Tissue-specific Heterogeneity in alpha -Dystroglycan Sialoglycosylation
SKELETAL MUSCLE alpha -DYSTROGLYCAN IS A LATENT RECEPTOR FOR VICIA VILLOSA AGGLUTININ B4 MASKED BY SIALIC ACID MODIFICATION*

(Received for publication, February 3, 1997, and in revised form, May 20, 1997)

James M. Ervasti Dagger , Annie L. Burwell and Aimee L. Geissler

From the Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Because the polypeptide core of alpha -dystroglycan is encoded by a single gene, the difference in apparent molecular mass between alpha -dystroglycans expressed in various tissues is presumably due to differential glycosylation. However, little is presently known about the tissue-specific differences in alpha -dystroglycan glycosylation and whether these modifications may confer functional variability to alpha -dystroglycan. We recently observed that laminin-1 binding to skeletal muscle alpha -dystroglycan was dramatically inhibited by heparin, whereas the binding of commercial merosin to skeletal muscle alpha -dystroglycan was only marginally inhibited (Pall, E. A., Bolton, K. M., and Ervasti, J. M. (1996) J. Biol. Chem. 3817-3821). In contrast to 156-kDa skeletal muscle alpha -dystroglycan, both laminin-1 and merosin binding to 120-kDa brain alpha -dystroglycan were sensitive to heparin. We have now examined the laminin binding properties of 140-kDa alpha -dystroglycan purified from cardiac muscle and observed that like skeletal muscle alpha -dystroglycan, heparin inhibited cardiac alpha -dystroglycan binding to laminin-1, but not to merosin. On the other hand, cardiac and brain alpha -dystroglycans could be distinguished from skeletal muscle alpha -dystroglycan by their reactivity with the terminal GalNAc-specific lectin Vicia villosa agglutinin. Interestingly, skeletal muscle alpha -dystroglycan became reactive with V. villosa agglutinin upon digestion with sialidase from Clostridium perfringens, Arthrobacter neurofaciens, or Streptococcus, but not Vibrio cholerae or Newcastle disease virus sialidase. While none of the sialidase treatments affected the laminin binding properties of alpha -dystroglycan, the sum of our results suggests that skeletal muscle alpha -dystroglycan contains a novel sialic acid residue linked alpha 2-6 to GalNAc. These properties are also consistent with the cellular characteristics of a GalNAc-terminated glycoconjugate recently implicated in neuromuscular synaptogenesis. Thus, variations in alpha -dystroglycan sialoglycosylation may prove as useful markers to further elucidate the role of alpha -dystroglycan glycoforms in different tissues and perhaps within a single cell type.


INTRODUCTION

alpha -Dystroglycan is a membrane-associated, extracellular glycoprotein (1) originally identified as a subunit of the dystrophin-glycoprotein complex (2) that is missing or abnormal in a number of muscular dystrophies (3, 4). In skeletal muscle, the dystrophin-glycoprotein complex is distributed throughout the sarcolemmal membrane (5) and is believed to serve as a transmembrane link between the cortical cytoskeleton and the extracellular matrix, based on the interaction of dystrophin with F-actin (6) and the binding of alpha -dystroglycan to the laminin family of proteins (7, 8). While these data certainly support a structural role for the dystrophin-glycoprotein complex in maintaining sarcolemmal membrane integrity (9-12), alpha -dystroglycan has also been implicated in more dynamic developmental processes in other tissues (13, 14) and even in muscle cells. For example, a highly similar complex of proteins (including alpha -dystroglycan) is associated with utrophin (15), an autosomal homologue of dystrophin (16). In contrast to the dystrophin-glycoprotein complex, the utrophin-glycoprotein complex is specifically localized to the neuromuscular junction (15, 17), suggesting that it may play a role in neuromuscular synapse formation. In support of this possibility, alpha -dystroglycan co-clusters with acetylcholine receptors in a heterologous expression system (18). alpha -Dystroglycan also binds agrins (19-22), a group of widely expressed and alternatively spliced basal lamina proteins that can induce acetylcholine receptor aggregation at the motor endplate (23, 24). However, alpha -dystroglycan antibodies that block agrin binding in vitro have yielded equivocal results in experiments testing their ability to perturb the clustering activity of agrin in cell culture (20-22). Furthermore, truncated agrins that fail to bind alpha -dystroglycan retain acetylcholine receptor clustering activity (25-27). Finally, Yancopoulos and co-workers (28, 29) have recently demonstrated that agrin acts via a novel receptor tyrosine kinase complex. Thus, a specific role for alpha -dystroglycan in neuromuscular synapse formation remains to be elucidated.

