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J Biol Chem, Vol. 273, Issue 52, 34667-34670, December 25, 1998
andFrom the Howard Hughes Medical Institute, Department of Physiology and Biophysics, and Department of Neurology, University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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Abstract
Introduction
Procedures
Results & Discussion
References
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Four unique transmembrane glycoproteins comprise
the sarcoglycan complex in striated muscle. The sarcoglycan complex
contributes to maintenance of sarcolemma integrity. A shared feature of
four types of autosomal recessive limb girdle muscular dystrophy (LGMD) is that mutations in a single sarcoglycan gene result in the loss of
all sarcoglycans at the sarcolemma. The mechanism of destabilization is
unknown. We report here our findings of sarcoglycan complex biosynthesis in a heterologous cell system. We demonstrate that the
sarcoglycans are glycosylated and assemble into a complex that resides
in the plasma membrane. Complex assembly was dependent on the
simultaneous synthesis of all four sarcoglycans. Mutant sarcoglycans
block complex formation and insertion of the sarcoglycans into the
plasma membrane. This constitutes the first biochemical evidence to
support the idea that the molecular defect in sarcoglycan-deficient LGMD is because of aberrant sarcoglycan complex assembly and
trafficking, which leads to the absence of the complex from the sarcolemma.
The dystrophin-glycoprotein complex
(DGC)1 is a group of integral
and membrane-associated proteins located in the sarcolemma of cardiac
and skeletal muscle fibers (for review see Refs. 1 and 2). The DGC
consists of dystrophin, the syntrophins, Mutations in Despite rapid advances regarding the genetic defects that cause LGMD,
little is known about the molecular defect underlying the disease.
Furthermore, the sarcoglycan-deficient limb girdle muscular dystrophies
are a heterogeneous group of diseases (15), and it will be important to
understand how individual mutations abrogate sarcoglycan complex
assembly. Mutations may result in the complete absence of messenger RNA
for that gene, or alternatively an aberrant protein may be synthesized.
In either case, protein trafficking miscues may result that block
sarcoglycan complex biosynthesis.
Maturation and trafficking of another integral membrane protein, the
cystic fibrosis transmembrane conductance regulator (CFTR) Cl Our goal was to develop a cell culture system for the analysis of
sarcoglycan complex biosynthesis. We reasoned that a heterologous cell
system that lacks muscle-specific proteins would be ideal for studying
both normal and mutant sarcoglycans. We report here expression of cDNA Expression Constructs--
The Myc-tagged human Cell Culture and Transient Transfection by
Electroporation--
CHO cells were maintained in Surface Biotinylation, Immunoprecipitation, and
Immunoblotting--
Surface molecules were biotinylated using
NHS-biotin (Pierce). Confluent cell monolayers were incubated with
NHS-biotin in PBS (0.5 mg/ml) for 30 min at room temperature. Unreacted
NHS-biotin was inactivated and removed by washing with Tris-buffered
saline. Lysates were prepared by solubilization in lysis buffer (50 mM HEPES, pH 7.8, 300 mM NaCl, 1% digitonin,
0.1% Nonidet P-40, 2.5 mM EDTA, 100 mM sodium
vanadate, 2 mM pepstatin, 0.5 trypsin inhibitory units of
aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 µM leupeptin). Clarified lysates were incubated with 20 µl of avidin-Sepharose (Pierce) at 4 °C with rotation overnight.
The avidin-Sepharose complexes were washed three times with lysis buffer and analyzed by SDS-polyacrylamide gel electrophoresis and
immunoblotting with the monoclonal 9E10 antibody. For
co-immunoprecipitations, lysates were prepared as above and incubated
overnight with monoclonal antibodies raised against Cytosolic versus Particulate Fractionation and Sucrose Gradient
Sedimentation--
Detergent-free cell extracts were separated into
soluble and particulate fractions as described previously (20). Cell
lysates were subjected to 5-30% linear sucrose gradient sedimentation (14).
Enzymatic Deglycosylation--
Cell lysates (20 µg) were
treated as recommended by the manufacturer (Oxford Glycosystems) and
analyzed by immunoblotting with 9E10 antibody.
Immunocytochemical Staining and Fluorescence
Microscopy--
Cells were fixed in 2% paraformaldehyde, 0.2% Triton
X-100 in PBS for 5 min at room temperature. The cells were blocked with 2% bovine serum albumin in PBS and then incubated with primary 9E10
antibody. Primary antibody was detected with fluorescently tagged
secondary antibody (Jackson Immunoresearch). Cells were imaged using a
Bio-Rad MRC-1024ES laser scanning confocal microscope.
