| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Neurobiology Department,*
University Medical
School, Newcastle upon Tyne, United Kingdom;
Généthon,
Évry, France;
Institut de Myologie,
Hôpital de la
Salpêtrière, Paris, France; Abteilung
Neuropädiatrie,§
Kinderklinik,
Humboldt-Universität, Berlin, Germany; Unitat de Genètica
Molecular,¶ Hospital de la Santa Creu i
Sant Pau, Barcelona, Spain; and Unitat de Patologia
Neuromuscular,||
Servei de Neurologia, Hospital Sant Joan
de Déu, Barcelona, Spain
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-, 
-, ß- or 
-sarcoglycan on
chromosomes 13q12, 17q12, 4q12, and 5q33, respectively.1-4
Among the dystrophies in which expression of the sarcoglycans is
normal, the gene responsible for LGMD2A has been identified as the
chromosome 15q15.1 to q21.1-encoded muscle-specific calcium-activated
neutral protease (CAPN3), calpain 3.5
The
genes for LGMD2B and LGMD2G have been localized to 2p136
and 17q11,7
and further recessive limb-girdle genes have
been inferred.7,8
The various forms of limb-girdle dystrophy are very difficult to
differentiate on clinical grounds alone.9
Thus, testing for
defective protein expression is a very useful way of determining which
gene or genes are at fault and therefore where to start the search for
gene mutations.10
Unlike the large multiexonic deletions
that are the most common type of mutation in Duchenne and Becker
dystrophy, the limb-girdle dystrophies typically have mutations that
are smaller and more difficult to detect. The sarcoglycan group has
very close interactions, and a mutation in one member protein may cause
an absence of protein expression for all four.4,11
As more
patient biopsies are examined, however, a common observation
(particularly in -sarcoglycanopathy) appears to be the deficiency or
absence of labeling for one member accompanied by a more moderate
reduction in the others.12-15
In these cases the protein
with the most severely altered expression has proved to be the one with
the mutated gene, indicating that protein analysis may be useful even
in the sarcoglycanopathies.10,15
Unlike the other forms of LGMD, LGMD2A is not caused by defective expression of a structural protein, but by mutations in the gene for a soluble proteolytic enzyme. The calpain family of proteins contains three ubiquitous enzymes that have been studied extensively (see reviews in 16 and 17 ). They were named µ-calpain (calpain 1), m-calpain (calpain 2), and intermediate µ/m-calpain, after their distinct sensitivities to Ca2+ ions. All are dimers composed of distinct large catalytic subunits (around 80 kd) plus a shared regulatory small subunit of 30 kd. The large calpain subunits have four functional domains.16 The N-terminal region of domain I is autocatalytically processed during activation by Ca2+ ions, suggesting that domain I is involved in the regulation of activity. Domain II is considered to be the cysteine protease module, and no function has yet been assigned to domain III. Domain IV contains structures for calcium binding, and thus this domain may be involved in the calcium activation of the calpains.
The founder members of this family have now been joined by an increasing number of tissue-specific calpains, but their precise roles remain uncertain.17 The muscle-specific form, calpain 3, differs in structure from the ubiquitous forms by having three unique insertions (designated NS at the beginning of domain I, IS1 in protease domain II, and IS2 in domain III) which increase the molecular mass from 80 to 94 kd. Calpain 3 was originally named "p94" from this size.18 The mRNA of a calpain 3 variant, predicted to code for an 82-kd protein, has recently been found in the lens of both rat19 and mouse (M. Herasse et al, manuscript in preparation). This appears to differ in that the NS, IS1, and IS2 muscle-specific sequences are spliced out.
The use of calpain 3 protein expression to detect mutations in the CAPN3 gene for the diagnosis of LGMD2A has just started. Spencer et al20 reported the use of polyclonal antibodies that recognized calpains 1, 2, and 3 in skeletal muscle to differentiate LGMD2A samples from others in a "blind" study. Here we report the first production of monoclonal antibodies to calpain 3, the characterization of their reactivity, and their use in analyzing protein expression in a group of LGMD2A patients with known mutations and clinical profiles.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two synthetic peptides from the published human CAPN3 sequence5 were conjugated to keyhold limpet hemocyanin via an additional C residue and used to immunize CD1 mice. One peptide contained amino acids 1 to 19 at the N terminus (MPTVISASVAPRTAAEPRS-C) in the calpain 3-specific NS domain, and the other consisted of amino acids 355 to 370 (C-RLRNPWGQVEWNGSWS) in protease domain II, which is a region of sequence conservation between calpains 1, 2, and 3. This peptide corresponded to the human version of the chicken sequence used previously to raise polyclonal antibodies.20 The mice were immunized over a period of 6 months, during which time several tail bleeds were taken, and mice were killed for unsuccessful fusion experiments. The experiments were conducted under a British Home Office license, and at the end of the specified 6-month time limit, the remaining mice had to be killed. The mice were therefore boosted before being killed, the splenocytes were frozen in medium containing 20% fetal calf serum + 10% dimethyl sulphoxide, and the cells were stored in liquid nitrogen. The final successful fusions, reported here, were performed on spleen cells that were rapidly thawed before immediate fusion with X63.Ag8.653 cells (assumed recovery of 5 x 107 viable splenocytes per spleen, with 5 x 106 myeloma cells). The antibody supernatants were screened on strips from Western blots of human muscle extracts. No significant labeling was obtained on unfixed frozen tissue sections with any of the antibodies. The cells in "positive" wells, which labeled bands of the correct size on Western blots, were cloned at least four times at 0.5 cells/well to ensure monoclonality. Specificity to calpain 3 was determined by loss of band reactivity in muscle from patients known to have null mutations in that gene.
