Characterization of the Transmembrane Molecular Architecture of
the Dystroglycan Complex in Schwann Cells*
Fumiaki
Saito
,
Toshihiro
Masaki§,
Keiko
Kamakura§,
Louise
V. B.
Anderson¶,
Sachiko
Fujita
,
Hiroko
Fukuta-Ohi
,
Yoshihide
Sunada
,
Teruo
Shimizu
, and
Kiichiro
Matsumura
From the
Department of Neurology and Neuroscience,
Teikyo University School of Medicine, Tokyo 173-8605, Japan, the
§ Third Department of Internal Medicine, National Defense
Medical College, Saitama 359-8513, Japan, and the
¶ University School of Neuroscience and Muscular Dystrophy Group
Laboratories, Regional Neuroscience Centre, Newcastle General Hospital,
Newcastle-upon-Tyne NE4 6BE, United Kingdom
 |
ABSTRACT |
We have demonstrated previously 1) that the
dystroglycan complex, but not the sarcoglycan complex, is expressed in
peripheral nerve, and 2) that
-dystroglycan is an extracellular
laminin-2-binding protein anchored to
-dystroglycan in the Schwann
cell membrane. In the present study, we investigated the transmembrane
molecular architecture of the dystroglycan complex in Schwann cells.
The cytoplasmic domain of
-dystroglycan was co-localized with Dp116, the Schwann cell-specific isoform of dystrophin, in the abaxonal Schwann cell cytoplasm adjacent to the outer membrane.
-dystroglycan bound to Dp116 mainly via the 15 C-terminal amino acids of its cytoplasmic domain, but these amino acids were not solely responsible for the interaction of these two proteins. Interestingly, the
-dystroglycan-precipitating antibody precipitated only a small fraction of
-dystroglycan and did not precipitate laminin and Dp116
from the peripheral nerve extracts. Our results indicate 1) that Dp116
is a component of the submembranous cytoskeletal system that anchors
the dystroglycan complex in Schwann cells, and 2) that the dystroglycan
complex in Schwann cells is fragile compared with that in striated
muscle cells. We propose that this fragility may be attributable to the
absence of the sarcoglycan complex in Schwann cells.
 |
INTRODUCTION |
Dystroglycan is encoded by a single gene and cleaved into two
proteins, an extracellular peripheral membrane glycoprotein
-dystroglycan and an integral membrane glycoprotein
-dystroglycan, by posttranslational processing (1). In skeletal
muscle,
-dystroglycan links laminin-2 and agrin in the basal lamina
with
-dystroglycan in the sarcolemma (1-4). On the cytoplasmic side
of the sarcolemma, on the other hand,
-dystroglycan is anchored to
the cytoskeletal protein dystrophin (5, 6). These findings
indicate that the dystroglycan complex, comprising
- and
-dystroglycans, spans the sarcolemma and links the basal lamina with
submembranous cytoskeleton, thus contributing to the mechanical
stability of the sarcolemma. The recent findings that
-dystroglycan
interacts with Grb2, an adaptor protein, and rapsyn, a peripheral
protein required for acetylcholine receptor clustering, also suggest
that the dystroglycan complex may have additional functions in skeletal
muscle (7, 8).
The dystroglycan complex is also expressed in nonmuscle
tissues. For instance, the dystroglycan complex, but not the
sarcoglycan complex, is expressed in peripheral nerve (9-11). In
peripheral nerve,
- and
-dystroglycans are expressed restricted
to the Schwann cell outer membrane apposing the endoneurial basal
lamina but not in the Schwann cell inner membrane or compact myelin, whereas laminin-2 and the nonneuronal isoform of agrin lacking acetylcholine receptor clustering activity are expressed in the endoneurial basal lamina (9-16). Recently, we have demonstrated that
Schwann cell
-dystroglycan is a mucin-type glycoprotein, which links
laminin-2 and agrin in the endoneurium with
-dystroglycan in the
Schwann cell outer membrane (11-13). Because peripheral myelination is
greatly disturbed in congenital muscular dystrophy patients and
dy mice deficient in laminin-2 (17-26), these findings implicate the interaction of the dystroglycan complex with laminin-2 in
peripheral myelinogenesis. This is further supported by the recent
revelation that Mycobacterium leprae invades Schwann cells by binding to
-dystroglycan via laminin-2, to cause leprosy, a
disease characterized by peripheral nerve degeneration (27, 28).
