Abstract
Background: Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by severe dystrophic muscle wasting from birth or early infancy with structural brain abnormalities. The gene for FCMD is located on chromosome 9q31, and encodes a novel protein named fukutin. The function of fukutin is not known yet, but is suggested to be an enzyme that modifies the cell-surface glycoprotein or glycolipids.
Objective: To elucidate the roles of fukutin gene mutation in skeletal and cardiac muscles and brain.
Methods: Immunohistochemical and immunoblot analyses were performed in skeletal and cardiac muscles and brain tissue samples from patients with FCMD and control subjects.
Results: The authors found a selective deficiency of highly glycosylated α-dystroglycan, but not β-dystroglycan, on the surface membrane of skeletal and cardiac muscle fibers in patients with FCMD. Immunoblot analyses also showed no immunoreactive band for α-dystroglycan, but were positive for β-dystroglycan in FCMD in skeletal and cardiac muscles.
Conclusion: The current findings suggest a critical role for fukutin gene mutation in the loss or modification of glycosylation of the extracellular peripheral membrane protein, α-dystroglycan, which may cause a crucial disruption of the transmembranous molecular linkage of muscle fibers in patients with FCMD.
Dystroglycan (DG) was originally identified as a component of dystrophin-associated proteins in skeletal muscle. It was suggested to play a crucial role in linking the extracellular basal lamina to the cytoskeletal proteins. DG consists of two subunits, α-DG and β-DG, that are generated by post-translational cleavage of proprotein encoded by a single gene mapped to chromosome 3p21.1 The molecular mass of the peptide moiety of α-DG is estimated to be 72 kDa from the complementary DNA sequence. Due to the possible tissue-specific glycosylation process, however, the molecular mass of heavily glycosylated α-DG is different in mammalian skeletal muscle (156 kDa), cardiac muscle (140 kDa), and brain and peripheral nerves (120 kDa). α-DG is a peripheral membrane protein that binds to extracellular matrix molecules such as laminin, perlecan, and agrin, and plays a key role in the assembly of the extracellular matrix.2-4⇓⇓ Conversely, 43-kDa β-DG spans the plasma membrane and links α-DG to the intracellular cytoskeletal proteins such as dystrophin and utrophin in skeletal muscle.5,6⇓
Fukuyama-type congenital muscular dystrophy (FCMD) is a severe CMD with CNS involvement.7 Our previous immunohistochemical study revealed a reduced expression of the laminin α2 chain of merosin (laminin-2 and -4) in the skeletal muscle fiber basal lamina of FCMD.8 With electron microscopy, the thin, disrupted muscle fiber basal lamina was observed in patients with FCMD.9,10⇓ Furthermore, in brain tissue from the FCMD fetus, breaches in the glia limitans–basal lamina complex with protrusion of neuroglial tissues were demonstrated and suggested as a primary cause of the cortical dysplasia.11-13⇓⇓ These findings suggest that the structural alteration of the basal lamina plays a key role in the pathologic process of muscle fiber damage and brain anomaly in FCMD.
Recently, the gene responsible for FCMD was identified on chromosome 9q31, which encodes a novel protein named fukutin.14 Fukutin messenger RNA is expressed ubiquitously, but is enriched in skeletal muscle, heart, and brain. Most patients with FCMD have a 3-kb retrotransposal insertion in the 3′ noncoding region, and fukutin messenger RNA is barely detectable in the lymphoblastoid cells from patients with FCMD. Fukutin is thought to be a secretory protein, although its function is not yet known. In the current study, we examined skeletal and cardiac muscles and brain tissue samples to elucidate the roles of fukutin gene mutation.
Methods.
Clinical materials.
All clinical materials were obtained for diagnostic purposes with informed consent. We analyzed a total of 39 biopsied limb-muscle specimens: 15 from patients with FCMD (aged 5 months to 4 years), two from normal subjects (14 and 17 years old) obtained at surgery, and 22 from diseased control subjects (aged 4 months to 25 years) including eight samples of primary merosin-deficient CMD, two of unclassified CMD, five of Duchenne muscular dystrophy, two of Becker muscular dystrophy, two of childhood dermatomyositis, one of limb-girdle muscular dystrophy type 1C, and two of spinal muscular atrophy. We also examined autopsied skeletal and cardiac muscle and brain tissue from four patients with FCMD (aged 15 to 25 years) and two control subjects with illnesses unrelated to neuromuscular disease (a 14-year-old with malignant lymphoma and a 64-year-old with lung cancer). The samples were flash-frozen in isopentane chilled with liquid nitrogen.
