Possible regulation of the conventional calpain system by skeletal muscle-specificcalpain, p94/calpain 3

Yasuko Ono‡§, Kazumi Kakinuma‡§, Fukuyo Torii‡, Akihiro Irie‡, Kazuhiro Nakagawa‡**,Siegfried Labeit¶, Keiko Abe‡, Koichi Suzuki||***, and Hiroyuki Sorimachi‡§†‡Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
Sciences, University of Tokyo, Tokyo 113-8657, Japan, §CREST, Japan Science and Technology,
Kawaguchi 332-0012, Japan, ¶Institut für Anästhesiologie und Operative Intensivmedizin,
Universitätsklinikum Mannheim, Germany, ||Tokyo Metropolitan Institute of Gerontology, Tokyo
173-0015, Japan.
†Corresponding author:

Laboratory of Biological Function, Department of Applied Biological

Chemistry,
Graduate School of Agricultural and Life Sciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
TEL/Fax: +81-3-5841-8118, e-mail: ahsori@mail.ecc.u-tokyo.ac.jp

Running Title: Regulation of conventional calpains by p94/calpain 3

1

SUMMARY
p94 (also called calpain 3) is the skeletal-muscle-specific calpain and is considered to be a“modulator protease” in various cellular processes. Analysis of p94 at the protein level is anurgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type
2A.

In this study, we enzymatically characterized one alternatively spliced variant of p94,

p94:exons 6-15-16- (p94∆), which lacks two of the p94-specific insertion sequences. In contrast
to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently
Ca2+-independent autolytic activity, p94∆ was stably expressed in COS and insect cells. p94∆
showed Ca2+-dependent caseinolytic and autolytic activities, and an inhibitor spectrum similar to
those of the conventional calpains. However, calpastatin did not inhibit p94∆, and is a substrate
for p94∆, which is consistent with the properties of p94, presenting p94 as a possible regulator of
conventional calpain system. We also established a semi-quantitative FRET assay using thecalpastatin sequence specifically to measure p94 activity. This method detects the activity of
COS-expressed p94 and p94∆, suggesting that it has potential to evaluate p94 activity in vivo andin the diagnosis of LGMD2A.

2

INTRODUCTION
Calpain (EC 3.4.22.17, clan CA, family C2) is a Ca2+-requiring cysteine protease representing
one of the most important families of the cysteine proteases (1-9). To date, various molecules
showing significant similarity to the calpain protease domain have been identified in almost allkinds of living organisms and constitute the “calpain superfamily” (6). Two representative
members, µ- and m-calpains, the so-called “conventional” calpains, are ubiquitously expressedand have been well characterized. These two calpains consist of a distinct larger catalytic
subunit containing a protease domain (µ- or m-calpain large subunit, abbreviated to µCL1 or
mCL, respectively) and a common smaller regulatory subunit (abbreviated to 30K according to
its molecular weight). On the basis of amino acid similarities, the large and small subunits havebeen described as consisting of four and two domains, respectively, which agrees with therecently resolved three-dimensional structure of m-calpain (10, 11; see Fig. 1A).
Conventional calpain has a specific endogenous proteinous inhibitor, calpastatin (12).
Calpastatin contains four repetitive inhibitory units, each of which inhibits equimolar amounts of
conventional calpain. The conserved reactive site interacts with the calpain protease domain,
whereas the flanking α-helical regions bind to domains IV and VI of the large and small subunits,
respectively. Synthetic oligopeptides (see Fig. 4D) corresponding to the calpastatin reactive site
specifically inhibit conventional calpain efficiently, although their inhibitory activity is weaker3

than that of the full-length inhibitory unit.
The primary structure of p94 (also called calpain 3) is very similar to those of µCL and mCL
throughout the entire molecule, beyond the p94-specific sequences, NS, IS1, and IS2 (13).
Previous studies have revealed several unique characteristics of p94 that diverge greatly fromthose of the conventional calpains. For instance, [1] p94 undergoes very rapid, thorough, andapparently Ca2+-independent autolysis in solution (half-life in vitro is less than 10 min) (14); [2]inhibitors of the conventional calpains, including calpastatin, EDTA, and EGTA, have no effecton p94 autolysis (14); [3] the gene for p94 produces several alternatively spliced products (15,16); and [4] p94 associates with the N2-line region and the C-terminus of connectin/titin, thegigantic filamentous molecule essential for myofibrils (17, 18; for a review of connectin/titin, see
19-21). The predominant expression of p94 in skeletal muscle, where its mRNA levels are ca.
10 times higher than those of µCL and mCL, indicates the physiological importance of p94 inthat tissue (13). Consistent with this, a defect in the p94 gene causes limb-girdle muscular
dystrophy type 2A (LGMD2A), suggesting that p94 functions are indispensable for propermuscle functions (22). Several studies, including ours, indicate that the loss of
substrate-processing activity, but not hyper-activation or a defect in the structural properties of
p94, causes LGMD2A (16, 23-26). Therefore, it has become an urgent issue to determine the invivo substrates of p94 to gain insight into the physiological functions of p94 and its relationships
4

to molecular mechanisms of LGMD2A (27).
However, the autolytic activity of p94 has hampered the study of p94 protein. Therefore, wehave focused on identifying the conditions that will allow us to analyse the proteolytic activity ofp94.

We have previously observed that deletion of either the IS1 or IS2 region, which aremainly encoded by exon 6 or exons 15+16, respectively, prevents the rapid autolysis of p94 (14).
In accord with this, we have recently shown that alternatively spliced variants of p94 that lackeither exon 6, exons 15+16, or all of these, are detected as non-autolyzed forms, unlike thefull-length p94, when they are recombinantly expressed in COS cells (16).
In this study, we chose one of the splicing variants, p94:exons 6-15-16- (abbreviated as p94∆ inthis paper, see Fig. 1A), which is produced from a transcript lacking exons 6, 15, and 16, andtherefore lacks most of the p94-specific insertion sequences, IS1 and IS2, for in vitro enzymaticcharacterization on the basis of its stability and expression efficiency. The enzymatic propertiesof p94∆ were compared with those of p94, and µ- and m-calpains. We thus identified several
novel enzymatic properties of p94. Notably, neither p94 nor p94∆ is inhibited by calpastatin,
and moreover, both hydrolyse calpastatin. These results led us to predict that p94 participates in
the regulation of the conventional calpain-calpastatin system in skeletal muscle. We used thesefindings to establish a calpastatin-based fluorescence assay specifically to measure p94 proteaseactivity, showing a possibility to application of this assay to a diagnostic tool for LGMD2A.
5

EXPERIMENTAL PREOCEDURES
Materials
Enzymes used for manipulating recombinant DNA were purchased from Takara Shuzo Inc.
(Kyoto, Japan).

The cDNAs for mouse p94/calpain 3 and p94:exons 6-15-16- (p94∆) (16), andSpodoptera frugiperda cells (Sf-9) were kindly provided by Dr. Muriel Herasse (Généthon, Evry,France), and Dr. Takeshi Nishino (Nippon Medical School, Tokyo, Japan), respectively. A
baculovirus expression vector, pFastBac1, culture media, and transfection reagent werepurchased from Invitrogen Inc. (CA, USA). Mammalian expression vectors, pECFP-N1 andpEYFP-N1, were purchased from BD Biosciences Clontech Inc. (CA, USA).
purchased from Merck (Frankfurt, Germany).

