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• W2092770107 abstract "O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction. O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy. In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction. O-Linked N-acetylglucosaminylation, termed O-GlcNAc, 5The abbreviations used are: O-GlcNAc, O-linked N-acetylglucosaminylation; MHC, myosin heavy chain; MLC, myosin light chain; WGA, wheat germ agglutinin; EDL, extensor digitorum longus; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid.5The abbreviations used are: O-GlcNAc, O-linked N-acetylglucosaminylation; MHC, myosin heavy chain; MLC, myosin light chain; WGA, wheat germ agglutinin; EDL, extensor digitorum longus; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid. is a regulatory post-translational modification that occurs in nuclear and cytosolic proteins, corresponding to the addition of a unique monosaccharide, N-acetyl-d-glucosamine, to a serine and threonine hydroxyl group by a β-linkage (1Torres C.R. Hart G.W. J. Biol. Chem. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar). Because of the existence of the UDP-GlcNAc-peptide-β-GlcNAc transferase, which transfers the monosaccharide into proteins (2Haltiwanger R.S. Holt G.D. Hart G.W. J. Biol. Chem. 1990; 265: 2563-2568Abstract Full Text PDF PubMed Google Scholar, 3Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar), and the N-acetyl-β-d-glucosaminidase, which removes it (4Dong D.L. Hart G.W. J. Biol. Chem. 1994; 269: 19321-19330Abstract Full Text PDF PubMed Google Scholar), O-GlcNAc is more similar to phosphorylation than classical glycosylation. The half-life of the monosaccharide is shorter than the half-life of the protein backbone (5Roquemore E.P. Chevrier M.R. Cotter R.J. Hart G.W. Biochemistry. 1996; 35: 3578-3586Crossref PubMed Scopus (149) Google Scholar), indicating that the O-GlcNAc cycle could rapidly respond to cellular signals (6Wells L. Vosseller K. Hart G.W. Science. 2001; 291: 2376-2378Crossref PubMed Scopus (791) Google Scholar). All of the known O-GlcNAc proteins described to date are also phosphoproteins (7Wells L. Whelan S.A. Hart G.W. Biochem. Biophys. Res. Commun. 2003; 302: 435-441Crossref PubMed Scopus (161) Google Scholar) (for review, see Ref. 8Whelan S.A. Hart G.W. Circ. Res. 2003; 93: 1047-1058Crossref PubMed Scopus (109) Google Scholar). Modifications by O-GlcNAc or phosphorylation could occur on the same site (9Cheng X. Cole R.N. Zaia J. Hart G.W. Biochemistry. 2000; 39: 11609-11620Crossref PubMed Scopus (150) Google Scholar) or at neighboring sites (10Comer F.I. Hart G.W. Biochemistry. 2001; 40: 7845-7852Crossref PubMed Scopus (234) Google Scholar); this competition between O-Glc-NAc and phosphorylation is called the “Yin-Yang” process. O-GlcNAc has been described to play a role in various cell functions: in nuclear transport (11Holt G.D. Snow C.M. Senior A. Haltiwanger R.S. Gerace L. Hart G.W. J. Cell Biol. 1987; 104: 1157-1164Crossref PubMed Scopus (309) Google Scholar, 12Hanover J.A. Cohen C.K. Willingham M.C. Park M.K. J. Biol. Chem. 1987; 262: 9887-9894Abstract Full Text PDF PubMed Google Scholar, 13Hanover J.A. FASEB J. 2001; 15: 1865-1876Crossref PubMed Scopus (249) Google Scholar); in protein degradation, with a reversible inhibition of the proteasome itself (14Zhang F. Su K. Yang X. Bowe D.B. Paterson A.J. Kudlow J.E. Cell. 2003; 115: 715-725Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) and a protection of the modified protein against proteasomal degradation (9Cheng X. Cole R.N. Zaia J. Hart G.W. Biochemistry. 2000; 39: 11609-11620Crossref PubMed Scopus (150) Google Scholar, 15Han I. Kudlow J.E. Mol. Cell. Biol. 1997; 17: 2550-2558Crossref PubMed Scopus (377) Google Scholar, 16Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar); and in regulation of protein expression, with the regulation of transcription (10Comer F.I. Hart G.W. Biochemistry. 