Genomic PCR.
DNA was isolated from either skeletal muscle or peripheral blood lymphocytes from 100 normal, healthy control subjects and the patients with FCMD using a standard technique after obtaining informed consent.15 The genomic PCR was performed using the primer sets designed for the human fukutin gene—F: 5′-AACGTTCCCTCT TTCCTCCTT G-3′ and R: 5′-GCCCTCAAATTTCTGTTTCTAT–3′ (858 bp). Fifty nanograms of the genomic DNA template was amplified under the following cycling conditions: 94 °C for 3 minutes; 30 cycles of 94 °C for 30 seconds, 58 °C for 60 seconds, and 72 °C for 60 seconds; and 72 °C for 7 minutes. The human dystrophin gene was also amplified simultaneously as a control using a primer set for exon 43, as described previously.15
Quantitative reverse transcriptase PCR.
The total RNA was extracted from 3 mg of human skeletal muscles using the guanidinium isothiocyanate phenol-chloroform method. Two micrograms of the total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase and oligo deoxythymidine as a primer, and 50 ng of the complementary DNA served as a template for reverse transcriptase PCR.16 Primers were designed for human fukutin complementary DNA and were labeled with fluorescent dye, 6-carboxyfluorescein (Applied Biosystems, Foster City, CA). The primers for fukutin complementary DNA were F: 5′-FAM-CTGCATTTGCACTGCAGTATCA-3′, and R: 5′-TTTG-GTCGTTATCCAGGAGC-3 (308 bp). To correct the relative quantity of messenger RNA in each muscle sample, human muscle-specific α-actin complementary DNA was coamplified as the internal standard. The primers for α-actin labeled with TET (472′7′-tetrachloro-6-carboxyfluorescein) (Applied Biosystems) were F: 5′-TET-TCGACATCAGGAAGGACC-TGT-3′ and R: 5′-GAGGTGGAGTGTGTCTAGAAG-3′ (282 bp). The PCR reactions contained 25 ng of each primer for fukutin, 0.2 μM of each deoxynucleotides (dNTP), and 10 units of Stoffel fragment (Applied Biosystems) in a 50-μL reaction volume. After the first 12 cycles, 25 ng of each primer for α-actin was added. After 20 cycles of PCR, the amplified products were loaded and analyzed automatically using capillary electrophoresis with the ABI PRISM 310 GeneScan (Applied Biosystems).
Antibodies.
The antibodies used in the current study were as follows. Monoclonal anti-α-DG antibody (VIA4–1: Upstate Biotechnology, Lake Placid, NY), affinity-purified sheep anti-α-DG antiserum,17 monoclonal anticranin (dystroglycan) antibodies (6C1 and 8B4; CHEMICON International, Temecula, CA), rabbit anti-β-DG antiserum,18 monoclonal antidystrophin,19 anti-α-, anti-β-, anti-γ-, and anti-δ-sarcoglycans (Novocastra Laboratories, Newcastle upon Tyne, UK), antilaminin subunit (α2, β1, γ1 chains) antibodies,8 and rabbit anti-type IV collagen antiserum (Advance, Inc., Tokyo, Japan). Affinity-purified fluorescein isothiocyanate–labeled goat F(ab′)2 antimouse IgG, goat F(ab′)2 antirabbit IgG, and rabbit F(ab′)2 antisheep IgG (all from TAGO Inc., Burlingame, CA) were used as second-layer antibodies. Preimmune rabbit and sheep sera and control mouse myeloma IgG served as controls.
Immunohistochemistry.