Casein wasProtease inhibitors were purchased from

Calbiochem Inc. (CA, USA), Peptide Institute Inc. (Osaka, Japan), or Sigma Inc. (MO, USA).
Other chemicals were obtained from Nacalai Tesque Inc. (Kyoto, Japan), or Wako Pure
Chemicals (Osaka, Japan).
p94-specific anti-pNS (antigen: PTVISPTVAPRTGAEPRS at the N-terminus) and anti-pIS2(identical to anti-pK-rich, antigen: NTISVDRPVKKKKNKPIIFV overlapping with the IS2
region) antisera have been described previously (14). Monoclonal antibodies 2C4 and 12A2,
which recognize N-terminus and the region around the catalytic residue Asn358 of p94,
respectively, were purchased from Novacastra Laboratories Ltd. (Newcastle, UK). Monclonal6

antibodies which recognize µCL and mCL were purchased from Biomol Research Laboratories
Inc. (PA, USA) and Chemicon International Inc. (CA, USA), respectively. Recombinantdomain 1 of human calpastatin protein and the peptides corresponding to its reactive site werepurchased from Takara Shuzo Inc. (7316 and SP007) and Sigma Inc. (C9181).

Recombinant proteins expressed in insect cells
The cDNA for p94∆ or p94:C129S, an active-site mutant, was inserted into the pFastBac1 vector
using appropriate restriction enzyme sites. Recombinant baculovirus was generated according
to the manufacturer’s instructions. Sf-9 cells were cultured and infected as previously described(28). The cells were then shaken vigorously at 27 °C for 50 h at a density of 1 × 106 cells/ml,
and collected by centrifugation at 600 × g for 5 min at 4 °C. Recombinant proteins were
purified following the procedures described previously (28), with minor modifications as follows.
The harvested cells were washed twice with ice-cold phosphate-buffered saline (PBS).

The

pellets were suspended in buffer A (20 mM Tris-Cl [pH 7.5], 5 mM EDTA, 10 mM
2-mercaptoethanol [2-ME]) containing 0.1 mM leupeptin, and the cells were homogenized with aFrench press (American Instrument Co., MD, USA). The cell lysate was then ultracentrifuged
at 150,000 × g for 40 min at 2 °C. Recombinant proteins were purified from the supernatant by
the AKTA system (Amersham Biosciences Inc., Amersham, UK) placed in a cold chamber, using
7

successive rounds of column chromatography: DEAE-Toyopearl (Tosoh Inc., Tokyo, Japan),
MonoQ (Amersham Biosciences Inc.), HiLoad 16/60 Superdex200, and MonoQ. Fractions
containing recombinant proteins were determined by western blotting analysis and caseinolytic
assays (for p94∆). Recombinant µ- and m-calpains were prepared in the manner described in
ref 28.

Bacterial expression and purification of recombinant proteins
cDNA fragments corresponding to two different N2A regions of connectin/titin, IgI80-81 andIgI82-83, were amplified by PCR from human skeletal muscle cDNA and cloned into the pET
vector.

Proteins were expressed in E. coli. BL21(DE3) and purified as previously described(29).
A bacterial expression vector for the fusion protein generated from enhanced cyan and yellow
fluorescence proteins (ECFP and EYFP, respectively) linked by a part of rabbit calpastatin
domain 1 (residues 175-255, GenBank accession number A26615), Y-C-CSTN, was created byfusing the PCR-amplified coding sequences of ECFP, EYFP, and calpastatin, and cloning theconstruct into the pET16b vector.

The recombinant protein, Y-C-CSTN, expressed in E. coli.

BL21(DE3), was purified by His-Bind Quick 900 Cartridges (Novagen, WI, USA), according to
the manufacturer’s protocol. Protein fractions eluted from the cartridge were desalted on a8

PD-10 column (Amersham Biosciences Inc.) and concentrated by Centricon (Millipore, MA,
USA).

Protease activity assay
Caseinolytic activity of p94∆ was measured essentially according to ref 30. In brief, thecomposition of the standard assay solution was 100 mM Tris-Cl (pH7.5), 25 mM 2-ME, 10 mM
CaCl2. CaCl2 was replaced with 10 mM EDTA for the control reaction. The reaction was
carried out by incubating either p94∆ (3 µg ), µ-calpain (0.2 µg), or m-calpain (0.4 µg) in 50 µlof the standard assay solution containing 3 mg/ml casein under the conditions indicated. The
reaction was stopped by the addition of 150 µl of 7% (w/v) trichloroacetic acid.

The tubes wereincubated on ice for 20 min, centrifuged at 15,000 × g for 10 min, and the A280 of the supernatantmeasured. An increase in A280 of 1.0 in 1 h was defined as 1 unit of caseinolytic activity.
The effect of Ca2+ on p94∆ was determined under the assay conditions described above, varying
either the concentration of Ca2+, temperature, or pH.

Tris-acetate buffer was used instead of

Tris-Cl buffer to measure the effect of pH. Protease inhibitors were added to the standard assay
solutions at the concentrations indicated. The reactions were carried out at 37 °C for 45 min andthe caseinolytic activity was determined as described. The effect of each inhibitor was
evaluated relative to the inhibitory effect of 10 mM EDTA set to 100.
9

To determine the substrate-hydrolysing activity of p94∆, various proteins were incubated withp94∆ in the standard assay solution. Hydrolysis of the proteins was examined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie
Brilliant Blue (CBB) staining or western blotting.
In the proteolytic assay using Y-C-CSTN as substrate, 2 µg of Y-C-CSTN was incubated withp94∆ in the presence of 15 mM Ca2+ for 0-45 min at 37 °C in a solution containing 0.1 M Tris-Cl(pH 7.5) and 10 mM 2-ME in a volume of less than 20 µl. The reaction was stopped by theaddition of 0.5 ml of 0.1 M EDTA (pH 8.0) or an equal volume of 2 × SDS sample buffer. The
fluorescence of the samples was measured by excitation at 434 nm (10 nm bandwidth), and theemission spectra were collected from 450 to 600 nm using an RF-1500 spectrofluorophotometer(Shimazu Inc., Kyoto, Japan).
define proteolytic activity.

The ratio of fluorescence at 477 nm to that at 527 nm was used to

To profile the dose-dependent changes in fluorescent signals, 0-2 µg

of p94∆ was incubated with Y-C-CSTN (0.6 µg) in a buffer consisting of 28 mM Ca2+, 1 mg/ml
bovine serum albumin (BSA), 0.1 mg/ml calpastatin (CSTN) domain 1, 20 mM 2-ME, and 100
mM Tris-Cl (pH 7.5) in a total volume of 15 µl for 5-45 min.

Cell culture and assays
COS-7 cells were maintained and transfected with each plasmid construct as described previously
10(24).

The cell lysate was prepared by sonicating harvested cells in a solution consisting of 50

mM Tris-Cl (pH 7.5), 25 mM 2-ME, 10 mM EDTA, and the proteinase inhibitors 10 µM
Z-D-CH2-DCB, 10 µM YVAD-CMK, 1 mM AEBSF, 2 µM calpastatin peptide C9181, and 2 mM
phenylmethylsulfonyl fluoride (PMSF).
To detect p94 activity in the cell lysate, the following protease inhibitors were added to thestandard assay mixture described above to inhibit the conventional calpains and other proteases:
10 µM Z-D-CH2-DCB, 10 µM YVAD-CMK, 1 mM AEBSF, 2 µM calpastatin peptide C9181,
and 2 mM PMSF. The same number of COS-7 cells (2-3 × 105) transfected with each plasmid
were incubated with Y-C-CSTN in the presence of 5 mM Ca2+ or 10 mM EDTA in total volumeof 20 µl for 90 min at 37 °C, after which 0.5 ml of 0.1 M EDTA was added. To detect the
autolysis of p94 and p94∆ by western blotting, the reaction was stopped by the addition of half avolume of 3 × SDS sample buffer.

