![]()
|
|
||||||||
J. Biol. Chem., Vol. 279, Issue 12, 11402-11407, March 19, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

¶













From the
Department of Neuromuscular Research, **Division of Radiation Protection, 
Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, and ||National Center Hospital for Mental, Nervous, and Muscular Disorders, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan and
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Received for publication, December 3, 2003 , and in revised form, December 30, 2003.
| ABSTRACT |
|---|
|
|
|---|
| INTRODUCTION |
|---|
|
|
|---|
Hereditary inclusion body myopathy (HIBM) is an autosomal recessive disorder that presents with adult-onset slowly progressive distal and proximal weakness and has characteristic pathological features in muscle tissue, including rimmed vacuoles and filamentous inclusions, that are similar to those seen in DMRV (4). Gene loci of both diseases have been mapped to chromosome 9 (5, 6). HIBM is caused by mutations in the UDP-GlcNAc 2-epimerase/ManNAc kinase gene (GNE gene) (7). Previously we identified homozygous and compound heterozygous mutations in the GNE gene in 27 DMRV patients (8), demonstrating that the two diseases are allelic.
UDP-GlcNAc 2-epimerase/ManNAc kinase is a dual functional enzyme catalyzing two initial steps in the biosynthesis of sialic acid (9). This enzyme catalyzes the conversions of UDP-GlcNAc to ManNAc and ManNAc to ManNAc 6-phosphate. Despite the identification of the GNE gene mutations, we still do not fully understand how these mutations contribute to the pathophysiology in DMRV/HIBM. Several questions remain unanswered. 1) What is the status of sialylation activity in the patients with GNE mutations? One would expect sialylation to be impaired but not completely absent in DMRV/HIBM patients, because sialic acid is essential for embryonic development (10). In fact, homozygous null mutations have never been identified in patients (8, 11). 2) Why are symptoms restricted to the skeletal muscles? GNE transcripts are expressed in various tissues and are especially predominant in the liver (12). 3) Why do mutant proteins not complement each other in patients who have heterozygous mutations in each of the two domains? The two domains in GNE protein have been reported to catalyze the enzymatic reactions separately and independently (13). To address these questions, we studied the relationships between mutations and enzymatic activity using in vitro expression and enzymatic assay systems. We also determined the levels of sialylation in sera, muscles, and primary cultured cells from DMRV patients and normal individuals.
| EXPERIMENTAL PROCEDURES |
|---|
|
|
|---|
Expression of Recombinant GNE ProteinsThe cDNA for wild-type GNE was obtained by reverse transcribed-PCR from normal muscle RNA and cloned into pCR-blunt vector (Invitrogen). The cDNAs for GNE mutants were obtained by reverse transcribed-PCR from skeletal muscle RNA of DMRV patients or by site-directed mutagenesis from wild-type cDNA. All cloned muscle cDNAs were sequenced by ABI cycle-sequencing procedures using an ABI 3100 (Applied Biosystems, Foster City, CA). The sequenced and inserted cDNAs were cut out with EcoRI and blunted, and the purified cDNA fragments were inserted in-frame into the expression vector, pCMV-Myc (Invitrogen). The expression constructs were transiently transfected into COS-7 cells using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. After 24 h, the Myc-tagged wild-type GNE and the mutant proteins were extracted from transfected cells. UDP-GlcNAc 2-epimerase activity was measured as described previously. The ManNAc kinase assay was performed with slight modification according the previous report (13).
Cross-linking of GNE Mutant ProteinsTo analyze the oligomer structure of wild-type and mutant GNE, cell lysates were subjected to a reaction with 10 mM MBS for 30 min at room temperature for cross-linking. The Myc-tagged cross-linked products were purified with anti-Myc-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and eluted by boiling in 2% SDS solution. The products were subjected to SDS-PAGE and Western blot using anti-Myc 9E10 antibody (Santa Cruz Biotechnology).
Lectin Staining and Protein Analysis of Skeletal Muscles from PatientsBiotin-labeled Maackia amurensis (MAM), soybean agglutinin (SBA), and Sambucus sieboldiana agglutinin (SSA) (Seikagaku Kogyo, Tokyo, Japan) and fluorescein isothiocyanate-labeled streptavidin (Vector Laboratories, Burlingame, CA) were used for the staining of muscle sections. Unfixed 10-µm-thick muscle sections were blocked in 2% casein/phosphate-buffered saline and stained with lectin solution for 2 h at room temperature. The proteins were extracted from the skeletal muscle sections using 8.5 M urea, 0.5% Nonidet P-40 and analyzed by two-dimensional PAGE. Monoclonal antibodies, H4A3 (for LAMP-1), 43DAG1/8D5 (for
-dystroglycan), and VIA4-I (for
-dystroglycan) were used for Western blotting. Laminin binding to
-dystroglycan was examined as described previously (14).
