Other Families in Clan CA | 455.
Calpain-2/m-Calpain
[82] Suzuki, K., Sorimachi, H., Yoshizawa, T., Kinbara, K., Ishiura, S.
(1995). Calpain: novel family members, activation, and physiologic
function. Biol. Chem. Hoppe Seyler 376(9), 523
529.
[83] Richard, I., Broux, O., Allamand, V., Fougerousse, F.,
Chiannilkulchai, N., Bourg, N., Brenguier, L., Devaud, C.,
Pasturaud, P., Roudaut, C., Hillaire, D., Passos-Bueno, M.-R.,
Zats, M., Tischfield, J.A., Fardeau, M., Jackson, C.E., Cohen, D.,
Beckmann, J.S. (1995). Mutations in the proteolytic enzyme calpain
3 cause limb-girdle muscular dystrophy type 2A. Cell 81(1), 27
40.
[84] Sorimachi, H., Kinbara, K., Kimura, S., Takahashi, M., Ishiura, S.,
Sasagawa, N., Sorimachi, N., Shimada, H., Tagawa, K.,
Maruyama, K., Suzuki, K. (1995). Muscle-specific calpain, p94,
responsible for limb girdle muscular dystrophy type 2A, associates
with connectin through IS2, a p94-specific sequence. J. Biol.
Chem. 270(52), 31158
31162.
[85] Donkor, I.O. (2011). Calpain inhibitors: a survey of compounds
reported in the patent and scientific literature. Expert Opin. Ther.
Pat. 21(5), 601
636.
[86] Portbury, A.L., Willis, M.S., Patterson, C. (2011). Tearin’ up my
heart: proteolysis in the cardiac sarcomere. J. Biol. Chem. 286(12),
9929
9934.
Hiroyuki Sorimachi
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
Email: sorimachi-hr@igakuken.or.jp
Shoji Hata
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
Yasuko Ono
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
Handbook of Proteolytic Enzymes, 3rd Edn
ISBN: 978-0-12-382219-2
Calpain-2/m-Calpain
DATABANKS
MEROPS name: calpain-2
MEROPS classification: clan CA, family C2, subfamily
C2A, peptidase C02.002
IUBMB: EC 3.4.22.53 (BRENDA)
Tertiary structure: Available
Species distribution: subphylum Vertebrata
Reference sequence from: Homo sapiens (UniProt:
P17655)
2007
[87] Sorimachi, H., Hata, S., Ono, Y. (2010). Expanding members and
roles of the calpain superfamily and their genetically modified ani-
mals. Exp. Anim. 59(5), 549
566.
[88] Murphy, R.M. (2010). Calpains, skeletal muscle function and exer-
cise. Clin. Exp. Pharmacol. Physiol. 37(3), 385
391.
[89] Liu, J., Liu, M.C., Wang, K.K. (2008). Calpain in the CNS: from
synaptic function to neurotoxicity. Sci. Signal 1(14), re1.
[90] Das, A., Guyton, M.K., Butler, J.T., Ray, S.K., Banik, N.L. (2008).
Activation of calpain and caspase pathways in demyelination and
neurodegeneration in animal model of multiple sclerosis. CNS
Neurol. Disord. Drug Targets 7(3), 313
320.
[91] Azuma, M., Shearer, T.R. (2008). The role of calcium-activated
protease calpain in experimental retinal pathology. Surv.
Ophthalmol. 53(2), 150
163.
[92] Dargelos, E., Poussard, S., Brule, C., Daury, L., Cottin, P. (2008).
Calcium-dependent proteolytic system and muscle dysfunctions:
a possible role of calpains in sarcopenia. Biochimie 90(2), 359
368.
[93] Davis, T.L., Walker, J.R., Finerty, P.J. Jr., Mackenzie, F.,
Newman, E.M., Dhe-Paganon, S. (2007). The crystal structures of
human calpains 1 and 9 imply diverse mechanisms of action and
auto-inhibition. J. Mol. Biol. 366(1), 216
229.
© 2013 Elsevier Ltd. All rights reserved.
DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00453-1
Chapter 455
Name and History
m-Calpain is an intracellular, nonlysosomal, ubiquitous
cysteine protease, whose activity requires mM levels of
Ca21 , at least in vitro. m-Calpain is a hetero-dimer con-
sisting of a smaller regulatory subunit (CAPNS1/30K) and
a larger catalytic calpain subunit (CAPN2/mCL), whose
amino acid sequence is ca. 60% identical to that of
CAPN1/μCL. As described in Chapter 454, there exist
many calpain homologs in biology. Thus, the account of
2008
the calpains in the present volume is divided into five
chapters (454
459) for μ-calpain (a complex of CAPN1/
μCL and CAPNS1/30K), m-calpain (a complex of
CAPN2/mCL and CAPNS1/30K), skeletal muscle-specific
calpain (CAPN3, also called p94), gastrointestinal-tract-
specific calpains (CAPN8, also called nCL-2, and CAPN9,
