Interaction between catalytically inactive calpain and
calpastatin
Evidence for its occurrence in stimulated cells
Monica Averna, Roberto Stifanese, Roberta De Tullio, Enrico Defranchi, Franca Salamino,
Edon Melloni and Sandro Pontremoli
Department of Experimental Medicine (DIMES), Section of Biochemistry and Centre of Excellence for Biomedical Research (CEBR),
University of Genova, Italy
In recent years, information has accumulated on the
3D structure of l-calpain and m-calpain [1–10], as well
as their isolated catalytic cores [11–16]. Much less is
known about the process by which calpain is activated
[3,4,6,17,18]. It is generally accepted that it is initiated
by the binding of calcium to several sites localized in
both calpain subunits and completed by a conforma-
tional change in domain II [19–29]. However, the role
of the two Ca
2+
-binding sites recently identified in this
catalytic domain is still to be defined [23]. More
intriguing is the possibility of detecting calpain activa-
tion in vivo, which has not been previously possible
because of the lack of reliable techniques for evaluat-
ing active calpain species and their intracellular local-
ization.
Identification of autolyzed calpain forms by means
of a specific monoclonal antibody does not seem to be
of a general use, as recent structural acquisitions have
suggested that calpain activation can also proceed
through a reversible process [1,2,4]. The proposed pro-
cedure involving the identification of calpain-degraded
target proteins appears not to be sufficiently specific

these conformational states showed catalytic activity and probably repre-
sent intermediate forms preceding the active enzyme state. In its native
inactive conformation, calpain shows very low affinity for this monoclonal
antibody, whereas, on binding to the ligands Ca
2+
, substrate or calpasta-
tin, the affinity increases up to 10-fold, with calpastatin being the most
effective. This methodology was also used to show that calpain undergoes
similar conformational changes in intact cells exposed to stimuli that
induce either a rise in intracellular [Ca
2+
] or extensive diffusion of calpast-
atin into the cytosol without affecting Ca
2+
homeostasis. The fact that the
changes in the calpain state are also observed under the latter conditions
indicates that calpastatin availability in the cytosol is the triggering event
for calpain–calpastatin interaction, which is presumably involved in the
control of the extent of calpain activation through translocation to specific
sites of action.
1660 FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS
transition to a conformation with significantly higher
affinity. The most extensive conformational change is
induced by calpastatin; the addition of substrate or
Ca
2+
proved to be less effective. Using this methodo-
logy, we have shown similar molecular transitions in
calpain in intact cells stimulated with agents known to
induce either a limited increase in intracellular [Ca

tion, is 50% of the maximal at 25 lm Ca
2+
and
maximal at 100 lm Ca
2+
, as occurs when soluble
native enzyme is used [3,4,9,20,30]. Furthermore,
inhibition of the immobilized enzyme by E64 or
calpastatin was retained (Fig. 1D). The efficiency of
both inhibitors was identical with that observed in a
control assay using soluble enzyme (data not shown).
Together these results indicate that immobilized cal-
pain is an appropriate tool for the study of the effects
of natural ligands in changing its conformation, and
that these can be monitored by evaluating the intensity
of the light signal generated by the binding of mAb
56.3.
To perform these investigations, the two preferential
ligands of calpain, Ca
2+
ions and calpastatin, were tes-
ted. In the presence of Ca
2+
concentrations ranging
from zero to 5 lm (close to physiological values), a
twofold increase in the intensity of the signal was
detected, in the absence of any appreciable proteolytic
activity (Fig. 2). The concentrations of Ca
2+
used in

M EDTA and the indicated amounts of mAb 56.3. The immu-
noreactive spots (see inset) were quantified with a Shimadzu
CS9000 densitometer and expressed as arbitrary units. (C) Immobil-
ized calpain (0.5 lg) was assayed as described in [33] using human
denatured globin as substrate for 60 min at 37 °C in the presence
of the indicated [Ca
2+
]. The nitrocellulose sheet was removed
before the addition of trichloroacetic acid (7% final concentration).
Calpain activity was quantified after the release of free NH
2
groups
as in [33]. (D) Activity of immobilized calpain was assayed as in (C)
in the presence of 1 m
M Ca
2+
and 10 lM E64 or 25 nmol rat brain
recombinant calpastatin RNCAST104.
M. Averna et al. Calpain–calpastatin interaction in stimulated cells
FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS 1661
light signal (7–8-fold) than when calpain was exposed
to Ca
2+
alone (Fig. 2). Moreover, the maximum effect
was reached with 10 nmol RNCAST104, which corres-
ponds approximately to a 1 : 1 protease ⁄ inhibitor
molar ratio. The addition of Ca
2+
even at a concen-
tration of 5 lm did not affect the RNCAST104-medi-

