Differential Vicia villosa Agglutinin Reactivity Identifies Three Distinct Dystroglycan Complexes in Skeletal Muscle
2001; Elsevier BV; Volume: 276; Issue: 37 Linguagem: Inglês
10.1074/jbc.m103843200
ISSN1083-351X
AutoresErin L. McDearmon, Ariana C. Combs, James M. Ervasti,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoWe present evidence for the expression of three α-dystroglycan glycoforms in skeletal muscle cells, including two minor glycoforms marked by either patent or latent reactivity with the N-acetylgalactosamine-specific lectinVicia villosa agglutinin. Both minor glycoforms co-isolated with β-dystroglycan, but not with other dystrophin/utrophin-glycoprotein complex components, suggesting that they may perform distinct or modified cellular functions. We also confirmed that both patent and latent V. villosaagglutinin-reactive α-dystroglycan glycoforms are expressed in C2C12 myotubes. However, we found that the combined effect of saturating concentrations of V. villosa agglutinin and laminin-1 were strictly additive with respect to acetylcholine receptor cluster formation in C2C12 myotubes, which suggests that laminin-1 and V. villosa agglutinin do not compete for the same binding site on the cell surface. Finally, although β-N-acetylhexosaminidase digestion dramatically inhibited agrin-, V. villosa agglutinin-, and laminin-1-induced acetylcholine receptor clustering in C2C12 myotubes, treatment with this enzyme had no effect on the amount of α-dystroglycan that was bound to V. villosa agglutinin-agarose. We conclude that α-dystroglycan is not the V. villosa agglutinin receptor implicated in acetylcholine receptor cluster formation. However, our data provide new support for the hypothesis that different glycoforms of α-dystroglycan may perform distinct functions even within the same cell. We present evidence for the expression of three α-dystroglycan glycoforms in skeletal muscle cells, including two minor glycoforms marked by either patent or latent reactivity with the N-acetylgalactosamine-specific lectinVicia villosa agglutinin. Both minor glycoforms co-isolated with β-dystroglycan, but not with other dystrophin/utrophin-glycoprotein complex components, suggesting that they may perform distinct or modified cellular functions. We also confirmed that both patent and latent V. villosaagglutinin-reactive α-dystroglycan glycoforms are expressed in C2C12 myotubes. However, we found that the combined effect of saturating concentrations of V. villosa agglutinin and laminin-1 were strictly additive with respect to acetylcholine receptor cluster formation in C2C12 myotubes, which suggests that laminin-1 and V. villosa agglutinin do not compete for the same binding site on the cell surface. Finally, although β-N-acetylhexosaminidase digestion dramatically inhibited agrin-, V. villosa agglutinin-, and laminin-1-induced acetylcholine receptor clustering in C2C12 myotubes, treatment with this enzyme had no effect on the amount of α-dystroglycan that was bound to V. villosa agglutinin-agarose. We conclude that α-dystroglycan is not the V. villosa agglutinin receptor implicated in acetylcholine receptor cluster formation. However, our data provide new support for the hypothesis that different glycoforms of α-dystroglycan may perform distinct functions even within the same cell. acetylcholine receptor neuromuscular junction N-acetylgalactosamine V. villosaagglutinin crude surface membrane(s) wheat germ agglutinin polyacrylamide gel electrophoresis The dystroglycan complex was originally identified as a component of the skeletal muscle dystrophin-glycoprotein complex, which spans the sarcolemma of muscle cells and physically couples the actin cytoskeleton with the extracellular matrix (1Straub V. Campbell K.P Curr. Opin. Neurobiol. 