also called nCL-4; and other calpains, respectively). For a
general introduction to the calpains, and the details of the
naming and history of m-calpain, see Chapter 455.
Activity and Specificity
The activity and specificity of m-calpain are almost iden-
tical to those of μ-calpain except for the Ca21 require-
ment (see Chapter 455). Most of the efficient inhibitors of
μ-calpain, including the endogenous inhibitor protein cal-
pastatin, also inhibit m-calpain efficiently [1].
m-Calpain undergoes N-terminal autolysis in what
was long believed to be an activation process. However, a
recent study indicated that autolysis of the N-terminus is
not required for activation, but is involved in the associa-
tion and dissociation of the two subunits, in some cases
[2]. That is, the autolysis may disrupt the salt bridge
between N-terminal Lys7 in CAPN1/mCL and Asp154 in
the 2nd EF-hand motif (EF-2) of CAPNS1/30K, and/or
the salt bridge between CAPN1/mCL’s Arg12 and
Glu260 in CAPNS1/30K’s EF-5 domain.
Structural Chemistry
As in the case of μ-calpain, mammalian m-calpain is a
heterodimer of about 110 kDa. At least in mammals, the
FIGURE 455.1 Schematic three-dimen-
sional structures of inactive and active
Ca2+-binding
CAPN1/mCL catalytic domains. Schematic
site (CBS-1)
three-dimensional ribbon structures of inac-
tive (Ca21 -free, dark) and active (Ca21 -
bound, light) forms of human and rat m-cal-
pain using PDB data, 1KFX and 3DF0,
respectively [4,8], are superimposed keeping
the PC1 domains of the both structures
aligned and fixed (RMSD 5 1.63 Å). The
active protease (CysPc) domain is formed
by fusion of the PC1 and PC2 domains after
the binding of a single Ca21 by each of the
core domains. This causes the rotation and
translation of His and Asn residues of the
active-site triad by 50 and 6 Å, respec-
tively. The active site is circled in yellow.
Blue balls represent Ca21. protease
core domain 1
(PC1)
Other Families in Clan CA | 455. Calpain-2/m-Calpain
catalytic subunits of μ- and m-calpains (CAPN1/μCL and
CAPN2/mCL, respectively) are similar but distinct,
whereas they form heterodimers with the same regulatory
subunit. Hence, the different Ca21 dependencies of
μ- and m-calpain must be ascribed to their catalytic subu-
nits. The domain structure of CAPN2/mCL is identical to
that of CAPN1/μCL, and is divided into four domains/
regions (see Chapter 455, and Figure 455.1B therein).
Since crystals of m-calpain were more easily achieved
than those of μ-calpain, three-dimensional structural stud-
ies using m-calpain or fragments containing certain
domains were the first to be performed. Comparison of
the m-calpain protease domains in the presence and
absence of Ca21 revealed that all the α-helices and
β-strands are conserved, consistent with previous CD
spectrum results [3] (see Chapter 455, and Figure 454.3
therein). In the presence and absence of Ca21 , the prote-
ase core domain 1 structures of both states can be aligned
very closely, whereas those of protease core domain 2
can be aligned only moderately [4
8]. As predicted from
the three-dimensional structure, Ca21 binding causes PC2
to be rotated and translated by 50 and 6 Å, if PC1
structures in the both states are aligned and fixed
(Figure 455.1). A Ca21 -induced Ca21 -binding site is
generated in each core domain, and Ca21 binding to these
sites causes the above-described PC2 rotation and transla-
tion for activation. These Ca21 -binding sites have a novel
and unique structure. The active-site cleft of m-calpain,
like that of μ-calpain, is deep and narrow compared with
that of papain.
Although the protease domains of CAPN1/μCL,
CAPN2/mCL, and CAPN9/nCL-4 are highly conserved
(59B71% aa identity), the Ca21 -bound three-dimensional
Ca2+-binding
site (CBS-2)
protease
Ca2+ core domain 2
(PC2)
Asn286 (inactive)
His262 (inactive)
activation
upon
Ca2+-binding
Asn286 (active)
Cys105 / Ser105 calpain
active site triad
(inactive / active)
(active)
protease His262 (active)
(CysPc) domain
(= PC1 + PC2)
Other Families in Clan CA | 455. Calpain-2/m-Calpain 2009

