SUPPRESSED DISASSEMBLY OF AUTOLYZING p94/CAPN3 BY N2A
CONNECTIN/TITIN IN A GENETIC REPORTER SYSTEM*
Yasuko Ono1, Fukuyo Torii2, Koichi Ojima1,3, Naoko Doi1,3, Katsuhide Yoshioka2, Yukiko
Kawabata2,3,§, Dietmar Labeit4, Siegfried Labeit4, Koichi Suzuki5, Keiko Abe2, Tatsuya Maeda3,6,and Hiroyuki Sorimachi1,31From Department of Enzymatic Regulation for Cell Functions, The Tokyo Metropolitan Instituteof Medical Science (Rinshoken), Tokyo 113-8613, Japan, 2Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Tokyo 113-8657, Japan, 3CREST, Japan Science and Technology(JST), Saitama 332-0012, Japan, 4Institut für Anästhesiologie und Operative Intensivmedizin,
Universitätsklinikum Mannheim, Mannheim 68167, Germany, 5New Frontiers ResearchLaboratories, Toray Industries Inc., Kanagawa 248-8555, 6Institute of Molecular and Cellular
Biosciences, The University of Tokyo, Tokyo 113-0032, Japan,§Present address: Department ofApplied Biological Science, Fukuyama University, Hiroshima 729-0292, Japan
Running title: Regulation of p94/calpain 3 stability by N2A connectin/titin
Address correspondence to: Hiroyuki Sorimachi, Department of Enzymatic Regulation for Cell Functions,
The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku,
Tokyo 113-8613, Japan, Tel./Fax. +81-3-3823-2181; E-Mail: sorimach@rinshoken.or.jp
connectin fragment that binds to p94 wasshown to suppress p94 autolytic disassembly.
The proximity of the IS2 autolytic andconnectin-binding sites in p94 suggested that
N2A connectin suppresses IS2-autolysis. These
data indicate the importance of p94–connectininteraction in the control of p94 functions byregulating autolytic decay of p94.

p94/calpain 3 is a skeletal muscle-specificmember of the Ca2+-regulated cytosolic cysteine
protease family, the calpains. Defective p94
protease activity originating from genemutations causes a muscular dystrophy called
calpainopathy, indicating p94’s indispensability
for muscle survival. Because of the existence ofthe p94-specific regions IS1 and IS2, p94
undergoes very rapid and exhaustive autolysis.
To elucidate the physiological relevance of thisunique activity, p94’s autolytic profiles and theeffect of the p94 binding protein, connectin/titin,on this process were investigated. In vitro
analysis of p94 autolysis showed that autolysis
in IS1 proceeds without immediate disassembly
into fragments, and that the newly identified
cryptic autolytic site in IS2 is critical fordisassembling autolyzed fragments. As a genetic
system to assay p94 autolysis semiquantitatively,
p94 was expressed in yeast as a hybrid protein
between the DNA binding and activation
domains of the yeast transcriptional activator
Gal4. Transcriptional activation by the Gal4–
p94:WT hybrid protein is precluded by p94
autolysis. Complete or partial loss of autolytic
activity by C129S active-site mutation,
LGMD2A pathogenic missense mutations, or
PCR-based random mutagenesis could be
detected by semiquantitative restoration of
Gal4-dependentβ–galactosidase
gene
expression. Using this system, the N2A

Calpains (named after the first-discovered andbest characterized member, Ca2+-dependent andpapain-like protease, EC 3.4.22.18, clan CA,
family C2) correspond to a diverse gene family
whose members all share the characteristic“calpain protease domain”, and comprise a uniquebranch of the cysteine proteases (1,2). To date,calpains have been identified in many different
mammalian tissues and in almost all types of livingorganism (3).
Studies on calpain protease activity, fromenzymatic characterization to clarification of its
roles in cellular phenomena, have mainly advanced
knowledge of the conventional calpains, the µ- andm-calpains. These ubiquitously expressed calpains
can be conveniently assayed in vitro using
established and reproducible methods, such as
casein hydrolysis assay, facilitating developmentof more specific inhibitors as well as moresensitive substrates.
In contrast, the skeletal-muscle-specific calpain,
p94/calpain 3, has remained poorly characterized1

terminus regions, of connectin, which are locatedin the sarcomeric N2- and M-lines, respectively(15,16); “A” in N2A represents a splice variant
predominant in skeletal muscle. In accordancewith this, p94 has been immunolocalized to theN2- and M-line regions in the skeletal muscle
sarcomere (17). The biochemical properties of p94
from skeletal muscle present features of connectinbound proteins, i.e., p94 is enriched in theconnectin-rich saline-insoluble fraction. Isolationof p94 from this myofibril fraction provokes rapid
autolysis and disappearance of full-length p94
protein (12). It was recently established that an inframe deletion in the connectin N2A region causesa recessively inherited severe muscular dystrophy
in mice: muscular dystrophy with myositis, mdm(18). The deletion caused by mdm results in a lossof p94-interacting activity of N2A connectin in theyeast two-hybrid system, and the amount of p94
protein shows a tendency to decrease in mdm/mdm
skeletal muscle (19,20). Together, theseobservations support the hypothesis that binding toconnectin is related to p94 stability. However, theactual interaction between p94 and the N2A
connectin fragment has not yet been studied under
the physiological context. Therefore, thesignificance of the interaction between p94 andN2A connectin and of the loss of interaction
between p94 and mdm-type N2A connectin
requires further investigation.
The focus of this study was to monitor the
autolytic activity of p94 and to evaluate thefactor(s) affecting this activity. We used the yeasttranscriptional activator Gal4 system, which was
originally described as “proteinase trapping” (21),to measure the autocatalytic activity of several
viral proteases such as the 3C protease ofcoxsackievirus B3, the 2A protease of poliovirus,
and the human immunodeficiency virus protease(22-24). In our system, suppression of p94
autolytic activity was detected as an increase intranscriptional activation using a hybrid protein of
two functional domains of Gal4 (Gal4bd andGal4ad) linked by p94. This is the first example inwhich a mammalian protease is applied toproteinase trapping. Using this methodology incombination with other in vitro analyses has been
beneficial in revealing the autolytic processes of
p94. This genetic system also could be used for
examining the effect of wild type and mdm-typeconnectin fragments on p94 autolysis, andwith regard to its protease activity. The expressionof p94 predominates over other calpain species inskeletal muscle, and a defect in p94 proteolytic
activity originating from gene mutations causes
muscular dystrophy (4-6). In this context, p94
exemplifies the critical importance of tissuespecific calpain species that must fulfill uniquefunctions, thereby, necessitating innovative
approach to investigate.
One characteristic of p94 is its very rapid andexhaustive autolytic activity, making it unfeasible
to carry out in vitro activity assays using purified
p94 protein (7). Previously, we took advantage ofthe relatively stable nature of one of the splicevariants of p94, p94∆, determined enzymatic
profiles, protease inhibitor sensitivity in vitro, andfound that calpastatin, an endogenous inhibitor
protein specific to calpains, is a substrate for p94(8). p94∆ lacks p94-specific insertion sequences,the IS1 and IS2 regions, encoded by exons 6 and15/16, respectively, and it is feasible to carry out
expression and purification of recombinant productat a scale sufficient for biochemical analysis.
Another group has also shown that the bacteriallyexpressed protease core of p94 serves as a useful
enzyme source for in vitro kinetic analysis (9,10).
Furthermore, a clan of alternatively spliced formsof p94, which is expressed in rodent lenses, wasalso shown to be useful for in vitro enzymatic
study (11). Together, these data strongly attributethe apparent autocatalytic instability of p94 to IS1
and IS2.
There is an interesting discrepancy between
recombinantly expressed p94 protein and native
p94 protein in skeletal muscle. Although too
unstable to completely purify (12), the full-length
p94 protein can be detected in freshly preparedskeletal muscle homogenates (13,14) in contrast to
recombinantly expressed p94, which is mainly
detected as autolyzed fragments (7). Therefore, itis predicted that skeletal muscle cells keep p94
from undergoing autolysis constitutively or
randomly. One of the candidate molecules
regulating p94 stability is connectin/titin, agigantic sarcomeric protein in striated muscle. In
both skeletal and cardiac muscles, a single
connectin molecule extends from the Z-line to the
M-line and therefore spans the entire halfsarcomeric distance of more than 1 µm. Previously,
our yeast two-hybrid studies showed that p94
binds to two distinct regions, the N2A and C2

provided by the manufacturer. Transformants wereselected on plates with SD-medium that lacked Trp(described as SD-W), or SD-LW. The expressionof reporter genes HIS3, ADE2, or both wasassessed by colony growth of AH109 on SD-LWH,
SD-LWA, or SD–LWHA plates, respectively.
Expression of the reporter gene lacZ was evaluatedusing CG1945 by liquid culture assay for βgalactosidase activity using ONPG as a substrate.
All the procedures were carried out following the“YeastProtocols
Handbook”(PT3024-1,
Clontech) with some modifications. One unit of βgalactosidase activity corresponds to 1 µmol of
ONPG hydrolyzed per min at 30°C. The amount ofyeast cells per reaction as well as the reaction time
was adjusted so that the measured A420 value
remains within the linear range of the assay.