Although alpha -dystroglycan is the product of a single gene (30), its apparent molecular mass varies dramatically in different tissues (7, 8, 19, 31, 32) presumably due to differences in glycosylation (7, 8), which is essential for its binding to laminin-1 (8) and agrin (20-22). Detailed carbohydrate analysis has been severely hampered by the exceedingly low abundance of alpha -dystroglycan in native tissues. None the less, characterization of alpha -dystroglycans purified from diverse tissues and species (1, 8, 19, 33-38) has identified many common structural features as well as potential tissue-specific differences in glycosylation (see Table I). Relevant to a putative role in neuromuscular synaptogenesis, neural forms of alpha -dystroglycan express terminal GalNAc residues (35, 36), whereas GalNAc-terminated glycoconjugate(s) have been implicated in acetylcholine receptor clustering (39-41). However, the GalNAc-terminated molecule(s) mediating acetylcholine receptor clustering are muscle cell-specific (40, 41), yet adult skeletal muscle and cultured myotubes apparently express alpha -dystroglycan lacking terminal GalNAc residues (33, 41). Of further concern is that all previously identified (35, 36) tissue-specific variations in alpha -dystroglycan glycosylation have relied solely on comparisons across species as well as tissues (see Table I). Since species-specific differences in glycosylation are prevalent in nature, but are not generally crucial to basic glycoprotein function (42), it is important to characterize the tissue-specific differences in alpha -dystroglycan glycosylation and to determine whether these modifications may confer functional variability to alpha -dystroglycan.

Table I. Characterization of alpha -dystroglycan glycosylation by lectin staining and glycosidase sensitivity


Lectin/enzyme Carbohydrate specificity Rabbit skeletal (1, 8)a Rabbit skeletal (33) Ovine brain (34, 35) Bovine sciatic nerve (36) Torpedo electric organ (19, 37)

ConAb Mannose + + + + +
PNA Galbeta 1-3GalNAc  + c  + c + + +
Jacalin Galbeta 1-3GalNAc + + + + ND
WGA GlcNAc,Neu5Ac + + + + +
MAA Neu5Acalpha 2-3Gal + ND + + ND
SNA Neu5Acalpha 2-6Gal  - ND +  - ND
Lotus Fucose  - ND  -  - ND
UEA-I Fucose  -  -  -  -  -
DBA Terminal GalNAc ND  - + ND +
VVA-B4 Terminal GalNAc ND ND + + ND
WFA GalNAc ND ND + ND ND
N-Glycosidase F N-Linked glycans + ND + + +
Neuraminidase Sialic acid + + + + +
O-Glycosidase O-Linked Galbeta 1-3GalNAc + ND + + +
Hepase HS  - ND  -  -  -
Nitrous acid HS  - ND  - ND ND
Chondroitinase CS  - ND  -  -  -
Keratanase KS  - ND  -  -  -

a References are given in parentheses.
b ConA, concanavalin A; MAA, M. amurensis agglutinin; UEA-I, Ulex europaeus agglutinin I; WFA, Wisteria floribunda agglutinin; HS, heparan sulfate; CS, chondroitin sulfate; KS, keratan sulfate; ND, not determined.
c PNA reactivity is evident only after neuraminidase digestion.

We recently observed (43) a significant difference in the heparin inhibition of skeletal muscle alpha -dystroglycan binding to laminin-1 versus merosin (a mixture of laminin-2 and -4), which may provide a basis for merosin-specific stabilization of cultured myotubes (44) and explain why laminin-1 up-regulation fails to compensate for merosin deficiency in some forms of muscular dystrophy (45, 46). Consistent with our findings with different laminins, alternatively spliced forms of agrin also bind alpha -dystroglycan in either a heparin-sensitive or -insensitive manner, dependent on the presence of a four-amino acid sequence at the A/Y splice site (27, 47). In contrast to our findings with 156-kDa skeletal muscle alpha -dystroglycan, we further observed that both laminin-1 and merosin binding to 120-kDa brain alpha -dystroglycan were sensitive to heparin (43), suggesting that tissue-specific differences in alpha -dystroglycan post-translational modification may influence its interactions with extracellular ligands. Cardiac muscle alpha -dystroglycan exhibits an electrophoretic mobility intermediate (140 kDa) to that of skeletal and brain alpha -dystroglycans (7, 8, 48), suggesting that it could exhibit laminin binding properties more similar to skeletal muscle alpha -dystroglycan, brain alpha -dystroglycan, or somewhere in between. Therefore, we have now examined the effect of heparin on the binding of purified cardiac muscle alpha -dystroglycan to laminin-1 and merosin. Our results suggest that cardiac and skeletal muscle alpha -dystroglycans are functionally related but distinct from nervous tissue alpha -dystroglycans with regard to merosin binding in the presence of heparin. However, we further demonstrate that cardiac muscle and brain alpha -dystroglycans appear to be similarly modified with terminal GalNAc residues, which are also present on skeletal muscle alpha -dystroglycan, but masked by a novel sialic acid modification. While sialic acid modification does not appear to be important for the laminin binding properties of alpha -dystroglycan, we discuss situations in which differences in sialoglycosylation may reflect alpha -dystroglycan glycoforms with unique or varied function, even in the same cell.


