(Received for publication, July 22, 1996, and in revised form, January 8, 1997)
From the Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada
The roles of N-terminal autolysis of the large
(80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in
lowering its Ca2+ requirement, and in reducing its
stability have been investigated with heterodimeric recombinant
calpains containing modified subunits. Both autolysis and
[Ca2+]0.5 were influenced by the ionic
strength of the buffers, which accounts for the wide variations in
previous reports. Autolysis of the small subunit (from 28 to 20 kDa)
was complete within 1 min but did not alter either the Ca2+
requirement ([Ca2+]0.5) or the stability of
the enzyme. Autolysis of the NHis10-80k large subunit at
Ala9-Lys10 is visible on gels, was complete
within 1 min, and caused a drop in [Ca2+]0.5
from 364 to 187 µM. The lower value of
[Ca2+]0.5 is therefore a property of the
9-80k large subunit. Autolysis at
Ala9-Lys10 of the unmodified 80-kDa large
subunit is not detectable on gels but was assayed by means of the fall
in [Ca2+]0.5. This autolysis was complete in
3.5 min and was inhibited by high [NaCl]. The autolysis product of
these calpains, which is essentially identical to that of natural
m-calpain, was unstable in buffers of high ionic strength. Calpain in
which the large subunit autolysis site had been mutated was fully
active but did not undergo a drop in
[Ca2+]0.5, showing that m-calpain is active
prior to autolysis. The main physiological importance of autolysis of
calpain is probably to generate an active but unstable enzyme, thus
limiting the in vivo duration of calpain activity.
The calpains (EC 3.4.22.17) are cytoplasmic cysteine proteinases, which are thought to be regulated by means of their Ca2+ dependence. While much work has been done on their biochemical properties, many aspects of autolysis, activation, and Ca2+ requirement remained unresolved (1-3). Two mammalian forms, µ- and m-calpain (calpain I and II), have been most studied, since they can be isolated from animal tissues. Some other calpain forms are known so far only from their mRNA (3, 4), and the chicken calpains are not further considered here since they appear to be slightly different (5, 6). Complete purification of calpain in adequate amounts from tissue extracts is difficult, and a bacterial expression system has been described, which with the aid of a His-tag provides larger amounts of pure enzyme in about 3 days and provides a means for mutational and structural work (7). A baculovirus-based expression system has also been reported (8).
The calpains consist of an 80-kDa catalytic subunit (the large subunit), containing four domains, I-IV, and a 28-kDa regulatory subunit (the small subunit), containing two domains, V and VI (9, 10). Domains I and V are involved in autolysis; domain II contains the most obvious active site residues (11); the function of domain III is not yet known, although it must also take part in the conformational changes induced by Ca2+; and the C-terminal domains of both subunits, IV and VI, contain putative E-F hand motifs, some of which bind Ca2+ (12-15). No structural information is available for the complete calpain heterodimer, but the crystal structures of domain VI, with and without bound Ca2+, have been solved recently.1
On exposure to sufficient Ca2+, the calpains are assumed to undergo a conformational change which permits the following four events: limited autolysis of the small subunit from 28 to 20 kDa; limited N-terminal autolysis of the large subunit; proteolysis of a substrate such as casein if it is present; and further inactivating proteolysis of the large subunit. However, the order of these events and the Ca2+ concentrations required have been difficult to establish. The Ca2+ requirements ([Ca2+]0.5) for casein hydrolysis by (initially) non-autolyzed calpains are usually reported to lie in the ranges of 5-50 µM Ca2+ for µ-calpain and 250-1000 µM Ca2+ for m-calpain. These ranges are lowered by prior autolysis to 1-5 µM Ca2+ for µ-calpain and 100-200 µM Ca2+ for m-calpain (16-25). The reported values vary widely for at least two reasons, first because the casein assay reflects the net effects of activation, autolysis, and inactivation, and is not well suited for kinetic studies (26); and second because, as shown here, the observed values are highly dependent on experimental conditions.
