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INTRODUCTION |
The dystrophin-associated glycoprotein complex
(DAGC)1 is a large array of
membrane and cytoskeletal proteins found in striated muscle, where it
is thought to function in force transmission and in protection of the
muscle membrane from contraction-induced damage (1, 2). The DAGC
consists of many proteins including syntrophins, dystroglycan,
sarcoglycans, and sarcospan that are directly or indirectly linked to
dystrophin (3-7). The importance of these proteins is evident from the
various forms of muscular dystrophy, in which primary or secondary
defects in DAGC components result in muscle tissue degeneration
(8-14).
The sarcoglycan complex (SGC) is a subcomplex of the DAGC (15). In
addition to playing a structural role, the SGC may have signaling
functions (16, 17). SGC consists of four transmembrane proteins:
-sarcoglycan, a type I transmembrane protein, and 
-, 
-, and
-sarcoglycans, which are type II transmembrane proteins. - and
-sarcoglycans are expressed exclusively in skeletal and cardiac
muscle, whereas - and -sarcoglycans are more widely distributed
(11, 18-21). Mutations in any one of these four sarcoglycans cause
autosomal recessive limb-girdle muscular dystrophy, and mutations in
the genes encoding individual sarcoglycans often lead to the
concomitant loss or reduction of all sarcoglycans from the sarcolemma,
suggesting that complex formation and localization of SGCs require all
four subunits (9). Direct interaction between the sarcoglycans has also
been demonstrated biochemically by co-immunoprecipitation (15, 22).
Transfection of Chinese hamster ovary cells with sarcoglycans indicates
that all four sarcoglycans must be co-expressed for proper cell surface
localization (23). Taken together, these data suggest that -, -,
-, and -sarcoglycans are subunits of a heterotetrameric molecule
(16).
A fifth sarcoglycan, 
-sarcoglycan, was identified recently (24, 25).
It is highly homologous to -sarcoglycan, but like - and
-sarcoglycans it is widely expressed in various tissues (24). It has
been reported that -sarcoglycan associates with - and
-sarcoglycans in smooth muscle (26). Despite its homology to
-sarcoglycan and its presence in skeletal muscle, endogenous -sarcoglycan is unable to rescue phenotypes associated with
-sarcoglycan loss (27). Therefore, in skeletal muscle
-sarcoglycan could exist as a monomer, form a complex with proteins
distinct from those that associate with -sarcoglycan, or associate
with -, -, and -sarcoglycans to form SGC that is only
partially able to compensate for the loss of -sarcoglycan-containing complexes.
Mice mutant in -, -, -, and -sarcoglycans have been
generated experimentally by homologous
recombination-dependent gene targeting (27-30). A
spontaneous null mutant, the -sarcoglycan-deficient hamster, also
exists (31). All sarcoglycan-deficient mice develop severe muscular
dystrophies with varying involvement of heart muscle. The
-sarcoglycan null mutant hamster succumbs from cardiomyopathy with
less severe involvement of skeletal muscle. The C2C12 mouse myoblast
cell line, a cell line widely used for studying myogenesis and muscle
differentiation, has been used as a model system to study biosynthesis
and assembly of sarcoglycans (21, 22, 32, 33).
Here we use gene targeting and C2C12 cells to demonstrate that
-sarcoglycan is indeed associated with -, -, and
-sarcoglycans in skeletal muscle in vivo and in
vitro. We generate -sarcoglycan-deficient mice and present
evidence, using immunofluorescence and protein fractionation, that SGCs
containing -, -, -, and -sarcoglycans are expressed in
striated muscle and that the localization of these complexes in the
muscle is similar to the -sarcoglycan-containing SGC. The existence
of an -sarcoglycan-containing SGC may explain why residual levels of
sarcoglycan expression are seen in mutant mice and patients with
-sarcoglycan deficiency (27, 34-36) and suggests that
-sarcoglycan may partially compensate for the loss of
-sarcoglycan.
