|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1997 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1996 : ¥1,600,000 (Direct Cost : ¥1,600,000)
This project was planned to determine the relationship between structures and functions of ER-60 protease which was isolated from the endoplasmic reticulum (ER) of rat liver and characterized by the head investigator.
In 1997, the experiments described below were carried out.
1.ER-60 protease is localized in ER in a soluble form but does not contain a C-terminal KDEL sequence, the well-known retention signal in ER.However, ER-60 protease has the unique tetrapeptide, QEDL,at its C-terminal position. To determine the function of the C-terminal QEDL sequence, we expressed the wild-type and mutant rat ER-60 proteases in COS cells, and observed the localization of the protease proteins in the cells by immunostaining. On immunostaining with anti-rat ER-60F serum, untransfected COS cells were not stained, but the COS cells transiently expressing the wild type rat ER-60 protease were stained, showing a typical ER profile. The mutant ER-60 protease, of which the QEDL sequence was deleted or repla
ced by a non-functional tetrapeptide, AAGL,were colocalized with the Golgi marker proteins. The ER of cells expressing the mutant ER-60 protease with KDEL substituted for QEDL was stained, as in the case of the cells epressing the wild-type ER-60 protease. The ER-60 proteases of the wild type and the KDEL-substituted mutant were not colocalized with the Golgi marker proteins. The secretion into the medium of the rat recombinant ER-60 protease pulse-labeled with [^<35>S] methinonine and-cysteine in transient transfectants of COS cells was determined by immunoprecipitation with the anti-rat ER60F serum. The wild-type enzyme mostly remained in cells after a 5-h chase. About 45% of the total mutant enzyme, of which the QEDL sequence was deleted or replaced by an AAGL sequence, was secreted into the medium after a 5-h chase. It is likely that the C-terminal QEDL sequence of ER-60 protease may function as a retention signal in ER.
2.The head investigator succeeded in the expression of the recombinant human ER-60 proteas, which had the proteolytic activity, in E.coli (BL21(DE3)). The activity was inhibited by pCMB.The recombinant enzyme was thus reconfirmed to be a cysteine protease. ER-60 protease contains seven cysteine residues, four of which constitute two CGHC motifs which were assumed to include an active center cysteine residue (s) of the proteases. From the experiment with the recombinant human ER-60 proteases with site-directed mutations of the C-terminal cysteine residues (Cys-60 and Cys-409) of the CGHC motifs to serine, these cysteine residues were suggested to be an active center cysteine residue (s) . The double-mutated enzyme with Cys-57 and Cys-406 both modified to alanine (C60A/C409A) showed no activity. The single-mutated enzymes, C60A and C409A exhibited activity. These results suggest the C-terminal cysteine residues of the CGHC motifs are responsible for the protease activity. Less