C-term
Calpastatin Calpain
P182(P4') Protease
Core domain 2
(PC2)
P181(P3')
N286
I180(P2')
T179(P1')
174-178 loop-out H262

S105
G174(distorted P1)

L173(P2)

Calpain
Protease A172(P3)
Core domain 1
(PC2)

N-term
FIGURE 455.2 Three-dimensional structure of the co-crystal of m-calpain, Ca21, and calpastatin repeat 1. Enlarged view of the interaction between
calpastatin (blue) and the catalytic cleft in the protease core domain PC1 (pink), PC2 (red) of m-calpain (the three-dimensional structure is from
3DF0). Calpastatin binds in the substrate orientation indicated by positions P3 to P30 . At P1, calpastatin distorts from the substrate path and projects
residues 174
178 or 613
617, which form a kink between the P2 and P10 anchor sites.

structures of these domains revealed that a reversible
intrinsic inhibitory/safety mechanism exists only for
CAPN2/mCL [6,9,10]. Trp106, which is adjacent to the
active-site Cys105 of CAPN2/mCL, is positioned to inter-
fere with substrates’ access to the active site; this mecha-
nism does not occur in rat CAPN1/μCL [6] (see Chapter
455 and Figure 455.4 therein). On the other hand,
CAPN9/nCL-4 has a novel intrinsic inhibitory mecha-
nism, i.e. the misalignment of the catalytic residues [10].
These findings together show that the activation/latency
mechanisms of calpains, regardless of their highly con-
served sequences, are somewhat specialized for each mol-
ecule, and are controlled by inter-domain interactions. In
the three-dimensional structure of the entire m-calpain
molecule [7,8], the above-mentioned Trp106 mechanism
is not found, indicating that the inter-domain interactions
impose dominant controls over the mechanisms of activ-
ity control specific to each calpain molecule above
mentioned.
When co-crystallized with calpastatin and Ca21, the
three-dimensional structure of m-calpain revealed one of
the mechanisms used by calpastatin to inhibit conven-
tional calpain activity [7,8]. Calpastatin interacts tightly
with calpain, but is not proteolyzed, because the calpasta-
tin sequence results in a small ‘kink’ very close to the cal-
pain active site (Figure 455.2). Mutant calpastatins in
which some residues involved in this kink are deleted are
proteolyzed by calpain [7,8].
In addition, humans have a second gene (CAPNS2)
for the calpain regulatory subunit, whose product is sim-
ilar to CAPNS1/30K [11]. CAPNS1/30K and CAPNS2
both belong to the PEF protein superfamily, but are sig-
nificantly more similar to each other (aa sequences are
73% identical), than to other PEF proteins. Moreover,
recombinant CAPNS2 and CAPN2/mCL form a hetero-
dimer that has a Ca21 -dependent activity identical to
that of m-calpain. The physiological function of
CAPNS2 is unclear as yet.
Preparation
Mammalian m-calpain can be purified by the same
method as μ-calpain (see Chapter 455). Mammalian skel-
etal muscle normally contains more m-calpain than μ-cal-
pain. In the first DEAE column chromatography step,
m-calpain is usually eluted at NaCl concentrations above
200 mM, whereas μ-calpain is co-eluted with calpastatin
at less than 100 mM NaCl. Recombinant m-calpain has
been successfully expressed in E. coli and an Sf-9/baculo-
virus system [12
14].
Biological Aspects
Mammalian m-calpain is expressed ubiquitously except in
erythrocytes, and it is thought to have essential and
2010
fundamental functions, like μ-calpain. As mentioned in
the previous chapter, however, disrupting the mouse gene
for CAPN1/μCL or CAPN2/mCL, i.e. Capn1 or Capn2,
respectively, leads to different results: Capn2 2 / 2 mice
die before the blastocyst stage, whereas Capn1 2 / 2 mice
appear normal and are fertile [15,16]. This suggests that
μ- and m-calpains differ in their functions and/or expres-
sion level, at least at specific developmental stage(s),
which is a very important and intriguing subject for
study.
Distinguishing Features
For Calpain portals, see The Calpain Family of Proteases
(http://ag.arizona.edu/calpains/), Calpain for Modulatory
Proteolysis (CaMP DB) (http://www.calpain.org/), and
The Calpain Research Portal (htttp://calpain.net).
Related Peptidases
See also Chapters 454
459 for accounts of μ-calpain
(CAPN1/μCL and CAPNS1/30K), muscle calpain
(CAPN3/p94), gastrointestinal calpain (CAPN8/nCL-2
and CAPN9/nCL-4), and other calpains, respectively.
Further Reading
For recent reviews on conventional calpain functions and
