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J. Biol. Chem., Vol. 277, Issue 25, 22883-22888, June 21, 2002
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From the
Received for publication, February 25, 2002, and in revised form, April 8, 2002
Mutations in dysferlin, a novel membrane protein
of unknown function, lead to muscular dystrophy. Myoferlin is highly
homologous to dysferlin and like dysferlin is a plasma membrane protein
with six C2 domains highly expressed in muscle. C2 domains are found in
a variety of membrane-associated proteins where they have been implicated in calcium, phospholipid, and protein-binding. We
investigated the pattern of dysferlin and myoferlin expression in a
cell culture model of muscle development and found that dysferlin is
expressed in mature myotubes. In contrast, myoferlin is highly
expressed in elongated "prefusion" myoblasts and is decreased in
mature myotubes where dysferlin expression is greatest. We tested
ferlin C2 domains for their ability to bind phospholipid in a
calcium-sensitive manner. We found that C2A, the first C2 domain of
dysferlin and myoferlin, bound 50% phosphatidylserine and that
phospholipid binding was regulated by calcium concentration. A
dysferlin point mutation responsible for muscular dystrophy was
engineered into the dysferlin C2A domain and demonstrated reduced
calcium-sensitive phospholipid binding. Based on these data, we propose
a mechanism for muscular dystrophy in which calcium-regulated
phospholipid binding is abnormal, leading to defective maintenance and
repair of muscle membranes.
The ferlin family is an emerging group of mammalian proteins
implicated in genetic disease. The ferlin family is named for its
homology to the Caenorhabditis elegans protein
fer-1 (1). fer-1 mutants exhibit fertility
defects because of aberrant membrane fusion in developing sperm that
results in an abnormal submembraneous vesicular accumulation (2). The
mutations in a human ortholog of fer-1, dysferlin, have been
associated with two different forms of muscular dystrophy in humans,
Miyoshi myopathy (MM)1 and
Limb Girdle muscular dystrophy 2B (LGMD2B) (2-4). The function of
dysferlin and the mechanism by which dysferlin mutations lead to muscle
dysfunction are unknown. It was recently reported (5, 6) that an
abnormal accumulation of submembraneous vesicles is a feature of
dysferlin mutant muscle.
Myoferlin is highly related to dysferlin and like dysferlin is also
found at the plasma membrane in heart and skeletal muscle (7). To date,
myoferlin has not been linked with any human disease, but its location
on human chromosome 10q24 and its expression pattern make it a
candidate gene for an autosomal dominant spastic paraplegia, SPG9, a
disorder associated with muscle wasting and weakness (8). Based on
homology to fer-1 and their predicted protein domain
structure, the mammalian ferlins may have evolved specialized roles for
cell type-specific membrane fusion. Myoferlin and dysferlin each have a
single carboxyl-terminal transmembrane domain and six C2 domains that
are predicted to reside in a large cytoplasmic domain (9).
Crystallographic studies have shown that the C2 domain is an
independently folding domain composed of eight Because of the predicted protein structure, domain function, and
expression of dysferlin and myoferlin at the sarcolemma, we
hypothesized that dysferlin and myoferlin are important for membrane
fusion, because they occur during muscle development and during muscle
repair. To assess this hypothesis, we studied the expression of ferlin
proteins in an in vitro model of muscle development (21).
Mature skeletal muscle is a syncytium that forms from the fusion of
mononucleated myoblasts to multinucleate myotubes. We found that
myoferlin was highly expressed in myoblasts that have elongated prior
to fusion to myotubes. After fusion, myoferlin expression was
decreased. Dysferlin expression increased concomitant with fusion and
maturation of myotubes. Given that ferlins are expressed in the correct
spatiotemporal pattern to play a role in membrane fusion events in
muscle, we studied the biochemical properties of myoferlin and
dysferlin C2 domains. We found that the first C2 domain, C2A, bound a
negatively charged phospholipid mixture similar to the phospholipid
composition of the inner surface of the plasma membrane (22, 23).