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MATERIALS AND METHODS |
Construction of Gene Targeting Vector--
The mouse
-sarcoglycan gene (asg) was isolated from a 129/SvJ mouse
genomic library (Stratagene, La Jolla, CA) by hybridization with a
mouse -sarcoglycan cDNA probe (21). The targeting vector was
designed to disrupt exons 1 and 2. A 15-kb fragment containing exons
1-6 of the -sarcoglycan gene was subcloned into pBluescript (Stratagene, La Jolla, CA), characterized by restriction mapping, and
sequenced. A 5.5-kb genomic fragment containing sequence upstream of
the ATG site in exon 1, was amplified using the ExpandTM
Long Template PCR System (Roche Molecular Biochemicals) with the
forward primer 5'-GAGGGTGCGAGGGTGACAAGGAC-3' and the reverse primer
5'-GGAAGTTACTGCTGCTGCCAT-3'. The 5.5-kb genomic PCR product was
subcloned, sequenced, and used as the 5' homologous region of the
targeting vector. A 2-kb genomic PCR fragment containing the 3' portion
of exon 2 through exon 6 was amplified with primers 5'-CTGCGCCTTCCAGAACAC-3' and 5'-ACCAGAGACACATTGCACCAG-3'. The PCR
product was subcloned, sequenced, and used as the 3' homologous region
of the targeting vector. A cassette containing the LacZ with
a nuclear localization signal and neo genes
(nlsLacZ/neo) was inserted 21 base pairs downstream of the
ATG initiation site. The diphtheria toxin A gene was inserted at the 3'
end of the targeting vector to select against random insertion events
(37). A map of the -sarcoglycan gene locus and the design of the
targeting vector are shown in Fig. 1.
Generation of -Sarcoglycan Null Mice--
SM38 ES cells,
derived from 129/SvEv mice,2
were transfected with the linearized targeting vector by
electroporation and selected for growth in the presence of G418 (38).
G418-resistant ES cell clones were screened by Southern blotting of
BamHI-digested genomic DNA using a probe shown in Fig. 1.
Two selected clones of targeted ES cells were injected separately into
C57BL/6J blastocysts, and the blastocysts were transferred into
pseudopregnant recipients. Chimeric male mice were identified by coat
color and bred to C57BL/6J females. Genotypes were determined by
Southern blot analysis of DNA from tail biopsies.
Cell Culture--
The C2C12 mouse myoblast cell line (39) was
obtained from the American Type Culture Collection (Rockville, MD).
Cells were cultured in dishes precoated with 50 µg/ml rat tail
collagen type I (Collaborative Biomedical Products, Bedford, MA) and
maintained in low glucose Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 20% fetal calf serum (HyClone
Laboratories, Logan, UT), 15 mM HEPES, and 20 mM glutamine at 37 °C in a humidified atmosphere of 5%
CO2. Antibiotics were not used (21, 40). The culture medium
was changed every 24 h. Myogenic differentiation was induced at
confluence by replacing the growth medium with Dulbecco's modified
Eagle's medium containing 2% horse serum (HyClone).
Antibodies--
Mouse monoclonal antibodies against
-sarcoglycan (NCL-a-sarc), -sarcoglycan (NCL-b-sarc), and
-sarcoglycan (NCL-g-sarc) were purchased from Novocastra
(Newcastle-upon-Tyne, United Kingdom). Affinity-purified rabbit
polyclonal antibody against -sarcoglycan was kindly provided by Dr.
E. M. McNally (University of Chicago, Chicago, IL) and rabbit
polyclonal antiserum against -sarcoglycan was kindly provided by Dr.
V. Nigro (Second University of Naples, Naples, Italy). Rabbit
polyclonal antisera against - and -sarcoglycans were made against
sequence-specific synthetic peptides coupled to keyhole limpet
hemocyanin (Sigma) (41) via an N-terminal cystein. The antibodies were
affinity-purified with the respective peptide antigen coupled to
UltraLink Iodoacetyl (Pierce). For some experiments, purified
antibodies were coupled to UltraLink Biosupport (Pierce).
