamelin was cloned. The porcine enamelin composed 1104 amino acid residues. Secreted enamelin having molecular weight of about 150 kDa were localized in immature enamel just adjacent to the plasma membrane of the ameloblast, and showed no affinity for apatite crystals. The N-terminal cleaved protein of the 150 kDa enamelin was 89 kDa enamelin which showed affinity for apatite crystals. The amino acid sequence of the 32kDa enamelin was located in the middle portion of the 89 kDa enamelin.4.In conclusion, three categories of enamel proteins play different roles in the amelogenesis. Amelogenin primarily form the shape of enamel, enamelin control the initial calcification and the growth of the apatite crystals, and the sheathlin produce prism sheaths which might act as a way through which enamel protein escape during enamel calcification.