Furthermore, C2A binding to phospholipids was calcium-sensitive. A
point mutation in the C2A domain of dysferlin that causes muscular
dystrophy showed abnormal calcium sensitivity and reduced phospholipid
binding. Together, these data suggest a role for the ferlin proteins in membrane fusion and a novel mechanism for muscular dystrophy.
C2C12 Expression--
C2C12 cells were cultured and harvested at
different timepoints in differentiation. Reducing serum content in the
medium to 2% induced differentiation. Cells were collected,
resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton
X-100) with protease inhibitors (Complete protease inhibitor mixture,
Roche Molecular Biochemicals), and incubated on ice for 10 min.
Cellular debris was removed by brief centrifugation at 8,000 × g for 5 min at 4 °C, and the protein concentration of the
supernatant was assayed with a Bio-Rad protein assay. Equal amounts of
protein were separated by SDS-PAGE followed by electrophoretic transfer
to polyvinylidene difluoride Immobilon-P membrane (Millipore).
The membrane was blotted with Myof2 or Dysf1 (7) at 1:1000 in
5% milk in 1× Tris-buffered saline plus 0.05% Tween 20. Secondary
antibody conjugated to horseradish peroxidase (Jackson Immunochemicals)
was used at 1:5000. ECL-Plus (Amersham Biosciences) substrate was used
for detection and visualized on Kodak Biomax MS film.
Immunofluorescence was performed on C2C12 cells grown on sterile glass
coverslips in tissue culture dishes. The coverslips were fixed in
methanol for 2 min, rehydrated in 1× phosphate-buffered saline, and
blocked in phosphate-buffered saline + 5% fetal bovine serum. They
were incubated with Myof2 at 1:100. After secondary antibody
conjugated to Cy3, the cells were mounted in Vectashield with
4',6-diamidino-2-phenylindole (Vector Labs), and images were captured
with an Axiophot microscope and Axiovision (Carl Zeiss) software.
Construction of Expression Vectors--
Sequence alignments to
identify C2 domains were performed with MacVector (Oxford Molecular
Group). The myoferlin C2A construct encoded for amino acids 1-124
(nucleotides 97-471 of GenBankTM accession number
AF182316), myoferlin C2B construct represents amino acids
197-327 (nucleotides 685-1079), myoferlin C2C construct represents
amino acids 357-505 (nucleotides 1165-1611), C2D construct represents
amino acids 1116-1254 (nucleotides 3442-3860), C2E construct
represents amino acids 1518-1685 (nucleotides 4648-5153), and C2F
construct represents amino acids 1771-1930 (nucleotides 5407-5888).
Primers corresponding to each of these nucleotide regions were designed
and used to amplify inserts from a human myoferlin cDNA template.
The inserts were ligated into the EcoRI and NotI
sites of pGEX4T-1 (Amersham Biosciences).
Dysferlin C2 Expression--
Human dysferlin C2A was amplified
using the following two primers: 350F Dys C2A-EcoR,
5'-GGAATTCATGCTGAGGGTCTTCATCCTCTATGCC-3', and 745R Dys
C2A-NotI, 5'-ATCAGCGGCCGCTGTGTAGGACACCTGCAGGACCAGCGAGG-3', using plasmid A15 generously provided by Drs. R. H. Brown and M. Ho. The dysferlin C2A domain was cloned into pCR2.1-TOPO (Invitrogen). The dysferlin V67D mutation was generated by PCR amplification using
the primers 5'-CAGGGCTCTGAGCTCCATGATGTGGTCAAAGACC-3' and 5'-GGTCTTTGACCACATCATGGAGCTCAGAGCCCTG-3', with Pfu
polymerase (Stratagene). The normal and mutant inserts were digested
with EcoRI and NotI and ligated into pGEX4T-1.
All sequences were verified by cycle sequencing.