Electrophoresis and Western Blotting
Standard buffers for electrophoresis and blotting were employed,21 although we now routinely use a biphasic system that is optimized to permit resolution of all the known muscular dystrophy proteins on a pair of gels/blots.22 Thus, the lower half of the gel contained 7% acrylamide (for resolving calpain 3, merosin, and the sarcoglycans, in the molecular mass range of 30 to 100 kd), whereas the upper half contained a gradient of 5.5 to 4% (for resolving myosin heavy chain and dystrophin in the range of 200 to 400 kd). A 3% stacking gel was used. The frozen tissue samples were weighed and kept frozen until homogenized with 19 volumes of electrophoresis treatment buffer containing 4% sodium dodecyl sulphate and 4 mol/L urea (no additional protease inhibitors). Lanes of control muscle (with no fat or fibrous connective tissue) typically contained 200 µg of protein.21 After electrophoresis the gels were blotted, labeled with the antibodies (Calp3c/11B3 used undiluted, Calp3d/2C4, and Calp3c/12A2 diluted 1:10) followed by a peroxidase-conjugated secondary antibody, and visualized with hydrogen peroxide and diaminobenzidine.21
Densitometric Analysis
Dried gels and blots were scanned at 400 dpi on an Epson GT8000
flatbed scanner using white light for gels and blue for blots. Each
image was stored as a bit map where 8 bits = 1 byte = 1
pixel. Each pixel was graded from 0 (pure black) to 255 (pure white) on
a 256 grayscale. Grayscale is not proportional to concentration, but
optical density (OD) is according to Beer's law, and is therefore the
proper measure for use in quantitation. Software that was written by
one of us (KD) for the Optimas v5.2 image analysis package was used for
the densitometric analysis. In this, grayscale values were converted to
OD units by scanning a Kodak SR-37 step wedge that has 37 steps of OD
values from 0.05 to 1.85. The grayscale values measured for each step
were then plotted against the true OD values, so that the scanner was
calibrated as a true densitometer. Subsequently, the grayscale value
for each pixel was automatically converted to an OD value. The outline
of each band was defined by a software algorithm involving background
measurements, to obtain a value for volume OD where:
 | (1) |
Pathological muscle samples contain a variable amount of fat and fibrous connective tissue. Therefore, myosin heavy chain staining on the postblotted gel was used as an indication of how much true muscle protein was loaded in each sample. Thus, the volume OD for each protein band in a sample was divided by the corresponding value for myosin heavy chain in that sample, to produce a density value that was "normalized volume OD."22
Human Muscle Samples from Normal and Disease Control Patients
Biopsies from patients with different neuromuscular diseases were obtained as part of routine diagnostic procedures at Newcastle General Hospital. These were stored in liquid nitrogen until required. More than 70 samples from patients with neuromuscular diseases have now been examined. Samples of rectus abdominis muscle were obtained, with informed consent, from normal control subjects undergoing a variety of abdominal operations. Control samples were also obtained from amputated legs. Altogether, samples from 33 different individuals without neuromuscular disease were examined.
LGMD2A Patients and Their Mutations
Nine patient biopsies from eight families with LGMD2A (from Réunion Island, Germany and Spain) were obtained for diagnostic procedures. Mutation analysis was undertaken at Généthon in Paris,5,23 Rudolf Virchow Klinikum, Humboldt-Universität, Berlin,24 and Unitat de Genètica Molecular, Hospital de la Santa Creu i Sant Pau, Barcelona. Clinical assessments were made by MF, CH, and JC.
Immunoreactivity of Calpain 3 Antibodies in Different Animals and Tissues
Because fresh human tissues are difficult to obtain, samples from a number of different animals were analyzed. Thus, skeletal muscle samples from human, mouse, rat, rabbit, dog, chicken, hamster, and pig were compared on the same blots labeled with calpain 3 antibodies. Subsequently, we examined samples of rabbit skeletal muscle, vas deferens, heart, brain, spinal cord, thymus, lung, kidney, spleen, and liver, and rat skeletal muscle, stomach, uterus, heart, brain, lung, kidney, spleen, and liver.