Despite these recent discoveries, there remain a number of
questions to be answered concerning the molecular organization of the
dystroglycan complex in Schwann cells. For instance, the submembranous
cytoskeletal system that anchors the dystroglycan complex in Schwann
cells remains elusive, because the full-length 427-kDa dystrophin is
not expressed in peripheral nerve (29). In the present study, we asked
whether dystrophin isoform Dp116 is a component of this system, for two
reasons. First, Dp116 is a Schwann cell-specific isoform of dystrophin,
composed mainly of the hinge 4, cysteine-rich, and C-terminal domains
containing the
-dystroglycan binding site (5, 6, 29, 30). Second, it
was reported recently that peripheral myelination was disturbed in a
peculiar Duchenne muscular dystrophy patient lacking Dp116 in
peripheral nerve because of a unique mutation at the 5' splice site of
intron 69 of the dystrophin gene (31). In this study, we also
investigated the stability of the interaction of the components of the
dystroglycan complex in Schwann cells and discussed its implications
for the pathogenesis of muscle cell degeneration in sarcoglycanopathy.
 |
EXPERIMENTAL PROCEDURES |
Miscellaneous--
Monoclonal antibodies 43DAG/8D5 against the C
terminus of
-dystroglycan and DYS2 against the C terminus of
dystrophin were characterized previously (9, 32). Alkaline and
detergent extraction of crude bovine peripheral nerve membranes (5 mg/ml) were performed as described previously (10-12). 3-12%
SDS-PAGE,1 immunoblotting,
laminin blot overlay, and immunohistochemical analysis were performed
as described previously (9-12).
Immunoelectron Microscopic Analysis--
All the following
procedures were performed at 4 °C unless otherwise stated. The
segments of bovine cauda equina were immersed for 3 h in a
solution of periodate-lysine-paraformaldehyde (33) or for 16 h in
4% paraformaldehyde in 0.1 M phosphate buffer. The
segments were then infiltrated with 30% sucrose in 0.1 M
phosphate buffer overnight, mounted in OCT compound, quickly frozen in
isopentane cooled in liquid nitrogen, and cut into 10-µm-thick
sections on a cryostat. The frozen sections were placed on
gelatin-coated slides and washed three times with phosphate-buffered
saline (PBS; 0.01 M phosphate buffer, PH 7.4, containing
0.15 M NaCl). After blocking with PBS containing 5% bovine
serum albumin and 0.05% Triton X-100 (buffer A) for 1 h, sections
were incubated for 16 h with 43DAG/8D5 at a dilution of 1:30 or
DYS2 at a dilution of 1:5 in buffer A. After washing five times in
buffer A, the sections were incubated for 16 h with
biotin-conjugated anti-mouse immunoglobulin (Pharmingen) at a dilution
of 1:200 in buffer A. After washing five times in PBS, the sections
were incubated for 4 h with Vectastain ABC reagent (Vector
Laboratories). After washing five times in PBS, the sections were
immersed in 1% glutaraldehyde in 0.1 M phosphate buffer.
After washing three times in PBS, the sections were incubated for 30 min in diaminobenzidine solution (50 mM Tris-HCl buffer, pH
7.4, containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride) at
room temperature and then incubated for 3 min in diaminobenzidine
solution supplemented with 0.01% H2O2. The
sections were washed twice in PBS and were postfixed for 2 h in
2% OsO4 solution. After dehydration at room temperature in a series of increasing concentrations of ethanol, they were embedded in
Epon 812. Ultrathin sections were made on an LKB Ultrotome and viewed
on a JEOL JEM-1010 electron microscope without any contrast staining.
Preparation of Fusion Proteins--
The
-dystroglycan binding
motif of dystrophin has most recently been localized to amino acids
3054-3271, but additional amino acids located downstream have been
shown to be important for maximum binding (5). Because these amino
acids are common to both dystrophin and Dp116 (29, 30), we prepared, as
a probe to study the interaction between Dp116 and
-dystroglycan in
peripheral nerve, the Dp116 fusion protein (Dp116C-Ter), which
corresponds to amino acids 3054-3685 of dystrophin. With regard to
-dystroglycan, fusion proteins corresponding to amino acids 654-750
(
DGExt), 775-895 (
DGCyt1), 775-880 (
DGCyt2), 775-860
(
DGCyt3), 775-840 (
DGCyt4), 775-820 (
DGCyt5), 775-800
(
DGCyt6), 801-895 (
DGCyt7), and 801-880 (
DGCyt8) were
prepared. The cleavage site of
- and
-dystroglycans is localized
to serine 654, and the transmembrane domain of
-dystroglycan is
localized to amino acids 751-774 (1, 34, 35) (Fig.
1).
DGExt thus corresponds to the
entire extracellular domain of
-dystroglycan, whereas
DGCyt1,
DGCyt2,
DGCyt3,
DGCyt4,
DGCyt5,
DGCyt6,
DGCyt7, and
DGCyt8 correspond to various regions of the cytoplasmic domain
of
-dystroglycan (Fig. 1).

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Fig. 1.
Alignment of the -dystroglycan fusion
proteins in the full length of bovine -dystroglycan. TM,
transmembrane domain.
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Human dystrophin cDNA corresponding to amino acids 3054-3685
(GenBank accession number M18533) and bovine
-dystroglycan cDNAs
corresponding to amino acids 654-750, 775-895, 775-880, 775-860,
775-840, 775-820, 775-800, 801-895, and 801-880 (GenBank accession
number AB009079) were amplified by polymerase chain reaction and
subcloned into pGEX-2TK expression vector (Amersham Pharmacia Biotech).