profiling
structure–function
relationships
underlying autolytic/protease activity unique to
p94.
EXPERIMENTAL PROCEDURES
cDNA constructs – The cDNAs for human andmouse p94/calpain 3 were kind gifts from Drs.
Muriel Herasse and Isabelle Richard. The cDNA
corresponding to the C-terminal transcriptional
activation domain (AD) of Gal4 was isolated from
pACT2, a yeast expression vector provided in the
MATCHMAKER Two-Hybrid System (Clontech,
CA, USA), and was subcloned into pAS2-1C,
which encodes the Gal4 DNA binding domain(BD), in tandem with human p94, resulting in theBD–p94–AD-expressing vector. Human connectin
cDNA clones CN48 and CN52, encoding N2A andC-terminal regions of connectin, respectively, havebeen reported previously (16). The mouse cDNA
corresponding to the human connectin CN48 clone(nt. 28858-29769 of NM_133378) was amplified
by PCR from mouse skeletal muscle cDNA using
Pfu DNA polymerase (Stratagene, CA, USA). The
mammalian expression vector pcDNA3.1/N-FLAG
and the yeast expression vectors pAS2-1C,
p415GPD, and p425GPD have been described
elsewhere (25). Human and mouse p94 cDNAs
were constructed into pSRD for protein expression
under the SRα promoter, as previously described(6,26). Mouse p94 (wild type or a splice variant
without exon 6) was also tagged with tandem myc
and 6×His epitopes at the C-terminus. Mutagenesis
using long PCR with Pfu or Pfu-Turbo DNA
polymerases was performed to introduce all themutations, as previously described (6). p94:N358D
was constructed by introducing an Asn358 to Asp
mutation into the human p94 cDNA and inserting
this into the BamHI–KpnI sites of the pFastBacHTb vector (Invitrogen, CA, USA), which adds
6×His at the N-terminus. Enzymes used for
manipulating recombinant DNA were purchasedfrom Takara Bio (Kyoto, Japan) or New England
Biolabs (MA, USA). All constructs were verified
by DNA sequencing.

Random mutagenesis by PCR – Random mutations
were introduced using human wild-type p94 cDNAand the primers, 5´–ATA CAT ATG CCG ACC
GTC ATT AGC GC–3´ and 5´–ATG TTG ATG
TAG GTT TTG CTC C–3´, which correspond to
NS and domain III of p94. The reaction was
performed in 50 µl of the solution [0.2 mM dGTP,
1 mM dATP/dCTP/TTP, 400 nM of each primer, 3mM MgCl2, 0.5 mM MnCl2 in 1 × rTaq buffer(Takara Bio)] with 50 ng of template DNA (humanwild-type p94 cDNA) and 1.25 units of rTaq using
the following cycles: 94°C, 5 min; [94°C, 30 sec;
55°C, 30 sec; 72°C, 120 sec] × 25 cycles; 72°C, 7
min. However, these conditions turned out to be
too mutation prone, and in the second set ofexperiments, the reaction was performed under“normal” PCR conditions, i.e., in 50 µl of thesolution [0.2 mM dGTP/dATP/dCTP/TTP, 500
nM of each primer in 1 × ExTaq buffer containing
2 mM Mg+ (Takara Bio)] with 50 ng of template
DNA and 1.25 units of ExTaq (Takara Bio) using
the same cycles as above. The mutagenized cDNA
fragments (20 µg) were cotransformed with 20 µgof the 6.7 kb Tth111I–PshAI fragment of the BD–
p94–AD expression vector into AH109 to cause invivo ligation by homologous recombination, as
previously described ((27), see Fig. 4A). About 2 ×
106 transformant were screened on selection
medium plates, SD–WHA, yielding about 3,400
colonies. Plasmid DNA was isolated from thesecolonies, and mutations were identified by DNA
sequencing.

Yeast transformation and assay for reporter gene
expression – Saccharomyces cerevisiae strains,
AH109 and CG1945, were transformed using the
FastTM-Yeast
Transformation
Kit
(Gene
Technology, MO, USA), according to the protocol
3

converted for densitometric evaluation. The outlineof each band was defined by the sharpness of thecontrast against background. The amount ofprotein in each band was represented by volume
OD and normalized against the value at 0 min of
incubation set to 1.

In vitro autolytic assay using recombinant protein– Spodoptera frugiperda cells (Sf-9) weregenerously provided by Dr. Takeshi Nishino(Nippon Medical School, Tokyo, Japan).
Recombinant p94:C129S and p94∆ were expressedand purified as previously described (8). Nterminally His-tagged p94:N358D was expressedin the Sf-9/baculovirus system in the same mannerand purified using a His-Bind Quick 900 Ni2+affinity column cartridge (Takara Bio) according
to the manufacturer’s instructions. Briefly, 5 × 107
infected Sf-9 cells were homogenized bysonication in 2 ml of 20 mM Tris-HCl, pH 7.5, 5
mM NaHCO3, 0.1 mM EGTA, 1 mM PMSF, and 5mM 2-mercaptoethanol. After supercentrifugation,
the supernatant was applied to the His-Bind Quick
900 cartridge preequilibrated with 1 mM His inbuffer A (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5mM 2-mercaptoethanol). The cartridge waswashed with 10 ml of 1 mM His in buffer A, andeluted with 2.5 ml of 0.2 M His in buffer A. The
eluent was immediately desalted by applying it to a
PD-10 gel filtration column (Amersham
Biosciences, NJ, USA) preequilibrated with 10
mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mM
DTT, and used for experiments within a few days.
Protease-inactive p94:C129S (final 1 mg/ml) andthe splice variant p94∆ were mixed at a molar ratioof 10:1 and incubated at 37°C for up to 240 min.
The assay solution contained 0.1 M Tris/Cl, pH 7.5,
25 mM 2-mercaptoethanol, and 25 mM Ca2+.
Autolysis of p94:N358D was performed byincubating it in 50 mM Tris-HCl, pH 7.5, 0.5 M
NaCl, 1 mM DTT, 1 mM PMSF, 1 µM pepstatin A,
and 10 µM TLCK at 37°C (p94:N358D protein:
final 10 µg/ml). The reaction was terminated at theindicated times by the addition of 5 × sample
buffer followed by 5 min incubation at 95°C. The
0 min sample was constituted by adding eachcomponent into a tube that already contained
sample buffer.

Protein expression in COS7 cells for western
blotting and immunoprecipitation – COS7 cells
were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum
heat inactivated prior to use (56°C, 30 min).
Electroporation was performed using a Bio-Rad
gene pulser, according to the manufacturer’s
instructions. Cells were harvested 72 hr after
electroporation and sonicated in a solution
consisting of 100 mM Tris-HCl, pH 7.5, 10 mM
EDTA, and 1 mM dithiothreitol, as previously
described (6). After SDS-PAGE, proteins weretransferred to PVDF membranes (Millipore, Tokyo,
Japan), which were, then, probed with appropriate
primary antibodies and horseradish peroxidasecoupled secondary antibodies followed byvisualization using POD immunostaining kit(Wako, Osaka, Japan).
For immunoprecipitation, cells transfected withexpression constructs for mouse p94 with orwithout a C-terminal myc-His tag were harvested
in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M
KCl, 1 mM EDTA-K, 1% (v/v) NP40, and 0.5%
Triton X-100) containing protease inhibitors as
follows: 1.5 µM aprotinin, 1 mM PMSF, 50 µM
leupeptin, 50 µM TLCK, and 2 mM
iodoacetoamide. After 30 min on ice withoccasional tapping, the lysate was centrifuged at
11,000 × g for 10 min, and the supernatant wasused for immunoprecipitation using anti-pIS2 oranti-myc antibodies.
The antibodies used in this study included ap94-specific anti-pIS2 antibody raised in goatagainst the epitope identical to the one used for thepreviously described anti-pKrich (7), an anti-pNS
antibody (7), an anti-dIII antibody (Triple Point
Biologics, OR, USA, RP3-calpain3), a mousemonoclonal anti-FLAG M2 antibody (Stratagene),
and a mouse monoclonal anti-myc antibody(Invitrogen, R950-25). An anti-p94-C-terminus
antibody
waselicited
using
CNVLEWLQLTMYA, corresponding to the Cterminus of rat p94, conjugated to KLH.