Stability of Calpain 3 Protein in Muscle
Three sets of experiments were conducted to investigate the stability of calpain 3 in normal human rectus abdominis muscle and to elucidate the nature of the lower molecular mass bands recognized by the antibodies.
Time before Freezing Fresh Tissue
A sample of fresh rectus abdominis muscle was taken and divided into eight portions (each ~50 mg) in a lidded Petri dish lined with filter paper moistened with phosphate-buffered saline. The samples were left at room temperature to emulate conditions that might occur if a biopsy sample were taken in a distant hospital and sent to our laboratory for processing and freezing. The fresh muscle samples were snap frozen, by dropping them in liquid nitrogen, at time points designated T0 (the quickest achieved, actually about 10 minutes after leaving the body), 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, and 8 hours.
Time Thawed before Homogenization
A sample of frozen rectus abdominis muscle was broken with a brass pestle and mortar chilled in liquid nitrogen. Pieces of tissue, each weighing between 25 and 30 mg, were left in tubes to thaw at room temperature for different lengths of time before homogenization. The time points were designated T0 (homogenized frozen), 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, and 2 hours.
Time as Homogenate in Saline
A sample of frozen rectus abdominis muscle was homogenized in an equal volume (1 g/1 ml) of sterile saline (Sterets Normasol, Seton Prebbles, Oldham, UK). The homogenate was left for different lengths of time at room temperature before 100-µl samples were taken and mixed with an equal volume of double-strength electrophoresis treatment buffer, before proceeding with boiling and centrifugation as normal. This resulted in a sample of 50% normal protein loading.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Three different monoclonal antibodies were isolated. One,
designated Calp3d/2C4, binds to an epitope within amino acids 1 to 19
in exon 1 of the human sequence and recognized a single band at ~94
kd plus an additional clear band at about 30 kd in blots of human
skeletal muscle (Figure 1)
. The other two
bind to epitopes within amino acids 355 to 370 in exon 8. One,
designated Calp3c/11B3, recognized at least four bands from 94 to 80 kd
(see expanded view in Figure 1
) corresponding to calpains 1, 2, and 3,
exactly like the polyclonal antiserum used in Spencer et
al.20
The other, designated Calp3c/12A2, only recognized
the uppermost calpain 3 band. Both of the exon 8 antibodies also
recognized a series of bands from a fairly clear 60 kd down to a more
fuzzy 45 kd (Figure 1)
. The immunoreactivity of each antibody was
abolished by adsorption with the appropriate immunizing peptide,
whereas adsorption with the other peptide had no effect. Analysis of
the clinical samples was undertaken with the two calpain 3-specific
antibodies only. The results with muscle from LGMD2A patients with null
mutations (see below) demonstrated that the bands at 94 kd, ~60 kd,
and 30 kd all represent products of the CAPN3 gene.
|
With the N-terminal antibody Calp3d/2C4, protein bands of the same
size as the 94-kd calpain 3 band in humans were observed in rabbit,
dog, and hamster, but immunoreactivity with a larger peptide
(approximately 110 kd) was also seen in hamster, and in rat. No
reactivity was seen in mouse, chicken, or pig (Figure 2
, top). The 30-kd fragment was clearly
visible in human muscle, but it was only weak in dog and rabbit. In the
various rabbit tissues, the 94-kd calpain 3 band was only observed in
skeletal muscle, although a variety of bands of other sizes were
detected in thymus, brain, kidney, and spleen (results not shown). The
~110-kd protein was only present in rat skeletal muscle and heart,
with a slightly larger form in brain (results not shown).
|
, middle).
The exon 8 antibody Calp3c/12A2, recognized the 94-kd calpain 3 band in
all species tested. It appeared to be specific in human, rabbit, dog,
and pig skeletal muscle, but some additional bands corresponding in
size to calpains 1 and/or 2 were detected in skeletal muscle from
mouse, rat, chicken, and hamster (Figure 2
, bottom). As with antibody
Calp3c/11B3, the group of calpain 3 breakdown bands starting at ~60
kd appeared to be present in all the animal muscle samples tested.
In the various rabbit tissues, the 94-kd calpain 3 band was only observed in skeletal muscle, although bands of other sizes were detected in thymus and spleen. Some clear immunoreactivity with myosin heavy chain was observed in the smooth muscle of rabbit vas deferens and more weakly in the skeletal muscle of all species examined (results not shown). The identity of the myosin heavy chain band was suggested by its very characteristic size (200 kd) and shape on blots.
Stability in Human Muscle
Protein bands that are smaller than expected are usually
degradation bands, and we undertook some deliberate muscle breakdown
experiments to determine whether there was a reciprocal relationship
between the abundance of the full-size and lower molecular mass bands.
