Stomach-specific Calpain, nCL-2/calpain 8, Is Active Without Calpain
Regulatory Subunit, and Oligomerizes Through C2-like Domains*
Shoji Hata1, Naoko Doi1,2, Fujiko Kitamura1, Hiroyuki Sorimachi1,21Department of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan

Institute of Medical Science (Rinshoken), Tokyo 113-8613, 2CREST, Japan Science and Technology
Agency (JST), Saitama 332-0012, Japan
Running title: Stomach-specific calpain oligomerizes via C2-like Domains
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; FAX: +81-3-3823-2359; E-mail: sorimach@rinshoken.or.jp.
ofanalysis revealed that nCL-2 exists in both

intracellular Ca -regulated cysteine proteases

monomeric and homo-oligomeric forms, butthat are indispensable in the regulation of a

not as a heterodimer with 30K or 30K-2, and

wide

that

Calpains

constituteafamily2+varietyofcellularfunctions.

The

the

oligomerization

occursthrough

improper activation of calpain causes lethality

domains other than the 5EF-hand domain IV,or various disorders, such as muscularmost probably through domain III, suggesting

dystrophies

a novel regulatory system for nCL-2.

and

tumor

formation.

nCL-2/calpain 8 is predominantly expressed in
the stomach, where it appears to be involved in

Calpain (Clan CA-C2, EC 3.4.22.17) is anmembrane trafficking in the gastric surface

intracellular Ca2+-regulated cysteine protease,mucus cells (pit cells). Although the primary

comprising a superfamily with members in almost

structure of nCL-2 is quite similar to that of the

all eukaryotes and some bacteria (1-3). Calpains

ubiquitoustheregulate a wide variety of cellular functions

enzymatic properties of nCL-2 have never been

including the cell cycle, signal transduction,

reported. Here, to characterize nCL-2, theapoptosis, and membrane trafficking, through therecombinant protein was prepared using an E.

limited proteolysis of their substrates (1,2,4,5).

coli

to

Dysregulation of calpain activity and/or defective

homogeneity. nCL-2 was stably produced as amutations in calpain genes cause lethality (6-8) or

solublethea variety of pathological phenotypes, whichconventional calpain regulatory subunit (30K).

include muscular dystrophies in mammals (9,10),

Ca2+-dependent

degeneration in the developing optic lobes of

m-calpain

expression
andPurifiedlargesystemactivenCL-2

subunit,andenzymeshowedpurifiedwithout

drosophila(11),mM Ca , similar to that of m-calpain, whereas

conditions

in

its

were

masculinization of nematode hermaphrodites (14),Immunoprecipitationand defective aleurone cell development in maize

activity, with half-maximal activity at about 0.3
2+

optimalcomparatively

pH

and

low.

temperature1

inadaptability
fungi

and

to

alkaline

yeasts(12,13),

(15). These observations clearly indicate calpain isshow full activity in the absence of 30K if

indispensable

thecorrectly folded (24), and cells from 30K

specific physiological functions of calpains andgene-deficient mice, which die at E10.5-11.5,

the molecular mechanisms underlying theirshow neither detectable μ- or m-calpain activity

functions remain unclear.

nor detectable μCL or mCL protein, even thoughphysiologically;

however,

The mammalian calpain family comprises

their mRNAs are expressed normally (7,8). These

products from 14 independent genes, and can be

results indicated that 30K functions as a molecularclassified into typical and atypical members

chaperon in vivo, at least for μCL and mCL.
nCL-2/calpain

according to their domain structures (2,3). The

8,

identified

asaubiquitous μ- and m-calpains, well-characterizedstomach-specific calpain, is a typical calpain withtypical calpains in mammals, are hetero-dimers

four domains (25) (see Fig. 3B). nCL-2 is mostcomposed of a distinct 80-kDa large catalytic

similar to mCL (amino acid identities in thesubunit (abbreviated here to μCL and mCL,

full-length protein and in domains II and IV are

respectively; also known as calpain 1 and 2) and a

61.5%, 73.4%, and 51.8%, respectively). Possible

common 30-kDa small regulatory subunit (30K).

functions for nCL-2 in the pituitary gland and in

The large and small subunits contain four (I–IV)

the embryogenesis of Xenopus laevis have been

and two (V and VI) domains, respectively: thereported (26,27). We recently showed that, in theregulatory N-terminal domain (I), the proteasestomach, nCL-2 as well as nCL-4/calpain 9, a

domaindigestive tract-specific calpain, are expressed

(II),

the

2+

Ca /phospholipid-binding

C2-domain-likedomain(III),

thespecifically in the mucus-secreting pit cells, quite

penta-EF-hand (PEF) domains (IV and VI), which

unlike the μ- and m-calpains, which are expressed

are missing in atypical calpains, and the

diffusely in all cells, and that nCL-2 may be

Gly-clustering hydrophobic domain (V). The

involved in the regulation of vesicle trafficking in

C-terminal fifth EF-hand motifs in domains IV

the gastric surface mucus cells (pit cells) (28).

2+and VI cannot bind Ca , and interact with each

These reports highlight potentially importantother to form the hetero-dimer. Domain IV is also

nCL-2 functions that cannot be compensated forreported to function in the recognition ofby μ- and m-calpains, not only in the stomach but

substrates and regulatory molecules (16-18).

also in other tissues.