1997; 10: 168-175Crossref Scopus (328) Google Scholar). The dystroglycan complex consists of α-dystroglycan, a highly glycosylated extracellular protein that binds to several extracellular ligands, and β-dystroglycan, a single-pass transmembrane protein that links cytoplasmic dystrophin with α-dystroglycan (2Henry M.D. Campbell K.P Curr. Opin. Cell Biol. 1999; 11: 602-607Crossref PubMed Scopus (253) Google Scholar). Both dystroglycan subunits are encoded by a single highly conserved pro-peptide that is proteolytically processed into α- and β-dystroglycan, which remain stably associated through noncovalent interactions (2Henry M.D. Campbell K.P Curr. Opin. Cell Biol. 1999; 11: 602-607Crossref PubMed Scopus (253) Google Scholar). Like muscle deficient in dystrophin or other core components in the dystrophin-glycoprotein complex (1Straub V. Campbell K.P Curr. Opin. Neurobiol. 1997; 10: 168-175Crossref Scopus (328) Google Scholar), deficiency of the dystroglycan complex in skeletal muscle results in compromised sarcolemmal integrity (3Cote P.D. Moukhles H. Lindenbaum M. Carbonetto S. Nat. Genet. 1999; 23: 338-342Crossref PubMed Scopus (201) Google Scholar). Thus, it is generally thought that the dystroglycan complex in skeletal muscle mainly functions to mechanically protect the sarcolemma against shear stresses imposed during muscle contraction. However, the dystroglycan complex has also been linked with more dynamic developmental or pathological processes (2Henry M.D. Campbell K.P Curr. Opin. Cell Biol. 1999; 11: 602-607Crossref PubMed Scopus (253) Google Scholar). In skeletal muscle, several studies (3Cote P.D. Moukhles H. Lindenbaum M. Carbonetto S. Nat. Genet. 1999; 23: 338-342Crossref PubMed Scopus (201) Google Scholar, 4Grady R.M. Zhou H. Cunningham J.M. Henry M.D. Campbell K.P. Sanes J.R Neuron. 2000; 25: 279-293Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 5Jacobson C. Cote P.D. Rossi S.G. Rotundo R.L. Carbonetto S. J. Cell Biol. 2001; 152: 435-450Crossref PubMed Scopus (170) Google Scholar) have implicated the dystroglycan complex in either the formation or maintenance of acetylcholine receptor (AChR)1-rich folds within the motor end plate of the neuromuscular junction (NMJ). However, it is presently unclear how the single, invariant dystroglycan protein may subserve different functions within a single cell type. Because α-dystroglycan displays tissue-specific differences in glycosylation (6Ervasti J.M. Burwell A.L. Geissler A.L J. Biol. Chem. 1997; 272: 22315-22321Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), it is possible that distinct α-dystroglycan glycoforms may perform different functions. With regard to a role in AChR cluster formation at the NMJ, we previously confirmed (6Ervasti J.M. Burwell A.L. Geissler A.L J. Biol. Chem. 1997; 272: 22315-22321Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) that neural α-dystroglycans express exposed (patent) terminal β-linkedN-acetylgalactosamine (GalNAc) residues, which bound to the GalNAc-specific lectin Vicia villosa agglutinin (VVA). Skeletal muscle α-dystroglycan displayed no patent binding to VVA but revealed latent VVA reactivity after digestion with neuraminidase (6Ervasti J.M. Burwell A.L. Geissler A.L J. Biol. Chem. 1997; 272: 22315-22321Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Interestingly, the VVA binding properties of neural and skeletal muscle α-dystroglycans were consistent with the characteristics of an unidentified VVA receptor previously implicated in AChR clustering (7Scott L.J.C. Bacou F. Sanes J.R J. Neurosci. 1988; 8: 932-944Crossref PubMed Google Scholar,8Martin P.T. Sanes J.R Neuron. 