Some selected time points from three experiments are shown in Figure 3
. For the N-terminal antibody,
Calp3d/2C4 (Figure 3
, top), there was little significant change in the
band profiles, with muscle kept up to 8 hours before freezing and up to
2 hours postthawing, with the 94-kd band showing no obvious drop in
abundance. In the saline homogenate experiment, the 94-kd and 30-kd
bands disappeared in parallel. For the exon 8 antibody, Calp3c/12A2
(Figure 3
, bottom), there was little change in the 94-kd band intensity
up to 8 hours before freezing and up to 2 hours postthawing. In both
experiments, however, there was a clear shift in the ~60-kd band
pattern, with bands around 45 kd becoming more intense. In the saline
homogenate experiment, the 94-kd band became progressively fainter,
whereas the lower bands (intense even at the T0
time point) shifted from ~60 kd down to ~45 kd.
|
Clinical Samples
Samples from 33 normal control subjects showed the described
profile of bands with almost no variation (see Figure 4
, lanes 1, 3, 5, 17, and 20). A similar
pattern of reactivity was found in 12 patients with Duchenne MD; 5 with
Becker MD; 7 with facioscapulohumeral MD; 4 with sarcoglycanopathy; 1
with Emery-Dreifuss MD; 3 with merosin-associated congenital MD; 4 with
myopathy accompanied by a secondary deficiency of laminin ß1 chain
expression (as in 25 and 26
), 6 adult limb-girdle patients
showing a secondary merosin deficiency on blots,8
and 8
patients with one of the inflammatory muscle diseases, polymyositis or
dermatomyositis. Some of these disease control samples are shown in
Figure 4
, lanes 7, 9, 12, 14, and 16.
|
, lane 15). No mutation has been found to date in this
patient, with all exons having been sequenced. No blood or tissue
samples were available from other family members to test for linkage to
chromosome 15, and there was no evidence of consanguinity.
A summary of the clinical details from the LGMD2A patients is shown in
Table 1
. The results of immunoanalysis
and gene mutation analysis are summarized in Table 2
, with Figure 4
illustrating the blot
lanes. Labeling of the 94-kd band was undetectable on visual inspection
in patients 1a, 1b, and 4 (Figure 4
, lanes 2, 13, and 11). In patients
2, 3, 6, and 8, full-size protein was represented by weakly labeled
bands (Figure 4
, lanes 4, 10, 6, and 19). The lower molecular mass
bands (30 kd and ~60 kd) were greatly reduced or absent in all these
patients, indicating that these bands represented calpain 3
polypeptides derived from the same CAPN3 gene and not
distinct cross-reactive proteins. A very different pattern of protein
expression was seen in patients 5 and 7 (Figure 4
, lanes 8 and 18).
Here the 94-kd band was indistinguishable from normal, but the lower
molecular mass bands were clearly reduced in intensity. Table 3
shows the densitometric analysis of the
calpain 3 bands labeled with the exon 1 antibody Calp3d/2C4. The
density of the 94-kd band in patient 5 (0.079) and patient 7 (0.155)
was within the range seen in control subjects (0.042 to 0.133). In
these controls, the values for 30-kd band density expressed as a
percentage of 94-kd band density ranged from 45 to 95%. In patients 5
and 7 this was reduced to 18% and 14%, whereas in the ?LGMD patient
(Figure 4
, lane 15), it was 9%. Thus, the protein profile of two
LGMD2A patients could not be differentiated from a ?LGMD patient.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This is the first time that potentially diagnostic monoclonal antibodies to human calpain 3 have been reported. This paper includes details of antibody characterization with respect to blot band patterns and species reactivity, because this is important for antibodies that will be available commercially for use with animal models as well as human diseases. There have been few reports of the immunological detection of calpain 3 in skeletal muscle, and early studies suggested that, although the mRNA was abundant in muscle, the protein had a very short half-life because of destruction by autolysis.27 More recently, polyclonal antibodies that recognize calpains 1, 2, and 3, have shown that calpain 3 is detectable in mouse28 and human20 skeletal muscle.