EXPERIMENTAL PROCEDURES

Purification of alpha -Dystroglycans

alpha -Dystroglycan was solubilized from rabbit skeletal muscle membranes (49) or cardiac membranes (7) by extraction with M urea and purified by sequential WGA1-Sepharose chromatography, DEAE-cellulose chromatography, and CsCl gradient centrifugation (43). alpha -Dystroglycan was also prepared from low ionic strength extracts of frozen rabbit brain, bovine brain, and rabbit sciatic nerve (Pel-Freez Biologicals) by laminin-1 affinity chromatography (31) followed by CsCl gradient centrifugation as described previously (43). Purified alpha -dystroglycans were dialyzed exhaustively against double distilled H2O and quantitated by A280 using E280 = 0.83 cm2/mg, calculated from the predicted amino acid sequence of alpha /beta -dystroglycan precursor (7) with the proteolytic cleavage site located between Gly-653 and Ser-654 (35).

SDS-Polyacrylamide Gel Electrophoresis and Blotting Assays

All samples were electrophoretically separated on 3-12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (1). Molecular mass standards were purchased from Sigma. alpha -Dystroglycan was identified on nitrocellulose membranes with 1 µg/ml specific polyclonal antibodies affinity-purified (1) from the yolk immunoglobulin fraction of White Leghorn chickens (50) immunized with the dystrophin-glycoprotein complex (51). Primary antibody labeling of alpha -dystroglycan was detected with a peroxidase-conjugated goat anti-chicken secondary antibody (Calbiochem) by chemiluminescence using SuperSignal CL-HRP (Pierce) as substrate.

To evaluate lectin binding to alpha -dystroglycan, nitrocellulose membranes were blocked for 1 h in phosphate-buffered saline (8 mM monobasic sodium phosphate, 42 mM dibasic sodium phosphate, pH 7.5, and 0.15 M NaCl) containing 0.05% Tween 20. Blocked membranes were incubated for 1 h in phosphate-buffered saline containing 0.05% Tween 20 and 1 µg/ml peroxidase-conjugated Vicia villosa agglutinin isolectin B4 (VVA-B4), Psophocarpus tetragonolobus agglutinin (PTA), Dolichos biflorus agglutinin (DBA), WGA, peanut agglutinin (PNA) (all from Sigma), or lima bean agglutinin (LBA) (EY Laboratories, San Mateo, CA). After two 10-min washes with phosphate-buffered saline, lectin staining was detected by chemiluminescence.

To assess laminin and merosin binding to alpha -dystroglycan, nitrocellulose membranes were blocked in phosphate-buffered saline containing 5% nonfat dry milk for 1 h at room temperature. Blocked membranes were rinsed briefly with TBS (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and incubated for 2 h at room temperature in TBS containing 3% bovine serum albumin, 1 mM CaCl2, 1 mM MgCl2, and 1 µg/ml native laminin (Upstate Biotechnology, Inc., Lake Placid, NY) or merosin (Life Technologies, Inc.) in the absence or presence of 1 mg/ml porcine mucosal heparin (Sigma). Laminin/merosin-binding proteins were detected with affinity-purified polyclonal laminin antibodies (Sigma) by chemiluminescence (43).

Solid-phase Binding Assay

A solid-phase microtiter assay using iodinated cardiac alpha -dystroglycan was performed as described previously (43). Briefly, Immulon 1 removable microtiter wells (Dynatech Laboratories Inc., Chantilly, VA) were coated with laminin or merosin in TBS; aspirated; and blocked with TBS containing 3% bovine serum albumin, 1 mM CaCl2, and 1 mM MgCl2. Triplicate wells were incubated for 2 h at room temperature with 1 nM 125I-labeled cardiac alpha -dystroglycan in 0.1 ml of TBS containing 3% bovine serum albumin, 1 mM CaCl2, and 1 mM MgCl2 in the absence or presence of the indicated concentration of heparin. After two brief washes with TBS containing 3% bovine serum albumin, 1 mM CaCl2, and 1 mM MgCl2, the wells were counted in a Packard 5650 gamma -counter.