Indirect Immunofluorescence--
Ten-µm transverse cryosections
of thigh muscle were prepared from 8-week-old wild type, heterozygous,
and homozygous -sarcoglycan mutant mice. Fluorescent staining was
performed by incubating sections for 1 h at 37 °C with
affinity-purified polyclonal antibodies. After washing with PBS three
times, sections were incubated with a fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (ICN,
Aurora, OH. 1:200) for 1 h at room temperature and then washed
with PBS. Dilutions of all the antibodies were made in PBS containing
3% bovine serum albumin. Sections were mounted with Vectashield
mounting medium for fluorescence (Vector, Burlingame, CA) and observed
under an Axiovert 405M fluorescence microscope (Zeiss). For
histological analysis, sections were stained with hematoxylin and eosin.
RT-PCR--
Total RNA was isolated using the Trizol reagent (Life
Technologies, Inc.). Five µg of total RNA were reverse transcribed
using RT-PCR kit (Stratagene, La Jolla, CA). The primers used were
against nucleotides 317-335 (5'-CAGCCTACAATCGAGACAG-3') and 554-574
(5'-GTCCCTCAATAGGAAGAGGGA-3') of mouse -sarcoglycan (21) and
nucleotides 459-481 (5'-CGAGGTTCTTGGAGACTTTCTCG-3') and 769-790
(5'-CTTCCTGATAGGTGGACACTTGC-3') of mouse -sarcoglycan (24). For
-sarcoglycan, primers were designed based on an expressed sequence
tag sequence (GenBank accession no. AA269841) representing mouse
-sarcoglycan. The sense primer was from nucleotides 98-117 (5'-GCTGAGGTTCAAGCAAGTG-3'), and the antisense primer was from 444-461
(5'-CTTCGCACAATTGCACGC-3'). For - and -sarcoglycans, nucleotide
sequences that are identical in man and hamster were chosen, namely for
human -sarcoglycan, nucleotides 352-374
(5'-TCAGAAGGGGAGGTCACAGGCAG-3') and 596-619
(5'-TTGGGGCATCCATGCTTAGACTCC-3'), and for human -sarcoglycan, nucleotides 139-162 (5'-CTGGTGAACTTGGCCATGACCATC-3') and 332-356 (5'-GTCTGGTCATTGAGAATGTTCACTG-3'). The number of PCR
amplification cycles (15, 25, and 30) was varied to allow a
semiquantitative estimate of levels of transcripts.
Biotinylation of Cell Surface Proteins and Preparation of Cell
Extracts--
C2C12 cells were washed three times with cold PBS and
incubated on ice for 15 min with 1 mg/ml NHS-LC-biotin (Pierce). The cells were washed once with PBS, and the residual NHS-LC-biotin was
inactivated by incubating the cells in 0.1 M glycine in PBS on ice for 5 min. Cells were washed three times with ice-cold PBS and
solubilized in lysis buffer (50 mM Tris, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture from
Roche Molecular Biochemicals) on ice for 20 min and centrifuged at
12,000 × g at 4 °C for 10 min. The protein
concentration of clarified lysates was measured with the BCA protein
assay kit (Pierce) using bovine serum albumin as the standard. For
isolation of biotinylated proteins, lysates were incubated overnight at
4 °C with 20 µl of streptavidin-Sepharose (Pierce). After washing
three times with lysis buffer, bound proteins were eluted in SDS-PAGE
sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting as
described below. In some experiments, cell extracts prepared from
biotinylated cultures were immunoprecipitated with Sepharose beads
coupled with either anti-- or anti--sarcoglycan antibodies as
described below.
Isolation of Skeletal Muscle Membranes--
Skeletal muscle
membrane proteins were prepared as described by Cohen et al.
(42). Three grams of mouse hindleg muscle were homogenized with a
polytron (Biospec Products, Bartlesville, OK) in 20 ml of
homogenization buffer (20 mM sodium pyrophosphate, 20 mM sodium phosphate monohydrate, 1 mM
MgCl2, 0.3 M sucrose, 0.5 mM EDTA,
pH 7.0) containing protease inhibitors. The homogenate was centrifuged
at 14,000 × g at 4 °C for 15 min. The pellet was extracted again with 10 ml buffer and centrifuged. The pooled supernatants were centrifuged at 30,000 × g at 4 °C
for 30 min to obtain the heavy microsome fraction. The pellet was
washed with a KCl solution (0.6 M KCl, 0.3 M
sucrose, 50 mM Tris-HCl, pH 7.4, containing protease
inhibitors) at 4 °C with agitation for 30 min, and then centrifuged
at 142,000 × g at 4 °C for 30 min. The pellet was
solubilized in lysis buffer by incubation on ice for 15 min, then
clarified by centrifugation at 12,000 × g at 4 °C
for 10 min.