2+

In the absence of Ca , the protease domain

However, the enzymatic properties of(II) is separated into two subdomains, IIa and IIb,

nCL-2, including its regulatory mechanisms, haveand residues in the catalytic triad are prevented

not been reported, except for one yeast two-hybrid

from assuming the correct positions for hydrolysis.

analysis suggesting that nCL-2 does not interact2+

to domains IIa, IIb, III, IV,with 30K despite the high similarity between

and VI induces conformational changes that allow

nCL-2 and mCL (29). Thus, in this study, using E.

domain II to form a single active domain (19-23).

coli–expressed and purified nCL-2, the detailed

Previous analyses revealed that μCL and mCL

biochemical properties of this enzyme were

The binding of Ca2examined, and several unique characteristics were

subcloned into appropriate vectors. For bacterial

revealed. Unexpectedly, nCL-2 was found to exist

expression, the amplified mouse nCL-2 cDNA

as both a monomer and as homo-oligomers, andwas ligated into the pCold I vector (TaKaRa) tothe domain responsible for oligomerization was,produce proteins fused with an N-terminal His-tag.

unlike in the μ- and m-calpains, the C2-like

For COS7 cell expression, the amplified cDNAs

domain (III), not the PEF domain (IV), suggesting

were ligated into a modified pSRD vector (31) tothat nCL-2 has a novel regulatory mechanism.

produce proteins fused with an N-terminal FLAG,
HA, or myc-tag. The nCL-2 deletion mutants wereconstructed by introducing termination codons atExperimental Procedures

the appropriate positions using a PCR-mediatedMaterials–

Recombinanthuman

m-calpains,anti-nCL2

polyclonal

and

μ-

site-directed mutagenesis method as previously
described (10).

antibody(previously called anti-nCL2/2’), and mouseProtein expression in bacterial cells

gastric mucosal protein were prepared as

purification– The constructed pCold I expression

described previously (28,30). RP2-calpain 8

plasmids

polyclonal antibody, anti-FLAG (clone M2),

BL21/pG-Tf2 (TaKaRa), and the transformants

anti-HA (clone 6E2), and anti-His monoclonal

were cultured at 37°C in 3 L of LB medium

antibodies were purchased from Triple Point

containing 0.1 mg/ml ampicillin until A600 = 0.6.

Biologics Inc. (OR, USA), Sigma (MO, USA),

Protein expression was induced by the addition of

Cell Signaling Technology Inc. (MA, USA), and

0.5 mM isopropyl-thiogalactoside and 5 ng/ml

Novagen Inc. (WI, USA), respectively. Proteasetetracycline (tet) for 24 h at 15°C. Harvested cells

inhibitors

I,

were washed once with phosphate-buffered saline,

calpastatin domain I, E64c, AEBSF, PMSF, TLCK,

and resuspended in 150 ml of lysis buffer (20 mM

pepstatin A) were purchased from Sigma, TaKaRa

Tris-Cl (pH 7.5), 150 mM NaCl, 5 mM imidazole,(Kyoto, Japan), and Peptide Institute Inc. (Osaka,and 1.4 mM 2-mercaptoethanol (2-ME)), andJapan).

pep42

lysed with a French Press (American Instruments(NH2-EVSDPMSSTYIEELGKREVTIPPKYREL

Inc., MA, USA). The cell lysate was spun at

LAKPIGPDDAIDAL-COOH), corresponding to

55,000 × g for 20 min, and the recovered

one of reactive sites of human calpastatin, wassupernatant was filtered through a 0.22-µm poresynthesized by Research Genetics (CA, USA).

filter (Millipore, MA, USA). The supernatant was

Cloning and construction of expression plasmids–

then applied by gravity to a 2-ml Ni2+-chelating

cDNAs encoding full-length mouse nCL-2,

agarose column (Qiagen, MD, USA) equilibrated

human and mouse 30K, and human and mousewith lysis buffer. The column was washed with 10

30K-2/CAPNS2 were amplified by PCR using Pfu

column volumes of lysis buffer, and then six

turbo DNA polymerase (Stratagene Inc., CA,

column volumes of wash buffer (20 mM Tris-ClUSA), and the sequences were verified after being(pH 7.5), 150 mM NaCl, 50 mM Imidazole, and(calpeptin,

calpain

CSTN

inhibitor

3

weretransformed

into

E.

and
coli

1.4 mM 2-ME). Protein was then eluted with 8 ml

recombinant nCL-2 was incubated in 10 μl of theof elution buffer (20 mM Tris-Cl, (pH 7.5), 150

reaction buffer with or without 10 mM CaCl2 at 25mM NaCl, 250 mM Imidazole, and 1.4 mM

or 37°C for the times indicated.

2-ME). The eluate was immediately dialyzed

Proteinagainst buffer A (20 mM Tris-Cl (pH 7.5), 1 mM

Immunoprecipitation–oexpressioninculture
COS7

cells
cells

and
were

EDTA, and 1 mM dithiothreitol (DTT)) at 4 C.

transfected with 5 μg of the expression plasmids

The dialyzed sample was further purified by a

by electroporation, as previously described (31).

MonoQ HR10/10 anion-exchange column (GEFor immunoprecipitation, the COS7 cells wereHealthcare, Tokyo, Japan) with a linear gradient of

harvested 48 h after transfection and lysed with

0–0.5 M NaCl in buffer A. For gel filtration, thelysis buffer (20 mM Tris-Cl (pH 7.5), 150 mM

sample was applied to a HiLoad 16/10 Superdex

NaCl, 5 mM EDTA, 1% TritonX-100, 0.2 mM

200 column (GE Healthcare) equilibrated with

PMSF, 10 μg/ml aprotinin, and 10 μM pepstatinbuffer A containing 150 mM NaCl. The purity of

A). The lysed cells were spun at 100,000 × g forthe protein was examined by SDS-PAGE analysis.

20 min and incubated with a 50% slurry of FLAG

The peak fraction was stored at 4°C until use.

(M2) agarose (Sigma) for 2 h, then theProteolysis assay– Proteolytic activity wasprotein-bound agarose was washed five times withmeasured

substrate,

1 ml of the lysis buffer. The immunoprecipitatedsuccinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-

proteins were eluted with 0.1 mg/ml FLAG

amide (Suc-LLVY-MCA) (Peptide Institute Inc.).

peptide (Sigma) and mixed with SDS sample

Recombinant nCL-2 (2 μg), μ-calpain (0.1 μg), or

buffer. For the in vitro proteolysis assay, 10 μl of

m-calpain (0.4 μg) was incubated with 0.1 mM

the eluted immunoprecipitated proteins were

Suc-LLVY-MCA in reaction buffer (0.1 M Tris-Cl

incubated with 2 μl of 6 × Ca-buffer (20 mM(pH7.5),

Tris-Cl (pH 7.5), 100 mM CaCl2, and 6 mM DTT)

using

20

a

mM

fluorogenic

2-ME

and

0.2%

3-[(3-cholamidopropyl)dimethyl-or 2 μl of 6 × EDTA-buffer (20 mM Tris-Cl (pHammonio]propanesulfonic acid (CHAPS)), with7.5), 100 mM EDTA, and 6 mM DTT) at 37°C.

