首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain
Authors:Takayuki Miyaji  Satoshi Murayama  Yoshiaki Kouzuma  Nobutada Kimura  Michael R Kanost  Karl J Kramer  Masami Yonekura  
Institution:a Laboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1, Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan;b Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan;c Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA;d Center for Grain and Animal Health Research, Agricultural Research Service, United States Department of Agriculture, 1515 College Avenue, Manhattan, KS 66502, USA
Abstract:A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (not, vert, similar9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu–Arg–MCA, Z-Phe–Arg–MCA and Boc–Val–Leu–Lys–MCA rapidly, whereas hydrolysis of Suc–Leu–Tyr–MCA and Z-Arg–Arg–MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with Ki values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.
Keywords:Cysteine protease inhibitor  Cystatin  Cathepsin F-like protease  Manduca sexta  Multifunctional domains
本文献已被 ScienceDirect 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号