pain substrate [4,9,30], induced a progressive increase
in the binding of mAb 56.3 to calpain, as revealed by
a 2.5–3-fold increase in the light signal at a concentra-
tion of 2 mgÆmL
)1
(Fig. 4).
The observations so far reported indicate that cal-
pain can exist in two freely convertible inactive confor-
mations. The former is mostly present in the absence
of any effector, and the other is induced, with different
degrees of efficiency, by interaction with micromolar
Fig. 2. Effect of Ca
2+
on binding of mAb 56.3 to nitrocellulose-
immobilized calpain. Immobilized calpain was incubated in 0.1 mL
50 m
M sodium borate buffer, pH 7.5, in the presence of 10 mM
EDTA or the indicated Ca
2+
concentration (d). After saturation,
mAb 56.3 was added and the binding of the mAb to calpain was
measured as described in Experimental procedures and the legend
to Fig. 1B and expressed as arbitrary units. Alternatively, immobi-
lized calpain was exposed to Ca
2+
in the presence of 10 lM E64
(s). Immobilized calpain was also incubated in 0.1 mL 50 m
M
sodium borate buffer, pH 7.5, containing the indicated Ca
2+

in Experimental procedures and quantified as reported in the
legend to Fig. 2.
Calpain–calpastatin interaction in stimulated cells M. Averna et al.
1662 FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS
(physiological amounts) Ca
2+
concentrations, a digest-
ible protein substrate, and finally calpastatin.
The finding that calpastatin was the most efficient
ligand at promoting calpain transition, detected as an
increase in mAb that bound to calpain, is consistent
with the fact that the protease has an affinity for its
protein inhibitor that is more than 10 000-fold higher
than that for its substrates.
When native calpain was replaced with the autolyzed
75-kDa form, identical results were obtained in all the
experimental conditions tested (data not shown). This
indicates that the removal of part of the DI and DV
domain from the calpain molecule does not abolish the
conformational transition described above and may
explain the Ca
2+
dependence of the autolyzed enzyme
[34].
We then explored whether, in stimulated cells, cal-
pain undergoes conformational changes that could be
detected by mAb 56.3 binding. For this purpose,
human neutrophils were stimulated with the chemotac-
tic peptide f-Met-Leu-Phe, which is known to promote
intracellular mobilization of Ca

scan. This further confirms that translocation to the
A
C
B
Fig. 5. Binding of mAb 56.3 to calpain in human neutrophils stimul-
ated with f-Met-Leu-Phe. Purified neutrophils (10
7
cells) were incub-
ated at 37 °C for 10 min in 10 m
M Hepes, pH 7.5, (10 mL),
containing 140 m
M NaCl, 5 mM MgCl
2
,5mM glucose and 50 lM
Ca
2+
in the absence (A) or presence (B) of 1 lM f-Met-Leu-Phe.
Cells were fixed and permeabilized as described in Experimental
procedures. They were then exposed to mAb 56.3, and its binding
to calpain was detected by confocal microscopy, after incubation
with fluorescein-labeled secondary antibody. (C) Fluorescence was
measured as described in Experimental procedures. The data repre-
sent the arithmetical mean ± SD of four different experiments.
A
B
Fig. 6. Binding of mAb 56.3 to calpain in MEL cells loaded with
Ca
2+
. MEL cells (10
7

action of calpain with calpastatin, we stimulated Jur-
kat cells with arachidonate, which is known to induce
apoptosis without producing, during the early phase
of stimulation, appreciable changes in intracellular
free [Ca
2+
] [49,50]. As previously observed (Fig. 7B)
in these cells, stimulation with ionophore A23187
promoted a calpain-mediated fluorescence increase of
7–8-fold. However, a sixfold increase in fluorescence
intensity was observed after brief stimulation with
arachidonate, indicating that a similar conformational
transition of calpain can be obtained in conditions in
which intracellular Ca
2+
homeostasis is almost unaf-
fected [49,50]. These findings excluded the involvement
of Ca
2+
in the conformational change in calpain,
strongly suggesting that it is the interaction with cal-
pastatin that is responsible for the observed effects.
Other ligands such as digestible substrates were exclu-
ded a priori because they would never be present in the
cytosol at suitable concentrations.
The different calpain fluorescence observed in control
(Fig. 7A) and arachidonate-stimulated (Fig. 7C) cells
after detection with the calpain mAb can be ascribed to
the presence of large amounts of calpastatin which,
after stimulation, becomes freely available in the cyto-