2+

varying Ca concentrations, temperatures, or pH.

The reactions were stopped by the addition of 5 ×

For pHs other than 7.5, Tris-acetate (pH5.5, 6.0, or

SDS sample buffer.

6.8) or Tris-Cl (pH7.0, 7.3, 8.0, 9.0, or 9.5) buffer

Protein sequencing – After SDS–PAGE, thewas used instead of Tris-Cl (pH7.5) buffer.

proteins were blotted onto a Pro-Blot membrane

Reactions in 40 μl were stopped by the addition of(Applied Biosystems, Tokyo, Japan). Target

40 μl of 10% SDS and 1.2 ml of 0.1 M Tris-Cl (pHprotein bands on the membrane were visualized

9.0). MCA release by proteolysis was monitored

by Coomassie Brilliant Blue (CBB) G-250

by(RF-1500,

staining, excised, and washed three times with

Shimadzu, Osaka, Japan), with excitation and

0.1% (v/v) trifluoroacetic acid in 50% (v/v)

emission wavelengths at 380 nm and 460 nm,

methanol and then with absolute methanol.

respectively. For the autolysis assay, 1 μg ofSequencing was performed using the ABI 491cLCaspectrofluorophotometer4

proteinsequencer(Applied

Biosystems),

Suc-LLVY-MCA,

according to the manufacturer’s instructions.

was

used

to

detecttheproteolytic activities. The activity of nCL-2 was
Ca2+ dependent, and was blocked by known
inhibitors of μ- and m-calpains but not by other

RESULTSinhibitors (Figs. 2A & B), demonstrating that
nCL-2

Preparation of recombinant mouse nCL-2

possesses

characteristics

typicalofusing an E. coli expression system. To investigate

conventional calpains. The concentration of Ca2+

the solubility and expression efficiency of nCL-2,

required for half-maximal activity was about 0.3

full-length mouse nCL-2 was expressed with or

mM, almost identical to that of m-calpain (Fig.

without 30K in E. coli BL21(DE3). nCL-2 was2A). The calculated specific activity was a 40thabundantly expressed in both cases, and noand a 25th of that of μ- and m-calpain, respectively.

significant difference was observed in its amountThe specific activity of the caseinolysis of nCL-2

or solubility (data not shown). Therefore, nCL-2

was also lower than those of μ- and m-calpain

was expressed without 30K for the purpose of(data not shown).

purification. To improve the solubility of the

To exclude a possibility that the proteolyticnCL-2 in E. coli, a tet-on chaperon-inducible

activity observed is due to host protease(s)BL21/pG-Tf2 strain was used, and active nCL-2

contaminating in the final nCL-2 fraction, thewas successfully recovered in a soluble fraction

Suc-LLVY-MCA assay was performed for thethat represented one-third to one-half of the totallysates of BL21/pG-Tf2 cells transformed with

nCL-2 (Fig.1A, lanes 1 and 2).

mock (pCold empty vector), or expression vectors
for inactive nCL-2 (nCL-2:C105S, in which the

Purification of recombinant nCL-2. As
described

inExperimental

active site Cys-105 was mutated to Ser), or WT

Procedures,

N-terminally His-tagged recombinant nCL-2 wasnCL-2.

expressed and purified to homogeneity by

nCL-2:C105S-transformed cell lysates showed2+

Ni -chelating

agarose

and

As

a

result,

the

mock-

and

MonoQ

virtually no activity (0.25 ± 0.01 and 1.97 ± 0.02

anion-exchange column chromatographies influorescence units (FU), respectively) in the

succession (Fig. 1A, lanes 3 and 4). The peak

presence of Ca2+, whereas WT nCL-2-transformed

elution of nCL-2 from the MonoQ column was atcell lysate showed >200 times their activities

around 0.2 M NaCl. Approximately 1 mg of(403.07 ± 0.81 FU) (Fig. 1B). Furthermore, “mockrecombinant nCL-2 was purified from 3 L of E.

purification” was also performed using thecoli culture, and subjected to further analyses.

mock-transformed cell lysate (Fig. 1C, S2) in the

Enzymatic properties of recombinant nCL-2.

same manner as that for WT nCL-2 (Fig. 1C, S1).

Using the nCL-2 prepared above, the enzymatic

As a result, the final Mono Q fractions (Fig. 1C,

characterization of recombinant nCL-2 waslanes E1–E3) around 0.2 M NaCl, where WT

performed, in comparison with recombinantnCL-2 was mainly eluted, showed no activity.

human μ- and m-calpains. A fluorogenic substrate,Thus, the possibility of host protease activities5was excluded.

anti-nCL2 antibody, respectively. This indicates

The optimal temperature for nCL-2 activity

that the 63-kDa and 41-kDa fragments extend

was 20°C (Fig. 2C), which was similar to that forfrom Ala-6 to the second EF-hand motif (EF-2) in

μ- and m-calpain under our assay conditions, anddomain IV and to the beginning of domain III,to that reported previously for nCL-4 (32,33). The

respectively (Fig. 3C, b and f). Autolytic

time courses of nCL-2 activity at various

fragments of 45 kDa and 44 kDa (Figs. 3A & 3B,

temperatures showed that the initial rates werearrowheads c and d) reacted with the anti-nCL2

maximal at 30 to 37°C, while maximal activity

and anti-His, but not the RP-calpain 8 antibodies,

was achieved at 15°C (Fig. 2D). The low optimal

indicating that these fragments extend from thetemperature for nCL-2 indicates that it is unstable