Escherichia coli DH5
cells were transformed with the
glutathione S-transferase (GST) fusion protein constructs. Overnight cultures were grown, and the fusion proteins were induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. The cell cultures were spun down, resuspended in 50 mM Tris-HCl, pH 8.0, 1%
Triton X-100, 50 mM NaCl, 1 mM dithiothreitol,
1 mM EDTA, 0.75 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride, and sonicated 10 times for 10 s. The sonicated material was centrifuged at 13,800 × g for 10 min. The supernatant containing the GST fusion
protein was incubated with glutathione-agarose beads. After extensive wash, the glutathione-agarose beads were eluted with 10 mM
glutathione in 50 mM Tris-HCl, pH 8.0.
Blot Overlay Assay--
Proteins were separated by 3-12%
SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes
(Immobilon). The PVDF transfers were blocked with 10 mM
triethanolamine, pH 7.6, 140 mM NaCl, 1 mM
CaCl2, and 1 mM MgCl2 containing
5% non-fat dry milk (MLBB) and then incubated with either the MLBB
containing 1 mM dithiothreitol and 0.2 µg/ml (unless
stated otherwise) fusion protein Dp116C-Ter overnight at room
temperature or the alkaline extracts of the crude bovine peripheral
nerve membranes for two nights at 4 °C. After extensive washing, the
Dp116C-Ter or the endogenous bovine peripheral nerve Dp116, which bound
to the PVDF transfers, was detected with monoclonal antibody DYS2.
Immunoprecipitation--
Immunoprecipitation was performed
using monoclonal antibody 43DAG/8D5. Two parts of digitonin or Triton
X-100 extracts of crude bovine peripheral nerve membranes (10-12) were
incubated with one part of crude culture medium of 43DAG/8D5 for 1 h at 4 °C. The resultant immune complex was precipitated with
protein G-Sepharose (Amersham Pharmacia Biotech). The precipitates and the voids were analyzed by 3-12% SDS-PAGE and immunoblotting.
 |
RESULTS |
To determine the localization of Dp116 and
-dystroglycan in
bovine peripheral nerve, we performed immunohistochemical and immunoelectron microscopic analyses using monoclonal antibodies DYS2
and 43DAG/8D5. We have reported previously that DYS2 detects Dp116 in
peripheral nerve (9-11). 43DAG/8D5 is directed against the C terminus
of
-dystroglycan and thus recognizes the cytoplasmic domain of
-dystroglycan (1, 32). The results of immunohistochemical analysis
are shown in Fig. 2. Dp116 and the
cytoplasmic domain of
-dystroglycan were both localized surrounding
the outermost layer of myelin sheath of peripheral nerve fibers (Fig.
2). The results of immunoelectron microscopic analysis are shown in
Fig. 3. Dp116 and the cytoplasmic domain
of
-dystroglycan were both localized in the abaxonal Schwann cell
cytoplasm (Fig. 3). The distribution of these proteins was most
prominent adjacent to the outer membrane (Fig. 3).

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Fig. 2.
Immunohistochemical analysis of
-dystroglycan and Dp116 in bovine peripheral nerve.
-Dystroglycan and Dp116 were detected with 43DAG/8D5 and DYS2,
respectively. Scale bar, 15 µm.
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Fig. 3.
Immunoelectron microscopic analysis of
-dystroglycan and Dp116 in bovine peripheral nerve.
-Dystroglycan and Dp116 were detected with 43DAG/8D5 and DYS2,
respectively. OM and CM, Schwann cell outer
membrane and compact myelin, respectively. Scale bar, 0.5 µm.
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To test whether Dp116 interacts with
-dystroglycan, we prepared
domain-specific fusion proteins of Dp116 and
-dystroglycan (Figs. 1
and 4). The PVDF transfer of
-dystroglycan fusion proteins was overlaid with Dp116C-Ter (Fig. 4,
a and c). Dp116C-Ter bound to
DGCyt1
corresponding to the cytoplasmic domain of
-dystroglycan but not to
DGExt corresponding to the extracellular domain of
-dystroglycan
or control GST (Fig. 4c). As we have reported previously (10, 11), Dp116 was enriched in the alkaline extracts of crude bovine
peripheral nerve membranes (Fig. 4b). To see whether the endogenous bovine peripheral nerve Dp116 binds to
-dystroglycan fusion proteins, the PVDF transfer of
-dystroglycan fusion proteins was overlaid with the alkaline extracts of crude bovine peripheral nerve membranes, and the Dp116, which bound to
-dystroglycan fusion
proteins on the PVDF membranes, was detected with DYS2. The endogenous
bovine peripheral nerve Dp116 bound to
DGCyt1 but not to
DGExt or
control GST (Fig. 4c). These results demonstrate that Dp116
binds to the cytoplasmic domain of
-dystroglycan.

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Fig. 4.
