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胱抑素（胱抑素S，胱抑素SA，Systatin SN，Cystatin C和Cystatin d），帕帕因家族的半胱氨酸蛋白酶的非竞争性抑制剂中，以便利用其生理功能。在该研究项目的第一阶段，开发了用于大规模生产的大肠杆菌S. s. Sn和SA。该系统创建的工程性半胱氨酸蛋白酶被认为等同于天然胱抑素。该系统还用于在胱抑素S和抑制蛋白蛋白酶之间生产半胱氨酸蛋白酶变体和嵌合胱抑素。动力学研究具有上述胱抑素和减小肽的动力学研究表明，囊肿的第一个和第二个头蛋白蛋白环路对于抑制胱胺蛋白酶的抑制作用很重要。创造嵌合囊蛋白的成功表明，可以通过蛋白质工程设计和生产人造胱抑素。试图寻求利用重组人囊蛋白的方法的尝试。作为结果，发现胱抑素可抑制牙龈毒性的生长，并通过人牙龈成纤维细胞诱导白细胞介丁蛋白（IL-6和IL-8）生产。此外，清楚地证明了胱抑素是组织蛋白酶K的强抑制剂，该抑制剂与骨质碎裂性抗菌体有关。这些发现强调，伴结用于骨质疏松症和牙周疾病的治疗可能性。