Studies on the production and practical use of engineered human salivary type cystatins.

本研究项目着眼于从动植物中提取的人唾液胱抑素和天然半胱氨酸蛋白酶抑制剂的生物活性的实际应用观点。此外，还研究了一种能够大规模生产数百至数千毫克水平的工程胱抑素的系统。基于这些概念和计划，利用本研究开发的反相酶谱法筛选了新型半胱氨酸蛋白酶抑制剂。结果表明：(1)大肠杆菌生产的重组胱他汀可通过细胞表面分子诱导人牙龈成纤维细胞产生白细胞介素-6和白细胞介素-8。(2)工程唾液型胱抑素还可抑制短杆菌等微生物对腋窝气味的形成。(3)一些实验数据表明，新泻泻光米糠提取物中存在抑制牙龈卟啉单胞菌生长的成分。目前，我们不仅在研究分子量为13 kDa的米胞他汀类药物，还在研究与半胱氨酸蛋白酶、木瓜蛋白酶和ficin紧密结合的50-70 kDa的候选蛋白。由于水稻半胱氨酸蛋白酶抑制剂可能是牙龈卟啉单胞菌和唾液胱抑素生长的有效抑制剂，因此这些物质的详细特征目前正在用反酶技术进行研究。(6)鱼类皮肤黏液中存在一种防御系统的机制可能有助于研究唾液蛋白在口腔中的生理作用。因此，我们试图在日本鳗鱼的黏液中寻找苍蝇防御蛋白，如蛋白酶、蛋白酶抑制剂和凝集素。结果表明，皮肤黏液含有新型半胱氨酸蛋白酶抑制剂，分子量约为13-15 kDa。然而，目前尚不清楚这些抑制剂是否属于胱抑素超家族成员。综上所述，可以得出结论，来自植物和动物的半胱氨酸蛋白酶抑制剂被认为是一种非常有用的生物材料，可用于维持口腔健康和牙周炎的临床试验。少

This research project focused on the viewpoint of practical use of biological activities of human salivary cystatins and natural cysteine proteinase inhibitors from plants and animals. In addition, a system enabling large-scale production of engineered cystatins at a Ievel of several hundreds to several thousands milligrams was investigated. Based on these concepts and plans, novel cysteine proteinase inhibitors were screened as well by the reverse zymography developed in this study. The results obtained are summarized as follows: (1) Recombinant cystatins produced by E. coli were demonstrated to induce interleukin-6 and interleukin-8 productions in the human gingival fibroblast cells via cell surface molecules. (2) The engineered salivary type cystatins were also found to depress the formation of axillary odors by microorganisms such as Brevibacterium. (3) Some experimental data have indicated that there are some components mat inhibit the growth of Porphyromonas gingivalis in the hea … More t-extract of the rice bran of Niigata-Koshihikari. At present, we have been studying not only oryzacystatins with molecular weight of 13 kDa but also several candidate proteins of 50-70 kDa that tightly bind to a cysteine proteinase, papain and ficin. The detail features of the substances are now under investigation by the reverse zymographical technique since rice cysteine proteinase inhibitors could be a potent inhibitor of the growth of P. gingivalis as well as salivary cystatins. (6) The mechanism of a defense system present in the skin mucus of fishes may be helpful to study physiological roles of salivary proteins in oral cavity. Hence, we have attempted to search for flie defense proteins such as proteainses, proteinase inhibitors, and the lectins in the mucus fluid of the Japanese eel. As the result, the skin mucus was shown to contain novel cysteine proteinase inhibitor, with molecular masses of about 13-15 kDa. However, it is obscure at present that the inhibitors may be the members of cystatin superfamily, or not. Taken together, it can be concluded that cysteine proteinase inhibitors from plants and animals are thought to be a considerably useful biomaterial for the maintenance of oral health and for the clinical trials of periodontitis. Less