Other protein experiments
SDS-PAGE, western blotting, and subsequent procedures were performed as previously described(14). Proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-P,
Millipore Inc.), as recommended by the supplier, and incubated with specific antisera as indicated.
The secondary antibody was horseradish peroxidase-coupled goat anti-rabbit or goat anti-mouse11

IgG (Nichirei Inc., Tokyo, Japan). The antibody complexes were visualized using peroxidase
substrate (POD Immunostaining Kit; Wako Inc., Osaka, Japan). To examine the autolytic
activity of p94∆, 1 µg of p94∆ was incubated in 15 µl of solution containing 100 mM Tris-Cl (pH7.5), 25 mM 2-ME, and 10 mM CaCl2, at either 0 °C or 30 °C for the indicated times. The
reaction was stopped by the addition of an equal volume of 2 × SDS sample buffer and subjected
to SDS-PAGE followed by CBB staining or western blotting, as described above.
Protein concentrations were measured using a DC Protein Assay Kit (Bio-Rad Inc. CA, USA)with BSA as the standard. The N-terminal amino acid sequence of the protein fragments wasdetermined on an Applied Biosystems protein sequencer type 477A/120A, according to
manufacturer’s instructions and the method described previously (31), with some modification.

12

RESULTS
Expression and purification of p94∆ and p94:C129S
Recombinantly expressed p94∆ and p94:C129S (a protease-inactive point mutant of p94/calpain3) were purified by four successive rounds of column chromatography, as described in Materials
and Methods (see Fig. 1A for structures of p94∆ and p94:C129S). The purity of these products
was estimated to be more than 90% based on a CBB-stained gel after SDS-PAGE, as shown in
Fig. 1B.

The N-terminal sequence of purified p94∆ revealed that, after two weeks storage at

2 °C, most of the purified p94∆ had been hydrolysed on the N-terminus of Ala34 (Fig. 1A, arrow[4]), and that anti-pNS antiserum, raised against a peptide corresponding to N-terminal residues
2-19 (14), no longer recognized the purified protein. The p94∆ sample was recognized byanti-pNS antiserum just after purification. p94:C129S, which was purified by a procedure
identical to that used for p94∆, showed no hydrolysis at the N-terminus. Therefore, we
concluded that the hydrolysis of p94∆ was autolytic.

This autolysis occurred in the presence of

5 mM EDTA, even at 2 °C, which is consistent with our previous observation that p94 rapidly
undergoes autolysis in the presence of excess EDTA (14).
The yields of totally purified p94∆ and p94:C129S proteins were ca. 1.2 and 2.4 mg/l culture,
respectively. Although the N-terminal autolysis of p94∆ did not require Ca2+, p94∆ showed
Ca2+-dependent caseinolytic activity (see Fig. 2; details are discussed later). Analysis of the13degraded casein by SDS-PAGE indicated that p94∆ degrades casein in a manner similar to thecaseinolysis by the conventional calpains (data not shown). The specific activity of p94∆
determined by caseinolysis was 9.0 unit/mg, which is ca. 1/80 of the value for recombinanthuman m-calpain determined with the same expression and purification systems (28, 32, 33).
However, because most of the purified p94∆ underwent N-terminal autolysis as described above,
we concluded that very low specific caseinolytic activity is one of the enzymatic properties ofp94∆, and does not result from partial denaturation or aggregation of the protein.

Enzymatic properties of p94∆: effects of temperature, pH, Ca2+-concentration, andprotease inhibitors
The caseinolytic activity of p94∆ was examined while varying either temperature, pH, or
Ca2+-concentration, and compared with the corresponding parameters for recombinant µ- andm-calpains. Fig. 2A shows the temperature-dependence of p94∆ caseinolytic activity. The
initial rate of p94∆ activity increased with temperature. In the range of temperatures examined
here, the activity of p94∆ increased persistently with time up to 60 min. The optimal
temperature for 20 min incubation of p94∆ was about 42 °C, which contrasts with the decrease inactivity observed for both µ- and m-calpains at temperatures above 25 °C (Fig. 2B). Althoughp94∆ shows maximum activity at around pH 7.5, as do other calpains, p94∆ maintains its activity
14even at pH 10 (Fig. 2C). Ca2+-dependence showed that a pCa for half maximal activity for
p94∆ was 2.9, which is lower than those of µ- and m-calpains (3.7 and 3.3, respectively) (Fig.
2D).
The effects of various inhibitors on the activity of p94∆ and µ- and m-calpains were investigated(Table 1). p94∆ has a spectrum of inhibitors very similar to those of µ- and m-calpains, except
calpastatin. The calpastatin fragment inhibited µ- and m-calpains completely at molar ratios ofalmost 1:1, whereas p94∆ was not inhibited by a 100-fold molar excess of the calpastatin
fragment or calpastatin-related peptides (Fig. 2E).

In fact, calpastatin was shown to be a

substrate for p94∆ (see below). Because the IS1 region is a target for p94 autolysis (31, 34), we
examined the possibility that synthetic peptides corresponding to IS1 would have a competitive
inhibitory effect on p94.

However, none of the peptides inhibited the caseinolytic activity ofp94∆, even at a molar ratio of 100:1 (data not shown).

Autolytic activity of p94∆
p94∆ is much more stable than full-length wild-type p94. However, as described above, limited
autolysis at the N-terminus of p94∆ occurs even in the presence of EDTA at 2 °C. Therefore,
the autolytic activity of p94∆ was analysed in more detail. p94∆, already lacking the N-terminal
33 amino acids of p94, underwent further autolysis in the presence of Ca2+. As shown in Figs.
15

2F and 2G, this second-phase autolysis generated an 83-kDa fragment very rapidly (t1/2 = 5 min at
30 °C, and 20 min at 0 °C). No change in caseinolytic activity or Ca2+-dependence was caused
by preincubation of p94∆ for 5, 10, 20, or 60 min at 30 °C (data not shown).

The N-terminal

sequence of the 83-kDa fragment was also from Ala34, indicating that the second autolysis
occurs at the C-terminus of p94∆ (data not shown).
To characterize further the autolytic activity of p94∆ relative to that of full-length wild-type p94,
p94:C129S, a good “intermolecular autolytic” substrate for p94, was proteolysed by p94∆ (24).
Co-incubation of p94:C129S (1 µg) and p94∆ (0.1 µg) resulted in the production of four major
fragments with approximate molecular weights of 93 kDa, 91 kDa, 58 kDa, and 33 kDa, as
shown in Fig. 2H. Interestingly, none of these fragments was recognized by anti-pNS antiserum,
and the full-length 94-kDa protein detected by anti-pNS antiserum decreased rapidly in thereaction. The N-terminal residue of the 93-kDa fragment (Fig. 2H, (a)) was Ala15 (Fig. 1A,
arrow [1]), whereas the N-terminal residues of the 91-kDa and 33-kDa fragments (Fig. 2H, (b)and (d), respectively) were identical to that of N-terminally autolyzed p94∆ (Ala34, Fig. 1A [2]).
These results indicate that p94∆ very efficiently hydrolyses the N-terminus of p94:C129S, as wellas that of p94∆ itself. The N-terminal residue of the 58-kDa fragment (Fig. 2H, (c)) was Glu323(Fig. 1A [3]), which corresponds to one of the previously determined autolytic sites in native p94(31). The 33-kDa fragment was not detected by either the monoclonal antibody 12A2 or
16

anti-pIS2 antiserum. These results indicate that p94:C129S is hydrolysed by p94∆ first at the
N-terminus and then in the region of IS1, which generates 58-kDa and 33-kDa fragments
corresponding to the C- and N-terminal parts of the 91-kDa fragment, respectively.