2001; 40: 7845-7852Crossref PubMed Scopus (234) Google Scholar, 17Kelly W.G. Dahmus M.E. Hart G.W. J. Biol. Chem. 1993; 268: 10416-10424Abstract Full Text PDF PubMed Google Scholar, 18Comer F.I. Hart G.W. Biochim. Biophys. Acta. 1999; 1473: 161-171Crossref PubMed Scopus (147) Google Scholar, 19Gao Y. Miyazaki J. Hart G.W. Arch. Biochem. Biophys. 2003; 415: 155-163Crossref PubMed Scopus (127) Google Scholar) and translation (20Datta B. Ray M.K. Chakrabarti D. Wylie D.E. Gupta N.K. J. Biol. Chem. 1989; 264: 20620-20624Abstract Full Text PDF PubMed Google Scholar). The involvement of O-GlcNAc in protein-protein interactions has been described in different biological systems. Indeed, many proteins playing a key role in organization and assembly of cytoskeleton are O-GlcNAc-modified, including cytokeratins 8, 13, and 18 (21Chou C.F. Smith A.J. Omary M.B. J. Biol. Chem. 1992; 267: 3901-3906Abstract Full Text PDF PubMed Google Scholar, 22Chou C.F. Omary M.B. J. Biol. Chem. 1993; 268: 4465-4472Abstract Full Text PDF PubMed Google Scholar), H, L, and M neurofilaments (23Dong D.L. Xu Z.S. Chevrier M.R. Cotter R.J. Cleveland D.W. Hart G.W. J. Biol. Chem. 1993; 268: 16679-16687Abstract Full Text PDF PubMed Google Scholar, 24Dong D.L. Xu Z.S. Hart G.W. Cleveland D.W. J. Biol. Chem. 1996; 271: 20845-20852Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), microtubule-associated proteins MAP1, -2, and -4 (25xDing M. Vandre D.D. J. Biol. Chem. 1996; 271: 12555-12561Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), crystallin (5Roquemore E.P. Chevrier M.R. Cotter R.J. Hart G.W. Biochemistry. 1996; 35: 3578-3586Crossref PubMed Scopus (149) Google Scholar, 26Roquemore E.P. Dell A. Morris H.R. Panico M. Reason A.J. Savoy L.A. Wistow G.J. Zigler Jr., J.S. Earles B.J. Hart G.W. J. Biol. Chem. 1992; 267: 555-563Abstract Full Text PDF PubMed Google Scholar), synapsin 1 (27Luthi T. Haltiwanger R.S. Greengard P. Bahler M. J. Neurochem. 1991; 56: 1493-1498Crossref PubMed Scopus (37) Google Scholar, 28Cole R.N. Hart G.W. J. Neurochem. 1999; 73: 418-428Crossref PubMed Scopus (95) Google Scholar), and Tau protein (29Arnold C.S. Johnson G.V. Cole R.N. Dong D.L. Lee M. Hart G.W. J. Biol. Chem. 1996; 271: 28741-28744Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Four sites of O-GlcNAc have been described on neurofilaments (23Dong D.L. Xu Z.S. Chevrier M.R. Cotter R.J. Cleveland D.W. Hart G.W. J. Biol. Chem. 1993; 268: 16679-16687Abstract Full Text PDF PubMed Google Scholar), these sites being localized on the head filament, which is involved in the polymerization of filament (30Wong P.C. Cleveland D.W. J. Cell Biol. 1990; 111: 1987-2003Crossref PubMed Scopus (92) Google Scholar). Moreover, the interaction between translation initiation factor eIF-2 and p67 protein is also dependent on O-GlcNAc (20Datta B. Ray M.K. Chakrabarti D. Wylie D.E. Gupta N.K. J. Biol. Chem. 1989; 264: 20620-20624Abstract Full Text PDF PubMed Google Scholar), the deglycosylation of p67 producing the dissociation of the complex. Key proteins involved in the skeletal muscle metabolism and in the contractile process have been recently identified as being O-GlcNAc-modified (31Cieniewski-Bernard C. Bastide B. Lefebvre T. Lemoine J. Mounier Y. Michalski J.C. Mol. Cell Proteomics. 2004; 3: 577-585Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The implication of O-GlcNAc in muscle physiology remains undetermined even if recent results suggest that O-GlcNAc variations could control the muscle protein homeostasis and are involved in the regulation of muscular atrophy (32Cieniewski-Bernard C. Mounier Y. Michalski J.C. Bastide B. J. Appl. Physiol. 2006; 100: 1499-1505Crossref PubMed Scopus (28) Google Scholar) as well as in the glucose metabolism (33Yki-Järvinen H. Virkamaki A. Daniels M.C. McClain D. Gottschalk W.R. Metabolism. 