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Volume 272, Number 50, Issue of December 12, 1997
pp. 31221-31224
(Received for publication, August 7, 1997, and in revised form, September 24, 1997)
From the ABSTRACT The dystrophin-glycoprotein complex is a
multisubunit protein complex that spans the sarcolemma and forms a link
between the subsarcolemmal cytoskeleton and the extracellular matrix.
Primary mutations in the genes encoding the proteins of this complex
are associated with several forms of muscular dystrophy. Here we report the cloning and characterization of sarcospan, a unique 25-kDa member
of this complex. Topology algorithms predict that sarcospan contains
four transmembrane spanning helices with both N- and C-terminal domains
located intracellularly. Phylogenetic analysis reveals that
sarcospan's arrangement in the membrane as well as its primary
sequence are similar to that of the tetraspan superfamily of proteins.
Sarcospan co-localizes and co-purifies with the dystrophin-glycoprotein complex, demonstrating that it is an integral component of the complex.
We also show that sarcospan expression is dramatically reduced in
muscle from patients with Duchenne muscular dystrophy. This suggests
that localization of sarcospan to the membrane is dependent on proper
dystrophin expression. The gene encoding sarcospan maps to human
chromosome 12p11.2, which falls within the genetic locus for congenital
fibrosis of the extraocular muscle, an autosomal dominant muscular
dystrophy.
INTRODUCTION In skeletal muscle fibers, the dystrophin-glycoprotein complex
(DGC)1 (1-5) is located at
the sarcolemma and is composed of both peripheral and integral membrane
proteins. Collectively, these proteins provide a physical connection
between the extracellular matrix and the intracellular cytoskeleton of
muscle cells. Disruption of this linkage eventually progresses to
muscle cell necrosis, as evidenced by the dystrophic muscle phenotypes
that result from defects in several of the DGC components (for review,
see Refs. 6 and 7). Although the DGC is known to be essential for
normal muscle function, the precise role of this multi-protein complex
remains to be determined.
Purification of the DGC has led to the identification of many of its
constituent polypeptides, which range in size from 25 kDa to over 400 kDa and include glycosylated as well as non-glycosylated proteins
(1-4). Characterization of these proteins has increased our
understanding of how the DGC is oriented in the sarcolemma and has
provided clues to its function. In addition to dystrophin, the DGC
consists of In the present work, we have determined the amino acid sequence of two
peptides derived from 25DAP and have isolated the corresponding human
cDNA. Previous analysis of the 25DAP indicated that it is an
integral membrane protein, since it reacts strongly with a probe for
protein hydrophobicity (4). We now report that 25DAP is a novel
component of the DGC and is predicted to span the sarcolemma four
times. This is unusual for integral membrane proteins of the DGC, which
possess only a single transmembrane span. We have renamed 25DAP
"sarcospan" in reference to its multiple sarcolemma-spanning domains. The predicted topology of sarcospan is similar to that of the
tetraspan superfamily of proteins (8, 9), in which all members have
four transmembrane domains and a large extracellular loop.
EXPERIMENTAL PROCEDURES The DGC was
extracted from rabbit skeletal muscle membranes (10) and then purified
by sWGA affinity chromatography followed by ion exchange on a
DEAE-column, as described previously (1, 2, 5). Purified DGC was
electrophoresed on 3-15% gradient gels (0.7 mm thick) and stained
with Coomassie Brilliant Blue as described by Ervasti et al.
(5). The 25-kDa band was cut from the gel and sent to the Biopolymers
Laboratory at the Massachusetts Institute of Technology for in-gel
Lys-C digestion (11) and peptide microsequence analysis (12).
Sarcospan cDNA clones
were isolated by hybridization screening of a
CLONTECH human adult skeletal muscle cDNA
library (HL5002a) with a PCR-derived sarcospan cDNA probe encoding
exons 1 to 3. The exon structure of the cDNA clones was confirmed
by PCR, and the 5 Multiple sequence alignment of
sarcospan and the tetraspanins was accomplished as before (9) with the
GCG Wisconsin Sequence Analysis software programs. Predictions of
transmembrane regions and orientations were made with the TMpred
program.2 This algorithm is
based on the statistical analysis of Tmbase, a data base of naturally
occurring transmembrane proteins (14).
