From the Consiglio Nazionale delle Ricerche Unit for
Muscle Biology and Physiopathology and the ¶ Department of
Biomedical Sciences, University of Padova Medical School, Viale
Giuseppe Colombo 3, I-35121 Padova, Italy
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ABSTRACT |
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Dystrophin is a large cytoskeletal protein associated with a
complex of integral and peripheral membrane proteins collectively termed DAPs.1 Dystrophin is a
long filamentous protein comprising four distinct structural domains:
the amino-terminal domain, which binds F-actin, the rod-like central
domain; the cysteine-rich domain, which binds the cytoplasmic portion
of Dystrophin is defective in Duchenne muscular dystrophy (DMD) and Becker
muscular dystrophy. In patients with DMD and in the mdx
mouse, an animal model for DMD, all of the components of the DAPs are
severely reduced at the sarcolemma (13, 14), even though they are
almost normal at the neuromuscular junction (15).
Mutations in the Here we show that Isolation of Sarcolemma and Purification of the Dystrophin-DAPs
Complex--
Sarcolemma vesicles from rabbit fast-twitch muscles were
isolated as described previously (22). The purification of the dystrophin-DAPs complex was performed according to the digitonin, 0.5 M NaCl, wheat germ agglutinin protocol of Ervasti et
al. (23), as described previously (24), with the only difference
being that the dystrophin-DAPs purification was terminated after the DEAE-cellulose column chromatography. The 175 mM NaCl and
the first 500 mM NaCl eluates were collected, concentrated
by filtration using an Amicon system (model 75 PSI), and stored at
[ Immunological Methods--
A polyclonal antiserum against the
Ser-Ala-Gln-Val-Pro-Leu-Ile-Leu-Asp-Gln carboxyl-terminal peptide of
adhalin (Chiron Mimotopes, Clayton, Australia) was raised in New
Zealand White rabbits by subcutaneous injections. For the first
injection, 500 µg of peptide in PBS mixed 1:1 (v/v) with Freund's
complete adjuvant was used. After 2 weeks, rabbits were boosted four
times at 1-week intervals. Specificity of the polyclonal antibody was
checked onto the dystrophin-DAPs preparation (Fig. 1) by immunoblotting
as described below.
One hundred µg of protein from a dystrophin-DAPs preparation were
incubated with 0.4 µM [
SDS-PAGE was carried out according to Laemmli (26) using 5-15%
polyacrylamide linear gels. The gels were either stained with Coomassie
Brilliant Blue or dried. For Western blotting, the proteins were
transferred overnight to nitrocellulose sheets at 300 mA in 25 mM Tris, 192 mM glycine, 0.03% SDS, and 10%
methanol. The nitrocellulose was stained with Ponceau Red (0.2%, w/v)
in 3% (v/v) trichloroacetic acid, photographed, and destained in distilled H2O. For staining with the monoclonal antibody
against adhalin, nitrocellulose was first saturated for 1 h in 50 mM Tris, pH 8.0, 85 mM NaCl, and 2% bovine
serum albumin. The saturating solution was discarded, and the
nitrocellulose was incubated with a 1:300 dilution of the anti-adhalin
antibody in the same buffer. For staining with monoclonal anti-nNOS
antibody, nitrocellulose membranes were saturated for 1 h in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% low fat milk and incubated for 2 h with monoclonal
anti-nNOS antibody diluted 1:500 in the same buffer. In both cases,
after three washes with 50 mM Tris, pH 8.0, 85 mM NaCl, 0.1% bovine serum albumin, and 0.2% Tween 20, the nitrocellulose was incubated for 1 h with anti-mouse
antibodies conjugated with peroxidase diluted 1:2000 in saturation
buffer. After three washes, the reaction was developed with the
