Distal Myopathy with Rimmed Vacuoles
2005; Elsevier BV; Volume: 166; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62332-2
ISSN1525-2191
AutoresYouichi Tajima, Eiichiro Uyama, Shinji Go, Chihiro Sato, Nodoka Tao, Masaharu Kotani, H. Hino, Akemi Suzuki, Yutaka Sanai, Ken Kitajima, Hitoshi Sakuraba,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoDistal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Arachis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. α-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including α-dystroglycan, occurs in DMRV. Distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Arachis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. α-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including α-dystroglycan, occurs in DMRV. Distal myopathy with rimmed vacuoles (DMRV), also known as Nonaka distal myopathy (OMIM 605820), is an autosomal recessive muscular disease that was delineated in Japan. The disease is characterized by early-adult onset with weakness of the tibialis anterior muscles, sparing of the quadriceps muscles until the late stage, and the presence of numerous rimmed vacuoles in affected muscle fibers without infiltration by inflammatory cells.1Nonaka I Sunohara N Ishiura S Satoyoshi E Familial distal myopathy with rimmed vacuole and lamellar (myeloid) body formation.J Neurol Sci. 1981; 51: 141-155Abstract Full Text PDF PubMed Scopus (260) Google Scholar Previous linkage studies2Ikeuchi T Asaka T Saito M Tanaka H Higuchi S Tanaka K Saida K Uyama E Mizusawa H Fukuhara N Nonaka I Takamori M Tsuji S Gene locus for autosomal recessive distal myopathy with rimmed vacuoles maps to chromosome 9.Ann Neurol. 1997; 41: 432-437Crossref PubMed Scopus (85) Google Scholar, 3Mitrani-Rosenbaum S Argov Z Blumenfeld A Seidman CE Seidman JG Hereditary inclusion body myopathy maps to chromosome 9p1–q1.Hum Mol Genet. 1996; 5: 159-163Crossref PubMed Scopus (91) Google Scholar indicated that DMRV is allelic to hereditary inclusion body myopathy (HIBM), also known as IBM2 (OMIM 600737),4Asaka T Ikeuchi K Okino S Takizawa Y Satake R Nitta E Komai K Endo K Higuchi S Oyake T Yoshimura T Suenaga A Uyama E Saito T Konagaya M Sunohara N Namba R Takada H Honke K Nishina M Tanaka H Shinagawa M Tanaka K Matsushima A Tsuji S Takamori M Homozygosity and linkage disequilibrium mapping of autosomal recessive distal myopathy (Nonaka distal myopathy).J Hum Genet. 2001; 46: 649-655Crossref PubMed Scopus (5) Google Scholar a similar autosomal recessive disorder that was delineated among Jews originating from Middle Eastern countries.3Mitrani-Rosenbaum S Argov Z Blumenfeld A Seidman CE Seidman JG Hereditary inclusion body myopathy maps to chromosome 9p1–q1.Hum Mol Genet. 1996; 5: 159-163Crossref PubMed Scopus (91) Google Scholar, 5Argov Z Yarom R "Rimmed vacuole myopathy" sparing the quadriceps. A unique disorder in Iranian Jews.J Neurol Sci. 1984; 64: 33-43Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 6Eisenberg I Hochner H Shemesh M Levi T Potikha T Sadeh M Argov Z Jackson CL Mitrani-Rosenbaum S Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12–p13.Eur J Hum Genet. 2001; 9: 501-509Crossref PubMed Scopus (19) Google Scholar This disease is the most common form of ethnic-related familial degenerative myopathy in Israel, with a prevalence of 1:1500 in the Jewish-Persian community.7Argov Z Eisenberg I Mitrani-Rosenbaum S Genetics of inclusion body myopathies.Curr Opin Rheumatol. 1998; 10: 543-547Crossref PubMed Scopus (29) Google Scholar Recently, Eisenberg and colleagues8Eisenberg I Avidan N Potikha T Hochner H Chen M Olender T Barash M Shemesh M Sadeh M Grabov-Nardini G Shmilevich I Friedmann A Karpati G Bradley WG Baumbach L Lancet D Asher EB Beckmann JS Argov Z Mitrani-Rosenbaum S The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy.Nat Genet. 