1995; 14: 743-754Abstract Full Text PDF PubMed Scopus (74) Google Scholar). Therefore, we hypothesized the expression of two α-dystroglycan glycoforms in skeletal muscle cells: 1) an extrasynaptic form with latent VVA reactivity caused by further modification with terminal sialic acid residues and 2) a motor end plate-specific glycoform marked with exposed β-linked GalNAc residues that may specifically participate in AChR cluster formation (6Ervasti J.M. Burwell A.L. Geissler A.L J. Biol. Chem. 1997; 272: 22315-22321Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Here we show that three α-dystroglycan glycoforms are expressed in skeletal muscle, including two minor glycoforms marked by either patent or latent VVA reactivity. Both minor glycoforms co-isolated with β-dystroglycan but not other dystrophin/utrophin-glycoprotein complex components, suggesting they may perform distinct cellular functions. Although we confirmed that both patent and latent VVA reactive glycoforms are also expressed in C2C12 myotubes, other experiments indicate that neither glycoform is the receptor that mediates VVA-induced AChR clustering. We conclude that the VVA receptor important in AChR cluster formation remains to be identified. C2C12 and L6 cell lines were obtained from American Type Culture Collection and used for three to seven passages. Proliferating cells were grown in 10-cm dishes or on poly-l-lysine-coated coverslips with Dulbecco's modified Eagle's medium (Cellgrow; Fisher) containing 10% fetal bovine serum (Hyclone, Logan, UT) plus 1% antibiotic/antimycotic (Sigma) at 37 °C in a humid atmosphere of 5–10% CO2. After reaching confluency, the medium was switched to Dulbecco's modified Eagle's medium containing 2% equine serum (Hyclone) plus 1% antibiotic/antimycotic. The cells were incubated until full differentiation to multi-nucleate myotubes was observed morphologically (with C2C12, 4 days; with L6, 9–11 days) with fresh medium exchanged every 2 days. Crude surface membranes (CSM) were isolated from rabbit skeletal muscle as described previously (9Ohlendieck K. Ervasti J.M. Snook J.B. Campbell K.P J. Cell Biol. 1991; 112: 135-148Crossref PubMed Scopus (241) Google Scholar). For small scale lectin chromatography, 1 mg/ml CSM was solubilized in 1% Triton buffer (50 mm Tris-HCl, pH 7.4, 1 mm CaCl2, 1 mm MgCl2, 100 μg/ml benzamidine, 40 μg/ml phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 0.5 μg/ml aprotinin, 1 mmiodoacetamide, 0.5 μg/ml pepstatin A, 1% Triton X-100) plus 150 mm NaCl, with end-over-end mixing for 1 h at 4 °C. Solubilized proteins were recovered in the supernatant fraction after centrifugation for 30 min at 100,000 × g. For large scale chromatography, 500–600 mg of CSM were solubilized in 1% Triton buffer plus 250 mm NaCl and 0.5 m sucrose and centrifuged as described above. Fully differentiated C2C12 or L6 myotubes were rinsed twice with 37 °C phosphate-buffered saline and scraped off the dish in 5 ml/dish ice-cold phosphate-buffered saline containing the following protease inhibitors: 100 μg/ml benzamidine, 40 μg/ml phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 0.5 μg/ml aprotinin, 1 mm iodoacetamide, and 0.5 μg/ml pepstatin A. The cells were pelleted by brief centrifugation at 50–200 ×g and solubilized in 0.5 ml/dish 1% Triton buffer containing 150 mm NaCl. The resuspended cell pellet was incubated at 4 °C for 1 h with end-over-end mixing followed by centrifugation as described above. For small scale lectin chromatography, 0.5 ml of Triton solubilate from CSM was incubated overnight at 4 °C with 100 μl of either wheat germ agglutinin (WGA)-agarose or VVA-agarose beads (from Sigma and EY Labs, San Mateo, CA, respectively) that