Cell CulturesCOS-7 cells were cultured in 10% fetal bovine serum/Dulbecco's modified Eagle's medium in 5% CO2. Primary fibroblasts and myoblasts from DMRV patients and normal individuals were cultured in 10% fetal bovine serum, Dulbecco's modified Eagle's medium, and Ham's F-12 medium in 5% CO2. The myoblasts were induced to myogenic differentiation by switching the medium to 5% horse serum, Dulbecco's modified Eagle's medium, and Ham's F-12 medium. At 24 h before lectin staining or sialic acid determination, the medium was replaced with serum-free Dulbecco's modified Eagle's medium and Ham's F-12 medium with or without 5 mM GlcNAc, ManNAc, or NeuAc. Cells were fixed and permeabilized as described previously (15). Biotin-labeled SBA, wheat germ agglutinin (WGA) (Seikagaku Kogyo), an antibody against desmin (ICN Pharmaceuticals, Costa Mesa, CA), and Alexa Fluo 594-labeled secondary antibodies (Molecular Probes, Eugene, OR) were used for staining the cells.
| RESULTS |
|---|
|
|
|---|
T, 89G
C, and 2173G
A (Table I). These novel mutations were absent in 100 control chromosomes from normal Japanese individuals.
|
206256) caused by exon 4 skipping. The abnormal protein was degraded in COS cells (Fig. 1A). We determined the specific activities of UDP-GlcNAc 2-epimerase and ManNAc kinase of the mutant proteins relative to wild type (Fig. 1B). The endogenous activities in mock transfected COS cells were determined to correct for the background enzyme activity. UDP-GlcNAc 2-epimerase activities in mutants C13S, H132Q, D176V, D177C, V331A, and D378Y were reduced to less than 20% of the control. In contrast, the I472T and G708S mutants each revealed an
50% reduction, and V572L, A630T, and A631V each showed only a 2030% reduction in activity as compared with wild-type cells. ManNAc kinase activity was retained in the N-terminal mutants C13S, H132Q, D176V, D177C, V331A, and D378Y, whereas the C-terminal mutants I472T, V572L, A630T, A631V, and G703S showed dramatic reductions in activities. These data were essentially compatible with a prior report (13). Interestingly, the A524V mutant preferentially affected UDP-GlcNAc 2-epimerase activity, although the mutation is in the kinase domain. None of the DMRV mutants showed complete loss of UDP-GlcNAc 2-epimerase or ManNAc kinase activities.
|
Sialic Acid Contents in Skeletal Muscles and Primary Cells from DMRV PatientsGNE gene mutations reduced the enzymatic activity of GNE protein. These results led us to hypothesize that sialylation should be affected in the tissues of DMRV patients. We measured the sialic acid content in sera and muscles from DMRV patients (Fig. 2A). In sera, no difference was detected between patients and normal controls, whereas in skeletal muscle, a 25% reduction of sialic acid was observed in DMRV muscles. We also assessed the status of sialylation in DMRV muscles by lectin staining. We used three lectins: SSA for detection of Sia
26Gal/GalNAc, MAM for Sia
23Gal, and SBA for GalNAc
13Gal (1618) (Fig. 2B). The results of lectin staining are summarized in Table II. SSA uniformly stained sarcolemma in control muscle, whereas it faintly stained sarcolemma and strongly stained interstitial tissues in DMRV muscle (Fig. 2B, panels df). MAM strongly stained sarcolemma and interstitial tissues in both the control and patient muscles. We did not observe any reduction in MAM staining in patients, which may be attributed to the strong intensity in our staining condition (data not shown). In contrast, SBA strongly highlighted the rimmed vacuoles containing fibers and the surrounding atrophic fibers in the patients both in sarcolemma and cytoplasm (Fig. 2B, panels h and i; see arrows), whereas it did not stain myofibers in the control (Fig. 2B, panel g). These data suggest that sialylation, other glycosylation, or both are at least partly disturbed in some myofibers in DMRV. Furthermore, we examined the expression of glycosylated
-dystroglycan in DMRV muscles using an antibody (VIA4-I) that recognizes a carbohydrate epitope. The
-dystroglycan staining was negative in rimmed vacuoles containing fibers and the surrounding atrophic fibers in one DMRV patient (Fig. 2B, panel l). However, positive staining in another patient demonstrated that
-dystroglycan expression varies among patients (Fig. 2B, panel k). Therefore, we concluded that the loss of
-dystroglycan staining is an extreme down-stream phenomenon in DMRV muscles. We also analyzed muscle sialylated glycoproteins (LAMP-1, and
- and
-dystroglycans) by one- or two-dimensional polyacrylamide gel electrophoresis, but when they were extracted in whole amounts, there was no significant change in the electrophoretic patterns of these proteins between control and patients (data not shown). Furthermore, we analyzed the laminin-binding property of
-dystroglycan from DMRV patients, and the
-dystroglycan showed a strong binding as the control (data not shown).