2001; 29: 83-87Crossref PubMed Scopus (447) Google Scholar identified mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) (EC 5.1.3.14/2.7.1.60) gene (GNE) in HIBM families. Then, mutations were identified in the same gene in Japanese patients with DMRV, as expected.9Kayashima T Matsuo H Satoh A Ohta T Yoshiura K Matsumoto N Nakane Y Niikawa N Kishino T Nonaka myopathy is caused by mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase gene (GNE).J Hum Genet. 2002; 47: 77-79Crossref PubMed Scopus (97) Google Scholar, 10Tomimitsu H Ishikawa K Shimizu J Ohkoshi N Kanazawa I Mizusawa H Distal myopathy with rimmed vacuoles: novel mutations in the GNE gene.Neurology. 2002; 59: 451-454Crossref PubMed Scopus (61) Google Scholar, 11Arai A Tanaka K Ikeuchi T Igarashi S Kobayashi H Asaka T Date H Saito M Tanaka H Kawasaki S Uyama E Mizusawa H Fukuhara N Tsuji S A novel mutation in the GNE gene and a linkage disequilibrium in Japanese pedigrees.Ann Neurol. 2002; 52: 516-519Crossref PubMed Scopus (46) Google Scholar, 12Nishino I Noguchi S Murayama K Driss A Sugie K Oya Y Nagata T Chida K Takahashi T Takusa Y Ohi T Nishimiya J Sunohara N Ciafaloni E Kawai M Aoki M Nonaka I Distal myopathy with rimmed vacuoles is allelic to hereditary inclusion body myopathy.Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (201) Google Scholar UDP-GlcNAc 2-epimerase has been shown to be the key enzyme in the sialic acid biosynthetic pathway.13Keppler OT Hinderlich S Langner J Schwartz-Albiez R Reutter W Pawlita M UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation.Science. 1999; 284: 1372-1376Crossref PubMed Scopus (282) Google Scholar This enzyme is bifunctional, having epimerase activity, in the amino-terminal region, and kinase activity, in the carboxy-terminal region,14Hinderlich S Stasche R Zeitler R Reutter W A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.J Biol Chem. 1997; 272: 24313-24318Crossref PubMed Scopus (253) Google Scholar and is involved in the first two steps of the biosynthesis of N-acetylneuraminic acid (NeuAc).15Stäsche R Hinderlich S Weise C Effertz K Lucka L Moormann P Reutter W A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Molecular cloning and functional expression of UDP-N-acetyl-glucosamine 2-epimerase/N-acetylmannosamine kinase.J Biol Chem. 1997; 272: 24319-24324Crossref PubMed Scopus (162) Google Scholar NeuAc is a common sialic acid in humans and the precursor of sialic acid compounds. Sialic acids are carbohydrates found on the cell surface, where they mediate a lot of cellular functions, eg, cell-cell or cell-matrix interactions. As the modification of sialic acid-containing glycoproteins and glycolipids on the cell surface is essential for biological functions, a genetic defect in the enzyme might cause a myopathy such as DMRV/HIBM. Mutations associated with DMRV/HIBM have been identified in both the epimerase and kinase domains.8Eisenberg I Avidan N Potikha T Hochner H Chen M Olender T Barash M Shemesh M Sadeh M Grabov-Nardini G Shmilevich I Friedmann A Karpati G Bradley WG Baumbach L Lancet D Asher EB Beckmann JS Argov Z Mitrani-Rosenbaum S The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy.Nat Genet. 2001; 29: 83-87Crossref PubMed Scopus (447) Google Scholar Recent studies revealed that GNE mutations including a common one for Japanese DMRV patients, V572L, cause reduction of UDP-GlcNAc 2-epimerase/ManNAc kinase activity.16Noguchi S Keira Y Murayama K Ogawa M Fujita M Kawahara G Oya Y Imazawa M Goto Y Hayashi YK Nonaka I Nishino I Reduction of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase activity and sialylation in distal myopathy with rimmed vacuoles.J Biol Chem. 2004; 279: 11402-11407Crossref PubMed Scopus (139) Google Scholar This enzyme is known to be a key enzyme in the sialic acid biosynthetic