Serial cryostat sections (6 μm) were thawed on gelatinized cover slips, fixed in 100% ice-cold acetone for 5 minutes, air-dried for 30 minutes, and then preincubated with phosphate-buffered saline containing 2% bovine serum albumin and 5% heat-inactivated normal goat or rabbit serum at pH 7.4. The sections were then incubated with primary antibodies for 2 hours at 37 °C or overnight at 4 °C, and affinity-purified secondary antibodies (10 μg/mL) at room temperature for 1 hour. The sections were mounted in a glycerol-based medium and observed under a Zeiss Axiophot2 microscope (Oberkochen, Germany) with epifluorescence. Cryosections of the skeletal muscles were also examined under additional fixation procedures including 4% paraformaldehyde for 15 minutes with or without 0.05% glutaraldehyde, methacarn solution (60% methanol, 30% chloroform, and 10% acetic acid) for 15 minutes, or 4% paraformaldehyde for 15 minutes followed by treatment with 0.1% sodium dodecyl sulfate for 15 minutes.
Immunoblotting.
Immunoblotting analyses for α-DG and β-DG were performed using samples dissected and homogenized in 10 × volume of homogenization buffer (phosphate-buffered saline containing 0.2% Triton X-100, 2 mM phenylmethanesulfonylfluoride and protease inhibitors). For α-DG, additional samples were also examined that prepared using the following steps: 1) proteins bound to the wheat-germ agglutinin (WGA) column, 2) unbound proteins to the WGA column, and 3) whole extract from 0.2% Triton X-100 (polyoxyethylene-p-isooctylphenol) and WGA-eluted proteins digested with a series of glycosidases.
For the WGA-Sepharose column procedure, the homogenates were centrifuged at 15,000 g for 10 minutes and the supernatants were circulated for 2 hours with an equal volume of WGA lectin-Sepharose 6MB (Amersham Pharmacia Biotech, Arlington Heights, IL). Then, the affinity beads were washed and eluted with an equal volume of 0.3 M N-acetylglucosamine.
For glycosidase treatment, samples were incubated with one or a series of the following glycosidases overnight at 37 °C; 1 mU glycopeptidase F (Takara Shuzo Co., Tokyo, Japan) in 2.5 μL denature buffer (1 M TRIS-HCl, pH 8.6 with 1% sodium dodecyl sulfate and 0.2 M 2-mercaptoethanol), Arthrobacter ureafacience sialidase (50 mU) (Boehringer, Mannheim, Germany) in 95 μL of 0.2 M sodium acetate buffer (pH 5.0), β-galactosidase from bovine testis (40 mU) (Sigma Chemical Corp., St. Louis, MO) in 60 μL of 0.2 M sodium acetate buffer (pH 5.0), and Acremonium sp. α-N-acetylgalactosaminidase (260 mU) (Seikagaku Corp., Tokyo, Japan) in 80 μL of sodium citrate buffer (pH 4.5). Heating the reaction mixture in boiling water for 3 minutes terminated digestions.
Samples were separated on 6% sodium dodecyl sulfate–polyacrylamide gels, transferred to a polyvinyl difluoride membranes (Nihon Eido, Tokyo, Japan), and allowed to react overnight with anti-α-DG and β-DG antibodies at 4 °C. The immunoreactive bands were then visualized using the Enhanced Chemiluminescence kit (Amersham Pharmacia). The optical area of each immunoreactive band for α-DG and β-DG was measured by densitometer with Quantity One (PDI Inc., Huntington Station, NY) and the ratios of α-DG/β-DG were calculated.
Results.
Genomic PCR and quantitative reverse transcriptase PCR.
The primer set for the fukutin gene amplified an 858-bp PCR product from control DNA samples, but did not amplify the fukutin gene in patients with FCMD because of the presence of 3-kb retrotransposal insertion. In all patients with genetically confirmed FCMD, quantitative reverse transcriptase PCR analysis revealed a drastic reduction of fukutin messenger RNA (from 0 to 21% of that of the control subjects) in the skeletal muscle (data not shown), suggesting a deficiency of fukutin.
Immunohistochemical analysis of skeletal muscles.