Luminol-based Boehringer BM chemiluminescence kit. Autoradiography was
carried out at ATPase Activity--
ATPase activity was measured
spectrophotometrically at 37 °C with an enzyme-coupled ADP release
assay (27) by measuring the oxidation of NADH at 340 nm (28). The assay
solution contained in 1 ml: 20 mM histidine, pH 7.2, 0.1 M KCl, 5 mM MgCl2, 2 mM ATP, 0.15 mM NADH, 0.5 mM
phospho(enol)pyruvate, 5 units of pyruvate kinase/L-lactate
dehydrogenase, and 10 µg of dystrophin-DAPs. For
Ca2+-ATPase activity measurements, basal ATPase activity
was checked first in the presence of 1 mM EGTA. Then, 1 mM CaCl2 (to obtain a final free
Ca2+ concentration of 10 µM) was added. The
inhibition of ATPase activity by BzATP was performed by UV irradiation
of 10 µg protein from a dystrophin-DAPs preparation in the Ca-Mg
buffer (see above) without DTT for 5 min, in the presence of 100 µM BzATP. In control experiments, the dystrophin-DAPs
preparation was irradiated in the absence of
[ Reagents--
[ The physiological role of Photoaffinity labeling experiments using [ To confirm the identity of the 50-kDa ATP-labeled protein as
-Sarcoglycan is a component of the sarcoglycan
complex of dystrophin-associated proteins. Mutations of any of the
sarcoglycan genes cause specific forms of muscular dystrophies,
collectively termed sarcoglycanopathies. Importantly, a deficiency of
any specific sarcoglycan affects the expression of the others. Thus, it
appears that the lack of sarcoglycans deprives the muscle cell of an
essential, yet unknown function. In the present study, we provide
evidence for an ecto-ATPase activity of
-sarcoglycan.
-Sarcoglycan binds ATP in a
Mg2+-dependent and
Ca2+-independent manner. The binding is inhibited by
3'-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis
reveals the existence of a consensus site for nucleotide binding in the
extracellular domain of the protein. An antibody against this sequence
inhibits the binding of ATP. A dystrophin·dystrophin-associated
protein preparation demonstrates a Mg-ATPase activity that is inhibited
by the antibody but not by inhibitors of endo-ATPases. In addition, we
demonstrate the presence in the sarcolemmal membrane of a P2X-type
purinergic receptor. These data suggest that
-sarcoglycan may
modulate the activity of P2X receptors by buffering the extracellular
ATP concentration. The absence of
-sarcoglycan in
sarcoglycanopathies leaves elevated the concentration of extracellular
ATP and the persistent activation of P2X receptors, leading to
intracellular Ca2+ overload and muscle fiber death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-dystroglycan and syntrophins; and the
carboxyl-terminal domain (1). The DAPs complex is composed of three
subcomplexes: syntrophins, dystroglycans, and sarcoglycans (2, 3).
Syntrophins are peripheral membrane proteins of unknown function that
bind the carboxyl terminus of dystrophin (4, 5). Dystroglycans consist
of two proteins derived from a common precursor protein:
-dystroglycan, a peripheral glycoprotein that binds extracellular
matrix proteins like laminin-2 (merosin) and, in the neuromuscular
junction, laminin-4 (agrin); and
-dystroglycan, an intrinsic
membrane protein that binds dystrophin at its cytoplasmic tail and
-dystroglycan at the opposite end (6, 7). Therefore, the
dystroglycans represent the link between the subsarcolemmal actin
cytoskeleton and the extracellular matrix through dystrophin. Five
sarcoglycans have been described:
-sarcoglycan (adhalin, 50 kDa),
-sarcoglycan (43 kDa),
- and
-sarcoglycans (35 kDa) (8-10),
and
-sarcoglycan (11, 12). The function of the sarcoglycans remains unknown.