various biological phenomena, see Donkor [1], Portbury
et al. [17], Sorimachi et al. [18,24,25], Murphy [19], Liu
et al. [20], Das et al. [21], Azuma & Shearer [22], and
Dargelos et al. [23].
References
[1] Donkor, I.O. (2011). Calpain inhibitors: a survey of compounds
reported in the patent and scientific literature. Expert Opin. Ther.
21(5), 601
636.
[2] Nakagawa, K., Masumoto, H., Sorimachi, H., Suzuki, K. (2001).
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), 605
611.
[3] Tsuji, S., Ishiura, S., Takahashi-Nakamura, M., Katamoto, T.,
Suzuki, K., Imahori, K. (1981). Studies on a Ca21 -activated neu-
tral proteinase of rabbit skeletal muscle. II. Characterization of
sulfhydryl groups and a role of Ca21 ions in this enzyme. J.
Biochem. 90(5), 1405
1411.
[4] Strobl, S., Fernandez-Catalan, C., Braun, M., Huber, R.,
Masumoto, H., Nakagawa, K., Irie, A., Sorimachi, H.,
Bourenkow, G., Bartunik, H., Suzuki, K., Bode, W. (2000). The
crystal structure of calcium-free human m-calpain suggests an elec-
trostatic switch mechanism for activation by calcium. Proc. Natl.
Acad. Sci. USA 97(2), 588
592.
Other Families in Clan CA | 455. Calpain-2/m-Calpain
[5] Hosfield, C.M., Elce, J.S., Davies, P.L., Jia, Z. (1999). Crystal
structure of calpain reveals the structural basis for Ca21 -dependent
protease activity and a novel mode of enzyme activation. EMBO J.
18(24), 6880
6889.
[6] Moldoveanu, T., Hosfield, C.M., Lim, D., Jia, Z., Davies, P.L.
(2003). Calpain silencing by a reversible intrinsic mechanism. Nat.
Struct. Biol. 10(5), 371
378.
[7] Hanna, R.A., Campbell, R.L., Davies, P.L. (2008). Calcium-bound
structure of calpain and its mechanism of inhibition by calpastatin.
Nature 456(7220), 409
412.
[8] Moldoveanu, T., Gehring, K., Green, D.R. (2008). Concerted mul-
ti-pronged attack by calpastatin to occlude the catalytic cleft of
heterodimeric calpains. Nature 456(7220), 404
408.
[9] Moldoveanu, T., Hosfield, C.M., Lim, D., Elce, J.S., Jia, Z.,
Davies, P.L. (2002). A Ca21 switch aligns the active site of cal-
pain. Cell 108(5), 649
660.
[10] Davis, T.L., Walker, J.R., Finerty, P.J. Jr., Mackenzie, F.,
Newman, E.M., Dhe-Paganon, S. (2007). The crystal structures of
human calpains 1 and 9 imply diverse mechanisms of action and
auto-inhibition. J. Mol. Biol. 366(1), 216
229.
[11] Schad, E., Farkas, A., Jekely, G., Tompa, P., Friedrich, P. (2002).
A novel human small subunit of calpains. Biochem. J. 362(Pt. 2),
383
388.
[12] Graham-Siegenthaler, K., Gauthier, S., Davies, P.L., Elce, J.S.
(1994). Active recombinant rat calpain II. Bacterially produced
large and small subunits associate both in vivo and in vitro. J. Biol.
Chem. 269(48), 30457
30460.
[13] Hata, S., Ueno, M., Kitamura, F., Sorimachi, H. (2012). Efficient
expression and purification of recumbinant human m-calpain using
an Escherichia Coli expression system at low temperature.
J. Biochem. 151(4), 417
422.
[14] Meyer, S.L., Bozyczko-Coyne, D., Mallya, S.K., Spais, C.M.,
Bihovsky, R., Kaywooya, J.K., Lang, D.M., Scott, R.W., Siman, R.
(1996). Biologically active monomeric and heterodimeric recombi-
nant human calpain I produced using the baculovirus expression
system. Biochem. J. 314(2), 511
519.
[15] Dutt, P., Croall, D.E., Arthur, S.C., De Veyra, T., Williams, K.,
Elce, J.S., Greer, P.A. (2006). m-Calpain is required for preimplan-
tation embryonic development in mice. BMC Dev. Biol. 6(3)
10.1186/1471
213X-6-3.
[16] Azam, M., Andrabi, S.S., Sahr, K.E., Kamath, L., Kuliopulos, A.,
Chishti, A.H. (2001). Disruption of the mouse mu-calpain gene
reveals an essential role in platelet function. Mol. Cell. Biol. 21(6),
2213
2220.
[17] Portbury, A.L., Willis, M.S., Patterson, C. (2011). Tearin’ up my
heart: proteolysis in the cardiac sarcomere. J. Biol. Chem. 286(12),
9929
9934.
[18] Sorimachi, H., Hata, S., Ono, Y. (2010). Expanding members and
roles of the calpain superfamily and their genetically modified ani-
mals. Exp. Anim. 59(5), 549
566.
[19] Murphy, R.M. (2010). Calpains, skeletal muscle function and exer-
cise. Clin. Exp. Pharmacol. Physiol. 37(3), 385
391.
[20] Liu, J., Liu, M.C., Wang, K.K. (2008). Calpain in the CNS: from
synaptic function to neurotoxicity. Sci. Signal 1(14), re1.
[21] Das, A., Guyton, M.K., Butler, J.T., Ray, S.K., Banik, N.L. (2008).
Activation of calpain and caspase pathways in demyelination and
Other Families in Clan CA | 455. Calpain-2/m-Calpain 2011