We engineered cytomegalovirus expression vectors encoding
full-length human
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
- and 
-dystroglycan, and
sarcoglycan, which is composed of four distinct transmembrane
glycoproteins named -, -, 
-, and 
-sarcoglycan (SG) (for
review see Refs. 1 and 2). Sarcospan (25 kDa), a recently identified
member of the DGC, is unique in that it is predicted to contain four
transmembrane domains (3). The DGC spans the sarcolemma and forms a
functional link between the extracellular matrix and the cytoskeleton
via the laminin-binding protein -dystroglycan and the actin-binding
protein dystrophin. The DGC is thought to stabilize the membrane
against contraction-induced damage (4).
-, -, -, and -SG cause autosomal recessive
limb girdle muscular dystrophy (LGMD) types LGMD2D (5), LGMD2E (6, 7),
LGMD2C (8), and LGMD2F (9), respectively. In each of these diseases a
mutation in any one sarcoglycan gene results in the deficiency of the
entire sarcoglycan complex. Genetic studies identified the BIO14.6
hamster as an animal model for sarcoglycan-deficient LGMD2F because of
a deletion in the -SG gene (10). In this animal, dystrophic features
are evident in skeletal and cardiac muscle (11), and the sarcoglycan
complex is missing (12, 13). We reported the first example of
sarcoglycan gene transfer and successful intervention of disease
progression in the BIO14.6 hamster, employing a recombinant -SG
adenovirus as a vehicle for gene transfer (14). Our work demonstrated
that the sarcoglycan complex is a requisite component of the DGC for stable anchorage of -dystroglycan to the extracellular face of the
sarcolemma, as well as maintenance of an intact sarcolemma.
channel, have been extensively studied. Cystic
fibrosis (CF) is an autosomal recessive disorder characterized by
defects in epithelial ion transport, often because of the loss of
functional CFTR from the cell surface (16). The majority of CF
mutations affect maturation of the CFTR by altering the secondary
structure and normal processing. The misfolded protein is targeted for
degradation via the ubiquitin-proteasome pathway (17-19). A similar
defect in sarcoglycan biosynthesis and trafficking may underlie LGMD, although no studies have been performed to date to address this issue.
-,
-, -, and -SG in Chinese hamster ovary (CHO) cells utilizing a
high efficiency transfection protocol. Biochemical studies demonstrate
that the sarcoglycans are glycosylated and assemble into a tight
complex that resides in the plasma membrane. Individually expressed
sarcoglycans were likewise glycosylated; however, they were located in
internal membrane pools. Strikingly, mutant sarcoglycan constructs
engineered to recapitulate known human mutations were found to abrogate
sarcoglycan complex assembly and targeting to the plasma membrane.
These results strongly suggest that the molecular defect in
sarcoglycan-deficient LGMD results from the aberrant assembly and
trafficking of the sarcoglycan complex to its final destination, the
sarcolemma. Furthermore, these results provide compelling support for
the idea that sarcoglycan complex assembly requires concomitant
synthesis of all four sarcoglycans.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
-,
-, -, -, and 
SG pcDNA3 (Amersham
Pharmacia Biotech) expression constructs have been described (14). The
Grb2myc expression construct has been described (20). For the
-SG and -SG deletion mutants, stop
codons were introduced immediately following amino acids 150 and 173, respectively. All constructs were sequenced by the DNA Core Facility at
the University of Iowa.
-minimal Eagle's
medium supplemented with nucleosides and 10% fetal bovine serum.
Electroporation was performed as described previously (20). Optimal
expression was achieved using approximately 5 µg of each plasmid DNA.
-SG (Ad1/20A6,
Ref. 14) or -SG (Sarc1/5B1, Ref. 14). Immune complexes were
resolved by SDS-polyacrylamide gel electrophoresis and analyzed by
immunoblotting using the 9E10 monoclonal antibody. For immunoblotting
with the 9E10 monoclonal antibody, proteins were visualized using
enhanced chemiluminescence (Pierce).
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-, -, -, and -SG, as well as sarcoglycan
deletion constructs that recapitulate mutations found in LGMD patients. All constructs contain a Myc epitope tag at the intracellular tail. A
schematic representation of these proteins is shown in Fig.
1. Overall, the sarcoglycans share a
similar topology with a single transmembrane domain. The sarcoglycans
are all glycosylated and possess clusters of cysteine residues in their
extracellular domains.
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Fig. 1.
Human sarcoglycan expression constructs.
A schematic of the sarcoglycan expression constructs is shown.
Transmembrane domains (TM), cysteine clusters
(cysteines), consensus sites for asparagine-linked
glycosylation (N), and the signal sequence
(signal) are indicated. Expected molecular masses are in
parentheses. All constructs contain a Myc epitope tag on the
intracellular tail. Disease-causing missense mutations in -SG (R77C
(23, 24), denoted by asterisk) and -SG (T151R (7),
denoted by asterisk), as well as frameshift mutations that
result in truncations in -SG (deletion of nucleotide 645 (8),
denoted by arrow) and -SG (deletion of nucleotide 656 (9), denoted by arrow) are shown.
