In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II

Biochimie (1992) 74, 565-570 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Pads 565 In vitro digestion of dystrophin by calci...

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Biochimie (1992) 74, 565-570 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Pads

565

In vitro digestion of dystrophin by calcium-dependent proteases,

calpains I and II P C o t t i n l, S P o u s s a r d 1, D M o m e t 2, JJ Brustis l, M M o h a m m a d p o u r I, J L e g e r 2, A D u c a s t a i n g l IISTAB, Laboratoire de Biochimie et Technologie des Aliments, Universit~ Bordeattr I, 33405 Talence Cedex; 21NSERM, U 300, Facult~ de Pharmacie, Avenue Charles.Flahaut, 34060 Montpellier Cedex, France (Received l0 February 1992; accepted 16 April 1992)

Summary - - Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca 2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro. The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173-1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43760) or the C-terminal (residues 3357-3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250-370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes

and before any conclusion as to the presenceor absence of dystrophincan be deducedfrom results obtainedusing immunoanalysesof muscle biopsies.

ealpain I / calpain !I / Ca2+.dependent proteolysis / eytoskeletal proteins / dystrophin Introduction Calcium-dependent neutral proteases, also called calpains I and II isolated from the cytosolic fraction of muscular tissue but which may be translocated to the cell membrane under certain conditions, seem to have distinct catalytic 80-kDa subunits and an identical regulatory 30-kDa subunit [1-5]. Their precise biological functions are still poorly understood despite the current increase in knowledge on their structural and enzymatic properties [6-131. Many reports have already shown that in vitro some membranous structures, myofibrillar and cytoskeletal proteins, are substrates for calpain I and II [14-21]. It is known that rapid protein degradation in vivo is always an important feature in several muscle wasting diseases. Three years ago the normal muscle cell membrane was found to contain a large molecule, named dystrophin, whose absence in Duchenne muscular dystrophy leads to rapid muscle degeneration in patients without the gene coding for dystrophin [22-25]. In some other myopathies, such as a

dystrophic myotony, dystrophin appears to be abnormal [26, 27]. In these specific cases it is not yet known if the abnormality results from repression of synthesis or from a putative degradation process of this protein. Elevated free calcium levels found in some pathological muscles [28] can promote an activation of the proteolytic activity of both calpains I and II. In the present study we investigated the degradation of dystrophin to determine whether this molecule is a potential substrate for these calpains. Dystrophin antibodies specific to different regions of the protein have been used to examine breakdown fragments. The results provide evidence that caution must be exercised before concluding from immunoanalyses that the detection of dystrophin, in its native form, becomes negative as the result of such a proteolytic process. Materials and methods Unless otherwise indicated, all chemicals and reagents were from Sigma in the highest grade and purity available.

566 Preparation of proteins Calpains I and II were purified from 2 kg of fresh rabbit muscle as previously described [29] and stored in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 2 mM dithiothreitol, 1 mM NaN s at 2°C. Calpastatin (calpain-specific inhibitor) was purified from fresh muscle as previously described [30] and stored lyophilized at -80°C. The dystrophin-enriched extract was prepared according to [3 !] and the procedure terminated by elution on a Gzs (Pharmacia) column to eliminate ammonium sulfate and the excess of protease inhibitors added during dystrophin extraction.

Digestion with calpains The dystrophin-enriched extract in 20 mM Tris-HCI, pH 7.5, 2 mM dithiothreitol, 1 mM NaN3 was incubated with purified calpain I and I! at the same weight ratio (1000 dystrophin]! calpain) for 0 to 10 min at 30°C. Proteolysis was initiated by the addition of 0.01 mM calcium (final concentation) for calpain I and 1 mM for calpain II. At the indicated times, the proteolytic digestion was stopped

by boiling the sample in 4% SDS denaturing solution before the SDS-PAGE analysis. Controls were carried out under various experimental conditions (ie no calpain, calpain in presence of calpastatin in excess) and dystrophin-enriched preparations were found to be stable for 30 rain at 30°C. Controls were also performed to determine whether calpain solutions were still active at the end of the proteolytic time-course studies. Anti-dystrophin antibodies

Rabbit polyclonal antibodies were produced using fusion protein as immunogens [31]. The three sera used were obtained from different parts of dystrophin molectde ie the N-terminal segment (residues 43-760), the central region (residues 11731728) and the C-terminal segment (residues 3357-3660). SDS-PAGE and immunobiot analyses

Enzymatic digests were separated on a 2.5-9.5% gradient resolving polyacrylamide gel containing 25% glycerol and no stacking gel. The relative molecular mass markers were as follows: myosin heavy chain (200 kDa), beta-galactosidase (I 16 kDa), phosphorylase B (97 kDa), bovine serum albumin (68 kDa). The fractionated proteins were electro-transferred onto lmmobilon P sheets in a transfer buffer containing 0.1% SDS and incubated with each polyclonal antibody used at 1/50 dilution, then revealed with anti-rabbit lgG coupled to alkaline phosphatase.

