Hitomi J, Yamaguchi K, Kikuchi Y, Kimura T, Maruyama K, Nagasaki K
Growth Factor Division, National Cancer Center Research Institute, Tokyo, Japan.
J Cell Sci. 1996 Apr;109 ( Pt 4):805-15. doi: 10.1242/jcs.109.4.805.
We found by using a 45Ca2+ overlay technique a large amount of Ca(2+)-binding activity in bovine amniotic fluid from which a novel calcium-binding protein (CaBP) was purified and is referred to as CAAF1 (calcium-binding protein in amniotic fluid-1), with an apparent molecular mass of 8 kDa determined by N-tris(hydroxymethyl)-methylglycine/ SDS-PAGE. It was structurally homologous with MRP/calgranulin proteins (MRP8/calgranulin A and MRP14/calgranulin B), members of the S100 protein family, which are abundantly found in the cytoplasm of granulocytes and macrophages. CAAF1 lacked the predicted signal peptide sequence, which is consistent with other CaBPs. The tissue and cellular distribution of CAAF1 was determined by monoclonal antibodies developed against this protein. Its immunoreactivity was found in squamous epithelial cells, neutrophils, and some macrophages throughout the fetal body. An especially characteristic staining pattern was obtained in the squamous epithelium, including that of the esophagus, skin and amnion: CAAF1 was detected in the suprabasal squamous epithelial cells undergoing differentiation, but not in the cells in the proliferating basal layer. Northern blot analysis also showed that CAAF1 mRNA was highly expressed in bovine fetal esophagus and skin. On the other hand, our ELISA studies showed that CAAF1 protein was present in amniotic fluid at a concentration of about 120 nM, which was over 30 times as high as that in the fetal serum. These results suggested that CAAF1 is one of the stage-specific proteins in the differentiation of squamous epithelial cells, and that CAAF1 is preferentially produced by fetal squamous epithelial cells, including epidermal keratinocytes and amniotic epithelial cells, and it is stored in the amniotic fluid during embryogenesis.
我们通过使用45Ca2+覆盖技术发现，牛羊水具有大量的Ca(2+)结合活性，从中纯化出一种新型钙结合蛋白（CaBP），称为CAAF1（羊水中钙结合蛋白-1），通过N-三（羟甲基）甲基甘氨酸/SDS-PAGE测定其表观分子量为8 kDa。它在结构上与MRP/钙粒蛋白家族成员（MRP8/钙粒蛋白A和MRP14/钙粒蛋白B）同源，后者是S100蛋白家族的成员，大量存在于粒细胞和巨噬细胞的细胞质中。CAAF1缺乏预测的信号肽序列，这与其他CaBP一致。CAAF1的组织和细胞分布通过针对该蛋白产生的单克隆抗体来确定。在整个胎儿体内的鳞状上皮细胞、中性粒细胞和一些巨噬细胞中发现了其免疫反应性。在鳞状上皮，包括食管、皮肤和羊膜的鳞状上皮中获得了一种特别典型的染色模式：在正在分化的基底上层鳞状上皮细胞中检测到CAAF1，但在增殖的基底层细胞中未检测到。Northern印迹分析还表明，CAAF1 mRNA在牛胎儿食管和皮肤中高度表达。另一方面，我们的ELISA研究表明，羊水中CAAF1蛋白的浓度约为120 nM，比胎儿血清中的浓度高30倍以上。这些结果表明，CAAF1是鳞状上皮细胞分化过程中的阶段特异性蛋白之一，并且CAAF1优先由胎儿鳞状上皮细胞产生，包括表皮角质形成细胞和羊膜上皮细胞，并且在胚胎发生过程中储存在羊水中。