SDS-PAGE and native-PAGE analyses –Proteolytic fragments were subjected to SDS- or
native-PAGE followed by CBB-staining andwestern blotting. SDS-PAGE was performed
according to the standard Laemmli method using
10% gels. Native-PAGE analysis was carried out
essentially in the same manner but without SDS,
and electrophoresed by a constant current 5-10 mA
at 4°C. Scanned images of CBB-stained gels were4

compared to those detected by native-PAGE bydensitometry (Fig. 1D). When the samples were
subjected to SDS-PAGE, the rapid decline wasobserved (t1/2 = about 140 min; Fig. 1D, closed
circles). In native-PAGE analysis, however, morethan 60% of the initial density was maintained for
up to 4 hours (Fig. 1D, open circles). Western blot
analysis with an anti-pIS2 antibody following
SDS- or native-PAGE showed the same trend, i.e.,
slower decrease of the signal intensity for the fulllength and the N-terminally cleaved p94:C129S
under non-denaturing condition (Figs. 1B and C,
anti-pIS2).
These observations indicate that intermolecular
autolytic cleavages of p94 do not simultaneously
generate separate autolyzed fragments, and thatthesefragments
maintain
theirinitialintramolecular interaction for more than twice thetime predicted by SDS-PAGE analysis. This
population of autolyzed p94 might be described as“IS1-nicked” molecules, because the 55 kDa
fragment detected in SDS-PAGE (Fig. 1B, theuppermost arrow) corresponds to the C-terminal
product generated by autolysis in IS1 (Fig. 1A,
arrows [2]).

RESULTSInteraction of autolyzed p94 fragments – The
autolysis of p94 was reconstituted in vitro bymixing p94:C129S and p94∆, a splicing variant
which lacks IS1 and IS2 (8), in a ratio of 10:1.
Previously, it was shown that p94∆ recapitulates
the substrate-specificity of p94 and retains
autolytic activity toward p94:129S, albeit Ca2+dependently (8). The condition used here allows
the detection of the autolyzed fragments from
p94:C129S
generatedby“intermolecular
autolytic” activity of p94∆ (8).
In SDS-PAGE analysis, a slight mobility shiftof p94:C129S was detected after 15 min of
incubation in the presence of Ca2+ (Fig. 1B, open
and closed arrowheads); it is caused by very rapid
cleavages at the N-terminus (Fig. 1A, arrows [1]).
Subsequently, 55 kDa and additional smaller
autolyzed fragments were generated (Fig. 1B,
arrows) (16). Since N-terminal cleavages do not
impair the proteolytic activity of p94∆ (8), it was
assumed that both the full-length and N-terminallyclipped populations of p94:C129S (Fig. 1B, open
and closed arrowheads, respectively) represent
structures of protease-active p94 species.
The same set of samples was analyzed bynative-PAGE to examine the change of molecularconformation. After 15 min of incubation,
p94:C129S slightly shifted toward the lower end of
the gel (Fig. 1C, CS+∆, lanes 15´ to 240´, open andclosed arrowheads). This mobility shift mostprobably reflects the N-terminal cleavage detected
in Fig. 1B (open and closed arrowheads), because
incubation of p94:C129S alone did not cause asignificant change in mobility or signal intensity(Fig. 1C, CS), and the mobility of p94∆ does notoverlap with that of p94:C129S in native-PAGE(Fig. 1C, arrows).
By size-exclusion chromatography, we havepreviously shown the possibility that purified
recombinant p94:C129S forms a homodimer (12).
The bands detected by the native-PAGE may
correspond to p94:C129S homodimer. It is not
likely, however, that the observed mobility shift of
p94:C129S is caused by monomer-dimer
conversion as the shift is probably too small for thechange of molecular mass between 94 kDa(monomer) and 188 kDa (dimer).
The sum of the full-length and N-terminallyclipped fragments detected by SDS-PAGE was

A cryptic autolytic site of p94 near the N2A
connectin binding region – In COS7 cell
expression system, the amount of wild type p94
detected as the 55 kDa fragment is considerably
smaller than that of the 94 kDa fragment for theprotease-inactive p94:C129S mutant (Fig. 2A, WT
and CS). This observation indicates that NS and
IS1 are dominant, but not the only sites forautolysis, and that wild-type p94 further undergoes
autolysis at several unidentified sites.
To reveal other autolytic sites, p94:ex6–, asplice variant that lacks the IS1 region encoded byexon 6 (26), was used. p94:ex6– is detectable as
full-length molecules of the calculated size (89kDa; Fig. 2A, arrow), and does not undergo
autolysis as exhaustively as does wild-type p94(open arrowhead). This variant undergoes autolytic
cleavages in its NS region, and also proteolyzes
p94:C129S to generate the 55 kDa fragment (datanot shown). Therefore, it is suggested thatp94:ex6– retains an autolytic profile similar towild-type p94 except for altered autolysis of itself
in IS1. An autolytic fragment of ca. 32 kDa (Fig.
2A, closed arrowhead) was abundantly detected
for p94:ex6– by anti-pIS2 antibody. This indicates5

bands of around 32 kDa (Fig. 2C, c) were faintly
detected by the anti-pIS2 antibody. The fragment
sizes suggest that at least one of these bands
corresponds to the C-terminal autolytic fragment
generated by a cut in the proximity of IS2 as
identified above. Although the identical bandshould be detected by the anti-C-terminal antibody,the titer of the anti-C-terminal antibody, about
1/100 that of anti-pIS2, fell short of the detection
limit.
Presuming that p94:N358D has weak otherwise
normal autolytic/protease activity, this mutant
illustrates the proceeding of intramolecular
autolysis of p94, i.e., cuts in IS1 occur very rapidly,
followed by a relatively slow cut in IS2, whereas a
cut in NS does not happen significantly under theconditions used. These features are in contrast to
those in intermolecular autolysis, i.e., between
active p94 variants, such as p94∆ and p94:ex6–,
and protease inactive p94:C129S, where NS
proteolysis happens first, indicating that NS isprone to being cut intermolecularly by other p94
molecules rather than intramolecularly.
In both cases, a cut in IS2 is likely todisassemble the p94 molecule. After the cut in IS2,
the N-terminal part, domain II + III with nicks in
IS1, may retain the structure for proteolytic
activity until it is subjected to further proteolysis.

the existence of another cryptic autolytic site in the
C-terminal half of p94. Very small or no amount of
the 32 kDa fragment was detected in wild type p94
or the C129S mutant, respectively.
To detect autolytic cleavage in the C-terminal
region of p94 more efficiently, wild-type p94 andp94:ex6– with C-terminal myc and 6×His epitope
tags (together causing an increase of about 3 kDa)
wereexpressed
in
COS7
cells
and
immunoprecipitated using anti-pIS2 (data notshown) or anti-myc antibodies (Fig. 2B). The 55
kDa autolyzed fragment of wild-type p94 wasdetected as a 58 kDa C-terminal fragment for mycHis-tagged wild-type p94, consistent with thecalculated +3 kDa size (Fig. 2B, open arrowhead).
Smaller fragments of ca. 35 kDa, was detected for
p94:WT- and p94:ex6–-myc-His, but not forprotease-inactive p94:ex6–:C129S-myc-His (Fig.
2B, closed arrowhead). Since the 35 kDa bands
contain a myc C-terminal epitope, the proteolytic
site for these autolyzed fragments was predicted tobe the N-terminus of the IS2 region of p94 (Fig.
1A, arrow [3]).
Analysis of intramolecular autolysis using the
p94:N358D mutant – To understand further the
autolytic processes of p94, autolysis was examinedusing an active site mutant, p94:N358D. The
substitution of active site Asn to Asp was reported
to result in very low, but not zero, specific activity
by affecting the interaction between active site
residues (28). Similarly, p94:N358D showed
weakened autolytic activity, unlike p94:C129S
whose activity is zero. Partially purified
recombinant p94:N358D protein was incubated at
37°C, and the generation of several different
autolytic fragments was detected by a panel ofanti-p94 antibodies (Figs. 1A and 2C). Based on
the molecular size as well as the reactivity to eachantibody, the major autolytic fragments werecategorized into three groups, labeled as a, b, and c(Fig. 1A, bidirectional arrows; Fig. 2C, open
arrowheads). During the first 5 min of autolysis,
generation of fragments “a” and “b”, as initially 35
kDa and 58 kDa bands, respectively, were detected(Fig. 2C, arrowheads a and b), and, concomitantly,
most of the full-length protein disappeared (Fig.
2C, black arrowhead). The fragments “a” retained
reactivity to anti-pNS antibody, indicating that the
autolysis in IS1 occurs very fast, whereas that in
NS does not. In the late phase of autolysis, several