pathway. However, what the enzyme defect causes is obscure. In this study, we examined the carbohydrate moieties of muscular glycoproteins from DMRV and control cases, using lectins and glycosidases to elucidate what a genetic defect in GNE results in. Seven affected individuals from five unrelated Japanese families with DMRV [DMRV-1: a 34-year-old male; DMRV-2: a 31-year-old male; DMRV-3: a 53-year-old female; DMRV-4: an 18-year-old female; and DMRV-5 brother (B): a 31-year-old male; DMRV-5 sister-1 (S-1): a 30-year-old female; and DMRV-5 sister-2 (S-2): a 29-year old female; the latter three being siblings from the same family], a patient with facioscapulo humeral muscular dystrophy (FSHD, a 59-year-old male), a patient with oculopharyngeal muscular dystrophy (OPMD, a 50-year-old female), a patient with Emery-Dreifuss muscular dystrophy (EDMD, a 41-year-old male), a patient with myotonic dystrophy (MD, a 45-year-old male), and four normal controls (N1, a 42-year-old female; N2, a 35-year-old female; N3, a middle-aged female; and N4, a 61-year-old male) were studied, with their informed consent and the approval of our local ethics committee. The five DMRV families were diagnosed based on the following findings: 1) weakness of the tibialis anterior muscles beginning after the age of 20; 2) sparing of the quadriceps muscles until the late stage; 3) the presence of numerous rimmed vacuoles in affected muscle fibers; and 4) identification of mutations in GNE; patients DMRV-1 to DMRV-4 are homozygous for the V572L mutation,11Arai A Tanaka K Ikeuchi T Igarashi S Kobayashi H Asaka T Date H Saito M Tanaka H Kawasaki S Uyama E Mizusawa H Fukuhara N Tsuji S A novel mutation in the GNE gene and a linkage disequilibrium in Japanese pedigrees.Ann Neurol. 2002; 52: 516-519Crossref PubMed Scopus (46) Google Scholar, 17Uyama E Hino H Obayashi K Arai A Tanaka K Tsuji S Uchino M Distal myopathy with rimmed vacuoles: phenotype/genotype relationship in Japanese families.Ann Neurol. 2003; 54: S73PubMed Google Scholar and the three siblings, DMRV-5B, DMRV-5S-1, and DMRV-5S-2, are compound heterozygous for G295D/A631V.17Uyama E Hino H Obayashi K Arai A Tanaka K Tsuji S Uchino M Distal myopathy with rimmed vacuoles: phenotype/genotype relationship in Japanese families.Ann Neurol. 2003; 54: S73PubMed Google Scholar The clinical manifestations are more severe in DMRV-1 to DMRV-4 than in DMRV-5B, DMRV-5S-1, and DMRV-5S-2. The primary antibodies used in these studies were a mouse monoclonal antibody to α-dystroglycan (α-DG) (250-fold diluted, clone IIH6 kindly provided by Dr. K.P. Campbell, Howard Hughes Medical Institute, University of Iowa, Iowa City, IA),18Ervasti JM Campbell KP Membrane organization of the dystrophin-glycoprotein complex.Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1119) Google Scholar an affinity-purified sheep polyclonal antibody directed against the 20-amino acid C-terminal sequence of chick α-DG (500-fold diluted; kindly provided by Dr. S. Kröger, University of Mainz, Mainz, Germany),19Herrmann R Straub V Blank M Kutzick C Franke N Jacob EN Lenard HG Kroger S Voit T Dissociation of the dystroglycan complex in caveolin-3-deficient limb girdle muscular dystrophy.Hum Mol Genet. 2000; 9: 2335-2340Crossref PubMed Scopus (122) Google Scholar a mouse monoclonal antibody against β-dystroglycan (β-DG) (clone 8D5, 100-fold diluted), a mouse monoclonal anti-dystrophin antibody (clone 6C5, 100-fold diluted; Novacastra Laboratories, Newcastle, UK), a mouse monoclonal anti-laminin α2 chain antibody (clone 5H2, 100-fold diluted; Chemicon, Temecula, CA), a mouse monoclonal anti-CD63 antibody (200-fold diluted) (Immunotech, Marseille, France),20Nieuwenhuis HK van Oosterhout JJ Rozemuller E van Iwaarden F Sixma JJ Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation.Blood. 