From the Institute of Biological Chemistry, University of Genoa,
Viale Benedetto XV, 1-16132 Genoa, Italy
A natural calpain activator protein has been
isolated from bovine brain and characterized in its properties and
molecular structure. The protein is a homodimer with a molecular mass
of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were
also observed, whereas a lower degree of similarity was found with a
family of heat-responsive proteins named YJGF and YABJ from
Haemophilus influenzae and Bacillus subtilis,
respectively.
The brain activator expresses a strict specificity for the µ-calpain
isoform, being completely ineffective on the m-calpain form. As
expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A
protein showing the same calpain-activating activity has been also
isolated from human red cells, indicating that this factor is widely
expressed. All these activators are efficient on µ-calpain independently from the source of the proteinase.
The high degree of specificity of the calpain activator for a single
calpain isoform may be relevant for the understanding of sophisticated
intracellular mechanisms underlying intracellular proteolysis. These
data are indicating the existence of a new component of the
Ca2+-dependent proteolytic system, constituted
of members of a chaperonin-like protein family and capable of promoting
intracellular calpain activation.
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INTRODUCTION |
Calpains are a family of dimeric proteinases all characterized by
an absolute dependence on Ca2+ (1-7). In the absence of
this metal ion, calpains are stabilized in an inactive conformational
state, by inter- and intramolecular constraints (8, 9). Binding of
Ca2+ to the proteinase molecules produces both dissociation
of the heterodimers (10) and conformational changes of the 80-kDa
catalytic subunits, triggering the enzyme activation that is completed
by an autoproteolytic event (11, 12). The concentrations of
Ca2+ inducing the conformational changes required for the
activation of both µ- and m-calpain are at least one order of
magnitude higher than the actual concentrations of this metal ion in
cells. Experiments designed to identify possible mechanisms effective
in reducing the calcium requirement of calpains have demonstrated that
association of the proteinase to phospholipid vesicles (12) or to
nuclei (13) are effective in increasing its affinity for
Ca2+. A more relevant physiological significance is
represented by a calpain activator protein recently identified in human
red blood cells (14) and in rat skeletal muscle (15). This protein
factor, which is significantly effective in reducing the requirement of the proteinase for calcium ions, binds Ca2+ with high
affinity (10) and associates to the particulate fraction of the cells;
in fact, it is recovered in the soluble fraction only when cell lysis
is performed by a medium containing metal chelators.
Furthermore, the activator-Ca2+ complex interacts with
calpain and thereby induces those conformational changes required to trigger the activation process of the proteinase. On the basis of these
results, the protein factor could be visualized as a physiological mean
to obtain a site-directed activation of calpain elicited in response to
an increased intracellular concentration of free Ca2+.
In this study we report the purification of a bovine brain calpain
activator. Taking advantage of new strategies for the purification procedure, we have obtained sufficient amounts of protein to establish its amino acid sequence. The primary structure of the rat brain calpain
activator is almost completely identical to that of a known protein
called UK114 (16), similar to that of a 23-kDa protein from rat liver
(17), corrected as reported by Ceciliani et al. (16) and to
that of HR12 heat-shock YABJ and YJGF proteins (SwissProt sequence data
base). The calpain activator is highly selective for the µ-isoforms
of the proteinase and does not reveal any apparent species specificity.
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EXPERIMENTAL PROCEDURES |
Superose 12 and Source 15 Q columns were purchased from Amersham
Pharmacia Biotech. Purified UK114 (10) was kindly provided by Dr.
Severino Ronchi, Istituto di Fisiologia Veterinaria e Biochimica, University of Milan, Milan, Italy.
Calpain Activator Purification--
Freshly collected bovine
brain was suspended in 4 volumes of 50 mM sodium acetate
buffer, pH 5.8, containing 1 mM EDTA and 1 mM
-mercaptoethanol and disrupted by using Potter-Elvehjem homogenizer.