Blot overlay analysis of the binding of Dp116
fusion protein or the endogenous bovine peripheral nerve Dp116 with
domain-specific -dystroglycan fusion proteins. a,
preparations of Dp116 fusion protein Dp116C-Ter were separated by
3-12% SDS-PAGE and transferred to a PVDF membrane. CB and
Anti-Dp116, SDS gel stained with Coomassie Blue and the PVDF
transfer reacted with DYS2, respectively. The 110-kDa band that reacted
with DYS2 strongly had the expected molecular size of the fusion
protein, indicating that it is Dp116C-Ter. b, the crude
bovine peripheral nerve membranes were extracted at pH 11 as described
previously (10, 11). The extracts (Alk Ext) and pellets
(Alk Plt) were separated by 3-12% SDS-PAGE, transferred to
a PVDF membrane and reacted with DYS2 (Anti-Dp116).
c, domain-specific -dystroglycan fusion proteins were
separated by 3-12% SDS-PAGE and transferred to PVDF membranes. The
PVDF transfers were overlaid with Dp116C-Ter (Dp116C-Ter
O/L) or the alkaline extracts of crude bovine peripheral nerve
membranes (Alk Ext O/L). The Dp116C-Ter or the endogenous
bovine peripheral nerve Dp116 that bound to the -dystroglycan fusion
proteins on the PVDF membranes was detected with DYS2. GST,
control GST fusion protein having no inserts; CB, SDS gel
stained with Coomassie Blue. Molecular mass standards (Da × 10 3) are shown on the left.
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Dystrophin has been reported to bind to the 15 C-terminal amino acids
of
-dystroglycan (5). We asked whether these amino acids were also
responsible for the binding of Dp116. Surprisingly,
DGCyt2
corresponding to the cytoplasmic domain of
-dystroglycan but lacking
the 15 C-terminal amino acids bound both Dp116C-Ter and the endogenous
bovine peripheral nerve Dp116, albeit with much lower binding affinity
than
DGCyt1 (Fig. 4c). The difference between
DGCyt1
and
DGCyt2 binding to the endogenous bovine peripheral nerve Dp116
appeared less marked than that to Dp116C-Ter (Fig. 4c). It
is possible that the native protein might have slightly altered binding
properties from the fusion protein or that the binding properties of
Dp116 might be modified in the presence of the Dp116-binding proteins
other than
-dystroglycan in the peripheral nerve extracts (discussed
below; also see Fig. 7).
We tested the effects of a synthetic peptide corresponding to the 15 C-terminal amino acids of
-dystroglycan on the binding of
Dp116C-Ter. The binding of Dp116C-Ter with
DGCyt1 was greatly reduced, but not completely abolished, by the peptide, whereas the
binding with
DGCyt2 was not affected (Fig.
5). In the presence of the peptide in the
overlay medium, furthermore, Dp116C-Ter bound to
DGCyt1 and
DGCyt2 with similar affinity (Fig. 5). The results indicate that the
15 C-terminal amino acids of
-dystroglycan are involved in the high
affinity binding of Dp116 but that they are not solely responsible for
the interaction of
-dystroglycan with Dp116. It was suggested
recently that the sequence of PPPY in the 15 C-terminal amino acids of
-dystroglycan might directly interact with the WW domain in hinge 4 of dystrophin (36, 37). However, a synthetic peptide, PPPY, did not
significantly affect the binding of Dp116C-Ter with
DGCyt1 (Fig. 5),
suggesting that these amino acids may not be crucial for the
binding.

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Fig. 5.
Inhibition of the binding of Dp116 fusion
protein with domain-specific -dystroglycan fusion proteins by
synthetic peptides. Domain-specific -dystroglycan fusion
proteins were separated by 3-12% SDS-PAGE and transferred to PVDF
membranes. The PVDF transfers of DGCyt1 and DGCyt2 were overlaid
with Dp116C-Ter in the presence (15 C-Ter Peptide (+)) or
absence (15 C-Ter Peptide ( )) of 500,000 times molar
excess of synthetic amino acids corresponding to the 15 C-terminal
amino acids of -dystroglycan. The PVDF transfers of DGCyt1 were
overlaid with Dp116C-Ter in the presence (PPPY Peptide (+))
or absence (PPPY Peptide ( )) of 500,000 times molar excess
of synthetic amino acids PPPY. The Dp116C-Ter that bound to the
-dystroglycan fusion proteins on the PVDF membranes was detected
with DYS2. CB, SDS gel stained with Coomassie Blue.
Molecular mass standards (Da × 10 3) are shown on
the left.
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The above results suggest the presence of a Dp116 binding sequence
other than the 15 C-terminal amino acids of
-dystroglycan. To see
whether this is true, we prepared fusion proteins corresponding to
-dystroglycan cytoplasmic domains lacking various lengths of
C-terminal amino acids and tested whether they bind Dp116C-Ter. Dp116C-Ter bound to all of
DGCyt3,
DGCyt4,
DGCyt5, and
DGCyt6 (Fig. 6). In addition, the
binding affinity of Dp116C-Ter with
DGCyt3,
DGCyt4,
DGCyt5,
and
DGCyt6 was similar to that with
DGCyt2 and much weaker than
that with
DGCyt1 (Fig. 6), suggesting that the 26 N-terminal amino
acids of the cytoplasmic domain of
-dystroglycan may also be
involved in the binding of Dp116. To confirm this, we prepared the
-dystroglycan cytoplasmic domain fusion protein lacking the 26 N-terminal amino acids (
DGCyt7) or lacking both the 26 N-terminal
and 15 C-terminal amino acids (
DGCyt8). The binding of Dp116C-Ter
with
DGCyt7 was slightly weaker than that with
DGCyt1, and the
binding with
DGCyt8 was not detectable (Fig. 6), indicating that the
26 N-terminal amino acids of the cytoplasmic domain of
-dystroglycan
are involved in the binding of Dp116.