Establishment of an assay system for p94 autolytic
activity using fusion proteins with Gal4 functional
domains – As an approach to assessing theautolytic processes of p94 semiquantitatively, theyeast transcriptional activator Gal4 system was
modified according to a previous study on
autolytic processing of 3C protease (24).
Transcriptional activation by Gal4 requires that its
two functional domains, Gal4bd (BD) and Gal4ad(AD), stay proximal to each other. Those domains
are linked by p94 to be expressed as a BD–p94–
AD hybrid protein. A hybrid protein containing
wild-type p94 (BD–p94:WT–AD) is anticipated to
fail to activate reporter genes, as BD and AD
become separated from each other upon p94
autolysis, especially in IS2, as discussed above(Fig. 3A, upper). In contrast, a hybrid protein
composed of protease-inactive p94:C129S mutant(BD–p94:C129S–AD) remains as the full-length
fusion protein, and, therefore, is expected to becapable of activating transcription (Fig. 3A, lower).
6

culture assay for β-galactosidase activity was
performed. Under the conditions where themeasured value for p94:C129S remained within
the linear range, the following values wereobtained; 1.64 ± 0.48, 172 ± 26, 60.7 ± 18, and30.0 ± 1.8 units for WT, C129S, R490W, andR572Q, respectively. The results were presented
relative to the activity of BD–p94:C129S–AD,
which was set to 100 (Fig. 3C). Transcriptional
activation by the hybrid BD–p94–AD with R490W
or R572Q mutation was significantly more
efficient than that by BD–p94:WT–AD (P < 0.001,
n = 4), but was not as strong as that by BD–
p94:C129S–AD. Together, it was shown that
reduced autolytic activity by the mutations R490W
and R572Q can be recapitulated as an increased
efficiency in transcriptional activation by hybrid
proteins with those mutations.
In summary, the presented system is competentto assay the autolytic activity of p94 by assessing
the deceleration of the autolysis of p94. Evaluationof the accelerating effects on p94 autolytic activity
was difficult as the value for wild-type hybrid
proteins was close to the lower end of themeasurable range for liquid culture assays.

Using the S. cerevisiae AH109 and CG1945
strains as hosts, transcriptional activation of thereporter genes HIS3, ADE2, and LacZ by BD–
p94–AD hybrid proteins was examined. As
expected, transformants expressing the hybrid
protein for p94:C129S but not for wild-type p94
were able to grow on plates without His and Ade,i.e., expression of HIS3 and ADE2 was activated(Fig. 3B, SD–WHA). Liquid culture assays for βgalactosidase activity, an index for reporter gene,
LacZ, expression, gave an essentially identicalresult: 12.9 ± 3.7 units for WT and 770 ± 175 units
for C129S.
Coexpression of intact wild-type p94 with BD–
p94:C129S–AD resulted in a slight reduction, but
not a complete loss, of the reporter gene
expression by BD–p94:C129S–AD (data notshown). Since the viability of yeast cells
expressing BD–p94:WT–AD on SD–W plates iscomparable to that expressing BD–p94:C129S–AD(Fig. 3B, SD–W), it is not likely that the proteaseactivity of p94 against yeast proteins is a dominantfactor causing the phenotypes observed above,
unlike poliovirus protease 2A (23). Together, it
was concluded that rapid autolysis of p94 causes
the dissociation of BD and AD, and is detected as
the lack of reporter gene expression in yeast cells.

Screening of mutations that inactivate p94 – Oneof important application of the above system could
be to study structure-function relationships
regarding p94 autolytic activity. The applicability
was tested by screening of p94 mutants with
reduced autolytic activity. Random mutations wereintroduced into domain II of the BD–p94:WT–AD
hybrid protein by PCR and in vivo homologous
recombination, and the mutations that allowed theexpression of reporter genes, HIS3 and ADE2,
were identified (Fig. 4A).
About 2 × 106 transformed colonies werescreened on selection medium plates, SD–WHA,
resulting in about 3,400 grown colonies. Amongthem, 276 colonies were picked, and plasmidDNAs were rescued from 23 colonies. Each
plasmid encoded the BD–p94–AD hybrid proteinwith up to 5 missense mutations in a region of
about 300 amino acid residues, corresponding
mainly to domain II (Table 1 and Fig. 4B).
It should be noted that among the 34 missense
mutations identified in 23 clones, (i) a W369R
mutation, where W369 is a highly conserved
residue in the calpain family (3), was found in 10
clones; and (ii) six loci, T184, R386, G329, Y336,

Detection of altered autolytic activity by point
mutations – To test the applicability of Gal4–p94
hybrid constructs for semiquantitative analysis of
p94 autolysis, point mutations in domain III,
R490W and R572Q, were introduced to the BD–
p94:WT–AD fusion protein. These mutations wereoriginally identified as pathogenic missense
mutations of p94 found in LGMD2A patients, andboth mutant p94s undergo autolysis but not as
exhaustively as wild-type p94 does when
expressed in COS7 cells (6).
The yeast cells expressing BD–p94:R490W–
AD were able to grow on plates that lack His andAde, however, more slowly than those expressing
BD–p94:C129S–AD (Fig. 3B, SD–WHA, 2dayand 3day). Therefore, it is suggested that reduced
autolytic activity of R490W mutant allowed theexpression of reporter genes, HIS3 and ADE2,
albeit less efficiently compared to C129S mutant.
The same trend was observed for BD–p94:R572Q–
AD (data not shown).
To compare the activity of the hybrid proteins
with different mutations more quantitatively, liquid7

Detailed mapping of the N2A connectinbinding region in p94 by conventional yeast twohybrid assay revealed that the region between
V573 and L580, just upstream of the IS2 region, is
required for interaction with N2A connectin (Fig.
1A). This region coincides with the cryptic
autolytic site identified above, which is predicted
to be critical for autolytic disassembly of the p94
molecule.
Together, these data indicate that the CN48
connectin fragment, which binds to p94, is able toretain BD and AD of the hybrid protein proximal
to each other, against the p94 autolytic activity.
This is the first demonstration that shows
suppressive effect of N2A connectin on autolytic
disassembly of p94, presenting the biologicalsignificance of p94–N2A connectin interaction.

G367, and N449, were where missense mutations
are found in LGMD2A patients, among which four
substitutions, T184M, R386C, G329R, and G367S
were exactly identified in LGMD2A patients (29).
For each clone, most probable mutation(s)responsible for inactivating p94 was(were)deduced considering the extent how well theresidue is conserved and/or whether the identicalmutation is found in LGMD2A patients (Table 1).
Five mutations, Q123H, W376C, W369R, W365R,
and L387P, are not included in the 133 pathogenic
LGMD2A missense mutations reported so far, andwere considered to be responsible for inactivationof p94 in our random mutagenesis experiments.
These results indicate that our system is adequate
for revealing critical amino acid residues for p94
activity, with large scale-screening planned in the
future.

A mutant connectin fragment corresponding to
the mdm deletion was not able to bind p94 or
stabilize p94 – The mouse N2A connectinfragment (denoted as mCN48) with pathogenic
mutation (mdm) was examined as to its effect on
p94 autolysis. Recessively inherited mousemuscular dystrophy, mdm, is caused by an 83-aa
deletion in N2A connectin (18,30). In theconventional yeast two-hybrid system, the mdm
deletion abolished the p94-connectin interaction as
we previously reported (Fig. 5A, mCN48–mdm,(20)). Coexpression of mCN48–mdm with Gal4–
p94 hybrid protein failed to restore transcriptional
activation of reporter genes by BD–p94:WT–AD,
but did not interfere with transcriptional activation
by BD–p94:C129S–AD (Fig. 5C, SD–LWHA).
This shows that the pathogenic mdm deletion
abolished N2A connectin’s ability to regulate
autolytic process of p94, which indicates further
impairment in p94 functions under mdm conditions.