Enzyme Treatments

Purified alpha -dystroglycans in 1% SDS were incubated at 100 °C for 5 min; diluted 10-fold into 0.5 M acetate, pH 5.5, and 1% Triton X-100; and incubated for 24 h at 37 °C in the absence or presence of sialidase (10 milliunits/µg alpha -dystroglycan) from Vibrio cholerae, Clostridium perfringens, Arthrobacter neurofaciens (all from Boehringer Mannheim), Newcastle disease virus (Oxford GlycoSystems Inc., Rosedale, NY), or nonpathogenic Streptococcus strain 6646K (Seikagaku America Inc., Ijamsville, MD). Purified brain alpha -dystroglycan was digested with jack bean beta -N-acetylglucosaminidase, chicken liver alpha -N-acetylgalactosaminidase, or Escherichia coli beta -galactosidase (all from Sigma) as described previously (40). Control and enzyme-digested samples were analyzed by SDS-polyacrylamide gel electrophoresis and the blotting assays described above.


RESULTS

The laminin-1 and merosin binding properties of cardiac muscle alpha -dystroglycan were first qualitatively compared in the blot overlay assay (Fig. 1A). As previously observed for 156-kDa skeletal muscle alpha -dystroglycan (43), laminin-1 and merosin binding to 140-kDa cardiac muscle alpha -dystroglycan were similarly inhibited by 10 mM EDTA or 0.5 M NaCl (data not shown). However, the addition of 1 mg/ml heparin significantly inhibited laminin-1 binding to skeletal and cardiac muscle alpha -dystroglycans, but had negligible effects on merosin binding to either alpha -dystroglycan (Fig. 1A). Cardiac muscle alpha -dystroglycan binding to laminin and merosin was further compared by examining the concentration dependence of heparin inhibition (0-2 mg/ml) using a solid-phase microtiter assay (Fig. 1B). 125I-Labeled cardiac alpha -dystroglycan binding to laminin-1 was significantly more sensitive to heparin over the range of 0.1-2 mg/ml in comparison with merosin, which was notably insensitive to heparin at all concentrations tested (Fig. 1B). Thus, cardiac and skeletal muscle alpha -dystroglycans share the capacity to bind merosin in a heparin-insensitive manner. In contrast, the binding of laminin-1 and merosin to 120-kDa alpha -dystroglycans purified from peripheral nerve (32) or brain (43) was significantly inhibited by heparin. The sum of these results supports our hypothesis that tissue-specific post-translational modifications alter the capacity of some alpha -dystroglycans to discriminate between different forms of laminin.


Fig. 1. Effect of heparin on cardiac alpha -dystroglycan binding to laminin-1 and merosin. A, shown are identical nitrocellulose transfers containing purified skeletal (SKEL) and cardiac (CARD) alpha -dystroglycans overlaid with laminin-1 (LAM) or merosin (MER) in the absence (-HEP) or presence (+HEP) of 1 mg/ml heparin. B, alpha -dystroglycan purified from rabbit cardiac muscle was iodinated (125I alpha -DG), and its binding to laminin (bullet ) and merosin (black-square) in the presence of the indicated concentration of heparin was measured in the solid-phase microtiter assay described under "Experimental Procedures." Binding data were normalized as a percent of control for experiments performed in triplicate, and the graph represents the mean ± S.E. of three experiments.
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Because alpha -dystroglycan is extensively glycosylated (1, 8, 19, 33, 35-37), carbohydrate modifications seemed most likely to account for the differences in laminin binding properties observed between muscle and neural alpha -dystroglycans. The lectin reactivities reported for alpha -dystroglycans prepared from different tissues and species (Table I) suggested the presence of terminal GalNAc residues on neural forms of alpha -dystroglycan, which were not apparent on skeletal muscle alpha -dystroglycan. To determine whether the expression of terminal GalNAc residues was a tissue- or species-specific feature of alpha -dystroglycan, we compared alpha -dystroglycans purified from rabbit skeletal muscle, cardiac muscle, sciatic nerve, and brain for staining by GalNAc-specific lectins. As previously demonstrated for alpha -dystroglycan purified from ovine brain (35) or bovine peripheral nerve (36), alpha -dystroglycan purified from rabbit brain or sciatic nerve reacted strongly with the terminal GalNAc-specific lectin VVA-B4 in a manner that was inhibited completely by an excess of GalNAc (Fig. 2). GalNAc concentrations as low as 0.05 mM completely inhibited VVA-B4 binding to brain alpha -dystroglycan, whereas 50 mM N-acetylglucosamine, alpha -methylmannoside, alpha -methylglucoside, sucrose, galactose, and mellibiose were without effect (data not shown). In contrast to the results obtained with brain and sciatic nerve alpha -dystroglycans, VVA-B4 reacted weakly or not at all with alpha -dystroglycan purified from rabbit skeletal muscle (Fig. 2). On the other hand, cardiac muscle alpha -dystroglycan was strongly stained by VVA-B4 (Fig. 2). Taken together, the results presented in Figs. 1 and 2 suggest that cardiac muscle alpha -dystroglycan has characteristics that resemble both nervous tissue and skeletal muscle alpha -dystroglycans: cardiac alpha -dystroglycan displays laminin binding properties most like those of skeletal muscle alpha -dystroglycan (Fig. 1), but also expresses a terminal GalNAc modification (Fig. 2) previously found only on nervous tissue alpha -dystroglycans.


