Identification and characterization of inhibitory sequences in four repeating domains of the endogenous inhibitor for calcium-dependent protease.
We reported previously the cDNA cloning of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli. The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues. The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions. The inhibitory activities of various fragments expressed in E. coli suggest the involvement of the regions around the well-conserved sequences. In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides. The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site. A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none. The inhibitory sequence is suggested as LGXKDREXTIPPXYRXLL. The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor. The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.
Kawasaki
H
H
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science.
Emori
Y
Y
Imajoh-Ohmi
S
S
Minami
Y
Y
Suzuki
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification; D010957:Plasmids; D011817:Rabbits; D012091:Repetitive Sequences, Nucleic Acid
D010446:Peptide Fragments; D004247:DNA; D002154:Calpain