While it is fairly clear that autolysis lowers [Ca2+]0.5 of both µ- and m-calpain, it has not been clear whether large and/or small subunit cleavage is required for, or responsible for, the fall in [Ca2+]0.5 and whether the non-autolyzed calpains are intrinsically active against substrates such as casein. In this work, we have answered some of these questions about autolysis and Ca2+ requirement in m-calpain, using a variety of recombinant large and small subunits with N- and C-terminal modifications.
Escherichia coli strain BL21(DE3) and the plasmids pET-16b(+), pET-20b(+), and pET-24d(+), were obtained from Novagen Inc., Madison, WI. pTRXFUS was a generous gift of Dr. LaVallie (see LaVallie et al. (27)). The plasmid pACpET-24, used for expression of small subunits, has been described previously (7).
N-terminal Amino Acid SequencingAll of the subunit modifications described were checked by DNA sequencing, and in some cases at the amino acid level. Amino acid sequences were obtained by automated Edman degradation, either from individual subunits following gel electrophoresis and blotting onto polyvinylidene difluoride membrane or from calpain solutions containing both subunits following concentration on a ProSpin cartridge (Applied Biosystems).
Calpain Large SubunitcDNA for the rat m-calpain 80-kDa subunit and for this subunit with a C-terminal His-tag (80k-CHis6), in the plasmid pET-24d(+), has been described previously (7). Natural calpain large subunits are N-terminally blocked, presumably with an acetyl group, but in E. coli the subunits are expressed unblocked, with or without an initiating methionine residue, according to the nature of the second residue (28). The novel constructs described below are listed in Tables I and II.
|
|
To provide an N-terminal
His-tag, the NcoI site at the initiation codon of 80 kDa was
converted to an NdeI site by site-directed mutagenesis. The
888-bp2 MluI-NdeI
fragment from pET-16b(+) was then ligated into these sites in
NdeI-80k. The resultant cDNA
(NHis10-80k) codes for an m-calpain large subunit with
an N-terminal leader peptide of 21 amino acids
(MGH10SSGHIEGRH-).
NHis10-80k-CHis6 was cloned by ligating the
1030-bp MluI-BamHI 5-fragment from
NHis10-80k into the same sites in
80k-CHis6.
To create a thioredoxin (TRX)
fusion protein with the m-calpain large subunit, a KpnI
linker (5-catgggtacc, which hybridizes to itself to generate
NcoI ends), was introduced at the NcoI site of
80k-CHis6. The 360-bp NdeI-KpnI
fragment from pTRXFUS (coding for thioredoxin (27)), the 2107-bp
KpnI-HindIII fragment from KpnI-80k-CHis6 (coding for m-calpain), and the
5246-bp NdeI/HindIII plasmid backbone from
pET-24b(+) were ligated, resulting in a thioredoxin-m-calpain-CHis6 construct
(TRX-80k-CHis6).
DNA coding for the N-terminal amino acid sequence of 80k-CHis6, MAGIAMKKDRE ... , was converted to sequence coding for MAGIAMKKDRE ... by means of site-directed mutagenesis, which introduced a DraI restriction site (tttaaa) into the cDNA at the new Phe9-Lys10 position. The initial purpose of this construct was to abolish the expected autolysis site at Ala9-Lys10 (23), but the construct was also used to make deletion mutants. The construct L8S, A9F-80k-CHis6 was generated in the same way.
Large Subunit N-terminal TruncationsThe construct L8F,
A9F-80k-CHis6 was digested with NcoI and
DraI, removing a (28 + 24-nucleotide) fragment, followed by
blunt ending with the Klenow fragment of E. coli DNA
polymerase and religation. The product codes for a large subunit
N-terminal sequence of MKDRE (M9-80k-CHis6) in which
the initiating methionine residue was retained on expression (as shown
by amino acid sequencing). This truncation was designed to approximate
the first autolysis product of m-calpain,
9-80k, which has the
N-terminal sequence KDRE (23). Several other N-terminally truncated
forms were made by digestion of L8F, A9F-80k-CHis6 with
NcoI and DraI, followed by insertion of pairs of
complementary oligonucleotides, giving rise to the N-terminal amino
acid sequences shown in Table II.