Fig. 2. V. villosa agglutinin reacts with cardiac, brain, and sciatic nerve alpha -dystroglycans, but not with skeletal muscle alpha -dystroglycan. Shown are identical nitrocellulose transfers of SDS-polyacrylamide gels loaded with alpha -dystroglycan purified from rabbit skeletal muscle (SKEL), cardiac muscle (CARD), brain, or sciatic nerve and stained with affinity-purified chicken polyclonal antibodies to alpha -dystroglycan (alpha -DG Ab; upper left panel) or stained with 1 µg/ml peroxidase-conjugated VVA-B4 in the absence (upper right panel) or presence (lower panel) of 50 mM GalNAc.
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VVA-B4 can bind either beta -linked or Ser/Thr-linked terminal GalNAc residues (40). Therefore, we tested additional lectins reactive with different anomers of GalNAc for binding to rabbit brain alpha -dystroglycan. Rabbit brain alpha -dystroglycan also bound DBA (reactive with alpha -linked, beta -linked, and Ser/Thr-linked GalNAc residues), but was not stained by either LBA or PTA (Fig. 3), which react with alpha -linked or Ser/Thr-linked terminal GalNAc residues (40). Cardiac muscle alpha -dystroglycan exhibited a similar pattern of staining with the various lectins, whereas skeletal muscle alpha -dystroglycan did not react with any GalNAc-specific lectins (data not shown). Whereas alpha -dystroglycan staining by VVA-B4 should be sensitive to linkage-specific N-acetylhexosaminidases, VVA-B4 staining of brain alpha -dystroglycan was not significantly diminished by jack bean beta -N-acetylglucosaminidase, chicken liver alpha -N-acetylgalactosaminidase, or E. coli beta -galactosidase. We are currently examining several possible explanations for this discrepancy. In the meantime, the results presented in Fig. 3 suggest that VVA-B4 reacted with terminal beta -linked GalNAc residues on brain and cardiac alpha -dystroglycans.


Fig. 3. V. villosa agglutinin appears to recognize terminal beta -linked GalNAc residues on brain alpha -dystroglycan. Shown are identical nitrocellulose transfers of SDS-polyacrylamide gels containing electrophoretically separated alpha -dystroglycan purified from rabbit brain and stained with 1 µg/ml peroxidase-conjugated VVA-B4, LBA, PTA, or DBA. The reactivity of each lectin for different anomers of N-acetyl-D-galactosamine is indicated by a plus or minus sign.
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The lack of reactivity of VVA-B4 with skeletal muscle alpha -dystroglycan could be due to the absence of GalNAc moieties present on alpha -dystroglycans expressed in other tissues. Alternatively, subterminal GalNAc-containing oligosaccharides on skeletal muscle alpha -dystroglycan might be blocked from interacting with VVA-B4 by further modification with terminal sialic acid (41, 42). To address this possibility, we tested the ability of various sialidases to expose cryptic VVA-B4-binding sites on skeletal muscle alpha -dystroglycan. Digestion of the skeletal muscle dystrophin-glycoprotein complex with sialidase from V. cholerae results in an increased electrophoretic mobility of alpha -dystroglycan with concomitant loss of staining by WGA and Maackia amurensis agglutinin (specific for Neu5Acalpha 2-3Gal) and exposure of latent binding sites for PNA (1), but no loss of laminin binding activity (8). We confirmed these results with purified skeletal muscle alpha -dystroglycan, yet found that it exhibited no reactivity with VVA-B4 upon digestion with V. cholerae sialidase (Fig. 4). Consistent with results obtained using V. cholerae sialidase, skeletal muscle alpha -dystroglycan digested with C. perfringens sialidase exhibited an increased electrophoretic mobility with concomitant loss of staining by WGA and exposure of latent binding sites for PNA (Fig. 5). However, alpha -dystroglycan digested with C. perfringens sialidase also became strongly reactive with VVA-B4 (Fig. 5) and DBA, but not with LBA or PTA (data not shown). Skeletal muscle alpha -dystroglycan also stained strongly with VVA-B4 after digestion with sialidases from A. neurofaciens and Streptococcus strain 6646K, but not Newcastle disease virus sialidase (Table II). Thus, skeletal muscle alpha -dystroglycan contains sialic acid moieties resistant to V. cholerae and Newcastle disease virus sialidases, but labile to digestion with C. perfringens, A. neurofaciens, and Streptococcus strain 6646K sialidases. In addition, these results suggest that like cardiac and brain alpha -dystroglycans (Fig. 2), skeletal muscle alpha -dystroglycan appears to express GalNAc-terminated oligosaccharides that are blocked from interacting with VVA-B4 by sialic acid modification (Fig. 5).


