(Received for publication, September 10, 1996, and in revised form, November 4, 1996)
From the Department of Glycobiology, Tokyo
Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku,
Tokyo 173, Japan, ¶ Department of Neurology and Neuroscience,
Teikyo University, School of Medicine, Itabashi, Tokyo 173, Japan,
Research Department, Noguchi Institute, Itabashi, Tokyo 173, Japan, and § Department of Neurology, Institute for Brain
Research, Faculty of Medicine, University of Tokyo, Bunkyo,
Tokyo 113, Japan
-Dystroglycan is a heavily
glycosylated protein, which is localized on the Schwann cell
membrane as well as the sarcolemma, and links the transmembrane protein
-dystroglycan to laminin in the extracellular matrix. We have shown
previously that sialidase treatment, but not N-glycanase
treatment, of bovine peripheral nerve
-dystroglycan greatly reduces
its binding activity to laminin, suggesting that the sialic acid of
O-glycosidically-linked oligosaccharides may be essential
for this binding. In this report, we analyzed the structures of the
sialylated O-linked oligosaccharides of bovine peripheral
nerve
-dystroglycan by two methods.
O-Glycosidically-linked oligosaccharides were liberated by
alkaline-borotritide treatment or by mild hydrazinolysis followed
by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion
exchange column chromatography that eluted at a position corresponding
to monosialylated oligosaccharides were converted to neutral
oligosaccharides by exhaustive sialidase digestion. The sialidases from
Arthrobacter ureafaciens and from Newcastle disease virus
resulted in the same degree of hydrolysis. The neutral oligosaccharide
fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9
glucose units upon gel filtration, and its reducing terminus was
identified as a mannose derivative. Based on the results of sequential
exoglycosidase digestion, lectin column chromatography, and
reversed-phase high-performance liquid chromatography, we concluded
that the major sialylated O-glycosidically-linked oligosaccharide of the
-dystroglycan was a novel
O-mannosyl-type oligosaccharide, the structure of which was
Sia
2-3Gal
1-4GlcNAc
1-2Man-Ser/Thr (where Sia is sialic
acid). This oligosaccharide constituted at least 66% of the sialylated
O-linked sugar chains. Furthermore, a laminin binding
inhibition study suggested that the sialyl
N-acetyllactosamine moiety of this sugar chain was involved
in the interaction of the
-dystroglycan with laminin.
-Dystroglycan is an extracellular peripheral membrane
glycoprotein anchored to the cell membrane by binding to a
transmembrane glycoprotein,
-dystroglycan (1, 2). These two
dystroglycan subunits were originally identified as members of a
sarcolemmal dystrophin-associated (glyco)protein complex.
- and
-dystroglycans are encoded by a single gene and cleaved into two
proteins by posttranslational processing (3). Based on recent
amino-terminal sequencing of
-dystroglycan, it was proposed that the
serine residue at position 654 of the precursor protein of dystroglycan is the cleavage site (4, 5). The
-dystroglycan·
-dystroglycan complex is widely expressed in many tissues (6) and thought to
act as a transmembrane linker between the extracellular matrix and
intracellular cytoskeleton (7), because
-dystroglycan binds
extracellular matrix components, laminin-1 and -2 with high affinity (7-10), and the intracellular domain of
-dystroglycan binds cytoskeletal proteins, dystrophin or its autosomal homologue, utrophin (2, 11, 12).
-Dystroglycan is heavily glycosylated. Whereas the deduced amino
acid sequence predicts a ~74-kDa core peptide,
-dystroglycan is
identified as a broad smear in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 156 kDa in skeletal muscle (1), 120 kDa in brain and peripheral nerve (4, 13, 14), and 190 kDa in
postsynaptic membrane of Torpedo electric organ (15). The differences
in the molecular masses of
-dystroglycans obtained from different
tissues seem to be due, not to differences in the primary structure,
but to tissue-specific differential glycosylation of the core protein
(6). Although the nature of the carbohydrate moiety of
-dystroglycan
has not been fully clarified yet, chemical modification by treatment
with periodic acid or trifluoromethanesulfonic acid resulted in the
loss of laminin binding (7, 16), suggesting that the sugar moiety is
essential for this activity. The deduced amino acid sequence shows
three potential N-glycosylation sites, and enzymatic
cleavage of N-linked sugar chains reduced the molecular mass
by approximately 4 kDa without affecting the laminin binding activity
(1). There are also two conserved serine-glycine dipeptides (6), which are one type of potential glycosaminoglycan attachment site (17). However, nitrous acid treatment to degrade heparin or heparan sulfate
or glycosaminoglycan lyase digestion affected neither the molecular
mass nor laminin binding (7, 10, 13, 15, 16, 18). Although
-dystroglycan from the C2 cell line (S27), which is defective in
glycosaminoglycan synthesis (19), was found to bind laminin poorly
(20), there is no direct evidence that N-linked
oligosaccharides (or glycosaminoglycans, if present) are required for
laminin binding.