1998; 47: 449-455Abstract Full Text PDF PubMed Scopus (92) Google Scholar). The functional significance of the O-GlcNAc modification of myosin heavy chain (MHC) could be its implication in protein-protein interaction, resulting in polymerization of the thick filament, or its involvement in the development of the muscle contraction by interacting with other contractile proteins. Therefore, the purpose of the present study was (i) to define whether other contractile proteins could be O-GlcNAc-modified and (ii) to determine a potential role of O-GlcNAc post-translational modification in skeletal muscle contraction. Biochemicals—Agarose-immobilized wheat germ agglutinin (WGA), GlcNAc-immobilized beads, and all chemical reagents were purchased from Sigma; sequencing grade modified trypsin was from Promega (Madison, WI); the mixture of antiproteases was from Amersham Biosciences; Zip-TipC18 pipette tips were from Millipore (Bedford, MA); Vivaspin concentrators were from Vivascience (Hannover, Germany); the nitrocellulose sheet was from Advantec MFS (Pleasanton, CA); and the MicroBCA protein assay reagent kit was from Pierce. Animals and Muscle Preparation—Experiments were carried out on skeletal muscles of adult male Wistar rats. The experiments as well as the maintenance conditions of the animals received authorization from the Ministry of Agriculture and the Ministry of Education (Veterinary Service of Health and Animal Protection, authorization 03805). Soleus (a typical slow postural muscle), extensor digitorum longus (EDL, a typical fast muscle), and gastrocnemius (a mixed composition of fibers) muscles were freshly removed from male Wistar rats (n = 10) anesthetized with an intraperitoneal injection of pentobarbital sodium (3 mg·kg-1), quickly frozen, and pulverized in liquid nitrogen. All samples were kept at -80 °C until analyzed. Contractile Protein Preparation—Muscle powder was resuspended in a 5 m EDTA solution, pH 7.0, containing antiproteases. Sample was homogenized for 5 min and then centrifuged at 4 °C for 10 min at 13,000 rpm. Pellet was washed twice using a 50 mm KCl solution containing anti-proteases and centrifuged at 4 °C for 10 min at 13,000 rpm. Purification of O-GlcNAc-modified Proteins—The pellet of contractile proteins described above was resuspended in the binding buffer (20 mm Tris/HCl, 200 mm KCl, 1 mm CaCl2, 1 mm MgCl2, antiproteases, pH 7.8), homogenized by ultrasonic waves (Cell Disruptor B-30). Protein quantification was performed using the MicroBCA protein assay. Isolation of O-GlcNAc proteins was obtained by passing contractile proteins through a WGA-immobilized column. For each experiment, 500 μg of contractile proteins were used. The column was washed with 50 column volumes of binding buffer, followed by washing with 10 column volumes of binding buffer containing 0.2 m GalNAc. Elution was finally performed with 20 column volumes of 0.2 m GlcNAc in the same buffer. Fractions were desalted and concentrated using centrifugation on a Vivaspin concentrator. Samples were resuspended in Laemmli buffer just prior to their electrophoretic analysis. To determine the glycosylation of the muscle biopsies, a similar protocol was applied using the batch technique. For this, 500 μg of proteins were incubated in the presence of 50 μl of WGA-agarose beads. After 1 h of incubation followed by centrifugation, the supernatant was eliminated, and four washes of beads were realized with the binding buffer. Mass Spectrometry—MALDI-TOF analysis was performed according to Ref. 31Cieniewski-Bernard C. Bastide B. Lefebvre T. Lemoine J. Mounier Y. Michalski J.C. Mol. Cell Proteomics. 