Three different monoclonal antibodies have been produced. One is to an
epitope within amino acids 1 to 19 in exon 1 in the calpain 3-specific
NS domain. This sequence shows few differences between species, and the
lack of immunoreactivity to rat, mouse, and pig calpain 3 suggests that
the epitope may span amino acids 7 and 8 or 14, which are the only
differences in the human sequence (Figure 5)
. The other antibodies are to two
different epitopes in exon 8 in protease domain II, a region of
sequence homology common to all the calpains. The exon 8 immunogen
contained a sequence of 16 amino acids (355 to 370), 11 of which are
common to calpains 1, 2, and 3. Thus, the epitope recognized by
antibody Calp3c/11B3 might span amino acids 357 to 361 (five contiguous
residues common to human calpains 1, 2, and 3), whereas the epitope
recognized by antibody Calp3c/12A2 might involve amino acids 366 to 370
(a group of five amino acids with two or three differences between
calpain 3 and calpains 1 or 2) (Figure 5)
. It is quite unusual to
isolate common and specific antibodies from immunization with a single
peptide. However, the degree of homology is similar to that in the
C-terminal region of dystrophin and utrophin, and immunization with
sequences from these proteins has also resulted in specific and
cross-reactive antibodies.29,30
|
Band Patterns on Blots
Studies on mouse muscle28 have shown that antibodies to an epitope shared by calpains 1, 2, and 3 in the common protease domain II produce a pattern of four or more related bands on blots. Apart from the 94-kd band of calpain 3, there are bands at 84 kd (representing intact calpain 1), 80 kd, and 78 kd. The latter two represent a mixture of the catalytic subunits of calpain 2 (both pre- and postautolysed) and autolysed calpain 1.28 Adjacent strips from blots of human muscle labeled with these polyclonal antibodies and monoclonal antibody Calp3c/11B3 showed an identical pattern of grouped bands.
Two of the monoclonal antibodies produced recognized calpain 3 alone,
but the protein was present as more than one band on blots of human
skeletal muscle. In addition to the full-sized 94-kd band, a clean
30-kd fragment was detected with the exon 1 NS domain antibody
Calp3d/2C4 (at 45 to 95% the density of the 94-kd band in control
muscle; see Table 3
), and a group of degradation bands running from
about 60 kd down to 45 kd was detected with the exon 8 domain II
antibody Calp3c/12A2. The total loss of immunoreactivity in LGMD2A
patients demonstrated that these additional polypeptides were related
to calpain 3 and the CAPN3 gene and not other unrelated
proteins.
The identity of these bands is not really certain, but they are most
likely to be proteolytic products of the full-size protein, possibly
with the 30-kd and 60-kd fragments arising from a single proteolytic
cut of the 94-kd protein. If this is the case, they may have been
caused by ex vivo degradation after tissue was removed from
the body or be normally present in muscle, having been generated as
part of the in vivo calpain 3 physiological process. The
30-kd band was clearly present in human muscle but only weakly visible
in muscle from dog and rabbit. This was not related to the time taken
to freeze the muscle initially, because this was comparable between the
species. In the degradation experiments we undertook there was no
simple reciprocal relationship between the full-size and smaller
polypeptides: the abundance of the 94-kd protein did not decrease in
parallel to an increase in the smaller fragments. Both 30-kd and 60-kd
bands were present from the earliest time points obtainable (Figure 3)
,
although they were slightly darker at the subsequent time points. In
these experiments, the 60-kd band did appear to degrade further into a
ladder of smaller bands down to about 45 kd, but there were no smaller
bands than the 30-kd one. The N-terminal region of domain 1 is
autocatalytically processed during Ca2+-dependent
activation in calpains 1 and 2,16
but the N terminus in
calpain 3 (exemplified by the epitope recognized by antibody
Calp3d/2C4) appears to be very stable in whole muscle, and it may be
that the calpain 3-specific NS sequence insertion blocks the autolysis
that occurs in calpains 1 and 2.
It is possible that the 30-kd fragment is a separate polypeptide product in its own right, expressed from exons 1 to 5 of the CAPN3 gene. Another tissue-specific calpain, which is expressed only in stomach, has two alternative gene products. The full-size stomach calpain is designated nCL-2, whereas the peptide corresponding to the N-terminal part is designated nCL-2'.31 No function has yet been allocated to this half-size polypeptide, which is missing the calcium binding domain characteristically required by the calpain family for activation. It is interesting to note, however, that the tissue-specific calpains, unlike their ubiquitous relatives, do not interact with the small regulatory subunit, which is also 30 kd.31,32 It may just be possible that the tissue-specific calpains use N-terminal products from their own genes to fulfill a regulatory role.
