Ecto-ATPase Activity of alpha -Sarcoglycan (Adhalin)*

Romeo BettoDagger §, Luigi Senter, Stefania Ceoldo, Elena Tarricone, Donatella BiralDagger , and Giovanni Salviatidagger Dagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

alpha -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 alpha -sarcoglycan. alpha -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 alpha -sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of alpha -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

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 beta -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: alpha -dystroglycan, a peripheral glycoprotein that binds extracellular matrix proteins like laminin-2 (merosin) and, in the neuromuscular junction, laminin-4 (agrin); and beta -dystroglycan, an intrinsic membrane protein that binds dystrophin at its cytoplasmic tail and alpha -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: alpha -sarcoglycan (adhalin, 50 kDa), beta -sarcoglycan (43 kDa), gamma - and delta -sarcoglycans (35 kDa) (8-10), and epsilon -sarcoglycan (11, 12). The function of the sarcoglycans remains unknown.

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 alpha -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, beta - and gamma -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 beta -, gamma -, and delta -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.

Here we show that alpha -sarcoglycan is a sarcolemma ecto-ATPase and that sarcolemma expresses a P2X-type purinergic receptor, a nonspecific cationic channel. We speculate that alpha -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

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 -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, beta -dystroglycan, and alpha - and gamma -sarcoglycans were used (not shown).


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Fig. 1.   Photoaffinity labeling of the dystrophin-DAPs preparation with [alpha -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 [alpha -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 alpha -sarcoglycan was selectively labeled. Lane a, molecular mass standards. Dys, dystrophin; alpha - and beta -DG, alpha - and beta -dystroglycan; alpha -, beta -, delta -, and gamma -SG, alpha -, beta -, delta -, and gamma -sarcoglycan; alpha -, beta 1- and beta 2-Syn, alpha -, beta 1-, and beta 2-syntrophin. The dystrophin-DAPs were identified by comparison with data in the literature and by immunoblot staining (not shown) with commercial antibodies.

[alpha -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 [alpha -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 [alpha -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 [alpha -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.

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 [alpha -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.

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 -80 °C by exposing dried gels or nitrocellulose sheets to Kodak XAR-5 films.

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 [alpha -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).

Reagents-- [alpha -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

The physiological role of alpha -sarcoglycan (adhalin), the protein of the sarcoglycan complex that is missing in LGMD-2D, is unknown. In the present study, we demonstrate that alpha -sarcoglycan 1) binds ATP, 2) has an ATPase activity not inhibitable by known inhibitors of endo-ATPases, and 3) is not a purinergic receptor.

Photoaffinity labeling experiments using [alpha -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-[alpha -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).

To confirm the identity of the 50-kDa ATP-labeled protein as alpha -sarcoglycan, we have used different immunological approaches with monoclonal and polyclonal anti-alpha -sarcoglycan antibodies (Fig. 2A, lanes c and d, respectively). Among the dystrophin-DAPs, the same protein band that bound [alpha -32P]ATP (Fig. 2B, lane f) was also selectively stained by the monoclonal anti-alpha -sarcoglycan antibody (Fig. 2B, lane g). Furthermore, the binding of [alpha -32P]ATP to the 50-kDa protein was inhibited by incubation of the dystrophin-DAPs preparation with the monoclonal anti-alpha -sarcoglycan antibody prior to photoactivation (Fig. 2C, lane i). Finally, polyclonal anti-alpha -sarcoglycan antibodies immunoprecipitated the 50-kDa [alpha -32P]ATP-labeled protein (Fig. 2C, lane j).