Lipid Binding Assays--
Fusion proteins were expressed in
Escherichia coli. Log phase E. coli were lysed in
lysis buffer (50 mM Tris, pH 7.4, 5 mM EDTA,
1.5 mg/ml lysozyme, 0.35 M NaCl, 0.75% Nonidet P-40) and sonicated for 3 × 10 s with 15-s rest intervals. Inclusion
bodies were pelleted by centrifugation at 15,000 rpm for 30 min. The supernatant containing soluble fusion protein was recovered. Fusion proteins were immobilized on glutathione-Sepharose, washed four times
in buffer A (50 mM HEPES, pH 7.4, 0.1 M NaCl, 2 mM EGTA), and used for lipid binding assays essentially as
described previously (15). Liposomes were made from a total of 1 mg of
lipid (Avanti Lipids, Alabaster, AL) plus 10 µCi of
[3H]phosphatidylcholine (Amersham Biosciences), lipids
were mixed, dried under nitrogen, dried under vacuum overnight, and
resuspended in 2 ml of buffer A. The lipid suspension was extruded
through a 0.1-µm filter in an extruder (Avanti Lipids) to give
uniform-sized vesicles. 7.5 × 105 cpm of lipids were
used per assay with 15 µg of protein. Protein was quantified by
Bio-Rad protein assay. Free calcium concentrations were calculated with
WEBMAXC version 2.10 (www.stanford.edu/~cpatton/webmaxc2.htm) (24). All experiments were carried out in MilliQ water (Millipore) at
room temperature, and a 0.1 M calcium standard (Orion) was used for making all of the solutions. Proteins were washed three times
in the specified calcium buffer, and lipid mixtures were brought to the
appropriate free calcium concentration before mixing for the assay.
Protein and lipid were incubated for 15 min at 24 °C with continuous
vortexing. Sepharose fusion protein was collected by centrifugation at
14,000 rpm for 1 min and washed three times in the appropriate calcium
buffer. 3H-Lipid was quantified by scintillation counting.
Assays were performed in triplicate with at least two separate lipid
and protein preparations. Assays performed in the presence of 1 mM KCl, 1 mM MgCl2, or 1 M NaCl were treated in the same manner. Curve-fitting and
binding constant calculations were performed using Origin 6.1 software
(Origin Lab Corp., Northampton, MA). For dysferlin C2A lipid binding
assays, 4 × 105 cpm of lipids were used, and results
were normalized to 15 µg of protein.
Expression of Dysferlin and Myoferlin in Myoblast
Differentiation--
To investigate the potential role of dysferlin
and myoferlin in skeletal muscle membrane fusion, we examined the
expression of dysferlin and myoferlin during myoblast differentiation
in C2C12 cells, a cell line that undergoes differentiation from
mononucleate myoblasts to multinucleate syncytial myotubes (21).
Cultures representing different phases of fusion and differentiation in C2C12 cells were harvested and tested for dysferlin and myoferlin expression (Fig. 1A).
Immunoblotting with an antibody specific to dysferlin (7) showed that
dysferlin was expressed at the greatest levels in mature myotubes (Fig.
1A, bottom, and B, lane 3).
An antibody specific to myoferlin (7) demonstrated that myoferlin is
expressed at the greatest levels in cultures with high numbers of
prefusion myoblasts (Fig. 1A, top, and
B, lane 1). The level of myoferlin protein
decreased after fusion of myoblasts to myotubes had occurred (Fig.
1B, lane 3).
To elucidate further the timing of myoferlin expression, we tested
C2C12 cultures at different stages of myoblast differentiation for
expression of myoferlin using indirect immunofluorescence and an
antibody specific to myoferlin. We noted high levels of myoferlin
protein expression in those myoblasts that had become elongated prior
to myoblast fusion into myotubes (Fig. 2,
A-D, arrows). These single-celled myoblasts
retained high level myoferlin expression, whereas those myoblasts that
had undergone cell-cell contact, fusion, and differentiation had
greatly reduced myoferlin protein expression (asterisk).
High magnification views of myoferlin expression in C2C12 cells during
myocyte differentiation demonstrated that myoferlin is expressed in
vesicles within the myoblasts (Fig. 2, E and F,
arrow).