were pre-equilibrated in wash buffer (50 mm Tris-HCl, pH 7.4, 1 mm CaCl2, 1 mm MgCl2, 250 mm NaCl, 100 μg/ml benzamidine, 40 μg/ml phenylmethylsulfonyl fluoride, 0.1% Triton X-100). The beads were pelleted, and the void volumes were removed. Lectin-bound proteins were then washed extensively and eluted with the appropriate eluting buffer (for WGA, wash buffer containing 0.3m N-acetylglucosamine (GlcNAc); for VVA, wash buffer containing 50 mm GalNAc). Small scale chromatography was performed in essentially the same way for Triton-solubilized C2C12 or L6 myotubes except that the wash buffer contained 150 mmNaCl and an extra 100 mm NaCl was added to the VVA eluting buffer. For large scale chromatography, ∼100 ml of CSM solubilate was loaded onto a 3-ml VVA-agarose column that was pre-equilibrated with wash buffer containing 250 mm NaCl. The column was washed and eluted with wash buffer containing 50 mm GalNAc. The eluates were concentrated by methanol precipitation. CSM solubilates were incubated with or without 0.1 unit/ml of Clostridium perfringens neuraminidase (Roche Molecular Biochemicals) for 2 h at 37 °C and used in small scale VVA chromatography as described above. Fully differentiated C2C12 or L6 myotubes were incubated with or without 0.1 unit/ml neuraminidase diluted in medium for 2 h at 37 °C. C2C12 myotubes were also treated for 6 h at 37 °C with 1 unit/ml β-N-acetylhexosaminidase from beef kidney (Roche Molecular Biochemicals), 1 unit/ml α-N-acetylgalactosaminidase from chicken liver (Sigma), or 0.05 unit/ml α-N-acetylgalactosaminidase from Acremonium sp. (Calbiochem, San Diego, CA), each diluted in medium. Following incubation, the cells were analyzed for AChR clustering as described below or rinsed and solubilized for lectin chromatography as described above. The samples were separated by SDS-PAGE under reducing or nonreducing conditions and transferred to nitrocellulose as described previously (10Ervasti J.M. Campbell K.P Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1112) Google Scholar). Western blotting was performed with the following antibodies: monoclonal antibody IIH6 to α-dystroglycan (10Ervasti J.M. Campbell K.P Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1112) Google Scholar), affinity purified chicken polyclonal antibody against α-dystroglycan (6Ervasti J.M. Burwell A.L. Geissler A.L J. Biol. Chem. 1997; 272: 22315-22321Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), monoclonal antibody XIXC2 to dystrophin (11Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P Nature. 1990; 345: 315-319Crossref PubMed Scopus (819) Google Scholar), monoclonal antibody SYN1351 to syntrophin (12Froehner S.C. Murnane A.A. Tobler M. Peng H.B. Sealock R. J. Cell Biol. 1987; 104: 1633-1646Crossref PubMed Scopus (121) Google Scholar), anti-dystrobrevin monoclonal antibody 13H1 (13Carr C. Fischbach G.D. Cohen J.B J. Cell Biol. 1989; 109: 1753-1764Crossref PubMed Scopus (82) Google Scholar), monoclonal antibody NCL-β-DG to β-dystroglycan, monoclonal antibody NCL-α-SG to α-sarcoglycan (both from Vector Laboratories, Burlingame, CA), Rb 1715 antiserum to ε-sarcoglycan (14Ettinger A.J. Feng G. Sanes J.R J. Biol. Chem. 1997; 272: 32534-32538Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), Rb 56 antiserum to utrophin (15Ohlendieck K. Ervasti J.M. Matsumura K. Kahl S.D. Leveille C.J. Campbell K.P Neuron. 