K. Nakagawa et al., Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation, J BIOCHEM, 130(5), 2001, pp. 605-611
Calpain is a heterodimeric, intracellular Ca2+-dependent, "bio-modulator" t
hat alters the properties of substrates through site-specific proteolysis.
It has been proposed that calpains are activated by autolysis of the N-term
inus of the large subunit and/or its dissociation into the subunits. It is,
however, unclear whether the dissociation into subunits is required for th
e expression of protease activity and/or for in vivo function. Recently, th
e 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 s
ubunit (mCL) makes contact with the 30K subunit, suggesting that autolysis
of the N-terminus of mCL changes the interaction of both subunits. To exami
ne the relationship between autolysis, dissociation, and activation, we mad
e and analysed a series of N-terminal mutants of mCL that mimic the autolys
ed 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
(Delta 19), but not those lacking only nine residues (Delta9), dissociated
into subunits even in the absence of Ca2+. Moreover, both Delta9 and Delta
19 mutants showed an equivalent reduced Ca2+ requirement for protease acti
vity. These results indicate that autolysis is necessary for the dissociati
on of the m-calpain subunits, and that the dissociation occurs after, but i
s not necessary for, activation.