Adult human multiple tissue Northern
blots (CLONTECH) containing 2 µg of
poly(A+) RNA per lane were probed with PCR-amplified probes
representing the full-length cDNA (exons 1 to 3) of sarcospan
(13).
The strategy for generating polyclonal
antibodies has been described previously (15). For generating sarcospan
antibodies, we injected two New Zealand White rabbits (rabbits 216 and
217; Knapp Creek Farms) at intramuscular and subcutaneous sites with 500 µg of an N-terminal sarcospan-2 synthetic peptide,
CAADRQPRGQQRQGDAAGPD (Research Genetics), coupled to keyhole limpet
hemocyanin in an emulsion of Freund's complete adjuvant (Sigma). The
amino acids Cys-Ala-Ala were added to the peptide for coupling
purposes. Affinity purification of sarcospan antibodies was
accomplished using Immobilon-P (Millipore) strips containing the
N-terminal peptide coupled to bovine serum albumin (15). This antibody
did not react with mouse tissue.
Immunofluorescence on muscle biopsies
from control and several DMD patients was accomplished as described
(15) except that muscle sections were incubated with affinity-purified
rabbit 216 sarcospan antibody (1:10) at room temperature for 12 h.
The sections were observed under a Bio-Rad MRC-600 laser scanning
confocal microscope, and the digitized images were captured under
identical conditions.
DGC fractions from the
DEAE-column were pooled and concentrated to 800 µl. The samples were
separated by centrifugation through a 5-30% sucrose gradient (2, 5).
The gradients were fractionated into 1-ml fractions, and 80 µl of
each fraction was resolved by 3-15% SDS-PAGE and either stained with
silver or transferred to nitrocellulose. Immunoblot staining with
antibodies to DGC (goat 20, 1:500) and sarcospan (rabbit 216 and 217, 1:20) was performed as described previously (15).
RESULTS AND DISCUSSION To determine the identity of the 25DAP (1-3) (also called A5
(3)), proteins of the DGC were purified from rabbit skeletal muscle and
separated using preparative SDS-PAGE. Peptide fragments of the 25DAP
were obtained by Lys-C digestion. Microsequencing of the
resultant peptides gave the following sequences: peptide I,
KHRYQVFYVGV; peptide II, KDRQPRGQQRQGDAAGPDDPG.
The amino acid sequences of these two peptides were used to search the
GenBankTM data base with the tBLASTn program (16). This
analysis revealed a homology between peptide I and Kirsten
ras-associated gene (KRAG) from human and mouse (accession numbers
X89105 and U02487, respectively). Murine KRAG was first identified
based on coamplification of its transcript with Kirsten ras in Y1
adrenal carcinoma cells (17). Similarly, the human KRAG gene was also
identified through analysis of transcripts co-amplified with KRAS2 in
lung and ovarian carcinoma cells (13). Although peptide II was not
found in GenBankTM, TIGR, or dbEST data bases, sequencing
of additional KRAG clones from a human skeletal muscle cDNA library
identified at least four cDNAs that do encode peptide II.
Translation of these cDNAs predicts a protein identical to the KRAG
sequence deposited in GenBankTM including an additional 26 residues at the N terminus (Fig. 1, A and B). This 26-amino acid extension includes a
perfect match with 17 out of 21 amino acids found in peptide II
isolated from rabbit (see above). We suspect that the amino acid
differences between the rabbit peptide and the human cDNA are due
to species variation. As verification of this, partial sequencing of
the 5
[View Larger Version of this Image (70K GIF file)]
The primary amino acid sequence deduced from the human cDNA
predicts a protein with four transmembrane spanning domains and a
topology similar to that of the tetraspan proteins (Fig.
1B). Based on the predicted membrane topology of the human
protein and its residence in the sarcolemma, we have named this protein sarcospan. The tetraspans, also referred to as the transmembrane 4 superfamily (TM4SF) and the tetraspanins, have four transmembrane spanning helices that are conserved among family members (8, 9).