The sarcoglycan expression constructs were introduced into CHO cells by electroporation (estimated transfection efficiency of 90%). To evaluate the extent of glycosylation, cell extracts were subjected to enzymatic deglycosylation with PNGaseF and analyzed by immunoblotting with monoclonal antibody 9E10 (Fig. 2A). All of the sarcoglycans are glycosylated, as evidenced by a decrease in molecular weight following PNGaseF treatment. Based on this crude estimation, the extent of glycosylation appears similar whether the proteins are expressed individually or all four together (Fig. 2A). These results indicate that the proteins are properly folded and competent to move through the glycolytic pathways. Next, to determine where the sarcoglycans reside within the cell, we examined the cytosol versus membrane distribution of the proteins. The sarcoglycans were found exclusively in the membrane fractions (Fig. 2B, lanes 2-6). As a control, the cytosolic adapter protein Grb2 was found in the soluble fraction (Fig. 2B, lane 7).
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One remarkable feature of the sarcoglycan complex isolated from native
tissue is that the complex is resistant to disassembly under extreme
conditions of detergent or alkaline pH (21, 22). To that end, we
performed biochemical experiments to determine whether the sarcoglycans
expressed in CHO cells assemble into a stable molecular complex. Cells
transfected with individual sarcoglycans or with all four sarcoglycans
were treated with NHS-biotin, a hydrophilic probe for surface
molecules. This probe forms a covalent bond with free amines in surface
proteins of living cells, and the biotin moiety then functions as a tag
for precipitation with avidin-Sepharose. Fig.
3A demonstrates that in cells
expressing -, -, -, and -SG, the sarcoglycan complex is
resident at the cell surface (lane 11). In contrast, in
cells expressing any single sarcoglycan, little if any sarcoglycan
protein appears at the cell surface, even in this overexposed blot
(Fig. 3A, lanes 7-10). This experiment has been
replicated in at least six independent experiments. As a control,
lysates were examined by 9E10 immunoblotting to confirm that similar
amounts of sarcoglycan proteins were synthesized in all cells (Fig. 3A,
lanes 2-6).
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To demonstrate formation of a stable molecular sarcoglycan complex, we
performed co-immunoprecipitations using monoclonal antibodies against
individual sarcoglycans. Immune complexes were analyzed by 9E10
immunoblotting, and as shown in Fig. 3A (lane 12), -, -, and -SG co-immunoprecipitate as a complex with
-SG. Specific immunoprecipitation using monoclonal antibodies
against -, -, and -SG also resulted in the
co-immunoprecipitation of all other sarcoglycans (data not shown).
Finally, we performed linear sucrose gradient fractionation of lysates
from cells expressing -, -, -, and -SG. As shown in Fig.
3B, the sarcoglycans migrate as a complex that peaks in
fractions 3-5. Together these data provide strong evidence that
simultaneous expression of -, -, -, and -SG in CHO cells
results in the formation of a tight molecular complex. Furthermore,
these results suggest that surface membrane localization of
sarcoglycans depends on the coordinate synthesis and assembly of -,
-, -, and -SG into a molecular complex.
Most missense and deletion mutations known to cause LGMD are
concentrated in the extracellular domains of the sarcoglycan proteins.
With this in mind, we engineered sarcoglycan extracellular deletion
mutants. The -SG mutant construct was designed to
remove a large portion of the extracellular domain (see Fig. 1). In
addition, the - and -SG constructs
recapitulate known mutations causing LGMD2C and LGMD2F, respectively
(8, 9). For completeness, an -SG deletion mutant was also
constructed; however, expression was too low for use in this study.
Lysates prepared from cells transfected with three normal sarcoglycans
and the corresponding single deletion mutant were fractionated into
soluble and membrane fractions and analyzed by 9E10 immunoblotting. As
seen in Fig. 4A, the deletion mutants were expressed at levels comparable with the normal
sarcoglycans, and like the normal proteins, the mutants were highly
enriched in membranes. This was expected as the deletion mutants retain their transmembrane domains. In the case of the -SG,
the large region removed from this protein includes the potential consensus site for N-linked glycosylation. In fact, this
protein is not glycosylated nor does it interfere with glycosylation of the other normal sarcoglycans as shown in Fig. 4A
(lanes 4 and 5).
