Hu J C, Ryu O H, Chen J J, Uchida T, Wakida K, Murakami C, Jiang H, Qian Q, Zhang C, Ottmers V, Bartlett J D, Simmer J P
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 78284-7888, USA.
J Dent Res. 2000 Jan;79(1):70-6. doi: 10.1177/00220345000790011301.
Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.
釉基质丝氨酸蛋白酶1（EMSP1）是一种从猪牙发育中的釉质中分离出来的蛋白水解酶。其明显功能是降解有机基质，为釉质成熟做准备。除猪以外，尚未在其他生物体中研究过EMSP1的表达，并且从未在成釉细胞中特异性证明过釉器中EMSP1的表达。在此，我们报告了重组猪EMSP1（rpEMSP1）的表达、针对rpEMSP1的兔多克隆抗体的产生、通过蛋白质印迹和免疫组织化学分析对抗体和EMSP1表达的表征、编码小鼠EMSP1的全长cDNA的克隆和表征，以及通过原位杂交对小鼠第14天第一和第二磨牙成釉细胞中EMSP1表达的定位。小鼠EMSP1全长cDNA克隆有1237个核苷酸，不包括聚腺苷酸（A+）尾，编码一个255个氨基酸的前原蛋白。小鼠EMSP1与猪EMSP1的氨基酸同一性为75%，有三个潜在的N-连接糖基化位点，其中两个在猪同源物中保守。蛋白质印迹分析表明，多克隆抗体对EMSP1具有特异性，不与胰蛋白酶发生交叉反应。猪切牙的免疫组织化学显示，在成熟阶段最早的时期，表面釉质有离散染色。在小鼠磨牙中，原位杂交在成熟阶段的成釉细胞以及牙齿萌出后的结合上皮中给出明显且特异的信号。我们得出结论，在釉质形成的早期成熟阶段，猪和小鼠的成釉细胞表达EMSP1。