A second NcoI site was introduced into 80k-CHis6
at Gly19 (converting the expressed amino acid sequence
GLGSHERAIK to the sequence GLGSHERAIK) by site-directed
mutagenesis. A 60-bp fragment was removed by NcoI digestion,
and the plasmid was religated, generating a large subunit N-terminal
amino acid sequence expressed as AHERAIK (A20-80k-CHis6). This also was designed to approximate
a second reported autolysis product,
19-80k, which has the
N-terminal sequence SHERAIK (16, 23). All of these constructs had
C-terminal His-tags but some of them were cloned also without a
C-terminal His-tag.
Isolation of cDNA for the full-length
rat calpain small subunit and its expression both as an unmodified
28-kDa subunit and as an NHis10-28-kDa subunit have been
described elsewhere.3 The C-terminal domain
VI fragment of the rat calpain small subunit (Met87-Ser270), previously described as the
21-kDa subunit, is now referred to as 86.
The rabbit calpain small subunit was expressed with and without an NHis10 group, using a cDNA clone kindly provided by Dr. C. Crawford (University of Oxford).
Expression and AssayExpression, purification, assay, electrophoresis, and immunoblotting of calpain were performed as described previously (30).3
Titration of Ca2+ RequirementFor measurement
of the Ca2+ dependence of calpain activity, a modification
of the standard casein assay was used. The duplicate assays contained 2 mg of casein in a final volume of 0.5 ml of 0.2 M NaCl, 10 mM -mercaptoethanol, 50 mM Tris-HCl, pH 7.6, net final CaCl2 concentrations from zero µM
to 3.8 mM, and 20 µl of enzyme sample (containing in most
cases 5-7 units of activity). The mixtures were incubated at 25 °C
for 30 min; reaction was terminated by addition of 0.35 ml of ice-cold
10% trichloroacetic acid, and the A280 nm
values of the supernatants were recorded. For Ca2+
titration, the enzymes were freshly re-purified by chromatography on
MonoQ, in buffers containing 50 mM Tris-HCl and 2 mM EDTA. Precise comparisons were valid only between
enzymes purified on the same day and titrated in the same buffers. The
dissociation constant for Ca2+-EDTA at pH 7.6 was taken to
be 1.22 × 10
8
M.4 The Ca2+
concentration required for half-maximal activity with casein as
substrate is given as [Ca2+]0.5. This value
was calculated by fitting the normalized activity data to the equation
y = xn/(kn + xn), where y is the fraction of maximum
activity, k = [Ca2+]0.5,
n is the Hill constant, and x is
[Ca2+].
Calpain samples (0.5-2
µM, 50-200 µg/ml) in 2 mM EDTA, 10 mM -mercaptoethanol, 0.05-0.5 M NaCl, 50 mM Tris-HCl, pH 7.6, at 20 °C were adjusted with
CaCl2 to a net final concentration of 2.5 mM
Ca2+, and shaken, normally for 30-120 s, followed by
addition of EDTA to a net final concentration of 2 mM free
EDTA. In some cases, casein (5 mg/ml) was also present. Control samples
received the same volumes of EDTA first and CaCl2 second.
Samples were taken for Ca2+ titration, gel electrophoresis,
and blotting, or re-chromatography on a MonoQ column. Autolysis
conditions were chosen so that not more than 25% of the original
activity was lost.
Stability of calpain is defined here in a special sense, in terms of recovery of activity from the MonoQ column. Control and autolyzed calpain samples were assayed to determine the loss of total activity due to autolysis and then chromatographed on the MonoQ column in the presence of 2 mM EDTA. Non-autolyzed m-calpains were eluted in high yield from this column at approximately 0.4 M NaCl, but for some constructs the activity remaining after brief autolysis was recovered only in poor yield from the column.