Fig. 4. Effect of V. cholerae neuraminidase digestion on skeletal muscle alpha -dystroglycan. Shown are identical nitrocellulose transfers of SDS-polyacrylamide gels loaded with control (-) or V. cholerae neuraminidase-digested (+) skeletal alpha -dystroglycan stained with affinity-purified chicken polyclonal antibodies to alpha -dystroglycan (alpha -DG Ab), peroxidase-conjugated VVA-B4, or WGA or overlaid with laminin-1 (LAM).
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Fig. 5. Skeletal muscle alpha -dystroglycan reacts strongly with V. villosa agglutinin after digestion with C. perfringens neuraminidase. Shown are identical nitrocellulose transfers of SDS-polyacrylamide gels loaded with control (-) or C. perfringens neuraminidase-digested (+) skeletal alpha -dystroglycan stained with affinity-purified chicken polyclonal antibodies to alpha -dystroglycan (alpha -DG Ab) or with 1 µg/ml peroxidase-conjugated V. villosa agglutinin isolectin B4 in the absence (VVA-B4) or presence (+GalNAc) of 50 mM GalNAc, WGA, or PNA. The molecular mass standards (in kilodaltons) are indicated on the left.
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Table II. VVA-B4 reactivity and laminin binding activity of skeletal muscle alpha -dystroglycan digested with different sialidases


Sialidase Linkage specificitya Expose VVA-B4 reactivity? Effect on laminin binding

V. cholerae  alpha 2 -3,6,8  -  -
C. perfringens  alpha 2 -3,8,6 +  -
A. ureafaciens  alpha 2 -6,3,8 +  -
Newcastle disease virus  alpha 2 -3,8  -  -
Streptococcus 6646K  alpha 2 -3,6,8 +  -

a Given in order of linkage preference.

Finally, the effect of digestion with C. perfringens sialidase on alpha -dystroglycan binding to laminin-1 and merosin was examined using the blot overlay assay (Fig. 6). As reported for alpha -dystroglycan purified from ovine brain (35), the staining intensity of VVA-B4 for rabbit brain (Fig. 6), sciatic nerve (data not shown), and cardiac (data not shown) alpha -dystroglycans increased measurably after digestion with C. perfringens sialidase. However, consistent with most (1, 19, 33, 34), but not all (36) previous studies, C. perfringens sialidase digestion had no effect on the laminin-1 or merosin binding properties of rabbit skeletal muscle (Fig. 6), cardiac muscle (data not shown), sciatic nerve (data not shown), or brain (Fig. 6) alpha -dystroglycan, either in the presence or absence of heparin. Similar results were obtained using sialidases from A. neurofaciens, V. cholerae, Streptococcus strain 6646K, and Newcastle disease virus (Table II) and alpha -dystroglycan purified from bovine brain (data not shown). In the study reporting a loss of laminin binding upon digestion with sialidase (36), it was possible that the extreme alkaline conditions used to solubilize alpha -dystroglycan from sciatic nerve membranes may have degraded O-acetylated sialic acid derivatives (52, 53) on alpha -dystroglycan that are otherwise resistant to sialidase digestion. However, the laminin binding activity of saponified rabbit or bovine brain alpha -dystroglycan was also not diminished after sialidase digestion (data not shown). Thus, the sum of our results indicates that the sialic acid moieties of alpha -dystroglycan that are labile to a variety of sialidases do not appear to be necessary for alpha -dystroglycan binding to laminin-1 or merosin.


Fig. 6. Effect of C. perfringens neuraminidase digestion on the laminin binding properties of skeletal and brain alpha -dystroglycans. Shown are nitrocellulose transfers containing control (-) and neuraminidase-digested (+) rabbit skeletal muscle (SKEL) and brain alpha -dystroglycans stained with affinity-purified chicken polyclonal antibodies to alpha -dystroglycan (alpha -DG Ab) or with 1 µg/ml peroxidase-conjugated VVA-B4. Identical transfers were also overlaid with laminin (LAM) or merosin (MER) in the absence or presence of 1 mg/ml heparin (HEP) . While laminin binding to sialidase-digested brain alpha -dystroglycan appears to be decreased in this experiment, the result was not reproducible.
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DISCUSSION