2004; 3: 577-585Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar. Briefly, SDS-PAGE bands corresponding to proteins purified on WGA affinity chromatography were excised from the gel. Gel pieces were destained using a solution of 30 mm potassium ferricyanide, 100 mm sodium thiosulfate, and washed with water. Proteins were reduced at 56 °C for 30 min with 10 mm dithiothreitol in 0.1 m NH4HCO3 and submitted to alkylation in 55 mm iodoacetamide in 0.1 m NH4HCO3 for 30 min. Gel pieces were washed with 0.1 m NH4HCO3 for 15 min and were then dehydrated and shrunk with acetonitrile in a vacuum centrifuge. For “in-gel” digestion, gel pieces were rehydrated in the digestion buffer containing 0.1 m NH4HCO3, 5 mm CaCl2, and 5 ng/μl trypsin at 4 °C for 30 min. The supernatant was removed, and the gel pieces were covered with the digestion buffer without trypsin. Digestion was performed overnight at 37 °C. After “in gel” trypsic digestion, peptides were extracted by the addition of 50 μl of 25 mm NH4HCO3, gel pieces were shaken for 15 min, and the supernatant was collected. Two successive extractions were performed with acetonitrile/HCOOH/water solution (45/10/45, v/v/v) for 20 min. The last extraction was realized with acetonitrile/HCOOH (95/5, v/v) for 20 min with shaking. Extracts were pooled and dried. Samples were desalted using Zip-TipC18 pipette tips, dried in a vacuum centrifuge, and resuspended in 0.1% trifluoroacetic acid in water prior to the mass spectrometry analysis. Protein identification was carried out using peptide mass fingerprinting on a MALDI-TOF mass spectrometer (Voyager DE-STR PRO). Peptide mass fingerprint spectra were recorded in reflectron positive ion mode. On average, 150–200 laser shots were accumulated per spectrum. Each spectrum was internally calibrated using the monoisotopic mass of the fragments resulting from trypsin autoproteolysis at 842.5100, 1045.5642, and 2211.1046 Da, respectively. Proteins were identified with an error tolerance of 50 ppm using the MS-Fit module of the Protein Prospector program (available on the World Wide Web at prospector.ucsf.edu/) from the NCBI and Swiss-Prot data bases. Tandem Mass Spectrometry Analysis—Analyses were performed on a Q-Star Pulsar mass spectrometer (hybrid quadrupole liquid chromatography/MS/MS mass spectrometer; PE Sciex Instruments) fitted with a nanoelectrospray ion source (Protana, Odense, Denmark). Peptide solution was sprayed from gold-coated “medium-length” borosilicate capillaries (Protana). A potential of -800 V was applied to the capillary tip. The focusing potential was set at -220 V, and the declustering potential varied between -15 and -50 V. For the recording of conventional mass spectra, TOF data were acquired by accumulation of 10 multiple channel acquisition scans over mass ranges of m/z 500–2000. In the collision-induced dissociation MS/MS analyses, multiple charged ions were fragmented using nitrogen as a collision gas (4 × 10-5 torr, 1 torr = 133.3 pascals) with a collision energy between -40 and -55 eV, according to the sample, to obtain optimal fragmentation. The collision-induced dissociation spectra were recorded on the TOF analyzer over a range of m/z 150–2000. All signals were resolved monoisotopically. Confirmation of the Glycosylation Type of Contractile Proteins by Immunoblotting—Contractile proteins (30 μg) of soleus and EDL muscles were separated using a linear SDS-PAGE gradient gel (10–20%) and were electroblotted on nitrocellulose membrane. Membranes were saturated for 45 min with 5% bovine serum albumin in TBS-Tween buffer (50 mm Tris/HCl, 150 mm NaCl, 0.05% Tween, pH 8.0). RL2 anti-O-GlcNAc monoclonal antibodies were incubated overnight at 4 °C at a dilution of 1:1000. Three washes of 10 min each were performed with TBS-Tween. Anti-mouse horseradish peroxidase-labeled secondary antibodies were used at a dilution of 1:10,000 for 1 h at room temperature. After three washes, the detection was carried out using the Western lightning chemiluminescence reagents kit (PerkinElmer Life Sciences). Gas Chromatography and Mass Spectrometry Analysis— Contractile proteins of soleus and EDL muscles were separated using a linear SDS-PAGE gradient gel (10–20%) and were then transferred on polyvinylidene difluoride membrane. We