N-terminus to the acidic loop in domain III and toat higher temperatures in the presence of Ca2+. The

the beginning of domain III, respectively (Fig. 3C,

optimal pH for nCL-2 activity was 6.0, which isc and d).

lower than the optimal pHs for conventional

In summary, the autolytic sites of nCL-2

calpains (Fig. 2E).

include the N-terminus, the N-terminal parts ofAutolytic properties of nCL-2. The autolysis

domain III, and EF-2 in domain IV.

of nCL-2 was examined by SDS-PAGE analysis,nCL-2

existsasamonomerandas shown in Fig. 3A. Various breakdown products

homo-oligomer. The finding that 30K was notappeared in a time-dependent manner, and wererequired for nCL-2 activity prompted us toconfirmed to be autolytic fragments of nCL-2 byinvestigate the tertiary structure of nCL-2. To

western blot analysis (Fig. 3B). By N-terminal

examine whether nCL-2 exists as a monomer orsequencing of a 43-kDa fragment (Fig. 3A,

homo-oligomer, the purified nCL-2 was subjected

arrowhead e), one of the autolysis sites wasto gel filtration column chromatography. As

determined to be between Ala-5 and Ala-6. Givenshown in Fig. 4A, nCL-2 was eluted with peaks at

its molecular weight, the C-terminus of thispositions corresponding to about 80 kDa and 160

fragment was in or near the acidic loop in domainkDa. This result indicates that nCL-2 is present as

III (Fig. 3C, e).

both a monomer and homo-oligomers, and that the

The autolytic fragment just below the 81-kDa

monomer and homodimer forms predominate.

full-length band (Figs. 3A & 3B, arrowhead a)

Suc-LLVY-MCA hydrolyzing activities of bothreacted with the anti-nCL2 but not the anti-His

fractions were measured, and the calculated

antibody. Based on this and its molecular weightspecific activity of the peak fraction at about 160(ca. 79 kDa), this autolytic fragment probably

kDa was about 1/15 of that at about 80 kDa.

spanned from Ala-6 to the C-terminus (Fig. 3C, a).

To confirm the homo-oligomerization ofAutolytic fragments of 63 kDa (Figs. 3A & 3B,

nCL-2,

immunoprecipitation

analysiswasarrowhead b) and 41 kDa (arrowhead f) reacted

performed. Full-length mouse nCL-2 fused with

with

8

an N-terminal FLAG, HA, or myc-tag were(anti-domain III) antibodies, and with only theco-expressed in COS7 cells; the cell lysate wastheanti-nCL2

andRP2-calpain

6

then

full-length nCL-2 or its ΔEF5 mutant, the ΔEF5

appropriate

mutant was co-precipitated with the ΔEF5 mutant

antibodies. The m-calpain subunits, FLAG-30K

as well as with full-length nCL-2 (Fig. 4F, lanesand HA-mCL, were used as controls. As shown in

6–8; Table 1), indicating that EF-5 is not required

Fig. 4C, HA-mCL was co-precipitated withfor the homo-oligomerization of nCL-2.

spun

in

an

ultracentrifuge

immunoprecipitated

using

and

the

FLAG-30K (lane 2), whereas HA-nCL-2 was not

Therefore, to identify the novel domain(s)(lane 5), consistent with the yeast two-hybrid

critical for the homo-oligomer formation, domain

results described above. These results indicatedIV- and domain III/IV-truncated mutants (Fig. 4B,

that this method was applicable for detecting theΔdIV and ΔdIII+IV, respectively) were tested. The

protein interactions of interest.

ΔdIV mutant was co-precipitated with the ΔdIV

When FLAG-nCL-2 and HA-nCL-2 weremutant (Fig. 5A, lane 3) as well as with full-length

co-expressed in COS7 cells, HA-nCL-2 wasnCL-2 (Fig. 5B, lanes 3 and 8; Table 1). On the

co-precipitated with FLAG-nCL-2 (Fig. 4D, lanes

other hand, the ΔdIII+IV mutant showed almost

1–3). To confirm this interaction, the combinationno interaction with the ΔdIII+IV mutant or withof FLAG-nCL-2 and myc-nCL-2 was also tested,full-length nCL-2 (Fig. 5A, lane 6; 5B, lanes 5 andand yielded consistent results (Fig. 4D, lanes 4 and

10; Table 1). Consistent with these results,5). Furthermore, to exclude a possibility that

HA-domain IV of nCL-2 (Fig. 4B, dIV) was

observed co-precipitation is an artifact due toco-precipitated with neither FLAG-full-length

overexpression, FLAG-nCL-2 and HA-nCL-2

nCL-2 nor FLAG-dIV of nCL-2 (Fig. 5C, lanes 3

were co-expressed in COS7 cells at very low

and 5; Table 1). These results indicate that domain

levels, even lower than the physiological levels

IV is dispensable and domain III is necessary for(Fig. 4E, anti-nCL2, lanes 1 vs 2 and 3). Even

the oligomer formation of nCL-2.

under

this

condition,

co-precipitationwas

To confirm the involvement of domain III in

observed (Fig. 4E, IP: FLAG, anti-HA, lane 3).

nCL-2 oligomerization, FLAG-domain III ofthrough

nCL-2 (Fig. 4B, dIII) was co-expressed with

Domain IV but through Domain III. To investigate

HA-full-length nCL-2 or HA-dIII of nCL-2. The

which domain is required for the homo-oligomerdIII expression was very faint (Fig. 5C, lysate,

formation of nCL-2, several truncation mutants oflanes 6 and 7), probably because domain III alone

nCL-2

nCL-2

oligomerization

(Fig.

4B)

not

in

the

is unstable in nature, as previously reported for rat

Since

the

mCL domain III (34). As shown in Fig. 5C, lanes 8

C-terminal fifth EF-hand motif (EF-5) in domains

and 10, FLAG-full-length nCL-2 and FLAG-dIII

IV and VI is critical for the heterodimer formation

co-precipitated HA-dIII, although the signal wasof μ- and m-calpains, we first tested whether the

not as clear as in the co-precipitation experiments

EF-5 of nCL-2 was necessary for oligomerization.

using full-length nCL-2 or the ΔdIV mutants (Figs.

