Scholarly article on topic 'Glycobiology of neuromuscular disorders'

Glycobiology of neuromuscular disorders Academic research paper on "Biological sciences"

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Academic research paper on topic "Glycobiology of neuromuscular disorders"

Glyeobiology vol. 13 no. 8 pp. 67R-75R, 2003 DOI: 10.1093/glycob/cwg077


Glyeobiology of neuromuscular disorders

Paul T. Martin1'2 and Hudson H. Freeze3

2Department of Neuroscience, Glyeobiology Research and Training Center, University of California, San Diego, School of Medicine, La Jolla, CA 92093-0691, and 3Glycobiology and Carbohydrate Chemistry Program, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037

Accepted on April 24, 2003

There has been a recent explosion in the identification of neuromuscular diseases caused by mutations in genes that affect carbohydrate metabolism or protein glycosylation. A number of these findings relate to defects in the glycosylation of a dystroglycan. a Dystroglycan is an essential component of the dystrophin-glycoprotein complex' and aberrant glycosylation of a dystroglycan is associated with multiple forms of muscular dystrophy in mice and humans. We review the evidence that defects in dystroglycan glycosylation cause muscular dystrophy. In addition' we review evidence that glycobiology is important in other disorders that affect muscle' including hereditary inclusion body myopathy type II and congenital disorders of glycosylation. Finally' we discuss the long-term potential of glycotherapies for muscle disorders.

Key words: congenital disorders of glycosylation/ dystroglycan/laminin/muscular dystrophy/skeletal muscle


The biochemical evidence that glycosylation is important for mediating dystroglycan's interactions with the muscle extracellular matrix has been confirmed by the recent identification of forms of congenital muscular dystrophy in which the aberrant glycosylation of dystroglycan is the primary molecular correlate (Figure 1; Table I). These include the muscle-eye-brain disease (MEB), which can be caused by mutations in POMGnTl and Walker-Warburg syndrome (WWS), which can be caused by mutations in POMT1. In addition, two other genes, fukutin and fukutin-related protein, cause aberrant glycosylation of dystroglycan in humans, and one gene, LARGE, does so in the mouse. Although the evidence for altered glycosyla-tion of dystroglycan hinges entirely on antibody binding, the fact that these disorders can arise from mutations in enzymes involved in the synthesis the O-linked glycans on a

'To whom correspondence should be addressed; e-mail:

dystroglycan strongly suggests that these are all congenital disorders of glycosylation.

Alterations in a dystroglycan glycosylation in congenital muscular dystrophies

A wise computational biologist once said that you can't make a model that fits all of the data because not all of the data are correct. So it is with studies that have attempted to define the glycosylation state of a dystroglycan in congenital muscular dystrophies. Most of this is not due to poor scientific technique but to a paucity of good reagents that can identify the various forms of the protein. The loss of dystroglycan glycosylation in these disorders is often entirely defined by the absence of staining and/or blotting

Posited Known Unknown ♦

«2,3 G1,4

| POMGnTl B1,2

pomti q

Fig. 1. Summary of defects in a dystroglycan glycosylation. Mutations in any of five proteins can cause defects in the glycosylation of a dystroglycan, resulting in muscular dystrophy. These include POMGnT1, where mutations are known to inhibit its UDP-GlcNAc:ManaO p1,2 N-acetylglucosaminyltransferase activity, and POMT1, which is posited to be a protein-O-mannosyltransferase. Mutations in LARGE, fukutin, and fukutin-related protein all likely affect the same glycan structure, but the mechanism for their effect is unknown.

Table I. Congenital muscular dystrophies with alterations in the glycosylation of dystroglycan

Disease Gene defect Reference

FCMD Fukutin Kobayashi et al., 1998

MEB POMGnTl Yoshida et al., 2001

WWS POMT1 Beltran-Valero de Bernabe et al., 2002

MDC1C FKRP Brockington et al., 2001a

LGMD2I FKRP Brockington et al., 2001b

myd LARGE Grewal et al., 2001

LARGE Fukutin

Fukutin-Related Protein

Glycobiology vol. 13 no. 8 © Oxford University Press 2003; all rights reserved.

with one of two carbohydrate-dependent monoclonal antibodies (IIH6 and VIA4-1). The glycan structures required for binding by either of these antibodies is still unknown. Given that these antibodies are very specific for a dystro-glycan, they may require a multivalent presentation of gly-cans within the mucin region for binding. Alternatively, glycans could be required merely to induce a protein conformation that allows antibody binding via a peptide epi-tope or a peptide-glycan epitope. Regardless, the absence of antibody binding does not necessarily signify a complete absence of the glycan in question but could merely reflect a lowered density. In addition, lectin-enriched material is often analyzed in these experiments. This opens up the possibility that not all dystroglycan is being studied.

What is entirely clear is that enzymes that either directly or indirectly modify O-linked mannose structures on a dystroglycan are defective in a subset of these disorders and that glycosylation of a dystroglycan is perturbed and perhaps severely reduced. Given the recent data suggesting that O-mannosylation may be more common than previously thought (Chai et al., 1999), however, one should be careful before attributing all of the phenotypes in these diseases to dystroglycan directly.