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Fig. 6.
Blot overlay analysis of the binding of Dp116
fusion protein with domain-specific -dystroglycan fusion
proteins. Domain-specific -dystroglycan fusion proteins were
separated by 3-12% SDS-PAGE and transferred to PVDF membranes. The
PVDF transfers were overlaid with Dp116C-Ter. The Dp116C-Ter that bound
to the -dystroglycan fusion proteins on the PVDF membranes was
detected with DYS2. CB, SDS gel stained with Coomassie Blue.
Molecular mass standards (Da × 10 3) are shown on
the left.
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The binding of Dp116C-Ter with
DGCyt1 and
DGCyt2 was maximal at
the physiological salt concentration (results not shown). The binding
of Dp116C-Ter with
DGCyt1 and
DGCyt2 was not inhibited by EDTA,
indicating that it is independent of divalent cations (results not
shown). We have also observed that
DGCyt1 and
DGCyt2 in solution
do not bind to Dp116C-Ter separated by SDS-PAGE and transferred to PVDF
membranes (results not shown). Together with the aforementioned
results, this suggests that the
-dystroglycan binding site of Dp116
is vulnerable to denaturation with SDS, whereas the Dp116 binding site
of
-dystroglycan is not.
To identify the endogenous bovine peripheral nerve proteins that
interact with Dp116, we prepared the PVDF transfers of crude bovine
peripheral nerve membranes. When the PVDF transfer was reacted with
43DAG/8D5, 43- and 30-kDa bands were detected (Fig. 7). As reported previously, the 43-kDa
band corresponds to the full-length
-dystroglycan, whereas the
30-kDa band is presumed to correspond to the unglycosylated form or the
proteolytic fragment of
-dystroglycan (11). When the identical PVDF
transfer was overlaid with Dp116C-Ter, these 43- and 30-kDa bands bound
Dp116C-Ter, in addition to the 60- and 35-kDa bands of undetermined
origin (Fig. 7). Furthermore, the 43- and 30-kDa
-dystroglycan bands immunoprecipitated by 43DAG/8D5 bound Dp116C-Ter (Fig.
8a). These results demonstrate
that Dp116 binds to the endogenous bovine peripheral nerve
-dystroglycan.

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Fig. 7.
Blot overlay analysis of the binding of Dp116
fusion protein with bovine peripheral nerve membrane proteins. The
crude bovine peripheral nerve membranes were extracted with Triton
X-100 as described previously (10-12). The extracts (Triton
Extract) and pellets (Triton Pellet) were separated by
3-12% SDS-PAGE, transferred to PVDF membranes, and overlaid with 25 µg/ml Dp116C-Ter. The Dp116C-Ter that bound to bovine peripheral
nerve membrane proteins on the PVDF membranes was detected with DYS2
(Dp116C-Ter O/L). Arrows, bands that bound
Dp116C-Ter. CB, Anti- DG, and
Anti-Dp116, SDS gel stained with Coomassie Blue and the
identical PVDF transfers reacted with 43DAG/8D5 and DYS2, respectively.
As reported previously, the 43-kDa band (43 DG)
corresponds to the full-length -dystroglycan, whereas the 30-kDa
band (30 DG) is presumed to correspond to the
unglycosylated form or the proteolytic fragment of -dystroglycan
(11). Molecular mass standards (Da × 10 3) are shown
on the left.
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Fig. 8.
Immunoprecipitation of -dystroglycan from
crude bovine peripheral nerve membranes. a, the extracts of
crude bovine peripheral nerve membranes were incubated with 43DAG/8D5.
The resultant immune complex was precipitated with protein G-Sepharose,
separated by 3-12% SDS-PAGE, and transferred to PVDF membranes
(Anti- DG Beads). Anti- DG, Lam
O/L, Anti-Dp116, and Dp116C-Ter O/L, PVDF
transfer reacted with 43DAG/8D5, overlaid with laminin-1, reacted with
DYS2, and overlaid with Dp116C-Ter, respectively; DG,
43 DG, and 30 DG, -dystroglycan and the
43- and 30-kDa bands of -dystroglycan, respectively.
Arrows, bands of mouse IgG detected by anti-mouse IgG
secondary antibody. b, the extracts of crude bovine
peripheral nerve membranes were incubated with or without 43DAG/8D5
(Anti- DG Beads and Control Beads,
respectively). After precipitation with protein G-Sepharose, the voids
were separated by 3-12% SDS-PAGE and transferred to PVDF membranes.
Anti- DG, Lam O/L, and Anti-Dp116,
PVDF transfer reacted with 43DAG/8D5, overlaid with laminin-1, and
reacted with DYS2, respectively; CB, SDS gel stained with
Coomassie blue; Lam, bands of laminin subunits detected by
anti-laminin antibody used in laminin-1 overlay assay. Molecular mass
standards (Da × 10 3) are shown on the
left.