We found a barely detectable immunoreaction to both monoclonal anti-α-DG antibody (VIA4–1) (figure 1) and sheep anti-α-DG antiserum (data not shown) on the surface membrane of skeletal muscle fibers exclusively in patients with FCMD, although β-DG was normally preserved (see figure 1). The immunoreaction to α-DG (VIA4–1) at the neuromuscular junctions (NMJ) was also drastically reduced in the FCMD muscles, whereas the reactivity for α-bungarotoxin was preserved (figure 2). The other proteins examined in the current study, including laminin α2 chain, dystrophin, and sarcoglycans, showed findings similar to those reported previously (see figure 1).8,20,21⇓⇓
Figure 1. Immunohistochemical analysis of skeletal muscles. Consecutive frozen sections of skeletal muscles from control subjects, two patients with Fukuyama-type congenital muscular dystrophy (FCMD), and patients with primary merosin-deficient CMD (Merosin-def. CMD) and Duchenne muscular dystrophy (DMD) immunostained with antilaminin α2 chain, α-dystroglycan (α-DG) (VIA4–1), β-DG, and dystrophin antibodies. Bar = 50 μm.
Figure 2. Immunohistochemical analysis of neuromuscular junctions of skeletal muscles. Sections from unclassified congenital muscular dystrophy (CMD) and Fukuyama-type CMD (FCMD) muscles are double-immunostained with anti-α-dystroglycan (anti-α-DG) antibody (VIA4–1) and α-bungarotoxin (α-BTX). Note the selective loss of α-DG on the surface membrane of each muscle fiber and the neuromuscular junctions in the FCMD samples. Bar = 50 μm.
The skeletal muscle tissue from patients with primary merosin-deficient CMD showed normal immunoreaction for anti-α-DG antibody (VIA4–1) (see figure 1), but greatly reduced staining for sheep anti-α-DG antiserum (data not shown). Duchenne and Becker muscular dystrophy tissue showed variable abnormalities in dystrophin, sarcoglycans, and DG (see figure 1), as described previously.22,23⇓ The specimens from the other diseased control patients, including the one with limb-girdle muscular dystrophy type 1C, showed normal immunoreactions for both α-DG and β-DG (see figure 1).
To determine whether the tissue localization of α-DG was altered or the reactivity to the antibody was changed in FCMD skeletal muscles, we immunostained the cryosections after carrying out a series of different fixation procedures and a treatment with detergent, as described in the “Methods” section. However, we could not identify a positive immunoreaction for α-DG (VIA4–1) on the surface membrane or in the cytoplasmic area of skeletal muscle fiber (data not shown).
Immunoblot analysis of skeletal muscles.
We obtained consistent results in immunoblot analysis. Skeletal muscle specimens from patients with FCMD showed loss of a 156-kDa band for α-DG (VIA4–1) in both whole extracts and WGA lectin–bound fraction, but showed a normal 43-kDa immunoreactive band for β-DG in the whole extracts (figure 3a). Because WGA lectin binds specifically to a cluster of sialic acids and bisecting N-acetylglucosamine, processing of glycans in α-DG may be altered and affect the binding of α-DG to the WGA column in FCMD muscles. To examine whether the glycosylation pattern of α-DG was altered in FCMD skeletal muscle, we performed immunoblot analysis using muscle samples of WGA column unbound proteins and digestions with glycosidases, as described in the “Methods” section. However, no immunoreactive band against α-DG (VIA4–1) was observed (data not shown). These findings suggest that α-DG is selectively lost in FCMD skeletal muscles in presence of the deficiency of fukutin by either rapid degradation or dissociation and elution from skeletal muscle. We cannot exclude the possibility that α-DG in FCMD muscles loses immunoreactivity against the antibody used.
Figure 3. Immunoblot analysis. (a) Control skeletal muscle samples (C) show a 156-kDa band for α-dystroglycan (α-DG) (VIA4–1) that is undetectable in all patients with Fukuyama-type congenital muscular dystrophy (FCMD) (F1 to F6) in wheat-germ agglutinin lectin–bound fraction. In contrast, β-DG is detected as a 43-kDa band in both control and FCMD samples. (b) Control cardiac muscle (C) shows a 140-kDa band for α-DG (VIA4–1), but muscle from patients with FCMD (F1 to F3) does not. β-DG is detected as a 43-kDa band in all samples examined. (c) All samples show a 120-kDa band for α-DG (VIA4–1,) with reduced amounts in one patient with FCMD (F2).
Cardiac muscles.