Substrates for p94∆
Previously, we found that p94 causes a decrease in a 60-kDa protein in vivo, when expressed in
COS cells (14). Peptide sequencing revealed that it corresponds to heat shock protein 60(HSP60). Consistent with this, an in vitro assay showed that p94∆ proteolyses HSP60 (Fig. 3A).
Moreover, HSP60 is proteolysed by m-calpain much more rapidly than by p94∆, suggesting that
HSP60 is also a possible in vivo target of the conventional calpains. Although HSP60 is highly
conserved from bacteria to eukaryotes (ca. 50% primary sequence identity between human andEscherichia coli proteins), the E. coli HSP60 homologue, GroEL, is very poorly hydrolysed byp94∆. Its co-chaperonin, GroES, and combinations of GroEL and ES with or without ATP werealso examined, but none was proteolysed by p94∆ (data not shown).
Several other proteins were incubated with p94∆ to test whether they are substrates. Two
different N2A fragments of connectin/titin, IgI80-81 and IgI82-83, were hydrolysed by p94∆ aswell as µ- and m-calpains. The connectin constructs IgI80-81 and IgI82-83 contain the two
N-terminal and the two C-terminal Ig motifs of the N2A region, respectively. IgI82-83 includes17

a p94-binding site for connectin (Fig. 3B) (17). IgI80-81 was more rapidly degraded than
IgI82-83 by p94∆ and µ- and m-calpains, as shown in Fig. 3C. N-terminal sequencing of thedegradation fragments of IgI80-81 revealed that one of the proteolytic sites susceptible to calpain
is on the N-terminus of Gly9434 (GenBank accession number NP_596869; ref 35; Fig. 3B,
arrow) in I80.

These results strongly suggest that not only µ- and m-calpains but also p94

proteolytically degrade connectin/titin.
Protease-inactive m-calpain, m-calpain:C105S, was proteolysed by p94∆, as shown in Fig. 3D,
whereas p94:C129S was not hydrolysed by m-calpain (data not shown). It should be mentionedthat anti-pIS2 detects p94∆ since a part of the epitope is retained in p94∆. Notably, the additionof the calpastatin domain 1 fragment (Fig. 3D, +CSTN) abolished m-calpainolysis by p94∆, but
not autolysis of p94∆ or caseinolysis by p94∆ (Fig. 2E), suggesting that the m-calpain-calpastatincomplex acquires resistance to proteolysis by p94∆ in the presence of Ca2+.

A widely usedfluorescent substrate for the conventional calpains, Suc-LLVY-MCA, was not significantly
hydrolysed by p94∆ (data not shown).
These results show that some of the substrates for p94∆ (and for p94) can be hydrolysed by theconventional calpains as well (casein, HSP60, connectin/titin N2A fragments, andm-calpain:C105S). On the contrary, calpastatin and p94:C129S are hydrolysed more efficiently
and exclusively, respectively, by p94∆ and p94.
18

Proteolysis of calpastatin by p94∆
Proteolysis of the calpastatin domain 1 fragment (14.1 kDa, residues 27-159 of human calpastatin,
GenBank accession number NP_775085; Takara Shuzo Inc. No. 7316) by p94∆ was examined byreversed-phase column chromatography. The calpastatin fragment was rapidly degraded
proteolytically by p94∆ into several fragments, as shown in Figs. 4A and 4B, and the N-terminal
sequences were determined.

Six cleavage sites were identified (Fig. 4C, lines i-vi; four of theseare shown in Fig. 4D), one of which is located close to the reactive site (i).

The others (ii-vi), if

cleaved, result in the loss of one of the helical regions on the C-terminal side of the reactive site,which interacts with domain VI of the conventional calpains.

All six cleavage sites are different

from the cleavage sites of the caspases (Fig. 4D, open triangle; 36).

These results indicate thatthe cleavage of calpastatin by p94 inactivates the calpain-inhibitory activity of calpastatin. The
most susceptible sites are on the N-terminus of Ala105 and Gly124 (accession number
NP_775085; ii and iv in Figs. 4C and 4D), which are detectable in the fractions already appearing
after 20 min digestion (Fig. 4A, closed triangles). These sites both have Pro at the P3' position
and a small amino acid (Ala and Gly) at the P1' position. Moreover, five out of six cleavage
sites in the calpastatin domain 1 fragment have two Pro residues between P2' and P8'.

These

features may reflect the proteolytic preference of p94. One of the autolytic sites in p94:C129S
19

and p94∆, the N-terminus of Ala15, also has Pro at the P3’ position and a small amino acid (Ala)at the P1’ position (Fig. 4C, N-1). However, there are no further significant similarities foundbetween the amino acid sequences surrounding the cleavage sites in calpastatin and those forautolytic sites in p94:C129S and p94∆ (Fig. 4C, N-2 and IS1-1~3) (31).
To test whether p94∆ proteolytically degrades short peptides, a 27-mer peptide corresponding to
the reactive site of calpastatin (Sigma C9181; Fig. 4D) was incubated with p94∆. Although theefficiency of cleavage was very low (less than 5% after 120 min incubation), the peptide wascleaved on the N-terminus of Lys99 (Figs. 4C and 4D, vii). The site has Pro residues at the P1
and P2 positions but not in the P' positions, suggesting that short peptides are cleaved by p94∆
differently from the cleavage of calpastatin domain 1 fragment, which is a much better substrate
for p94∆.

Assay system for p94 utilizing calpastatin as substrate
As observed above, calpastatin is the best substrate for p94∆ among the protein substrates
examined in this study except for p94:C129S. Therefore, we developed an assay for p94 using
the calpastatin fragment and fluorescent resonance energy transfer (FRET). When the purified
substrate, Y-C-CSTN (Fig. 5A), was incubated with p94∆, it was rapidly degraded into several
fragments (Fig. 5B). Before the substrate was cleaved by p94∆, the fluorescence spectrum of20

Y-C-CSTN excited at 434 nm, the CFP excitation wave length, showed a peak at 527 nm, which
is an emission wavelength of YFP, indicating that YFP is excited by the CFP emission by FRET
(Fig. 5C, 0 min).

The emission spectrum of Y-C-CSTN excited at 434 nm was measured during

the proteolytic process. A time-dependent increase in CFP emission at 477 nm and acorresponding decrease in YFP emission at 527 nm was observed, demonstrating that theproteolysis of Y-C-CSTN by p94∆ can be monitored as the change in the emission spectrum
caused by the loss of FRET between those two fluorescent units (Fig. 5C). Various amounts ofp94∆ were tested for Y-C-CSTN digestion and the ratio of YFP emission to CFP emission, 477
nm/527 nm, was plotted for each time point (Fig. 5D).

The increase in the fluorescence ratio

477/527 was time- and dose-dependent, and the activity of as little as 250 ng of p94∆ could beassayed with an incubation of 45 min. µ-calpain produced no increase in the fluorescence ratio
nor proteolysis of Y-C-CSTN over this period of time or in this range of protein concentrations(data not shown), suggesting that Y-C-CSTN is a p94∆-specific substrate but not for theconventional calpains under our assay condition.

Activity of COS-expressed full-length wild-type p94 detected by Y-C-CSTN
To test whether the assay described above can detect the proteolytic activity of full-length
wild-type p94, as well as the activity of p94∆, among one hundred other co-occurring proteases21

in vivo, the proteolytic activity of p94 expressed in COS7 cells was measured using Y-C-CSTN as
substrate. COS cells are known to express considerable amounts of m-calpain and other cellular
proteases, such as caspases. Ca2+-dependent proteolysis of Y-C-CSTN was detected in thelysates of cells expressing full-length wild-type p94 (Fig. 5E, f.l.p94) or p94∆ (Fig. 5E, p94∆) inthe presence of protease inhibitors for major proteases including conventional calpains, caspases,
and serine proteases. On the other hand, the lysates of cells expressing protease-inactive
full-length p94:C129S (Fig. 5E, CS) or µ-calpain (Fig. 5E, µ-calpain) showed no activity above
background levels, i.e., above the value determined for cells transfected with empty vector,
regardless of enough amount of the proteins detected (Fig. 5F, CS and µ-calpain).