The amino acid sequence of -dystroglycan shows one feature of a
mucin type O-glycosylation site (21) in the central region of the molecule. Threonine, serine, and proline are densely distributed between the 317th and the 488th amino acid residues, often clustering, and over one-half of the proline residues in this region are at positions
1 or +3 relative to the threonine or serine residues. In
addition, the susceptibility of
-dystroglycan to
O-sialoglycopeptidase supports the hypothesis that
-dystroglycan is a sialylated mucin-type glycoprotein (4). Recently,
we demonstrated that exhaustive sialidase treatment of
-dystroglycan
or the addition of sialic acid to the incubation medium diminished the
laminin binding activity of
-dystroglycan, suggesting that the
sialic acid residues of
-dystroglycan, which are probably attached
to O-linked oligosaccharides, were essential for this
binding (10).
In this study, we have analyzed the structures of sialylated
O-linked oligosaccharides of bovine peripheral nerve
-dystroglycan and demonstrate that a novel O-linked
mannose-type oligosaccharide, Sia
2-3Gal
1-4GlcNAc
1-2Man-Ser/Thr,1
is the major component. The results of a binding-inhibition study suggest that this unique oligosaccharide contributes to the laminin binding activity of
-dystroglycan.
NaB3H4 (1000 mCi/mmol)
was purchased from DuPont NEN; 1,2-diamino-4,5-methylenedioxybenzene
(DMB) from Dojindo Laboratories (Kumamoto, Japan); 2AB labeling kit
from Oxford Glycosystems (Oxon, United Kingdom); bicinchoninic acid
protein assay reagent from Pierce; Silver Stain DAIICHI reagent kit
from Daiichi Pure Chemicals (Tokyo, Japan); avidin-biotin complex (ABC)
kit (horseradish peroxidase-labeled) from Vector (Burlingame, CA);
polystyrene microtiter plate from Costar (Cambridge, MA); AG 3-X4 and
AG 50-X12 from Bio-Rad; and Sep-Pack C18 from Waters
(Milford, MA). Mono Q and Superdex Peptide columns were obtained from
Pharmacia Biotech Inc.; Cosmosil 5C18-AR column from Nacalai Tesque
(Kyoto, Japan); and Shodex SUGAR SP-1010 column from Showa Denko
(Tokyo, Japan). Arthrobacter ureafaciens sialidase was
purchased from Nacalai Tesque; Newcastle disease virus sialidase,
diplococcal -galactosidase, and diplococcal
-N-acetylhexosaminidase was from Boehringer Mannheim.
Jack bean
-N-acetylhexosaminidase was prepared from jack
bean meal by the method of Li and Li (22). Psathyrella
velutina lectin (PVL)-Affi-Gel 10 was generously provided by Dr.
Naohisa Kochibe (Gunma University). Wheat germ agglutinin-Sepharose was
obtained from Pharmacia. Biotinylated laminin-1 was prepared using
sulfosuccinimidyl-6-(biotinamido) hexanoate sodium salt (Vector
Laboratories), and laminin-1 was obtained from Biomedical Technologies
(Stoughton, MA). N-Acetylneuraminic acid,
N-glycolylneuraminic acid, 3
-sialyllactose, and
6
-sialyllactose were obtained from Sigma;
N-acetyllactosamine, 3
-sialyl
N-acetyllactosamine, and 6
-sialyl
N-acetyllactosamine were from Seikagaku Kogyo (Tokyo, Japan), and GlcNAc
1-2Man was from Dextra Laboratories (Reading, UK). Gal
1-3GalNAc was prepared from fetal calf serum fetuin
(Sigma) by mild hydrazinolysis (23) and A. ureafaciens sialidase digestion. GlcNAc
1-3Man,
GlcNAc
1-4Man, and GlcNAc
1-6Man were chemically synthesized as
described previously (24), and their structures were confirmed by
1H and 13C NMR.