Effect of connectin–p94 interactions during the
p94 autolytic process – Two distinct regions ofconnectin, N2A and the C-terminus, have been
identified as binding sites for p94 in the sarcomere(16) (Fig. 5A, CN48 and CN52). These fragments
were coexpressed in yeast cells with BD–p94–AD
to test whether or not autolytic activity of p94 isaffected by the interaction with connectin. The
rationale was that a partial, if not the complete,interference in the autolytic process of p94 could
yield detectable expression of reporter genes if thepresence of a hybrid protein over a certain
threshold amount was achieved.
Expression of BD–p94:WT–AD in yeast cells
transformed with the empty vector resulted in thelack of growth on SD-LWHA, which is identical toand consistent with the result shown in Fig. 3B(Fig. 5B, SD–LWHA, BD–WT–AD + mock).
Coexpression of CN48, but not CN52, restoredtranscriptional activation of reporter genes, HIS3
and ADE2, by BD–p94:WT–AD as shown by
growth on SD–LWHA plates (Fig. 5B, SD–LWHA,
BD–WT–AD + CN48 or CN52). Yeast cells
expressing the protease-inactive hybrid, BD–
p94:C129S–AD, which is by itself functional as atranscriptional activator (Fig. 3B and 3C), was able
to grow on a SD–LWHA plate independently of
coexpressed connectin fragments (Fig. 5B, SD–
LWHA, BD–C129S–AD). The protease-inactive
hybrid without the C-terminal AD domain, was notcapable of activating transcription (Fig. 5B, SD–
LWHA, BD–C129S).

DISCUSSION
In this study, the interaction between the
autolyzed fragments of p94 and the effects of theconnectin–p94 interaction during autolysis wereanalyzed. Using the in vitro reconstituted autolytic
reaction of full-length p94, it was suggested thatp94 retains functional conformation as a proteaseduring autolysis. Furthermore, we have developed
a genetic method of studying the autolytic
properties of p94. The validity of the system fordetecting changes in p94 autolytic activity and8the autolytic site revealed in this study. The datasuggests that IS2, which has been considered toplay a role for modulating the Ca2+-dependence ofp94 (32,33), is also involved in the autolysis
together with NS and IS1.
Autolytic
reaction
reconstitutedusing
p94:C129S and p94∆ as substrate and active
enzyme, respectively, proceeds too rapidly tocapture the sequential involvement of NS, IS1, andIS2 in the process. Therefore, another p94 mutant,
p94:N358D was used for comparing the generationof different autolytic fragments using much lower
concentration of p94 protein ([p94:N358D]2 is ca.
1/1,000 of [p94:C129S][p94∆], as theirconcentrations are 0.01, 1, and 0.1 mg/ml,
respectively) to make the intramolecular autolysis
predominant. It has been shown that the mcalpain:N286D mutant has very weak activity
determined by caseinolytic assay; the weakprotease activity is because of the impairment of
the interaction between active site residues rather
than the alteration in Ca2+-sensitivity (28). Native
p94 partially purified from rabbit skeletal muscle
first autolyzes at the N-terminus of G275 in IS1,
yielding ca. 60 kDa fragments, which eventuallyconverges to a 55 kDa fragment by a further cut in
the N-terminus of E323 (12). The autolytic profile
of p94:N358D essentially reproduced the trend(Fig. 2C, anti-pIS2), supporting the idea that thismutant has weak but otherwise unaltered autolytic
activity, and, therefore, is appropriate for theexperiment here.
It has been observed that autolysis in the NS
region is very rapid in intermolecular reactions but
is relatively slow in intramolecular reactions; theratio of both reactions depends on theconcentrations of p94. This would explain why thein vitro autolysis of p94:C129S by p94∆ showed
quick NS proteolysis (Fig. 1B), whereas the
autolysis of lower concentrated p94:N358D did not(Fig. 2C). It is, however, the cleavage in IS1 that isobserved in the early phase of autolysis in bothcases. The small C-terminal autolyzed fragment
cut in the proximity of IS2 is generated later.
Therefore, it was concluded that a cut in IS2 isslow but critical for disassembly of autolyzed p94
fragments. As summarized in Fig. 6A, thespeculated procedure of autolytic cleavage is,intermolecularly, the order of proteolysis is NS (b)→ IS1(c) → IS2(d), and intramolecularly, it isIS1(e) → IS2(f) → NS.

examining the effects of interacting molecules,
particularly connectin/titin fragments, in autolytic
process has been demonstrated. These data provide
a model for a molecular mechanism how connectin
is involved in the regulation of p94.
It has been anticipated that p94 autolysis would
result in disruption of the protease core, i.e., ininactivation of p94, as dominant autolytic sites of
p94 resides in the IS1 region between the active
site Cys129 and His334/Asn358 (Fig. 1A, arrows[2]). Thus, the amount of active p94 was expected
to be quickly reduced during autolysis.
Nevertheless, several proteins besides p94 itself
were substantially proteolyzed in COS7 cells
expressing p94 (6), casting a doubt about theassumption that autolysis of p94 immediately
inactivates p94.
Several different approaches using fragments of
p94 argue for the association of autolyzed
fragments of p94 (10,31). The protease core of p94,
corresponding to the region NS–IIa–IS1–IIb (Fig.
1A upper panel), undergoes Ca2+-dependent
autolytic processing in the NS and IS1 regions.
The cleavages in NS and IS1 were demonstrated toprecede the gain in protease activity against othersubstrate molecules, and the interaction between
autolyzed fragments was biochemically shown(10). These reports, however, have not shown
information about the whole structure of p94.
In this study, association of autolyzed
fragments shown by native-PAGE analysis, inwhich p94:C129S was intermolecularly autolyzed
by p94∆, clearly indicates that not only theprotease core but also almost whole structure of
p94 retain its intact conformation for some timeafter autolysis. Therefore, these results including
ours indicate that p94 expresses its proteaseactivity even with nicks in IS1 by autolysis, whichanswers the question of why endogenous proteinssuch as HSP60 and calpastatin were properly
proteolyzed regardless of full-length p94 being
hardly detected when expressed in COS7 cells (6).
Furthermore, another autolytic site in p94 was
identified at the N-terminus of IS2. This may
explain the fact that the 55 kDa autolytic fragment
corresponding to the structure from IS1 to the Cterminus is detected much less than expected.
Lp82, a lens-specific splicing variant of p94, wasreported to have an autolytic site between domains
III and IV corresponding to the N-terminus of
R592 of p94 (11), which is probably identical to9The above hybrid protein assay in yeast clearly
demonstrated for the first time that N2A connectin,
encoded by the clone CN48, interacts directly with
p94 to suppress autolytic process of p94. The
molecular mechanisms of how N2A connectin
affects p94 autolysis could be inferred as“suppression” rather than “inhibition” of autolysis,
as coexpression of the CN48 fragment with p94 incultured cells did not significantly inhibit the p94
autolysis (12). Considering that N2A connectinbinding site in p94 is in the vicinity of the autolytic
site proximal to IS2, presumably between D591
and R592, N2A connectin is likely to affect the
autolysis in this site by structural hindrance (Fig.
1A and Fig. 6A (g)). It is unlikely that the above
autolysis disrupts connectin binding sequence in
p94, because the p94:S581term mutant, which
does not have a C-terminal part after S581, canbind N2A connectin (Fig. 1A).
Alternatively, weak interactions between CN48
and IS2/domain IV that could not have been
detected by the conventional yeast two-hybrid
system may delay the complete dissociation of BD
and AD, even after a cut in/near IS2 (Fig. 6A (h)).
In contrast, the interaction between p94 and the
C-terminus of connectin (CN52) detected in theconventional yeast two-hybrid system (15,16)
failed to be evaluated in the hybrid protein assay.
As CN52 requires full-length p94 structure (15),
CN52 also must suppress autolysis in IS2. If
p94:WT were autolyzed at IS2, it would cause
dissociation of the C-terminal part, resulting ininability in binding CN52. A possible account forthis apparent discrepancy is that the full-length p94
structure, which is required for CN52 binding (15),might be compromised by simultaneous additionof BD and Ad. The alternative approach is
requiredfor
determining
thebiologicalsignificanceof
p94–connectin
C-terminus
interaction.
An in-frame deletion of mouse N2A connectinhas been shown to cause severe muscular
dytrsophy, mdm, and the binding of CN48 to p94
was suggested to be disrupted by this mutation(Fig. 5A) (18,20). In this study, one of thephysiological consequences of such a deletion was
suggested to be the lack of regulation of p94
autolysis, which can explain decreased p94 protein
levels in mdm homozygous skeletal muscle
reported by several studies (18,19,34).