Effects of Subunit Modifications on Formation of Active Heterodimeric Calpains
Many combinations of modified m-calpain large and small subunits
have been co-expressed. The yields of activity derived from 4 liters of
E. coli, as observed after the first purification step, are
given in Table I. The table also shows the specific activities of the purified calpains, which with one or two exceptions were all close to 1800 units/mg protein. The subunit compositions of
the purified calpains were checked by immunoblotting and by Coomassie
staining of SDS/Tris-Tricine gels, to establish their heterodimeric
character. The differing yields of activity in each co-expression
experiment appear to reflect primarily the relative levels of
expression of the two subunits, but the fairly constant specific
activities of the purified products show that the subunit modifications
in most cases did not affect heterodimer assembly or catalytic
function. However, some of the active heterodimers had properties that
were useful in the study of autolysis and [Ca2+]0.5. Much of the work has been done
with calpains having a C-terminal His-tag, since they were more highly
expressed, but the results have been duplicated in several cases with
calpains lacking the C-terminal His-tag. Table I also includes the
fully active construct L8F, A9F-80k-CHis6/86, in which
the autolysis site at Ala9-Lys10 has been
abolished, for comparison with L8S, A9F-80k-CHis6/
86, which was surprisingly inactive.
Steric Factors in Co-expression
As described elsewhere,3 both the unmodified 28-kDa or
NHis10-28k were less well expressed than 86 but were
able to form active enzymes with 80 kDa. Conversely,
NHis10-80k formed active enzyme with
86. However when
both large and full-length small subunits contained N-terminal
extensions (NHis10-80k/NHis10-28k or
TRX-80k-CHis6/NHis10-28k), very little activity
was formed and was not stable to further purification (Table I).
Immunoblots of the crude extracts showed that the subunits were
sufficiently expressed, and particularly the TRX-construct was highly
expressed as predicted (not shown) (27). These results indicate that
the two N-terminal extensions caused a steric interference to
heterodimer formation and suggest therefore that the N termini of both
subunits must be close to each other in normal m-calpain. It may be
noted here also that the structure of domain VI1 strongly
suggests that the C termini of the large and small subunits are also
close together.
Measurement of Ca2+ Requirement for Casein Hydrolysis
Fig. 1 shows measurements of
[Ca2+]0.5 of 80k-CHis6/86 for
casein hydrolysis, as a function of the concentration of NaCl and of
Tris buffer at pH 7.6. The effects of KCl were identical to those of
NaCl. The increases in buffer and salt concentrations raised the value
of [Ca2+]0.5 progressively from 175 to 436 µM. A very similar dependence of
[Ca2+]0.5 on ionic strength was observed with
several other recombinant calpains that were tested and also with
partially purified natural rat m-calpain. Variation of pH between 7.2 and 7.8 (around the pH optimum for casein hydrolysis (30)) had little
effect, but the value of [Ca2+]0.5 was
greatly increased at pH 8.5 (not shown). The Ca2+
concentrations of the solutions were checked by flame photometry, and
we confirmed the observation (19) that dialysis of the casein substrate
against EDTA had no effect on [Ca2+]0.5
values. All subsequent Ca2+ titrations were therefore
performed in 50 mM Tris-HCl, 0.2 M NaCl, pH
7.6.
Small Subunit Autolysis, [Ca2+]0.5, and Heterodimer Stability
Expression of m-calpain with an NHis10-28k small
subunit (in fact a mixture of partially degraded small subunits) and
autolysis of this heterogeneous small subunit to a homogeneous 20-kDa
small subunit have been described previously.3 In
80k-CHis6/NHis10-28k, small subunit autolysis
was complete within 1 min (as shown later, Fig. 4) and did not cause a
fall in [Ca2+]0.5 (Fig.
2a). Since this autolysis was done in 0.4 M NaCl, as discussed later, large subunit autolysis can be
neglected in this experiment; the result therefore demonstrates that
small subunit autolysis alone does not contribute to the fall in
[Ca2+]0.5 in this m-calpain.
The same conclusion can be drawn from a separate experiment. The
recombinant 86 small subunit and the natural 20-kDa autolysis product of the small subunit are essentially identical
(31).3 80k-CHis6/
86 therefore closely
resembles the product expected if only the small subunit were autolyzed
on exposure of 80k-CHis6/NHis10-28k to
Ca2+, and there was no significant difference in
[Ca2+]0.5 between 80k-CHis6/
86
and non-autolyzed 80k-CHis6/NHis10-28k (Fig.
2b).