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We performed immunoprecipitation of bovine peripheral nerve membrane
extracts using 43DAG/8D5. Both the 43- and 30-kDa bands of
-dystroglycan were precipitated completely by 43DAG/8D5 (Fig. 8,
a and b). As expected,
-dystroglycan was also
precipitated by 43DAG/8D5 (Fig. 8a). However, the amount of
-dystroglycan remaining in the voids after precipitation was only
slightly reduced compared with control (Fig. 8b), indicating
that only a small fraction, but not the vast majority, of
-dystroglycan was precipitated. To our surprise, laminin was not
precipitated by 43DAG/8D5 (Fig. 8, a and b) (Note
that the bands of laminin subunits are not detectable in the
immunoprecipitates in Fig. 8a, Lam O/L.) Dp116 was not precipitated either (Fig. 8, a and b), but this
could be attributable to the fact that 43DAG/8D5 is directed against
the 15 C-terminal amino acids of
-dystroglycan involved in the high
affinity binding of Dp116 as described above. The results were
identical using digitonin (Fig. 8) or Triton X-100 (results not shown)
for extraction of peripheral nerve membranes.
 |
DISCUSSION |
When combined together, the data presented here indicate that
Dp116 is a component of the submembranous cytoskeletal system that
anchors the dystroglycan complex in Schwann cells. We have found that
the 15 C-terminal amino acids of the cytoplasmic domain of
-dystroglycan are involved in the high affinity binding of Dp116 but
that these amino acids are not solely responsible for the interaction
of
-dystroglycan with Dp116. We have found that the 26 N-terminal
amino acids of the cytoplasmic domain of
-dystroglycan are also
involved in the low affinity binding of Dp116. In this respect, it is
noteworthy that the 26 N-terminal, as well as the 15 C-terminal, amino
acids of the cytoplasmic domain of
-dystroglycan are completely
conserved among all the species investigated so far, human, cow, rabbit
and mouse, suggesting that this sequence may have an important
function. Our finding is consistent with this expectation. In addition
to
-dystroglycan, furthermore, we have found that the 60- and 35-kDa
bovine peripheral nerve proteins bind Dp116. Although the
identification of these proteins awaits future studies, our results
suggest that they may interact with Dp116 in the submembranous
cytoskeletal system in Schwann cells. Recently, it was reported that
M. leprae invades Schwann cells by binding to the
dystroglycan complex via the G domain of laminin
2 chain (28).
Interestingly, the dystroglycan complex starts aggregating on the
Schwann cell membrane in response to the challenge with the laminin
2 chain-coated M. leprae, suggesting that the
submembranous cytoskeleton anchoring the dystroglycan complex is
reorganized to permit the entry of bacteria into the cytoplasm (28).
Our results raise the possibility that Dp116 may play a role in this process.
In this study, we have found that the interaction of the components of
the dystroglycan complex is unstable in Schwann cells. First, the
finding that the
-dystroglycan-precipitating antibody 43DAG/8D5
precipitated a small fraction, but not a major fraction, of
-dystroglycan from the peripheral nerve membrane extracts indicates
that a substantial amount of
-dystroglycan was dissociated from
-dystroglycan during biochemical procedures. This is in sharp
contrast to the striated muscle cells, where
- and
-dystroglycans are tightly complexed (2, 38, 39). Second, the finding that laminin was
not precipitated by 43DAG/8D5 at all indicates that laminin was
completely dissociated from
-dystroglycan during biochemical
procedures. In this respect, it is noteworthy that the sarcoglycan
complex, comprising
-,
-,
-, and
-sarcoglycans as a whole,
is not expressed in peripheral nerve (9-11). Recent evidence indicates
that, by binding to both
- and
-dystroglycans, the sarcoglycan
complex reinforces their interaction in striated muscle cells (40). We
presume that, because of the absence of the sarcoglycan complex, the
interaction between
- and
-dystroglycans in Schwann cells is not
as stable as in striated muscle cells. The absence of the sarcoglycan
complex may somehow destabilize the interaction of laminin with
-dystroglycan as well. In any case, the link between the basal
lamina and submembranous cytoskeleton via the dystroglycan complex is
presumed to be fragile in Schwann cells.
 |
ACKNOWLEDGEMENTS |
Bovine dystroglycan and human dystrophin
cDNAs were kindly provided by Drs. Haruaki Ninomiya (Kyoto
University) and Michihiro Imamura (National Center for Neurological and
Psychiatric Disorders), respectively.
 |
FOOTNOTES |
*
This work was supported by the grants from Kato Memorial
Bioscience Foundation, the Cell Science Research Foundation, and the
Science Research Promotion Fund from Japan Private School Promotion
Foundation, Research Grants 8A-1 and 8A-2 for Nervous and Mental
Disorders from the Ministry of Health and Welfare, a Health Sciences
research grant for research on brain science from the Ministry of
Health and Welfare, and Research Grants 08457195, 09470156, 09770460, 09877121, and 10044319 from the Ministry of Education, Science, Sports
and Culture.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Neurology, Teikyo University School of Medicine, 2-11-1 Kaga,
Itabashi-ku, Tokyo 173-8605, Japan. Tel.: 81-3-3964-1211; Fax:
81-3-3964-6394; E-mail: k-matsu{at}med.teikyo-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PAGE, polyacrylamide
gel electrophoresis;
PBS, phosphate-buffered saline;
PVDF, polyvinylidene difluoride;
GST, glutathione
S-transferase.
 |
REFERENCES |
-
Ibraghimov-Beskrovnaya, O.,
Ervasti, J. M.,
Leveille, C. J.,
Slaughter, C. A.,
Sernett, S. W.,
and Campbell, K. P.