Fukutin and Fukuyama-type congenital muscular dystrophy

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive muscular dystrophy that is most often seen in Japanese populations. In Japan, its incidence is roughly 1 per 10,000 births, a frequency equivalent to that for Duchenne muscular dystrophy (DMD) in the worldwide population. Most Japanese patients with the disease have a 3 kbp retrotransposonal insertion in the 3' noncoding region of the fukutin gene, and this correlates with a near absence of mRNA in lymphoblastic cells isolated from patients (Kobayashi et al., 1998). Point mutations can also cause the disease. The fukutin gene contains a single transmembrane domain. Introduction of green fluroescent protein (GFP)-fukutin fusion constructs into cells showed that fukutin had a different pattern of intracellular distribution in different cell types. In COS cells (Kobayashi et al., 1998) and Chinese hamster ovary cells (Esapa et al., 2002) fukutin-GFP or fukutin-myc constructs colocalize with Golgi markers, whereas it is present in secretory-type granules in C2 muscle cells (Kobayashi et al., 1998). Thus the precise subcellular localization of the fukutin protein in muscle is unclear. Fukutin mRNA is ubiquitously expressed but is concentrated in skeletal muscle, heart, and brain (Kobayashi et al., 1998). Patients have severe muscular dystrophy and severe central nervous system (CNS) pathology, including cortical dysplasia (Fukuyama et al., 1984). The basal lamina in skeletal muscle is disrupted, as is the glia limitans-basal lamina complex in the brain.

Hayashi et al. (2001) first showed that patients with this disorder have reduced expression of normally glycosylated a dystroglycan using the VIA4-1 antibody. Expression of dystrophin and p dystroglycan, however, were normal. Laminin a2 expression was reduced somewhat, but it was still present in the basal lamina. This makes FCMD quite different than merosin-dependent congenital muscular

dystrophy, which results from mutations in the laminin a2 gene (LAMA2). In patients with LAMA2 mutations, lami-nin a2 protein is not expressed in the basal lamina surrounding muscle fibers. Neuromuscular junctions also appeared normal in FCMD muscle biopsies (Hayashi et al., 2001), though these were only analyzed in cross-section, which would not provide much information about defects in synaptic structure. VIA4-1 expression was also almost completely absent in heart, whereas expression in the brain appeared normal. Immunoblots of whole cell or wheat germ agglutinin-enriched cell lysates using VIA4-1 failed to show binding of the antibody to a dystroglycan in skeletal muscle and heart, but a protein of normal molecular weight was identified in brain. The finding of a normally expressed a dystroglycan in brain is inconsistent with the severe brain pathology in these patients.

Michele et al. (2002) significantly extended these findings in three ways. First, they showed that IIH6, a second carbohydrate-dependent antibody, was also reduced or absent in FCMD muscle. Second, they showed that a polyclonal antiserum against the a dystroglycan polypeptide recognizes a dystroglycan in FCMD muscle. a Dystroglycan migrated at a molecular weight that was reduced by about 50 kDa with respect to normal muscle. This suggested that a major deficit in a dystroglycan glycosylation had occurred but that a dystroglycan was still expressed. Indeed, immuno-staining with this same antiserum suggested that a dystroglycan protein was present along the sarcolemmal membrane. Third, laminin, agrin, and neurexin binding to a dystroglycan was severely reduced in FCMD muscle. Thus aberrant glycosylation of a dystroglycan correlates with reduced (almost absent) ligand binding. Reduced lami-nin binding may account for the secondary changes in laminin a2 protein expression in FCMD muscle. At least some of the brain pathology in FCMD patients can be mimicked by mice lacking a dystroglycan specifically in the nervous system (Moore et al., 2002), again suggesting that aberrant glycosylation of a dystroglycan is likely to be responsible for muscle and brain pathology.

How defects in fukutin alter a dystroglycan glycosylation is not known. Aravind and Koonin (1999) have suggested that fukutin could be involved in phosphoryl-sugar attachment to cell surfaces. This hypothesis is based on amino acid homology between fukutin and bacterial proteins involved in polysaccharide/phosphorylcholine modifications and yeast proteins involved in mannosyl phos-phorylation of oligosaccharides. All of these proteins have an aligned DxD motif with several evenly spaced amino acids preceding it. This sequence homology is extremely minimal, however, with the DxD motif being the primary structural similarity. Thus this idea is highly speculative but would be consistent with the glycosylation defects seen. FCMD patients have abnormal ganglioside composition (Izumi et al., 1995), which might also support such a concept. However, the rather dramatic CNS changes that occur in FCMD patients could also alter ganglioside composition.


MEB disease is a severe autosomal recessive disorder characterized by congenital muscular dystrophy, ocular

abnormalities, and brain malformation (Cormand et al., 2001). Patients present with hypotonia, severe myopia, and mental retardation. Serum creatine kinase levels are highly elevated, electromyographic recordings are myopathic, and muscle biopsies show evidence of muscular dystrophy. Brain pathology includes type II lissencephaly, with an almost complete disorganization of the lamination of both the cerebellar and cerebral cortices. This likely reflects an abnormality in the migration of neurons that populate the cortex, which normally form six layers of cells in a very stereotyped pattern. MEB brains have coarse gyri or folds with abnormally nodular surfaces. This pattern is typically referred to as a "cobblestone cortex." Ocular manifestations include a pronounced glial preretinal membrane, glaucoma, pallor of the optic disks, and retinal hypoplasia.