1991; 7: 499-508Abstract Full Text PDF PubMed Scopus (321) Google Scholar), α7CDB2 antiserum to α7B integrin cytoplasmic domain (16Song W.K. Wang W. Sato H. Biesler D.A. Kaufman S.J J. Cell Sci. 1993; 106: 1139-1152Crossref PubMed Google Scholar), anti-β1 integrin monoclonal antibody MAB1997 (Chemicon, Temecula, CA), anti-AChR β subunit monoclonal antibody (BD Transduction Laboratories, Franklin Lakes, NJ), and monoclonal antibody HNK-1 (Becton Dickinson, Bedford, MA). To assay for AChR clustering, C2C12 and L6 myotubes were grown on poly-l-lysine-coated glass coverslips. After treatment with laminin-1 (the gift of Dr. Hynda Kleinman, NIDR), VVA lectin (Sigma or EY Labs), C-Ag4,8 conditioned medium (17O'Toole J.J. Deyst K.A. Bowe M.A. Nastuk M.A. McKechnie B.A. Fallon J.R Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7369-7374Crossref PubMed Scopus (67) Google Scholar) (the gift of Drs. Mark Bowe and Justin Fallon, Brown University), and/or glycosidases, the myotubes were washed with 37 °C phosphate-buffered saline and incubated for 1 h at 37 °C with 500 ng/ml Alexa 488-conjugated bungarotoxin (Molecular Probes, Eugene, OR) diluted in Dulbecco's modified Eagle's medium. The cells were washed and fixed in 4% paraformaldehyde for 10 min at room temperature. Fixed cells were rinsed in phosphate-buffered saline and mounted on slides for immunofluorescence analysis. Images were collected with a Bio-Rad MRC 1000 confocal microscope (Keck Center for Biological Imaging) using a 40× oil immersion objective. AChR clusters were counted from five randomly chosen fields for each experiment. 7-μm cross-sections of control andmdx mouse hind limb muscle were adhered to Plus slides (Fisher), fixed in 4% paraformaldehyde for 10 min at room temperature, rinsed, and blocked in 1% bovine serum albumin for 2 h at room temperature. Blocked sections were rinsed and incubated either with Rab 56 antiserum to utrophin for 2 h at 37 °C followed by 20 μg/ml Alexa 568-conjugated rabbit secondary antibody (Molecular Probes) for 1 h at 37 °C or with 500 ng/ml rhodamine-conjugated bungarotoxin (Molecular Probes) for 1 h at 37 °C. All sections were double-labeled with 50 μg/ml VVA-fluorescein isothiocyanate lectin (ICN, Costa Mesa, CA), which was included during the incubation with secondary antibodies or bungarotoxin. The images were collected as described above. To determine whether a VVA-reactive glycoform of α-dystroglycan is expressed in adult skeletal muscle, we attempted to isolate α-dystroglycan from detergent-solubilized rabbit skeletal muscle CSM by small scale VVA-agarose chromatography. The chromatography protocol was first tested with WGA-agarose, a lectin that has previously been shown to bind α-dystroglycan isolated from a wide variety of tissues and species, including rabbit skeletal muscle (11Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P Nature. 1990; 345: 315-319Crossref PubMed Scopus (819) Google Scholar). We observed that all α-dystroglycan in 1 mg of solubilized CSM protein quantitatively bound to WGA-agarose and was eluted with buffer containing 0.3 m GlcNAc (Fig.1 a). In addition, several other dystrophin-glycoprotein complex constituents were observed to co-purify with α-dystroglycan in the WGA eluate (Fig. 1 a). However, no α-dystroglycan was detected in the GalNAc eluate from VVA-agarose using the small scale chromatography protocol (Fig.1 b). Because the motor end plate makes up less than 0.1% of the entire muscle cell plasma membrane (18Burden S.J Genes Dev. 1998; 12: 133-148Crossref PubMed Scopus (170) Google Scholar), it seemed probable that a VVA-reactive species of α-dystroglycan might be present in such low abundance that we could not detect it by small scale chromatography. Therefore, we performed a large scale VVA chromatography protocol in which 500–600 mg of CSM solubilate was loaded onto a 3-ml VVA-agarose column. The GalNAc eluate from the 3-ml VVA-agarose column was then concentrated 300–400-fold, relative to the CSM solubilate. On this scale of amplification, we readily detected two distinct species of α-dystroglycan in the concentrated VVA eluate with molecular weights of ∼156,000 and 110,000 (Fig. 2). Both proteins were reactive with the α-dystroglycan-specific monoclonal antibody IIH6 and an affinity-purified polyclonal antibody to α-dystroglycan (Fig. 2).Figure 2Amplification of distinct α-dystroglycan glycoforms from skeletal muscle membranes by large scale VVA chromatography. 