Previous studies have demonstrated that most, if not all, alpha -dystroglycans express Galbeta 1-3GalNAc-Ser/Thr moieties variably modified by alpha 2-3-sialic acid in a tissue-specific fashion (1, 35, 36). Ovine brain alpha -dystroglycan uniquely expresses an alpha 2-6-sialic acid associated with an Asn-linked oligosaccharide based on its reactivity with Sambucus nigra agglutinin (SNA) that is removed by digestion with N-glycosidase F (35). More recently, the structure Siaalpha 2-3Galbeta 1-4GlcNAcbeta 1-2Man-Ser/Thr was shown to compose two-thirds of the sialylated, Ser/Thr-linked oligosaccharides of bovine peripheral nerve alpha -dystroglycan (38). Based on our present results, we propose a novel sialic acid modification of skeletal muscle alpha -dystroglycan, SiaXalpha 2-6GalNAc, using the nomenclature suggested by Varki (53) to indicate a sialic acid with an unidentified substitution. We have shown that digestion of skeletal muscle alpha -dystroglycan with any of three sialidases that hydrolyze alpha 2-3-, alpha 2-6-, or alpha 2-8-sialic acid linkages unmasks a terminal GalNAc residue that is normally exposed in alpha -dystroglycans prepared from a variety of other tissues (Table II). Newcastle disease virus sialidase, which cleaves alpha 2-3- and alpha 2-8-linkages, but not alpha 2-6-linkages, failed to expose the terminal GalNAc residue, suggesting an alpha 2-6-linkage. While both V. cholerae sialidase and the lectin SNA would be expected to react with Siaalpha 2-6GalNAc, neither is reactive on substrates in which the carboxylate group in the sialic acid moiety is derivatized (54, 55). Thus, the failure of SNA to recognize skeletal muscle alpha -dystroglycan (Table I) and of V. cholerae sialidase to expose the terminal GalNAc residue (Fig. 4 and Table II) can be best explained by the presence of one or more SiaXalpha 2-6GalNAc moieties on skeletal muscle alpha -dystroglycan. Similar structures may also be present on other tissue forms of alpha -dystroglycan, as we have observed that VVA-B4 staining of alpha -dystroglycans purified from rabbit cardiac muscle, sciatic nerve, and brain increases with sialidase digestion. However, only skeletal muscle alpha -dystroglycan displays a complete lack of VVA-B4 reactivity prior to digestion with appropriate sialidases.

The functional role(s) of tissue-specific differences in alpha -dystroglycan sialoglycosylation remain to be elucidated. However, it should be noted that unusual terminating carbohydrate structures (most notably derivatives of sialic acid and GalNAc) are often critical tissue-specific determinants in the function of biologically important glycoconjugates (42, 56). Sanes (39, 40) and co-workers demonstrated that the adult neuromuscular junction is specifically stained with lectins that recognize terminal GalNAc residues. The GalNAc-specific lectin VVA-B4 was further shown to modestly induce acetylcholine receptor clustering by itself and significantly potentiate the clustering activity of agrin in cultured myotubes (41). However, the GalNAc-terminated molecule(s) mediating these effects remained to be identified since VVA-B4 did not appear to bind directly to agrin, acetylcholine receptors, or myotube alpha -dystroglycan (41). Interestingly, digestion of myotubes with C. perfringens neuraminidase was shown to dramatically expose latent VVA-B4-binding sites distributed throughout the sarcolemma and to stimulate acetylcholine receptor clustering in the absence of agrin (41). The sum of these data leads us to hypothesize the coexpression of two alpha -dystroglycan glycoforms in skeletal muscle: 1) an abundant extrasynaptic form containing a terminal beta -linked GalNAc residue that is blocked from interacting with VVA-B4 by further modification with sialic acid and 2) a low abundance motor endplate-specific form with an exposed GalNAc residue involved in neuromuscular synaptogenesis. Because the motor endplate makes up only 0.1% of the total sarcolemmal membrane area, this hypothesis may be exceedingly difficult to test further in skeletal muscle using biochemical methods. The strong staining of cardiac muscle alpha -dystroglycan with VVA-B4 also raises the question of whether VVA-B4-reactive alpha -dystroglycan could play a more general role in establishing and/or maintaining the necessary postsynaptic organization required for appropriate nerve-muscle communication. The heart is extensively innervated by both sympathetic and parasympathetic neurons with numerous varicosities that appear to overlie a significantly greater fraction of cardiac sarcolemmal surface area than is the case with motor neurons in skeletal muscle (57-59). On the other hand, it is of some concern that VVA-B4 failed to label the surface of Schwann cells (40), which express a VVA-B4-reactive form of alpha -dystroglycan (36). It is possible that the binding of native ligands to Schwann cell alpha -dystroglycan in tissue sections hinders its binding to VVA-B4. Alternatively, VVA-B4-reactive alpha -dystroglycan may be one of several GalNAc-terminated glycoconjugates concentrated at the neuromuscular junction (40), but too diffusely distributed in the Schwann cell membrane to be detected with VVA-B4 by histochemical analysis. All of these issues will be more directly addressed with the availability of probes specific for different glycoforms of alpha -dystroglycan.