1987; 70: 838-845Crossref PubMed Google Scholar a rabbit polyclonal anti-LIMP 2 antibody (200-fold diluted; kindly provided by Dr. Y. Tanaka, Kyushu University, Kyushu, Japan),21Kuronita T Eskelinen EL Fujita H Saftig P Himeno M Tanaka Y A role for the lysosomal membrane protein LGP85 in the biogenesis and maintenance of endosomal and lysosomal morphology.J Cell Sci. 2002; 115: 4117-4131Crossref PubMed Scopus (121) Google Scholar and mouse monoclonal anti-endolyn/CD164 antibodies (clone N6B6.2, 100-fold diluted; BD-PhaMingen, San Diego, CA; clones 103B2 and 105A5, 100-fold diluted, kindly provided by Dr. A.C.W. Zannettino, University of Adelaide, Adelaide, Australia).22Zannettino AC Buhring HJ Niutta S Watt SM Benton MA Simmons PJ The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis.Blood. 1998; 92: 2613-2628Crossref PubMed Google Scholar Immunohistochemical analysis was performed on frozen tissue cryosections of biopsied skeletal muscle specimens from normal controls (N1 and N2), pathological controls (OPMD, EDMD, and MD), and the DMRV patients (DMRV-1 to DMRV-5S-2). In brief, frozen sections (8 μm) were mounted on glass slides, dried at room temperature for 1 hour, fixed in cold acetone or 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 minute at room temperature, and then incubated with 1% bovine serum albumin-PBS. The sections were then incubated with the above primary antibodies overnight at 4°C, followed by incubation with fluorescein isothiocyanate-labeled (200-fold diluted), Cy3-labeled (300-fold diluted) (Jackson ImmunoResearch, West Grove, PA), Alexa Fluor 488-labeled (1000-fold), or Alexa Fluor 594-labeled (1000-fold) secondary antibodies (Molecular Probes, Eugene, OR) as described previously.23Kotani M Osanai T Tajima Y Kato H Imada M Kaneda H Kubo H Sakuraba H Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells mouse central nervous system.J Neurosci Res. 2002; 67: 595-606Crossref PubMed Scopus (30) Google Scholar For lectin staining, frozen skeletal muscle tissue sections from the patients with DMRV and the normal controls were incubated with the following fluorescein isothiocyanate-labeled or rhodamine isothiocyanate-labeled lectins: Wheat germ agglutinin (WGA, 1 μg/ml), Arachis hypogaea (peanut) agglutinin (PNA, 1 μg/ml), Glycine max (soybean) agglutinin (SBA, 1 μg/ml), Datura stramonium agglutinin (DSA, 1 μg/ml), Ricinus communis agglutinin120 (RCA120, 1 μg/ml) (Vector Laboratories Inc., Burlingame, CA), and Maackia amurensis agglutinin (MAA, 1 μg/ml) (EY Laboratories, San Mateo, CA) in 1% bovine serum albumin-PBS for 1 hour at room temperature.26Pena SD Gordon BB Karpati G Carpenter S Lectin histochemistry of human skeletal muscle.J Histochem Cytochem. 1981; 29: 542-546Crossref PubMed Scopus (63) Google Scholar The stained sections were observed under a microscope (Axiovert 1000M; Carl Zeiss, Oberkochen, Germany) equipped with a laser-scanning confocal imaging system, LSM510 (Carl Zeiss). Digitized images were captured under identical conditions. Frozen skeletal muscle sections from the DMRV patients and controls including normal subjects were sonicated in 100 μl of Triton X lysis buffer (1% Triton X, 150 mmol/L NaCl, and 20 mmol/L Tris-HCl, pH 7.5). The tissue extracts were centrifuged at 10,000 × g for 5 minutes at 4°C to remove the insoluble debris. Then, the supernatants were treated with trichloroacetic acid, followed by centrifugation at 10,000 × g for 5 minutes at 4°C. The trichloroacetic acid pellets were resuspended in 20 μl of Triton X lysis buffer, and then neutralized with 1 mol/L Tris-HCl (pH 9.0). Sample protein concentration was performed using micro BCA protein assay reagent (Pierce, Rockford, IL). Twenty μg aliquots of protein of the tissue extracts were solubilized with an equal volume of sodium dodecyl sulfate (SDS) sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% β-mercaptoethanol). Then, they were separated on a 4 to 20% Tris-glycine polyacrylamide gel (PAG mini; Daiichi Pure Chemical Co., Ltd., Tokyo, Japan), and then electrotransferred to polyvinylidene difluoride (PVDF) Immobilon-P membranes (Millipore Corp., Bedford, MA). For Western blotting, the blots were blocked in Tris-buffered saline (20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl) containing 5% skim milk for 15 minutes at room temperature. Then, they were probed with an anti-α-DG antibody (clone IIH6) at a dilution of 1:500 in Tris-buffered saline containing 5% skim milk for 1 hour at room temperature. After the blots had been washed with Tris-buffered saline three times, then probed with a peroxidase-conjugated donkey anti-mouse secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 hour at room temperature. Then, the blots were washed with Tris-buffered saline three times, and then developed with ECL (Amersham Pharmacia Biotech) as the chemiluminescent substrate on Hyperfilm ECL (Amersham Pharmacia Biotech) for varying times (1 to 10 minutes). Specific binding of lectins was used for identification of the carbohydrate moieties in extracts of the frozen skeletal muscle tissues, according to the manufacturer's specifications (DIG glycan differentiation kit; Roche Molecular Biochemicals). PVDF membranes with the extracts that had been separated by SDS-polyacrylamide gel electrophoresis (PAGE) were incubated for 1 hour with PNA and MAA lectins conjugated to the steroid hapten digoxigenin (DIG). After washing the membranes, glycoproteins were detected with anti-DIG Fab fragments conjugated with alkaline phosphatase, followed by detection with CDP-star (Roche Molecular Biochemicals) as the chemiluminescent substrate. The sialic acid content was determined by fluorometric analysis using the α-keto acid-specific reagent 1,2-diamino-3,4-methylenedioxybenzene (DMB).24Hara S Yamaguchi M Takemori Y Nakamura M Ohkura Y Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection.J Chromatogr. 1986; 377: 111-119Crossref PubMed Scopus (103) Google Scholar Skeletal muscle glycoproteins from the DMRV patients and controls including normal controls blotted onto PVDF membranes were analyzed by the mild acid hydrolysis-fluorometric HPLC method, as described previously.25Sato C Fukuoka H Ohta K Matsuda T Koshino R Kobayashi K Troy II, FA Kitajima K Frequent occurrence of pre-existing alpha 2−>8-linked disialic and oligosialic acids with chain lengths up to 7 Sia residues in mammalian brain glycoproteins. Prevalence revealed by highly sensitive chemical methods and anti-di-, oligo-, and poly-Sia antibodies specific for defined chain lengths.J Biol Chem. 2000; 275: 15422-15431Crossref PubMed Scopus (103) Google Scholar Sections were treated with 50 mU sialidase from Clostridium perfringens (Roche, Mannheim, Germany) in 50 mmol/L sodium acetate (pH 5.0) for 6 hours at 37°C or with 5 U N-glycosidase F (Roche) in PBS for 12 hours at 37°C, respectively. Then, samples were analyzed by means of lectin staining and lectin blotting. Frozen skeletal muscle sections were solubilized in 100 μl of Triton X lysis buffer. The extracted proteins were added to 20 μl of PNA-agarose (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) overnight with shaking at 4°C. The precipitated proteins were washed extensively in Triton X lysis buffer and then boiled in SDS sample buffer. The supernatant (not binding PNA-agarose) was treated with trichloroacetic acid, followed by centrifugation at 10,000 × g for 5 minutes at 4°C, and then solubilization in SDS sample buffer. The proteins were separated and blotted as described above, and then detected with anti-α-DG antibody IIH6. The data in this study were presented as means ± SD. Statistical comparisons were conducted