-Dystroglycan was purified as described
previously (25). Crude bovine peripheral nerve membranes were suspended
at a protein concentration of 5 mg/ml in 50 mM Tris-HCl, pH
7.4, containing 0.5 M NaCl and a mixture of protease
inhibitors: benzamidine (0.75 mM), phenylmethylsulfonyl
fluoride (0.1 mM), pepstatin A (0.7 µM),
aprotinin (76.8 mM), and leupeptin (1.1 µM).
The suspension was titrated to pH 12 by slowly adding 1 N
NaOH, extracted for 1 h, and then centrifuged at 140,000 × g for 30 min at 25 °C. The supernatant was titrated to pH
7.4 and centrifuged at 140,000 × g for 30 min at
4 °C. The supernatant was circulated over the wheat germ
agglutinin-Sepharose overnight at 4 °C. After extensive washing with
buffer A (50 mM Tris-HCl, pH 7.4, 0.75 mM
benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride), the
proteins bound to the wheat germ agglutinin-Sepharose were eluted with
the buffer A containing 0.3 M
N-acetylglucosamine. The eluate was then circulated over
laminin-Sepharose (14) overnight at 4 °C in the presence of 1 mM CaCl2 and 1 mM
MgCl2. After extensive washing with buffer A containing 1 mM CaCl2 and 1 mM
MgCl2, the proteins bound to the laminin-Sepharose were
eluted with buffer A containing 10 mM EDTA. The eluate was
extensively dialyzed against 50 mM
NH4HCO3 and used for the following analyses.
The protein concentration was measured by using the bicinchoninic acid
protein assay reagent. The purity of the sample was determined by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant
Blue staining and by silver staining using the Silver Stain DAIICHI
reagent kit. In the latter staining, to increase the sensitivity of
glycoprotein detection, the periodic acid-silver stain method (26) was
used with a modification; the gel was treated with 1% periodic acid for 10 min at room temperature prior to fixation instead of with 0.2%
periodic acid treatment for 1 h at 4 °C after fixation in the
original method. With both staining methods,
-dystroglycan was
detected as a prominent broad band approximately 116 kDa (data not
shown).
The DMB labeling method (27)
was used with some modifications. Sialic acids were released from
-dystroglycan (155 ng of protein) in 60 µl of solution by A. ureafaciens sialidase digestion (250 mU for 18 h at 37 °C
with 0.8% Triton X-100) or by acid hydrolysis (0.1 N HCl
for 1 h at 80 °C) (28). To this was added 240 µl of DMB
solution (4 mM DMB, 2 M acetic acid, 0.45 M
-mercaptoethanol, and 11 mM
Na2S2O4), and the mixture was
incubated for 2.5 h at 50 °C in the dark. A 60-µl aliquot of
the sample was applied to the Cosmosil 5C18-AR reversed-phase HPLC
column (4.6 × 250-mm), and the column was eluted with
acetonitrile:methanol:water (9:7:84, v/v) at a flow rate of 0.75 ml/min
at room temperature. The fluorescence was monitored at 448 nm
(excitation, 373 nm). Authentic N-acetylneuraminic acid
treated in the same way was used to make a standard curve.
The sugar chains of
-dystroglycan were converted to oligosaccharide derivatives by two
chemical methods. In the first procedure, an
-dystroglycan sample
(31.0 µg of protein) was thoroughly dried and subjected to
hydrazinolysis for 5 h at 60 °C according to the method of
Patel et al. (23). The sample was subjected to N-acetylation followed by paper chromatography using
1-butanol:ethanol:water (4:1:1, v/v) for 18 h. The area of the
paper from the origin to the position of authentic lactose was
extracted with water, and the extracted oligosaccharides were labeled
with 2-aminobenzamide (2AB) using the 2AB labeling kit (29). As for the
2AB-labeled oligosaccharides, fluorescence was monitored at 430 nm
(excitation, 330 nm).