Intermolecular Autolysis of the Small Subunit
To investigate the relative contributions of inter- and
intramolecular reaction to autolysis of the small subunit, autolysis experiments were performed with mixtures of active and inactive calpains. In this experiment only, the full-length small subunits were
from rabbit, and it should be noted that the monoclonal antibody (kindly provided by Dr. R. Mellgren, University of Toledo) used to
observe the small subunit does not react with the rat small subunit. No
other difference has been detected between calpains composed of the rat
m-calpain large subunit and rabbit or rat calpain small
subunits.5 As shown in Fig.
3, in a mixture of equal molar amounts of the inactive
mutant
C105S-80k-CHis6/rabbit-NHis10-28k
with active 80k-CHis6/86, the heterogeneous small
subunits of the inactive calpain were converted to a homogeneous 20-kDa
form within 1 min, and this reaction could only be intermolecular
(tracks 1-3). Autolysis of the small subunit in active
80k-CHis6/rabbit-NHis10-28k was also
complete within 1 min (tracks 4-6), but in this case it is not
possible to distinguish between inter- and intramolecular reaction. In
the control experiment,
C105S-80k-CHis6/rabbit-NHis10-28k alone was not autolyzed (tracks 7-9).
Effect of Casein on Subunit Autolysis
Fig. 4 shows the autolysis for 0-8 min in 0.25 M NaCl with and without casein of (a) the large
subunit in NHis10-80k/86 and (b) the
heterogeneous small subunit in
80k-CHis6/NHis10-28k. In each case it is
evident that autolysis was slightly retarded by the presence of 5 mg/ml
casein (but was still essentially complete after 1 min), which is
consistent with some contribution by intermolecular reaction to
autolysis of both subunits. This effect of casein on autolysis of the
large subunit was evident also in the Ca2+ titrations
described below.
Large Subunit Autolysis and [Ca2+]0.5
In the study of large subunit autolysis, conditions were chosen so that the calpains did not lose more than 25% of their starting activity, since it was felt that normalization of Ca2+ titration data obtained after very extensive autolysis might be misleading. It was found that the effects of autolysis of the various calpains on their [Ca2+]0.5 for casein hydrolysis depended both on the experimental conditions during autolysis and also on the differences in the recombinant large subunits.
Effect of High [NaCl]Autolysis of 80k/86 in 0.4 M NaCl (as eluted from the Q-Sepharose column, the final
step in purification) caused loss of activity, showing that the calpain
was active, but did not change [Ca2+]0.5. The
titration curves are not shown since they were identical to those in
Fig. 2. This result was unexpected but was highly reproducible. The
activity remaining after autolysis of 80k-CHis6/
86 in
0.4 M NaCl was recovered in high yield from
re-chromatography on MonoQ, and this material had clear N-terminal
sequence showing that both subunits were intact. This result shows that
m-calpain is active without large subunit autolysis, a point that was
established even more rigorously with the L8F, A9F mutant (see below).
Partially purified natural rat m-calpain also showed no change in
[Ca2+]0.5 on autolysis in 0.4 M
NaCl (data not shown).
When 80k/86 was autolyzed in 0.18 M NaCl, the value of
[Ca2+]0.5 showed the expected fall; in the
presence of 5 mg/ml casein, about 270 s of autolysis were required
to reach the lowest value of [Ca2+]0.5,
falling from 324 to 186 µM (Fig.
5a). A comparison of autolysis of 80k/
86
for 120 s in 0.18 M NaCl with and without casein
showed that casein slightly retarded the conversion to a
low-Ca2+ requiring form, from 338 µM at time
0 to 292 µM in the presence of casein and to 246 µM in the absence of casein (Fig. 5b).
N-terminal sequencing of 80k-CHis6/
86 after incubation
with Ca2+ for 10 min gave a mixture of three sequences,
representing approximately 33% intact large subunit (AGIAMKL), 66%
autolyzed large subunit (KDREAA), and 100% intact small subunit. This
cleavage at Ala9-Lys10 is in agreement with a
previous report (23).