-sarcoglycan gene, which is located on chromosome
17q21 (10), were demonstrated in limb girdle muscular dystrophy-2D
(LGMD-2D), an autosomal recessive muscular dystrophy that affects both
females and males (16, 17). In LGMD-2D,
- and
-sarcoglycan were
also absent or greatly reduced, whereas dystrophin and the dystroglycan
complex were preserved (18). Similar modifications were also found in
the skeletal muscle of the cardiomyopatic hamster, an animal model of
this disease (19). Recently, mutations in the genes that encode for
-,
-, and
-sarcoglycan, located on chromosomes 4q12, 13q12,
and 5q33-34, were discovered in LGMD-2E, -2C, and -2F, respectively
(9, 20, 21). These mutations caused the absence not only of the
respective protein product but also of the other three components of
the sarcoglycan complex. Thus, mutations causing loss of one component
result in the disruption of the whole sarcoglycan complex, although
dystrophin and the dystroglycan complex are preserved. These findings
suggest that the subcomplexes of the dystrophin-DAPs have distinct
physiological roles. Dystrophin and the dystroglycan complex, by
linking the actin membrane cytoskeleton to the extracellular matrix,
organize the membrane cytoskeleton and protect the sarcolemma from
mechanical stress during muscle contraction; syntrophins and the
sarcoglycan complex, apart from a suggested stabilizing effect on the
dystrophin-glycoprotein complex, have a function that is as yet unknown.
-sarcoglycan is a sarcolemma ecto-ATPase and that
sarcolemma expresses a P2X-type purinergic receptor, a
nonspecific cationic channel. We speculate that
-sarcoglycan, by
controlling the extracellular concentration of ATP, may modulate the
activity of these receptors providing an attractive pathogenetic mechanism for cell death in sarcoglycanopathies.
EXPERIMENTAL PROCEDURES
80 °C until used. The obtained dystrophin-DAPs preparation
displays a protein pattern very similar to that published elsewhere
(Fig. 1; see Refs. 4, 7, and 23). To
verify the composition and the level of purification of the
dystrophin-DAPs preparation, commercial antibodies against dystrophin,
-dystroglycan, and
- and
-sarcoglycans were used
(not shown).
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Fig. 1.
Photoaffinity labeling of the dystrophin-DAPs
preparation with [ -32P]ATP.
Twenty µg of dystrophin-DAPs in 200 µl of 0.025 M Tris,
pH 7.4, 2.5 mM MgCl2, 10 µM
CaCl2, and 1 mM DTT were UV-photoactivated in
the presence of 0.4 µM [
-32P]ATP as
described under "Experimental Procedures." A sample of lane
c contained an excess of cold ATP (1 mM). After
SDS-PAGE, the gel was dried and autoradiographed (lanes b
and c). Only a protein band co-migrating with
-sarcoglycan was selectively labeled. Lane a, molecular
mass standards. Dys, dystrophin;
- and
-DG,
- and
-dystroglycan;
-,
-,
-, and
-SG,
-,
-,
-, and
-sarcoglycan;
-,
1- and
2-Syn,
-,
1-, and
2-syntrophin. The dystrophin-DAPs were identified by
comparison with data in the literature and by immunoblot staining (not
shown) with commercial antibodies.
-32P]ATP Photoaffinity
Labeling--
Photoaffinity labeling was carried out by the UV
irradiation method previously described (25). Dystrophin-DAPs
preparations (14-21 µg of protein) or sarcolemma membranes (28 µg
of protein) were equilibrated with 0.4 µM ATP (containing
2 and 4 µCi of [
-32P]ATP, respectively) in 100 µl
of the following different assay environments: 1) Ca-Mg buffer: 25 mM Tris, pH 7.4, 2.5 mM MgCl2, 10 µM CaCl2, and 1 mM
dithiothreitol; 2) magnesium buffer: 25 mM Tris, pH 7.4, 2.5 mM MgCl2, 2.5 mM EGTA, and 1 mM dithiothreitol; 3) Ca/Mg-free buffer: 25 mM
Tris, pH 7.4, 2.5 mM EGTA, 2.5 mM EDTA, and 1 mM dithiothreitol. Competitive binding assays were carried
out on the dystrophin-DAPs preparation (20 µg of protein) equilibrated with 0.4 µM [
-32P]ATP
either in Ca-Mg buffer in the presence of one of the following nucleotides, each at 50 µM:
3'-O-(4-benzoyl)benzoyl ATP (BzATP), adenosine
5'-triphosphate-2',3'-dialdehyde, ADP, UTP, and GTP; or in calcium-free
buffer with 50 µM BzATP. The inhibition of ATP binding by
the monoclonal anti-adhalin antibody was measured by incubating 37.5 µl of the antibody with 10 µg of protein from a dystrophin-DAPs
preparation in 100 µl of Ca-Mg buffer without DTT. After incubation
at room temperature for 2 h with constant stirring, 0.4 µM [
-32P]ATP was added. UV irradiation
was carried out by direct exposure to UV light (254 nm) for 20 min in a
flat dish refrigerated on ice. The UV lamp (8 watts) was kept at a
distance of about 5 cm from the dish surface. The reaction was stopped
by the addition of 30 µl of SDS-PAGE sample buffer.