We also examined autopsied cardiac muscle samples from patients with definite FCMD. The immunoreaction of α-DG (VIA4–1) was barely detectable at the surface membrane of cardiac muscles in patients with FCMD, whereas the control cardiac muscles expressed α-DG clearly at the surface membrane (figure 4). Interestingly, in FCMD, material immunopositive for α-DG was observed in the cytoplasmic area of a few cardiac muscle cells (see figure 4, arrows). Immunoreaction for β-DG, however, was clearly positive at the plasma membrane and T tubules of cardiac muscles from control and FCMD tissue (see figure 4). Immunoblot analysis showed no immunoreactive band for 140-kDa α-DG in either WGA-column–bound or –unbound proteins, but was positive for 43-kDa β-DG in the whole extract in FCMD (see figure 3b). These findings indicate that α-DG is lost or cannot localize properly on the surface membrane of the cardiac muscle fibers in fukutin-deficient FCMD.
Figure 4. Immunohistochemical analysis of cardiac muscles. Frozen sections of cardiac muscles from control subjects and patients with Fukuyama-type congenital muscular dystrophy (FCMD), immunostained with anti-α-dystroglycan (anti-α-DG) (VIA4–1) and β-DG antibodies. Selective deficiency of α-DG but not β-DG is observed in cardiac muscle specimen from a patient with FCMD. Immunopositive materials for α-DG are observed in the cytoplasm of a few cardiac muscle fibers in the FCMD tissue (arrows). Lipofuscins are observed as dots with strong intensity. Bar = 50 μm.
Brain tissues.
The immunohistochemical study was performed using the cerebellum specimens from one patient with FCMD and one control patient with lung cancer. Both the control and FCMD patient showed strong immunoreaction of blood vessels to α-DG (VIA4–1) with no marked difference. In addition, punctate α-DG labeling was observed mainly in the molecular layer and around the cell surface of the Purkinje cells (figure 5). This punctate labeling, however, was reduced in FCMD. On the other hand, anticranin (dystroglycan) antibodies showed positive immunoreaction of the Purkinje cell bodies, fiberlike structures, and blood vessels with no difference between control and FCMD tissue (see figure 5). The discrepancy of immunostaining patterns between anti-α-DG (VIA4–1) and cranin antibodies was unclear, although Tian et al.24 reported similar punctate immunolabeling of α-DG in the rat cerebellum, and Smalheiser and Kim25 showed labeling of Purkinje cell bodies and fibers. Immunoblot analysis using anti-α-DG antibody (VIA4–1) showed similar 120-kDa immunoreactive bands in WGA column bound fractions from both control and FCMD autopsied brain samples (see figure 3c). The ratios of α-DG/β-DG protein amounts obtained by densitometric scanning were reduced in one FCMD sample but not in the other (C = 5.1 and 5.8; F1 = 12.3; F2 = 2.1). The proteins unbound to the WGA column showed no immunoreactive band for α-DG (data not shown).
Figure 5. Immunohistochemical analysis of cerebella. Frozen sections of cerebella from control subjects and patients with Fukuyama-type congenital muscular dystrophy (FCMD), immunostained with two anti-α-dystroglycan (anti-α-DG) antibodies (VIA4–1 and cranin 6C1). VIA4–1 shows strong immunoreaction of blood vessels (bv) in both control and FCMD tissues. Widespread punctate labeling is observed, mainly in the molecular layer (ml) and around the cell surface of the Purkinje cells (pu) in the control tissue. These punctate immunopositive materials are greatly reduced in FCMD tissue. Cranin shows positive immunoreaction of Purkinje cells, fiberlike structures, and blood vessels with no difference between control and FCMD tissues. Bar = 100 μm.
Discussion.
In the current study, we showed the selective deficiency of α-DG, a highly glycosylated cell surface protein, but not β-DG, in skeletal and cardiac muscles from patients with FCMD. The discrepancy in the immunostaining pattern between α-DG and β-DG is of note, because both arise from a single transcript by post-translational cleavage.1 Similar findings were reported in a patient with limb-girdle muscular dystrophy type 1C, who had a mutation in the caveolin-3 gene.17 However, in the current study, no muscle specimen other than FCMD showed such a discrepancy in the immunostaining pattern between α-DG and β-DG, including a patient with limb-girdle muscular dystrophy type 1C. The skeletal muscle from patients with merosin-deficient CMD showed normal immunoreaction to anti-α-DG antibody (VIA4–1) although they had greatly reduced immunostaining for sheep antiserum to α-DG. Further study is needed to clarify the reason of this staining discrepancy.
