(Received for publication, February 3, 1997, and in revised form, May 20, 1997)
From the Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706
Because the polypeptide core of -dystroglycan
is encoded by a single gene, the difference in apparent molecular mass
between
-dystroglycans expressed in various tissues is presumably
due to differential glycosylation. However, little is presently known about the tissue-specific differences in
-dystroglycan glycosylation and whether these modifications may confer functional variability to
-dystroglycan. We recently observed that laminin-1 binding to
skeletal muscle
-dystroglycan was dramatically inhibited by heparin,
whereas the binding of commercial merosin to skeletal muscle
-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
-dystroglycan, both laminin-1 and merosin binding to 120-kDa brain
-dystroglycan were sensitive to heparin. We have now examined the
laminin binding properties of 140-kDa
-dystroglycan purified from
cardiac muscle and observed that like skeletal muscle
-dystroglycan,
heparin inhibited cardiac
-dystroglycan binding to laminin-1, but
not to merosin. On the other hand, cardiac and brain
-dystroglycans
could be distinguished from skeletal muscle
-dystroglycan by their
reactivity with the terminal GalNAc-specific lectin Vicia
villosa agglutinin. Interestingly, skeletal muscle
-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
-dystroglycan, the sum of our results suggests
that skeletal muscle
-dystroglycan contains a novel sialic acid
residue linked
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
-dystroglycan sialoglycosylation may prove as
useful markers to further elucidate the role of
-dystroglycan glycoforms in different tissues and perhaps within a single cell type.
-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
-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),
-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
-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,
-dystroglycan co-clusters with
acetylcholine receptors in a heterologous expression system (18).
-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,
-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
-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
-dystroglycan in neuromuscular
synapse formation remains to be elucidated.
Although -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
-dystroglycan in native tissues. None
the less, characterization of
-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
-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
-dystroglycan lacking terminal GalNAc residues (33, 41). Of
further concern is that all previously identified (35, 36)
tissue-specific variations in
-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
-dystroglycan glycosylation and to
determine whether these modifications may confer functional variability
to
-dystroglycan.
|
We recently observed (43) a significant difference in the heparin
inhibition of skeletal muscle -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
-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
-dystroglycan, we
further observed that both laminin-1 and merosin binding to 120-kDa
brain
-dystroglycan were sensitive to heparin (43), suggesting that
tissue-specific differences in
-dystroglycan post-translational
modification may influence its interactions with extracellular ligands.
Cardiac muscle
-dystroglycan exhibits an electrophoretic mobility
intermediate (140 kDa) to that of skeletal and brain
-dystroglycans
(7, 8, 48), suggesting that it could exhibit laminin binding properties
more similar to skeletal muscle
-dystroglycan, brain
-dystroglycan, or somewhere in between. Therefore, we have now
examined the effect of heparin on the binding of purified cardiac
muscle
-dystroglycan to laminin-1 and merosin. Our results suggest
that cardiac and skeletal muscle
-dystroglycans are functionally
related but distinct from nervous tissue
-dystroglycans with regard
to merosin binding in the presence of heparin. However, we further
demonstrate that cardiac muscle and brain
-dystroglycans appear to
be similarly modified with terminal GalNAc residues, which are also
present on skeletal muscle
-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
-dystroglycan, we discuss situations in which differences in
sialoglycosylation may reflect
-dystroglycan glycoforms with unique
or varied function, even in the same cell.
-Dystroglycan was
solubilized from rabbit skeletal muscle membranes (49) or cardiac
membranes (7) by extraction with 8 M urea and purified by
sequential WGA1-Sepharose
chromatography, DEAE-cellulose chromatography, and CsCl gradient
centrifugation (43).
-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
-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
/
-dystroglycan precursor (7) with the proteolytic cleavage
site located between Gly-653 and Ser-654 (35).
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. -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
-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 -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 -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).
A solid-phase microtiter assay
using iodinated cardiac -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
-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
-counter.
Purified -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
-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
-dystroglycan was digested with jack
bean
-N-acetylglucosaminidase, chicken liver
-N-acetylgalactosaminidase, or Escherichia
coli
-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.