-32P]ATP (20 µCi/ml) in 500 µl of Ca-Mg buffer without DTT and photoactivated as
described above. After photoactivation, 500 µl of polyclonal anti-adhalin antibody cross-linked to immobilized protein A resin were
added and incubated at 0 °C for 2 h. The mixture was then centrifuged to sediment the protein bound to the resin. The pellet was
washed exhaustively with Ca-Mg buffer without DTT and finally solubilized in the SDS-PAGE sample buffer.
80 °C by exposing dried gels or nitrocellulose
sheets to Kodak XAR-5 films.
-32P]ATP. ATPase activity was measured as above.
Inhibition of ATPase activity by antibodies was performed by
preincubating the dystrophin-DAPs preparation with either monoclonal or
polyclonal anti-adhalin antibody in the Ca-Mg buffer for 2 h at
room temperature. 2 mM ATP was then added, and the
incubation was continued for a further 10 min. Pi release
was measured according to Lanzetta et al. (29).
-32P]ATP (10 µCi/mmol) was
obtained from NEN Life Science Products. The monoclonal anti-adhalin
antibody was obtained from Yelem-Novocastra (Rome, Italy), and the
monoclonal anti-nNOS antibody was from Transduction Laboratories
(Lexington, KY). Anti-mouse IgG antibody conjugated with peroxidase,
phospho(enol)pyruvate, wheat germ agglutinin-agarose macrobeads, BzATP,
and adenosine 5'-triphosphate-2',3'-dialdehyde were from Sigma. The
immobilized protein A was from Pierce (ImmunoPure IgG orientation kit,
Rockford, IL). Pyruvate kinase, L-lactate dehydrogenase,
and the BM chemiluminescence kit were from Boehringer Mannheim. All
other chemicals were analytical grade.
RESULTS AND DISCUSSION
-sarcoglycan (adhalin), the protein
of the sarcoglycan complex that is missing in LGMD-2D, is unknown. In
the present study, we demonstrate that
-sarcoglycan 1) binds ATP, 2)
has an ATPase activity not inhibitable by known inhibitors of
endo-ATPases, and 3) is not a purinergic receptor.
-32P]ATP
demonstrate that one protein in a purified dystrophin-DAPs preparation
bound ATP. As shown in Fig. 1, the incubation of a dystrophin-DAPs
fraction with 0.4 µM Mg-[
-32P]ATP
followed by UV photoactivation caused the labeling of a protein with an
apparent molecular mass of 50 kDa (lane b). Labeling was
specific, as it was prevented by excess cold ATP (lane
c).
-sarcoglycan, we have used different immunological approaches with
monoclonal and polyclonal anti-
-sarcoglycan antibodies (Fig. 2A, lanes c and
d, respectively). Among the dystrophin-DAPs, the same
protein band that bound [
-32P]ATP (Fig. 2B,
lane f) was also selectively stained by the monoclonal anti-
-sarcoglycan antibody (Fig. 2B, lane g).