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Fig. 2.   Identification of the [alpha -32P]ATP-labeled protein as alpha -sarcoglycan. A, the monoclonal antibody raised against a fusion peptide containing the ATP-binding site located in the extracellular domain of alpha -sarcoglycan (lane c) and the polyclonal antibody raised against a carboxyl-terminal peptide of alpha -sarcoglycan (lane d) were tested on 20 µg of a dystrophin-DAPs preparation (lane b). Both antibodies selectively labeled the alpha -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 [alpha -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-alpha -sarcoglycan antibody (lane g), as described under "Experimental Procedures." This antibody recognizes the same 50-kDa protein labeled by [alpha -32P]ATP. C, in a second set of experiments, the monoclonal anti-alpha -sarcoglycan antibody was added to 20 µg of protein from the dystrophin-DAPs preparation before (lane i) and after (lane h) the addition of [alpha -32P]ATP and UV irradiation. The antibody inhibited the labeling of alpha -sarcoglycan by [alpha -32P]ATP (lane i). On the other hand, incubation of the [alpha -32P]ATP-labeled alpha -sarcoglycan with the polyclonal anti-alpha -sarcoglycan antibody allowed the immunoprecipitation of the [alpha -32P]ATP-labeled protein (lane j).

Analysis of the deduced amino acid sequence of rabbit alpha -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 alpha -sarcoglycan sequence. It should be noted that this sequence is conserved between rabbit, mouse, hamster, and human alpha -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 alpha -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 alpha -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) alpha -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 [alpha -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 alpha 1-syntrophin (34) (compare, for example, Fig. 2, lane h with lane f).


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Fig. 3.   Photoaffinity labeling of dystrophin-DAPs and sarcolemma membranes preparations with [alpha -32P]ATP. A, 20 µg of dystrophin-DAPs (lanes b-e) in 200 µl of 25 mM Tris, pH 7.4, and 1 mM DTT in the presence of 2.5 mM MgCl2 and 10 µM CaCl2 (lane c), or 2.5 mM MgCl2 and 2.5 mM EGTA (lane d), or 2.5 mM EGTA and 2.5 mM EDTA (lane e) were UV-photoactivated in the presence of 0.4 µM [alpha -32P]ATP as described under "Experimental Procedures." B, 50 µg of sarcolemma membrane proteins (lanes g-k) in 200 µl of 25 mM Tris, pH 7.4, and 1 mM DTT in the presence of either 2.5 mM MgCl2 and 10 µM CaCl2 (lane h), or 2.5 mM MgCl2 and 2.5 mM EGTA (lane i), or 2.5 mM EGTA and 2.5 mM EDTA (lane j) were UV-photoactivated in the presence of 0.4 µM [alpha -32P]ATP as described under "Experimental Procedures." The proteins were then separated by SDS-PAGE and stained with Coomassie Brilliant Blue (lanes b and g). Gels were then dried and autoradiographed (lanes c-e and h-j). Lanes a and f, molecular mass standards. Lane k, immunoblot staining with the monoclonal anti-alpha -sarcoglycan antibody of a sarcolemma membrane preparation.

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 [alpha -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 [alpha -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 [alpha -32P]ATP has been labeled.

To verify whether native alpha -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 [alpha -32P]ATP caused labeling of the alpha -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 [alpha -32P]ATP4-, alpha -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 alpha -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 [alpha -32P]ATP to alpha -sarcoglycan. As indicated by the results shown in Fig. 3, because alpha -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 [alpha -32P]ATP to alpha -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 [alpha -32P]ATP to alpha -sarcoglycan, further suggesting that alpha -sarcoglycan is an ATPase. On the other hand, BzATP also inhibited the binding of [alpha -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 [alpha -32P]ATP to alpha -sarcoglycan, whereas GTP had a very little effect (Fig. 4, lane i). The inability of UTP to influence the labeling of [alpha -32P]ATP to alpha -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 [alpha -32P]ATP to alpha -sarcoglycan (Fig. 4, lane h) indicates that this protein is not a P2Y receptor.