Dysferlin and Myoferlin Contains Six C2 Domains--
We analyzed
the sequence of dysferlin and myoferlin using the SMART protein domain
analysis program (smart.embl-heidelberg.de/) (25, 26) in which at least
six C2 domains were predicted (Fig. 3A). In myoferlin, there is a
potential seventh C2 domain (amino acids 1302-1409,
GenBankTM accession number AF182316) with a high
E value indicating a low confidence level and lacking the
majority of the conserved residues found in other C2 domains.
Therefore, the sequence analysis is most consistent with six C2 domains
in dysferlin and myoferlin. The position of each of these C2 domains in
the linear dysferlin and myoferlin protein sequence is indicated in the
schematic in Fig. 3A. Of note, there is high conservation
between homologous C2 domains of dysferlin and myoferlin (an average of
74% homology). That is, C2A of dysferlin is more related to C2A of
myoferlin than it is to other C2 domains in dysferlin (an average of
15% homology).
Fig. 3B shows the alignment of consensus C2 domain with the
C2A domains of dysferlin and myoferlin. This alignment demonstrates the
conserved residues found in many C2 domains as well as the predicted
calcium binding residues. Based on sequence alignments, we predicted
that these domains have type II topology similar to those seen in
cytosolic phospholipase A2, the non-classical protein
kinase C isoforms A Single Myoferlin C2 Domain Binds to Phospholipids--
All six
myoferlin C2 domains were generated as GST fusion proteins (Fig.
4A). Each purified fusion
protein was tested for its ability to bind to phospholipid vesicles
containing lipids found in the cytoplasmic surface of the cell membrane
in muscle (22, 23). Lipid vesicles containing 50% phosphatidylserine (PS) in phosphatidylcholine (PC) bound myoferlin C2A in the presence of
1 mM calcium (Fig. 4B). The lipids composed of
50% PS did not show binding to any of the other five myoferlin C2
domains, C2B, C2C, C2D, C2E, and C2F (Fig. 4B). None of the
six myoferlin C2 domains demonstrated binding to 100% PC vesicles in
the presence or absence of calcium (Fig. 4C). These findings
are consistent with the phospholipid binding properties of a number of
homologous C2 domains that similarly do not bind neutral phospholipid
(15) but instead demonstrate binding to the negatively charged PS (14, 27). The lipids containing 25% PS in PC showed no binding to myoferlin
C2A, C2C, or C2F regardless of calcium concentration (data not shown),
suggesting that a significant presence of negatively charged
phospholipid is required for the interaction with myoferlin C2A. Also,
50% phosphatidylinositol and 50% phosphatidylethanolamine vesicles
were similarly tested and did not bind to any of the six myoferlin C2
domains (data not shown).
Binding Properties of Myoferlin C2A to Phosphatidylserine--
A
range of calcium concentrations was tested to determine the binding
constant of myoferlin C2A for 50% PS. C2A was unable to bind
PS-containing vesicles in the presence of normal intracellular calcium
levels (0.1 µM); however the ability to bind rapidly
increased with half-maximal lipid binding observed at 1 µM (Fig. 5A).
The interaction of myoferlin C2A with PS is abolished in the presence of high salt, consistent with the predicted electrostatic interaction (Fig. 5B). The requirement for calcium is also specific,
because in the presence of Mg2+ or K+,
phospholipid binding did not occur (Fig. 5B).