Yoshida et al. (2001) identified mutations in the human POMGnT1 gene, which encodes a UDP-GlcNAc:Mana-O p1,2-N-acetylglucosaminyltransferase, as a cause of MEB. Point mutations can lead to the production of mutant protein, but it is almost completely devoid of normal enzymatic activity. As with fukutin, POMGnT1 is expressed ubiquitously, despite the preponderance of muscle and nervous system abnormalities in MEB patients. POMGnT1 is only expressed in mammals and is more closely related to the GnT1 (UDP-GlcNAc:a3-D-mannoside p1,2-N-acetylglucosaminidase I) gene of Caenorhabditis elegans than it is to the human GnT1 gene. Yoshida et al. (2001) argue that this must mean that the POMGnT1 gene, which has a far more complex genomic structure than human GnT1 (22 exons versus 1 exon), must have arisen via a gene duplication long ago in evolution.

There is very good evidence that POMGnT1 possesses a UDP-GlcNAc; Mana-O p1,2GlcNAc transferase activity (Zhang et al., 2002; Yoshida et al., 2001), but it remains to be seen just how specific the enzyme really is. This is especially true in light of the fact that no studies have been done on changes in N-linked glycans in any of these disorders. A soluble recombinant form of the POMGnT1 protein expressed in HEK293T cells has activity against a Mana-O-Thr linked peptide but not against mannose or parani-trophenyl-a-D-mannosylpyranoside (Yoshida et al., 2001). The Km for the mannosyl-peptide was 1.85 mM and 0.73 mM for UDP-GlcNAc. Yoshida et al. (2001) went on to show that the same enzyme had no activity against glycoside substrates typical of N-linked glycans, including Man(a1,6)-[Man(a1,3)]Man(a1,6)[Man(a1,3)]Manp1,4GlcNAcp1, 4GlcNAc-pyridylaminate (M5-PA) and Man(a1,6)-[Man(a1,3)]Manp 1,4GlcNAcp 1,4GlcNAc-pyridylaminate (M3-PA). Thus POMGnT1 appears to be specific for O-linked mannose. This is slightly contradicted in the study by Schachter and colleagues (Zhang et al., 2002), who showed that a recombinant human POMGnT1 protein produced in baculovirus sf9 cells had some activity against substrates typical of N-linked glycans when higher concentrations of substrates were used. POMGnT1 could convert Man(a1,6)[Mana1-3](Man(p1-)O-octyl (M3-octyl) to Man(a 1,6)[GlcNAcp 1,2Mana 1,3)](Manp 1)-O-octyl (GnM3-octyl), for example. The enzyme also had activity against GnM3-octyl and Man(a1,3)Man(p1-)-octyl (M2-octyl). POMGnT1 (called GnT1.2 in this study) also had activity against O-linked mannose substrates, including Man(a1)O-benzyl and a Mana-O-Thr-linked peptide.

The mannose-linked peptide had the highest activity in both studies, and this likely reflects its true specificity in vivo.

As with FCMD, Michele et al. (2002) have shown that muscles from MEB patients do not express the IIH6 or VIA4-1 antigens. Immunoblots and immunostaining using antibodies against a dystroglycan polypeptide, however, show that a dystroglycan protein is still produced and is present along the muscle membrane. This is in contrast to the statement in Kano et al. (2002) that there is no a dystroglycan expressed in MEB tissue. In that study, however, no data were provided to support their contention. Because a dystroglycan is half glycan by molecular weight, use of antipeptide antibodies is fraught with danger, as there are few peptide epitopes that are not masked by glycosylation. Thus one is inclined to believe the studies where such antipeptide antibodies were actually shown to work. In MEB, the molecular weight of a dystroglycan is reduced by ~50 kDa, an amount similar to that observed in FCMD muscle, suggesting a severe loss of glycosylation (Michele et al., 2002). It is odd, however, that the same change in molecular weight would occur in FCMD and MEB, as these disorders are caused by defects in different proteins. It is possible that some of the loss in protein weight has resulted from proteolytic cleavage. This could unmask particularly good antipeptide antigens, allowing a strong signal on immunoblots of this type. As with FCMD, a dystroglycan from MEB muscle shows little or no binding to laminin, agrin, or neurexins in gel overlays or to laminin-1 in solid-state binding assays. These data strongly suggest that the lack of POMGnT1 activity leads to loss of O-linked glycosylation on a dystroglycan and that this in turn abrogates ligand binding and causes muscular dystrophy.


WWS is a disorder with properties similar to FCMD and MEB. Patients with WWS have muscular dystrophy, ocular abnormalities, type II lissencephaly, and cortical dysplasia. It is one of the most severe of the congenital muscular dystrophies, with most patients living only a year or less (Cormand et al., 2001). WWS and MEB patients both have ocular abnormalites, which are missing or are muted in FCMD. In some WWS patients, the corpus collosum has been reported to be absent.