Furthermore, the binding of [
-32P]ATP to the 50-kDa
protein was inhibited by incubation of the dystrophin-DAPs preparation
with the monoclonal anti-
-sarcoglycan antibody prior to
photoactivation (Fig. 2C, lane i). Finally, polyclonal anti-
-sarcoglycan antibodies immunoprecipitated the 50-kDa [
-32P]ATP-labeled protein (Fig. 2C,
lane j).
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Fig. 2.
Identification of the
[ -32P]ATP-labeled protein
as
-sarcoglycan. A, the
monoclonal antibody raised against a fusion peptide containing the
ATP-binding site located in the extracellular domain of
-sarcoglycan
(lane c) and the polyclonal antibody raised against a
carboxyl-terminal peptide of
-sarcoglycan (lane d) were
tested on 20 µg of a dystrophin-DAPs preparation (lane b).
Both antibodies selectively labeled the
-sarcoglycan protein.
Lane a, molecular mass standards. B, 20 µg of
dystrophin-DAPs in 0.025 M Tris, pH 7.4, 2.5 mM
MgCl2, 10 µM CaCl2, and 1 mM DTT were UV-photoactivated in the presence of 0.4 µM [
-32P]ATP. After SDS-PAGE, the
protein bands were transferred to nitrocellulose, stained with Ponceau
Red (lane e), autoradiographed (lane f), and
probed with the monoclonal anti-
-sarcoglycan antibody (lane
g), as described under "Experimental Procedures." This
antibody recognizes the same 50-kDa protein labeled by
[
-32P]ATP. C, in a second set of
experiments, the monoclonal anti-
-sarcoglycan antibody was added to
20 µg of protein from the dystrophin-DAPs preparation before
(lane i) and after (lane h) the addition of
[
-32P]ATP and UV irradiation. The antibody inhibited
the labeling of
-sarcoglycan by [
-32P]ATP
(lane i). On the other hand, incubation of the
[
-32P]ATP-labeled
-sarcoglycan with the polyclonal
anti-
-sarcoglycan antibody allowed the immunoprecipitation of the
[
-32P]ATP-labeled protein (lane j).
Analysis of the deduced amino acid sequence of rabbit -sarcoglycan
(19) for ATP binding consensus sequences revealed the presence of two
sequences at amino acids 163-171
(Gly-(Leu-Trp-Glu-Pro)-Gly-Glu-Leu-Lys) and amino acids 221-234
(Arg-(Cys-Ala-Arg)-Gly-(Gln-Pro-Pro)-Leu-(Leu-Ser-Cys-Tyr)-Asp) that are similar to the consensus sequences
Gly-(X)4-Gly-Lys-(Thr) and
Arg/Lys-(X)3-Gly-(X)3-Leu-(hydrophobic)4-Asp
present in several ATPases (30). Both sequences are located in the
extracellular domain of the protein. Interestingly, the peptide used to
produce the monoclonal antibody is a fusion protein encompassing amino acids 217-289 of the rabbit
-sarcoglycan sequence. It should be
noted that this sequence is conserved between rabbit, mouse, hamster,
and human
-sarcoglycans and that it contains residues 221-234 of
the putative ATP-binding domain, a finding that explains the ability of
the monoclonal antibody to prevent the binding of ATP to
-sarcoglycan.
Several extracellular proteins are known to bind ATP. Among these are
the ecto-ATPases, the protein kinases, and the purinergic receptors.
Ecto-ATPases are transmembrane enzymes that catalyze the hydrolysis of
extracellular ATP. They have been identified at the surface of numerous
cell types in many different species (31-33). When purified, these
ecto-enzymes generally show an activity that is dependent on
Mg2+ or Ca2+, although it is insensitive to
specific inhibitors of endo-ATPases (33). ATP binding to
-sarcoglycan was Mg2+-dependent and
Ca2+-independent (Fig. 3),
because after incubation of the dystrophin-DAPs preparation in the
absence of Mg2+ (Fig. 3A, lane e)
-sarcoglycan was not labeled. It can be noted that, in the presence
of Mg2+ (2.5 mM), a protein of about 150 kDa
was also labeled by [
-32P]ATP (Fig. 3A, lanes
c and d). This protein was tentatively identified as
neuronal-type nitric oxide synthase (nNOS) by immunoblot using monoclonal anti-nNOS antibody (data not shown). In fact, this enzyme is
known to be a nonstructural component of the dystrophin complex, so
that in some preparations it may copurify with dystrophin because of a
direct interaction with
1-syntrophin (34) (compare, for example,
Fig. 2, lane h with lane f).