cut off of the membrane individually each protein band corresponding to the different contractile proteins and submitted them to a reductive β-elimination (0.1 n NaOH, 1 m BH4Na) for 18 h at 45 °C. The reaction was stopped on ice by the addition of acetic acid to bring it to pH 5–6. Methylborates were eliminated by co-distillation with methanol followed by evaporation under N2. Dried samples containing alditols were treated with acetic anhydride at 100 °C for 4 h. Peracetylated alditols were then extracted in chloroform. Chloroformic phase was analyzed by gas chromatography on a 30 m × 0.32-mm BPX 70 column, with a cyanopropyl (polysilphenylene-siloxane) stationary phase. A peracetylglucosaminitol standard was analyzed using the same conditions. For gas chromatography/mass spectrometry analysis, the gas chromatography separation was performed on a Carlo Erba GC 8000 gas chromatograph equipped with a 25 m × 0.32-mm CP-Sil5 CB low bleed/MS capillary column and 0.25-μm film phase (Chrompack France, Les Ullis, France). The temperature of the Ross injector was 260 °C, and the samples were analyzed using the following temperature program: 90 °C for 3 min, next 5 °C/min up to 260 °C, followed by a plateau of 20 min at 260 °C. The column was coupled to a Finnigan Automass II mass spectrometer. Analyses were performed in the EI mode (ionization energy 70 eV; source temperature 150 °C) and in the positive CI mode, in the presence of ammonia (ionization energy 150 eV, source temperature 100 °C). A reference standard sample of peracetylated glucosaminitol was used to secure these identifications. Skinning Procedure and Isometric Tension Determination— Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (3 mg·kg-1), and muscles were immediately removed. After removal, the biopsies were immediately chemically skinned by exposure to a skinning solution containing EGTA, 10 mm MOPS, 170 mm potassium propionate, 2.5 mm MgAc, and 5 mm K2 EGTA (34Mounier Y. Holy X. Stevens L. Pflugers Arch. Eur. J. Physiol. 1989; 415: 136-141Crossref PubMed Scopus (38) Google Scholar). This procedure permeabilized the sarcolemmal and transverse tubular membranes and allowed the application of activating solutions of various calcium and strontium concentrations (pCa and pSr, with pCa = -log[Ca2+] and pSr =-log[Sr2+]) directly to the contractile proteins. The skinned biopsies were stored at -20 °C in a 50:50 (v/v) glycerol/skinned solution (storage solution) for up to 2 months. Protease inhibitors, leupeptin and pepstatin, were added to the storage solution (10 μg·ml-1) to prevent loss of contractile proteins and preserve fiber tension (35Reiser P.J. Kasper C.E. Greaser M.L. Moss R.L. Am. J. Physiol. 1988; 254: C605-C613Crossref PubMed Google Scholar). Solutions—The composition of all solutions was calculated by the Fabiato computer program (36Fabiato A. Methods Enzymol. 1974; 157: 378-417Crossref Scopus (973) Google Scholar). The program calculation was used with stability constants listed for Ca2+ (37Orentlicher M. Brandt P.W. Reuben J.P. Am. J. Physiol. 1977; 233: C127-C134Crossref PubMed Google Scholar) and for Sr2+ (38Moisescu D.G. Thieleczek R. Biochim. Biophys. Acta. 1979; 546: 64-76Crossref PubMed Scopus (50) Google Scholar) to keep a final ionic strength at 200 mm. pH was adjusted to 7.0, and ATP was added to each solution at a final concentration of 2.5 mm. The following solutions were used for the experimental procedure: a washing solution (W) composed of 10 mm MOPS, 185 mm potassium propionate, and 2.5 mm MgAc; a relaxing solution (R) identical to the skinning solution; and pCa-activating solutions consisting of W solution added to various concentrations of free Ca2+ from CaCO3, buffered with EGTA and added in proportions to obtain the different pCa values (7.0 to 4.2). In order to eliminate a hypothetic influence of the sarcoplasmic reticulum on the tension developed by the myofilaments, each