Cells were lysed by sonication (6 bursts of 10 s each), and the
particulate material was removed by centrifugation. The pH of the
supernatant was adjusted to 5.8, and the sample was heated at 90 °C
for 3 min and centrifuged, and the clear supernatant was collected. The
proteins were concentrated by adding ammonium sulfate at the 80%
concentration, suspended in 50 mM sodium borate, pH 7.5, containing 0.1 mM EDTA (Buffer A), dialyzed against the same buffer, then submitted to ion exchange chromatography on the
Source 15 Q column (1.6 × 5 cm) equilibrated in buffer A. The
unabsorbed proteins, also containing the calpain activator, were
collected and applied to a butyl-agarose column equilibrated in Buffer
A. The unretained material, containing calpain activator activity, was
collected and submitted to gel chromatography on the Superose 12 column
(1.8 × 50 cm) equilibrated in buffer A.
Calpain activator from rat skeletal muscle was purified from 120 g
of fresh tissue, suspended in 10 volumes of cold 0.25 M sucrose containing 0.1 mM EDTA and 1 mM
-mercaptoethanol and homogenized in a tissue grinder. All
purification steps were carried out as reported above for bovine brain
activator. The activator protein was also isolated from human
erythrocytes. Erythrocytes were deprived of leukocytes and platelets
and washed three times with 10 mM sodium phosphate solution
containing 0.15 M NaCl. Cells were then packed and lysed
with 5 volumes of 50 mM sodium borate, pH 8.3, containing 1 mM EDTA. The membranes were discarded by centrifugation and
the clear supernatant was heated at 90 °C for 3 min. The human
erythrocyte activator protein was further purified to homogeneity
as described for bovine brain activator.
Assay of Calpain Activator Activity--
The activator protein
activity was assayed by adding the appropriate amounts of the activator
sources to the routine calpain assay mixture containing 2 µM Ca2+ (18). One unit of calpain activator
activity is defined as the amount causing the appearance of one unit of
calpain activity in the presence of 2 µM Ca2+
(14).
Sequence Analysis of Bovine Brain Activator--
Purified
activator protein (100 pmol) was lyophilized in the presence of 0.05%
SDS to avoid the irreversible denaturation and insolubilization of the
protein, submitted to 16%
SDS-PAGE,1 and then
transblotted on a polyvinylidene difluoride sheet. The region of
membrane-containing activator protein, localized by staining with
Ponceau S, was cut, washed with H2O, saturated at 37 °C
for 30 min with 0.5% polyvinylpyrrolidone, and then minced (19). The
fragments were suspended on 0.6 ml of 50 mM sodium borate
buffer, pH 8.5, containing 5% CH3CN and 1 µg of trypsin and incubated at 37 °C for 18 h. After digestion, the soluble material was lyophilized, suspended in 50 µl of H2O, and
the pH was acidified by addition of 0.1% trifluoroacetic acid. The
sample was submitted to chromatography on a C-18 column (1 × 100 mm) equilibrated with distilled water containing 0.1% trifluoroacetic acid. Absorbed peptides were eluted with a linear gradient of CH3CN (from 0 to 70%) and fractions of 30 µl were
collected. The fractions containing the tryptic peptides were directly
loaded on the Beckman LF3000 protein sequencer to determine primary
structure.
A comparison of primary structure of rat brain activator and other
protein sequences was done using the SwissProt sequence data base.
Alignment of the sequences of brain activator and goat liver UK114 was
performed using the PALIGN program. The alignment of the amino acid
sequence of the calpain activator protein with a rat liver 23-kDa
protein (16), HR12 (HR12 MOUSE), YJGF (YJGF HAEIN), and YABJ
(YABJ BACSU) proteins was obtained using the CLUSTAL program.
Purification of µ- and m-Calpain from Different
Sources--
µ-Calpain and m-calpain from rat brain were purified as
reported (20), and those from skeletal muscle were obtained as
described previously (21). Human and bovine erythrocyte µ-calpain and m-calpain were purified as reported (10).