When a tagged EF-5 deletion mutant (Fig. 4B,

5A & B; Table 1). These results strongly suggest

FLAG- or HA-ΔEF5) was co-expressed withthat nCL-2 oligomerizes through domain III, andimmunoprecipitation

were

is

used

experiment.

7

that domain I and/or II may also be required forexpressed in combination with HA-nCL-2 or

the proper conformation for binding (Table 1).

HA-mCL, HA-mCL but not HA-nCL-2 was

Effect of oligomerization on nCL-2 activity. To

co-precipitated with FLAG-30K-2 (Fig. 7, lanes 3

examine the effect of homo-oligomer formation

and 6). The interaction of 30K-2 and mCL wason nCL-2 autolysis, HA-nCL-2 was co-expressed

weaker than that of 30K and mCL, consistent with

with FLAG-nCL-2, immunoprecipitated using an

previously reported results (35).

anti-FLAG antibody, and incubated in the absenceor presence of Ca2+, followed by western blot

DISCUSSION

analysis using anti-FLAG or anti-HA antibodies.
As shown in Fig. 6A, both FLAG-nCL-2 andIn this study, we purified E. coli-expressed

HA-nCL-2 showed Ca2+-dependent autolysis inrecombinant stomach-specific calpain, nCL-2,

the same manner.

and demonstrated that it can be present as a2+

Next, to investigate whether or not Ca

monomer or form a homo-oligomer throughinduces dissociation of the nCL-2 homo-oligomer,

domain III. This is the first description of thethe HA- and FLAG-nCL-2 oligomers were boundenzymatic and biochemical properties of nCL-2.

to anti-FLAG agarose, incubated with or without

As expected from the high sequence similarity

2+

Ca , washed, and eluted with FLAG-peptide to

between nCL-2 and mCL, several enzymatic

see if HA-nCL-2 was associated with orproperties of nCL-2 resembled those of m-calpain,

dissociated from the bound FLAG-nCL-2 protein.

such as the Ca2+-dependency and the inhibitor

In this experiment, to avoid the autolysis of nCL-2

profile of its enzyme activity. However, several

2+

inactive

other properties were unique to nCL-2, which

nCL-2:C105S (nCL-2CS) was used. As shown inmight reflect its specific expression and functions

Fig. 6B, lanes 3 and 6, HA-nCL-2CS was detectedin the stomach that cannot be compensated for byat the same level in the absence or presence ofthe μ- and m-calpains. First, nCL-2 showed a low

duringincubation

Ca ,

with

2+

Ca . These results indicate that the nCL-2

optimal temperature in a long incubation assay,

homo-oligomer

which was similar to that of nCL-4 (33) but

possesses

protease

activity

without dissociating in the presence of Ca2+.

different from that of p94Δ (37). Second, theNo interaction of nCL-2 with 30K-2. Recently,

optimal pH for nCL-2 activity was lower than that

30K-2/CAPNS2, a novel small subunit that sharesfor the μ- and m-calpains. These properties

about 70% amino acid identity with 30K, wassuggest that

identified, and E. coli-expressed 30K-2 wasstomach-specific conditions. Alternatively, theseshown to form a hetero-dimer with mCL that had

findings may suggest the existence of certain

enzymatic

as-yet unidentified stabilizing factor(s) specificproperties

similartom-calpain(mCL+30K) (35,36). Therefore, it was possible

nCL-2

evolved

to

adapt

to

for nCL-2 in the stomach.

that 30K-2 interacts with and functions as a

The specific activity of nCL-2 was much

regulator of nCL-2. When FLAG-30K-2 waslower than the specific activities of the μ- and8m-calpains. The strong autolytic activity (see Fig.

the stomach. Considering that PEF domain of

3A), however, indicated that the low specificmCL is more similar to nCL-2 in its primaryactivity was not due to partial denaturation of thesequence than to that of nCL-4, but that nCL-4,

purified recombinant nCL-2. Moreover, thebut not nCL-2, forms a hetero-dimer with 30K, thenCL-2 autolysis was very rapid and extensive; i.e.,

dimer formation between PEF domains appears tofull-length nCL-2 decayed with a t1/2 of less thanbe governed by complicated structural properties

10 sec at 25°C, and it was extensively degradedthat cannot be predicted from the primary

within 3 min (see Fig. 3A). These properties aresequence.

distinct from the μ- and m-calpains and from

Importantly, nCL-2 exists not only as anCL-4+30K (33,38,39), but similar to p94,

monomer, but also as a homo-oligomer, and thealthough p94 apparently does not require Ca2+ for

oligomerization is most likely through domain III.

autolysis (31). Taken together, these findings

The suppressive effect of oligomerization on

suggest that 30K suppresses the autolysis ofnCL-2 activity is possible to be related tocalpains with which it forms a hetero-dimer, andregulation mechanisms of nCL-2 activity. In othermay indicate that nCL-2 and p94 need to bewords, as 30K and 30K-2 act as activity

self-down-regulated very rapidly after theirsuppressors for conventional calpains and nCL-4,

activation, probably to play specific roles inoligomerization of nCL-2 may function for thehighly differentiated cells like pit cells and muscle

same purpose without 30K or 30K-2. This

cells, respectively.

property has never been reported for any kind of

Alternatively, the low specific activity of

calpain

under

any

experimental

condition.

nCL-2 for Suc-LLVY-MCA and casein, despite its

However, although the oligomerization wasstrong autolytic activity, may indicate that theobserved even in cells exogenously expressing

substrate specificity of nCL-2 is rather different

nCL-2 near the physiological levels, further

from that of the μ- and m-calpains. In this regard,

analysis is required to conclude whether or not

the identification of in vivo substrates for nCL-2,

native nCL-2 forms oligomers in the stomach.

including the candidate molecule β-COP (28), is aThevery important and emerging future issue.