The exon 1 antibody detected a band at about 110 kd in skeletal muscle from rat and hamster. It was also observed in rat heart, with a slightly larger form in brain. These bands may represent reactivity with unrelated gene product(s) or may represent novel isoforms of calpain 3. There is increasing evidence for tissue-related alternative splicing in this gene. The mRNA of an isoform, predicted to be an 82-kd protein, has been described in the lens of both rat19 and mouse (M. Herasse et al, manuscript in preparation). There is also evidence for alternative splicing of the CAPN3 gene in the digestive tract smooth muscle of human fetuses early in development.33
Stability of Calpain 3 in Muscle
Calpain 3 is stable in intact human muscle, there being little decrease in the abundance of full-size protein detected in biopsy samples rested at room temperature for up to 8 hours after leaving the body. This is in contrast to early reports that seemed to indicate rapid autolysis.27 It appears that it is critical for the muscle to be unfractionated and simply applied straight to gels. There may also be variation between samples, given that in these experiments we also found little change up to 2 hours postthaw, whereas a single freeze/thaw cycle had previously been shown to reduce the abundance of full-size protein within 10 to 15 minutes.20 The difference may also be accentuated by the presence of moisture. Our samples were thawed "dry" in the homogenization tube, whereas previously the samples were left on a damp gauze in a Petri dish.20 Here, homogenization in saline was the only way of producing degradation with a clear reduction in full-size protein within 30 minutes. The breakdown under these conditions was in agreement with short half-life reported by Sorimachi et al.27
The stability of calpain 3 in whole muscle may be mediated by its binding to titin, a giant molecule that stretches between the M and Z lines, and may act as a template around which other myofibrillar proteins are assembled.32,34 So far, titin is the only candidate protein that appears to bind calpain 3; other potential substrates and functions for calpain 3 remain elusive. The association with titin may indicate a function in maintaining myofibrillar integrity, and studies on rat myoblast cultures have shown that blocking calpain 3 expression with antisense oligonucleotides leads to muscle fiber instability and Z-line disorganization.35 However, patients lacking calpain 3 are reported not to show dramatic ultrastructural changes,35,36 and it does not seem likely that the pathogenesis of the human MD involves titin instability.
Protein Expression in Patients with LGMD2A
Three types of mutation are represented in this group of patients:
homozygous null, homozygous missense, and heterozygous combinations of
null/missense and null/in-frame deletion. As expected, the homozygous
null mutation produced the most profound effect on protein expression,
with undetectable calpain bands (see Figure 4
, lanes 2, 11, and 13).
Evolution of the disease was considered to be severe in these patients.
Patients heterozygous for a null mutation and a missense change in exon
21 (S744G or R748, in an EF loop that might indicate calcium binding)
had intermediate or mild disease progression but severely reduced
calpain 3 expression (Figure 4
, lanes 4, 10, and 19). Thus, missense
mutations in this region may affect either protein production or
protein stability but presumably preserve some residual enzymatic
activity. Thus, this result also indicates that milder clinical
phenotypes can exist in the presence of greatly reduced enzyme levels.
Calpain 3 protein was also severely reduced in one patient with a null
mutation plus a small in-frame deletion within exon 4 in a highly
conserved region (Figure 4
, lane 6). A small in-frame deletion might
have resulted in translation of a slightly smaller protein in normal
abundance, so again this result appears to confirm a region is
important for protein stability and/or function. In this instance, the
patient had a severe clinical phenotype. In summary, very low levels of
calpain 3 were found in patients who varied in the classification of
their disease severity from "mild" to "severe."
One patient (no. 7) had a homozygous missense mutation situated in exon
5, in the protease domain II within a region of sequence conservation
between species. In this mutation the 222nd amino acid was changed from
glycine to arginine, thereby converting a nonpolar side chain to one
that is very basic. Expression of the full-size protein appeared to be
unaffected, but the band intensity (indicating abundance) of the 30-kd
and ~60-kd bands was very severely reduced (Figure 4
, lane 18; Table 3
). Disease progression in this patient was classed as "severe"
compared with the others. Thus, it appears that this mutation had
affected calpain 3 function, and that the inactive protein was not
removed from, or accumulated within, the muscle as a result of this
dysfunction.
Exactly the same pattern of calpain 3 bands was observed in a patient
(no. 5) in whom progression of the disease was mild (Figure 4
, lane 8).
In this case the underlying mutations were a null mutation plus a
missense mutation in exon 4 (the 184th amino acid changed from
threonine to methionine, thereby converting a polar side chain to a
nonpolar one). Again, the smaller fragments (30 kd and ~60 kd) were
clearly reduced in abundance (see Table 3
). In summary, normal levels
of the full-size calpain 3 band were found in patients who varied in
disease severity and progression from "mild" to "severe." In
these cases, however, the normal full-size band was accompanied by
obvious changes to the abundance of the smaller bands.
Given that the only apparent change in protein expression in patients 5
and 7 was the reduced abundance of the 30-kd and ~60-kd bands, is the
generation of these smaller bands part of normal calpain 3 function,
and is their reduction a specific (ie, diagnostic) indication of loss
of enzyme function? Possibly not: although this protein profile has not
been seen in any other patients, it was observed in one ?LGMD sample in
which mutations in the CAPN3 gene had not been found (Figure 4
, lane 15; Table 3
). We need to discover a lot more about the
physiology of calpain 3 and the circumstances under which the smaller
bands are generated before any firm conclusions about the diagnostic
usefulness of these minor changes in protein expression can be
assessed.