Assay of µ- and m-Calpain Activity--
Calpains were
routinely assayed as previously reported (18).
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RESULTS |
Following ion exchange chromatography of the heat-treated bovine
brain homogenate on Source Q resin, which binds calpastatin with high
efficiency, the protein factor is recovered in the washing material.
When tested for its activity, it is found to promote activation of
calpain at a concentration of calcium ineffective to promote
activity of the native proteinase. The solution is then filtered
through a column of butyl-agarose, which removes the residual traces of
calpastatin activity and other contaminating proteins. Finally, the
solution containing calpain activator activity is submitted to gel
chromatography on a Superose 12 column. As shown in Fig.
1, three major protein peaks are eluted:
the first one showing an approximate molecular mass of 30 kDa (emerging immediately before carbonic anhydrase, 29 kDa); a second one with a
mass of approximately 22 kDa; and a third one with a mass below 10 kDa
and identified as ubiquitin by its N-terminal amino acid sequence (data
not shown). Only the first peak contains the calpain activator
activity. All fractions under this peak are collected and submitted to
SDS-PAGE (Fig. 2). A single protein band
is detected with a mobility corresponding to a molecular mass of
approximately 15 kDa, indicating that in the native active form the
activator is present in a dimeric structure.

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Fig. 1.
Separation of bovine brain calpain activator
by gel filtration on a Superose 12 column. Bovine brain calpain
activator obtained following the hydrophobic chromatographic step (see
"Experimental Procedures") was submitted to gel chromatography on a
Superose 12 column previously equilibrated in Buffer A. The activator
activity ( ) was assayed using bovine erythrocyte µ-calpain in the
presence of 2 µM Ca2+ (see "Experimental
Procedures"). The continuous line indicates the absorbance at 280 nm.
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Fig. 2.
SDS-PAGE of bovine brain activator. A
sample (200 µl) of fractions (from 13 to 16, see Fig. 1) containing
activator activity obtained by Superose 12 column chromatography was
lyophilized and suspended into 0.05 ml of 0.1 M Tris/HCl,
pH 8.8, containing 2% 2-mercaptoethanol and 2% SDS and submitted to
16% SDS-PAGE. At the end of the electrophoretic run, the protein bands
were identified by silver staining. The arrows indicate the
molecular mass of standard proteins (bovine serum albumin, 67 kDa;
chicken egg albumin, 45 kDa; carbonic anhydrase, 29 kDa; and cytochrome
c, 13 kDa); lanes 1-4 refer to fractions from 13 to 16 of the chromatography shown in Fig. 1.
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To further characterize the protein, the material recovered from ten
separate chromatographic analyses, each one corresponding to that shown
in Fig. 1, is loaded on a SDS-PAGE and transblotted on a polyvinylidene
difluoride sheet. The position of the single protein band is identified
by staining with Ponceau S, cut, and destained as described under
"Experimental Procedures." The protein is then solubilized by
digestion with trypsin, and the peptides mixture separated on a C-18
reverse-phase chromatography (Fig. 3).
The peptides are eluted by a linear gradient of acetonitrile (from 0 to
70%) and each peptide is separately collected, sequenced, and analyzed
for its similarity with known proteins using SwissProt sequence data
base (program FASTA3). All peptides show a sequence almost completely
identical to the corresponding tryptic peptides from a known protein
named UK114. The sequences are then aligned following the primary
structure of UK114 protein, and the results are shown in Fig.
4. The differences between the two amino
acid sequences are confined at residues 7, 43, and 91 at the C-terminal end. The calpain activator protein also shows structural similarities with a 23-kDa protein from rat liver (16) with the mouse
heat-responsive protein HR12, and with two families of proteins,
members of which (YJGF and YABJ) are reported in Fig.
5. The similarity of this protein factor
with heat-shock proteins is very suggestive and might indicate a
"chaperonin-like function."

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Fig. 3.