by means of Student's t-test under the conditions of two-tailed distribution and two-sample equal variance. To identify carbohydrate moieties in muscular glycoproteins of both the DMRV patients and control subjects, lectin-staining of frozen sections from normal controls (N1 and N2), pathological controls (OPMD, EDMD, and MD), and the DMRV patients (DMRV-1 to DMRV-5S-2) was analyzed. We used five lectins in this study, ie, PNA (specific for Galβ1-3GalNAc), SBA (specific for GalNAcα1-3Gal), RCA120 (specific for Gal), WGA (specific for GlcNAc and Neu5Ac), and MAA (specific for Neu5Acα2-3Gal). PNA lectin reacted strongly with the sarcolemma and connective tissues in skeletal muscle sections from all seven DMRV patients (Figure 1), whereas it hardly stained those in normal controls and the OPMD patient under the described conditions (Figure 1A). Furthermore, we double-stained skeletal muscle sections with PNA lectin and an antibody against dystrophin, a classical sarcolemma marker, to determine the localization of PNA reactivity in the DMRV muscles. The merged images clearly show that there is co-localization of PNA-reactive materials and dystrophin in the DMRV muscle, although weak PNA reactivity is also present in the connective tissues (Figure 1B). In contrast, PNA stained the connective tissues of the MD, EDMD, and normal control muscles (Figure 1B). These results indicate an increase in the disaccharide Galβ1-3GalNAc unit in the plasma membranes of the DMRV skeletal muscles. SBA lectin stained the connective tissues in the DMRV muscles stronger than those in a control (Figure 2A). Rimmed vacuoles, which were confirmed by means of phase contrast microscopy, showed increased reactivity with SBA (Figure 2A). The sarcoplasm of several atrophic fibers in DMRV sections also showed increased reactivity with SBA (Figure 2A). The SBA reactivity was stronger in the rimmed vacuoles and sarcoplasm of the atrophic fibers in DMRV than in connective tissues. We next double stained the DMRV muscles with SBA lectin and an antibody against dystrophin. The localization of SBA-reactive materials was apparently different from that of dystrophin (Figure 2B). This suggested that the SBA-reactive materials are not localized in the sarcolemma. RCA120 lectin reacted with the sarcolemma, connective tissues, and blood vessels in the DMRV skeletal muscles, although it also reacted with those in controls. The rimmed vacuoles and sarcoplasm of the atrophic fibers in DMRV were stained strongly (Figure 2A). These results indicate that terminal Gal and GalNAcα1-3Gal carbohydrate moieties are increased in the rimmed vacuoles and sarcoplasm of atrophic fibers in DMRV. In contrast, MAA and WGA stained the sarcolemma, connective tissues, and blood vessels in both the control and DMRV patient muscles (Figure 2). These results indicate the presence of Neu5Acα2-3Gal and (GlcNAc)n carbohydrate moieties in both the DMRV and control skeletal muscles. To confirm the results of lectin staining, we performed lectin blotting analysis with a highly specific DIG-labeled lectin-CDP-star ECL system. MAA reacted strongly with a major band at 250 kd and several minor bands for both control and DMRV muscle cell extracts (Figure 3A). In contrast, PNA reacted with highly glycosylated broad bands at ∼200 kd for cell extracts of the DMRV skeletal muscle specimens, but not in control specimens (Figure 3B). The increase in PNA reactivity to DMRV skeletal muscle glycoproteins could be because of an increase in Galβ1-3GalNAc moieties. Alternatively, subterminal Galβ1-3GalNAc residues on skeletal muscle glycoproteins might be prevented from interacting with PNA by further modification with terminal sialic acids in normal controls. To address this possibility, we examined the ability of sialidase to expose the cryptic PNA-binding sites on