Immunoprecipitation and Immunoblotting--
One hundred µg of
protein from extracts of cells or microsomes were incubated with 15 µl of Sepharose-bound, affinity-purified antibodies against - or
-sarcoglycan at 4 °C overnight. The Sepharose beads were washed
three times with lysis buffer, and bound proteins were resolved by
SDS-PAGE on 4-20% gradient gels (Novex, San Diego, CA). Proteins were
transferred from SDS-PAGE gels to nitrocellulose membranes (Bio-Rad).
The MultiMark standard was used for markers (Novex). In immunoblotting
experiments, antisera against -, -, and -sarcoglycans were
used at 1:100 dilution, and antisera against -sarcoglycan at
1:10,000 dilution. Goat anti-mouse IgG conjugated with peroxidase
(Bio-Rad) was used at a 1:2,000 dilution, and goat anti-rabbit IgG
conjugated with peroxidase (Calbiochem, San Diego, CA) was used at a
1:5,000 dilution. For detecting cell surface localization of SGCs,
streptavidin conjugated with peroxidase (Pierce) was used at a 1:1,000
dilution. Immunoreactive bands were visualized by enhanced
chemiluminescence (ECL+PLUS system; Amersham Pharmacia Biotech).
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RESULTS |
Generation of -Sarcoglycan-deficient Mice--
In order to
design a targeting vector to generate -sarcoglycan-null mice, we
cloned a large portion of the mouse asg gene. Exons 1 and 2 were chosen for targeting to delete the signal sequence and disrupt the
gene. Homologous recombination replaced the major portions of exons 1 and 2 and the entire first intron with the nlsLacZ/neo. The
disruption of the asg gene in one allele of ES cells was
verified by Southern blots of BamHI-digested genomic DNA
with a flanking probe (Fig. 1). In
properly targeted clones, this probe hybridized to two fragments: an
11-kb BamHI fragment, indicating the disrupted allele, and a
7.5-kb fragment from the wild type allele. In order to confirm a single
integration of the nlsLacZ/neo gene, digested DNA from
mutant clones were also hybridized to neo and
LacZ gene sequences. Two ES cell clones with the correct
genotype were injected into C57BL/6J blastocysts to generate chimeric
mice. These mice were bred to obtain two lines of asg 
/
mice.
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Fig. 1.
Targeted disruption of the mouse
asg gene by homologous recombination. Intron one
and parts of exons 1 and 2 were replaced with nlsLacZ/neo
cassette. The 5'-flanking probe used for Southern blot analysis is
indicated. The targeted allele gives an 11-kb BamHI-digested
fragment of genomic DNA, whereas wild type gives a 7.5-kb band. +/+,
wild type; +/, heterozygous; /, homozygous mutant.
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Sarcoglycans in asg-Null Mice--
Immunofluorescence analysis
showed no expression of -sarcoglycan in homozygous mutant mice (Fig.
2b), and absence of
-sarcoglycan was confirmed by immunoblotting (see below and Fig.
3A), indicating that the
targeting vector used had created a null allele. Immunofluorescence analysis showed that the expression and localization of -sarcoglycan were not affected in null mutant mice (Fig. 2d). This is in
agreement with previous work (27). Immunofluorescence analysis further showed that -sarcoglycan was present but reduced (Fig.
2c). In agreement with other reports, - and
-sarcoglycans were also reduced in amounts as measured by
immunofluorescence and/or immunoblotting (data not shown). The presence
of -sarcoglycan and the residual expression of -, -, and
-sarcoglycans suggested that a complex containing -, -, -, and -sarcoglycans might exist in the mutant mouse muscle.
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Fig. 2.