Although there is no clear function for the TM4SF members, many of them
are associated with integrins and have been implicated in the
regulation of cell proliferation (9). Using phylogenetic analysis, we
find that sarcospan is more closely related to the divergent family
members Rom-1, peripherin, and uroplakin (data not shown). We refer to
the two cloned cDNAs as sarcospan-1 (spn1; GenBankTM accession number X89105) and sarcospan-2
(spn2; GenBankTM accession number AF016028),
where Spn2 includes 26 amino acids at its N terminus. In light of the
identification of sarcospan, we have redesignated KRAG (13) as the
spn1 gene.
To examine the tissue distribution of sarcospan, we performed RNA
hybridization analysis. We probed human multiple tissue Northern blots
with a full-length spn1 cDNA probe. As shown in Fig.
2, a 6.5-kb transcript is present
exclusively in skeletal and cardiac muscles. However, a 4.5-kb
transcript is also found in skeletal and cardiac muscle, as well as in
thymus, prostate, testis, ovary, small intestine, colon, and spleen.
Our data suggest the presence of tissue-specific sarcospan transcripts.
The expression of sarcospan in a broad array of tissues is most similar
to that of dystroglycan (18, 19), suggesting that these proteins play important roles in muscle as well as non-muscle tissues.
[View Larger Version of this Image (91K GIF file)]
We confirmed that the 25-kDa Coomassie-stained band in the DGC was
sarcospan by staining immunoblots containing purified DGC with
sarcospan antibodies. Although a 25-kDa band is present in purified DGC
from rabbit skeletal muscle, it does not elicit an antibody response
when DGC preparations were used to immunize either sheep or goat (Fig.
3A). Thus, we generated a
polyclonal antibody to sarcospan by immunizing rabbits with peptide II.
This affinity-purified antibody specifically stains a 25-kDa band on immunoblots of purified DGC from rabbit skeletal muscle membranes (Fig.
3A).
[View Larger Version of this Image (71K GIF file)]
As a first demonstration that sarcospan is an integral component of the
DGC, we show that sarcospan enriches in purification of the complex.
Immunoblots of purified DGC stained with DGC antibodies show a clear
enrichment of these purified proteins relative to the levels in rabbit
skeletal muscle membranes (Fig. 3A). Identical immunoblots
illustrate that sarcospan also enriches in DGC prepared from rabbit
membranes (Fig. 3A).
As another test for association between sarcospan and the DGC, we
examined sarcospan expression in muscle from DMD patients. We examined
several DMD patients with primary mutations in dystrophin, resulting in
loss of the entire dystrophin protein. Indirect immunofluorescence assays with antibodies against dystrophin's N and C termini and the
central rod domain verify that dystrophin is completely absent from the
DMD sarcolemma (data not shown). Without dystrophin, the other members
of the complex are reduced, perhaps the result of premature protein
degradation, improper assembly of the complex, or aberrant
transportation to the sarcolemma. We show that sarcospan is present at
the sarcolemma in normal muscle and is dramatically reduced in DMD
muscle (Fig. 3B). The reduced staining of sarcospan in DMD
muscle provides further evidence that this protein is complexed with
the DGC.
The tight association of sarcospan with the DGC is illustrated by
centrifugation of the DGC through sucrose gradients. Sucrose gradient
sedimentation separates the DGC from any proteins which might bind with
low affinity to the complex. Proteins from the sucrose gradient
fractions were separated by SDS-PAGE. The resultant polyacrylamide gels
were stained with silver to better visualize sarcospan (Fig.
4), which stains weakly with Coomassie
Brilliant Blue (Fig. 3A). Immunoblotting with sarcospan
antibodies confirms that the intensely silver-stained band at 25 kDa is
sarcospan. The peak of DGC proteins migrates in fractions 9 and 10 as
seen by the silver-stained gels and Western blotting of these same fractions with anti-DGC antibodies (Fig. 4). Sarcospan migrates in the
same fractions as the DGC during sedimentation through sucrose
gradients, confirming that sarcospan is an integral member of this
complex (Fig. 4).
[View Larger Version of this Image (37K GIF file)]
The structural connection between the extracellular matrix and the
intracellular actin network is dependent on the integrity of the DGC.