normal skeletal muscle glycoproteins. Digestion of frozen sections from normal controls with C. perfringens sialidase resulted in strong reactivity with PNA on both lectin staining (Figure 4A) and lectin blotting analysis (Figure 4B). This indicates exposure of latent binding sites for PNA in the sialidase-treated normal muscle sections as well as in the nontreated DMRV skeletal muscle sections. To characterize the glycoforms that reacted with PNA, MAA, and WGA in the DMRV skeletal muscle specimens, DMRV skeletal muscle sections were treated with N-glycosidase F. N-Glycosidase F is known to cleave N-linked high mannose and hybrid and complex oligosaccharides. Digestion of frozen skeletal muscle sections from the DMRV patients with N-glycosidase F did not alter the reactivity with PNA (Figure 5), whereas it reduced the reactivity with MAA and WGA (Figure 5). These results indicate that the oligosaccharides that reacted with PNA in the DMRV skeletal muscle specimens were mainly O-linked glycans, whereas those that reacted with MAA and WGA were mainly N-linked glycans. In DMRV patients, mutation of the GNE gene reduces the enzymatic activity of UDP-GlcNAc 2-epimerase, according to a report by Noguchi and colleagues.16Noguchi S Keira Y Murayama K Ogawa M Fujita M Kawahara G Oya Y Imazawa M Goto Y Hayashi YK Nonaka I Nishino I Reduction of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase activity and sialylation in distal myopathy with rimmed vacuoles.J Biol Chem. 2004; 279: 11402-11407Crossref PubMed Scopus (139) Google Scholar Therefore, we measured the sialic acid contents of skeletal muscle specimens from the DMRV patients. Homogenates prepared from the skeletal muscle specimens were subjected to SDS-PAGE before transfer to PVDF membranes. The membranes were cut into eight equal pieces according to decreasing Mr, and then analyzed for sialic acid content by the mild hydrolysis-fluorometric HPLC method. No difference was detected in each membrane slice (Mr 20,000 to >250,000) between the patients and normal controls without glycosidase treatment (data not shown). Then, the skeletal muscle extracts were treated with N-glycosidase F to detect O-linked sialic acid components. A 20% reduction in the total sialic acid content (calculated as sum of the sialic acid contents of membrane slices 1 to 8) was observed in the DMRV muscles on treatment with N-glycosidase F (controls, 4.27 ± 0.46 pmol/μg versus DMRV patients, 3.51 ± 0.26 pmol/μg). Furthermore, a 20 ∼ 40% reduction was observed for each of membrane slice 2 (Mr, 120,000 to 200,000; P = 0.13), slice 3 (Mr, 80,000 to 120,000; P < 0.05), slice 4 (Mr, 60,000 to 80,000; P < 0.05), and slice 6 (Mr, 30,000 to 40,000; P < 0.05) (Figure 6). Because the reactivity of PNA was significantly increased in the DMRV patients, we tried to identify the sarcolemmal glycoproteins that reacted with PNA. We obtained two fractions for the skeletal muscle extracts according to PNA-binding activity by means of PNA agarose; PNA-binding and PNA-nonbinding fractions. The PNA fraction of the DMRV muscle extracts reacted with a monoclonal antibody, IIH6, that recognizes the carbohydrate epitope of α-DG, but that of a normal control extract did not (Figure 7). By contrast, the PNA-nonbinding fraction of the normal control extract reacted with IIH6 (Figure 7). These data suggest that α-DG contains PNA-reactive sugar chains in the DMRV patients. Furthermore, we examined the expression of glycosylated α-DG in the biopsied muscle specimens from the DMRV patients with IIH6. Positive staining was demonstrated in all of the DMRV patients as well as the normal controls. This suggests that the basic structure of the α-DG sugar chain recognized by the IIH6 antibody was not altered in the DMRV patients (data
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