As an approach complementary to in vitro
study of p94 autolysis described above, we haveestablished a system for monitoring p94 autolytic
activity using a Gal4-based transcriptional
activation system. Developing such a system was
expected to make it feasible to capture thebehavior of p94 by evaluating its autolysis andthen to screen mutations/factors influencing p94
autolytic activity. Experiments using LGMD2A
mutants clearly showed that the system iscompetent for detecting changes in p94 autolytic
activity semiquantitatively. Furthermore, using thissystem for screening as performed here has
following advantages: (i) non-sense mutations,which terminate polypeptide chain somewhere in
p94, i.e., between BD and AD, are intrinsicallyeliminated; and (ii) specific domains can beselected to introduce mutations for structure–functional studies. These features greatly improvethe efficiency of identification and characterizationof the mutants, which can be applied to primary, if
not decisive, diagnosis for LGMD2A. One of thesignificant applications of the system would bescreening of conditionally inactive mutants of p94,
e.g., temperature-sensitive mutants of p94 that
could be beneficial for protein expression andpurification.
The BD–p94:WT–AD hybrid protein caused no
detectable transcription of the reporter genes,
indicating that autolysis is fast enough to result ina state where the major parts of BD and AD are
dissociated. Based on the scheme for autolytic
process of p94 in Fig. 6A, there are two
possibilities that can dissociate BD from AD; a cut
in NS to liberate N-terminally fused BD, and/or acut in IS2 to dissociate the C-terminal fragment of
p94 fused to AD. The former and the latter,
respectively, happen preferentially in inter- andintra-molecular autolyses (Fig. 6A, (b) and (f)).
Coexpression of BD–p94:C129S–AD and p94:WT
reduced the reporter gene activation to some extent,
but not to zero, indicating that the intramolecular
reactions predominates over the intermolecular
reactions in yeast cells, and/or that NS became lessaccessible because of the fusion with BD. In bothcases, the IS2 cut is more likely to casuse BD/AD
dissociateion. Although the in vitro analyses
suggest that autolysis in IS2 occurs relatively
slowly after that in IS1, our results indicate that
autolysis in IS2 of BD–p94:WT–AD is fast enoughnot to provoke reporter gene expression in yeast.
10

endogenous inhibitor protein for the conventional
calpains. Besides the direct suppressive effect ofN2A connectin on p94 autolytic disassembly, it ispossible that the adjacent PEVK region ispositioned so that it modulates p94 activity, forexample, by serving as a pseudosubstrate, because
calpastatin has been identified as one of the best
substrates for p94 so far (8).
Our hybrid protein assay demonstrated the
suppressive effect of N2A connectin on p94
autolysis; however, it seems that additional cellularcomponents, which could be specific to skeletal
muscle cells, are required for maintaining the fulllength p94 protein at an amount sufficient for its
function.
Immunodetection
shows
that
considerable amounts of the full-length p94 protein
exist as the major molecular species in skeletal
muscle tissue ((13), Ojima et al., unpublishedresults). Furthermore, the autolytic properties of
p94 are probably relevant under those cellular
context for maintaining the proper amounts of its
activity. For example, unlike wild-type p94,
overexpression of one of the splice variants of p94
lacking IS1, p94:ex6-, which is rather resistant toautolytic degradation, interfered with normal
muscle maturation (35). In addition to directlyinteracting molecules, it is possible that there arefactors that potentiate the proper amount of p94
activity, e.g., through changing the local Ca2+
concentrations or regulation of the expression
spectra of p94 splice variants. Further studies are
required for complete elucidation of the regulationof p94 activity, which will lead to anunderstanding of the true roles of p94 in skeletal
muscle and its involvement in muscular dystrophy
when defective.

The relationship between the activity of p94
and mdm pathology has been studied using mutant
mice that have the connectin mdm mutation
combined with p94 gene knockout oroverexpression of wild-type p94 (34). Transgenic
overexpression of p94 in mdm homozygous(Ttnmdm/mdm) mice caused exacerbation of thedystrophic phenotype (34), while it yields almost
no phenotype in wild type mice (35). Moreover,gait activity is slightly altered in mdm
heterozygous (Ttn+/mdm) mice, and transgenic
overexpression of wild-type p94 corrected thisfeature. Together with the results presented in thisstudy and the fact that N2A connectin is present instoichiometric excess over p94 in skeletal muscle,these phenotypes can be explained as follows: p94
overexpression (i) shows no toxicity in wild-type
mice, because N2A connectin absorbs excess p94
and suppresses p94 activity; (ii) was detrimental in
Ttnmdm/mdm mice, because free p94, which cannot be
absorbed due to the mdm mutation, attacks
myofibril proteins; and (iii) corrected mild
phenotypes in Ttn+/mdm mice, because a sufficientamount of p94 was supplied to cancel theimbalance between molecular components at theN2A region; e.g., in mdm homozygous skeletal
muscle, another binding protein in N2A region ofconnectin,
CARP(cardiac
ankyrin-repeat
protein)/MARP1 (muscle ankyrin-repeat protein 1)
is upregulated (20). These highlight the functionalcrosstalk between p94 and other muscle proteins in
the N2A region.
The minimum p94-binding region in N2A
connectin corresponds to two tandem IG domains,denoted as I82 and I83, at the C-terminus of N2A(20). As shown in Fig. 6B, mdm deletion causes
truncation of the last two β-strands of the Cterminal IG domain (I83); the 3D structure of I82–
I83 in Fig. 6B is deduced from the reported
structure of Z1–Z2, other IG repeat modules in theconnectin molecule. The deletion may destroy the
structure of I83 necessary for binding andstabilizing p94.
It should be noted that the p94-binding region
region is in the vicinity of the so-called PEVK
region, rich in Pro, Glu, Val, and Lys, andcomposed of random coils. The PEVK region has
been considered to play a key role in expressing
passive elasticity. This region is recently describedas “intrinsically unstructured protein (IUP)” (36),and is similar to another IUP, calpastatin, an
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13

FOOTNOTES* We are grateful to all of our laboratory members for experimental support and valuable discussion. This
work was supported in part by MEXT.KAKENHI 16026209 (to HS), and 14656054 (to KA), by
JSPS.KAKENHI 15380089 (to HS), KAKENHI 14086203 (to TM), a grant (No.0349) from the Salt
Science Research Foundation (to TM), a grant from DFG (La 1969/1-1, to DL), and a Research Grant(17A–10) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare (to HS).
The abbreviations used are: LGMD2A, limb-girdle muscular dystrophy type 2A; NS, N-terminal sequence
unique to p94; IS1 (IS2), insertion sequence 1 (2) unique to p94; CN48 (CN52), connectin/titin clone No.
48 (52); AD, activation domain of Gal4; BD, DNA binding domain of Gal4; CARP (MARP), cardiac(muscle) ankyrin repeat protein; mdm, muscular dystrophy with myositis; PEVK, flexible region ofconnectin rich in Pro, Glu, Val, and Lys; SD–W etc., synthetic minimal medium with dextrose minus
tryptophan etc.