(1992)
Nature
355,
696-702[CrossRef][Medline]
[Order article via Infotrieve]
-
Ervasti, J. M.,
and Campbell, K. P.
(1993)
J. Cell Biol.
122,
809-823[Abstract]
-
Gee, S. H.,
Blacher, R. W.,
Douville, P. J.,
Provost, P. R.,
Yurchenco, P. D.,
and Carbonetto, S.
(1993)
J. Biol. Chem.
268,
14972-14980[Abstract/Free Full Text]
-
Fallon, J. R.,
and Hall, Z. W.
(1994)
Trends Neurosci.
17,
469-473[Medline]
[Order article via Infotrieve]
-
Jung, D.,
Yang, B.,
Meyer, J.,
Chamberlain, J. S.,
and Campbell, K. P.
(1995)
J. Biol. Chem.
270,
27305-27310[Abstract/Free Full Text]
-
Suzuki, A.,
Yoshida, M.,
Hayashi, K.,
Mizuno, Y.,
Hagiwara, Y.,
and Ozawa, E.
(1994)
Eur. J. Biochem.
220,
283-292[Abstract]
-
Yang, B.,
Jung, D.,
Motto, D.,
Meyer, J.,
Koretzky, G.,
and Campbell, K. P.
(1995)
J. Biol. Chem.
270,
11711-11714[Abstract/Free Full Text]
-
Cartaud, A.,
Coutant, S.,
Petrucci, T. C.,
and Cartaud, J.
(1998)
J. Biol. Chem.
273,
11321-11326[Abstract/Free Full Text]
-
Matsumura, K.,
Yamada, H.,
Shimizu, T.,
and Campbell, K. P.
(1993)
FEBS Lett.
334,
281-285[CrossRef][Medline]
[Order article via Infotrieve]
-
Yamada, H.,
Shimizu, T.,
Tanaka, T.,
Campbell, K. P.,
and Matsumura, K.
(1994)
FEBS Lett.
352,
49-53[CrossRef][Medline]
[Order article via Infotrieve]
-
Yamada, H.,
Chiba, A.,
Endo, T.,
Kobata, A.,
Anderson, L. V. B.,
Hori, H.,
Fukuta-Ohi, H.,
Kanazawa, I.,
Campbell, K. P.,
Shimizu, T.,
and Matsumura, K.
(1996)
J. Neurochem.
66,
1518-1524[Medline]
[Order article via Infotrieve]
-
Yamada, H.,
Denzer, A. J.,
Hori, H.,
Tanaka, T.,
Anderson, L. V. B.,
Fujita, S.,
Fukuta-Ohi, H.,
Shimizu, T.,
Ruegg, M.,
and Matsumura, K.
(1996)
J. Biol. Chem.
271,
23418-23423[Abstract/Free Full Text]
-
Chiba, A.,
Matsumura, K.,
Yamada, H.,
Inazu, T.,
Shimizu, T.,
Kusunoki, S.,
Kanazawa, I.,
Kobata, A.,
and Endo, T.
(1997)
J. Biol. Chem.
272,
2156-2162[Abstract/Free Full Text]
-
Burgeson, R. E.,
Chiquet, M.,
Deutzmann, R.,
Ekblom, P.,
Engel, J.,
Kleinman, H.,
Martin, G. R.,
Meneguzzi, G.,
Paulsson, M.,
Sanes, J.,
Timpl, R.,
Tryggvason, K.,
Yamada, Y.,
and Yurchenco, P. D.
(1994)
Matrix Biol.
14,
209-211[CrossRef][Medline]
[Order article via Infotrieve]
-
Leivo, I.,
and Engvall, E.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
1544-1548[Abstract]
-
Sanes, J. R.,
Engvall, E.,
Butkowski, R.,
and Hunter, D. D.
(1990)
J. Cell Biol.
111,
1685-1699[Abstract]
-
Arahata, K.,
Hayashi, Y. K.,
Koga, R.,
Goto, K.,
Lee, J. H.,
Miyagoe, Y.,
Ishii, H.,
Tsukahara, T.,
Takeda, S.,
Woo, M.,
Nonaka, I.,
Matsuzaki, T.,
and Sugita, H.
(1993)
Proc. Jpn. Acad. Ser. B Phys. Biol. Sci.
69,
259-264
-
Sunada, Y.,
Bernier, S. M.,
Kozak, C. A.,
Yamada, Y.,
and Campbell, K. P.