The anti-α-DG antibody (VIA4–1) used in the current study was raised against membrane preparation of rabbit skeletal muscle, and recognizes the 156-kDa glycoprotein copurified with dystrophin.26 Conversely, the affinity-purified sheep antiserum to α-DG was raised against 20 amino acids of C-terminus of chick α-DG, which contains a high degree of sequence identity to DG of several species.17 In FCMD muscles, these two anti-α-DG antibodies showed an immunoreaction deficiency that may suggest the instability of the protein of α-DG. We cannot exclude the possibility that these antibodies may only detect highly glycosylated forms of α-DG, and FCMD muscles could contain a less-glycosylated form of α-DG that is not recognized by the antibodies. However, if this is the case, the altered glycosylation pattern of α-DG in skeletal and cardiac muscles in FCMD may cause crucial functional disruption of the transmembranous molecular linkage.
The function of fukutin is not yet known, although detailed computer analyses of the fukutin protein sequence allow prediction of the function that may involve modifying the cell-surface glycoproteins or glycolipids.27 Thus, it has been suggested that fukutin deficiency affects the modification of glycosylation of α-DG in skeletal and cardiac muscles, and that the altered α-DG cannot localize or function properly on the extracellular surface membrane of the muscle fiber and in the NMJ even if α-DG is transported to the extracellular surface.
Deficiency of α-DG hampers the physiologic linkage between extracellular basal lamina and the intracellular cytoskeleton. This disruption could cause developmental, structural, and functional damage to skeletal and cardiac muscles in patients with FCMD. Skeletal muscles from patients with FCMD show small, round muscle fibers with disorganized myofibrils.7 Similar histologic changes were observed in skeletal muscles from patients with primary merosin-deficient CMD, caused by mutations in laminin α2 chain gene. An in vitro study using an antibody blocking α-DG and laminin interaction showed similar dystrophic phenotypes on differentiated cultured muscles, including reduced myotube size and disorganization of myofibrils.28 These findings indicate that the disruption of the interaction between α-DG and laminin caused specific histologic changes in the skeletal muscle.
DG are also thought to have important roles for clustering or stabilization of acetylcholine receptors through interaction with agrin and rapsyn at the NMJ.29,30⇓ In FCMD muscles, however, the clustering of acetylcholine receptors appeared to be maintained normally, as shown by α-bungarotoxin staining in the absence of α-DG (VIA4–1). More detailed examinations of NMJ in FCMD muscles will provide insights for the role of DG during formation of NMJ and their functions.
In brain tissue from patients with FCMD, an abnormal pattern of gangliosides was reported.31 Although it is unclear whether the modification of glycans of α-DG are altered in FCMD, we observed reduced punctate labeling for α-DG in the FCMD cerebellum by immunohistochemistry. One patient also showed a reduced amount of α-DG by immunoblotting. These findings suggest the possibility that fukutin participates in modifying not only glycolipids but also glycoprotein(s) in the CNS. Because cortical dysplasia and breaches of the glia limitans–basal lamina complex in the FCMD brain is known to occur during the fetal period,32 the expression pattern of α-DG in the fetal brain in control and FCMD would help clarify the role of fukutin during the developmental stage. Further studies are necessary to clarify the process of selective deficiency of α-DG in FCMD muscles, the role of fukutin, and the pathomechanism of FCMD.
Acknowledgments
Supported by grants-in-aid for Scientific Research for Center of Excellence (COE), Brain Sciences, from the Ministry of Health and Welfare, research grants from the Ministry of Education, Science, and Culture; Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Corporation; and the Uehara Memorial Foundation.
Acknowledgment
The authors thank Drs. F. Muntoni and H. Sugita for their helpful discussion and advice, and Drs. S. Kröger, E. Ozawa, and M. Imamura for providing antibodies.
- Received October 10, 2000.
- Accepted March 3, 2001.
References
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