Histological and immunofluorescence analysis
of skeletal muscle from two-month-old mice. a,
e, and i show hematoxylin and eosin staining of
thigh muscle from homozygous mutant (/), heterozygous (+/) and
wild type (+/+) mice. Muscle from homozygous mutants (/) shows
dystrophic changes compared with muscle from heterozygous (+/) and
wild type (+/+) mice. -Sarcoglycan is absent from homozygous mutants
(b), The intensity of -sarcoglycan staining is reduced in
the asg / mice (c), whereas the level of
-sarcoglycan is the same in heterozygous mice (g) as in
wild type mice (k). -Sarcoglycan is not affected by the
absence of -sarcoglycan (d, h,
l).
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Fig. 3.
A, co-isolation of sarcoglycans in
asg / mice and wild type littermates using
anti--sarcoglycan antibodies. Heavy microsomes were isolated from
wild type and mutant mouse muscle, extracted, and incubated with
-sarcoglycan antibodies. The isolates from 80 µg of extract were
analyzed by SDS-PAGE, and immunoblotting. -sarcoglycan is absent in
the isolates from mutant mice, whereas -sarcoglycan is associated
with -sarcoglycan in both wild type and mutant mice. B,
co-isolation of sarcoglycans in normal muscle using anti-- or
anti--sarcoglycan antibodies. One hundred µg of protein from
extracts of microsomes was immunoprecipitated with either anti-- or
anti--sarcoglycan antibodies. The immune complexes were analyzed by
SDS-PAGE and immunoblotted with antibodies against -, -, -,
-, and -sarcoglycans. -Sarcoglycan was undetectable in the
preparation isolated with anti--sarcoglycan antibodies and
vice versa.
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To determine whether -sarcoglycan in mutant mouse muscle is
associated with the other sarcoglycans, we used -sarcoglycan antibody beads to isolate SGCs from wild type and mutant mouse muscle
and analyzed such complexes for the presence of -, -, and
-sarcoglycans (Fig. 3A) as well as for -sarcoglycan
(data not shown). All these sarcoglycans co-isolated with
-sarcoglycan from extracts of wild type muscle, suggesting that -
and -sarcoglycans may be present in the same complex with
-sarcoglycan or form separate complexes with -sarcoglycan. From
asg null mutant mice, the -sarcoglycan antibody
co-isolated -, -, and -sarcoglycans, suggesting the existence
of an --- complex. To determine if an ---
complex is also present in normal muscle, we carried out
immunoisolation with antibodies against either - or -sarcoglycan, followed by immunoblotting analysis to detect the individual
sarcoglycans within the isolates (Fig. 3B).
Anti--sarcoglycan antibody co-isolated -, -, and
-sarcoglycans, but not -sarcoglycan. However, the anti--sarcoglycan antibody also co-isolated -, -, and
-sarcoglycans, but -sarcoglycan was not found in these isolates.
The data indicate that at least two different sarcoglycan complexes are
present in normal skeletal muscle, one composed of --- and
another of ---, and that when -sarcoglycan is absent,
the --- complex persists. To confirm the existence of two
such complexes, we next used a defined cell system, the C2C12 cells, to
study the association of sarcoglycans.
Differential Expression of Sarcoglycans in Vitro--
First, we
used RT-PCR to determine transcript levels of individual sarcoglycans
during myogenic differentiation. RNA was isolated from C2C12 cells at
different time points. By varying the number of cycles in RT-PCR, we
found that transcripts for - and -sarcoglycans, the striated
muscle-specific sarcoglycans, increased during differentiation. In
contrast, no difference in transcript levels of -, -, and -sarcoglycans was detected regardless of the number of PCR cycles used (Fig. 4A). Next, we
determined protein levels by immunoblotting using specific antibodies
against each sarcoglycan. - and -sarcoglycans were barely
detectable in C2C12 myoblasts (Fig. 4B, lane
1 day) but were present at high levels in
cultures of differentiated cells (Fig. 4B, lanes
2-5 days). The protein levels for -sarcoglycan also
increased slightly, while - and -sarcoglycans were present at
constant levels throughout the culture period (Fig. 4B).