(1994)
J. Biol. Chem.
269,
13729-13732[Abstract/Free Full Text]
-
Sunada, Y.,
Bernier, S. M.,
Utani, A.,
Yamada, Y.,
and Campbell, K. P.
(1995)
Hum. Mol. Genet.
4,
1055-1061[Abstract]
-
Xu, H.,
Christmas, P.,
Wu, X. R.,
Wewer, U. M.,
and Engvall, E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
5572-5576[Abstract]
-
Xu, H.,
Wu, X.-R.,
Wewer, U. M.,
and Engvall, E.
(1994)
Nat. Genet.
8,
297-302[Medline]
[Order article via Infotrieve]
-
Tomé, F. M. S.,
Evangelista, T.,
Leclerc, A.,
Sunada, Y.,
Manole, E.,
Estournet, B.,
Barois, A.,
Campbell, K. P.,
and Fardeau, M.
(1994)
C. R. Acad. Sci. III
317,
351-357[Medline]
[Order article via Infotrieve]
-
Helbling-Leclerc, A.,
Zhang, X.,
Topaloglu, H.,
Cruaud, C.,
Tesson, F.,
Weissenbach, J.,
Tomé, F. M. S.,
Schwartz, K.,
Fardeau, M.,
Tryggvason, K.,
and Guicheney, P.
(1995)
Nat. Genet.
11,
216-218[Medline]
[Order article via Infotrieve]
-
Shorer, Z.,
Philpot, J.,
Muntoni, F.,
Sewry, C.,
and Dubowitz, V.
(1995)
J. Child Neurol.
10,
472-475[Medline]
[Order article via Infotrieve]
-
Bradley, W. G.,
and Jenkison, M.
(1973)
J. Neurol. Sci.
19,
227-247
-
Madrid, R. E.,
Jaros, E.,
Cullen, M. J.,
and Bradley, W. G.
(1975)
Nature
257,
319-321[Medline]
[Order article via Infotrieve]
-
Rambukkana, A.,
Salzer, J. L.,
Yurchenco, P. D.,
and Tuomanen, E. I.
(1997)
Cell
88,
811-821[Medline]
[Order article via Infotrieve]
-
Rambukkana, A.,
Yamada, H.,
Zanazzi, G.,
Mathus, T.,
Salzer, J. L.,
Yurchenco, P. D.,
Campbell, K. P.,
and Fischetti, V. A.
(1998)
Science
282,
2079-2081[Abstract/Free Full Text]
-
Byers, T. M.,
Lidov, H. G. W.,
and Kunkel, L. M.
(1993)
Nat. Genet.
4,
77-78[Medline]
[Order article via Infotrieve]
-
Koenig, M.,
Monaco, A. P.,
and Kunkel, L. M.
(1988)
Cell
53,
219-228[Medline]
[Order article via Infotrieve]
-
Comi, G. P.,
Ciafaloni, E.,
Rohan de Silva, H. A.,
Prelle, A.,
Bardoni, A.,
Rigoletto, C.,
Robotti, M.,
Bresolin, N.,
Moggio, M.,
Fortunato, F.,
Ciscato, P.,
Turconi, A.,
Rose, A. D.,
and Scarlato, G.
(1995)
Hum. Mol. Genet.
11,
2171-2174
-
Cullen, M. J.,
Walsh, J.,
and Nicholson, L. V. B.
(1994)
Acta Neuropathol.
87,
349-354[CrossRef][Medline]
[Order article via Infotrieve]
-
McLean, I. W.,
and Nakane, P. K.
(1974)
J. Histochem. Cytochem.
22,
1077-1083[Medline]
[Order article via Infotrieve]
-
Deyst, K. A.,
Bowe, M. A.,
Leszyk, J. D.,
and Fallon, J. R.
(1995)
J. Biol. Chem.
270,
25956-25959[Abstract/Free Full Text]
-
Smalheiser, N. R.,
and Kim, E.
(1995)
J. Biol. Chem.
270,
15425-15433[Abstract/Free Full Text]
-
Einbond, A.,
and Sudol, M.
(1996)
FEBS Lett.
384,
1-8[CrossRef][Medline]
[Order article via Infotrieve]
-
Yuasa, K.,
Miyagoe, Y.,
Yamamoto, K.,
Nabeshima, Y.,
Dickson, G.,
and Takeda, S.
(1998)
FEBS Lett.
425,
329-336[CrossRef][Medline]
[Order article via Infotrieve]
-
Ervasti, J. M.,
Ohlendieck, K.,
Kahl, S. D.,
Gaver, M. G.,
and Campbell, K. P.
(1990)
Nature
345,
315-319[CrossRef][Medline]
[Order article via Infotrieve]
-
Ervasti, J. M.,
and Campbell, K. P.
(1991)
Cell
66,
1121-1131[Medline]
[Order article via Infotrieve]
-
Sakamoto, A.,
Ono, K.,
Abe, M.,
Jasmin, G.,
Eki, T.,
Murakami, Y.,
Masaki, T.,
Toyo-oka, T.,
and Hanaoka, F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13873-13878[Abstract/Free Full Text]
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