Mutation in Dysferlin C2A Abolishes Lipid Binding--
Mutations
in dysferlin lead to two forms of muscular dystrophy, MM and LGMD2B (3,
4). A large number of mutations have been described to date (28-34),
and there is a phenotypic range of symptoms that varies from mild to
severe associated with these mutations. We tested the ability of
dysferlin C2A to bind lipid vesicles containing 50% PS and found that
dysferlin C2A demonstrated similar binding properties to myoferlin C2A
(Fig. 6A). Dysferlin C2A bound
50% PS with a half-maximal lipid binding at 4.5 µM
calcium. A mutation in dysferlin was recently described (V67D,
GenBankTM accession number AF075575) that is associated
with LGMD (31). Individuals with the V67D mutation exhibited a range of
phenotypic severity consistent with both MM and LGMD2B. This mutation
alters a residue within the C2A domain of dysferlin and introduces an acidic residue for a conserved non-polar residue within a Similar to most C2 domain-containing proteins, myoferlin and
dysferlin are found associated with membranes. The predicted topology
of dysferlin and myoferlin places the six C2 domains within the
cytoplasm anchored by their carboxyl-terminal transmembrane regions. In
a cell culture model of myoblast differentiation, dysferlin was
expressed at low levels in myoblasts and increased its expression upon
differentiation as myoblasts fused to myotubes. In contrast, myoferlin
was expressed highly in the prefusion myoblast. These prefusion
myoblasts were distinguished from quiescent myoblasts by their
elongated appearance and an accumulation of myoferlin-containing vesicles. The fusion of myoblasts to myotubes is known to involve vesicular structures (35). Similarly, the repair of torn surface membranes uses a number of mechanisms that may also include vesicular structures (36). Because of their intracellular location, the spatiotemporal pattern of expression, and domain structure, we hypothesize a coordinated role of myoferlin and dysferlin where myoferlin may be specialized for myoblast fusion and dysferlin may be
important for membrane fusion and repair in the mature myotube. To
examine the potential role of ferlin proteins in membrane fusion
events, we studied the biochemical properties of ferlin C2A domains and
found that myoferlin and dysferlin C2A domains bind phospholipid in a
calcium-sensitive fashion.
The ferlin proteins are the only known C2 domain-containing proteins
that have more than three C2 domains, and the ferlin proteins are
unique with six C2 domains. In other C2 domain-containing proteins, a
single C2 domain is often responsible for calcium-dependent phospholipid binding. We tested all six C2 domains of myoferlin and
found that only C2A demonstrated the binding to phospholipids under our
experimental conditions. The role of the remaining ferlin C2 domains is
unknown, but similar to other C2 domain-containing proteins, these
domains may be involved in protein-protein interactions. The
requirement for the negatively charged phospholipid may be influenced
by the number of positively charged residues in the calcium binding
loops of the dysferlin/myoferlin C2A domain. A negatively charged lipid
membrane domain is necessary for membrane fusion to occur (37, 38).
Additionally, the myoblast membrane is associated with an unusual
composition of phospholipids that is distinct from fibroblasts and
consistent with the phospholipid binding profile of C2A (22, 23). The
requirement for 50% PS for C2A binding suggests that C2A may respond
to lipid clustering in the membrane or to specific regions of the
membrane with higher concentrations of PS.
Myoblast fusion is critical during muscle development and in the repair
of existing muscle fibers damaged through regular use and exercise and
in degenerative disorders like muscular dystrophy. Myoblast fusion has
been shown to require an influx of calcium from the extracellular space
(39). It has been estimated that the fusion of myoblast membranes
requires calcium concentrations in the range of 1.4 µM
(40), and localized pools of increased calcium are present near the
regions of membrane fusion (41). Dysferlin and myoferlin C2A
phospholipid binding occurred at calcium concentrations above 1 µM, consistent with calcium requirements for myoblast
fusion. The repair of membrane tears also requires calcium (36). It is
known that in animal models for muscular dystrophy, these membrane
tears occur more frequently (42, 43). An elevation of myoferlin has
been observed in microsomal fractions from this same animal model (7),
potentially reflecting an increased need for membrane repair machinery
in this damage-susceptible muscle.
The first C2 domain of dysferlin demonstrated similar calcium and
phospholipid binding properties to myoferlin C2A. The cooperativity of
binding of these two C2 domains is consistent with binding multiple
calcium ions. The C2A domains are found at the extreme amino termini of
dysferlin and myoferlin, most remote from the transmembrane domain at
the carboxyl terminus. We speculate that this placement may indicate a
role for the C2A domain in localizing the amino terminus to the plasma
membrane or possibly in the attraction of ferlin-containing vesicles to
the plasma membrane. In support of this finding, it has been noted that
dysferlin mutant muscle displays an increase in vesicle accumulation
under the membrane (5, 6). Additionally, dysferlin interacts with
caveolin, another membrane-associated protein important for
intracellular vesicular trafficking (44).