Recently, Beltran-Valero de Bernabe et al. (2002) have identified mutations in the gene encoding protein O-man-nosyltransferase I (POMT1) in 6 of 30 unrelated WWS cases. Based on homology with the seven yeast O-manno-syltransferases, four human candidate O-mannosyltrans-ferases have been identified in the human genome, POMT1, POMT2, SDF2, and SDF2L1 (Hamada et al., 1996;, Jurado et al., 1999; Fukuda et al., 2001). Only POMT1 and POMT2 have a putatitive catalytic domain. Therefore there are likely to be only two O-mannosyltrans-ferases in humans. None of the thirty cases studied had mutations in POMT2. Because both POMT1 and POMT2 are highly expressed in skeletal muscle (Jurado et al., 1999), this suggests that the POMT2 cannot compensate for the loss of POMT1. As with MEB and FCMD, a carbohydrate-dependent antibody to a dystroglycan (VIA4-1) did not stain skeletal muscle from WWS patients. An antipeptide

antisera to a dystroglycan was also reported to be severely reduced, whereas p dystroglycan and laminin a2 were still expressed. Unfortunately, no immunoblot was provided to support the notion that a dystroglycan is absent and not just aberrantly glycosylated. The discovery of POMT1 mutations in WWS again links defects in O-linked mannose on a dystroglycan in congenital muscular dystrophies. The fact that cases exist in which POMT1 and POMT2 are not mutated suggests that other as yet unidentified genes are also essential for this type of glycosylation to occur.

Large and the myodystrophy mouse

The myodystrophy (myd) mouse is a muscular dystrophy model for which there is currently no known human equivalent. These mice have abnormal gait and posture, elevated serum creatine kinase levels, myopathy indicative of muscular dystrophy, and sensorineural deafness (Lane et al., 1976; Mathews et al., 1995). Myd mice also have defects in cortical and cerebellar lamination that are likely to result from defects in neuronal migration, defects in retinal transmission (abnomal b-wave electroencephalograms), and dystrophic pathology in the heart (Michele et al., 2002; Holzfeind et al., 2002). Thus myd mice phenotypically mimic many aspects of MEB disease in humans.

Grewel et al. (2001) showed that the phenotype of this mouse is due to an intragenic deletion of exons 4-7 in the LARGE gene, which causes a frame shift mutation resulting in premature termination of the protein coding sequence. The coding sequence for LARGE suggests that it is a glycosyltransferase. The LARGE gene encodes a protein with a transmembrane domain followed by a coiled-coil domain and two DxD-containing catalytic domains. LARGE protein has a 22% identity in catalytic domain 1 to WaaJ, an Escherichia coli protein in the family of putative a-glycosyltransferases (Heinrichs et al., 1998) and a 28% identify in catalytic domain 2 to human UDP-GlcNAc:Galp1,3-N-acetylglucosaminyltransferase (iGnT) (Sasaki et al., 1997), which is required for synthesis of poly-N-acetyllactosamine backbone (Galp1,4GlcNAcp1,3)n in the erythrocyte i antigen. Thus the myd phenotype could be due to loss of this tandem glycosyltransferase activity, but these activities have yet to be defined. Given that there are bisubstituted O-linked mannose chains in the brain (Chai et al., 1999), one possibility would be that LARGE is a UDP-GlcNAc:Mana-O p1,6-N-acetylglucosaminyl-transferase capable of synthesizing the GlcNAcp1, 6(GlcNAcp1,2)Mana-Ser/Thr moiety. It is important to point out that a partial protein containing the transmembrane and coiled-coil regions of LARGE protein could still be expressed in myd mice, and this could also have a dominant negative effect on other glycosyltransferases. Analysis of mice with a complete deletion of the LARGE gene should clarify this scenario.

As in FCMD and MEB, Grewal et al. (2001) reported that the myd mouse has no expression of the VIA4-1 antigen on a dystroglycan, whereas p dystroglycan and sarco-glycans were expressed (and may be elevated) (Holzfeind et al., 2002). Grewal et al. (2001) report that a polyclonal antiserum to a dystroglycan blots a protein of the normal molecular weight in myd skeletal muscle. This data, coupled

with the absence of VIA4-1 expression, suggested that a dystroglycan was glycosylated but that this glycosylation was not of the correct type. In contrast, Michele et al. (2002) show that an antipeptide antiserum recognizes an a dystro-glycan protein of reduced molecular weight in myd muscle, suggesting that a severe loss of glycosylation has occurred. They also report the loss of carbohydrate-dependent antibody staining and blotting, which is in agreement between the two studies. Thus these studies agree in their demonstration that carbohydrate-dependent antibodies do not bind to a dystroglycan isolated from myd muscle, but they disagree on the extent to which a dystroglycan glycosylated or not. Again, these disparities likely result from difficulties in developing antipeptide antibodies against the densely gly-cosylated a dystroglycan protein.

Myd mice show brain defects that are analogous to those seen in FCMD and MEB patients (Michele et al., 2002; Holzfeind et al., 2002), and these also mimic the defects seen in mice lacking dystroglycan in the brain (Moore et al., 2002). Myd mice appear to have abnormal neuronal migration in the cerebral cortex, hippocampus, and cerebellum. The cortical layering is very disorganized and is reminiscent of type II lissencephalic changes in FCMD and MEB. Laminin a2 expression is reported to be normal in myd muscle but is mislocalized in the cortex and in the glial limiting membranes (Michele et al., 2002). Thus myd mice appear to mimic many of the brain as well as muscle pathologies seen in FCMD and MEB patients, even though the gene involved is not the same.