The laminin-1 and merosin binding properties of cardiac muscle
-dystroglycan were first qualitatively compared in the blot overlay
assay (Fig. 1A). As previously
observed for 156-kDa skeletal muscle
-dystroglycan (43), laminin-1
and merosin binding to 140-kDa cardiac muscle
-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
-dystroglycans, but had negligible effects on merosin binding
to either
-dystroglycan (Fig. 1A). Cardiac muscle
-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
-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
-dystroglycans share the capacity to
bind merosin in a heparin-insensitive manner. In contrast, the binding
of laminin-1 and merosin to 120-kDa
-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
-dystroglycans to discriminate between different forms of
laminin.
Because -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
-dystroglycans. The lectin reactivities reported for
-dystroglycans prepared from different tissues and species (Table
I) suggested the presence of terminal GalNAc residues on neural forms of
-dystroglycan, which were not
apparent on skeletal muscle
-dystroglycan. To determine whether the
expression of terminal GalNAc residues was a tissue- or
species-specific feature of
-dystroglycan, we compared
-dystroglycans purified from rabbit skeletal muscle, cardiac muscle,
sciatic nerve, and brain for staining by GalNAc-specific lectins. As
previously demonstrated for
-dystroglycan purified from ovine brain
(35) or bovine peripheral nerve (36),
-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
-dystroglycan, whereas 50 mM
N-acetylglucosamine,
-methylmannoside,
-methylglucoside, sucrose, galactose, and mellibiose were without
effect (data not shown). In contrast to the results obtained with brain
and sciatic nerve
-dystroglycans, VVA-B4 reacted weakly
or not at all with
-dystroglycan purified from rabbit skeletal
muscle (Fig. 2). On the other hand, cardiac muscle
-dystroglycan was
strongly stained by VVA-B4 (Fig. 2). Taken together, the
results presented in Figs. 1 and 2 suggest that cardiac muscle
-dystroglycan has characteristics that resemble both nervous tissue
and skeletal muscle
-dystroglycans: cardiac
-dystroglycan
displays laminin binding properties most like those of skeletal muscle
-dystroglycan (Fig. 1), but also expresses a terminal GalNAc
modification (Fig. 2) previously found only on nervous tissue
-dystroglycans.
VVA-B4 can bind either -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
-dystroglycan. Rabbit brain
-dystroglycan also bound DBA (reactive with
-linked,
-linked, and Ser/Thr-linked GalNAc
residues), but was not stained by either LBA or PTA (Fig.
3), which react with
-linked or
Ser/Thr-linked terminal GalNAc residues (40). Cardiac muscle
-dystroglycan exhibited a similar pattern of staining with the
various lectins, whereas skeletal muscle
-dystroglycan did not react
with any GalNAc-specific lectins (data not shown). Whereas
-dystroglycan staining by VVA-B4 should be sensitive to
linkage-specific N-acetylhexosaminidases, VVA-B4
staining of brain
-dystroglycan was not significantly diminished by
jack bean
-N-acetylglucosaminidase, chicken liver
-N-acetylgalactosaminidase, or E. coli
-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
-linked GalNAc residues on brain and cardiac
-dystroglycans.
The lack of reactivity of VVA-B4 with skeletal muscle
-dystroglycan could be due to the absence of GalNAc moieties present on
-dystroglycans expressed in other tissues. Alternatively, subterminal GalNAc-containing oligosaccharides on skeletal muscle
-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
-dystroglycan. Digestion of the skeletal muscle
dystrophin-glycoprotein complex with sialidase from V. cholerae results in an increased electrophoretic mobility of
-dystroglycan with concomitant loss of staining by WGA and
Maackia amurensis agglutinin (specific for Neu5Ac
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
-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
-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,
-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
-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
-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
-dystroglycans (Fig. 2), skeletal muscle
-dystroglycan
appears to express GalNAc-terminated oligosaccharides that are blocked
from interacting with VVA-B4 by sialic acid modification
(Fig. 5).
|
Finally, the effect of digestion with C. perfringens
sialidase on -dystroglycan binding to laminin-1 and merosin was
examined using the blot overlay assay (Fig.