Effect of the N-terminal His-tag
In contrast to the behavior
of 80k/86, the fall in [Ca2+]0.5 on
autolysis of the N-terminally extended NHis10-80k/
86 at
Ala9-Lys10 was equally rapid in high or low
[NaCl], or with or without casein (Fig. 6), and the
small drop in molecular mass of the large subunit could be readily
detected on gels (see above, Fig. 4). The large subunit of autolyzed
NHis10-80k/
86 was N-terminally sequenced after gel
electrophoresis and blotting, giving the sequence KDREAA which results
from cleavage at Ala9-Lys10 (strictly
Ala30-Lys31 in this construct). Over longer
periods of autolysis, the remaining activity correlated with
densitometry of the large subunit (Fig. 7), and since
the large subunit after 30-60 s is 100% cleaved at
Ala9-Lys10, the activity after this time was
therefore clearly a property of the autolyzed calpain. These results
show that the lower [Ca2+]0.5 in m-calpain is
a property of the
9-80k large subunit.
Effect of Abolishing the Autolysis Site
Calpain does not
possess a strong consensus cleavage sequence to direct our choice of
mutation, but there are very few examples of cleavage with a
phenylalanine residue in the P1 position (1). In accordance
with this, the autolysis site mutant L8F, A9F-80k/86 showed no
significant change in [Ca2+]0.5 under any
autolysis conditions (Fig. 8). This result demonstrates clearly, as also suggested earlier, that calpain with an intact large
subunit is active in casein hydrolysis without autolysis.
Large Subunit Autolysis and Calpain Stability
After quenching of the autolysis reaction with excess EDTA, the
remaining activity of the various calpains was stable for some hours at
0 °C. The results in Table III suggest, however, that the autolysis
product 9-80k/
86 is relatively unstable during purification on
MonoQ, which includes elution at approximately 0.4 M NaCl.
Incubation of 80k-CHis6/
86 with 2.5 mM
Ca2+ in 0.4 M NaCl causes loss of activity with
time but does not involve cleavage at
Ala9-Lys10, and the subsequent MonoQ recovery
was high (84%); autolysis of several constructs in conditions where a
relatively slow fall in [Ca2+]0.5 occurs and
where the conversion to
9-80k/
86 was therefore incomplete was
followed by intermediate recoveries from MonoQ (40-60%); and
autolysis of NHis10-80k-CHis6/
86, which was
complete, gave much lower recoveries (20%). In the latter case, it was
shown that the active material recovered in low yield from MonoQ was
9-80k-CHis6/
86 (Fig. 9).
|
N-Terminal Truncation of the Large Subunit in m-Calpain
The treatment of the initiating methionine residue in E. coli does not permit expression of the previously suggested large subunit autolysis products, 9-80k and
19-80k, with the desired free N-terminal amino acids (28). As a close approximation, the
constructs M
9-80k-CHis6/
86,
8-80k-CHis6/
86, and
A
20-80k-CHis6/
86 were all expressed but were
inactive (Table II), although the proteins could be
isolated as heterodimers in average amounts (not shown). The excised
N-terminal residues were then progressively reintroduced but did not
restore activity until
2-80k-CHis6/
86. The presence
or absence of the C-terminal His-tag had no effect on these
observations. At present we have no explanation for these results.
At the beginning of this work we found it difficult to reconcile
the extensive but sometimes inconsistent literature on
[Ca2+]0.5 and autolysis in m-calpain. It has
now become clear that the observed values of
[Ca2+]0.5 are highly dependent on
experimental conditions. It should be noted also in the following
discussion that autolysis of calpain involves not only the generation
of the 93-small subunit and the
9-large subunit, in which form
the enzyme is active and has a lower
[Ca2+]0.5, but also involves cleavage of the
large subunit to smaller fragments that are no longer active (32, 33).
The relative rates of limited N-terminal autolysis and of inactivating
autolysis are also dependent on the construct and on the ionic
strength. Together these observations appear to explain much of the
wide variation in [Ca2+]0.5 reported over the
last 15 years from different laboratories.
With
80k-CHis6/86,in the absence of any prior autolysis, it
was found that [Ca2+]0.5 was highly dependent
on the concentration of Tris buffer and of NaCl (Fig. 1). This was true
also for several different calpain constructs and for partially
purified natural rat m-calpain. Within the range of pH 7.2-7.8, the
effect of pH was not significant, but at higher pH values
[Ca2+]0.5 increased significantly. These
effects of ionic strength and pH on [Ca2+]0.5
for m-calpain have not, to our knowledge, been reported previously.