Separation of tryptic peptides of bovine
brain calpain activator. Bovine brain activator, obtained from 10 chromatographic steps as shown in Fig. 1, was submitted to SDS-PAGE
(see Fig. 2) and then the proteins were transblotted on a
polyvinylidene difluoride sheet (see "Experimental Procedures").
Protein bands were stained with Ponceau S, and the bands with a
mobility corresponding to a mass of 14 kDa were cut and destained. The
bound protein was solubilized by digestion with 1 µg of trypsin. The
peptides were separated by reverse-phase chromatography on a C-18
column. Each number refers to the peptide used for the determination of
the activator protein sequence.
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Fig. 4.
Amino acid sequence of calpain activator and
its similarity to that of the UK114 protein. Peptides, isolated as
shown in Fig. 3, were collected, and their amino acid sequences were
determined automatically with a Beckman LF 3000 protein sequencer. The
calpain activator (C. A.) amino acid sequence is compared
with that of the UK114 protein. The arrows indicate
differences between the two sequences.
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Fig. 5.
Structural similarities among bovine brain
calpain activator (C. A.), HR12, 23-kDa rat liver protein,
and YJGF and YABJ proteins. The amino acid sequences of HR12,
YJGF, and YABJ proteins were taken from the SwissProt data base. The
sequence of the 23-kDa protein was taken from the work of Ceciliani
et al. (16). *, indicate a perfectly conserved residue; ·,
indicates a well conserved residue.
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For comparison we have analyzed the functional properties of UK114 on
calpain activity, due to the almost complete identity between the
calpain activator protein and UK114. A sample of UK114 protein is
submitted to Superose 12 column chromatography in the conditions used
for the isolation of the brain calpain activator (Fig.
6). Two protein peaks are separated; the
major is eluted in an identical volume to that of the calpain
activator, the second one in an elution volume corresponding to
ubiquitin. The first peak is found to possess a potent activating
effect on calpain, almost indistinguishable from that observed with the
authentic bovine brain calpain activator.

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Fig. 6.
Activity of UK114 protein on
µ-calpain. A sample of UK114 protein (20 µg) dissolved in
water was submitted to Superose 12 chromatography in the same
conditions used for brain activator (see Fig. 1 and "Experimental
Procedures"). Aliquots of the eluted fractions (30 µl) were assayed
for calpain activator activity using bovine erythrocyte µ-calpain
(see "Experimental Procedures").
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As shown in Table I, addition of the
bovine brain activator to µ-calpains, isolated from different
sources, promotes a more than 10-fold decrease in the Ca2+
requirement of the proteinases without any modification of their Vmax. On the contrary, the activator is
ineffective on rat brain or skeletal muscle m-calpain.
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Table I
Effect of bovine brain calpain activator on the Ca2+
requirement and on the Vmax of µ- and m-calpains isolated
from different sources
Ca2+ requirement was determined assaying calpain activity at
increasing concentration of Ca2+ in the absence or presence of
calpain activator in an amount causing the maximal effect (see Fig. 7).
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The activator effect is dose-dependent (Fig.
7) and reaches a maximum on
activator/calpain molar ratio of approximately 1 to 1. Rat skeletal
muscle µ-calpain shows a slightly lower affinity for the activator
and requires a 1.1-1.2 factor excess.

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Fig. 7.
Effect of rat brain activator on the
catalytic activity of µ-calpain isolated from different sources.
µ-Calpain was purified from human or bovine erythrocytes and rat
brain or skeletal muscle as described under "Experimental
Procedures." Calpains (0.01 nmol) were incubated with increasing
amounts of activator at the indicated molar ratio. Calpain activity was
assayed in the presence of 2 µM Ca2+ and is
expressed at the percentage of total proteolytic activity measured in
the presence of 1 mM Ca2+.
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In Table II, the properties of calpain
activators from human erythrocyte and goat liver sources are compared
with those of the bovine brain protein factor. The three activators are
equally efficient in promoting expression of catalytic activity of
homologous and heterologous µ-calpains. All of these protein factors
are ineffective on m-calpains.