Disruption of this linkage eventually progresses to muscle cell
necrosis, as evidenced by the dystrophic phenotype that results from
defects in several of the DGC components, including the dystrophin and
the sarcoglycans (for review, see Refs. 6 and 7). Mutations in
sarcospan would feasibly also give rise to a dystrophic phenotype. The
gene encoding sarcospan maps to human chromosome 12p11.2 (13).
Congenital fibrosis of the extraocular muscle (CFEOM) is an autosomal
dominant disorder which primarily affects the ocular muscles, rendering
patients with little or no eye movement. Linkage analysis of two large
unrelated families with CFEOM indicate that this disease locus lies
within 12p11.2-q12 (20, 21). We propose that sarcospan is a prime
candidate gene for this disease.
Sarcospan is the first identified member of the DGC that spans the
membrane more than once. In fact, over 60% of sarcospan's amino acids
are predicted to be within the membrane and this unique characteristic
may be providing clues to sarcospan's function. For instance,
sarcospan's transmembrane domains are expected to hold this protein
firmly within the lipid bilayer, in which case it could provide a solid
anchorage for the rest of the DGC. Additionally, the multiple
transmembrane regions of sarcospan might form a pore in the sarcolemma
and thereby serve as a membrane channel. The latter scenario is
particularly attractive in light of the emerging concept that the DGC
performs functional as well as structural roles in muscle. The charged
residues in sarcospan's transmembrane domains may be important for
protein-protein interactions, perhaps with integrins or other DGC
components. Finally, the observation that sarcospan expression is
amplified in some human tumors, in combination with the proposal that
tetraspan proteins play a role in cell growth, supports the idea that
sarcospan itself has essential functions that extend beyond its role in
muscle.
FOOTNOTES The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF016028. ACKNOWLEDGEMENTS We are indebted to Richard Cook at the
Biopolymers Facility at MIT. We thank the University of Iowa Diabetes
and Endrocrinology Research Center, funded through National Institutes
of Health Grant DK25295, and the University of Iowa DNA Sequencing Core Facility.
REFERENCES
COMMUNICATION:
Sarcospan, the 25-kDa Transmembrane Component of the
Dystrophin-Glycoprotein Complex*
§,
,, and**
Howard Hughes Medical Institute, Department
of Physiology and Biophysics and the Department of Neurology,
University of Iowa College of Medicine, Iowa City, Iowa 52242 and the
¶ Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Wilmslow Road, Manchester, M20 9BX, United Kingdom
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/
-dystroglycan, the sarcoglycans (, , 
, and
subunits), and the syntrophins. While most of the
"dystrophin-associated proteins" (DAPs) have been identified, one
of them, a 25-kDa protein (also called A5) (1, 3-5), has remained an
enigma.
sequence was determined by direct analysis of
biotinylated PCR products, as described previously (13). Sequencing
analysis of the clones was performed using dye terminator cycling and
analyzed on a 373A Stretch Fluorescent Automated sequencer (Applied
Biosystems). The 5 end of rabbit sarcospan was sequenced from a
CLONTECH rabbit skeletal muscle cDNA library,
as described for the human clone.
sarcospan cDNA from rabbit skeletal muscle was performed.
Translation of this region demonstrates an exact match between the
amino acids deduced by the rabbit cDNA (KDRQPRGQQRQGDAAGPDDPG) and
the amino acid sequence of peptide II (shown above). Furthermore, we
confirmed sarcospan expression in rabbit skeletal and cardiac muscle by Northern analysis with a full-length cDNA probe (data not shown).
Fig. 1.
Primary structure and predicted membrane
topology of sarcospan. A, the deduced amino acid sequence of
human sarcospan is shown in single-letter code. Homology
between the human sarcospan sequence and the Lys-C-digested fragments
of rabbit sarcospan are shown (underlined). Predicted
transmembrane domains are shaded with gray boxes. Consensus
phosphorylation sites for cAMP-dependent protein kinase
(#), protein kinase C (
), and casein kinase II (*) are indicated.
B, schematic representation of membrane topology of
sarcospan. Predictions of the topology and transmembrane helix orientation were made with the program TMpred (14).
Fig. 2.
Expression of sarcospan in human
tissues. Northern blots containing 2 µg of poly(A+)
RNA from the indicated human tissues were hybridized with a sarcospan-1
cDNA probe. Equal RNA loading was confirmed by hybridizing blots
with an actin probe (not shown). Molecular size markers are indicated
on the left (kb).