In the second procedure, a lyophilized -dystroglycan sample (201.5 µg of protein) was dissolved in 400 µl of 0.05 N NaOH and 1 M NaBH4 containing 25 mCi of
NaB3H4 and incubated for 16 h at 45 °C
(30, 31). After adjusting the pH to 6 by adding acetic acid, the
solution was passed through a column containing 1 ml of AG 50-X12
(H+), and the column was washed with 10 ml of water. The
effluent and the wash volume were combined and evaporated to dryness.
After the remaining borate was removed by repeated evaporation with methanol, the residue was subjected to paper chromatography using 1-butanol:ethanol:water (4:1:1, v/v) for 18 h. The area of the paper from the origin to the position of authentic
GlcNAc
1-2ManOT was extracted with water.
The oligosaccharide samples dissolved in distilled water were applied to the Mono Q HR5/5 column. After elution of neutral oligosaccharides with 10 ml of water, acidic oligosaccharides were eluted with a 0-1 M gradient of ammonium acetate, pH 4.0, at a flow rate of 1 ml/min at room temperature. Neutral oligosaccharides were applied to the Superdex Peptide HR10/30 gel filtration column (1 × 60-cm) and eluted with distilled water at a flow rate of 0.4 ml/min at 60 °C. PVL-Affi-Gel 10 column chromatography was performed as described previously (32). Reducing termini of tritium-labeled oligosaccharides were determined by using the Shodex SUGAR SP-1010 column (8 × 300-mm) (33). Reversed-phase HPLC was carried out on the Cosmosil 5C18-AR column by eluting with a 50 mM acetate-acetonitrile gradient solvent system at a flow rate of 1.0 ml/min at 40 °C. The 50 mM acetate-acetonitrile ratio was changed linearly from 100:0 to 95:5 (v/v) over 50 min from 5 min after injection for 2AB-labeled disaccharide analysis and from 100:0 to 98.5:1.5 (v/v) over 50 min from 10 min after injection and then maintained at that ratio for 2AB-labeled monosaccharide analysis.
Glycosidase DigestionOligosaccharides were incubated with
one of the following mixtures for 18 h at 37 °C: (i) A. ureafaciens sialidase (200 mU) in 80 µl of 0.5 M
sodium acetate or ammonium acetate buffer, pH 5.0; (ii) Newcastle
disease virus sialidase (10 mU) in 40 µl of 50 mM sodium
acetate buffer, pH 5.5; (iii) diplococcal -galactosidase (5 mU) in
40 µl of 0.3 M citrate phosphate buffer, pH 6.0; and (iv)
jack bean
-N-acetylhexosaminidase (0.5 U) in 55 µl of
0.3 M citrate phosphate buffer, pH 5.0. One drop of toluene
was added to all reaction mixtures to inhibit bacterial growth during
incubation. Digestions were terminated by heating the reaction mixture
in a boiling water bath for 3 min. Digested samples were desalted using
ion exchange resin (300 µl of AG3-X4(OH
) and 300 µl
of AG50-X12(H+)) for neutralized tritium-labeled
oligosaccharides or using the Sep-Pack C18 (washing with 8 ml of distilled water and elution with 6 ml of acetonitrile:water (2:8,
v/v)) for neutralized 2AB-labeled oligosaccharides. As for samples
after sialidase digestions, ammonium acetate was removed by extensive
evaporation, and sodium acetate was removed by passing the sample
through a AG50-X12(H+) column (300 µl) and
evaporation.
Sixteen ng of
-dystroglycan were dispensed to each well of a polystyrene
microtiter plate and dried under blowing cold air. After rinsing with
LBB (10 mM triethanolamine, pH 7.6, 140 mM NaCl, 1 mM CaCl2, and 1 mM
MgCl2), the wells were blocked with LBB containing 3%
bovine serum albumin (3% BSA-LBB). Fifty µl of the biotinylated
laminin-1 (2 nM in 3% BSA-LBB) containing various
saccharides were added to the wells and incubated overnight at room
temperature. After rinsing with LBB, the binding of laminin-1 was
detected using the avidin-biotin complex (ABC) kit.
o-Phenylenediamine dihydrochloride was used as a coloring
substrate, and absorbance was measured at 492 nm. The experiments were
done in triplicate for each added saccharide.