To consider first small subunit
autolysis, we have shown that incubation of
80k-CHis6/NHis10-28k with Ca2+
caused rapid autolysis of the small subunit. This was easily seen on
blots (Fig. 4) but did not alter [Ca2+]0.5
(Fig. 2a). In this experiment, since autolysis was performed in 0.4 M NaCl, the large subunit was not cleaved. The
result was confirmed by the finding that
80k-CHis6/NHis10-28k and its hypothetical small
subunit-only autolysis product, 80k-CHis6/86, had
identical [Ca2+]0.5 (Fig. 2b).
These results demonstrate unequivocally that autolytic removal of
domain V does not affect [Ca2+]0.5 of
m-calpain. Domain V of the small subunit is clearly not required for
calpain activity or for formation of active calpain as a foreign
protein in E. coli, although it presumably has some role in
calpain regulation in eukaryotic cells. It may be noted also that
removal of domain V does not by itself cause instability in m-calpain,
since 80k(±CHis6)/
86 is stable through several steps of
purification.
We have not studied the question of whether small subunit autolysis is strictly required, as seems likely, before large subunit autolysis or before casein hydrolysis. It would be interesting to attempt to generate mutant full-length small subunits that are resistant to autolysis to prove that removal of domain V is the essential first step. This may be difficult in practice, since domain V appears to be prone to hydrolysis at several sites.3
Small subunit autolysis has been described both as intramolecular, on the basis of kinetic observations (19, 34), and also as intermolecular (22, 35). The experiments reported here with inactive calpain showed that intermolecular autolysis of the small subunit can occur at least as rapidly as any other autolysis step in this work (Fig. 3). In active m-calpain, small subunit autolysis was slightly retarded in the presence of casein, which also suggests some contribution by an intermolecular mechanism (Fig. 4). While the intermolecular reaction is clearly feasible in vitro, intramolecular reaction may be more likely in vivo, where the calpains are "relatively dilute and surrounded by potential substrates" (35).
Large Subunit Autolysis in m-CalpainIt has frequently been reported that incubation of natural m-calpain with Ca2+ causes a fall in Ca2+ requirement for hydrolysis of casein or other substrate, without any apparent change in the large subunit that can be detected on gels (2, 23, 24). As a result of this difficulty, the fall in [Ca2+]0.5 was at first attributed to small subunit autolysis alone (34) and later to cleavage of the large subunit either at Ala19-Ser20 (16) or at Ala9-Lys10 (23).
The construct NHis10-80k/86 has the advantage that its
autolysis can be easily seen on gels by Coomassie staining or by
immunoblotting (Figs. 4, 9), in a manner highly reminiscent of
autolysis in µ-calpain (22, 26, 36). It should be noted that the pre-
and post-autolysis values of [Ca2+]0.5 are
the same for NHis10-80k/
86 as for 80k/
86 and that the autolysis product
9-80k/
86 is identical to the autolysis product of natural m-calpain, except for 7 N-terminal amino acids in the small
subunit that do not affect its properties.3 These results
demonstrate that the lower [Ca2+]0.5 in
m-calpain is a property of the
9-80k large subunit, so that the
value of [Ca2+]0.5 can be used as a reliable
index of large subunit autolysis at Ala9-Lys10.
Autolysis at Ala9-Lys10 in
NHis10-80k/86 is essentially complete in about 50 s
in any conditions (data not shown), although the autolysis shown in
Fig. 6 was for 2 min to permit comparison with other experiments. This
is significantly faster than autolysis of 80k/
86, as measured by the
fall in [Ca2+]0.5, which in the presence of
0.18 M NaCl and 5 mg/ml casein required approximately
270 s for completion (Fig. 5a). It has been reported
elsewhere that large subunit autolysis in m-calpain was extremely slow,
but the measurements depended on antipeptide antibodies that may not
have been sufficiently diagnostic of autolysis (22).