Fig. 3.
Association of sarcospan with the
dystrophin-glycoprotein complex. A, enrichment of sarcospan
with the DGC. 50 µg of crude surface membranes (lane 1)
and 80 µl of purified DGC (lane 2) were
electrophoretically separated on 3-15% SDS-polyacrylamide gels and
transferred to nitrocellulose. An SDS-polyacrylamide gel stained with
Coomassie Brilliant Blue is shown in the left panel.
Nitrocellulose transfers were separately stained with antibodies against DGC (middle panel) or affinity-purified rabbit
polyclonal antibodies against sarcospan (right panel).
Dys, dystrophin; -, -DG,
/-dystroglycan, respectively; -, -, -, and
-SG, -, -, -, and -sarcoglycans,
respectively; Syn, syntrophins; and SPN,
sarcospan. Molecular size standards are indicated on the left of each blot (× 103 Da). B,
immunohistochemical analysis of sarcospan in normal human control
(Control) and DMD (DMD) skeletal muscle.
Transverse cryosections were labeled by indirect immunofluorescence
with antibodies against dystrophin, sarcospan, and laminin-2.
Absence of dystrophin in the DMD patient was confirmed with three
separate dystrophin antibodies (not shown). Laminin-2 staining was
positive on both control and DMD muscle (not shown). Bar,
100 µm.
Fig. 4.
Isolation of the DGC by sedimentation through
linear sucrose gradients. DGC purified from rabbit skeletal muscle
membranes was centrifuged through sucrose gradients. DGC proteins in
fractions 7 to 15 from the sucrose gradient were electrophoresed on
3-15% SDS-polyacrylamide gels and stained with silver. Nitrocellulose transfers of identical samples were stained with DGC and sarcospan antibodies. Molecular size standards are indicated on the
right of each panel (× 103 Da).
§
Supported by the Robert G. Sampson postdoctoral research fellowship
from the Muscular Dystrophy Association.
Funded by the Cancer Research Campaign, UK.
**
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Institute, 400 Eckstein Medical Research Bldg., University of Iowa College of
Medicine, Iowa City, IA 52242. Tel.: 319-335-7867; Fax:
319-335-6957; E-mail: kevin-campbell{at}uiowa.edu; Web site address:
http://www-camlab.physlog.uiowa.edu.
1
The abbreviations used are: DGC,
dystrophin-glycoprotein complex; CFEOM, congenital fibrosis of the
extraocular muscle; DAP, dystrophin-associated protein; DMD, Duchenne
muscular dystrophy; KRAG, Kirsten ras associated gene; Spn1,
sarcospan-1; Spn2, sarcospan-2; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel eletrophoresis; kb, kilobase(s).
2
This program is available on the Internet
(http://ulrec3.unil.ch/software/TMPRED_form.html).
Volume 272, Number 50,
Issue of December 12, 1997
pp. 31221-31224
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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F. Duclos, V. Straub, S. A. Moore, D. P. Venzke, R. F. Hrstka, R. H. Crosbie, M. Durbeej, C. S. Lebakken, A. J. Ettinger, J. van der Meulen, et al. Progressive Muscular Dystrophy in alpha -Sarcoglycan-deficient Mice J. Cell Biol., September 21, 1998; 142(6): 1461 - 1471. [Abstract] [Full Text] [PDF]
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G. Piluso, M. Mirabella, E. Ricci, A. Belsito, C. Abbondanza, S. Servidei, A. A. Puca, P. Tonali, G. A. Puca, and V. Nigro gamma 1- and gamma 2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells J. Biol. Chem., May 19, 2000; 275(21): 15851 - 15860. [Abstract] [Full Text] [PDF]
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R. Barresi, S. A. Moore, C. A. Stolle, J. R. Mendell, and K. P. Campbell Expression of gamma -Sarcoglycan in Smooth Muscle and Its Interaction with the Smooth Muscle Sarcoglycan-Sarcospan Complex J. Biol. Chem., December 1, 2000; 275(49): 38554 - 38560. [Abstract] [Full Text] [PDF]
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