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We next performed biochemical experiments to evaluate what effects
mutations in individual sarcoglycan proteins might have on the assembly
and localization of the complex as a whole. Living cells expressing
normal copies of -, -, and -SG, as well as the mutant
-SG, were treated with the membrane-impermeant
surface label NHS-biotin. Biotinylated surface molecules were recovered
using avidin-Sepharose, and the complexes were analyzed by 9E10
immunoblotting. The sarcoglycan complex is readily detected at the
surface of cells expressing -, -, -, and -SG, as
demonstrated earlier (Fig. 4B, lane 4; Fig. 3A, lane 11). Dramatically, the sarcoglycan
complex is nearly absent from the surface of cells expressing -,
-, -, and -SG (Fig. 4B, lane
5). Lysates from these cells were examined by 9E10 immunoblotting
to confirm that similar amounts of each sarcoglycan were in fact
synthesized (Fig. 4B, lanes 2 and 3).
This observation, together with the fractionation data, implies that
the normal sarcoglycan proteins (-, -, and -SG) are trapped in
internal membrane pools because of the presence of the
-SG mutant protein.
To assess molecular interactions of sarcoglycans expressed in the
presence of a single deletion mutant, we performed
co-immunoprecipitation experiments. -SG immunoprecipitations were
performed from cells expressing -, -, -, and -SG (Fig.
4B, lane 6), -, -, -, and
-SG (Fig. 4B, lane 7), -, -,
-, and -SG (Fig. 4B, lane
8), or -, -, -, and -SG (Fig.
4B, lane 9), and the immune complexes were
examined by 9E10 immunoblotting. -, -, and -SG proteins were
co-precipitated with -SG from cells expressing all four normal
copies of -, -, -, and -SG (Fig. 4B, lane
6). In contrast, only -SG is efficiently immunoprecipitated
from cells expressing one mutant sarcoglycan and three normal
sarcoglycans (Fig. 4B, lanes 7-9). In each case,
similar amounts of all four sarcoglycans were present (Fig.
4A), so changes in protein expression levels because of the
co-expression of a mutant construct cannot account for the disruption
in sarcoglycan complex assembly. Interestingly, the normal proteins
expressed together with -SG appear fully glycosylated
(Fig. 4A, lanes 4 and 5), suggesting that the block in sarcoglycan complex assembly occurs after
glycosylation. This result clearly demonstrates that the assembly of a
sarcoglycan complex requires four normal copies of -, -, -,
and -SG.
Finally, to substantiate our idea that the mutant sarcoglycans block
sarcoglycan complex assembly and trafficking to the plasma membrane, we
looked at the localization of these proteins in cells by
immunofluorescence. In cells expressing -, -, -, and -SG, the 9E10 antibody detected the sarcoglycan complex at surface membrane
structures (Fig. 4C, panel a). In addition, there
was a considerable amount of signal in various intracellular membrane compartments, which we attribute to the high levels of expression. In
contrast, in cells expressing -, -, -, and
-SG, the 9E10 antibody did not detect sarcoglycans at the surface,
and instead the proteins were concentrated in regions around the
nucleus (Fig. 4C, panel c). Our interpretation of
this finding is that sarcoglycans that fail to assemble because of the
presence of the -SG are not competent for trafficking
to the plasma membrane. The cells were imaged using differential
interference contrast optics to clearly define the cell borders (Fig.
4C, panels b and d).
Despite recent advances in our understanding of the genetic defects in
individual sarcoglycan genes that underlie autosomal recessive
sarcoglycan-deficient LGMD, essentially nothing is known about the
molecular defect that results in loss of the entire sarcoglycan complex
from the sarcolemma. We have employed a heterologous cell system to
study sarcoglycan complex assembly. The strength of this system is that
mutations in individual sarcoglycans can be assessed in terms of their
impact on sarcoglycan complex assembly. Additionally, this model system
may be useful as a tool to discover pharmacological interventions to
overcome the blockade in complex assembly and trafficking. Details
about the molecular defect in sarcoglycan biosynthesis gained from this
work will help direct efforts aimed at developing therapies for LGMD.
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ACKNOWLEDGEMENTS |
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DNA sequencing services and cell culture
media were provided by the University of Iowa Diabetes and
Endocrinology Research Center (National Institutes of Health Grant
DK25295). We are greatly indebted to Louise Anderson for monoclonal
antibodies Ad1/20A6 and Sarc1/5B1. We thank members of the Campbell
laboratory for helpful discussions and critique of the manuscript.
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FOOTNOTES |
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* This research was supported in part by the Muscular Dystrophy Association.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.
Supported by the University of Iowa Diabetes and Endocrinology
Research Center.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence and requests for materials should be addressed: Howard Hughes Medical Inst., University of Iowa College of Medicine, 400 EMRB, Iowa City, IA 52242. Tel.: 319-335-7867; Fax: 319-335-6957; E-mail: kevin-campbell{at}uiowa.edu.
The abbreviations used are: DGC, dystrophin-glycoprotein complex; SG, sarcoglycan; LGMD, limb girdle muscular dystrophy; CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.
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REFERENCES |
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Results & Discussion
References
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