neurodegeneration in animal model of multiple sclerosis. CNS
Neurol. Disord Drug Targets 7(3), 313
320.
[22] Azuma, M., Shearer, T.R. (2008). The role of calcium-activated
protease calpain in experimental retinal pathology. Surv.
Ophthalmol. 53(2), 150
163.
[23] Dargelos, E., Poussard, S., Brule, C., Daury, L., Cottin, P. (2008).
Calcium-dependent proteolytic system and muscle dysfunctions:
a possible role of calpains in sarcopenia. Biochimie 90(2),
359
368.
[24] Sorimachi, H., Hata, S., Ono, Y. (2011). Calpain chronicle
an
enzyme family under multidisciplinary characterization. Proc. Jpn.
Acad Ser B Phys Biol. Sci. 87(6), 287
327.
[25] Sorimachi, H., Hata, S., Ono, Y. (2011). Impact of genetic insights
into calpain biology. J. Biochem. 148(1), 150(1), 23
37.

Hiroyuki Sorimachi
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
Email: sorimachi-hr@igakuken.or.jp

Shoji Hata
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.

Yasuko Ono
Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.

Handbook of Proteolytic Enzymes, 3rd Edn © 2013 Elsevier Ltd. All rights reserved.
ISBN: 978-0-12-382219-2 DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00454-3