These results

clearly indicate that our assay system specifically detects and distinguishes the protease activityof p94 in the presence of interfering proteases.
To our surprise, full-length wild-type p94 showed Ca2+-dependent calpastatinolytic activity (Fig.
5E, f.l.p94).

The autolysis of recombinantly expressed p94, however, was Ca2+-independent(Fig. 5F, f.l.p94; grey arrow head) as shown previously (14), which is consistent with the fact thatmost COS-expressed p94 had already disappeared at harvest. These results indicate that the
autolysis of p94 does not require Ca2+, whereas calpastatinolysis occurs in a Ca2+-dependent
manner, suggesting different substrate specificities for p94 in the absence and presence of Ca2+.

22

DISCUSSION
In this study, the enzymatic properties of one of the alternatively spliced variants of p94/calpain 3
were examined, to understand better the unique characteristics of p94 and their relevance to its
physiological functions. Isolating significant amounts of the proteins is a prerequisite for invitro enzymatic studies, and this has been almost impossible owing to the very rapid autolytic
activity of p94 (31). On the other hand, some natural splice variants of p94 expressed in skeletal
muscle are somewhat stable. After several different isoforms were examined, p94∆, whichlacks both IS1 and IS2, was the most promising variant for large-scale preparation. The
structure of p94∆ is almost identical to that of µCL and mCL, except that it has the NS. It
shows proteolytic characteristics similar to those of µ- and m-calpains, such as Ca2+-dependence,
insofar as IS1 and IS2 are involved in Ca2+-independent protease and autolytic activity, ahallmark of p94.
However, there are several traits distinguishing p94∆ from the conventional calpains: (1) p94∆
shows specific caseinolytic activity much lower than those of the conventional calpains, (2) the
optimal temperature for p94∆ activity is higher (ca. 42°C) than those of the conventional calpains(25-30°C), and (3) p94∆ has a lower pCa for half maximal activity than µ- or m-calpains. At
present, the possibility cannot be excluded that these properties originate from the lack of IS1 or
IS2. It should be emphasized, however, that many features of p94∆ are shared with full-length23p94: neither is inhibited by, but proteolytically degrades calpastatin; both exhibit proteolytic
activity without 30K; both bind connectin/titin at the N2A and M-line regions (16); both cut
p94:C129S at identical sites; and both undergo autolysis in the presence of EDTA. Therefore, it
is conceivable that the properties of p94∆ described above reflect the nature of p94 and are
related to the functions of p94 in the context of the physiology of skeletal muscle.

Stable and active without 30K
The co-expression of 30K is required for recombinant µ- and m-calpains and nCL-4 in the same
Sf-9/baculovirus system (28, 32, 33, 37, 38). However, proteolytically active p94∆ and inactive
p94:C129S were isolated in the soluble and stable fraction without 30K. Furthermore, nointeraction between p94∆ and 30K was detected in the yeast two-hybrid system, as was also thecase for p94 (17, 16). These data strongly suggest that p94∆ and p94 do not form heterodimers
with 30K. It awaits further analyses on the tertiary structures of p94∆ and p94 to determine ifthey form homodimers which was predicted from the elution positions of p94:C129S and p94∆
on the gel-filtration column (31 and data not shown).

Autolytic activity of p94 and p94∆
Our previous results on the in vitro translation of p94 indicated that the autolysis of p94 proceeds

24

in the presence of excess EDTA (14), which was also observed for the N-terminal autolysis of
p94∆. Branca et al., however, reported Ca2+-dependent proteolytic fragmentation ofrecombinant p94 (39). They isolated a substantial amount of full-length wild-type p94 using theSf-9/baculo virus system.

In our hands, however, p94:C129S was expressed abundantly andstably, and was successfully purified almost to homogeneity using the same Sf-9/baculo virus
system (Fig. 1B), whereas wild-type p94 expressed in the same system was detected only as a
55-kDa autolyzed fragment (data not shown). At present, no clear explanation is available forthese discrepancies, but p94 may show Ca2+-dependent autolysis under certain conditions.
Indeed, Ca2+-dependent hydrolytic activity was detected for full-length wild-type p94 as well as
for p94∆ using a calpastatin-based FRET substrate in this study.
Hydrolysis of p94:C129S by p94∆, but not by m-calpain, strongly suggests that the substratespecificities of p94∆ and p94 are the same.

In addition to the autolytic site in IS1 (31), p94∆

hydrolyses p94:C129S at the N-termini of Ala15 and Ala34. In this study, one of theintramolecular autolytic sites of p94∆ was also demonstrated to be Ala34. Ca2+-independent
autolysis at the N-terminus of Ala34 generates a rather stable autolytic fragment of p94∆ since nofurther autolysis occurred during the storage of p94∆ for over a year (data not shown). Recently,
Rey and Davies reported that one of the autolytic sites in a recombinant protein corresponding to
the protease domain of p94 was Ala15 (34), which is consistent with our result.
25

They also

reported Ala45 and Thr316 as autolytic sites, which were not identified in our experiments.

It

can be reasoned that the lack of domain III and regions thereafter in their construct exposed sites
potentially susceptible to autolytic cleavage. Previously, we have observed that theCa2+-independent autolytic activity of recombinant p94:exon6- was much weaker than that offull-length p94, resulting in the stabilization of p94:exon6- (16 and unpublished data).
Therefore, it is inferred that the lack of exon 6, which constitutes most of IS1, contributes to thestability of p94∆ by abrogating the pivotal autolytic site. Rey and Davies also reported
Ca2+-dependent autolysis of their protein, which can be ascribed to the lack of IS2 based on ourprevious observations (16 and unpublished data). Very recently, Fukiage et al. reported thequalitative characterization of Lp82 (40), another alternative splice variant of p94, which is
specifically expressed in the lens. They showed that the lack of IS2 does not effectively
stabilize the product when NS is replaced by AX1, a lens-specific N-terminal sequence (41).
Considering these data together, we conclude that NS, IS1, IS2, and AX1, unique sequences
specific to CAPN3 proteins, have independent functions, and that different combinations of themconfer specific characters, e.g., different autolytic activities, upon each splice variant.

p94-specific assay towards a diagnosis of LGMD2A
Because the loss of the substrate-processing activity of p94 causes LGMD2A (24), we have26

focused on identifying its substrates.

One of the major problems we faced was how to

distinguish the activity of p94 from that of other proteases, including the conventional calpains,in a physiological context. The enzymatic properties determined for p94∆ in this study showedthat there are conditions favourable for p94 but not for the conventional calpains, such as hightemperature and the presence of calpastatin. Consequently, we established a specific assay
system for p94 and demonstrated its ability to identify COS-7-expressed p94, as well as p94∆.
In theory, 0.3 mg muscle, which corresponds to about three 10 µm cryostat slices of a muscle
biopsy, contains at least 300 ng of p94 (42).

Our system can assay as little as 250 ng of p94∆.

The activity of the COS-7-expressed p94 and p94∆ detected in this study corresponds to aminimum of approximately 10 ng of protein.

Therefore, we anticipate that the activity ofendogenous p94 in tissues (biopsy samples) will be measurable using this assay, and that thismethodology will be applicable to the diagnosis of LGMD2A by screening for the loss of p94
activity.