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It has been demonstrated that P2-type purinergic receptors in several
cell systems bind ATP4 (35, 36). To determine whether the
50-kDa protein is a muscle isoform of the P2-type purinergic receptor,
we have incubated a dystrophin-DAPs preparation with 0.4 µM [
-32P]ATP4
in the
absence of both Ca2+ and Mg2+ (calculated
according to Fabiato (37)). Fig. 3A, lane e, shows that,
under these conditions, two proteins of about 260 and 130 kDa, but not
the 50-kDa species, were intensely labeled. Boiling the
[
-32P]ATP-labeled samples in SDS sample buffer before
electrophoresis resulted in a great reduction of the intensity of the
260-kDa protein band, suggesting that the 260-kDa protein is probably a
dimer of the 130-kDa species (data not shown). Interestingly, no
protein corresponding to this 130-kDa band was visible in the Coomassie
Blue-stained gel (Fig. 3), suggesting that a minor protein component
with very high affinity for [
-32P]ATP has been labeled.
To verify whether native -sarcoglycan is also able to bind ATP, we
performed photoaffinity labeling experiments using a sarcolemma membrane preparation. In agreement with the results obtained by using
the dystrophin-DAPs preparation, photoaffinity labeling of sarcolemmal
vesicles with [
-32P]ATP caused labeling of the
-sarcoglycan protein band. Again, the labeling was
Mg2+-dependent and Ca2+-independent
(Fig. 3B, lanes h and i, respectively). On the
other hand, when the sarcolemma vesicles were incubated with
[
-32P]ATP4-,
-sarcoglycan was not
labeled (Fig. 3B, lane j). It should be noted that under the
latter condition, in addition to the high molecular mass proteins
identified in the dystrophin-DAPs preparations (i.e. the
260- and 130-kDa proteins in Fig. 3A, lane e), an additional protein of about 100 kDa was also labeled.
Extracellular ATP can be either the agonist of purinergic
P2 receptors or the substrate of ecto-ATPases. To elucidate
the protein family to which -sarcoglycan belongs, we have analyzed the effects of a number of agonists and antagonists of P2-type receptors, and also of nucleotides that are substrates of
ecto-nucleotidases, on the binding of [
-32P]ATP to
-sarcoglycan. As indicated by the results shown in Fig. 3, because
-sarcoglycan is not labeled by ATP4
, it is not a
P2-type purinergic receptor. This fact is further demonstrated by the
inability of adenosine 5'-triphosphate-2',3'-dialdehyde, a
P2X7-type receptor antagonist (38), to affect the binding of [
-32P]ATP to
-sarcoglycan (Fig.
4, lane e). BzATP is an
agonist of P2X7 purinergic receptors (35, 36, 39), but it
is also a photoaffinity probe that binds covalently to the nucleotide
sites of ATPases (40, 41). As shown in Fig. 4, lane d,
preincubation of the dystrophin-DAPs preparation with BzATP completely
inhibited the binding of [
-32P]ATP to
-sarcoglycan,
further suggesting that
-sarcoglycan is an ATPase. On the other
hand, BzATP also inhibited the binding of
[
-32P]ATP4
to the 130-kDa protein (Fig.
