Functions of characteristic Cys-Gly-His-Cys (CGHC) and Gln-Glu-Asp-Leu (QEDL) motifs of microsomal ER-60 protease.
The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b(+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.
Urade
R
R
Research Institute for Food Science, Kyoto University, Uji.
Oda
T
T
Ito
H
H
Moriyama
T
T
Utsumi
S
S
Kito
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C077733:ER-60 protease