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Studies on Production of Human Salivary Type Cystatins by Genetic Engineering and their Application to Dental Medicine

基因工程生产人唾液型胱抑素及其在牙科医学中的应用研究

基本信息

批准号：
09671910
负责人：
SAITOH Eiichi
金额：
$ 1.98万
依托单位：
Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671910/
关键词：
Saliva Cysteine proteinase inhibitor Recombinat cystatin Chimeric cystatin Site directed mutagenesis Utilization of recombinant cystatin Protein engineering Structural and functional relationship of protein ヒト唾液シスタチンカテプシンK シスタチンSA変異体

项目摘要

The head investigator took the focus to human cystatins (cystatin S, cystatin SA, systatin SN, cystatin C, and cystatin D), noncompetitive inhibitors for cysteine proteinases of the papain family, in order to utilize their physiological functions. In the first stage of this research project, the E. coli system for the large-scale production of cystatins S. SN, and SA was developed. The engineered cystatins created by this system were considered to be equivalent to the natural cystatins. This system was also employed for the production of cystatin variants and chimeric cystatins between cystatin S and cystatin C. The kinetic study with above cystatins and minicking peptides showed that the first and second hairpin loops of the cystatins are important in the inhibition of cysteine proteinases. The success in creation of chimeric cystatins suggested that it is possible to design and produce artificial cystatins by protein engineering.The attempts for seeking ways to utilize recombinant human cystatins were finally made. As the results, the cystatins were found to inhibit the growth of Porphyromonas gingivalis and to induce interleukins (IL-6 and IL-8) productions by human gingival fibroblasts. In additions, the cystatins were clearly demonstrated to be a strong inhibitor for cathepsin K, which is associated with osteoclastic born resorpiton. These findings emphasize that the cystatins possess possibility of use in therapy for osteoporosis and periodontal disease.

首席研究员将重点放在人半胱氨酸蛋白酶抑制剂（半胱氨酸蛋白酶抑制剂S、半胱氨酸蛋白酶抑制剂SA、半胱氨酸蛋白酶抑制剂SN、半胱氨酸蛋白酶抑制剂C和半胱氨酸蛋白酶抑制剂D），木瓜蛋白酶家族半胱氨酸蛋白酶的非竞争性抑制剂，以利用其生理功能。在本研究的第一阶段，E。大肠杆菌系统用于大规模生产半胱氨酸蛋白酶抑制剂S. SN和SA。由该系统产生的工程化半胱氨酸蛋白酶抑制剂被认为等同于天然半胱氨酸蛋白酶抑制剂。该系统还用于产生半胱氨酸蛋白酶抑制剂变体和半胱氨酸蛋白酶抑制剂S与半胱氨酸蛋白酶抑制剂C之间的嵌合半胱氨酸蛋白酶抑制剂。对上述半胱氨酸蛋白酶抑制剂和minicking肽的动力学研究表明，半胱氨酸蛋白酶抑制剂的第一和第二发夹环在半胱氨酸蛋白酶的抑制中是重要的。嵌合型半胱氨酸蛋白酶抑制剂的成功构建表明，利用蛋白质工程技术设计和生产人工半胱氨酸蛋白酶抑制剂是可能的，并为寻求利用重组人半胱氨酸蛋白酶抑制剂的途径进行了尝试。结果发现，半胱氨酸蛋白酶抑制剂抑制牙龈卟啉单胞菌的生长，并诱导人牙龈成纤维细胞产生白细胞介素（IL-6和IL-8）。此外，明确证明半胱氨酸蛋白酶抑制剂是组织蛋白酶K的强抑制剂，组织蛋白酶K与骨细胞产生的再吸收相关。这些发现强调了半胱氨酸蛋白酶抑制剂具有用于治疗骨质疏松症和牙周病的可能性。