Dysferlin missense mutations may alter dysferlin function. Illarioshkin
et al. (31) described a mutation associated with both mild
and severe phenotypes that changed a single amino acid (V67D) within
the C2A domain of dysferlin. This amino acid substitution occurs within
one of the eight There are two important membrane fusion events in skeletal muscle.
Myoblasts fuse into myotubes during muscle development in the embryonic
state, and in the adult, this process occurs to repair and regenerate
muscle fibers that have been damaged. The increased expression of
myoferlin specifically in the prefusion myoblast suggests that this
protein plays a critical role in this event. By demonstrating the
ability of myoferlin and dysferlin to bind calcium and phospholipids,
two of the key players in muscle membrane fusion, we have now placed
ferlin family members in the correct temporal and spatial position for
a role in membrane fusion in the development and repair of muscle.
We thank Donna Fackenthal for sequencing,
Stephen Meredith and David Gordon for assistance with liposome
preparation, Jihong Bai and Edwin Chapman for advice on lipid binding
assay protocols, and Dorothy Hanck for helpful discussions.
*
This work was supported by the Muscular Dystrophy
Association, the American Heart Association, and the National
Institutes of Health (to E. M. M.); a National Institutes of Health
Cardiovascular Pathophysiology training grant, an American Association
for University Women American dissertation fellowship, and the Medical
Scientist Training Program (to D. B. D.); and a National Science
Foundation fellowship (to K. R. D.).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 Medicine, The
University of Chicago, 5841 S. Maryland, Rm. G611, MC 6088, Chicago, IL
60637. Tel.: 773-702-2672; Fax: 773-702-2681; E-mail: emcnally@medicine.bsd.uchicago.edu.
Published, JBC Papers in Press, April 16, 2002, DOI 10.1074/jbc.M201858200
The abbreviations used are:
MM, Miyoshi
myopathy;
LGMD2B, Limb Girdle muscular dystrophy 2B;
GST, glutathione
S-transferase;
PC, phosphatidylcholine;
PS, phosphatidylserine.
Calcium-sensitive Phospholipid Binding Properties of Normal and
Mutant Ferlin C2 Domains*
,
**
Department of Pathology, the
§ Department of Molecular Genetics and Cell Biology, the
¶ Department of Medicine, and the Department of Human
Genetics, The University of Chicago, Chicago, Illinois 60637
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strands forming a
sandwich structure (10, 11). Calcium binding loops reside at one
end of the sandwich structure, and calcium binding is mediated
through a conserved group of aspartic acid residues. C2 domains are
present in many membrane-associated proteins (12, 13). Proteins
directly implicated in membrane fusion such as the synaptotagmins
contain two C2 domains. The first C2 domain of synaptotagmin binds
calcium and anionic phospholipids (14, 15). The second synaptotagmin C2
domain aids in protein-protein interactions and homo-oligomerization
(16) and more recently was shown to bind phospholipids (17, 18). Most
recently, data have emerged supporting the role of synaptotagmins as
calcium sensors regulating the process of fast exocytosis (19, 20). In
synaptotagmins, up to three calcium ions are known to bind at three
loops at the end of the sandwich structure.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of myoferlin in C2C12 cells
undergoing differentiation. A,
phase-contrast images of C2C12 cultures at three different stages of
differentiation. The top panel (1) represents the
early stages of myoblast differentiation, whereas the lower
panel (3) represents fully differentiated myotubes.
B, the expression of dysferlin (D) and myoferlin
(M) is shown using antibodies specific to each of these
proteins, Myof2 and Dysf1 (7). Lanes 1, 2, and
3 in B correspond to the phase contrast images
1, 2, and 3 in A. Dysferlin
expression, like many muscle-specific proteins, is up-regulated and
remains highly expressed in the differentiated myotube. Myoferlin is
expressed earlier when there is a greater number of prefusion and
fusing myoblasts. Myoferlin expression decreases in fully
differentiated cultures.
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Fig. 2.