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Table II
Isozyme and tissue specificity of calpain activator
Calpain activator effect is expressed as the percentage of calpain
activity measured in the presence of 2 µM Ca2+
and equimolar amounts of activators. As 100% was taken the calpain
activity assayed in the presence of 1 mM Ca2+. The
effect of the activator on m-calpains was determined assaying the
enzyme activity at 20, 50, and 100 µM and 1 mM Ca2+ with or without equimolar amounts of the
indicated protein factors.
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Taken together, these findings indicate that the µ-calpain activator
protein is not a characteristic of brain tissue, and it is probably
widely distributed.
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DISCUSSION |
It is generally recognized that the soluble
calcium-dependent proteolytic system is composed by one or
more proteinases (calpains) and by their natural inhibitor, calpastatin
(1-6, 22-25). In the past years, we have observed that a possible
third component of this system, effective in reducing the
Ca2+ requirement of calpains and thereby in promoting
activation of these proteinases, is also present in different human and
rat tissues (14, 15). Its molecular characterization and degree of
identity with other proteins have never been defined, probably due to
difficulties in the preparation procedures and in the concentration of
the samples as well as in the chromatographic analyses. This protein,
in fact, binds to many inert supports or membranes particularly to
those employing hydrophobic materials. These difficulties have been
overcome by the use of new purification strategies; on the basis of
which it was possible to obtain a sufficient amount of pure calpain
activator to determine the primary structure.
The molecular mass resulted in being slightly higher than that of
cytochrome c, approximately 15 kDa in denaturing conditions and around 30 kDa in native conditions, indicating that the native active form is a dimer. Sequence analysis has revealed its almost complete identity with a previously characterized protein isolated from
the acid-soluble material of goat liver and called UK114 (16). In
addition, the bovine brain calpain activator shows structural
similarities with a 23-kDa protein from goat liver (16, 17), a high
degree of similarity with the mouse heat-responsive protein HR12, or
with heat-shock proteins (YJGF and YABJ), suggesting that the mechanism
of action may also include a chaperonin-like activity directed at
promoting conformational changes of target proteins. This hypothesis is
in agreement with the previously described functional properties of the
calpain activator and consistent with a sequential mechanism of
activation involving at first an interaction of the
Ca2+-activator complex with calpain, followed by a
conformational change of the proteinase, resulting in its activation at
very low [Ca2+].
Since the activator interacts with intracellular membrane structures
upon binding of Ca2+ (data not shown), it could also be
suggested that the overall mechanism consists of promoting a
site-directed activation of calpain and thereby a site-directed
specificity for digestion of protein substrates. This model can be
considered to operate with a high degree of specificity in different
cells in which the soluble calcium-dependent proteolytic
system requires a sophisticated regulatory efficiency. However, we have
observed that the calpain activator isolated from rat skeletal muscle,
although showing a similar activating mechanism, displays different
specificity in activating m-calpain (26). The presence of multiple
activator forms, together with the observed structural similarities
among the protein factor and other protein molecules, suggests the
existence of a family of calpain-activating proteins with specific
properties operating in various tissues in response to different
stimuli. As revealed by the Garnier analysis method (27), the calpain activator and the proteins showing a high degree of similarity with
this factor contain a central region rich in helical
conformation-preferring residues (residues 52-72 in the calpain
activator protein), suggesting the existence of common structural
features that could be involved in similar intracellular functions.
Furthermore, these results represent the first demonstration for an
intracellular function of UK114 protein related to the modulation of
the soluble calcium-dependent proteolytic system and
thereby stressing the role of the calcium-mediated proteolysis in cell
functions.
We thank Dr. Severino Ronchi, Istituto di
Fisiologia Veterinaria e Biochimica, University of Milan, Milan, Italy,
for the gift of a sample of pure UK114 and Roberto Minafra for
technical assistance.