4, lane j). This result suggests that the 130-kDa protein is
a P2X7 purinergic receptor. It is well known that GTP, UTP,
and ADP may be substrates of ecto-nucleotidases (33, 42). Fig. 4,
lane f, shows that ADP completely prevented the binding of
[
-32P]ATP to
-sarcoglycan, whereas GTP had a very
little effect (Fig. 4, lane i). The inability of UTP to
influence the labeling of [
-32P]ATP to
-sarcoglycan
suggests it has a different specificity to nucleotides relative to
other ecto-ATPases (31, 33). On the other hand, the inability of UTP, a
P2Y receptors agonist (43), to affect the binding of
[
-32P]ATP to
-sarcoglycan (Fig. 4, lane
h) indicates that this protein is not a P2Y receptor.
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Plesner et al. (44) have demonstrated that increasing
concentrations of monovalent cations up to 20 mM caused the
increase of the ecto-ATPase activity of an enzyme isolated from
mesenteric arteries. Further increase of monovalent cation salts
decreased this activity. We have therefore tested the effects of
varying concentrations of NaCl on the photoaffinity labeling of
-sarcoglycan by [
-32P]ATP. Fig.
5 shows that ATP labeling of
-sarcoglycan was increased by NaCl up to 20 mM, whereas
higher concentrations were inhibitory. These data indicate that
-sarcoglycan share the same sensitivity to monovalent cations as
known ecto-ATPases.
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These photoaffinity labeling experiments suggested that -sarcoglycan
might be an ecto-ATPase. To prove this point conclusively, we tested
the ATPase activity of our dystrophin-DAPs preparation. We found that
the dystrophin-DAPs preparations had an ATPase activity (0.39 ± 0.01 µmol/min/mg protein, n = 6) that was
Mg2+-dependent and Ca2+-independent
and that was highly reduced upon covalent binding with BzATP after UV
irradiation (Table I). Furthermore, like other ecto-ATPases (33, 45, 46), the ATPase activity of dystrophin-DAPs
preparations was not inhibited by inhibitors of ion-translocating
ATPases such as thapsigargin, cyclopiazonic acid, and vanadate (data
not shown). It appears likely that the relatively low specific activity
is because of the presence of digitonin, used to purified the DAP
complex (22, 23). Indeed, it has been demonstrated that detergents
inhibit the activity of other ecto-ATPases (31, 33). To ascertain
whether the ATPase activity could be attributed to
-sarcoglycan
only, we incubated the dystrophin-DAPs preparation with the monoclonal
anti-
-sarcoglycan antibody (that is that raised against the putative
ATP binding site of the protein) before measuring the ATPase activity.
Under these conditions this antibody was able to reduce the ATPase
activity (Fig. 6). The inhibitory action
of the monoclonal antibody was not the result of nonspecific effects,
because the polyclonal antibody raised against the last 10 amino acids
of the C terminus of
-sarcoglycan (a portion of the protein without
critical sites for the ATPase activity) had no effect. At variance from
the almost complete inhibition of the binding of
[
-32P]ATP to
-sarcoglycan, inhibition of ATPase
activity by the monoclonal anti-
-sarcoglycan antibody was only
partial. One possible explanation is that our preparation was
contaminated by trace amounts of T-tubule ecto-ATPase, a 56-kDa protein
which is characterized by a high specific activity (6.6 mmol/min/mg
protein, Ref. 32). Although labeling of a 56-kDa protein was not
detected, the rabbit T-tubule ecto-ATPase has a relatively low affinity
for ATP (the apparent Km at 25 °C for Mg-ATP is
170 µM (31)). Therefore, amounts of the contaminant below
the threshold of detection in [
-32P]ATP binding could
still be responsible for the fraction of ATPase activity that is not
inhibited by the anti-
-sarcoglycan antibody. Consistent with this
explanation, polyclonal antibodies directed against the T-tubule
Mg2+-ATPase (a generous gift of T. Kirley, University of
Cincinnati) have revealed the presence of a 56-kDa protein in our
preparations (data not shown).