homo-oligomer

formation

throughdomain III may be supported by the fact that theIt was shown that nCL-2 is not co-precipitated

3D structure of domain III resembles tumor

with 30K or 30K-2, and that it is stable and has

necrosis factor α (TNFα) (20), which forms afull proteolytic activity without 30K, strongly

homotrimer

suggesting that no interaction occurs between

C2-domain has not been reported.

nCL-2 and 30K or 30K-2 in vivo. As mentioned

Very

(40),
recently,

although
it

was

the

trimerizing

reported

that

above, although it cannot be excluded that aminicalpains 1, 2, and 9 (domain II) in theregulatory subunit(s) other than 30K and 30K-2

presence of Ca2+ are structurally different despite

may exist, these results strongly suggest that

their highly conserved sequences, suggesting thatnCL-2 functions without a regulatory subunit intypical calpains have rather divergent mechanisms
9for their activation and regulation (41). It is

molecule could bind directly to scaffold proteins

possible that there are structural modifications

at the membrane, while another nCL-2 in thespecific to nCL-2 oligomerization involving

oligomer, could appropriately proteolyze its

inter/intra-domain interactions among domains I,

substrates at the membrane, such as β-COP. One

II, and III of nCL-2.

of the most important issues to be resolved is

Our recent study highlighted a role of nCL-2

whether the monomer-oligomer transition isin membrane trafficking via its location at theconstitutive or inducible in vivo, and, if inducible,

Golgi and interaction with a coatomer subunit ofwhen it is induced. The answers to such questions

vesicles derived from the Golgi (28). This

will shed light on the physiological functions offunction implies a possible physiological function

nCL-2 and are emerging future issues.

of the nCL-2 oligomerization: one nCL-2
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FOOTNOTES*We would like to thank Hiroyuki Kawahara (Hokkaido University), Noriko Toyama-Sorimachi(International Medical Center of Japan), Shoichi Ishiura, Tatsuya Maeda, Keiko Abe (The University ofTokyo), Koichi Suzuki (Toray Inc.), and all of our laboratory members for experimental support andvaluable discussion. This work was supported in part by MEXT.KAKENHI 17780115 (to S.H.),
17028055, and 18076007 (to H.S.), by JSPS.KAKENHI 18380085 (to H.S.), by a Sasagawa Scientific
Research Grant from The Japan Science Society (to S.H.), by a Research Grant (14B-4) for Nervous andMental Disorders from the Ministry of Health, Labor and Welfare, and by a Takeda Science Foundation
Research Grant (to H.S.).
The abbreviations used are: nCL-2, novel calpain large subunit-2; nCL-4, novel calpain large subunit-4;
SDS, sodium dodecyl sulfate; WT, wild type.

12

FIGURE LEGENDS
Fig. 1. Purification of recombinant mouse nCL-2. (A) SDS-PAGE analysis. nCL-2 was expressedin E. coli and purified on Ni2+-chelating and MonoQ columns. Samples were separated by SDS-PAGE andstained by CBB. nCL-2 bands are indicated by arrowheads. Lanes: 1, total lysate of nCL-2-expressing E.
coli BL21/pG-Tf2 cells; 2, its supernatant after centrifugation; 3, an nCL-2 fraction (5 μg) eluted from theNi2+-chelating column; 4, the final nCL-2 fraction (1.5 μg) after MonoQ column chromatography. An
asterisk indicates a host protein still included in the final nCL-2 fraction. (B) Suc-LLVY-MCA
hydrolyzing activities of total lysate supernatants (100 μg) of BL21/pG-Tf2 cells transformed with empty
pCold vector (mock), or expression vector for inactive nCL-2:C105S (nCL-2CS) or WT nCL-2 (nCL-2).
Fluorescence units in vertical axis represent increase in emission at 460 nm ± Ca2+ at 20oC for 30 min. (C)“Mock purification” using mock-transformed BL21/pG-Tf2 cell lysate. Samples were separated by
SDS-PAGE and stained by CBB. Lanes: S1 and S2, nCL-2- and mock-transformed cell lysate
supernatants, respectively; E1–E3, the final Mono Q fractions eluted around 0.2 M NaCl. nCL-2 band isindicated by an arrowhead.
Fig. 2. Characterization of nCL-2. In (A), (C), and (E), closed circles, open squares, and triangles
represent recombinant nCL-2, μ-calpain, and m-calpain, respectively. (A) Ca2+-dependency of the
Suc-LLVY-MCA hydrolyzing activity. The activities were standardized by defining the values at pCa=2
for nCL-2 and m-calpain and pCa=3 for μ-calpain as 100%. (B) Effect of inhibitors on nCL-2 activity. The
activities were standardized by defining the activity in the presence of 5 mM EDTA and in the absence of
inhibitors as 0% and 100%, respectively. CSTN DI, calpastatin domain I; CSTN pep42, an inhibitory
peptide derived from one of four calpastatin-reactive domains. (C) Temperature dependency with a
20-min incubation. Activities were standardized with the values at 20°C defined as 100%. (D) (left) Timecourse of nCL-2 activity at the indicated temperatures. Activities were standardized with the value at 15°C
for 60 min defined as 100%. (right) Amplified view of the left graph in the range from 0 to 2 min of
incubation. (E) pH dependency. Activities were standardized with the values at pH=6.0 for nCL-2, pH=7.0
for μ-calpain, and pH=7.3 for m-calpain defined as 100%.
Fig. 3. Characterization of autolysis of nCL-2. nCL-2 (0.1 mg/ml) was incubated with or without2+