Fukutin-related protein and congenital muscular dystrophy MDC1C and limb girdle muscular dystrophy 21

The fukutin-related protein (FKRP) is mutated in congenital muscular dystrophy MDC1C, a severe muscular dystrophy characterized by early onset, inability to achieve independent ambulation, muscle hypertrophy, and highly elevated serum creatine kinase levels (Brockington et al., 2001a). Unlike FCMD, there is no apparent brain involvement. Mutations in FKRP also cause Limb-Girdle muscular dystrophy (LGMD) 2I, which has a later onset (Brockington et al., 2001b). The mutations in FKRP that cause LGMDI do not appear to overlap with those that cause MDC1C. At the severe end of the spectrum, LGMD2I patients have a progression akin to DMD, with loss of ambulation in the teens, whereas the course of progression in other patients is far milder.

The FKRP gene was identified based on its sequence homology with fukutin (Brockington et al., 2001a). Both share a DxD motif suggestive of a glycosyltransferase. FKRP, like fukutin, has a predicted transmembrane region and is expressed in most tissues but is highly concentrated in skeletal muscle. Esapa et al. (2002) have shown localization of FKRP in transfected cells to the medial Golgi. Cotrans-fection of FKRP with dystroglycan altered dystroglycan processing in such cells. Moreover, expression of FKRP containing a mutation found in MDC1C caused incorrect targeting of the protein; this correlated with a failure to alter dystroglycan migration on sodium dodecyl sulfate (SDS) gels. The suggestion of such experiments is that mutations in FKRP inhibit its targeting to the Golgi and that therefore

it no longer can modify dystroglycan. It remains to be seen if any of these results would occur in an actual muscle. Muscles from MDC1C patients have binding of the VIA4-1 antibody, but a dystroglycan migration is reduced on SDS-polyacrylamide gel electrophoresis gels. p Dystro-glycan and a sarcoglycan are still expressed, as is laminin a2, though laminin a2 levels appear reduced by both immunostaining and immunoblotting (Brockington et al., 2001a,b).

It should be noted that despite the fact that these genes have similar names, the differences between the diseases caused by fukutin and FKRP, at both the clinical and molecular levels, are quite significant. Clinically, fukutin defects are associated with severe brain pathology and mental deficits, whereas FKRP mutations cause neither of these, at least in some cases. Although no immunblot data exists showing a dystroglycan migration in the brains of FKRP-mutated individuals, one assumes, based on the lack of brain pathology, that it is normal. Rather, FKRP mutations appear to make muscle dystroglycan migrate at a molecular weight more consistent with that of the brain form. It is tempting to speculate that FKRP is responsible for modifications that are specific to muscle and heart and that the lack of such deficits in brain is due to the fact that brain dystroglycan is not affected in these patients.

Hereditary inclusion body myopathy: a disease of sialic acid biology?

There are four loci identified for hereditary inclusion body myopathy (HIBM). There are three autosomal dominant forms (IBM1, IBM3, and a new as yet unnamed form) and an autosomal recessive form (IBM2) (for a review, see Askanas and Engel, 2001; Oldfors and Lindberg, 1999). IBM3 is associated with joint contractures and ophthalmoplegia in addition to myopathy and has been mapped to 17p13.1, a region with a myosin heavy chain cluster. Some individuals have missense mutations in the motor domain of MYHC2A, a major myosin isoform in abnormal muscles. IBM2 has been mapped to 9p13-p12, and recently the mutations in the GNE gene, which encodes the UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase, have been shown to be the cause of this adult-onset disorder (Eisenberg et al., 2001). Another autosomal dominant IBM that is associated with Paget disease of the bone and fron-totemporal dementia has also been mapped to 9p13.3-p12.

UDP-GlcNAc 2-epimerase/ManNAc-6 kinase is a bifunc-tional enzyme that catalyzes the first two steps in the biosynthesis of sialic acids (Hinderlich et al., 1997). Site-directed mutagenesis studies have shown that the two enzymatic functions of this protein are carried out by spatially segregated domains (Effertz et al., 1999). The N-terminal domain contains the epimerase activity, and the C-terminal region encodes the kinase activity. Dominant missense mutations in the epimerase domain cause sialuria (Seppala et al., 1999). These mutations cause a loss of product inhibition of the enzyme, causing constitutive overexpression of sialic acid. Mutations associated with IBM2 occur in both the epimerase and the kinase domain (Eisenberg et al., 2001). These mutations do not overlap with those causing

sialuria, and IBM2 patients have no increased sialic acid in their urine. Eisenberg et al. (2001) suggest that the relatively broad distribution of mutations in GNE associated with IBM2 is consistent with a loss of function. A recent abstract (Huizing et al., 2002) showed a substantial decrease in epimerase activity in fibroblasts (30% of normal) and muscle (10% of normal) from a patient with one mutation in the epimerase domain and one mutation in the kinase domain. Another abstract reported that an IBM2 patient with a homozygous mutation in the epimerase domain had only slightly decreased (down 20%) activity in fibroblasts (Hinderlich et al., 2002). Clearly, the relationship of residual enzyme activity to the development of this disease is not yet resolved. In fact, loss of GNE in mice results in embryonic lethality (Schwarzkopf et al., 2002). Therefore, if mutations in GNE affect enzyme activity, they would most likely reduce activity rather than abrogate it. Until a detailed analysis of the carbohydrate composition of proteins and lipids is done in IBM2 patients, we will not know whether this disorder is due to changes in sialic acid biology; however, given that mutations in GNE cause the disease, it is likely that lack of appropriate glycosylation has something to do with the phenotype.