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Extracellular ATP is an important neurotransmitter in a wide variety of
tissues. Its action is regulated by enzymatic degradation by several
ecto-ATPases (33, 36, 42, 47). Thus, ecto-ATPases appear to have an
important role in modulating purinergic neurotransmission. In skeletal
muscle a functional role of extracellular ATP in modulating the opening
time of acetylcholine receptors at the end plate region has been
described (48). Our results indicate that a P2X-type purinergic
receptor is expressed in skeletal muscle, and this is likely to
represent the hypothetical receptor for ATP suggested by Lu and Smith
(48). Furthermore, our results indicate that -sarcoglycan is an
ecto-ATPase. Thus, it appears possible that
-sarcoglycan
modulates the activity of P2X-type purinergic receptors.
Mutations in the -sarcoglycan gene have been demonstrated in LGMD-2D
(10), a group of diseases that shares some features of DMD. Although in
both cases the disease is characterized by muscle fiber necrosis, LGMD
and DMD are caused by mutations of different genes leading to different
alterations in the two subcomplexes of the dystrophin-associated
proteins: the sarcoglycans and the dystroglycans (49, 50). In LGMD,
only the sarcoglycan complex is lacking (18). In DMD, dystrophin and
the dystroglycan complex are missing, and the sarcoglycans are greatly
reduced in amount (14, 15). Therefore, it is possible that the primary
molecular mechanisms involved in the degeneration and necrosis of the
muscle fibers are different in the two diseases.
Today, the more widely accepted theory on the role of dystrophin in
skeletal muscle fibers is the mechanical theory. Dystrophin, by acting
as a link between the actin membrane cytoskeleton and the extracellular
matrix via - and
-dystroglycan, could transmit the local stresses
generated during contraction across the sarcolemma to the extracellular
matrix. The absence of dystrophin, by weakening the mechanical
resistance of the membrane, could therefore predispose to physical
disruption of the sarcolemma during muscle activity (2, 51, 52)
allowing the entry of Ca2+. The elevated intracellular free
Ca2+ level could then activate intracellular degradation
processes (53, 54).
In LGMD-2D, -2C, -2E, and -2F, on the other hand, mutations cause the
absence of the sarcoglycans, whereas dystrophin and dystroglycans are
preserved at the sarcolemma (3, 49, 50). Thus, the mechanical
resistance of the membrane should not be affected, suggesting that cell
necrosis in these diseases has a different origin. One physiological
role of ecto-ATPases is the control of ATP concentration at the surface
of cells that express ATP receptors, thereby attenuating the magnitude
and/or the duration of ATP-induced signals (41, 46). We have
demonstrated that skeletal muscle fibers express P2X7-type
receptors that are activated by extracellular ATP in the micromolar
range, forming nonselective pores that mediate a generalized
bidirectional increase in plasma membrane permeability to molecules up
to 900 Da (35, 36). It appears that the absence of -sarcoglycan, and
therefore the absence of ecto-ATPase activity of the sarcolemma, could
cause a persistent increase of extracellular ATP concentration. The consequent prolonged stimulation by ATP of P2X7 receptors
may in turn lead to intracellular Ca2+ overload and cell
death, as has been demonstrated for most mammalian cells that express
P2X7/P2X-subtype receptors (55, 56). In DMD the low amount
of the sarcoglycan complex may represent an additional molecular
mechanism that contributes to muscle fiber necrosis.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Paolo Bernardi and Prof. Roger Sabbadini for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by institutional funds from the Consiglio Nazionale delle Ricerche and by Grant 692 from the Fondazione Telethon of Italy (to G. S. and R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 39-49-8276027; Fax.: 39-49-8276040; E-mail: betto{at}civ.bio.unipd.it.
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ABBREVIATIONS |
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The abbreviations used are: DAPs, dystrophin-associated proteins; DMD, Duchenne muscular dystrophy; LGMD, limb girdle muscular dystrophy; BzATP, 3'-O-(4-benzoyl)benzoyl ATP; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; NOS, nitric oxide synthase; nNOS, neuronal-type nitric oxide synthase.
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REFERENCES |
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