600 mg of Triton-solubilized rabbit skeletal muscle CSM were circulated overnight on a 3-ml VVA-agarose column. After washing, the column was eluted with buffer containing 50 mm GalNAc, and the eluates were concentrated by methanol precipitation. The samples were separated by SDS-PAGE, transferred to nitrocellulose, and stained with antibodies that recognize the following proteins: α-dystroglycan (α-DG polyclonal antibody (pAb) andIIH6), α-dystrobrevins-1 and -2 (Db), β-dystroglycan (β-DG), dystrophin (DYS), utrophin (UTR), α-sarcoglycan (α-SG), ε-sarcoglycan (ε-SG), and syntrophin (Syn). Note that the eluates were concentrated ∼300-fold relative to the CSM solubilate (SUP).View Large Image Figure ViewerDownload Hi-res image Download (PPT) One or both of the VVA-reactive α-dystroglycan species could have originated from small amounts of blood vessel or peripheral nerve tissues that may contaminate the muscle membrane preparations used. Indeed, we observed that the 110-kDa species, but not the 156-kDa species, reacted with a monoclonal antibody to the HNK-1 epitope (data not shown). Because HNK-1 antibodies have been observed to react with peripheral nerve but not skeletal muscle α-dystroglycan (19Yamada H. Chiba A. Endo T. Kobata A. Anderson V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Crossref PubMed Scopus (100) Google Scholar) 2A. Combs and J. Ervasti, unpublished results. and because peripheral nerve α-dystroglycan has a molecular weight of ∼120,000 (19Yamada H. Chiba A. Endo T. Kobata A. Anderson V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Crossref PubMed Scopus (100) Google Scholar), we concluded that the 110-kDa α-dystroglycan species in the large scale VVA eluate likely originated from peripheral nerve tissue. However, α-dystroglycan from visceral and lung smooth muscle is not reactive with monoclonal antibody IIH6 (20Straub V. Ettinger A.J. Durbeej M. Venzke D.P. Cutshall S. Sanes J.R. Campbell K.P J. Biol. Chem. 1999; 274: 27989-27996Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 21Durbeej M. Campbell K.P J. Biol. Chem. 1999; 274: 26609-26616Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In addition, ε-sarcoglycan, a component of the smooth muscle dystrophin-glycoprotein complex (20Straub V. Ettinger A.J. Durbeej M. Venzke D.P. Cutshall S. Sanes J.R. Campbell K.P J. Biol. Chem. 1999; 274: 27989-27996Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), did not co-purify with α-dystroglycan in the large scale VVA eluates (Fig. 2). These results suggested that the 156-kDa VVA-reactive α-dystroglycan species likely originated from skeletal muscle cells. Based on the densitometric intensities of IIH6 staining in the solubilate and VVA eluate and taking into account the difference in sample load, we estimated that the 156-kDa VVA-reactive species comprised ∼0.1% of total skeletal muscle α-dystroglycan (TableI).Table Iα-Dystroglycan glycoforms in skeletal muscle cellsTissue/cell typeMrVVA reactivityEstimated abundance%Rabbit156,000No98Skeletal156,000Yes (patent)0.11Muscle156,000Yes (latent)1.7110,000Yes (patent)aNot quantifiable because no IIH6 immune signal was detected in CSM solubiliates.C2C12134,000Yes (patent)80Myotubes134,000Yes (latent)20L6 Myotubes136,000No100a Not quantifiable because no IIH6 