Ca

for 0, 10, 30, 60, and 180 sec at 25°C and for 600 sec at 37°C. (A) (left) SDS-PAGE analysis of

autolytic fragments stained with CBB. (right) The band intensity of full-length nCL-2 (closed arrowhead)
was quantified by densitometry. An asterisk indicates the impurity as also shown in Fig. 1A. (B) Western
blot analyses with anti-nCL2 (left), anti-His (middle), and RP2-calpain 8 (right) antibodies. Closed
arrowheads and open arrowheads (a-f) indicate full-length and autolytic fragments of nCL-2, respectively.
(C) Schematic illustration of the recognition sites for the anti-nCL2, anti-His, and RP2-calpain 8
13

antibodies, the autolytic sites, and positions of the autolytic fragments obtained above (a-f).
Fig. 4. nCL-2 forms homo-oligomers. (A) Elution profile of purified nCL-2 through a gel filtration
column. The solid line represents A280 of the eluate from the column. Fractions were subjected to
SDS-PAGE with CBB staining, and are shown in the upper panel. (B) Schematic illustration of thestructures of epitope-tagged wild-type or deletion mutants of nCL-2, mCL, 30K, and 30K-2 used for the
immunoprecipitation analyses. (C) HA-mCL (lanes 1 and 2), HA-nCL-2 (lanes 4 and 5), or mock (lane 3)
was co-expressed with FLAG-30K (lanes 2, 3, and 5) or mock (lanes 1 and 4) in COS7 cells. The celllysates were immunoprecipitated (IP) with anti-FLAG agarose and subjected to western blot analysis
using anti-FLAG and anti-HA antibodies. (D) FLAG-nCL-2 (lanes 1, 3, and 5) or mock (lanes 2 and 4)
was co-expressed with HA-nCL-2 (lanes 2 and 3), myc-nCL-2 (lane 4 and 5), or mock (lane 1) in COS7
cells. The cell lysates were immunoprecipitated (IP) with an anti-FLAG or anti-myc antibody as indicated,and subjected to western blot analysis using anti-FLAG, anti-HA, and anti-myc antibodies. (E) (left)
FLAG-nCL-2 (lane 3) or mock (lane 2) was co-expressed with HA-nCL-2 (lanes 2 and 3) in COS7 cells.
In this experiment, 0.1 μg of each DNA was used for the transfection. The cell lysates were
immunoprecipitated (IP) with anti-FLAG agarose and subjected to western blot analysis using anti-FLAG
and anti-HA antibodies. To confirm the expression level of nCL-2 in the transfected cell lysates, 30 μg ofeach cell lysate was analyzed by western blot using anti-nCL2 antibody (lanes 2 and 3), compared with thesame amount of cell lysate prepared from mouse gastric mucosa (lane 1). (right) CBB staining of the celllysates used. (F) HA-ΔEF5 (lanes 5–7) or HA-nCL-2 (lane 8) was co-expressed with FLAG-ΔEF5 (lanes
6 and 8), FLAG-nCL-2 (lane 7), or mock (lane 5) in COS7 cells. The cell lysates were immunoprecipitatedwith anti-FLAG agarose and subjected to western blot analysis using anti-FLAG and anti-HA antibodies.
Data in lanes 1–3 are essentially the same as lanes 1–3 in (D), but from independent experiments forcomparison with lanes 4–8.
Fig. 5. Domain IV is not required for nCL-2 oligomerization. Various truncation mutants with a
FLAG or HA tag at the N-terminus were co-expressed in COS7 cells in the combinations indicated. Mockwas used as a partner for single protein expression. The cell lysates were immunoprecipitated with
anti-FLAG agarose and subjected to western blot analysis using anti-FLAG and anti-HA antibodies. (A)The nCL-2 oligomer formed without dIV (lane 3), but was barely detectable when dIII and dIV weredeleted (lane 6). (B) Full-length nCL-2 oligomerized with ΔdIV (lane 3), but not with ΔdIII+IV (lane 5),
regardless of the types of tag used. (C) dIV of nCL-2 did not form oligomers (lane 3). The expression ofdIII of nCL-2 was not as strong as that of other constructs (lanes 6–8, and 10), but the faint dIII
co-precipitated with dIII (lane 8) or full-length nCL-2 (lane 10).
Fig. 6. Effect of oligomerization on nCL-2 activity. (A) HA-nCL-2 was co-expressed with
14

FLAG-nCL-2 in COS7 cells, and the cell lysates were immunoprecipitated with anti-FLAG agarose. The
precipitant was then incubated for 0 (lane 1), 30 (2), 60 (3), and 120 min (4 and 5) with or without Ca2+ as
indicated, and subjected to western blot analysis using anti-FLAG and anti-HA antibodies. Closed andopen arrowheads indicate full-length and autolyzed nCL-2, respectively. (B) HA-nCL-2CS (lanes 2, 3, 5,
and 6) or mock (lanes 1 and 4) was co-expressed with FLAG-nCL-2CS (lanes 3 and 6) or mock (lanes 2
and 5) in COS7 cells. The cell lysates were immunoprecipitated with anti-FLAG agarose, and theprotein-bound anti-FLAG agarose was incubated with (lanes 4–6) or without (lanes 1–3) Ca2+, thenwashed with the same buffer. The protein remaining on the agarose was subjected to western blot analysis
using anti-FLAG and anti-HA antibodies.
Fig. 7. nCL-2 does not bind 30K-2. HA-nCL-2 (lanes 2 and 3), HA-mCL (lanes 5 and 6), or mock(lanes 1 and 4) was co-expressed with FLAG-30K-2 (lanes 1, 3, 4, and 6) or mock (lanes 2 and 5) in COS7
cells. The cell lysates were immunoprecipitated with anti-FLAG agarose and subjected to western blot
analysis using anti-FLAG and anti-HA antibodies.