6). As reported for
-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)
-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)
-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
-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
-dystroglycan from sciatic nerve
membranes may have degraded O-acetylated sialic acid
derivatives (52, 53) on
-dystroglycan that are otherwise resistant
to sialidase digestion. However, the laminin binding activity of
saponified rabbit or bovine brain
-dystroglycan was also not
diminished after sialidase digestion (data not shown). Thus, the sum of
our results indicates that the sialic acid moieties of
-dystroglycan
that are labile to a variety of sialidases do not appear to be
necessary for
-dystroglycan binding to laminin-1 or merosin.
Previous studies have demonstrated that most, if not all,
-dystroglycans express Gal
1-3GalNAc-Ser/Thr moieties variably modified by
2-3-sialic acid in a tissue-specific fashion (1, 35,
36). Ovine brain
-dystroglycan uniquely expresses an
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 Sia
2-3Gal
1-4GlcNAc
1-2Man-Ser/Thr
was shown to compose two-thirds of the sialylated, Ser/Thr-linked
oligosaccharides of bovine peripheral nerve
-dystroglycan (38).
Based on our present results, we propose a novel sialic acid
modification of skeletal muscle
-dystroglycan, SiaX
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
-dystroglycan with any of three sialidases that
hydrolyze
2-3-,
2-6-, or
2-8-sialic acid linkages unmasks a
terminal GalNAc residue that is normally exposed in
-dystroglycans
prepared from a variety of other tissues (Table II). Newcastle disease
virus sialidase, which cleaves
2-3- and
2-8-linkages, but not
2-6-linkages, failed to expose the terminal GalNAc residue,
suggesting an
2-6-linkage. While both V. cholerae
sialidase and the lectin SNA would be expected to react with
Sia
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
-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 SiaX
2-6GalNAc moieties on skeletal
muscle
-dystroglycan. Similar structures may also be present on
other tissue forms of
-dystroglycan, as we have observed that
VVA-B4 staining of
-dystroglycans purified from rabbit
cardiac muscle, sciatic nerve, and brain increases with sialidase
digestion. However, only skeletal muscle
-dystroglycan displays a
complete lack of VVA-B4 reactivity prior to digestion with
appropriate sialidases.
The functional role(s) of tissue-specific differences in
-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
-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
-dystroglycan glycoforms in
skeletal muscle: 1) an abundant extrasynaptic form containing a
terminal
-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
-dystroglycan with VVA-B4 also raises the
question of whether VVA-B4-reactive
-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
-dystroglycan
(36). It is possible that the binding of native ligands to Schwann cell
-dystroglycan in tissue sections hinders its binding to
VVA-B4. Alternatively, VVA-B4-reactive
-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
-dystroglycan.
With regard to the carbohydrate structures important for
-dystroglycan binding to laminin, Matsumura and co-workers (36) recently reported that prolonged sialidase digestion of bovine sciatic
nerve
-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
-dystroglycan by 65% (36) or 33% (38), respectively. These results
were interpreted to indicate that Sia
2-3Gal
1-4GlcNAc is an
essential determinant for laminin binding by
-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
-dystroglycan binding (31, 34). We observed that
sialidase digestion had no effect on the laminin binding activity of
-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
-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
-dystroglycan, we cannot rule out the possibility that bovine
sciatic nerve
-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
-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
-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
-dystroglycan.2 Since
laminin, merosin, and agrin all compete with one another for binding to
-dystroglycan (21, 27, 43), these results suggest that an exposed
terminal GalNAc residue is not an important determinant in
-dystroglycan binding to extracellular ligands. Third,
Torpedo electric organ
-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
-dystroglycan (22, 27). Fourth,
Gesemann et al. (27) noted that the binding of laminin,
c95A4B8, and c95A0B0 to
-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
-dystroglycan glycoforms with different extracellular ligands. However, variations in
-dystroglycan sialoglycosylation may
prove as useful markers to further elucidate the role of
-dystroglycan glycoforms in different tissues and perhaps within a
single cell type.
We are grateful to Dr. Marion Greaser for
advice in preparing chicken yolk immunoglobulins to -dystroglycan
and Brian Renley and Matthew Sdano for expert and enthusiastic
technical assistance. We thank Kurt Amann for many helpful
discussions.