Dissociation of m-Calpain Subunits Occurs after Autolysis of the N-Terminus of the Catalytic Subunit, and Is Not Required for Activation

• Published in: Journal of biochemistry (Tokyo) Vol. 130; no. 5; pp. 605 - 611
• Main Authors: Nakagawa, Kazuhiro, Masumoto, Hajime, Sorimachi, Hiroyuki, Suzuki, Koichi
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.11.2001
• Subjects: activation; Amino Acid Motifs; Amino Acid Sequence; Animals; Autolysis; Calcium - metabolism; calpain; Calpain - chemistry; Calpain - genetics; Calpain - metabolism; Catalytic Domain; Cell Line; dissociation; EF-hand; Enzyme Activation; Enzyme Stability; Humans; Insecta - cytology; Models, Molecular; Molecular Weight; Mutagenesis, Site-Directed; Protein Subunits
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a003025

Calpain is a heterodimeric, intracellular Ca2+-dependent, “bio-modulator” that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-tenninus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into sub-units is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca2+ has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (ç19), but not those lacking only nine residues (ç9), dissociated into subunits even in the absence of Ca2+. Moreover, both ç9 and ç19 mutants showed an equivalent reduced Ca2+ requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.