immune signal was detected in CSM solubiliates. Open table in a new tab We next examined the large scale VVA eluates for co-purification of other components in the dystrophin-glycoprotein complex. As expected, β-dystroglycan was observed to co-purify with VVA-reactive α-dystroglycan (Fig. 2). However, other components of the dystrophin-glycoprotein complex were not detected in the large scale VVA eluates (Fig. 2). Of particular relevance, we did not detect the co-purification of utrophin with VVA-reactive α-dystroglycan, as might be expected if VVA-reactive α-dystroglycan was expressed at the neuromuscular junction (15Ohlendieck K. Ervasti J.M. Matsumura K. Kahl S.D. Leveille C.J. Campbell K.P Neuron. 1991; 7: 499-508Abstract Full Text PDF PubMed Scopus (321) Google Scholar). Although it is possible that Triton X-100 may have dissociated VVA-reactive dystroglycan complex from other dystrophin-associated proteins (22Fuhrer C. Gautam M. Sugiyama J.E. Hall Z.W J. Neurosci. 1999; 19: 6405-6416Crossref PubMed Google Scholar), we observed the same results when large scale VVA chromatography was performed with CSM solubilized in 1% digitonin (data not shown). We also observed the same results when large scale VVA chromatography was performed using Triton-solubilized rat skeletal muscle (data not shown). The presence of AChRs in both solubilized CSM and rat skeletal muscle was verified by Western blotting with an antibody against AChR β-subunit (data not shown). These results indicate that a small amount of VVA-reactive α-dystroglycan is expressed in skeletal muscle cells in association with β-dystroglycan but is not stably associated with other proteins in the dystrophin-utrophin glycoprotein complexes. The dystrophin homologue utrophin is normally restricted to the adult NMJ (15Ohlendieck K. Ervasti J.M. Matsumura K. Kahl S.D. Leveille C.J. Campbell K.P Neuron. 1991; 7: 499-508Abstract Full Text PDF PubMed Scopus (321) Google Scholar). Immunofluorescence analysis of skeletal muscle from the dystrophin-deficient mdx mouse previously revealed an up-regulation in utrophin expression and its redistribution to the extrasynaptic sarcolemma normally occupied by dystrophin (23Matsumura K. Ervasti J.M. Ohlendieck K. Kahl S.D. Campbell K.P Nature. 1992; 360: 588-591Crossref PubMed Scopus (444) Google Scholar, 24Khurana T.S. Watkins S.C. Chafey P. Chelly J. Tome F.M.S. Fardeau M. Kaplan J.C. Kunkel L.M Neuromuscular Disorders. 1991; 1: 185-194Abstract Full Text PDF PubMed Scopus (240) Google Scholar). More recently, proteins stably associated with utrophin in biochemical assays were also shown to redistribute with utrophin to the extrasynaptic sarcolemma of mdx muscle (25Lumeng C. Phelps S. Crawford G.E. Walden P.D. Barald K. Chamberlain J.S Nat. Neurosci. 1999; 2: 611-617Crossref PubMed Scopus (131) Google Scholar, 26Peters M.F. Adams M.E. Froehner S.C J. Cell Biol. 1997; 138: 81-93Crossref PubMed Scopus (211) Google Scholar). To determine whether VVA receptors in skeletal muscle associate with utrophin in vivo, we double-labeled control andmdx mouse skeletal muscle cross-sections with VVA lectin and utrophin antibody (Fig. 3). From analysis of several fields with identifiable neuromuscular junctions (n = 7), we observed that VVA receptors do not redistribute with utrophin in mdx mouse skeletal muscle. In addition, bungarotoxin-labeled AChRs remained strictly localized at the neuromuscular junctions in mdx mouse skeletal muscle (data not shown and Refs. 27Man N. Ellis J.M. Love D.R. Davies K.E. Gatter K.C. Dickson G. Morris G.E J. Cell Biol. 1991; 115: 1695-1700Crossref PubMed Scopus (246) Google Scholar and 28Peters M.F. Sadoulet-Puccio H.M. Grady R.M. Kramarcy N.R. Kunkel