Feasibility of Immunodiagnosis
This study has shown very clearly that the stability of calpain 3 protein in human muscle biopsies is not a problem for immunodetection. Very little change was observed in human muscle left on moist filter paper for up to 8 hours before freezing. Thus, biopsies taken in outlying hospitals and sent to a major center for analysis would yield useful results. During the course of this study, we found that samples prepared for electrophoresis showed no obvious loss of immunoreactivity when they sat for several hours before analysis, or even after four or five freeze/thaw cycles. It should be noted, however, that the experiments on protein stability were undertaken on "wild-type" calpain 3 in normal control muscle: mutated forms of the protein might behave differently.
A limitation on the widespread use of immunodiagnosis for calpain 3 is that the current antibodies only work on Western blots and not on tissue sections. Work is currently underway to obtain antibodies that will work for both techniques. Nevertheless, if the generation of the 30-kd and ~60-kd bands has some functional significance, this could only be examined on blots. However, if the different fragments/isoforms have a different subcellular distribution, immunocytochemistry on sections would still be useful.
How accurate is immunodiagnosis likely to be? Absence of calpain 3 protein probably indicates the presence of null mutations (as the absence of protein expression does for other recessive diseases). Similarly, significantly reduced levels of protein are usually associated with pathological mutations. False-negative results might arise in circumstances in which calpain 3 is broken down (eg, after transporting a biopsy to a referral center in liquid, or prolonged thawing of a muscle sample in transit), but this would probably be detected by an increase in lower molecular mass bands on the gel and the loss of all the proteins to be analyzed. The use of another protein as an internal control for labeling would be useful in this context, and the multiplex blotting system is ideal.22 From this study, false-positive results appear to be much more of a problem. Among nine patients, two were found to have normal levels of full-size calpain 3 protein. The diagnostic significance of the observed reduction in the 30-kd and ~60-kd bands in these two patients is uncertain.
Prognosis based on dystrophin analysis was generally easy for patients with Duchenne and Becker dystrophy. It was much less easy in the sarcoglycanopathies, although there was a general rule that null mutations were associated with absence of detectable protein and, often, a severe phenotype. Most other mutations resulted in reduced protein expression, so that there was little correlation between clinical severity and figures for protein abundance. In LGMD2A, in which the gene product is a proteolytic enzyme, the situation is likely to be more complicated. Different mutations might result in enzyme levels that were reduced, normal, or even increased.37 From these initial results, it seems very unlikely that calpain 3 protein expression alone will be of value for the prediction of clinical severity in LGMD2A.
In summary, these results indicate that immunodiagnosis for some patients is feasible, and this represents an important advance, because it has been estimated that 50% of patients with autosomal recessive LGMD actually have LGMD2A.38 However, caution will need to be exercised with the interpretation of near-normal protein profiles. The confidence with which immunodiagnosis is used will only improve when many more patients with defined mutations have been analyzed. It is quite clear that this is an urgent priority for the future.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the Muscular Dystrophy Group of Great Britain and the Association Française contre les Myopathies.
Accepted for publication July 18, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-sarcoglycan in chromosome 13 muscular dystrophy. Science 1995, 270:819-822-sarcoglycan gene. Nat Genet 1996, 14:195-198[Medline]
-, ß- and -sarcoglycan in patients with limb-girdle muscular dystrophy. Neuromusc Disord 1996, 6:467-474[Medline]
-sarcoglycanopathy): clinical, pathologic, and genetic correlation in 20 patients with autosomal recessive muscular dystrophy. Neurology 1997, 48:1227-1234-sarcoglycans in 20 -sarcoglycan (adhalin)-deficient patients: immunohistochemical analysis and clinical aspects. Acta Neuropathol 1997, 94:28-35[Medline]
This article has been cited by other articles:
 |
|
 |
|