Calpain network and LGMD2A
The apparently Ca2+-independent proteolysis of p94:C129S and calpastatin is p94-specific, which
is not observed for the conventional calpains (17). m-calpain:C105S was also degraded
proteolytically by p94∆ in a Ca2+-dependent manner, although p94:C129S was not degraded by27

m-calpain. These results imply, for the first time, that p94 has a certain role in regulating theconventional calpains directly and indirectly by proteolytic degradation of the calpains andcalpastatin, respectively.
It has been reported that there are several biological context where several different proteasesystems keep cross-talking. For example, calpain and caspase proteolytic systems functionsynergically in apoptosis, where proteolytic inactivation of calpastatin by caspase facilitatescalpain activation and calpain activates caspase by limited proteolysis (43). It has not yet been,
however, shown whether there is a cross-talk between p94 and the conventional calpains, theso-called “calpain network” in skeletal muscle where the expression level of p94 predominates.
It is already clear that the function of p94 cannot be completely compensated by other calpains
since a defect of p94 function causes LGMD2A. It is, therefore, of our interest to investigate
mechanisms how p94 could intervene in the conventional calpain system, which would becompromised in LGMD2A.

28

ACKNOWLEDGEMENTS
We are very grateful to Dr. Muriel Herasse (Généthon, Evry, France), Dr. Takeshi Nishino(Nippon Medical School, Tokyo, Japan), and Dr Hajime Masumoto (Toray Inc., Kanagawa,Japan) for providing us with mouse cDNAs for p94 and its variants, Sf-9 cells, and recombinantµ- and m-calpains, respectively. We would also like to thank Prof Shoichi Ishiura, Dr Tatsuya
Maeda, Dr Ichiro Matsumoto, and Dr Akihito Yasuoka of the University of Tokyo, Dr Hiroyuki
Kawahara of the University of Hokkaido, Dr Takaomi C. Saido of the Riken Institute, Dr Noriko
Sorimachi of Tokyo Medical and Dental University, and all the other “Team Calpain” members(Dr Shoji Hata, Dr Eiichi Kimura, Ms Yukiko Kawabata, Mr Katsuhide Yoshioka, Mr Suguru
Koyama, and Mr Yoshitaka Yamamuro) of the Graduate School of Agricultural and Life Sciencesin the University of Tokyo, for their generous gifts of experimental materials, valuable
discussions, and encouragement. This work was supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas (Cell Cycle) from the Ministry of Education, Science,Sports and Culture, a Grant-in-Aid for Scientific Research and Research Fellowships for Young
Scientists from JSPS (the Japan Society for the Promotion of Science), a Research Grant (14B-4)
for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, the Deutsch
Forschungsgemeinschaft (La668/7-1), and “Ground-based Research Announcement for Space
Utilization” promoted by the Japan Space Forum.
29

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34

FOOTNOTES**Present address: Glyco-Chain Functions Laboratory, Frontier Research System, RIKEN,
Wako 351-0198, Japan***Present address: New Frontiers Research Laboratories, Toray Industries Inc., Kamakura248-8555, Japan1

The abbreviations used are: µCL, µ-calpain large subunit; mCL, m-calpain large subunit;

p94∆, p94:exons 6-15-16-; LGMD2A, limb-girdle muscular dystrophy type 2A; CBB,
Coomassie Brilliant Blue; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; PBS, phosphate-buffered saline; 2-ME, 2-mercaptoethanol; BSA, bovine
serum albumin; CSTN, calpastatin; ECFP, enhanced cyan fluorescence protein; EYFP,
enhanced yellow fluorescence protein; FRET, fluorescence resonance energy transfer.

35

FIGURE LEGENDS
FIG. 1.

Schematic structure of p94:C129S, p94∆, and purified proteins(A) Schematic structure of p94:C129S and p94∆. NS, IS1, and IS2 are specific sequences foundin p94 but not in the conventional (µ- and m-) calpain large subunits (µCL and mCL). Domains
I, III, and V are α-helical, C2-like Ca2+-binding and Gly-rich hydrophobic domains, respectively.
Domains IV and VI contains five EF-hand motifs and are very similar to each other.
Subdomains IIa and IIb, kept apart in the inactive state, comprise the protease domain when
activated. Thick horizontal bars indicate the positions of epitopes for the antibodies used in thisstudy. Note that a part of the epitope for anti-pIS2 is retained in p94∆. Arrows (1) to (4)indicate the proteolytic sites determined in this study. (B) Purified p94:C129S and p94∆ used inthis study. Lane 1, molecular weight marker; lanes 2 and 3, p94:C129S (5 µg) and p94∆ (5 µg),respectively, in the final MonoQ fractions (open triangles) were separated electrophoretically andstained with CBB.

FIG. 2.

Characterization of p94∆ caseinolytic activity(A) Temperature and incubation-time dependence of activity. Activities were standardized withthe value at 37°C for 30 min taken as equal to 1.

(B) Temperature dependence of activity with20 min incubation. Closed circles, open lozenges, and open squares represent p94∆, µ-calpain,36and m-calpain, respectively. Activities were standardized as the maximum value (determined at
42°C for p94∆, and at 21°C for µ- and m-calpains) equal to 1. (C) pH dependence of activity.
Activities were standardized with the maximum values equal to 1 (values at pH 7.5 for p94∆ andm-calpain, and at pH 7.4 for µ-calpain). (D) Ca2+ dependence of activity. Activities werestandardized with the maximum value equal to 1 (at pCa = 2.0). (E) Activity in the presence ofvarious concentrations of calpastatin (CSTN) domain 1 or related peptides. Sequences of CSTN
peptides C9181 and SP007 are given in Fig. 7. Activities were standardized with the value
without inhibitor equal to 1. (F) The second-phase autolysis of p94∆ in the presence of Ca2+.
p94∆ (1 µg) was incubated for 0-90 min on ice or at 30 °C, and visualized by SDS-PAGE and
CBB staining.

(G) Quantification of autolysis fragments of p94∆. Band intensity of the 84-(full-length, F.L.) and 83- (autolyzed) kDa bands in (F) were determined, and plotted after
standardizing the value at 0 min as equal to 100%. (H) “Intermolecular autolysis” of
p94:C129S by p94∆. p94:C129S (1 µg) and p94∆ (0.1 µg ) were co-incubated in the presenceof 10 mM CaCl2 for 0-60 min. Samples were subjected to SDS-PAGE, followed by CBB
staining, or western blotting using the antibodies indicated. For the positions of the antibody
epitopes, see Fig. 1A. The N-terminal sequences of fragments (a)-(d), indicated by triangles, are
Ala15, Ala34, Glu323, and Ala34, respectively. These correspond to the proteolytic sites
specified by arrows [1], [2], [3], and [2] in Fig. 1, respectively. Arrow heads represent p94∆37

detected by antibody 12A2.

FIG. 3.

In vitro proteolysis of possible substrates of p94∆

(A) HSP60 degraded proteolytically by p94∆ or m-calpain. HSP60 (0.6 µg) was incubated with
0.3 µg of p94∆ or 0.04 µg of m-calpain in the presence of 10 mM CaCl2 (+Ca2+) or 20 mM
EDTA (+E) for 0-120 min and visualized by CBB staining.

(B) Schematic structure of the N2A

region of connectin/titin and the binding site for p94. Part of the N2A region of connectin/titin
is shown, and the bidirectional arrows indicate the regions corresponding to IgI80-81 andIgI82-83. IgI80-IgI83 are motifs with similarity to immunoglobulin superfamily (I-line region
subset); “is” is the intervening region between IgI80 and IgI81; and PEVK is the region rich in
Pro, Glu, Val, and Lys. The vertical arrow indicates one of the m-calpain-susceptible proteolytic
sites in connectin/titin. CN48 is the original N2A clone isolated by yeast two-hybrid screening
using p94 as bait (17), and CN48-∆1 to -∆6 were generated from CN48. Each construct wasco-transformed with bait plasmid bearing p94 into AH109, and binding was evaluated by growth
on SD-LWA plates and β-galactosidase activity (shown in the right column). (C) Proteolysis of
N2A connectin/titin fragments by p94∆, µ-calpain, or m-calpain.