Hence, the expression of - and -sarcoglycans, which are striated
muscle-specific, appeared to correlate with myogenic differentiation at
both transcriptional and translational levels, while -, -, and
-sarcoglycans were expressed constitutively.
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Fig. 4.
Expression of sarcoglycans during myogenic
differentiation of mouse C2C12 cells. A, mRNA
expression of sarcoglycans. Total RNA was purified from C2C12 cells on
day 0, 1, and 5 of differentiation and subjected to 30 cycles of PCR.
Amplified PCR products were resolved on 1% agarose gels and stained
with ethidium bromide. mRNA levels of - and -sarcoglycans
increased during differentiation, while no differences were detected in
mRNA levels of -, -, and -sarcoglycans. B,
analysis of protein expression levels in differentiating C2C12
cultures. Total protein (30 µg/well) prepared from C2C12 cell
extracts on day 1, 2, 3, and 5 of differentiation was separated by
SDS-PAGE under reducing condition, transferred to nitrocellulose
membranes, and blotted with specific antibodies against each
sarcoglycan. - and -sarcoglycans increased from day 2 of
differentiation, -sarcoglycan increased slightly, while no change
was detected in levels of - and -sarcoglycans.
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- and -Sarcoglycans Characterize Separate Complexes in Muscle
Cells--
The -, -, -, and -sarcoglycans have been shown
to exist in a stable complex, as they can be co-isolated from rabbit
skeletal muscle (15, 43) and mouse C2C12 myocytes (22). To test whether -sarcoglycan also exists in such a complex with other sarcoglycans in C2C12 cells, lysates prepared from C2C12 cells were used for immunoisolation with antibodies against either - or -sarcoglycan, followed by immunoblotting analysis of bound proteins using antibodies specific for each sarcoglycan. As shown in Fig.
5A, the anti--sarcoglycan antibody co-isolated -, -, and -sarcoglycans but not
-sarcoglycan. The amount of the complex, as judged from the
intensity of each sarcoglycan band, increased dramatically around day 3 after induction of differentiation (data not shown). The -, -,
and -sarcoglycans were also co-isolated by the anti--sarcoglycan
antibody (Fig. 5A), but -sarcoglycan was not detected in
such preparations even after prolonged exposure of the immunoblot.
These results confirm in cultured muscle cells that -, -, and
-sarcoglycans associate with either - or -sarcoglycan, forming
distinct complexes.
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Fig. 5.
A, sarcoglycan complexes in
differentiated C2C12 cells analyzed by co-immunoprecipitation using
anti-- or anti--sarcoglycan antibodies. One hundred µg of
cellular protein, prepared from cultured C2C12 cells on day 5 of
differentiation, were immunoprecipitated with either anti-- or
anti--sarcoglycan antibodies. The immunocomplexes were analyzed by
SDS-PAGE and immunoblotted with antibodies against -, -, -,
-, and -sarcoglycans. -Sarcoglycan was undetectable in
anti--sarcoglycan isolates and vice versa. B,
cell surface expression of sarcoglycan subcomplexes. Cultured C2C12
cells were labeled with NHS-LC-biotin. One hundred µg of protein from
cell lysates were immunoprecipitated with antibodies against either
- or - sarcoglycan, and precipitated proteins were analyzed by
SDS-PAGE. After proteins in the gel were transferred to nitrocellulose,
biotin-labeled proteins were detected by streptavidin conjugated with
peroxidase.
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Cell Surface Localization of Two Sarcoglycan Complexes--
The
SGC containing -sarcoglycan is present in the membrane fraction of
muscle tissue and differentiated mouse myotubes (22, 32, 43).
Furthermore, transfection of Chinese hamster ovary cells has
demonstrated that cell surface localization of -, -, -, and
-sarcoglycans requires co-expression of all four sarcoglycans (23).