fiber was bathed for 20 min at the beginning of an experiment in a Brij solution made up of R solution with 2% Brij 58 (polyoxyethylene 20 cetyl ether). Experimental Protocol—For each experiment, a 5-mm fiber segment was isolated from the skinned biopsy of soleus. A silk thread was tied at each extremity, allowing the mounting of the fiber in an experimental chamber with constant stirring, initially filled with R solution. The fiber was held at one end by small fixed forceps and at the other end by a clamp connected to a strain gauge (force transducer Fort 10 (World Precision Instruments), sensitivity 10 V/g). The resting sarcomere length was measured by means of a helium/neon laser (Spectra Physics) directed perpendicular to the long axis of the fiber. Next, the fiber was stretched to ∼120% of resting length to allow maximal isometric tension development upon ionic activation. The resulting sarcomere length (2.6 ± 0.04 μm) was subsequently regularly controlled and readjusted if necessary. The output of the force transducer was amplified and recorded on a graph recorder (Gould model 6120). Force Measurements and Effects of GlcNAc on Calcium Properties—All experiments were performed in a thermostatically controlled room (20 ± 1 °C). In our study, two experimental protocols were used. In the first protocol, single fibers were bathed for 1 h in the R solution. Next, the same fibers were bathed for 1 h in the R solution containing 0.2 m GlcNAc, 0.2 m GalNAc, or 0.2 m glycerol. The muscular fibers were incubated in a solution of free N-acetyl-d-glucosamine to suppress protein-protein interactions through the O-GlcNAc moiety. It is known that incubation of free GlcNAc allows a rise in protein-protein interaction depending on the O-GlcNAc moiety (39Verbert A. Montreuils J. Bouquelet S. Cacan R. Debray H. Fournet B. Michalski J.C. Spik G. Strecker G. Methods on Glycoconjugates: A Laboratory Manual. Harwood Academic Publishers, Chur, Switzerland1995Google Scholar). The concentration 0.2 m was chosen, since it is the optimal concentration to elute glycoproteins bearing O-GlcNAc by affinity chromatography on immobilized WGA, a lectin that recognizes N-acetyl-d-glucosamine. In the second protocol, single fibers were submitted to two successive baths of 1 h each. One set of experiments corresponded to two baths in 0.2 m glycerol, and another set corresponded to a first bath in 0.2 m GalNAc followed by a second bath in 0.2 m GlcNAc. After preliminary bathings according to the two protocols described above or in the absence of bathing (control experiments), each single fiber was bathed in W solution to eliminate EGTA traces from the previously applied R solution. Next, the fiber was activated at a level P with various pCa solutions (from 7.0 to 4.8, with a step ordinarily equal to 0.2 pCa units). Each steady state submaximal tension P was followed immediately by a maximum contraction Po ensured by pCa 4.2 solution that contained enough calcium to saturate all troponin C sites. The tensions P were expressed as a percentage of the maximal tension Po and reported as tension/pCa (T/pCa) relationships. Finally, the fiber was relaxed in R solution. All solutions contained the tested molecules (i.e. GlcNAc, GalNAc, or glycerol) except for the control experiments. For some experiments, fibers were washed, and a new control T/pCa relationship was performed to determine the reversibility of the effects observed in the presence of GlcNAc. Fibers were rejected if force declined during a sustained contraction or decreased by more than 20% during the whole experiment and if T/pCa were not completely achieved. The T/pCa relationship provided information about the affinity of the contractile apparatus for Ca2+ that was represented by the pCa50 value (50% of maximal Ca2+ tension responses). Two other important parameters could be determined from the T/pCa relationship: the threshold for activation by Ca2+ as an indicator of the calcium sensitivity of the contractile system and the steepness of the T/pCa reflecting the cooperativity between the different regulatory proteins within the thin filament. The steepness of the T/pCa was determined by the Hill coefficients n1 and n2, calculated according to the following equation (40Brandt P.W. Cox R.N. Kawai M. Robinson T. J. Gen. Physiol. 