IgI80-81 (1 µg; upper) or

IgI82-83 (1 µg; lower) connectin/titin N2A recombinant peptide was incubated with 2, 1, 0.5, 0.2,
or 0.1 µg (left to right in each panel) of p94∆ (left), m-calpain (middle), or µ-calpain (right) in the38

presence of 15 mM CaCl2 for 60 min at 37 °C for p94∆, or for 20 min at 30 °C for m- andµ-calpains. The lanes labelled “+E” contain 2 µg of p94∆ (left), m-calpain (middle), orµ-calpain (right) incubated with each peptide in the presence of 20 mM EDTA where no autolytic
degradation occurred. Closed triangles indicate p94∆, mCL, or µCL and open triangles indicate
uncleaved connectin/titin peptide, IgI80-81 or IgI82-83. Protein fragments were visualized by
CBB staining.

(D) m-calpain:C105S degraded proteolytically by p94∆.

p94∆ (2 µg) andm-calpain:C105S (1.5 µg) were co-incubated for 0-120 min in the presence of 15 mM CaCl2, 20
mM EDTA (+E), or 15 mM CaCl2 and 0.7 µg of calpastatin domain 1 (+CSTN).

Samples were

subjected to SDS-PAGE, and proteins detected by CBB staining or western blotting using
anti-human m-calpain or anti-pIS2 antisera.

FIG. 4.

Proteolysis of calpastatin by p94∆

(A) Processes of calpastatin proteolysis by p94∆ determined by reversed-phase column
chromatography. Calpastatin domain 1 (30 µg) and p94∆ (1 µg) were co-incubated in thepresence of 15 mM CaCl2 for 0-120 min at 37 °C.

The reaction was stopped at each time pointby adding 1 ml of 0.1% TFA, and the products were subjected to C18 reversed-phase column
chromatography (RPC) with a 10-50% acetonitrile gradient. Closed triangles in the 20 min
digestion represent fractions of peptides degraded at the most susceptible sites in calpastatin,
39

judged by the earliest appearance of these peaks. Lower-case letters on open triangles in the 60
min digestion correspond to the fractions in (B). (B) Tris-tricine gel electrophoretic analysis ofproteolytic fragments of calpastatin (digested for 60 min).
panel correspond to fractions labelled in (A).

Lower-case letters on the top of the

T and U stand for total protein before separation

by RPC, and undigested calpastatin domain 1, respectively. (C) Comparison of sites of
calpastatin cleavage by p94∆.

All six N-terminal sequences identified from the digestion of

calpastatin domain 1 (i-vi) and one from the digestion of calpastatin peptide C9181 (vii) are
shown.

The autolytic sites at the N-terminus (N-1 and -2) and in the IS1 region (IS1-1 to -3) arealso shown for comparison (31).
residues are reversed.

The vertical arrow indicates the proteolytic site. Proline(D) Alignment of human and rabbit calpastatin inhibitory domains 1-4

and positions of the proteolytic sites for p94∆. Amino acid sequences around the inhibitory
reactive site (indicated by the bidirectional arrow) are shown, and highly and moderately
conserved residues are reversed and shaded, respectively. The open triangle indicates the
position of the caspase proteolytic site (36).

Thick lines indicate the sequence corresponding to

the commercially available calpastatin peptides, C9181 and SP007, used in this study. Verticalarrows with numbers correspond to the sequence labels in (C).

40

FIG. 5.

Assay for p94∆ utilizing calpastatin and FRET of fluorescence proteins(A) Schematic structure of p94∆-specific substrate (Y-C-CSTN) used in this study. CSTN
represents residues 157-255 of rabbit calpastatin (GenBank/EMBL/DDBJ accession number
A26615). (B) Steps in substrate processing by p94∆. Y-C-CSTN (2 µg) was incubated alone(-p94∆) or with 1.5 µg of p94∆ (+p94∆) in a buffer composed of 15 mM Ca2+, 1 mg/ml BSA, 0.1
mg/ml CSTN, 10 mM 2-ME, and 100 mM Tris-Cl (pH 7.5) in a volume of 15 µl for 0-45 min at
37 °C. CBB staining. (C) Changes in the fluorescence spectrum of the substrate during
proteolytic digestion by p94∆. Reactions in (B) (+p94∆) were terminated by the addition of 0.5
ml of 50 mM EDTA (pH 8.0) and the fluorescence emission spectra from 450 to 600 nm excitedby 434 nm were scanned. (D) Dose-dependent fluorescent signals in this assay system.
p94∆ (0-2 µg) was incubated with 0.6 µg of Y-C-CSTN under the same conditions as in (B), but
in the presence of 28 mM Ca2+. Reactions were stopped by adding 0.5 ml of 50 mM EDTA (pH8.0), and the fluorescence emission of 476 nm and 527 nm excited by 434 nm light was measured.
The ratio of fluorescence at 477 nm to that at 527 nm was plotted. (E) Activity of
COS-expressed full-length wild-type p94 and p94∆.

The lysate from COS-7 cells (2.5 × 105)transfected with either the pSRD vector, or vectors expressing full-length wild-type p94 (f.l.p94),
p94∆, full-length p94:C129S (CS), or µ-calpain (µ-calpain) was prepared by sonicating the cells
in 50 mM Tris-Cl (pH 7.5) buffer supplemented with 25 mM 2-ME, 10 mM EDTA, and the41

following protease inhibitors: 10 µM Z-D-CH2-DCB, 10 µM YVAD-CMK, 1 mM AEBSF, 2 µM
calpastatin peptide C9181, and 2 mM PMSF. The lysate was incubated with Y-C-CSTN in thepresence of 5 mM Ca2+ (+) or 10 mM EDTA (-) at 37 °C for 90 min. Measurements andcalculations were as in (D). The values shown are representative of three independent
experiments and are averages calculated using duplicate measurements. (F) Autolysis of
COS-expressed p94 simultaneous with substrate hydrolysis. Reactions were carried out
essentially as in (E) except that they were stopped by the addition of half a volume of 3 × SDS
sample buffer.

Western blot analysis of p94 (f.l.p94), p94∆, and full-length p94:C129S (CS)with the antibody 12A2 and of µ-calpain (µ-calpain) with the antibody MAB3082 showed
Ca2+-independent and Ca2+-dependent autolysis only for p94 and p94∆, respectively.