The delay in autolysis in the presence of casein (Fig. 5b) suggested that large subunit autolysis in m-calpain could also be at least to some extent an intermolecular reaction, just as for the small subunit. In keeping with this idea, it has recently been shown that µ-calpain could autolyze m-calpain in vitro, suggesting a cascade mechanism for in vivo activation of m-calpain (37). In that report, m-calpain activation was stated to be due to small subunit hydrolysis, and a fall in [Ca2+]0.5 of m-calpain was observed from 160 to 64 µM (measured in 10 mM HEPES, compare the value of 175 µM in 50 mM Tris in Fig. 1). We have shown, however, that small subunit autolysis does not affect [Ca2+]0.5, and in our view this fall in [Ca2+]0.5 was undoubtedly due to large subunit autolysis that could not be detected on gels.
Activity of Intact m-CalpainIt is evident that intact
m-calpain must be active in the first steps of its own autolysis, and
it has been suggested previously that intact m-calpain (at least with
an intact large subunit) could hydrolyze substrates such as casein (17,
20, 24). This point has now been rigorously proven with the mutant L8F, A9F-80k/86, which hydrolyzes casein with the same specific activity and [Ca2+]0.5 as the other calpains
described; on exposure to Ca2+ it loses activity (by
inactivating cleavage of the large subunit) but does not undergo a fall
in [Ca2+]0.5 and therefore does not undergo
N-terminal autolysis (Fig. 8). The same conclusion could be drawn from
the activity of 80k/
86 in 0.4 M NaCl, where N-terminal
autolysis also does not occur.
It was reported previously
that autolyzed m-calpain was less stable to dialysis at high ionic
strength than at low ionic strength (23). This is reflected in our
finding that 9-80k/
86 formed by autolysis of various constructs
was very poorly recovered from the MonoQ column, which requires elution
at 0.4 M NaCl (Table III). We have made a
related observation that the stability of the heterodimer containing an
intact large subunit and an N-terminally truncated small subunit
(80k/NHis-
116) was also reduced at high ionic strength.3
Both these observations imply that salt links contribute to subunit binding. Autolyzed calpain is clearly not exposed to 0.4 M
NaCl in vivo, but the loss of stability may still have
in vivo implications that remain to be explored. Loss of
9-80k/
86 in the presence of EDTA suggests that it is prone to
processes such as subunit dissociation (3) and aggregation. The
constructs M
9-80k/
86 and
8-80k/
86 were designed as models
of the natural autolysis product and were expressed at average levels
as heterodimers. They were, however, inactive and stable to
purification and therefore differ in some way from the natural
autolysis product. They may be in some way improperly folded, or the
free N-terminal lysine residue at position 10 may be especially
important, but this point requires further work.
These studies have clarified a number of aspects of m-calpain biochemistry. For purified m-calpain in vitro, the pathways and rates of autolysis, and also the observed values of [Ca2+]0.5, are highly dependent on the experimental conditions. Autolysis of the small subunit in m-calpain in the presence of Ca2+ is rapid, is slightly retarded in the presence of casein, and does not affect either [Ca2+]0.5 or the stability of calpain. m-Calpain is fully active prior to autolysis of the large subunit, but in vitro at an approximately physiological ionic strength the intact large subunit has a short half-life in the presence of Ca2+, and it is assumed that the same is true in vivo. Autolysis of the large subunit at Ala9-Lys10 may be a little slower than that of the small subunit and is also slightly retarded by the presence of casein, showing that at least part of the large subunit autolysis reaction is intermolecular. Although autolysis of both subunits can occur by intermolecular reaction, there are some grounds for believing that the reaction in vivo may be mainly intramolecular. Cleavage at Ala9-Lys10 is responsible both for the fall in [Ca2+]0.5 and for the reduction in stability of the autolysis product. It is clear, however, that factors either in addition to, or other than, autolysis are involved in lowering [Ca2+]0.5 in vivo (1, 2), and calpain autolysis products cannot be detected in normal tissue extracts and must, therefore, be very rapidly cleared after activation (20). In our view, the physiological importance of autolysis is probably that it generates forms of active calpain with short half-lives, resulting from their lower [Ca2+]0.5, their instability, and their further auto-degradation, thus limiting the duration of calpain activity in vivo.
We are grateful to Dr. P. L. Davies for frequent helpful discussions and to S. Gauthier for highly skilled assistance.