14

FIGURE LEGENDS
Fig. 1. Connectin binding and autolytic profiles of p94.
A, Upper panel; A schematic of the molecular structure of p94. NS, IS1, and IS2 are p94-specific insertion
sequences. Subdomains IIa and IIb compose the calpain protease domain. Domains III and IV are C2-like
Ca2+-binding and 5EF-hand motif domains, respectively. Arrows [1] to [3] indicate proteolytic sites forautolysis. The positions of the epitopes for the antibodies used in this study and the deduced positions of
the fragments indicated by open arrowheads in Fig. 2C (a–c) are shown by the bracketed lines and thebidirectional arrows, respectively. The bars under the schematic structure represent the result from yeast
two-hybrid assay for surveying the sites in p94 required for interaction with N2A connectin. A series of Cterminal truncations (bars) were introduced into p94, and their effect on the binding activity to N2A
connectin, clone CN48, was evaluated. Deletion of most of IS2 did not compromise the connectin-binding
activity of p94.
B, The autolytic profile of p94:C129S. The intermolecular autolytic reaction was reconstituted in vitro
using p94:C129S as a substrate for p94∆. The reaction was stopped at each indicated time and analyzed by
SDS–PAGE followed by CBB-staining or immunoblotting using anti-pIS2 antibody. Open and closed
arrowheads suggest the full-length and N-terminally clipped molecules, respectively. Arrows indicate theautolyzed fragments of p94:C129S by further proteolysis in IS1 and other regions. A grey arrowhead
incicates p94∆.
C, Native-PAGE analysis of the set of samples shown in B. As controls, p94:C129S, after 240 min
incubation with and without Ca2+, and p94∆ were electrophoresed separately. Open and closed arrowheads
indicate the mobility corresponding to the full-length and the N-terminally clipped fragments of
p94:C129S. The mobility of those two populations is compared at the right side of the panel. Note that themobility of p94∆ does not overlap with either (arrow).
D, Changes in the sum of the full-length and the N-terminally autolyzed fragments. Signal intensity
corresponding to the full-length and the N-terminally autolyzed fragments (open and closed arrowheads in
Fig. 1B and C) was measured and plotted against incubation time. The values are standardized with thevalue at 0 min set to 1. Closed and open circles indicate the values obtained for SDS–PAGE and nativePAGE analysis, respectively.
Fig. 2. Autolysis in the vicinity of the IS2 region
A, Comparison of autolytic fragments. Wild-type p94 (WT), a splice variant that lacks the exon 6-encoded
IS1 region (p94:ex6–), and p94:C129S (CS) expressed in COS7 cells were compared by western blotting
using an anti-pIS2 antibody. Wild-type p94 is mainly detected as the autolyzed 55 kDa fragment (openarrowhead). For p94:ex6–, the full-length product (arrow) is detected along with autolyzed fragments, inwhich the 32 kDa fragment is dominant (closed arrowhead).
B, Detection of C-terminal autolyzed fragments. Myc epitope and tandem His hexamer sequences wereintroduced to the C-terminus of wild-type p94 and p94:ex6–. Constructs were expressed in COS7 cells andimmunoprecipitated with an anti-myc antibody. The calculated molecular weight increase caused by theadded epitope sequences is about 3 kDa. An autolyzed fragment of calculated molecular size
corresponding to 32 kDa in Fig. 2A was recovered for both wild-type p94 and p94:ex6– (closed
arrowhead). The 55 kDa autolyzed fragment generated by proteolysis in IS1 was detected as an
approximately 58 kDa fragment only for wild-type p94 with the C-terminal epitope sequence (openarrowhead). Asterisks indicate degradation products unrelated to autolysis. An arrow points the full-length
molecule of p94:ex6– with the C-terminal tag.
C, Analysis of the autolytic processes of p94 by p94:N358D. The active site Asn358 to Asp mutant,
p94:N358D, was incubated at 37°C for the indicated times (min). Autolysis of p94 was detected bywestern blotting using anti-pNS, anti-dIII, anti-pIS2, or anti-C-term antibodies against p94 as indicated.
The locations of the fragments marked by the open arrows (a–c) are indicated by the bidirectional arrows
in Fig. 1A, upper panel. Fragments a, b, and c correspond to the regions, from the N-terminus to the N15

terminal half of IS2, from the C-terminal half of IS1 to the C-terminus, and from the IS2 to the C-terminus,
respectively.
Fig. 3. Autolytic activity of p94 coupled to transcriptional activation by a Gal4–p94 hybrid protein.
A, Schematic overview of transcriptional activation by the Gal4–p94 hybrid protein. Two distinct
functional domains of Gal4, the DNA-binding domain (BD) and the transcription-activating domain (AD)were fused to the N-terminus and C-terminus of p94, respectively. A hybrid bearing the wild-type but not
the C129S mutant p94 failed to activate transcription because BD and AD are dissociated upon p94
autolysis. Arrows indicate the reported autolytic sites of p94.
B, Growth of yeast AH109 cells transformed with the expression vectors for hybrid proteins. Yeast cells
expressing Gal4–p94 hybrid proteins, BD–WT–AD, BD–C129S–AD, or BD–R490W–AD were examinedfor growth on selection medium. After 2 days on non-selective SD–W plate, expression of those hybrid
proteins did not affect the growth of yeast cells. On selective medium without histidine and adenine, SD–
WHA, the C129S mutant allowed growth of yeast cells by transcriptional activation of reporter genes, HI3
and ADE2. Yeast cells expressing wild-type hybrid protein did not grow under the same conditions. The
R490W mutation caused slow growth of yeast cells, an intermediate phenotype between wild-type and theC129S mutant (compare SD–WHA, 2day and 3day) .
C, Effect of mutations affecting autolytic activity on transcriptional activation by hybrid proteins.
Transcriptional activation by a hybrid protein with R490W or R527Q mutations was measured by liquid
culture β-galactosidase assay to evaluate the expression of another reporter gene, lacZ. Yeast cells
expressing C129S produced significant activity. Both R490W and R572Q mutations increased theefficiency of transcription of the reporter gene but did not exceed the efficiency of the C129S mutant. The
activity was normalized relative to the value for the C129S mutant protein. *P < 0.001 (n = 4).
Representative results from three independent assays are shown. As expected, there was almost no activity
in cells expressing hybrid protein with wild-type p94.
Fig. 4. Screening of p94 mutants defective in autolytic activity.
A, A scheme for screening of mutations that inactivate p94. (a and b) Domain II of the BD–p94:WT–AD
hybrid protein was exchanged with domain II fragments harboring random mutation(s). (c) The yeast
colonies viable on the selection medium SD–WHA were considered to express hybrid proteins with
mutation(s) inactivating p94 autolytic activity. (d) Plasmids for a hybrid protein were rescued from those
viable yeast colonies, and the introduced mutations were determined by DNA sequencing.
B, Schematic overview of missense mutations of p94 identified by the screening. The positions ofmutations identified by the screening are shown. Based on the definition as described in Table1, the lociare categorized into four (from the upper to the lower); highly conserved in the calpain super family,conserved among p94, µCL, and mCL, conserved among p94, and divergent. *Identical substitutions havebeen found in LGMD2A patients. **Identical loci have been subjected to point mutations in LGMD2A
patients.
Fig. 5. Effect of the connectin–p94 interaction on the autolytic instability of p94.
A, p94 binding regions in connectin. Clones CN48 and CN52 encode the N2A and M-line regions ofhuman connectin, respectively, and they were identified by conventional yeast two-hybrid screening.
mCN48 is the same region from mouse connectin. mCN48-mdm has a C-terminal truncation that
corresponds to a deletion in connectin produced from the mdm allele connectin gene. Bidirectional
arrowheads show the relative location in the sarcomere of a connectin molecule. The bracket indicates theregion deleted from N2A connectin by the mdm mutation. “I” and “M” together with the numbers
indicates the IgC2 motif defined in I-band region and M-line region connectin, respectively.
B, Effect of coexpressed connectin CN48 and CN52 fragments. Gal4–p94 hybrid proteins for wild type
(BD–WT–AD) or the C129S mutant (BD–C129S–AD), and BD domain fused to the C129S mutant (BD–C129S) or without any fused peptide (BD–), were coexpressed with connectin CN48 or CN52 fragments
using yeast expression vector p415GPD (+ CN48 or CN52). As the negative control, coexpression with an16

empty vector was included (+ mock). Coexpression with CN48 fragment, but not CN52 fragment, restoredtranscriptional activity of BD–WT–AD (SD–LWHA, BD–WT–AD). Hybrid constructs of p94:C129S
without the C-terminal AD domain (SD–LWHA, BD–C129S) or BD domain alone (SD–LWHA, BD–)
showed no transcriptional activity in the presence of either CN48 or CN52. Cells expressing the C129S
mutant hybrid protein were capable of grow on selection medium independent of coexpressed fragments(SD–LWHA, BD–C129S–AD). All the combination of coexpression resulted in growth on non-selective
medium comparable to each other (SD–LW).
C, Effect of C-terminal truncation in connectin CN48 fragment on p94 hybrid protein activity. Connectinfragments, mCN48 and mCN48-mdm, were expressed using the p425GPD yeast expression vector
together with Gal4-p94 hybrid protein as described in B. Coexpression of the mCN48-mdm fragment,
which has a C-terminal truncation, with wild type hybrid protein did not result in the growth of yeast cells,suggesting the fragment failed to resotre transcriptional activity of the p94 hybrid protein (SD–LWHA,
BD–p94–AD + mCN48-mdm).
Fig. 6. Potential mechanism for N2A connectin binding on p94.
A, A schematic of the autolytic processes of p94. (a) The three arrows and the bidirectional arrow indicate
the autolytic sites in the NS, IS1, and IS2 regions and the site for interaction with CN48, respectively. d.II,
d.III, and d.IV stand for domains II, III, and IV, respectively. In intermolecular autolysis (e.g., p94:C129S
cut by p94∆), NS is cut very fast (b), followed by cutting of IS1 (c), and finally the molecule dissociates
into two after IS2 is cut (d). In intramolecular autolysis (e.g., p94:N358D autolysis at low concentrations),
IS1 is cut very fast (e), followed by cutting of IS2 (f), which causes molecular dissociation. If a CN48
fragment exists, suppression of IS2 cutting (g) and/or dissociation after IS2 cutting (h) occurs, resulting inreporter gene expression if the N- and C-terminal ends of the p94 molecule are linked to BD and AD,
respectively.
B, Schematic 3D structures of the connectin–p94 interaction. The reported crystal structures of the humanm-calpain catalytic subunit (from 1DKV) and Z1–Z2 connectin IG domains (from 1YA5) were aligned inan equal scale to model p94 and I82–I83 IG domains in N2A connectin. The ribbon scheme shown was
drawn using MolFeat Ver.2.2 3D imaging software (FiatLux Inc., Tokyo, Japan). The sequence deleted by
the mdm mutation (indicated in red) composes the last two β-strands of I83 (Z2) structure and extends tothe adjacent PEVK region. The spatial size of the two IG domains drawn in this view substantiates their
impeditive effect against proteolysis in the IS2 region. Two IG domains are large enough to be an obstacle
to proteolysis in the IS2 region.