To determine if the -sarcoglycan-containing SGC, like the -SGC,
is localized to the cell surface in C2C12 cells, cell surface proteins
were labeled with biotin before lysis of the cells. SGCs were then
isolated with either anti-- or anti--sarcoglycan antibodies, and
labeled polypeptides visualized with streptavidin-conjugated peroxidase
following separation on SDS-PAGE. A strong band at 35 kDa, which
corresponds to the migration position of - and -sarcoglycans, and
a weaker band at 43 kDa, corresponding to -sarcoglycan, were
observed in anti--sarcoglycan isolates (Fig. 5B). We did
not detect a band corresponding to -sarcoglycan (~50 kDa). There
are five lysine residues within the extracellular portion of mouse
-sarcoglycan (21), but -sarcoglycan may be poorly labeled by
biotin. Anti--sarcoglycan isolates contained the same 35- and 43-kDa
bands as were present in anti--sarcoglycan isolates, and in addition
a band at ~47 kDa corresponding to the expected
Mr of -sarcoglycan (Fig. 5B).
-Sarcoglycan has 12 lysine residues, which may potentially be
labeled with biotin (25). The reverse experiment, using
streptavidin-Sepharose as the primary reagent for isolation, followed
by immunoblotting with antibodies against -, -, -, -, and
-sarcoglycans, confirmed the expression of -sarcoglycan at the
cell surface (data not shown). Taken together, these results indicate
that both - and -SGCs are expressed at the cell surface.
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DISCUSSION |
The importance of sarcoglycans in skeletal muscle is evident from
the various forms of muscular dystrophy that result from their absence.
In order to analyze the molecular events in the pathogenesis of
-sarcoglycanopathy, we generated an -sarcoglycan-deficient mouse
by homologous recombination in ES cells. The homozygous mutant mice
generated from the mutant ES cells expressed no detectable -sarcoglycan mRNA or protein. Although not described in detail in this report, the mutant mice we generated are similar in phenotype to the ones previously reported (27). They are outwardly normal, similar to dystrophin-deficient mdx mice but different from
mice with laminin 2-deficiency (38) that have a much more severe phenotype affecting their behavior and viability at an early age. However, the -sarcoglycan-deficient mice do show the hallmarks of
muscular dystrophy, including early stage muscle hypertrophy and
regeneration and fibrosis at later stages.
Immunofluorescent analysis of sarcoglycans in the muscle of the
asg / mice showed a reduction in staining intensity for the -, -, and -sarcoglycans relative to wild type and
heterozygous mice. In contrast, the staining intensity for
-sarcoglycan was similar in mutant and wild type mice. A reduction
in the staining intensity for -, -, and -sarcoglycans in
-sarcoglycan deficiency has been reported for numerous patients
(34-36). We show here that the residual amount of -, -, and
-sarcoglycans is due to their presence in a complex with
-sarcoglycan. Although -SGC do not seem to be more abundant in
our -sarcoglycan mutant mice than in wild type mice, it is likely
that the -SGC, even at low levels, play some compensatory role in
skeletal muscle of both mutant mice and human patients with a primary
-sarcoglycan defect. Varying levels of -sarcoglycan expression,
and consequently of -SGC, in the skeletal muscle of these patients
could be a determinant of disease severity, i.e. low levels
of -sarcoglycan could mean more severe disease. In fact, great
variability in disease severity has been noted in patients (36). That
- and -sarcoglycans form similar complexes and presumably
substitute for each other to some extent in skeletal muscle is in
agreement with their high homology. At this point, we do not know if
-sarcoglycan also exists as a monomer or in association with
unidentified proteins.
-Sarcoglycan is expressed at higher levels in blood vessels and
nerves than in skeletal muscle fibers (24). However, the sarcoglycans
co-isolated with -sarcoglycan from muscle extracts in our
experiments must have originated, at least in part, from muscle cells,
because -sarcoglycan, which was present in the isolates, is specific
for striated muscle (11). To confirm the existence of an -SGC of
muscle origin, we also isolated such a SGC from clonal C2C12 cells,
which originate from skeletal muscle. SGCs from these cells also
included complexes composed of --- SGC in addition to the
well known --- SGC. We also show in C2C12 cells that SGCs
containing -sarcoglycan in place of -sarcoglycan are present at
the cell surface, a location that suggests that they are functional.