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Fig. 4.   Effects of modulators of P2 purinergic receptors and of substrates of ecto-ATPases on the photoaffinity labeling of alpha -sarcoglycan by [alpha -32P]ATP. Twenty µg of dystrophin-DAPs were UV-photoactivated in a buffer containing either 0.025 M Tris, pH 7.4, 1 mM DTT, 0.4 µM [alpha -32P]ATP, 2.5 mM MgCl2, and 10 µM CaCl2 (lanes c-i) or 0.025 M Tris, pH 7.4, 1 mM DTT, 0.4 µM [alpha -32P]ATP, 2.5 mM EGTA, and 2.5 mM EDTA (lane j) in the absence (lanes c and g) or the presence of the indicated compounds at 50 µM. The dystrophin-DAPs sample of lane e was incubated with the oxidized ATP for 2 h at 0-4 °C, whereas all other samples were incubated for 10 min at 24 °C before UV photoactivation. Proteins were then separated by SDS-PAGE, transferred to nitrocellulose, stained with Ponceau Red (lane b), and autoradiographed, as described under "Experimental Procedures." Lane a, molecular mass standards.

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 alpha -sarcoglycan by [alpha -32P]ATP. Fig. 5 shows that ATP labeling of alpha -sarcoglycan was increased by NaCl up to 20 mM, whereas higher concentrations were inhibitory. These data indicate that alpha -sarcoglycan share the same sensitivity to monovalent cations as known ecto-ATPases.


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Fig. 5.   Effect of sodium ions on the photoaffinity labeling of dystrophin-DAPs by [alpha -32P]ATP. A and B, 50 µg of sarcolemma membrane proteins resuspended in 0.025 M Tris, pH 7.4, 1 mM DTT, 0.4 µM [alpha -32P]ATP, 2.5 mM MgCl2, and 10 µM CaCl2 in the absence or in the presence of the indicated concentrations of NaCl were UV-photoactivated as described under "Experimental Procedures." After SDS-PAGE, the gel was stained with Coomassie Brilliant Blue (A), dried, and autoradiographed (B; only the alpha -sarcoglycan region is shown). C, values of the intensity of [alpha -32P]ATP labeling of alpha -sarcoglycan shown in B were plotted against the NaCl concentration.

These photoaffinity labeling experiments suggested that alpha -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 alpha -sarcoglycan only, we incubated the dystrophin-DAPs preparation with the monoclonal anti-alpha -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 alpha -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 [alpha -32P]ATP to alpha -sarcoglycan, inhibition of ATPase activity by the monoclonal anti-alpha -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 [alpha -32P]ATP binding could still be responsible for the fraction of ATPase activity that is not inhibited by the anti-alpha -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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Table I
ATPase activity of dystrophin-DAPs preparations
The effect of BzATP, a photoaffinity probe that covalently binds to the nucleotide site of ATPases (41, 42), on the basal ATPase activity (see "Experimental Procedures") of the dystrophin-DAPs preparation (10 µg) was determined. Preincubation of the preparation with 100 µM BzATP (+BzATP) before measuring ATPase activity was without effect, whereas irradiation for 5 min at 254 nm (+UV) partially reduced the activity. On the contrary, irradiation in the presence of 100 µM BzATP (+BzATP/UV) almost abolished the ATPase activity. Data are from two different dystrophin-DAPs preparations. The Mg2+ dependence and Ca2+ independence of the ATPase activity of alpha -sarcoglycan was determined in five different dystrophin-DAPs preparations (mean ± S.E.) incubated under basal conditions followed either by the stepwise addition of 5, 10, and 20 mM EDTA or by the removal of Ca2+ by the addition of 1 mM EGTA (*, p < 0.001; **, p < 0.002).


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Fig. 6.   Inhibition of the ATPase activity of dystrophin-DAPs preparation by monoclonal anti-alpha -sarcoglycan antibody. Inhibition of ATPase activity was performed by preincubating the dystrophin-DAPs preparation for 2 h at room temperature with either the monoclonal () or the polyclonal (open circle ) anti-alpha -sarcoglycan antibodies (Ab) at the indicated ratio in 0.025 M Tris, pH 7.4, 2.5 mM MgCl2, and 10 µM CaCl2. The reaction was started by the addition of 2 mM ATP. After 10 min the Pi released was measured by the method of Lanzetta et al. (29). DGC, dystrophin-glycoprotein complex.