Congenital disorders of glycosylation and myopathies

Fourteen types of congenital disorders of glycosylation (CDGs) have been defined (for a review, see Freeze, 2001). Ten of these (type I), affect the biosynthesis and transfer of the Dol-PP-oligosaccharide to proteins. Five type I disorders (CDG-Ic, -Id, -Ih, -Ii, and -Ij) are specific to the N-linked pathway, and the other five impair the synthesis of precursor molecules for multiple glycosylation pathways. Type II CDGs affect remodeling or extension of protein-bound oligosaccharides. Two of these (CDG-IIa and -IIb) are specific to the N-linked pathway, and CDG-IIc and -IId would affect multiple pathways.

Almost all CDGs are associated with hypotonia. The prevalence of hypotonia in most of these different types makes it unlikely that there is just one underlying cause, but defects in muscle could be a contributing factor. Nevertheless, no studies have been done of muscle glycosylation in CDG patients. These patients seldom have muscle biopsies performed because CDG diagnosis is based on detecting abnormal glycosylation of a serum protein, such as transferrin. Other pathologies lead to many hospitalizations and sometimes become life-threatening. Parents are often reluctant to consent to an invasive muscle biopsy. Studying muscle glycosylation is an obvious direction for future work in CDG.

The O-mannose based oligosaccharides of a dystroglycan probably use Dol-P-Man as a donor substrate. CDG-Ie is caused by mutations in DPMI, the catalytic subunit of Dol-P-Man synthase, and the patients have very severe hypotonia. CDG-If is caused by defects in MPDU1, which is thought to enable efficient transfer of Man from Dol-P-Man to both glycoprotein and glycolipid molecules. CDG-Ia patients are defective in synthesis of Man-1-P from Man-6-P and have lower amounts of GDP-Man, the donor for Dol-P-Man synthesis. All of these could reduce

the amount of Dol-P-Man transferred to a dystroglycan. CDG-Ib patients, who are deficient in PMI (Fruc-6-P ^ Man-6-P) are not hypotonic, perhaps because they salvage small amounts of mannose from exogenous sources. The hypotonia seen in CDGs whose defects are specific to mutations in the N-linked pathway suggest that the absence of entire sugar chains may also cause hypotonia. However, it is difficult to determine whether the effect is due to the absence of entire chains or critical terminal sugars they support.

Absence of entire N-linked sugar chains would also reduce sialylation of the voltage-gated sodium channel. Voltage-gated sodium channels in skeletal muscle are heavily glycosylated, with glycans contributing between 20-40% of their molecular weight. This is true of the large a subunit but is also true for the smaller p subunits (Messner and Catterall, 1985). Neuraminidase treatment of the eel elec-troplax sodium channel reduces its molecular weight from 290 to 220 kDa (Recio-Pinto et al., 1990). Treatment of the rat sodium a chain with neuraminidase and Endo F or with TFMS gives a similar reduction in molecular weight (Messner and Catterall, 1985). Sialic acid can contribute to the conductance properties of these channels. Neuramini-dase treatment of eel electroplax or rat skeletal muscle sodium channels causes a large shift in steady-state activation, shifting the midpoint by 31 mV toward depolarizing potentials (from —71 mV to —40 mV) (Recio-Pinto et al., 1990). This means that a much higher depolarization of the membrane is needed for the channel to conduct sodium. Treatment of human cardiac or skeletal muscle sodium channels with glycosidases or enzyme inhibitors shifts the excitation properties in the same direction (Recio-Pinto et al., 1990; Fermini and Nathan, 1991; Bennett et al., 1997; Zhang et al., 1999). In these studies, there was also a small (+4 mV) but significant hyperpolarizing shift in steady-state inactivation. Thus neuraminidase made these channels less excitable and more readily inactivated. Both of these findings point toward a less electrically responsive myofiber, which could contribute to a hypotonic state in CDGs.

Potential glycotherapies for myopathies

The number of diseases of skeletal muscle that have a direct connection to defects in glycosylation has increased dramatically over the past several years. Given that this is the case, it does not seem overly optimistic to begin to discuss means by which glycobiologists might use their unique perspective and skills not only to help define the causes of these disorders but to discuss ways they might create novel glycotherapies to treat them. Such glycotherapies could involve monosaccharide replacement via diet, which has had success in some CDGs, delivery of synthetic glycans or glycoproteins that bypass defective biosynthetic steps, stimulation of glycosyltransferase activities or expression, or delivery of glycosyltransferase genes via cell or gene therapy techniques.