The
enzymatic cleavage and acid hydrolysis released about 14.9 and 13.8 mol
of free sialic acids from 1 mol of -dystroglycan, respectively. When
the sialic acid residues thus obtained were analyzed by HPLC,
N-acetylneuraminic acid and N-glycolylneuraminic acid were detected in a ratio of 4:1 by both methods. Although the DMB
labeling method can distinguish O-acetyl derivatives of sialic acid from N-acetyl or N-glycolyl ones
(27), no O-acetyl derivative was detected in
-dystroglycan.
O-Linked
oligosaccharides of bovine peripheral nerve -dystroglycan, released
by mild hydrazinolysis and labeled with 2AB, were subjected to Mono Q
column chromatography at pH 4.0 (Fig. 1A).
The acidic fraction was exhaustively digested by A. ureafaciens sialidase, and the neutral oligosaccharides, thus
obtained, were subjected to Superdex Peptide gel filtration column
chromatography. Three major peaks eluting at 3.9 glucose units (GU)
(F-I), 2.9 GU (F-II), and 0.9-1.0 GU (F-III) were detected (Fig.
1B). Their percentage molar ratios calculated on the basis
of their peak areas were 54, 30, and 16%, respectively.
The peak F-I released one galactose residue upon incubation with
diplococcal -galactosidase, which cleaves only the Gal
1-4GlcNAc linkage (34) (Fig. 2A), and subsequently one
N-acetylhexosamine residue was released upon jack bean
-N-acetylhexosaminidase digestion (Fig. 2B).
The product eluted with 0.8 GU in Fig. 2B was identified to
be Man-2AB by 2AB-derived monosaccharide analysis using reversed-phase HPLC (Fig. 3A). The component in Fig.
2A was eluted at the same retention time as that of
authentic GlcNAc
1-2Man-2AB standard in the reversed-phase HPLC
(Fig. 4A), and more than 90% of it was
retarded in the PVL-Affi-Gel 10 column, which specifically interacts
with GlcNAc residues but not with GalNAc residues in the nonreducing
terminus (32, 35) (Fig. 5).
The fraction F-II was eluted with the same retention time as that of
authentic Gal1-4GlcNAc-2AB on reversed phase HPLC (Fig. 4B). This fraction released one galactose residue upon
diplococcal
-galactosidase digestion (Fig. 2C). The
digestion product corresponded to authentic GlcNAc-2AB in the
monosaccharide-2AB analysis (Fig. 3B). The fraction F-III
was separated into two peaks corresponding to Gal-2AB and Glc-2AB,
respectively, in the monosaccharide-2AB analysis (Fig. 3C).
Based on these data, the following structures were proposed for the
components of F-I, F-II and F-III: F-I, Gal
1-4GlcNAc
1-2Man-2AB;
F-II, Gal
1-4GlcNAc-2AB; F-III, Gal-2AB and Glc-2AB.
The above results showed
that the major sialylated O-linked oligosaccharide in bovine
peripheral nerve -dystroglycan was an O-mannosyl-type
oligosaccharide. However, the oligosaccharides in the fractions F-II
and F-III, except for Glc-2AB, may be the peeling reaction products of
the major component found in the fraction F-I. This raised a question
whether the major component of the apparent O-mannosyl-type
oligosaccharide also might be a peeled product of larger
oligosaccharides. To solve this problem, we investigated whether
oligosaccharides with the same composition could be obtained from the
same material by the conventional
-elimination method, which
releases O-linked oligosaccharides from the polypeptide backbone.
Tritium-labeled O-linked oligosaccharides released by
alkaline -elimination from the bovine peripheral nerve
-dystroglycan were separated by Mono Q column chromatography at pH
4.0 into neutral and acidic fractions (21 and 79%, respectively, based on the radioactivities). Three main peaks of the acidic fractions (T-A1, T-A2, and T-A3) were obtained (Fig.
6A). By exhaustive A. ureafaciens
sialidase digestion, the peak T-A1 was completely converted to neutral
oligosaccharides, whereas the peaks T-A2 and T-A3 were not digested
(Fig. 6B), indicating that most of the sialylated
O-linked oligosaccharides of the bovine peripheral nerve
-dystroglycan are monosialylated and do not contain any other
anionic residues such as sulfated sugars and uronic acids. Sialidases
from both A. ureafaciens and Newcastle disease virus gave
the same results (data not shown), indicating that the sialic acid
residues are linked at the C-3 position of the galactose (36). The
predominance of
2-3-linked sialic acid is compatible with the
previous results of a lectin blot study in which bovine peripheral
nerve
-dystroglycan was stained with Sia
2-3Gal-specific Maackia amurensis agglutinin but not with
Sia
2-6Gal-specific Sambucus nigra agglutinin (10).