15

Table 1. Summary of interactions between nCL-2 and its mutants

FL(HA-tagged)

FL++

FLC105SnFL-C105S

n

++*

ΔEF5

++

n

ΔdIV

++

ΔdIII+IV
dIV
dIII(FLAG-tagged)
ΔEF5
ΔdIV
ΔdIII+IV

dIV

dIII

++

++

-

-

+

n

n

n

n

++

n

n

n

n

n

n

++

n

n

n

-

n

n

n

-

n

n

-

n

n

n

n

-

n

+

n

n

n

n

n

+

FL: full-length; n: not tested++, +, and -: abundantly, weakly, and not detected, respectively, in the absence of Ca2+*: both in the presence and absence of Ca2+

16

Hata et al. Fig. 1
(A)
1

(kDa)
97

2

3

4

66
45

*

30

CBB

(B)
fluorescence unit

500
400
300
200
100
0

mock

nCL-2CSnCL-2cell lysate

(C)

mock
lysate fraction
S1 S2 E1 E2 E3

(kDa)
97
66
45

30

CBB

Hata et al. fig.2(A)

relative activity (%)

100
nCL-2

80
60

m
40
20
0

7

∞

6

5

pCa

4

3

2

(B)

remaining activity (%)

100
80
60
40
20

SF

PM

BS

TA

AE

ED

mM

M

1m

0.1

ne

no

M

5m

0

F

Hata et al. fig.2 (continued)(C)

relative activity (%)

100

m

80

nCL-2

60
40
20
0

15

20

25

30

temperature (oC)

35

40

100

15oC

80

20oC

60
25oC

40

30oC

20

0

10

20

30

40

50

time (min)60

relative activity (%)

nCL-2
m

80
60
40
20
0

37oC

10
30oC
25oC
42oC

5

20oC

37oC
42oC

(E)
100

relative activity (%)

relative activity (%)

(D)

6.0

7.0

pH

8.0

9.0

15oC

0

1

time (min)2

Hata et al. fig. 3(A)2+(sec) 0

+Ca
0

2+

-Ca

2+

10 30 60 180 600 600

(kDa)
83

a

62

b
c

*

100

47.5

e

d

f

32.5

Remaining amount offull-length nCL-2 (%)

-Ca

80
60
40
20
0

200

25

400

600

time (sec)

CBB

(B)

Western blot
anti-nCL2-Ca2+(sec) 0anti-His

+Ca2+
0

-Ca2+

-Ca2+

10 30 60 180 600 600 (sec) 0

RP2-calpain 8

+Ca2+

-Ca2+

-Ca2+

10 30 60 180 600 600 (sec) 0

0

+Ca2+
0

-Ca2+

10 30 60 180 600 600

(kDa)
83

a

a

62

b

b

47.5

c
d

c
e

d

f

32.5
25

(C)

A6GISKQRA...
f
e
d
c
b
a
EF-1 2MNHKV(H)6IEGRNI- I

YY

anti-His

IIa

IIb

III

Cys

His Asn

Acidic loop

YY

anti-nCL2YY

RP2-calpain 8

3 4 5

IV

Hata et al. fig. 4(A)
fractions

A280 (mAU)
nCL-2

40

CBB

20

0

20

estimated MW
(kDa)

(B)

40
440

232

HAFLAGHA-

nCL-2

FLAGHA-

I
I

I

80

158

67

elution100 (ml)

EF-1 2 3 4 5FLAGfull-length HA- I
mycFLAG-

60

IIa

IIb

C or S

H N

IIa

IIb

C

H N

IIa

IIb

C

H N

IIa

IIb

C

H N

III

IV
EF-1 2 3 4

III

IV

III

FLAG-

dIV

EF-1 2 3 4 5IV

HAFLAG-

dIII

III

HA-

EF-1 2 3 4 5

mCL

HA- I

IIa

IIb

C

H N

FLAGHA-

30K, 30K-2

mCL

(C)

1
anti-FLAG
lysate2

IV

III
EF-1 2 3 4 5

VI

V

nCL-2
3

4

5
30K

anti-HA

anti-FLAGIP:
FLAG
anti-HA

30K

Hata et al. fig. 4 (continued)
1(D)

lysate

IP:
FLAG

2

3

4

anti-FLAG

lysateanti-FLAG

anti-HA

anti-myc

anti-FLAG

anti-FLAG

IP:
FLAG

anti-HA

anti-myc

IP:
myc

1

(E)

2

5

3

anti-myc
anti-FLAG

1

2

3

anti-nCL2

(kDa)
175
83

lysate anti-FLAG

62
anti-HA

IP:
FLAG

47.5

anti-FLAG

32.5

anti-HACBB(F)

FLAG-full-length
HA-full-length
FLAG-∆EF5
HA-∆EF5+

1
anti-FLAG
lysateanti-HAIP:
FLAG

anti-FLAG
anti-HA

+
2

+
+
3

+
+
4

+
5

+
+
6

+
7

+
+
8

Hata et al. fig. 5(A)

FLAG-∆dIV
HA-∆dIV

+
1

+
2

+

+
+

2

3

FLAG-∆dIII+IV +HA-∆dIII+IV
4

+
+
3

+
5

+
+
6

anti-FLAG
lysateanti-HA

anti-FLAGIP:
FLAG
anti-HA(B)
FLAG-full-length +HA-∆dIV
HA-∆dIII+IV
1

+
+
4

+
5

+

+

4

+
5

FLAG-∆dIV
FLAG-∆dIII+IV
HA-full-length +
6

+

+

7

+
8

+
9

+
+
10

+

+

9

+
10

anti-FLAG
lysateanti-HA

anti-FLAGIP:
FLAG
anti-HA(C)FLAG-full-length
FLAG-dIV +HA-dIV1anti-FLAG
lysateanti-HAIP:
FLAG

anti-FLAG

anti-HA

+
2

+
+
3

FLAG-full-length
FLAG-dIII +HA-dIII
6

+
7

+
+
8

Hata et al. fig. 6

(A)
+Ca2+
(kDa)
83

1

2

3

-Ca2+
4

5

+Ca2+
1

2

3

-Ca2+
4

5

62
47.5

32.5
anti-FLAG

anti-HA(B)-Ca2+

FLAG-nCL-2CS
HA-nCL-2CS+
1

anti-FLAG
lysateIP:
FLAG

anti-HA

anti-FLAG
anti-HA

+
2

+Ca2+

+
+
3

+
4

+
5

+
+
6

Hata et al. fig.7

HA-nCL-2
HA-mCL
FLAG-30K-2 +

+

+

1

2

+
3

anti-FLAG
lysateanti-HAIP:
FLAG

anti-FLAG
anti-HA

+
4

+
5

+
+
6