  C. A. Goodman, D. Horvath, C. Stathis, T. Mori, K. Croft, R. M. Murphy, and A. Hayes Taurine supplementation increases skeletal muscle force production and protects muscle function during and after high-frequency in vitro stimulation J Appl Physiol, July 1, 2009; 107(1): 144 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Murphy and G. D. Lamb Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch J. Biol. Chem., March 20, 2009; 284(12): 7811 - 7819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Groen, R. Charlton, R. Barresi, L. V. Anderson, M. Eagle, J. Hudson, M. S. Koref, V. Straub, and K. M. D. Bushby Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy 2A Brain, December 1, 2007; 130(12): 3237 - 3249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Fanin, L Nardetto, A C Nascimbeni, E Tasca, M Spinazzi, R Padoan, and C Angelini Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A J. Med. Genet., October 1, 2007; 44(10): 609 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nguyen, G. Bassez, M. Krahn, R. Bernard, P. Laforet, V. Labelle, J. A. Urtizberea, D. Figarella-Branger, N. Romero, S. Attarian, et al. Phenotypic Study in 40 Patients With Dysferlin Gene Mutations: High Frequency of Atypical Phenotypes Arch Neurol, August 1, 2007; 64(8): 1176 - 1182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Fanin, A C Nascimbeni, and C Angelini Screening of calpain-3 autolytic activity in LGMD muscle: a functional map of CAPN3 gene mutations J. Med. Genet., January 1, 2007; 44(1): 38 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. H. Geesink, S. Kuchay, A. H. Chishti, and M. Koohmaraie {micro}-Calpain is essential for postmortem proteolysis of muscle proteins J Anim Sci, October 1, 2006; 84(10): 2834 - 2840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ono, F. Torii, K. Ojima, N. Doi, K. Yoshioka, Y. Kawabata, D. Labeit, S. Labeit, K. Suzuki, K. Abe, et al. Suppressed Disassembly of Autolyzing p94/CAPN3 by N2A Connectin/Titin in a Genetic Reporter System J. Biol. Chem., July 7, 2006; 281(27): 18519 - 18531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Murphy, R. J. Snow, and G. D. Lamb {micro}-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans Am J Physiol Cell Physiol, January 1, 2006; 290(1): C116 - C122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. H. Geesink, R. G. Taylor, and M. Koohmaraie Calpain 3/p94 is not involved in postmortem proteolysis J Anim Sci, July 1, 2005; 83(7): 1646 - 1652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zatz and A. Starling Calpains and Disease N. Engl. J. Med., June 9, 2005; 352(23): 2413 - 2423. [Full Text] [PDF]
|
|
|
|
 |
|
 E. Verburg, R. M. Murphy, D. G. Stephenson, and G. D. Lamb Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres J. Physiol., May 1, 2005; 564(3): 775 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Saenz, F. Leturcq, A. M. Cobo, J. J. Poza, X. Ferrer, D. Otaegui, P. Camano, M. Urtasun, J. Vilchez, E. Gutierrez-Rivas, et al. LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene Brain, April 1, 2005; 128(4): 732 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fanin, A. C. Nascimbeni, L. Fulizio, C. P. Trevisan, M. Meznaric-Petrusa, and C. Angelini Loss of Calpain-3 Autocatalytic Activity in LGMD2A Patients with Normal Protein Expression Am. J. Pathol., November 1, 2003; 163(5): 1929 - 1936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Vainzof, F de Paula, A M Tsanaclis, and M Zatz The effect of calpain 3 deficiency on the pattern of muscle degeneration in the earliest stages of LGMD2A J. Clin. Pathol., August 1, 2003; 56(8): 624 - 626. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. GOLL, V. F. THOMPSON, H. LI, W. WEI, and J. CONG The Calpain System Physiol Rev, July 1, 2003; 83(3): 731 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Spencer, J. R. Guyon, H. Sorimachi, A. Potts, I. Richard, M. Herasse, J. Chamberlain, I. Dalkilic, L. M. Kunkel, and J. S. Beckmann Stable expression of calpain 3 from a muscle transgene in vivo: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation PNAS, June 25, 2002; 99(13): 8874 - 8879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Haravuori, A. Vihola, V. Straub, M. Auranen, I. Richard, S. Marchand, T. Voit, S. Labeit, H. Somer, L. Peltonen, et al. Secondary calpain3 deficiency in 2q-linked muscular dystrophy: Titin is the candidate gene Neurology, April 10, 2001; 56(7): 869 - 877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Fanin, E. Pegoraro, C. Matsuda-Asada, R.H. Brown Jr., and C. Angelini Calpain-3 and dysferlin protein screening in patients with limb-girdle dystrophy and myopathy Neurology, March 13, 2001; 56(5): 660 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Talim, A. Ognibene, E. Mattioli, I. Richard, L.V.B. Anderson, and L. Merlini Normal calpain expression in genetically confirmed limb-girdle muscular dystrophy type 2A Neurology, March 13, 2001; 56(5): 692 - 693. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tagawa, C. Taya, Y. Hayashi, M. Nakagawa, Y. Ono, R. Fukuda, H. Karasuyama, N. Toyama-Sorimachi, Y. Katsui, S. Hata, et al. Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A Hum. Mol. Genet., May 22, 2000; 9(9): 1393 - 1402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. D. Bushby The limb-girdle muscular dystrophies--multiple genes,multiple mechanisms Hum. Mol. Genet., September 1, 1999; 8(10): 1875 - 1882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. D. Bushby Making sense of the limb-girdle muscular dystrophies Brain, August 1, 1999; 122(8): 1403 - 1420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. V. B. Anderson and K. Davison Multiplex Western Blotting System for the Analysis of Muscular Dystrophy Proteins Am. J. Pathol., April 1, 1999; 154(4): 1017 - 1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-L. Chou, C. Angelini, D. Daentl, C. Garcia, C. Greco, I. Hausmanowa-Petrusewicz, A. Fidzianska, H. Wessel, and E. P. Hoffman Calpain III mutation analysis of a heterogeneous limb-girdle muscular dystrophy population Neurology, March 1, 1999; 52(5): 1015 - 1015. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