We found that -, -, and -sarcoglycans are expressed in
undifferentiated C2C12 myoblasts, and that only the expression of the
two striated muscle-specific sarcoglycans, - and -sarcoglycans, is induced during differentiation of the cells into myotubes. This
suggests that some of the sarcoglycans play a role in the undifferentiated cells as well as in the differentiated cells, and that
skeletal myoblasts contain a SGC consisting of -, -, and
-sarcoglycans. An -- SGC has been described in smooth muscle cells (26). Interestingly, as most studies indicate the necessity for a heterotetrameric SGC as a functional unit, the skeletal
myoblast and the smooth muscle SGCs may either contain an additional
copy of one of the sarcoglycans, or they may contain a homologue of the
missing -sarcoglycan.
An important question is whether a SGC contains four proteins, or
whether the complex is a protein composed of four subunits? Implicit in
the latter view are two assumptions: (i) that the sarcoglycan
functional unit is the tetramer rather than any single sarcoglycan, and
(ii) that the presence of one sarcoglycan in a cell signals the
presence of a tetrameric sarcoglycan assembly. The subunit hypothesis
suggests that cell types expressing fewer than four of the known
sarcoglycans must contain unidentified subunits. The subunit hypothesis
is supported by the many observations showing that a deficiency of one
sarcoglycan reduces the levels of other sarcoglycans (27, 34-36);
sarcoglycans unable to form a complex due to lack of a partner subunit
may be unstable. Our experiments support the subunit hypothesis in that
four sarcoglycans remained assembled with each other throughout all
extraction and fractionation procedures, and that - and
-sarcoglycans were never found in the same assembly. In contrast, in
immunoprecipitation experiments with an antibody raised to the
extracellular domain of -sarcoglycan, Chan et al. (22)
detected only free -sarcoglycan and were unable to
co-immunoprecipitate other known sarcoglycans. Previous studies have
shown that sarcoglycans interact with each other primarily via their
extracellular domains (44). Since the antibody used by Chan et
al. binds to a site in -sarcoglycan involved in interaction
with -, -, and -sarcoglycans, this antibody may have
dissociated SGCs after their extraction (22). In our study, the
antibodies used for immunoisolation were raised against the
intracellular domain of sarcoglycans and are less likely to interfere
with protein-protein interactions between individual sarcoglycans.
Although the functions of sarcoglycans are largely unknown, the current
focus is on their association with the DAGC, and on the muscular
dystrophy that develops in the absence of sarcoglycans. The
dystroglycan and associated DAGC components are proposed to link the
cytoskeleton with the extracellular basement membrane, thereby
stabilizing muscle and protecting it from contraction-induced damage.
However, dystroglycan apparently plays several non-structural roles; it
is essential for formation of the basement membrane in the early embryo
(45) and for a laminin-containing extracellular matrix in
vitro (46) and mediates a laminin-dependent assembly of a cytoskeletal network in cells (47). There is no firm evidence that
sarcoglycans play a structural role in muscle. In fact, the recent
report by Hack et al. (16) shows that there is no apparent structural damage to the skeletal muscle of -sarcoglycan-deficient mice, even with excessive exercise. Increased uptake of a dye into
sarcoglycan-deficient muscle indicates that the muscle cell membrane is
leaky, as it is in muscle deficient in dystrophin (48, 49). The leaky
membranes observed in sarcoglycan deficiencies may result from loss of
functional integrity of the cell membrane (16). In this regard, a
recent report that -sarcoglycan has ATPase activity is noteworthy
(17).
Only muscle-associated defects have been observed in sarcoglycan
deficiencies; however, sarcoglycans are likely to play important roles
in other tissues. Our preliminary data indicate that individual sarcoglycans are also expressed in endothelial cells, Schwann cells,
and macrophages.3 These
observations suggest the presence of novel SGCs in these cells. Both
the Drosophila melanogaster and Caenorhabditis
elegans genomes contain single /-like, -like, and
/-like sarcoglycans. The observation that vertebrates contain up
to four genes for each gene present in Drosophila or
C. elegans (50) suggests that additional sarcoglycans await
discovery. In summary, we show here that -sarcoglycan is
functionally similar to -sarcoglycan in skeletal muscle, and suggest
that novel sarcoglycans may be responsible for sarcoglycan function in
non-muscle as well as muscle cells.
and
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ABSTRACT
INTRODUCTION