The cytotoxic T cell carbohydrate antigen and DMD

Recently, it has been shown that overexpression of the cytotoxic T cell (CT) GalNAc transferase (Galgt2) (Smith

and Lowe, 1994) in skeletal muscles of mdx mice can inhibit the formation of muscular dystrophy (Nguyen et al., 2002). Mdx mice are a model for DMD because they have a mutation that leads to loss of dystrophin protein expression in most myofibers. Like DMD patients, mdx mice have very reduced expression of many other members of the dystrophin-glycoprotein complex as well, including dystro-glycan and sarcoglycans. Utrophin, a dystrophin paralog, synaptic forms of laminin, and presumably synaptic forms of dystroglycan and sarcoglycans are still expressed at the neuromuscular junction in mdx muscle (Matsumura et al., 1992; Patton et al., 1999). These findings suggest that a synaptic scaffold composed of proteins not mutated in DMD exists at the neuromuscular junction and that this complex does not depend on dystrophin. Given the evidence that a dystroglycan binds laminin via O-linked glycans, it is likely that the synaptic form of a dystroglycan would be defined by a novel O-linked glycan linkage. The neuromuscular junction does contain uniquely synaptic glycans (Sanes and Cheney, 1982), and many of these contain terminal p-linked GalNAc (Martin et al., 1999).

Because utrophin was ectopically expressed in Galgt2 transgenic mice, Nguyen et al. (2002) crossed these animals into the mdx background to determine if such ectopic expression would inhibit muscular dystrophy in mdx animals. This was the case. No evidence of dystrophy was evident, even at 6 months of age, in Galgt2 transgenic mdx animals. This inhibition of dystrophy was as good as that previously observed by Davies and colleagues in transgenic mice made to overexpress utrophin (Deconinck et al., 1997; Tinsley et al., 1998). Thus although the mechanism for this effect remains to be deciphered, Galgt2 is a potential molecular target for the treatment of DMD.

Overexpression of a number of other molecules that are normally concentrated at the neuromuscular junction also have a beneficial effect in rodent models of muscular dystrophy. Utrophin overexpression can inhibit dystrophy in mdx mice (Deconinck et al., 1997; Tinsley et al., 1998), integrin a7 can ameliorate dystrophy in mdx/utrn-/- mice (Burkin et al., 2001), neuronal nitric oxide synthase can ameliorate dystrophy in mdx mice (Wehling et al., 2001), and an agrin minigene can inhibit dystrophy in dy mice (Moll et al., 2001). Altering synaptic glycosylation, which can affect the expression of multiple potential therapeutic molecules (Xia et al., 2002; Nguyen et al., 2002), may be the best means of exploiting these molecular targets. Indeed, Galgt2 overexpression increases the ectopic expression of not only utrophin but also synaptic forms of laminin. This suggests that Galgt2 may also be therapeutic in merosin-deficient congenital muscular dystrophy, in which the extrasynaptic form of laminin is absent.

Dystroglycan-based therapies in congenital muscular dystrophies

In congenital muscular dystrophies in which aberrant glycosylation of a dystroglycan is the primary molecular determinant, glycotherapy to replace a dystroglycan or its glycans are likely therapies. In MEB, FCMD, and WWS, it would seem that the logical therapy would be to simply replace the missing glycans causing the disease. Because a

dystroglycan is a peripheral membrane protein, one approach would be simply to inject native a dystroglycan. Because p dystroglycan is expressed at normal levels in these disorders, a dystroglycan may be able to bind when added back in trans. In addition, expression of dystroglycan in satellite cells can ameliorate the loss of its expression in myotubes and may be another means of delivering normal dystroglycan to muscle cells (Cohn et al., 2002). Another option would be to introduce glycan analogs of the NeuAca2,3Galp 1,4GlcNAcp 1,2Mana-O structure. These could be lipid-linked to allow incorporation into membranes. Such analogs could directly bind to laminin and replace the linkage normally provided by a dystroglycan. Analogs that also bind to p dystroglycan would be preferable, as this would reconstitute the linkage through the membrane. Analogs of carbohydrate metabolism may also be effective. For example, if POMT1 was able to incorporate GlcNAcp1,2Man-GDP onto proteins, one could add hydrophobic (perhaps peracetylated) versions of this structure to bypass defects in POMGnT1. Though it is unclear whether any of these approaches would be applicable to this entire group of disorders, roughly the same degree of change in a dystrolgycan glycosylation was reported for myd, MEB, and FCMD (Michele et al., 2002), suggesting a common molecular pathology.

Finally, gene therapy is another option. This technology would not be trivial to use in the brain, where many defects are found in these patients. A prerequisite to using such therapies would be an understanding of the degree to which the neuronal migration defects in the brain in these disorders is due to cell-autonomous phenomenon. If that is the case, such defects would be very difficult to cure with any therapy that involves introducing genes into cells. If, however, brain malformations result primarily from defects in glial adhesion to the vasculature, such an approach might ultimately be feasible using systemic delivery of viral vectors via the blood. Because most of these disorders involve defects in early brain development, a fetal test is paramount to the development and delivery of effective treatments.