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 alpha -sarcoglycan is an ecto-ATPase. Thus, it appears possible that alpha -sarcoglycan modulates the activity of P2X-type purinergic receptors.

Mutations in the alpha -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 alpha - and beta -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 alpha -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.

    ACKNOWLEDGEMENTS

We are grateful to Prof. Paolo Bernardi and Prof. Roger Sabbadini for critical reading of the manuscript.

    FOOTNOTES

* 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.

dagger Deceased March 12, 1998.

§ To whom correspondence should be addressed. Tel.: 39-49-8276027; Fax.: 39-49-8276040; E-mail: betto{at}civ.bio.unipd.it.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Winder, S. J. (1997) J. Muscle Res. Cell Motil. 18, 617-629[CrossRef][Medline] [Order article via Infotrieve]
  2. Michalak, M., and Opas, M. (1997) Curr. Opin. Neurol. 10, 436-442[Medline] [Order article via Infotrieve]
  3. Straub, V., and Campbell, K. P. (1997) Curr. Opin. Neurol. 10, 168-175[Medline] [Order article via Infotrieve]
  4. Suzuki, A., Yoshida, M., Hayashi, K., Mizuno, Y., Hagiwara, Y., and Ozawa, E. (1994) Eur. J. Biochem. 220, 283-292[Abstract]
  5. Ahn, A. H., Freener, C. A., Gussoni, E., Yoshida, M., Ozawa, E., and Kunkle, L. M. (1996) J. Biol. Chem. 271, 2724-2730[Abstract/Free Full Text]
  6. Suzuki, A., Yoshida, M., Yamamoto, M., and Ozawa, E. (1992) FEBS Lett. 308, 154-160[CrossRef][Medline] [Order article via Infotrieve]
  7. Henry, M. D., and Campbell, K. P. (1996) Curr. Opin. Cell Biol. 8, 625-631[CrossRef][Medline] [Order article via Infotrieve]
  8. Yoshida, M., Suzuki, A., Yamamoto, H., Noguchi, S., Mizuno, Y., and Ozawa, E. (1994) Eur. J. Biochem. 222, 1055-1061[Abstract]
  9. Nigro, V., de Sá Moreira, E., Piluso, G., Vainzof, M., Belsito, A., Politano, L., Puca, A. A., Passos-Bueno, M. R., and Zatz, M. (1996) Nat. Genet. 14, 195-198[Medline] [Order article via Infotrieve]
  10. McNally, E. M., Yoshida, M., Mizuno, Y., Ozawa, E., and Kunkel, L. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9690-9694[Abstract/Free Full Text]
  11. Ettinger, A. J., Geng, G., and Sanes, J. R. (1997) J. Biol. Chem. 272, 32534-32538[Abstract/Free Full Text]
  12. McNally, E. M., Ly, C. T., and Kunkel, L. M. (1998) FEBS Lett. 422, 27-32[CrossRef][Medline] [Order article via Infotrieve]
  13. Ohlendieck, K., and Campbell, K. P. (1991) J. Cell Biol. 115, 1685-1694[Abstract]
  14. Ohlendieck, K., Matsumura, K., Ionasescu, V. V., Towbin, J. A., Bosch, E. P., Weinstein, S. L., Sernett, S. W., and Campbell, K. P. (1993) Neurology 43, 795-800[Abstract]
  15. Matsumura, K., Ervasti, J. M., Ohlendieck, K., Kahl, S. D., and Campbell, K. P. (1992) Nature 360, 588-591[CrossRef][Medline] [Order article via Infotrieve]