42

Table 1

Effects of various protease inhibitors on p94∆, µ- and m-calpain

Values are relative to the inhibitory activity of 0 and 10 mM EDTA, which are set to 0 and 100,
respectively.
Name

conc.

p94∆

µ-calpain

m-calpain

EDTA

10 mM

100

100

100

Leupeptin

1 mM

102

101

100

0.1 mM

86.7

98.7

97.8

0.1 mM

101

101

76.6

10 µM

87.0

91.2

90.7

Chymostatin

1 mM

85.1

90.7

84.1

Calpeptin (ZLNal)

10 µM

76.1

98.7

98.1

1 µM

19.0

88.9

87.6

1 mM

65.2

57.4

73.1

ALLNal (MG-101, calpain inhibitor I) 10 µM

64.1

90.1

88.7

MG-132 (ZLLLal)

1 µM

14.9

35.9

56.6

PD150606

10 µM

11.7

55.0

73.4

1 µM

6.52

14.3

44.5

Calpastatin domain 1

0.14 µM

4.36

77.2

76.1

Pepstatin A

10 µM

3.26

1.44

3.85

AEBSF

10 mM

0

0

0

Aprotinin

15 µM

0

0

0

Bestatin

1 µM

0

0.160

0

1-10-Phenanthrolin

20 mM

0

0

0

Phosphoramidon

0.1 mM

0

0

0

PMSF

10 mM

0

0

0

TLCK

1 mM

0

0

7.97

E-64c

TPCK

43

Ono, Y. et al., Fig. 1

A[1] [2]

p94/calpain 3[3]

Cys129

IIa

NS

His
Asn

Cys

His AsnIIb

IIaRegulatory
Protease Domain
DomainµCL, mCL(calpain large subunit)

I

IIa
Cys

IIb
His Asn

30K(conventional calpain small subunit)

B

1
(Mr
x10-3)

94
67

43

30

20

2

IV

IS2

12A2

NS

p94∆

III

IS1 IIb

pNS,2C4
[4]

pIS2

3

IV
III
C2-like
Domain5-EF-Hand
DomainIII
Gly-rich
DomainV

IV
VI

Ono, Y. et al., Fig. 2
E

A

p94∆

1.0

1.0

37oC
Relative Activity

Relative Activity (37oC, 60 min. = 1)

0.8

42oC
0.8

30oC

µ-calpain

0.6

0.4

0.2

21oC

0.6

m-calpain

in in
pa pa
4∆ cal cal
p9 µ- mCSTN Domain 1
CSTN peptide-C9187
CSTN peptide-SP007µ-, m-calpain

0.0

0

1

10

45oC
0.4

100

1000

Molar Ratio

16oC

F

0'

5'

10' 20' 45' 60' 90'

0'5'

10' 20' 45' 60' 90'

84K (F.L.)
83K(Autolyzed)

0.2

0 oC

30oC

G
0

10

20

30

40

50

100

60

Incubation time (min.)

B

p94∆

80

0.8

o

30

C oC
0
F.L. (84K)
Autolyzed (83K)

0.6

0.4

Relative Amount (%)

Relative Activity to Maximum

1.0µ-calpain
m-calpain

0.2

0.0
10

20

30

40

60

40

50

Temperature (oC)C

Autolyzed, 30oC

1.0

Autolyzed, 0oC

20
Relative Activity to Maximum

0.8

F.L., 0oC
p94∆

F.L., 30oC
0.6

0

20

40

60

80

100

Time (min.)µ-calpain

0.4

m-calpain

H

0.2

0'

10'

Incubation time with 10 mM CaCl2
0'
10'
60'
0'
10'
60'

60'

0'

10'

60'

Mrx10-3
0.0
6

7

8

9

10

94
p94∆

pH

D

(a)
(b)

(a,b)

67

(c)
1.0

(c)

43

µ-calpain

(d)

30

CBB

0.6

0.4

0.2

0.0
8

Relative Activity to Maximum

(d)
p94∆

m-calpain

0.8

7

6

5

4

pCa

3

2

1

anti-pNS(N-term)

12A2(Dm.IIb)

anti-pIS2(IS2)

Ono, Y. et al., Fig. 3

A
Mrx10-3

0'

5'

10'

p94∆
20'

60'

120' +E0'm-calpain
10' 20'

5'

60'

+E
p94∆
or mCL

94
67

Hsp60
43
30

B

IgI80-81

IgI82-83
p94 binding region

N-termC-term

IgI80

is

IgI81

IgI82

IgI83

PEVK
Binding

50 aar

CN48+

CN48-∆1-

CN48-∆2-

CN48-∆3+

CN48-∆4+

CN48-∆5-

CN48-∆6-

C
Mrx10-3

p94∆
2 1 .5 .2 .1 +E

m-calpainµ-calpain
2 1 .5 .2 .1 +E 2 1 .5 .2 .1 +E(µg)
Calpain

94
67

IgI80-81

43

30

94

Calpain

67
43

IgI82-83

30

D
Mrx10-3
94
67

0'

30'

60' 120' +E +CSTN
p94∆
and
mCL

43
CBB

30

20

80

antihuman
m-calpain

83
anti-pIS2

58

Ono, Y. et al., Fig. 4

A

B
T[AcCN]
(%)

a

b

c

d

e

f

g

h

i

U

CSTN Domain 1

50

Mrx10-3

40

14.0

30
20

6.5

10
0

3.5

0'
20'

g
f
a

b

cd

e

h

40'

C

i

60'

120'

D

i

vii

8 7 6 5 4 3 2 1 1' 2' 3' 4' 5' 6' 7' 8'i. 83 S T Y I E E L G K R E V T I P P
ii. 97 P P K Y R E L L A K P I G P D Diii. 98 P K Y R E L L A K P I G P D D A
iv. 116 A L S S D F T C G S P T A A G K
v. 141 V L K A Q S A G T V R S A A P P
vi. 144 A Q S A G T V R S A A P P Q E Kvii. 91 K R E V T I P P K Y R E L L A K
N-1
2ASVAPRTAAEPRSPGP
N-2 26 P A Q S K A T E A G G G N P S G
IS1-1 267 D D G T N M T Y G T S P S G L N
IS1-2 289 R M V R N M D N S L L Q D S D L
IS1-3 315 R T I I P V Q Y E T R M A C G L

ii

iii

Caspasesiv

Dm.
Human 1. E V S D P M S S T Y I E E L G K R E V T I P P K Y R E L L A - - - - - K P I G P D D A I D A L S S D F T C G S

2. V D E A K A K E E K L E K C G E D D E T I P S E Y R L K P A T D K D G K P L L P E P E E K P K P R S E S E L I
3. K V K E K A K E E D R E K L G E K E E T I P P D Y R L E E V K D K D G K P L L P K E S K E Q L P P M S E D F L
4. K V K E K A K A E H R D K L G E R D D T I P P E Y R H L L D D N G Q D K P V K P P T K K S E D S K K P A D D Q
Rabbit 1. E I S D P M S S T Y I E E L G K R E V T I P P K Y R E L L E K K T G V A G P P P D S V T P L G P D D A I D A L
2. V A E A K R K E E K V E K C G E D D E T V P A E Y R L K P A T D K D G K P L L P E P A E K P K P R S E S E L I
3. K I K E K S K E E E R E K L G E K E E T I P P D Y R L E E A K D K D G K P L L P S E P T A Q L P A L S E D L L
4. K V K E R A K K E H K D K L G E R D D T I P P E Y R H L L D Q G E Q D K P E K P P T K K S K E I K K P A G D Q
C9187
SP007
Reactive Site

Ono, Y. et al., Fig. 5

A

D
eYFP

CSTN
157-255

10xHiseCFPB- p94∆
0'

45' 0'

Fluorescence Ratio (Em:λ476/l527; Ex=433 nm)

45'+ p94∆
5'

10' 25'45'(Mr x10-3)

94
83
67
48
43

1.6

30'

20'

1.4

10'

5'

1.2

1

33

0

0.5

1

1.5

2

Amount of p94∆ (µg)

E

C

1.0

450

476 nm (eCFP)

0.9

0'

Ratio Em:477/Em:527 at Ex:434

Fluorescence Intensity(Arbitrary Unit)

527 nm (eYFP)

5'
10'
25'
45'

500
550
Emission Wave Length (nm)

600

+EDTA
+Ca2+

0.8

0.7

0.6

0.5

pSRD

f.l.p94µ-calpain

p94∆

CS

p94∆
- +

CS µ-calpain+ - +F
pSRD f.l.p94
Ca
+
+
2+

time

0' 90' 0' 90' 0' 90' 0' 90' 0' 90' 0' 90' 0' 90' 0' 90'(Mr x10-3)

94
67

43
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16