Results

Comparative proteolytic patterns of dystrophinenriched preparation Limited proteolysis of dystrophin present in the dystrophin-enriched preparation of chicken smooth muscle was performed and compared using three dif-

ferent and specific sera (Materials and methods). The domain-specific polyclonal dystrophin antibodies allowed us to identify cleavage products relative to their original region. The gel pattern shown in figure 1A was revealed with antibodies directed against the dystrophin Cterminal domain. In the presence of calpain I (calcium: 0.01 mM) or calpain II (calcium: 1 raM) the degradation products were the same. The native dystrophin band (Mr = 420 kDa) was very quickly degraded (in 60 s) but no protein band corresponding to the breakdown products with sizes between Mr 420 kDa and 66 kDa were revealed (eg according to the migration of the markers). After 60 s of calpain proteolysis no protein band corresponding to the native form of dystrophin remained, thus indicating that the C-terminal region of the protein seemed to contain at least one or more proteolytie sites which were highly sensitive to the proteolytic activity. Using the antibody specific to the central part of dystrophin (fig 1B), the same proteolytic patterns were revealed either with calpain I or calpain II. Three main stable breakdown products were detected: these proteins have relative molecular masses of 370, 310 and 250 kDa, respectively. The comparative time-courses showed that with calpain I, cleavages occurred slightly faster than with calpain II. In our experimental conditions, the native 420 kDa protein decreased over the time of proteolysis and remained detectable after 10 rain of digestion by both calpains: this last observation (fig 1B) was due to the higher sensitivity of this antibody to the spectrin-like domain of dystrophin. The calpain proteolytic patterns (fig IC) revealed with the dystrophin antibodies directed against the Nterminal part of the molecule did not show significant differences between both proteolytic time courses with calpain I and calpain H. Two major proteolytic products were identified with Mr 370 and 310 kDa. Among these two products, only the 310-kDa breakdown product was found to be stable and resistant to further proteolytic degradation.

Discussion It was first concluded from this study that dystrophin was a good substrate, at least in vitro, for both calpains I and II. The use of domain specific dystrophin antibodies directed against the C-terminal part of the molecule clearly indicated that calpains could very rapidly degrade the C-terminal region of dystrophin. Different cleavage sites, determined according to the relative molecular mass of the main dystrophin fragments generated by the proteolytic activity of both

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Fig 1. Digestion of smooth muscle dystrophin by calpain I and calpain II. Smooth muscle dystrophin-enriched solution containing approximately 10 mg/ml of protein was incubated at 30°C in the presence of calpain I or calpain 1I (0.1 mg/ml) and free Ca ~+ (0.010 mM and 1 mM respectively). The times of incubation were: lanes a, e: 0 min; b, f: 0.5 min; c, g: 1 min; d, h: 10 min. Immunodetection of dystrophin and its breakdown products were performed with polyclonal antibodies and a secondary rabbit antibody coupled with alkaline phosphatase (as described in Materials and methods). A. Polyclonal antibody raised against the C-terminal domain (aminoacids 3357-3660). B. Polyclonal antibody raised against the spectrin-like domain (aminoacids 1173-1728). C. Polyclonal antibody raised against the actin-binding domain (aminoacids 43-760). The electrophoretic mobility of the molecular weight markers used is shown by black arrows in the right part of the gel.

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Fig 2. Localization of calpain I and lI cleavage regions within a smooth muscle dystrophin. A schematic drawing illustrating the different dystrophin domains as previously described [35]. Localization of the three regions used to obtain sites of recognition by the polyclonal antibodies for these experiments are shown in dashed area. -, Cleavage sites for calpains I and II. Z, preferential cleavage region; Y, secondary cleavage region; X, third cleavage region. The M, of the generated breakdown products are indicated in kilodaitons (kDa). calpains, are proposed in a linear schematic drawing of the dystrophin molecule in figure 2. It would certainly be preferable to identify the exact cleavage sites rather than guess at them by transferring individual blotted bands onto a sequencer, but accurate results have been difficult to obtain due to difficulties in obtaining pure dystrophin-enriched solutions. As shown in figure 2, the first cleavage site is denoted as Z at the C-terminal end. This result was suggested by the presence of a proteolytic product of M, = 370 kDa detected with both N-terminal and central-specific dystrophin sera. The 310-kDa breakdown product detected by the antibody directed against the N-terminal part of dystrophin must have been derived from the 370-kDa fragment shortened in its C-terminal region by a cleavage between the central spectrin-like domain and the cystein-rich region (cleavage site Y). In addition, the antibody directed against the central portion of the dystrophin molecule enabled detection of a 250-kDa protein band which was not detected by the antibodies directed against either of the distal pans of the molecule. This fragment must have been derived from another cleavage site splitting the N-terminal pan from the central spectrin-like domain (cleavage site X).

Although there are other possibilities for generating different 310- or 250-kDa fragments the cleavage sites X, Y and Z appear to be the most probable. In fact, there are no definitive ways of mapping these cleavage sites but the central region of dystrophin showed strong resistance towards calpain hydrolyses, which is an important result from this study. According to these results, the use of antibodies directed towards the central domain of dystrophin would be more optimal to prove the absence of dystrophin from immunohistochemical staining and immunoblot analyses of pathological muscles. The initial greater affinity for calpains towards the C-terminal domain is very important, because this region, via the glycoprotein complex, is thought to interact with the muscle membrane [32-34]. Moreover, the increase in free intracellular calcium ions described under some pathological conditions [28] could activate calpains and thus lead to dystrophin detachment from the muscle membrane. Some illnesses could therefore exhibit dystrophin-deficiency-like symptoms due to proteolytic dissociation of dystrophin from the muscle membrane. Experiments with artificial increases in calcium-ion concentration are being conducted in order to investigate whether the calcium stimulated proteolytic activities of both calpains I and I1 could effectively lead to dystrophin deficiency-like symptoms in vivo.

569

Acknowledgments We are grateful to Dr Jean J Leger for his helpful advice and discussions, P Lochet for technical assistance and S Oree for rereading the manuscript. This work was supported by Institut National de la Recherche Agronomique and ~.ssociation Fran~:aise contre les Myopathies.

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