  16. Roberds, S. L., Leturcq, F., Allamand, V., Piccolo, F., Jeanpierre, M., Anderson, R. D., Lim, L. E., Lee, J. C., Tomé, F. M. S., Romero, N. B., Fardeau, M., Beckmann, J. S., Kaplan, J.-C., and Campbell, K. P. (1994) Cell 78, 625-633[Medline] [Order article via Infotrieve]
  17. Piccolo, F., Roberds, S. L., Jeanpierre, M., Leturcq, F., Azibi, K., Beldjord, C., Carrié, A., Récan, D., Chaouch, M., Reghis, A., El Kerch, F., Sefiani, A., Voit, T., Merlini, L., Collin, H., Eymard, B., Beckmann, J. S., Romero, N. B., Tomé, F. M. S., Fardeau, M., Campbell, K. P., and Kaplan, J.-C. (1995) Nat. Genet. 10, 243-245[Medline] [Order article via Infotrieve]
  18. Kawai, H., Akaike, M., Endo, T., Adachi, K., Inui, T., Mitsui, T., Kashiwagi, S., Fujiwara, T., Okuno, S., Shin, S., Miyoshi, K., Campbell, K. P., Yamada, H., Shimizu, T., Matsumura, K., and Saito, S. (1995) J. Clin. Invest. 96, 1202-1207[Medline] [Order article via Infotrieve]
  19. Roberds, S. L., Ervasti, J. M., Anderson, L. D., Ohlendieck, K., Kahl, S. D., Zoloto, D., and Campbell, K. P. (1993) J. Biol. Chem. 268, 11496-11499[Abstract/Free Full Text]
  20. Lim, L. E., Duclos, F., Broux, O., Bourg, N., Sunada, Y., Allamand, V., Meyer, J., Richard, I., Moomaw, C., Slaughter, C., J. C., Tomé, F. M. S., Fardeau, M., Jackson, C. E., Beckmann, J. S., and Campbell, K. P. (1995) Nat. Genet. 11, 257-265[Medline] [Order article via Infotrieve]
  21. Noguchi, S., McNally, E. M., Othmane, K. B., Hagiwara, Y., Mizuno, Y., Yoshida, M., Yamamoto, H., Bönnemann, C. G., Gussoni, E., Denton, P. H., Kyriakides, T., Middleton, L., Hentati, F., Hamida, M. B., Nonaka, I., Vance, J. M., Kunkel, L. M., and Ozawa, E. (1995) Science 270, 819-822[Abstract]
  22. Luise, M., Presotto, C., Senter, L., Betto, R., Ceoldo, S., Furlan, S., Salvatori, S., Sabbadini, R. A., and Salviati, G. (1993) Biochem. J. 293, 243-247[Medline] [Order article via Infotrieve]
  23. Ervasti, J. M., Kahl, S. D., and Campbell, K. P. (1991) J. Biol. Chem. 266, 9161-9165[Abstract/Free Full Text]
  24. Senter, L., Ceoldo, S., Petrusa Meznaric, M., and Salviati, G. (1995) Biochem. Biophys. Res. Commun. 206, 57-63[CrossRef][Medline] [Order article via Infotrieve]
  25. Salvatori, S., Damiani, E., Barhanin, J., Furlan, S., Salviati, G., and Margreth, A. (1990) Biochem. J. 267, 679-687[Medline] [Order article via Infotrieve]
  26. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  27. Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 622-626[Abstract]
  28. Salviati, G., Volpe, P., Salvatori, S., Betto, R., Damiani, E., Margreth, A., and Pasquali-Ronchetti, I. (1982) Biochem. J. 202, 289-301[Medline] [Order article via Infotrieve]
  29. Lanzetta, P. A., Alvarez, P. A., Reinach, P. S., and Candia, O. A. (1979) Anal. Biochem. 100, 95-97[Medline] [Order article via Infotrieve]
  30. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951[Medline] [Order article via Infotrieve]
  31. Sabbadini, R. A., and Dahms, A. S. (1989) J. Bioenrg. Biomembr. 21, 163-213