incubated at 37 °C for 10 min in 10 m
M Hepes, pH 7.5 (10 mL),
containing 140 m
M NaCl, 5 mM MgCl
2
,5mM glucose and 50 lM
Ca
2+
in the absence (A) or presence of (B) 1 lM A23187Ca
2+
-iono-
phore or (C) 100 l
M arachidonate. Cells were fixed and permeabil-
ized as described in Experimental procedures. They were then
exposed to mAb 56.3, and its binding to calpain was detected by
confocal microscopy, after incubation with fluorescein-labeled sec-
ondary antibody. The data represent the arithmetical mean ± SD of
four different experiments. The arrow points to the calpain ring
around the cell.
Fig. 8. Effect of arachidonate on the intracellular distribution of cal-
pastatin in Jurkat cells. Jurkat cells were incubated with arachido-
nate as described in the legend to Fig. 7C. After 30 min of
incubation, cells were fixed, and calpastatin was probed with
7 lgÆmL
)1
mAb 35.23 [35] followed by a fluorescein-labeled second-
ary antibody. Fluorescence was quantified using the software as
described in Experimental procedures. Cell nuclei were stained
with propidium iodide. The arrows indicate the perinuclear calpasta-
tin aggregates. C ¼ control; Ar. ¼ arachidonate.

or a digestible protein substrate, or calpastatin is fol-
lowed by a change in its conformation, which can be
monitored by an increase in its affinity for its mAb.
Calpain does not show catalytic activity in any of these
conditions, indicating that this molecular transition
precedes the onset of the active enzyme form. Using a
Scatchard plot as a calibration curve, we established
that calpastatin promotes the conversion of almost all
the calpain molecules into the high-affinity conforma-
tion, whereas the other ligands promote the transition
of only 20–30% of the calpain molecules. Thus, this
procedure provides a tool for the identification of the
calpain states generated by its interaction with natural
ligands.
This methodology was successfully applied to intact
cells, and the results show that similar conformational
changes in calpain occur after stimulation with appro-
priate effectors.
In human neutrophils and MEL cells, even though
the limited increase in Ca
2+
could be regarded as the
event that promoted these changes, two relevant find-
ings suggest a different conclusion. The first concerns
the extent of the increase in calpain fluorescence, which
could not be induced by Ca
2+
alone as indicated in the
in vitro experiments (Figs 2 and 3). The second is rela-
ted to the availability of calpastatin in the cytosol, the

lar [Ca
2+
] also induces translocation of a small
amount of the protease to the plasma membrane.
These results are in agreement with the accepted evi-
dence that an increase in intracellular [Ca
2+
] induces
degradation of some proteins specifically localized at
the inner surface of the plasma membrane [3,4,30] and
with the observation that the extent of calpain activa-
tion at the plasma membrane is a function of the
amount of cytosolic calpastatin [43].
Taken together, these findings suggest that the for-
mation of a calpain–calpastatin complex before the
onset of calpain activation is functionally relevant, not
only for the modulation of calpain activation in the
cytosol, but also for controlling the amount of calpain
translocated to and activated at the plasma membrane.
Experimental procedures
Materials
Ca
2+
ionophore A23187, f-Met-Leu-Phe, arachidonic acid,
BSA, casein, nonfat skimmed milk powder, trypsin, and
E64 were purchased from Sigma Aldrich (Milan, Italy).
Purification of human erythrocyte calpain and
recombinant rat brain calpastatin
Human erythrocyte calpain was purified and assayed as
reported in [33]. Autoproteolyzed human erythrocyte cal-

perature with or without trypsin in a calpain ⁄ trypsin ratio of
1000 : 1 [14]. After 60 min, 50 lL 120 mm Tris ⁄ HCl,
pH 6.8, containing 4% SDS, 4% 2-mercaptoethanol and
20% glycerol was added to the incubation mixture and then
heated for 3 min at 100 °C. Samples (50 lL) were submitted
to SDS ⁄ PAGE (10% gel) [40], and western blot was per-
formed as indicated in [41]. Proteins were probed with mAb
56.3. The immunoreactive material was revealed as reported
in [42].
Calpain immobilization
The procedure for immobilization of human erythrocyte
calpain is summarized in Scheme 1. The purified enzyme
(0.5 lgin5lL50mm sodium borate buffer, pH 7.5, con-
taining 0.1 mm EDTA) was spotted on a nitrocellulose
sheet (0.5 cm · 0.5 cm; Bio-Rad Laboratories, Bio-Rad Ita-
lia, Milan, Italy) and left for 15 min at 4 °C in a humidified
chamber. The sheet was then washed with 1 mm EDTA
and saturated with 5% nonfat skimmed milk powder. The
nitrocellulose sheet was then incubated in 0.1 mL sodium
borate buffer, pH 7.5, containing the calpain ligands in the
conditions specified elsewhere. The mixtures were incubated
at 4 °C for 30 min. mAb 56.3 (0.2 lg) was then added.
Calpain was detected using a peroxidase-conjugated secon-
dary antibody [42] developed with an ECL
Ò
detection sys-
tem (Amersham Pharmacia Biotech).
Immunoreactive material was detected by subjecting the
probed nitrocellulose sheets to autoradiography and quanti-
fied with a Shimadzu CS9000 densitometer using a fixed

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