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

首席研究员将重点放在人半胱抑素（半胱抑素S、半胱抑素SA、系统抑素SN、半胱抑素C和半胱抑素D）上，这是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。在本课题的第一阶段，开发了大规模生产胱抑素S. SN和SA的大肠杆菌体系。该系统产生的工程胱抑素被认为与天然胱抑素相当。该系统还被用于生产半胱抑素变异和半胱抑素S和半胱抑素c之间的嵌合半胱抑素。对上述半胱抑素和半胱抑素缩氨酸的动力学研究表明，半胱抑素的第一和第二发夹环在抑制半胱氨酸蛋白酶中起重要作用。嵌合半胱氨酸抑制素的成功制备表明，利用蛋白质工程技术设计和生产人工半胱氨酸抑制素是可能的。最后对重组人胱抑素的利用方法进行了探索。结果发现，胱抑素类药物抑制牙龈卟啉单胞菌的生长，诱导人牙龈成纤维细胞产生白细胞介素（IL-6和IL-8）。此外，胱抑素被清楚地证明是组织蛋白酶K的强抑制剂，组织蛋白酶K与破骨细胞出生再吸收有关。这些发现强调了胱抑素在骨质疏松症和牙周病的治疗中具有应用的可能性。

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.