|
|
6074% of control cells when cells were cultured in serum-free medium (Fig. 3C). By adding ManNAc or NeuAc into the culture medium, sialic acid levels in the fibroblasts and myotubes were restored to normal levels (Fig. 3C, +ManNAc and +NeuAc). Furthermore, WGA staining of cells from patients also increased to normal levels, and particularly strong WGA staining was observed in the plasma membrane of myotubes. In contrast, the SBA staining in DMRV cells disappeared by the addition of either sugar (Fig. 3, A and B; +ManNAc and +NeuAc). The addition of GlcNAc into the medium had no effect on the staining pattern with either lectin (Fig. 3, A and C, +GlcNAc). These results suggest the potential therapeutic use of ManNAc and NeuAc.
|
| DISCUSSION |
|---|
|
|
|---|
C mutation accounts for 55% (36 of 66) of the abnormal alleles confirming the high frequency of this mutation in Japan. Haplotype analysis suggests that this common mutation is due to a founder effect (8). All of the mutations identified in DMRV patients caused reduction (but not total loss) of enzymatic activities of either UDP-GlcNAc 2-epimerase or ManNAc kinase. These results strongly suggest that DMRV is caused by partial loss of function of the gene product. Interestingly, we previously identified the compound heterozygous mutations D378Y and A631V in a North American DMRV patient of German and Irish origin; these mutations have also been identified in an Irish HIBM patient (13). D378Y reduced UDP-GlcNAc 2-epimerase activity, and A631V decreased ManNAc kinase activity. Together with clinical and pathological similarities, these biochemical and molecular genetic results suggest that DMRV and HIBM are actually the same disease. Through our study, we obtained information about novel molecular aspects in GNE. The two catalytic domains of the GNE molecule do not always work separately or independently in contrast to a published report (13). For example, the A524V mutation is within the predicted ManNAc kinase domain; however, it strongly inhibited UDP-GlcNAc 2-epimerase. Interestingly, this mutant did not form an oligomeric structure similar to the other N-terminal mutants. The failure of oligomerization in this A524V mutant is probably responsible for the reduced UDP-GlcNAc 2-epimerase activity as suggested previously (13).
Sialylation was decreased in muscle and in cultured cells from patients but was not completely lost, because all of the mutant proteins with missense mutations partially retained both enzymatic activities. Sialic acid levels in sera from DMRV patients were normal. Sialic acids are predominantly produced in the liver and transferred to synthesized glycoproteins. The sialylated proteins are released into the blood plasma, and free sialic acid in the plasma is derived from desialylation of these glycoproteins. GNE is expressed in the liver in large amounts; therefore, the reduction in enzymatic activities by mutations may not significantly affect the synthesis of sialic acid in the livers of DMRV patients, and sialic acids are present at concentrations comparable with normal blood levels. In contrast, in DMRV skeletal muscles, the sialic acid contents are reduced. The reduced enzymatic activities along with weak expression of GNE protein are probably responsible for the more serious reduction in sialic acid synthesis in muscle tissue compared with plasma. Lectin staining showed abnormal staining only in some fibers, indicating that a restricted number of myofibers has glycosylation abnormalities. This selective involvement may be due to muscle uptake of sialic acid, which can compensate for the defect of sialic acid synthesis in most fibers and explains why patients are normal at birth and develop late onset myopathies.
By feeding DMRV myotubes and fibroblasts with NeuAc as well as ManNAc, sialic acid concentrations in the cells increased to normal levels. As reported previously (21), treatment with NeuAc resulted in more rapid and potent effects on the restoration of sialylation than treatment with ManNAc. This strongly suggests that pharmacological therapy may be effective against DMRV/HIBM. Interestingly, even in myotubes harboring mutations that severely decrease ManNAc kinase activity, sialylation was restored by treatment with ManNAc. Schwarzkopf et al. (10) also reported similar observations in the embryonic stem cell culture in which the GNE gene was disrupted. They suggested that another sugar kinase may convert ManNAc to ManNAc-6-phosphate in those cells; therefore, ManNAc kinase activity of GNE may not be essential for sialic acid synthesis. If so, then why do the GNE mutations retaining UDP-GlcNAc 2-epimerase activity cause loss of sialylation and disease? One possible explanation is that these mutations may destabilize the GNE molecule resulting in decreased amounts of mutated proteins. However, we did not detect any reductions in the expressed amounts or defects in oligomerization of ManNAc-mutated recombinant proteins. Further analysis is necessary to clarify the mechanisms for the rescue resulting from the addition of ManNAc.