L.M. Sanes J.R. Sealock R. Froehner S.C J. Cell Biol. 1998; 142: 1269-1278Crossref PubMed Scopus (112) Google Scholar). The results of Figs. 2 and 3 suggested that neither VVA receptors at the neuromuscular junction nor VVA-reactive α-dystroglycan are specifically associated with utrophin. Because AChRs and residual dystroglycan are both retained at the NMJ of dystrophin/utrophin double knock-out mice (29Deconinck A.E. Rafael J.A. Skinner J.A. Brown S.C. Potter A.C. Metzinger L. Watt D.J. Dickson J.G. Tinsley J.M. Davies K.E. Cell. 1997; 90: 717-727Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 30Grady R.M. Teng H.B. Nichol M.C. Cunningham J.C. Wilkinson R.S. Sanes J.R Cell. 1997; 90: 729-738Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar), it remained possible that the small amount of VVA-reactive α-dystroglycan present in skeletal muscle may participate in AChR clustering independent of an interaction with dystrophin or utrophin. α-Dystroglycan in C2C12 myotubes was recently shown to bind to VVA-agarose (31Grow W.A. Ferns M. Gordon H. Dev. Neurosci. 1999; 21: 436-443Crossref PubMed Google Scholar). However, it remained to be determined what fraction of total C2C12 α-dystroglycan was patently reactive with VVA and whether VVA-reactive α-dystroglycan may also be expressed in other clonal myotube cell lines. Therefore, we analyzed both C2C12 and rat L6 myotubes for expression of distinct α-dystroglycan glycoforms. Both C2C12 and L6 myotubes were shown to express α-dystroglycan with similar average molecular weights of 134,000 and 136,000, respectively. Because the polypeptide core of α-dystroglycan is 67,000, the high molecular weights of C2C12 and L6 α-dystroglycan suggested that these cell lines express glycosylated α-dystroglycan. In addition, α-dystroglycan expressed in both cell lines reacted with IIH6 (Fig.4 a), which has been shown to recognize carbohydrate residues on α-dystroglycan (32Ervasti J.M. Campbell K.P J. Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1177) Google Scholar). Finally, both C2C12 and L6 myotubes expressed α-dystroglycan that bound WGA-agarose (Fig. 4 a). These results suggested that α-dystroglycan in C2C12 and L6 myotubes is glycosylated in a manner similar to that observed for skeletal muscle CSM α-dystroglycan (Fig. 1). Small scale VVA chromatography indicated that ∼80% of C2C12 myotube α-dystroglycan could bind to VVA-agarose (Fig. 4 a). β-Dystroglycan also co-purified with VVA-reactive α-dystroglycan from C2C12 myotubes, whereas other components of the dystrophin-glycoprotein complex were only detected in the VVA void (Fig. 4 b). To determine whether the small fraction of α-dystroglycan left in the VVA void (Fig. 4 a) was due to saturation of the lectin beads, we incubated the VVA void with a fresh aliquot of VVA-agarose overnight but did not detect any additional binding of α-dystroglycan to VVA (data not shown). In contrast to C2C12 myotube α-dystroglycan, L6 myotube α-dystroglycan showed no detectable binding to VVA-agarose (Fig. 4 a). These data confirm that C2C12 myotubes express two distinct α-dystroglycan glycoforms, one of which predominates and is marked by its patent reactivity with VVA-agarose. In addition, these results indicate that skeletal muscle cells are capable of expressing three distinct glycoforms of α-dystroglycan. Laminin-1 and VVA can each induce AChR clustering in C2C12 myotubes in a manner that is independent of and additive to the well established agrin/MuSK pathway (31Grow W.A. Ferns M. Gordon H. Dev. Neurosci. 1999; 21: 436-443Crossref PubMed Google Scholar, 33Sugiyama J.E. Glass D.J. Yancopoulos G.D. Hall Z.W J. Cell Biol. 1997; 139: 181-191Crossref PubMed Scopus
Referência(s)