The neutral fraction obtained by sialidase digestion was then subjected
to Superdex Peptide gel filtration column chromatography. A peak
eluting at 3.8 GU with a slight shoulder was detected (Fig. 7). It must be stressed here that no radioactive peak
around 1 GU was detected. The shoulder portion was separated into two
peaks of 3.8 and 2.7 GU by a second gel filtration. The combined
fraction of the peaks of 3.8 GU was designated as fraction T-I, and the peak of 2.7 GU was designated as fraction T-II. The percentage molar
ratios of T-I and T-II were 78 and 22%, respectively, on the basis of
their radioactivities.
The fraction T-I released one galactose residue upon incubation with
diplococcal -galactosidase (data not shown). When the digested
fraction was applied to the PVL-Affi-Gel 10 column, 84% of the
radioactivity was recovered in the retarded fraction (data not shown).
The retarded fraction was then heated in 4 N HCl for 3.5 h at 100 °C followed by N-acetylation, and the
tritium-labeled monosaccharide at the reducing terminus was determined
by SP-1010 column chromatography. A radioactive peak appeared at the
same retention time as that of authentic mannitol (data not shown). On
the other hand, the fraction T-II was resistant to diplococcal
-galactosidase digestion. By SP-1010 column chromatography after acid hydrolysis of the fraction T-II, a peak with a retention time
corresponding to N-acetylgalactosaminitol was detected (data not shown). Further structural studies of the oligosaccharides in
fraction T-II could not be performed because of the limited amount of
the sample.
Based on these results, the following structures were proposed for the
major components of the fractions T-I and T-II: T-I, Gal1-4GlcNAc-ManOT; and T-II,
hexose-GalNAcOT. Based on the radioactivities of the
fractions obtained by the Superdex Peptide and PVL-Affi-Gel 10 column
chromatographies, the amount of the Gal
1-4GlcNAc-ManOT was estimated to be at least 66% of the neutral oligosaccharides obtained by sialidase digestion.
We investigated the effect of sialic acid compounds,
especially those having configurations similar to that of the major
O-linked sialylated oligosaccharide of -dystroglycan, on
its laminin binding activity (Fig. 8). 3
-Sialyl
N-acetyllactosamine, Neu5Ac
2-3Gal
1-4GlcNAc, which is
the trisaccharide terminal portion of
Neu5Ac
2-3Gal
1-4GlcNAc
1-2Man, inhibited the binding of
laminin, whereas both 3
-sialyllactose and 6
-sialyllactose did not.
6
-Sialyl N-acetyllactosamine,
Neu5Ac
2-6Gal
1-4GlcNAc, which is an isomer of 3
-sialyl
N-acetyllactosamine in the sialic acid linkage, also reduced
the binding of laminin, but its effect was weaker than that of
3
-sialyl N-acetyllactosamine. These results indicate that
the interaction between laminin and sialylated oligosaccharides of
-dystroglycan are not simply dependent on the anionic charge of
sialic acid residues but also on the structure comprising the neutral
sugar portion and the sialic acid linkage.
By two different analytical methods, we identified a
sialidase-sensitive oligosaccharide with the same configuration as the major component. Based on these results, we conclude that the major
sialylated O-linked oligosaccharide of the bovine peripheral nerve -dystroglycan has the following structure: Neu5Ac (and Neu5Gc)
2-3Gal
1-4GlcNAc
1-2Man-Ser/Thr (where Neu5Gc is
N-glycolylneuraminic acid). Based on the radioactivity
incorporated by the
-elimination method, these oligosaccharides
constitute at least 66 molar percent of the total sialylated
O-linked sugar chains. The components corresponding to
fractions F-II and F-III (Fig. 1B), obtained by mild
hydrazinolysis, were not detected by the
-elimination method.
Therefore, Gal
1-4GlcNAc-2AB and Gal-2AB in these fractions were
considered to be the degradation products of the major oligosaccharide by a peeling reaction during hydrazinolysis. No reasonable explanation could be presented for the detection of Glc-2AB. Analysis of the fraction T-II was incomplete because of the limited amount of the
radioactive sample. In view of the previous reports that lectin blots
revealed the presence of a Gal
1-3GalNAc group in
-dystroglycan (4, 7, 10), the hexose-GalNAcOT in the fraction T-II may represent this disaccharide. Mild hydrazinolysis has not been applied
to various samples as frequently as conventional
-elimination to
release their O-linked oligosaccharides. Although our
results suggest the occurrence of a peeling reaction to some extent in the mild hydrazinolysis, this method has great advantages in that it
requires a smaller amount of sample and allows a greater choice of
labeling method. In the mild hydrazinolysis followed by 2AB derivatization, we could determine the major oligosaccharide structure by using less than one-sixth the amount of sample compared with the
conventional alkaline borotritide treatment method. The fluorescent derivatives generally allow us to apply reversed-phase HPLC analysis, by which we could separate the disaccharide derivatives of the four
possible GlcNAc
1-Man isomers (Fig. 4A), and determine the exact linkage between the N-acetylglucosamine and mannose
residues by using only about 2 pmol of the oligosaccharide
derivative.
O-Mannosyl-type linkages, Man-Ser/Thr, have been found in the cell
walls of microorganisms (37-39) and the skin collagen of the clam worm
(40), but they are all neutral or glucuronosyl oligosaccharides. Thus
far, a sialylated oligosaccharide with this type of linkage has been
reported only in rat brain proteoglycans, which have a configuration of
Neu5Ac2-3Gal
1-4GlcNAc
1-3Man1-Ser/Thr (31, 41, 42). The
major sialylated O-linked oligosaccharide in
-dystroglycan identified in this study resembles this structure, but
it is different in the linkage between the
N-acetylglucosamine and the mannose residues. The type of
linkage (GlcNAc
1-2Man or GlcNAc
1-3Man) would have a strong
influence on the three-dimensional structure of the disaccharide. In
the C1 conformer of D-mannose, the hydroxyl residue at C-2
takes an axial position, whereas that at C-3 is equatorial. Therefore,
the carbohydrate chains attached to the nonreducing side of the mannose
residue stretch out in completely different directions. Furthermore,
the result of the disaccharide-2AB analysis by reversed-phase HPLC
suggested that these different structures have different
hydrophobicities (Fig. 4A).
Neu5Ac2-3Gal
1-4GlcNAc
1-2Man is commonly found as a side
chain of complex type N-linked oligosaccharides. For
example, human chorionic gonadotropin and human placental fibronectin
contain such sugar chains (43, 44), but laminin binding of such
glycoproteins has not been reported. The deduced amino acid sequence of
-dystroglycan predicts a mucin domain with a dense clustering of
threonine, serine, and proline residues in the central region of the
molecule (21). Consistent with this, a recent electron microscopic
study of the cardiac muscle
-dystroglycan shows a rod-shaped segment with globular domains attached to both ends (45). Clustering of
O-linked oligosaccharides in a mucin domain is thought to
have important structural implications for the interaction between the
oligosaccharides and their ligands (46). On the other hand, conformational analyses revealed that the side chains of complex type
N-linked oligosaccharides do not always stretch out parallel to each other (47-49), suggesting that N-linked
oligosaccharides are not proper to make sugar chains clustered toward a
particular direction. Although the
sialyl-N-acetyllactosaminyl-mannose structure is common, the
functional significance may be different, depending on whether it links
to the mannosyl-chitobiosyl core of an N-linked oligosaccharide or whether it links O-glycosidically to the
core peptide directly.
We have recently demonstrated that both exhaustive sialidase treatment
of -dystroglycan and the addition of sialic acids to the incubation
medium diminish the binding of laminin to the bovine peripheral nerve
-dystroglycan, suggesting that the sialic acid residues of
-dystroglycan are essential for this binding (10). In this study, we
identified the structure of the major component of the sialylated
O-linked oligosaccharides. The results of the inhibition
study with sialylated oligosaccharides suggest that not only the
anionic charge of sialic acid residues but also the sialyl
N-acetyllactosamine configuration may be important in the
interaction with laminin. To verify whether this unique type of
O-linked oligosaccharide has a specific role in laminin binding, future studies need to clarify whether the
-dystroglycans of other species or tissues have the same structure of sialylated O-linked oligosaccharide as that found in the bovine
peripheral nerve, because it is well known that glycosylation of
proteins is both tissue- and species-specific (50).