  32. Treuheit, M. J., Vaghy, P. L., and Kirley, T. L. (1992) J. Biol. Chem. 267, 11777-11782[Abstract/Free Full Text]
  33. Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214[Medline] [Order article via Infotrieve]
  34. Brenman, J. E., Chao, D. S., Xia, H., Aldape, K., and Bredt, S. D. (1995) Cell 82, 743-752[Medline] [Order article via Infotrieve]
  35. Dubyak, G. R., and El-Moatassim, C. (1993) Am. J. Physiol. 265, C577-C606[Abstract/Free Full Text]
  36. Burnstock, G. (1996) CIBA Found. Symp. 198, 1-34[Medline] [Order article via Infotrieve]
  37. Fabiato, A. (1988) Methods Enzymol. 157, 378-417[Medline] [Order article via Infotrieve]
  38. Murgia, M., Hanau, S., Pizzo, P., Rippa, M., and Di Virgilio, F. (1993) J. Biol. Chem. 268, 8199-8203[Abstract/Free Full Text]
  39. Cusack, N. J. (1993) Drug Dev. Res. 28, 244-252
  40. Bar-Zvi, D., Bar, I., Yoshida, M., and Shavit, N. (1992) J. Biol. Chem. 267, 11029-11033[Abstract/Free Full Text]
  41. Pal, P. K., Ma, Z., and Coleman, P. S. (1992) J. Biol. Chem. 267, 25003-25009[Abstract/Free Full Text]
  42. Zimmermann, H. (1996) Drug Dev. Res. 39, 337-352[CrossRef]
  43. Brake, A. J., and Julius, D. (1996) Annu. Rev. Cell Dev. Biol. 12, 519-541[CrossRef][Medline] [Order article via Infotrieve]
  44. Plesner, L., Juul, B., Skriver, E., and Aalkjaer, C. (1991) Biochim. Biophys. Acta 1067, 191-200[Medline] [Order article via Infotrieve]
  45. Lin, S.-H., and Russel, W. E. (1988) J. Biol. Chem. 263, 12253-12258[Abstract/Free Full Text]
  46. Barbacci, E., Filippini, A., De Cesaris, P., and Ziparo, E. (1996) Biochem. Biophys. Res. Commun. 222, 273-279[CrossRef][Medline] [Order article via Infotrieve]
  47. Kennedy, C., Westfall, T. D., and Sneddon, P. (1996) Semin. Neurosci. 8, 195-199[CrossRef]
  48. Lu, Z., and Smith, D. O. (1991) J. Physiol. (London) 436, 45-56[Abstract]
  49. Beckmann, J. S., and Bushby, K. M. D. (1996) Curr. Opin. Neurol. 9, 389-393[Medline] [Order article via Infotrieve]
  50. Ozawa, E., Noguchi, S., Mizuno, Y., Hagiwara, Y., and Yoshida, M. (1997) Muscle Nerve 21, 421-438[CrossRef]
  51. Petrof, B. J., Shrager, J. B., Stedman, H. H., Kelly, A. M., and Sweeney, H. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3710-3714[Abstract]
  52. Pasternak, C., Wong, S., and Elson, E. (1995) J. Cell Biol. 128, 355-361[Abstract]
  53. Turner, P. R., Westwood, T., Regen, C. M., and Steinhardt, R. A. (1988) Nature 335, 535-538
  54. Carlson, C. G. (1998) Neurobiol. Dis. 5, 3-15[CrossRef][Medline] [Order article via Infotrieve]
  55. Pizzo, P., Zanovello, P., Bronte, V., and Di Virgilio, F. (1991) Biochem. J. 274, 139-144[Medline] [Order article via Infotrieve]
  56. Pizzo, P., Murgia, M., Zambon, A., Zanovello, P., Bronte, V., Pietrobon, D., and Di Virgilio, F. (1992) J. Immunol. 149, 3372-3378[Abstract/Free Full Text]


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