Enhanced staining with SBA lectin was observed in the sarcolemma and within the cytoplasmic area of some myofibers. These fibers were clustered and tended to be atrophic or have rimmed vacuoles. There is a report describing negative staining with SBA in Duchenne and Becker muscular dystrophies (22), suggesting that it is probably not because of dystrophic changes of myofibers but rather because of the lack of sialic acids. This abnormal glycosylation apparently preceded the formation of rimmed vacuoles, which is a pathological hallmark of DMRV. These rimmed vacuoles were electron-microscopically recognized as focal accumulations of autophagic vacuoles, which sometimes surround degenerated myofibrils and amyloid deposits. However, it is unknown whether the focal accumulations of autophagic vacuoles are the cause or result of the degeneration of myofibrils and amyloid deposits. Hypo-sialylation and abnormal glycosylation could cause the misfolding of some glycoproteins, and thus these misfolded glycoproteins may be targets of autophagic degradation and also behave as cores for formation of amyloid deposits. In our study, dystroglycan and SSA lectin staining was variable among patients. One possible explanation is that sialylation may not be the direct cause of the disease. For example, the loss of GNE enzymatic activity may induce the accumulation of the substrate, UDP-GlcNAc, leading to the abnormal O-GlcNAc modification of various proteins in the cells (23). Nevertheless, this possibility may also be unlikely because the overexpression of O-GlcNAc transferase did not cause any morphological abnormality in skeletal muscles in mice (24). In the next step, further analyses using animal models, as well as further testing of therapy with ManNAc or NeuAc, will be necessary to clarify the pathomechanism of DMRV and HIBM and the pathway from hyposialylation to rimmed vacuole formation and muscle atrophy.
| FOOTNOTES |
|---|
¶ To whom correspondence should be addressed. Tel.: 81-423412712; Fax: 81-423461742; E-mail: noguchi{at}ncnp.go.jp.
1 The abbreviations used are: DMRV, distal myopathy with rimmed vacuoles; HIBM, hereditary inclusion body myopathy; MAM, Maackia amurensis lectin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimido ester; SSA, Sambucus sieboldiana agglutinin; SBA, soybean agglutinin; GNE, UDP-GlcNAc 2-epimerase/ManNAc kinase; WGA, wheat germ agglutinin. ![]()
| ACKNOWLEDGMENTS |
|---|
| REFERENCES |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
M. C. V. Malicdan, S. Noguchi, I. Nonaka, Y. K. Hayashi, and I. Nishino A Gne knockout mouse expressing human GNE D176V mutation develops features similar to distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy Hum. Mol. Genet., November 15, 2007; 16(22): 2669 - 2682. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. C. V. Malicdan, S. Noguchi, I. Nonaka, Y. K. Hayashi, and I. Nishino A Gne knockout mouse expressing human V572L mutation develops features similar to distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy Hum. Mol. Genet., January 15, 2007; 16(2): 115 - 128. [Abstract] [Full Text] [PDF] |
||||
![]() |
Z. Wang, Z. Sun, A. V. Li, and K. J. Yarema Roles for UDP-GlcNAc 2-Epimerase/ManNAc 6-Kinase outside of Sialic Acid Biosynthesis: MODULATION OF SIALYLTRANSFERASE AND BiP EXPRESSION, GM3 AND GD3 BIOSYNTHESIS, PROLIFERATION, AND APOPTOSIS, AND ERK1/2 PHOSPHORYLATION J. Biol. Chem., September 15, 2006; 281(37): 27016 - 27028. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. Ricci, A. Broccolini, T. Gidaro, R. Morosetti, C. Gliubizzi, R. Frusciante, G. M. Di Lella, P. A. Tonali, and M. Mirabella NCAM is hyposialylated in hereditary inclusion body myopathy due to GNE mutations Neurology, March 14, 2006; 66(5): 755 - 758. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. E. Sparks, C. Ciccone, M. Lalor, E. Orvisky, R. Klootwijk, P. J. Savelkoul, M. C. Dalakas, D. M. Krasnewich, W. A. Gahl, and M. Huizing Use of a cell-free system to determine UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase activities in human hereditary inclusion body myopathy Glycobiology, November 1, 2005; 15(11): 1102 - 1110. [Abstract] [Full Text] [PDF] |
||||
![]() |
L-S Ro, G-J Lee-Chen, Y-R Wu, M Lee, P-Y Hsu, and C-M Chen Phenotypic variability in a Chinese family with rimmed vacuolar distal myopathy J. Neurol. Neurosurg. Psychiatry, May 1, 2005; 76(5): 752 - 755. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Tajima, E. Uyama, S. Go, C. Sato, N. Tao, M. Kotani, H. Hino, A. Suzuki, Y. Sanai, K. Kitajima, et al. Distal Myopathy with Rimmed Vacuoles: Impaired O-Glycan Formation in Muscular Glycoproteins Am. J. Pathol., April 1, 2005; 166(4): 1121 - 1130. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |