From the
Cranin was described in 1987 as a membrane glycoprotein
expressed in brain and many other tissues, which binds laminin with
high affinity in a calcium-dependent manner. Dystrophin-associated
glycoprotein (``dystroglycan'') is a laminin-binding protein
cloned in 1992 whose relation to cranin has remained uncertain. Here we
describe the purification of cranin to homogeneity from sheep brain,
show cranin to be a form of dystroglycan, and localize the N terminus
of
Dystrophin-associated glycoprotein, or dystroglycan, is
synthesized as a large precursor protein which is cleaved into two
distinct proteins,
Cranin
was described in 1987 as a heterogeneous membrane glycoprotein
expressed at low abundance in brain and many other tissues, which binds
laminin with high affinity in a calcium-dependent manner (18). When
Ibraghimov-Beskrovnaya et al.(1) cloned the cDNA of
dystroglycan, they noted its similarities to cranin, and suggested that
the two proteins might be related or identical. However, the relation
between these proteins has remained uncertain. For example, while
cranin was characterized as primarily an integral membrane
protein(18, 19) ,
The DEAE eluate was adjusted to 1 mM in
MnCl
The Jacalin eluate was concentrated to 2 ml on Centriprep
30 filters (Amicon), diluted to 30 ml in TEA buffer, and concentrated
again, to reduce the concentration of free sugar, then treated three
times with 0.5 ml of Bio-Beads SM2 (Bio-Rad) to reduce the detergent
concentration. Eluate was mixed batch-wise with laminin affinity beads
(3.5 ml, laminin coupled to Affi-Gel beads with a commercially
available hydrazide kit (Bio-Rad)) overnight at 4 °C. Beads were
rinsed and eluted with 12 ml of 1 M NaCl, 10 mM EGTA,
10 mM EDTA, 0.1% Triton X-100, TEA buffer. This first
``high salt/chelator'' eluate was concentrated to 100 µl,
diluted back to 10 ml in TEA buffer, and placed again over laminin
beads, rinsed, and the ``calcium chelator'' eluate was
obtained in 12 ml of 10 mM EGTA, 10 mM EDTA, 0.1%
Triton X-100, TEA buffer. The final eluate was concentrated to 100
µl and stored at -70 °C in 10-µl aliquots. Often, the
first laminin bead pass-through was put over laminin beads and eluted
twice again, to obtain additional cranin. (Laminin beads were
regenerated in buffer containing 1 M NaCl, 0.1% Triton X-100,
TEA buffer, and stored in TEA buffer with 0.05% azide.)
In one series of experiments, lectin-binding intensities of
untreated cranin were compared to that of neuraminidase-digested
cranin: cranin (9 µl) was added to 135 µl of TEA buffer, heated
to 85 °C for 5 min, adjusted to 10 mM in CaCl
Perfusion and
post-fixation of rats with 2% paraformaldehyde/periodate/lysine
fixative, infiltration with sucrose, and sectioning of adult rat brain
was performed essentially as described(26) . Sections were cut
at 45 µm and reacted as floated sections. After bleaching in 0.3%
H
Beginning with 36 sheep brains per preparation,
Thus,
cranin/
Taken together, these findings suggest that N-linked saccharides on cranin/
These findings suggest that O-linked
saccharides on cranin/
Several mucin-like recognition molecules expressed in
the immune system represent ligands for the selectins(32) ;
these express a set of fucose- and/or sulfate-dependent epitopes,
including Le
To
confirm that these laminin-binding bands represented authentic
dystroglycan, DEAE-enriched membrane extracts of CHO cells were
immunoblotted with anti-dystroglycan antiserum (Fig. 7).
Moreover, anti-dystroglycan peptide antibody 6C1 stained CHO wild-type
cells (Fig. 8), both CHO mutant cell lines (data not shown) and
adult rat cerebellum (Fig. 9) strongly and specifically by
immunocytochemistry.
Dystroglycan is a cell surface glycoprotein which binds
directly to extracellular matrix proteins and to the underlying
cytoskeleton (4). Dystroglycan is expressed very widely across tissues
and may be important generally in mediating or modulating many of the
effects of extracellular matrix on cells. Yet dystroglycan varies
widely in its association with other proteins. In different places,
dystroglycan associates with laminin(39) , merosin(40) ,
or agrin(7, 8, 9, 11) , and may
associate either with dystrophin (4) or utrophin(5) . In
muscle, dystroglycan also associates with a set of so-called
sarcoglycan complex proteins (3) which are either absent or are
antigenically different in non-muscle tissues(4) .
Dystroglycan isolated from different tissues has different
mobilities on SDS-PAGE gels ranging from 120 to 200
kDa(1, 12, 19, 20) . Since dystroglycan
is encoded by a single-copy gene(2) , and since only a single
mRNA and protein species has been reported to date(2) ,
saccharide modifications of dystroglycan are likely to underlie its
tissue-specific heterogeneity. Considerable indirect evidence suggests
that the O-linked saccharides on dystroglycan are critical for
its high affinity binding to laminin(4, 19) . However,
the functional significance of tissue-specific heterogeneity remains
unclear at present, for example, brain and muscle dystroglycan both
appear to bind laminin and agrin
Another laminin-binding membrane
protein, cranin, was described in brain and other tissues several years
prior to the discovery of dystroglycan(18) . In the present
paper, cranin has been purified to homogeneity from sheep brain,
identified as a form of dystroglycan, and characterized in several ways
which extend and reassess current knowledge of dystroglycan structure
and function.
An alternative view is that dystroglycan is not a
proteoglycan at all, but instead resembles a typical mucin-like
membrane protein(47) . Although Lasky et al.(48) have commented in passing on the mucin-like nature of
sequences present within dystroglycan, it bears emphasis that
dystroglycan exhibits a discrete domain extremely rich in Thr and Pro:
in a stretch of 169 residues(317-485) there are 40 Thr, many of
them clustered in short runs; 10 Ser; and 32 Pro, 24 of them adjacent
to Thr, Pro, or Ser. Such a pattern closely resembles peptide sequences
which have been shown to be good acceptors for GalNAc
transferase(49) . The susceptibility of brain and CHO cell
dystroglycan to O-sialoglycoprotease (Fig. 4) and its
lectin-binding profile (I) are consistent with the
properties of mucin-like proteins, as is the sulfatide-like manner in
which cranin and certain mucin-like proteins each bind their
ligands(19, 32, 50, 51) . Experiments
reported here in CHO mutant cells lacking xylosyltransferase and
galactosyltransferase I indicate that chondroitin sulfate and heparan
sulfate are not necessary for high affinity binding of dystroglycan to
laminin, and indeed these chains appear to be absent entirely from
wild-type CHO cells ( Fig. 6and Fig. 7). It remains
possible that dystroglycan might be a ``part-time''
proteoglycan expressing glycosaminoglycan chains in muscle.
Alternatively, tissue-specific expression of mucin-like chains (52) or differing levels of sulfation of these chains (53) could provide the basis for tissue-specific differences in
dystroglycan structure and function.
In
each step, yields were calculated by measuring protein content of
start, pass through, and eluate fractions. Recoveries were estimated by
comparing the relative intensity of laminin-binding bands obtained from
equal aliquots of start, pass through, and eluate fractions in the
ligand-blotting assay.
Purified cranin (
Lectin-blotting was performed
as described under ``Experimental Procedures,'' scoring band
intensities on a 0-5 scale relative to ConA and Jacalin binding
of untreated samples (set at 5). Binding to untreated samples was
tested for all lectins on at least two occasions under these
standardized comparative conditions, and most lectins which bound
cranin were tested further as well. A ``strong decrease'' in
lectin binding after N-glycanase treatment implies at least
50% loss of band intensity relative to incubated control sample.
Purified samples (
We thank Dr. Jeffrey Esko (University of Alabama at
Birmingham) for gifts of CHO wild-type and mutant cell lines; Dr.
Eugene Butcher (Stanford University, Stanford, CA) for a gift of
MECA-79 antibody; and Dr. Barbara Collins (Michael Reese Hospital,
Chicago, IL) for performing immunocytochemistry on rat cerebellum.
Thanks also to Drs. Steven Rosen, Nancy Schwartz, and Justin Fallon for
helpful advice. Monosaccharide analyses were performed at a facility
supervised by Dr. Adriana Manzi (University of California at San
Diego), and amino acid sequencing at a facility supervised by Dr. John
Leszyk (Worcester Foundation, Shrewsbury, MA).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dystroglycan to amino acid residue 654. We find that brain
-dystroglycan is tightly associated with membranes, and localizes
to regions of synaptic contact as assessed by immunocytochemistry of
rat cerebellum. Brain
-dystroglycan expresses high mannose/hybrid N-linked saccharides, terminal GalNAc residues, and the HNK-1
epitope. Although dystroglycan has previously been presumed to be a
proteoglycan, the amino acid sequence, pI, O-sialoglycoprotease susceptibility, lectin-binding profile,
and laminin-binding properties of brain dystroglycan are more typical
of mucin-like proteins. Furthermore, using CHO mutant cell lines
deficient in xylosyltransferase and galactosyltransferase I, which are
required for glycosaminoglycan biosynthesis, it is shown that
chondroitin sulfate and heparan sulfate are not critical for laminin
binding, and indeed are apparently not expressed at all in dystroglycan
from CHO cells.
-dystroglycan and
-dystroglycan, expressed
widely in many tissues(1, 2) .
-Dystroglycan
resides on the extracellular face of the cell surface associated with
-dystroglycan (3), which in turn spans the membrane and binds
directly to dystrophin (4) or utrophin (5) intracellularly.
Dystroglycan appears to be important for maintaining normal muscle
integrity, and it has been proposed that loss of dystroglycan from the
muscle surface in Duchenne/Becker and other congenital muscular
dystrophies is one of the primary events leading to muscle injury in
these diseases(6) . Recently it has been proposed that
dystroglycan mediates agrin-induced clustering of ACh receptors at the
neuromuscular
junction(7, 8, 9, 10, 11) .
Dystroglycan is also expressed in brain(12) , where its mRNA
largely co-localizes with dystrophin and utrophin(13) . Interest
in brain dystroglycan has been greatly stimulated by the observations
that cognitive deficits and mental retardation occur in many patients
with Duchenne/Becker and other dystrophies(14) , that matrix
proteins play important roles in neuronal migration and axonal
outgrowth(15) , that dystrophin is enriched at postsynaptic
densities in the central nervous system(16, 17) , and
that the gene for dystroglycan in mouse maps closely to the loci of
several genetic neurological diseases(2, 13) .
-dystroglycan has been reported
to be variously an integral membrane component(20) , a
peripheral membrane component(21) , or even freely soluble in
brain(12) . O-Linked saccharides on both proteins have
been proposed to be critical for binding to
laminin(4, 19) . However, cranin was described as a
mucin-like protein which binds in a sulfatide-like manner to the E3
domain of laminin (19); in contrast, dystroglycan has generally been
presumed to be a proteoglycan which binds in a heparin-like manner to
the same domain (4, 12). In the present paper, we describe the
purification of cranin to homogeneity from sheep brain, show that
cranin is a form of dystroglycan, and provide new biochemical analyses
that address some of these questions. Some of these results have been
presented in preliminary form(22) .
Purification of Cranin
The procedure previously
described for embryonic chick brain (19) was scaled-up for
batches of 36 adult sheep brains. Brains were obtained at a local
slaughterhouse and kept on ice for 2 h before homogenization of
whole brains or gray matter in a Waring blender (low setting 30 s, high
setting 1 min) in cold 50 mM Tris, 5 mM EDTA, 150
mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 10
mMN-ethylmaleimide, pH 7.6, adjusted with 6 N HCl, a ratio of 1.6 liters of buffer for each 12 brains. Crude
membranes were pelleted (GSA rotor, 20,000
g, 1 h, 4
°C), rinsed with an equal volume of fresh 10 mM Hepes, 0.5 M NaCl, 6 M urea, pH 7.4, adjusted with 5 N NaOH, then diluted 4-fold with 10 mM Hepes, pH 7.8, and
spun down again. Membranes were solubilized in 4 volumes of 2% Triton
X-100 in TEA
(
)buffer (0.01 M triethanolamine, 137 mM NaCl, 1 mM CaCl
, 1 mM MgCl
, pH 7.4, adjusted
with 5 N NaOH) containing 5 mg/liter aprotinin, on ice for
6-8 h. After pelleting again, the supernatant was incubated
batch-wise with DEAE-Sepharose CL-6B beads (7.2 liters of extract with
1.5 liters of beads) at 4 °C for at least 1 day on a rocker,
filtered, and rinsed on a large sintered glass funnel, and eluted with
4.5 liters of TEA buffer containing 0.5 M NaCl, 0.1% Triton
X-100. (Beads were regenerated with 2 M NaCl, 0.1% Triton
X-100, TEA buffer, and stored in TEA buffer containing 0.05% sodium
azide until reuse.)
, and passed in a continuous loop over a ConA-lectin
affinity column (100 ml, Bio-Rad) for several days, rinsed until a
plateau was reached by absorbance of the pass-through at 280 nm, and
eluted slowly overnight with 300 ml of 0.4 M
-methylmannoside, 0.1% Triton X-100, TEA buffer. The ConA eluate
was concentrated 10-fold using a Spectrum stirred cell and Type C
membrane (cutoff = 50 kDa), then diluted 10-fold in 175 mM Tris, pH 7.5, and passed in a continuous loop over a Jacalin
lectin affinity column (10 ml, Vector Labs) for 2 days. After rinsing
to a plateau, sample was eluted overnight in 30 ml of 0.8 M melibiose, 0.1% Triton X-100, TEA buffer. The high concentrations
of sugars and slow elution times were essential to obtain an optimal
yield, because of the very tight binding of cranin to the lectin
columns.
Electrophoretic Methods, Ligand-blotting Assay, and
Immunoblotting Assays
In general, these methods were performed
as described previously(18, 19) . Proteins were
separated on 7.5% SDS-PAGE gels under reducing conditions, transferred
to Immobilon-P membranes, and blocked overnight in 2% BSA, 0.1% Nonidet
P-40, TEA buffer at 4 °C. In ligand-blotting assays, blots were
incubated with laminin (from murine EHS sarcoma, Life Technologies,
Inc.; 0.2 µg/ml in 1% BSA, TEA buffer), rinsed, incubated with an
affinity-purified polyclonal anti-laminin antiserum (1:5,000), rinsed,
incubated with peroxidase-conjugated anti-rabbit IgG (1:5,000), rinsed,
and visualized with chemiluminescence (ECL, Amersham) usually with
exposures of 1 min or less. As negative controls, laminin was omitted,
or blots were incubated with laminin in the presence of 5-10
mM EGTA. In immunoblot assays, blots were blocked overnight,
incubated in the presence of primary antibodies diluted in 1% BSA, TEA
buffer for 1-2 h at room temperature, rinsed, incubated in the
presence of secondary antibodies (usually diluted 1:5,000 in 1% BSA
when the primary antibody was polyclonal, and 1:1,000 when the primary
antibody was monoclonal), rinsed, incubated in avidin-biotin conjugate
(Vectastain Elite ABC kit, Vector Labs, 1:4 to 1:10), rinsed, and
visualized either with chemiluminescence or with 3,3`-diaminobenzidine
(0.3 mg/ml) and HO
(30%, 8 µl/100 ml).
Isoelectric Focusing and Two-dimensional
Electrophoresis
These were carried out using a Protean II xi
cell (Bio-Rad), following the manufacturer's recommended
protocols and solutions exactly, with the exception that isoelectric
focusing tube gels were cross-linked with 1.6% bis-acrylamide instead
of 0.8%. Also, ampholytes used were Pharmalyte brand (Sigma, pH
4-6.5 and 3-10) in place of Bio-Lyte (Bio-Rad). Purified
native cranin (5 µl) was mixed with isourea solution (25 µl),
with or without two-dimensional SDS-PAGE standards (Bio-Rad) added to
provide an internal pI calibration. Alternatively, cranin (4 µl)
was denatured with 4 SDS-PAGE buffer (1 µl, final
concentration 1.6% SDS), placed on a boiling water bath for 3 min, and
cooled before adding isourea solution. To resolve embryonic day 14
chick brain ConA-enriched membrane extracts(19) , solid urea (24
mg) and dithiothreitol (5 µl, final concentration 1%) were added to
the extract (50 µl). To resolve soluble (not membrane-bound) chick
brain proteins, supernatant from brain homogenates was partially
enriched in cranin by passage over DEAE and ConA columns(19) ,
then eluate (10 µl) was mixed with urea (5 mg), 3-10
ampholytes (3 µl), and two-dimensional PAGE standards (Bio-Rad, 25
µl in isourea solution).
Lectin-binding Assays
For routine lectin-blotting
assays, blots were blocked overnight as above, incubated in
biotinylated lectin (from Vector or Sigma; 0.5 µg/ml) in TEA
buffer, rinsed, incubated in avidin-biotin conjugate, and visualized
with 3,3`-diaminobenzidine and HO
. However,
this method gave high backgrounds for certain lectins because of direct
binding to BSA present in the blocking and rinse solutions. In order to
compare the relative binding of a large number of different lectins,
the more sensitive and generally applicable method of Gravel et al. (24) was adopted, in which blots were blocked in 0.5% Tween 20,
TEA buffer for 30 min with rocking at room temperature;
lectin-containing and rinse solutions all contained 0.5% Tween 20, TEA
buffer; and bands were visualized with chemiluminescence, dipping all
treatment groups together and holding exposure times constant at 30 s.
,
and digested with neuraminidase (from Arthrobacter ureafaciens, Sigma,
1 unit/ml final concentration) for 2 h at 37 °C, stopped by adding
an equal volume of 4
SDS-PAGE buffer, plus 45 µl of 11%
dithiothreitol, boiled, and loaded onto 9 lanes of a SDS-PAGE gel.
These incubation conditions removed
80% of sialic acid residues as
assessed by the induced decrease in Mackia amurensis II lectin binding.
In a second series of experiments, lectin-binding intensities were
compared between samples of cranin that had been incubated with N-glycanase, and control samples incubated without enzyme.
Samples of cranin were reduced and denatured by adding dithiothreitol
and sodium dodecyl sulfate (1% final concentration of each), heated to
85 °C for 5 min, diluted 10-fold in 20 mM EDTA, 1% Nonidet
P-40, TEA buffer, and incubated for 16 h at 37 °C, in the presence versus absence of recombinant N-glycanase (Genzyme, 6
units/ml final concentration). Reactions were stopped by adding an
equal volume of 4
SDS-PAGE buffer plus dithiothreitol. These
incubation conditions removed
80% of N-linked saccharides
as assessed by the induced decrease in ConA-lectin binding.
Generation of Monoclonal Anti-dystroglycan
Antibodies
Peptides were synthesized as the free acid
corresponding to human dystroglycan amino acid residues 572-604
on an ABI model 430A peptide synthesizer using Fmoc/NMP/HObt chemistry.
Peptides were cleaved from resin with trifluoroacetic acid and
subsequently lyophilized, dissolved in water at 10 mg/ml, emulsified
with a 4-fold excess of Freund's complete adjuvant, and injected
intraperitoneal or subcutaneously into a mouse; subsequent injections
were made every three weeks in Freund's incomplete adjuvant. The
test bleed antiserum recognized the peptide by ELISA assay and purified
cranin by immunoblotting. Spleen cells were fused with mouse SP 2/0
myeloma cells(25) ; hybridomas were screened for their ability
to recognize the peptide in the ELISA assay, to stain purified cranin
on immunoblots, and to recognize dystroglycan within fixed frozen
sections of adult rat cerebellum. Monoclonal antibody 6C1, of the IgM
class, was positive in all three assays.
Immunocytochemistry
CHO wild-type or mutant cells
were plated on glass coverslips (Lab-Tek 4 well chambers, Nunc) in 10%
fetal calf serum, F-12 medium, plus gentamicin, and incubated overnight
at 5% CO, 37 °C. Cells were briefly rinsed in TEA
buffer, then fixed in 3.7% formaldehyde in TEA buffer for 20 min, room
temperature; rinsed again, and permeabilized and bleached in 0.3%
H
O
in MeOH for 20 min at room temperature.
Cells were rinsed again, blocked for several hours in 10% horse serum,
incubated in 6C1 (full-strength hybridoma conditioned medium) for at
least 1 h, rinsed twice in 1% serum or 3% BSA, incubated in secondary
antibody (biotinylated anti-mouse Ig, Vector, 1:1,000) for 1 h, rinsed,
incubated in avidin-biotin conjugate (1:3) for 1 h, rinsed, and
visualized with 3,3`-diaminobenzidine and H
O
.
As negative controls, an irrelevant monoclonal antibody
(``1A5'') was substituted which did not recognize
dystroglycan peptide 572-604 by ELISA assay, or primary or
primary/secondary antibodies were omitted.
O
, 30 min, sections were blocked in 5% horse
serum, 0.25% Triton X-100, phosphate-buffered saline, for several
hours. Sections were incubated with 6C1 (full-strength conditioned
medium or diluted up to 1:50) overnight at 4 °C with gentle
rocking, rinsed, incubated with biotinylated anti-mouse Ig (rat
adsorbed, Vector, 1:200), 1.5 h, rinsed, incubated with avidin-biotin
conjugate (1:2) for 1 h, rinsed, and visualized with Very Intense
Purple (Vector Labs). As negative controls, a variety of other IgG and
IgM antibodies were used in place of 6C1, or hybridoma feeding medium
was used.
Purification of Cranin
100
µg of purified cranin was obtained (). Silver staining
revealed a broad
115-120 kDa band plus a doublet at 43 kDa (Fig. 1). The 115-120 kDa band corresponded to cranin as
detected by ligand-blotting assay of this sample, while the 43-kDa
doublet did not bind laminin in this assay and appears to have
co-purified with the larger band. Ligand blotting was the most
sensitive method of detecting cranin, since a strong band was routinely
observed using 0.5-1% of a preparation in this assay, whereas
silver staining required loading 5% of a preparation on a lane to see
bands reliably (Fig. 1). The 115-120-kDa band stained
heterochromatically (blue) with Stains-all, but failed to stain visibly
with Coomassie Blue, even when 20% of a preparation was loaded on a
single lane, which showed good staining of the 43-kDa doublet.
Figure 1:
Purified cranin. A
sample of purified cranin (5% of one 24-brain preparation) was
separated on a 7.5% SDS-PAGE gel under reducing conditions and silver
stained, revealing a prominent band at 115-120 kDa and a doublet
at 43 kDa. Molecular mass markers (Sigma) are, from top, 205, 116, 97,
66, and 45 kDa.
Purified cranin was transferred to Immobilon-P membranes; the
110-kDa band was digested with endo-LysC peptide mapped by reverse
phase high performance liquid chromatography, and the three most
hydophobic peaks subjected to amino acid sequencing ().
Two nonoverlapping peptides each gave sequences identical (except for
uncertain residues) to the published sequences of rabbit, human, and
mouse muscle dystroglycan(1, 2, 13) . As well,
the two bands at 43 kDa were subjected to N-terminal sequencing (), and were again identical (except for uncertain
residues) to the published sequences of rabbit, human, and mouse
dystroglycan. Furthermore, the 110-kDa band was specifically recognized
by anti-dystroglycan peptide antibodies (see below). Purity of the
cranin band was confirmed by two-dimensional SDS-PAGE, revealing a
single band co-migrating with cranin as assessed by silver staining (Fig. 2), ligand-blotting assay, lectin blotting, and
immunoblotting (see Fig. 5, below). These findings demonstrate
that cranin is a form of dystroglycan. The data also identify the N
terminus of -dystroglycan as amino acid residue 654.
Figure 2:
Profile of cranin on two-dimensional PAGE
gels. An aliquot of cranin (eluted from laminin affinity beads in 1 M NaCl, 10 mM EGTA, 10 mM EDTA, TEA buffer)
was mixed with two-dimensional SDS-PAGE standards (Bio-Rad) to provide
an internal pI calibration, subjected to isoelectric focusing in 8 M urea containing CHAPS and Nonidet P-40, followed by
electrophoresis by SDS-PAGE on a 7.5% gel and silver staining. The
position of cranin (arrow) relative to conalbumin (A,
pI = 6.0, 6.3, 6.6), bovine serum albumin (B, pI
= 5.4-5.6), and bovine muscle actin (C, pI
= 5.0, 5.1) indicates a heterogeneous pI for cranin, ranging
from 5.3 to 6.0, and centered at 5.7. Molecular weight markers are at right.
Figure 5:
HNK-1 immunoreactivity. Aliquots of cranin
were subjected to two-dimensional PAGE exactly as described in the
legend to Fig. 2, followed by transfer to Immobilon membranes and
immunoblotting using HNK-1 antibody as described under
``Experimental Procedures.'' Blots were incubated in HNK-1
hybridoma conditioned medium (diluted 1:30), biotinylated anti-mouse
IgM (1:5,000), avidin-biotin conjugate (1:5), and visualized with
diaminobenzidine and HO
. Cranin is strongly
positive, whereas the Bio-Rad standards and molecular weight markers
are not visualized (cf. Fig. 2). No bands were observed when
primary antibody was omitted, or when a different IgM antibody (CD15 or
Le
) was employed.
The pI of
purified cranin was ascertained by two-dimensional SDS-PAGE. Cranin
exhibited a broad, heterogeneous pI profile from 5.3 to 6.0, centered
at 5.7 (Fig. 2). The pI values were similar whether cranin
was detected by silver staining, lectin-blotting, ligand-blotting, or
immunoblotting assays (e.g.Fig. 5below); whether
isoelectric focusing was carried out in the presence or absence of
urea; and whether pH gradients were assessed by using pI protein
standards mixed with cranin (Fig. 2) or by slicing isoelectric
focusing gels and measuring the pH values directly. Even when cranin
was fully dissociated from the 43-kDa proteins and denatured with
boiling in SDS-PAGE buffer under reducing conditions prior to carrying
out isoelectric focusing in the presence of 8 M urea, the pI
was still centered on 5.7. Furthermore, a similar pI value was obtained
when ConA-enriched membrane extracts (19) were employed instead
of purified cranin, and when embryonic day 14 chick crude brain
extracts were tested (both proteins enriched from the supernatant and
from the pellet of the crude homogenate were tested). Finally, to
verify that a more highly acidic glycoprotein of similar size could be
resolved in this system, partially purified membrane extracts were
separated and immunoblotted to detect the MAb 4/199
antigen(25) ; the pI of this antigen was 4.9-5.6, centered
on 5.2, in agreement with our previous findings using a different
isoelectric focusing apparatus(25) .
-dystroglycan appears to be only mildly acidic, which is in
keeping with its elution from DEAE columns at 0.5 M NaCl(18) . The pI value of 5.7 is much less acidic than the
value of
3.7 previously reported not only for 156-kDa muscle
dystroglycan (27) but for 120-kDa soluble chick and bovine brain
dystroglycan as well(12) . We do not currently have an
explanation for this discrepancy, although it should be noted that
chick and bovine brain dystroglycan were also reported to elute from
DEAE columns at 0.5 M NaCl(12) , which might not be
expected if it were extremely acidic.
Saccharide Modifications of Cranin
N-Linked Saccharides
The amino acid sequence of
rabbit and human dystroglycan predicted by cDNA cloning exhibits 3
potential consensus sites for N-glycosylation in
-dystroglycan, and a single potential site in
-dystroglycan
at residue 661(1, 2) . All four sites are conserved
between rabbit and human species. The
-dystroglycan bands bound
ConA and Lens culinaris lectins by lectin blotting (Fig. 3). As well, residue 661 (asparagine) could not be reliably
sequenced from either band of the 43-kDa doublet, even though the
adjacent asparagine at 662 was readily detected (). Each
of these findings indicate that residue 661 is glycosylated in the
purified protein.
Figure 3:
ConA binding sites are susceptible to endo
H. Aliquots of cranin (0.5 µg) were reduced and denatured by
adding dithiothreitol and sodium dodecyl sulfate (1% final
concentration of each), heated to 85 °C for 5 min, diluted 10-fold
in 1% Triton X-100, 0.1 M sodium acetate/acetic acid buffer,
pH 6.0, with 2 mM phenylmethylsulfonyl fluoride and 50 µg
of BSA added. Lane A, control, incubated for 16 h at 37
°C. B, incubated with recombinant endo H (S.
plicatus, Boeringer Mannheim, 50 milliunits/ml final
concentration). Samples were assayed for ConA-lectin binding by the
method of Gravel et al. (24) (see ``Experimental
Procedures''). Cranin/
-dystroglycan (upper arrow)
and the 43-kDa doublet/
-dystroglycan (lower arrow) both
bound ConA-lectin in lane A, whereas all staining was
abolished after endo H digestion (lane B). Jacalin lectin
blotting verified that cranin was still present in the endo H-digested
lane, exhibiting a slightly lower apparent molecular mass by
5 kDa
compared to the controls (data not shown; cf. Ref. 19). The
faint band seen in both lanes represents BSA. (Another band of greater
apparent mass than cranin is also seen in the lane at left,
representing an abundant protein found in the Jacalin eluate; this band
appears to bind laminin affinity columns weakly, and is present in the
eluate when cranin is eluted from laminin beads with high salt, but
usually is lost when these eluates are passed over laminin beads a
second time and eluted with chelators
alone.)
Lectin blotting profiles provided further
information regarding N-linked saccharides on
cranin/-dystroglycan. All ConA-lectin binding sites were
susceptible to endo H (Fig. 3), indicating that they represent
high mannose and/or hybrid structures. Several other lectins also bound
cranin/
-dystroglycan in a manner that was strongly decreased after N-glycanase digestion (I), indicating that they
bind N-linked saccharides (without precluding that they may
also bind O-linked saccharides to some extent as well).
According to the manufacturer's specificity guidelines, ricin I
recognizes terminal Gal or GalNAc, Dolichos biflorus recognizes terminal
-linked GalNAc, Sambucus nigra recognizes Sia-
2-6-Gal, Griffonia I recognizes
-linked Gal or GalNAc, and wheat germ agglutinin recognizes sialic
acid or GlcNAc (I). It is likely that wheat germ
agglutinin recognizes sialic acid here, rather than GlcNAc, because
succinylated wheat germ agglutinin bound cranin only faintly, and
because neuraminidase digestion abolished binding to both of these
lectins.
-dystroglycan are
primarily high mannose/hybrid structures, that at least some chains
express terminal GalNAc residues (and possibly Gal as well), and that
at least some of these are sialylated. In contrast, the single N-linked saccharide on the 43-kDa doublet/
-dystroglycan
was recognized strongly by ConA-lectin, and faintly by lens culinaris
lectin and wheat germ agglutinin, but not by any of the other lectins
indicated in I.
O-Linked Saccharides
The predicted amino acid
sequence of -dystroglycan includes a domain extremely rich in
threonine and proline, which is typical of mucin-like proteins (see
below). O-Sialoglycoprotease is an enzyme from Pasteurella
hemolytica that, to date, has been reported to attack only
glycoproteins expressing highly clustered, sialylated O-linked
saccharides(28, 29) . Cranin/
-dystroglycan proved
to be highly susceptible to O-sialoglycoprotease, both when
the purified protein was tested and when ConA-enriched membrane
extracts having cranin as a very minor component were tested (Fig. 4). Prolonged incubation with O-sialoglycoprotease
had no effect upon the MAb 4/199 antigen(25) , even though it is
a laminin-binding membrane glycoprotein of similar size present within
the same extract (Fig. 4, C and D), and even
though it expresses some O-linked saccharides(19) .
Figure 4:
Susceptibility to O-sialoglycoprotease. A and B, purified
cranin (1.5 µg) was incubated in TEA buffer (20 µl). Lane A, control, incubated for 1 h at 37 °C. B,
incubated with O-sialoglycoprotease (Cedar Lane, Ltd., 4
µl). Samples were assayed for laminin binding via ligand blotting
assay. Similar results were obtained using ConA-lectin blotting assays
(not shown). C and D, embryonic day 14 chick brain
membrane extracts were employed that were partially enriched in cranin
through DEAE and ConA column steps (19). Extract (25 µl) was mixed
with BSA (50 µg) and aprotinin (10 µg). Lane A,
control, incubated for 16 h at 37 °C. B, incubated with O-sialoglycoprotease, 2 µl. In the top panel,
ligand blotting assay revealed a complete loss of cranin in the treated
sample, whereas, in the bottom panel, immunoblotting with
monoclonal antibody 199 shows that the MAb 4/199 antigen was entirely
unaffected.
Lectin-blotting profiles also provided information regarding O-linked saccharides on cranin. Six lectins bound to
cranin/-dystroglycan in a manner that was not affected at all by N-glycanase digestion (I). Three of these have
previously been reported to bind
cranin/
-dystroglycan(4, 19, 21) : Jacalin
and peanut agglutinin recognize Gal
1-3GalNAc disaccharides
characteristic of mucins, and as expected, binding of these lectins was
increased after neuraminidase treatment (I). Mackia
amurensis II lectin recognizes
2,3-linked sialic acid,
particularly that linked to Gal-GlcNAc. Of particular interest are
three additional lectins, not previously reported: Vicia villosa lectin, which recognizes terminal GalNAc residues; Wisteria lectin, which preferentially recognizes GalNAc-Gal; and Griffonia
II lectin, which specifically recognizes terminal GlcNAc residues (I).
-dystroglycan strongly express saccharide
chains with terminal GalNAc residues, at least some of which are
sialylated (I). It is interesting to note that the entire
lectin-blotting profile described here is consistent with the presence
of complex mucin-like structures having variable elongation and
variable sialylation of the terminal sequence
GalNAc-[Gal]-Gal-GlcNAc(30) . However, direct
identification of saccharide chain lengths and sequences will have to
await more detailed structural characterization (e.g. by mass
spectrometry). In contrast to the prominence of O-linked
saccharides on cranin/
-dystroglycan, the 43-kDa
doublet/
-dystroglycan only exhibited faint binding of Jacalin and
no binding of the other five lectins described above.
Carbohydrate Epitopes
Cranin was well stained by
HNK-1 antibody (Fig. 5), which recognizes a sulfated epitope
expressed on many cell adhesion molecules within the nervous system.
This epitope may not be critical for laminin binding, however, since we
were unable to inhibit cranin-laminin binding by preincubating blots
containing purified cranin with full-strength HNK-1 conditioned medium.
Although it remains uncertain whether the HNK-1 epitope in
glycoproteins contains a terminal 3-sulfated glucuronic acid, it does
appear to be associated with sulfated carbohydrate
moieties(31) . HNK-1 antibody probably recognizes a sulfated
moiety in our immunoblots as well, since HNK-1 immunoreactivity of
brain membrane proteins was greatly diminished by desulfation
(methanolic HCl 50-100 mM, 24 h, 25-37 °C),
whereas Jacalin lectin binding was unaffected by this treatment (data
not shown).
, SLe
, and MECA-79(33) ,
which represent modifications of a lactosaminoglycan backbone in some
cases(34) . However, Le
, SLe
, and
MECA-79 antibodies did not recognize cranin on immunoblots, either
before or after neuraminidase digestion. These findings are consistent
with the lack of binding of Lotus and Ulex I lectins, which recognize
fucose within Le
structures, and with the lack of binding
of a set of lectins (Lycopersicon, Datura, Solanum, and pokeweed) which typically recognize
lactosaminoglycan structures (I).
Monosaccharide Composition
Neutral and amino
sugars from samples of purified cranin (containing both
-dystroglycan and
-dystroglycan) are shown in .
The composition is not remarkable, except that mannose is the most
abundant sugar. This may simply reflect the presence of high
mannose/hybrid structures as described above, although the presence of O-linked mannose (35) cannot be excluded at present.
Dystroglycan in CHO Wild-type versus
Glycosaminoglycan-deficient Cells
We examined the expression of
dystroglycan in CHO cells, since cranin has been reported to be
expressed in fibroblasts(18) , and since several mutant CHO cell
lines have well characterized single enzyme defects in
glycosaminoglycan biosynthesis(36, 37, 38) .
Ligand-blotting assays of wild-type CHO crude membrane extracts
revealed a single laminin-binding band at 110-120 kDa (Fig. 6); like dystroglycan isolated from brain, this band was
not removed by rinsing membranes with high salt and urea, and could be
partially purified on DEAE and ConA columns in a similar fashion. No
laminin-binding bands were observed if assays were carried out in the
absence of calcium, or if extracts were first digested with O-sialoglycoprotease.
Figure 6:
Expression of -dystroglycan in CHO
cells. CHO wild-type cells (A), xylosyltransferase-deficient
cells (B), and galactosyltransferase-deficient cells (C) were grown to confluency, scraped in homogenization buffer
(see ``Experimental Procedures'' for sheep brain
purification), and subjected to 30 strokes of a Dounce homogenizer.
Crude membranes were pelleted in a Sorvall SS-34 rotor at 20,000
g, 45 min, then rinsed in high salt/urea buffer,
pelleted again, and solubilized. Aliquots representing material from
approximately 4 confluent 100-mm tissue culture dishes were loaded onto
each lane (adjusted further so that equal amounts of protein were
loaded in each lane), and analyzed by ligand blotting assay. A broad
band at 110-120 kDa was observed in all three groups (large
arrow). This was the only laminin-binding band detected in these
extracts, since the other bands were still observed when laminin was
omitted from the assay (data not shown; the highest band (small
arrow) migrates at
220 kDa and presumably represents laminin
B chains produced by the CHO cells).
Wild-type CHO cells (K1 strain) were
grown in parallel with two mutant cell lines (a generous gift of Dr. J.
Esko), one deficient in xylosyltransferase (``745'') and one
deficient in galactosyltransferase I
(``761'')(36, 37, 38) . Both have been
characterized as failing to synthesize chondroitin sulfate and heparan
sulfate. After confirming that these mutants incorporated relatively
little radioactive sulfate into trichloroacetic acid-precipitable
material (<15% of wild-type levels), all three lines were grown to
confluency, membrane extracts were prepared, and the mobility and
laminin-binding properties of these mutants were compared with
wild-type cells. As shown in Fig. 6, all three cell lines
expressed a single broad laminin-binding band at 110-120 kDa. The
apparent size and yield of dystroglycan were similar in the wild-type
and xylosyltransferase mutant, whereas the galactosyltransferase I
mutant consistently expressed a higher yield than the wild-type (Fig. 6). In all three cell lines, no laminin-binding bands were
observed if assays were carried out in the absence of calcium, or if
extracts were first digested with O-sialoglycoprotease.
Figure 7:
Immunoblotting of CHO membrane extracts
with -dystroglycan antiserum. CHO cells from wild type and mutant
lines were grown to confluency in parallel and homogenized. In order to
obtain adequate amounts of dystroglycan for immunoblotting, crude
membrane extracts from 25 100-mm dishes were rinsed with high salt and
urea, solubilized, and enriched over DEAE beads with elution at 0.5 M NaCl; dystroglycan still represents only a very minor
component of this eluate. Equal amounts of protein were loaded on each
lane. A-C, immunoblotted with anti-dystroglycan peptide
antiserum (1:200). D-F, immunoblotted with normal mouse serum
as a negative control. A and D, wild-type. B and E, xylosyltransferase-deficient. C and F, galactosyltransferase I-deficient. A single broad band at
110-120 kDa is seen in each lane (arrow; cf.
Fig. 6). Molecular weight markers are at
left.
Figure 8:
Immunostaining of CHO cells with
-dystroglycan peptide antibody 6C1. Upper panel, CHO
wild-type cells were immunostained as described under
``Experimental Procedures.'' Particularly strong staining was
observed in a juxtanuclear region (presumably the Golgi region) within
cells that were well spread. In cells that were more rounded, discrete
punctate staining of the cell surface was also observed, apparently
corresponding to small microvillar protrusions of membrane. Lower
panel, as a negative control, no staining was observed when cells
were incubated with hybridoma conditioned medium 1A5 (which did not
recognize dystroglycan peptide 572-604 by ELISA
assay).
Figure 9:
Immunostaining of adult rat cerebellum
with -dystroglycan peptide antibody 6C1. Cerebellar sections were
immunostained as described under ``Experimental Procedures.''
Strong, selective staining was observed of fibers throughout the
molecular layer (M). Little staining is observed within the
granule cell layer (G) or white matter (W) except for
some blood vessels. At higher power, in favorable sections (inset), some of the stained fibers are seen to exhibit a
pattern of dots, short segments, and spines that closely follows the
trajectory of climbing fibers upon Purkinje cell dendrites. This
suggests that the immunoreactive product is localized to regions of
synaptic contact upon the dendrites. Although one cannot resolve
whether staining is presynaptic, postsynaptic or both, the findings are
consistent with: (a) an in situ hybridization study
(13) indicating that dystroglycan mRNA is restricted to Purkinje cells
in the adult mouse cerebellum; (b) an immunocytochemical study
(17) indicating that dystrophin is enriched at central nervous system
postsynaptic densities in general, and upon Purkinje cell dendrites in
particular; and (c) reports that dystroglycan is enriched at
postsynaptic membranes in electric organ (20) and muscle (Ref. 5; see
also Ref. 11). When sections were incubated with a variety of other IgG
or IgM antibodies, or with hybridoma feeding medium alone, some light
diffuse staining of Purkinje cell bodies was still observed, but the
characteristic pattern of fiber staining within the molecular layer was
absent. Also, fiber staining intensity was reduced when 6C1 (2.5 ml at
1:50 dilution) was preabsorbed with peptide 572-604 (4 mg/ml),
but not when 6C1 was preabsorbed with a different peptide (data not
shown).
(
)in a similar
manner(4, 7) .
Reassessing Properties of Brain Dystroglycan
In our
hands, brain dystroglycan is strikingly similar to dystroglycan
isolated from a modified synapse, the Torpedo electric
organ(7, 20) , in that the -subunits behave as
integral membrane components(18, 19) , and co-purify on
ligand affinity columns with a tightly (but noncovalently) associated
-dystroglycan subunit (see also Ref. 4). These findings are in
marked contrast to a report by Gee et al.(1993) which
described
80% of brain
-dystroglycan as recoverable from
ultrasupernatants of saline tissue homogenates, i.e. not
membrane bound, and not associated with
-dystroglycan after
purification(12) . Several other discrepant findings also exist
between our two laboratories (see ``Results'' and Ref. 19)
and the reasons remain unclear. However, monoclonal anti-dystroglycan
peptide antibody 6C1 localized dystroglycan in adult rat cerebellum
along Purkinje cell dendrites (Fig. 9), in a distribution which
co-localizes with dystrophin(17) . Such a pattern strongly
suggests that a significant portion of dystroglycan is membrane
associated, at sites of synaptic contact, in the brain in
situ.
N Terminus of
Analyses of
consensus sequences for proteolytic cleavage have suggested that the N
terminus of -Dystroglycan
-dystroglycan might lie at residues 457 (1) or
640(12) . Direct N-terminal sequencing of both 43-kDa doublet
bands now reveals the site to be residue 654 (), which is
somewhat surprising since it is not directly adjacent to a basic
residue.
N-Linked Saccharides
The N-linked
saccharides expressed on - and
-dystroglycan are shown to be
of the high mannose/hybrid variety (Fig. 3). These are not
necessary for laminin binding(4, 19) , nor does mannan
inhibit laminin binding(19) , but it is possible that they might
be involved in other recognition or binding events. For example,
oligomannosides have been reported to interact with cell surfaces to
promote cell spreading(41) , and NCAM and L1 are known to bind
to each other in cis on cell surfaces via a lectin-like domain
of NCAM binding to oligomannose residues on L1(42) . In this
regard, it is noteworthy that three of the four potential N-glycosylation sites in dystroglycan are located more
proximally to the membrane than the mucin-like domain, and hence would
be well located to interact in cis with sarcoglycan proteins
(3, 6) or other proteins on the cell surface.
Other Saccharide-based Recognition Motifs
Both N-linked and O-linked saccharides on
cranin/-dystroglycan are shown to bind a number of lectins
strongly which recognize terminal GalNAc residues (I). It
is worth noting that GalNAc-specific lectins are also known to bind
selectively to the neuromuscular junction, where they identify a number
of glycoproteins and glycolipids(43) . Moreover, synthetic
oligosaccharides such as GalNAc-
1,3-Gal-
1,4-Glc are
especially potent, nontoxic inhibitors of proliferation in neural
cells(44) . The HNK-1 epitope, expressed on brain
-dystroglycan (Fig. 5), has also been proposed to have a
potential recognition/binding role(45, 46) . Finally,
the presence of terminal GlcNAc residues (indicated by Griffonia II
lectin binding) suggests that
-dystroglycan may be an acceptor for
Gal- or GalNAc-specific glycosyltransferases.
Is Dystroglycan a Proteoglycan?
Most previous
discussions of dystroglycan have presumed that it is likely to be a
proteoglycan. The high carbohydrate content, Alcian blue
staining(4) , and low pI (3.7) of muscle dystroglycan (27) are all typical of proteoglycans. Moreover, binding of
dystroglycan to matrix proteins is inhibited by
heparin(4, 9, 12) . S27 muscle cells, which are
deficient in synthesizing chondroitin sulfate and heparan sulfate, make
a form of dystroglycan which is abnormally small and which binds agrin
and laminin poorly(8, 9, 11) . Finally, the
predicted amino acid sequence of dystroglycan contains two conserved
Ser-Gly consensus sequences for addition of
glycosaminoglycans(2) . On the other hand, at least four groups,
working with both muscle and brain dystroglycan, have been unable to
affect the mobility or laminin-binding properties of dystroglycan using
chondroitinases, heparitinases, or
keratanase(4, 7, 12, 19) , and cranin
was previously reported to resist nitrous acid treatment which degrades
heparin and heparan sulfate(18) . Moreover, the molecular defect
in S27 cells has not been identified, and it has not been ruled out
that O-linked saccharides might be generally abnormal in these
cells.
Future Prospects
Our data demonstrate the
purification of dystroglycan from mammalian brain in amounts which
should permit direct structural examination of its saccharide moieties.
The high and selective susceptibility of dystroglycan to O-sialoglycoprotease, together with its lack of toxicity for
living myeloid cells (29) and NG108-15 cells,(
)suggests exploring the use of this protease as a
novel experimental tool for identifying and analyzing the functions of
mucin-like proteins in neural cells. Monoclonal anti-dystroglycan
peptide antibody 6C1 is, to our knowledge, the first antibody described
which is suitable for carrying out immunocytochemical localization of
-dystroglycan in the central nervous system. Therefore, the
present studies open several new avenues of research.
Table: Purification of cranin from sheep brain
Table: Amino acid sequencing of cranin peptides and
N-terminal sequence of 43-kDa doublet
100
µg) was separated on 7.5% SDS-PAGE gels under reducing conditions,
transferred to Immobilon-P membranes (Millipore) as described (18), and
stained with Amido Black; the 120-kDa band was cut out and digested
with endo Lys-C followed by separation on reverse-phase HPLC columns as
described (7), and the three most hydrophobic peaks were sequenced as
described (7, 23). The upper and lower bands of the 43-kDa doublet were
cut out separately and subjected to N-terminal sequencing. Initial
yields of cranin peptides were
10 pmol, and for 43-kDa doublet
bands were
2 pmol each. Dystroglycan residues are numbered
relative to the predicted amino acid sequences of rabbit, mouse and
human species (1, 2, 13). Uncertain assignments are indicated in bold
face. Asterisks indicate asparagines which satisfy consensus for N-linked glycosylation.
Table: Lectin-blotting
profile of cranin/-dystroglycan
Table: Monosaccharide
composition (neutral and amino sugars)
50
µg in 60 µl; containing both cranin/
-dystroglycan and the
43-kDa doublet/
-dystroglycan) were treated with BioBeads SM
(Bio-Rad) to remove excess detergent. After hydrolyzing with
trifluoroacetic acid (2 M, 3 h, 100 °C), neutral and amino
sugars were separated by HPLC using a CarboPac PA-1 column, quantified
by peak areas, and identified relative to standards run in parallel.
The GlcNAc peak was normalized to 1.00, and other peaks compared to it.
Two different samples were tested; shown is the result from the sample
having the smaller amount of glucose contamination.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.