1982; 79: 997-1016Crossref PubMed Scopus (111) Google Scholar): P/Po = (([Ca2+]/K)n/(1 + ([Ca2+]/K)n)), where P/Po is the normalized tension and K is the apparent dissociation constant (pK =-log K = pCa50). n1 corresponds to P/Po > 50%, and n2 corresponds to P/Po < 50%. Curves were fitted with the Hill parameters (n1 for P/Po > 50%, and n2 for P/Po < 50%) according to the relation y = (1/(1 + 10-n × (pCa50 - pCa))) × 100. Electrophoresis—All fibers were dissolved in 20 μl of SDS lysis sample buffer and stored at -20 °C until electrophoretic analysis. The samples were heated at 90 °C for 3 min just prior to the electrophoresis. A 10–20% linear gradient SDS-PAGE was used to obtain a good separation of the contractile proteins. Proteins were silver-stained. Statistical Analysis—All of the data were reported as means ± S.E. The statistical significance of the difference between means was determined using the t test. Differences at or above the 95% confidence level were considered significant. In order to specifically study the O-GlcNAc modification of contractile proteins, we performed an enrichment of contractile proteins from a total extract of skeletal muscles before the purification of O-GlcNAc proteins by affinity chromatography. Skeletal muscle powder was homogenized in a high ionic solution allowing an extraction of contractile proteins (41Toursel T. Bastide B. Stevens L. Rieger F. Mounier Y. Exp. Neurol. 2000; 162: 311-320Crossref PubMed Scopus (29) Google Scholar). A pellet of contractile proteins was obtained by this protocol. Electrophoretic analysis of this pellet allowed easy identification of myosin heavy and light chains (essential and regulatory light chain), actin, and tropomyosin isoforms as previously reported (42Stevens L. Sultan K.R. Peuker H. Gohlsch B. Mounier Y. Pette D. Am. J. Physiol. 1999; 277: C1044-C1049Crossref PubMed Google Scholar, 43Kischel P. Bastide B. Stevens L. Mounier Y. J. Appl. Physiol. 2001; 90: 1095-1101Crossref PubMed Scopus (20) Google Scholar, 44Bastide B. Kischel P. Puterflam J. Stevens L. Pette D. Jin J.P. Mounier Y. Pflugers Arch. 2002; 444: 345-352Crossref PubMed Scopus (25) Google Scholar, 45Stevens L. Bastide B. Kischel P. Pette D. Mounier Y. Am. J. Physiol. 2002; 282: C1025-C1030Crossref Scopus (33) Google Scholar) and shown in Fig. 1. Contractile proteins obtained as described above from soleus (Fig. 2, lanes 1 and 3) and EDL (Fig. 2, lanes 4 and 6) muscles were purified by WGA-immobilized affinity chromatography to retain O-GlcNAc-modified proteins. Fractions corresponding to flow-through (lanes 1 and 4), GalNAc wash (lanes 2 and 5), and GlcNAc elution (lanes 3 and 6) were analyzed on 10–20% SDS-PAGE after silver staining (Fig. 2). The proteins that were retained in the WGA-immobilized affinity chromatography were also present in the flow-through fractions (Fig. 2, lanes 1 and 4), indicating that only a fraction of the contractile protein" @default.
• W2092770107 created "2016-06-24" @default.
• W2092770107 creator A5000688638 @default.
• W2092770107 creator A5014531122 @default.
• W2092770107 creator A5024636319 @default.
• W2092770107 creator A5027938286 @default.
• W2092770107 creator A5042905522 @default.
• W2092770107 creator A5077530613 @default.
• W2092770107 date "2007-04-01" @default.
• W2092770107 modified "2023-09-30" @default.
• W2092770107 title "O-Linked N-Acetylglucosaminylation Is Involved in the Ca2+ Activation Properties of Rat Skeletal Muscle" @default.
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