17

Table 1. Summary of mutations of p94 harbored in colonies that grew on SD–WHA plates
Bold italic: highly conserved residues in the calpain super family; Bold: conserved among p94, µCL, andmCL; italic: conserved among human, mouse, chicken, and fugu p94s; “quoted”: a residue position where
missense mutations are found in LGMD2A patients; underlined: a pathogenic missense mutation found in
LGMD2A patients. Most probable responsible mutation(s): estimation is based on the fact that a more
widely conserved residue is more crucial for protease activity, i.e., order of precedence is a bold italic,
bold, and italic residue included in the mutations found. If there was more than one residue of the samepriority, the underlined residue was chosen.
Mutation(s) identified
Corresponding
Most probable
Number of
domain(s)responsible
independent
mutation(s)clones identified
I121F/W373C*1/D403V/H407Y*1
IIa/IIb × 3
D403V
2
Q123H/”Y336”F
IIa/IIb
Q123H*2
1
A133T/G367S/S400Y
IIa/IIb × 2
G367S
1
L140P/M262V/W376C
IIa/IIb × 2
W376C
1
H151Y/W369R
IIa/IIb
W369R
1
T184M/W369R
IIa/IIb
T184M and/or
1
W369R
S202G/L387P
IIa/IIb
S202G and/or L387P
1
A227T/M294T*1
IIa/IS1
A227T
1
IIb × 2/linker
L261P
1
L261P/W373R*1/D424V*1
T314P*1/T324S/S337Y/L387P
IS1/IIb × 3
S337Y and/or L387P
1
G329R/T339M/L387Q
IIb × 3
G329R
1
W365R
IIb
W365R
1
W369R
IIb
W369R
8
S370R/R386C/I405V*3/D424G*1/N449D IIb × 3/linker/III
R386C*4
1*1
IIb/linker
L387P
1
L387P/D424G
23
Total*1: M294, T314, W373, H407, and D424 are divergent residues in the calpain family. M294/T314 andD424 locate, respectively, in the IS1 region and the linker region connecting domains II and III.
*2: Y336F was ignored because an identical substitution is found in human calpain 10.
*3: The I405V substitution is found in mouse p94.
*4: N449D was ignored because an identical substitution is found in two nematode calpains and plant
calpains.

18

Ono, Y. et al., Fig. 1

A

[1]

[2]

A15 A34
C129

p94

[3]

G275, S297, E323
H334 N358

IIa

NS

IS1 IIb

anti-pNS

R592?

III

IS2

anti-dIIIanti-pIS2ab

Binding to N2A connectin (CN48)+

C

p94:P601term
p94:K595term

V594

p94:S581term

L580

p94:V573term

R572

C
15' 60' 120' 240'

0'

+
+
+
-

K600

B
0'

anti-C-termcS

p94:C129S

IV

V573-L580

15'

CS
+
240' 240'

∆
0'

0'

CS + ∆+
15' 60' 120' 240'Ca2+

60' 120' 240'

(kDa)
97
66
43

CBB
30

CBB

anti-pIS2

anti-pIS2

D
1.0
Ratio to time=0'

native
0.8
0.6
0.4

SDS

0.2
0

50

100
150
200
Incubation time (min)

250

mock

W

A

T
p9
4:
e
CS x6 -

Ono, Y. et al., Fig. 2

kDa
94
67
55 K

43

32 K

30

kDa

mock

W

B

Tm
p9 ycH
4:
ex is
6
p9
4: -myexc
6 - -Hi
:C
s
Sm
yc
-H

is

anti-pIS2

94
*
*
*
*

67
43

58 K

*

35 K

IP: anti-myc
WB: anti-pIS2

30

C
0

1

5

10 15 60

0

1

5

10 15 60

0

1

5

10 15 60

0

1

5

10 15 60

(kDa)
94

b

67
43

p94:N358D
full-length

b

b

a

IS1-C-termcN-term-IS1,
IS2-C-term

30

anti-pNS

anti-dIIIanti-pIS2

anti-C-termOno, Y. et al., Fig. 3

A

p94:WTCys

His Asn

BD

dII

NS

IS1

Ser

dIII

dIV

AD

IS2

BD

AD
no transcription

His AsnBD

ADtranscriptional activation of reporter genes:
no autolysis

p94:C129S

B

autolysis

SD-W
2day

SD-WHA
2day
BD-WT-AD

BD-C129S-AD

BD-R490W-AD

C

100

Arbitrary Unit

100

39*

50

18*
0

0.96
WT R490W R572Q C129S

BD-p94-AD

3day

Ono, Y. et al., Fig. 4

A

(a)
Cys

(b)

p94:WT

PshAI
Cys

His AsnBD

AD

His AsnAD

p94:dIIw/ mutation(s)(c)
His Asn

p94:WT

pAS2-1C
restriction enz. digestion
BD

co-TF
Cys

His Asn

BD

PCR
Cys

Tth111I

pAS2-1C

in vivohomologous
recombination

BD

AD

(d)

AD

p94 w/ mutation(s) in dII
Cys

His AsnBD

pAS2-1C
yeast AH109 viable
on SD-WHA

pAS2-1C

plasmid
rescueB

N449D**

L387P/Q

S400Y
D403V

III

S370R
W373C/R
R386C*

T339M

T324S
M294T
T314P

M262V

A227T
T184M*

L140P
H151Y

Conserved among p94s
Divergent

IIb

L261P

S202G

I121F

Highly concervedin the calpain super family
Conserved among
p94, µCL and mCL

IS1

G329R*Y336F**
S337Y
W365R
G367S*
W369R
W376C

IIa

Q123H
A133T

NS

His334 Asn358

233

I405V
H407Y
D424G/V

Cys129Ono, Y. et al., Fig. 5A

Z-lineN2A

N-

M-line-C

PEVK
mdm deletion

is

I81

I82

I83

M7

PEVK

is6

p94 binding
+

CN48
mCN48
mCN48-mdm

+
-

M8

M9

is7

M10

p94 binding+

CN52
IG motif

B
BD-WT-AD

BD-C129S-AD

BD-C129S

BD-

SD-LWHA

BD-WT-AD

SD-LW

SD-LWHA

mock
CN48
CN52

SD-LW

C

+

BD-C129S-AD+

mock
mCN48-mdm
mCN48

Ono, Y. et al., Fig. 6

A

(b)

(c)

IS1

NH2

NS

d.IV

IS1
d.II

NS
NH2

d.III

CN48

HOOC
d.IV

IS2

intramolecular
IS1 cut(very rapid)

NS
d.III

HOOC
d.IV

IS2

(e)

(f)

IS1
d.II

d.III
HO

OC

NS
NH2

IS
2

IS1
d.II

NS
NH2

d.III

HOOC
IS2

d.III

IS2 cut(slow)
anddissociation

suppression+CN48
of IS2 cut
and/ordissociation

HOOC
d.IV

(g)

IS2

(h)

IS1
d.II

NS
NH2

and/
or
CN48

d.IV

IS1
d.II

d.III

HOOC

B

IS2

IS2 cut(slow)
anddissociation

d.I
V

d.IV

p94

d.II

NS
IS1 cut(rapid)HOOC

intermolecular
NS cut(very rapid)

IS1

d.II

d.II

d.III

(a)

(d)

IS1

NS
NH2

d.III

HOOC

CN48

d.IV

IS2

IS2

mdm deleted
COOH

NH2

interaction
Connectin/titin biding site
IS2 inserted

IS1 inserted

COOH

Domain IV

Domain IIb

NH2

NS attached
Domain III

Active site
Domain IIa