Inclusion body myopathy and sialic acid

If the genetic forms of inclusion body myopathy (IBM2) or the immune-based inclusion body myositis are truly defects in sialic acid metabolism, they may be very amenable to monosaccharide therapy. This would come in one of two forms. First, delivery of derivatized ManNAc-6-phosphate could bypass the lack of ManNAc-6-P caused by mutations in the UDP-GlcNAc epimerase/N-acetylmannosamine kinase, much as can be done with cells in culture (Hinderlich et al, 2001). Patients with mutations in the epimerase domain could presumably consume ManNAc, as the kinase is expressed on a separate domain and may therefore still be active. The second method would be to consume NeuAc and have these be incorporated into glycoproteins and glycolipids via conversion to CMP-NeuAc in muscle cells. Given the robust nature of the salvage pathway for sialic acid (Oetke et al., 2001), we may be able to obtain adequate sialic acid from diet already; however, these disorders do not develop for several decades, so the consequences of

localized deficits in NeuAc may arise very slowly over a number of years.

The kind of sialic acid one eats may be just as important. For example, most species of mammals make large amounts of NeuGc in addition to NeuAc. Humans have lost the ability to synthesize NeuGc because of an Alu insertion in the CMP-NeuAc hydroxylase (Chou et al., 1998). Thus if sialic acid salvage pathways are highly up-regulated in IBM2 patients, which one would assume if they are making less of their own sialic acid, it is possible that a high proportion of sialic acid in IBM2 muscles is derived from diet. If so, IBM2 muscles may build up large amounts of NeuGc obtained from eating NeuGc-rich foods. Because protein adhesion to sialic acids can be mediated by the type of N-acyl linkage present (for a review, see Varki, 2001), such changes could have adverse consequences for IBM2 patients. For example, calcium homeostasis in muscle may be altered. GM3(NeuAc) increases calcium uptake by voltage-gated calcium channels in rabbit skeletal muscle, and GM3(NeuGc) inhibits calcium uptake (Muthing et al., 1998). Other sialic acids, such as KDN, may also be increased in IBM2. KDN, which lacks the N-acyl group, is synthesized from mannose-6 phosphate, which would still be made in the absence of GNE activity. An insufficient amount of NeuAc derived from UDP-GlcNAc may adversely shift the balance of different types of sialic acids and may lead to muscle degeneration in time.

Dol-P-Man-deficient patients may have hypoglycosylated a dystroglycan with insufficient O-mannose-based glycans. As such, it is possible that generating hyperphysiological levels of GDP-Man in cells might drive this inefficient reaction to increase glycosylation. Providing fucose to CDG-IIc patients with a defective GDP-fucose transporter appeared to elevate their GDP-Fuc precursor pool and normalize the synthesis of Sialyl Lewis x (Marquardt et al., 1999). This correction also occurred when FX-null mice, who cannot synthesize GDP-Fuc from GDP-Man, were given oral fucose (Smith et al., 2002). In the case of Dol-P-Man-deficient CDG patients, providing a hydrophobic cell-permeable derivative of Man-1-P with esterified hydroxyl groups and nontoxic acetoxymethyl phosphates might increase the GDP-Man pool. Preliminary experiments show that this does indeed occur (Ichikawa et al., unpublished data). This type of approach would be analogous to providing HIBM2 patients with an analogous hydrophobic version of ManNAc-6-P. There is little understanding of what controls the utilization or preference of salvaged versus de novo synthesized sugar precursors.


The recent demonstration that mutations in glycosyltrans-ferases cause forms of muscular dystrophy leaves no doubt that muscle integrity is dependent on glycosylation. The association of multiple disorders with defects in the glyco-sylation of a dystroglycan shows that this protein is central to the maintenance of muscle cell integrity as well as for proper brain development. Because of this, it is imperative

that the glycobiology of this complex protein be fully deciphered in the coming years. Because there are still patients with congenital muscular dystrophy for whom no candidate gene exists, more genes will be identified that are involved in this process, and this should help us gain a more complete understanding of this type of glycosylation. Glycobiologists have much to offer here. Understanding the function and regulation of the glycosyltransferases in the O-linked man-nose pathway will be essential to the design of diagnostic tests and therapies for these disorders.


This work was supported in part by grants from Advancement of Research for Myopathies to H.F. and from the Muscular Dystrophy Association to P.T.M.


CDGs, congenital disorders of glycosylation; CNS, central nervous system; CT, cytotoxic T cell; DMD, Duchenne muscular dystrophy; FCMD, Fukuyama-type congenital muscular dystrophy; FKRP, fukutin-related protein; GFP, green fluorescent protein; GNE, UDP-GlcNAc 2-epimer-ase/N-acetylmannosamine kinase; GnTl, UDP-GlcNA-c:a3-D-mannoside ßl,2N-acetylglucosaminidase; HIBM, hereditary inclusion body myopathy; LGMD, Limb-Girdle muscular dystrophy; mEb, muscle-eye-brain disease; myd, myodystrophy; POMGnTl, UDP-GlcNAc:Mana-0 ßl,2N-acetylglucosaminidase; POMTl, protein O-manno-syltransferase I; WWS, Walker-Warburg syndrome.


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