With regard to the carbohydrate structures important for alpha -dystroglycan binding to laminin, Matsumura and co-workers (36) recently reported that prolonged sialidase digestion of bovine sciatic nerve alpha -dystroglycan diminished its laminin binding activity. It was further observed that 10 mM sialic acid or 3'-sialyl-N-acetyllactosamine inhibited the binding of nanomolar concentrations of laminin to bovine sciatic nerve alpha -dystroglycan by 65% (36) or 33% (38), respectively. These results were interpreted to indicate that Siaalpha 2-3Galbeta 1-4GlcNAc is an essential determinant for laminin binding by alpha -dystroglycan. Unfortunately, the inhibitor studies of laminin binding (36, 38) did not evaluate negatively charged control sugars or measure the effect of the control sugars used at any concentration higher than that reportedly effective for sialic acid inhibition. The examination of negatively charged control sugars such as glucose 6-phosphate or glucose 6-sulfate is relevant because these monosaccharides effectively block sulfatide binding to the laminin E3 domain (60, 61), which is also the site for alpha -dystroglycan binding (31, 34). We observed that sialidase digestion had no effect on the laminin binding activity of alpha -dystroglycan purified from a variety of tissues, regardless of enzyme source (Table II). Our results are consistent with all other previous studies that have evaluated sialidase digestion for an effect on alpha -dystroglycan binding to extracellular ligands (1, 19, 33, 34). The reason for this discrepancy is not presently clear. While we could not reproduce the observed loss of laminin binding upon prolonged sialidase digestion of rabbit sciatic nerve or bovine brain alpha -dystroglycan, we cannot rule out the possibility that bovine sciatic nerve alpha -dystroglycan expresses a unique sialic acid-containing structure that is critical for its laminin binding activity. However, it is worth noting that the dissenting study (36) did not address whether the loss of laminin binding activity of bovine sciatic nerve alpha -dystroglycan upon sialidase digestion may have been caused by another glycosidase or protease activity contaminating the sialidase preparation utilized.

Although we did not address the issue experimentally, we do not expect that any of the identified tissue-specific differences in sialoglycosylation influence the interaction between agrin and alpha -dystroglycan for several reasons. First, a recombinant carboxyl-terminal agrin fragment containing four- and eight-amino acid inserts at the A/Y and B/Z splice sites, respectively, failed to bind either GalNAc-agarose or VVA-B4-agarose (41). Second, we observed that a 600-fold molar excess of VVA-B4 had no effect on laminin or merosin binding to control or neuraminidase-digested skeletal muscle alpha -dystroglycan.2 Since laminin, merosin, and agrin all compete with one another for binding to alpha -dystroglycan (21, 27, 43), these results suggest that an exposed terminal GalNAc residue is not an important determinant in alpha -dystroglycan binding to extracellular ligands. Third, Torpedo electric organ alpha -dystroglycan contains exposed terminal GalNAc residues (37), but binds nerve agrin (Ag4,8/c95A4B8) and muscle agrin (Ag0,0/c95A0B0) with properties (22, 47) very similar to those reported for skeletal muscle alpha -dystroglycan (22, 27). Fourth, Gesemann et al. (27) noted that the binding of laminin, c95A4B8, and c95A0B0 to alpha -dystroglycan from C2C12 myotubes, bovine peripheral nerve, and chick skeletal muscle was very similar. Thus, we conclude that tissue-specific differences in sialoglycosylation do not have gross effects on the biochemical interaction of alpha -dystroglycan glycoforms with different extracellular ligands. However, variations in alpha -dystroglycan sialoglycosylation may prove as useful markers to further elucidate the role of alpha -dystroglycan glycoforms in different tissues and perhaps within a single cell type.


FOOTNOTES

*   This work was supported by a grant-in-aid from the American Heart Association and by Grants AR42423 and AR01985 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Physiology, University of Wisconsin, 120 Service Memorial Inst., 1300 University Ave., Madison, WI 53706. Tel.: 608-265-3419; Fax: 608-265-5512; E-mail: ervasti{at}facstaff.wisc.edu.
1   The abbreviations used are: WGA, wheat germ agglutinin; VVA-B4, V. villosa agglutinin isolectin B4; PTA, P. tetragonolobus agglutinin; DBA, D. biflorus agglutinin; PNA, peanut agglutinin; LBA, lima bean agglutinin; SNA, S. nigra agglutinin; TBS, Tris-buffered saline; Sia, sialic acid.
2   A. L. Burwell and J. M. Ervasti, unpublished results.

ACKNOWLEDGEMENTS

We are grateful to Dr. Marion Greaser for advice in preparing chicken yolk immunoglobulins to alpha -dystroglycan and Brian Renley and Matthew Sdano for expert and enthusiastic technical assistance. We thank Kurt Amann for many helpful discussions.


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