Expression of myoferlin in developing
myoblasts. A-D, C2C12 cultures early in myoblast
differentiation stained with a myoferlin-specific rabbit polyclonal
antibody (Myof2) and a Cy3-conjugated secondary antibody. Prefusion
myoblasts become elongated prior to fusion. It is these prefusion
myoblasts that show the highest level of myoferlin expression
(arrows). After fusion, myoferlin expression was reduced
(asterisk). 4',6-Diamidino-2-phenylindole (blue)
counterstaining demonstrates nuclei. The areas in the white
box from A and B are shown in C
and D, respectively. E and F, high
magnification views of myoblasts undergoing differentiation stained
with the antibody Myof2. Myoferlin is highly expressed in vesicles
(arrow).
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Fig. 3.
Alignment of myoferlin and dysferlin C2A with
synaptotagmin I C2A. A, a schematic representation of
dysferlin and myoferlin protein. Each of the six predicted C2 domains
are shown in gray boxes, and the transmembrane domain is
shown in black. B, Myoferlin and dysferlin C2A
domains are aligned with a consensus sequence for C2 domains. This
consensus sequence was obtained using evolutionarily divergent C2
domains (rat synaptotagmin II, IV, and VI, an Arabidopsis
putative C2 domain protein, and rat protein kinase C) from
the Conserved Domain Data Base
(www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) using the SMART version
3.3 alignment. Because the secondary structure of a number of these C2
domains has been solved (13), the likely position of calcium binding is
indicated (circles), and the positioning of the strands
is indicated (bars). Seven of the eight strands are
indicated; the eighth is positioned to the right of the
diagram and is less conserved. The capital letters indicate
those residues that are important for the C2 structure, particularly
those of the strands, and predicted calcium-coordinating residues.
The letters in gray boxes are invariant in
evolutionarily divergent C2 domains. The asterisk indicates
a dysferlin point mutant responsible for muscular dystrophy (31) whose
calcium and phospholipid binding properties we studied.
, 
, 
, and 
, and phosphoinositide-specific phospholipase C isoforms. This topology prediction is based on the
presence of the predicted calcium binding residues between the first
and second strands and between the fifth and sixth strands as
seen in all topology II C2 domains (13). The C2 domains at the amino
terminus of proteins typically use topology II (13). The calcium
binding sites of topology II C2 domains, similar to dysferlin and
myoferlin C2A, have been noted to often lack one or more of the five
conserved aspartic acid or glutamic acid residues and to contain
alternative residues capable of coordination (13).
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Fig. 4.
C2 domain expression and binding to lipid
vesicles. A is a Coomassie Blue-stained
SDS-polyacrylamide gel demonstrating all six myoferlin C2 domains A-F
expressed as GST fusion proteins and purified from E. coli.
Each fusion protein is the correct predicted size. B, the
binding of myoferlin C2A to phospholipid vesicles containing 50%
PS/50% PC. None of the other fusion proteins nor the GST control
demonstrated binding to 50% PS/PC. Fusion proteins were incubated with
3H-labeled vesicles in the presence of 0.2 mM
EDTA (no calcium) or the presence of 1 mM free calcium. The
ability to bind to 100% phosphatidylcholine vesicles was tested
similarly in the absence and presence of 1 mM calcium. No
binding to 100% PC was seen.
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Fig. 5.
Myoferlin C2A binds 50% PS with positive
cooperativity. Myoferlin C2A fusion protein was incubated with
50% PS vesicles at various calcium concentrations. The ability to bind
phospholipid was measured as the amount of 3H-labeled
vesicles that remain bound to the protein after three washes.
A, myoferlin C2A bound 50% PS at calcium concentrations
greater than 1 µM. All assays were performed in
triplicate, and error bars represent mean ± S.D.
B, myoferlin C2A phospholipid binding is
calcium-dependent and involves electrostatic interactions.
Myoferlin C2A was unable to bind 50% PS in the presence of
Mg2+ or K+, demonstrating specificity for
calcium. Also, the presence of high salt (1 M NaCl)
prevented phospholipid binding even in the presence of 1 mM
calcium, demonstrating that myoferlin C2A phospholipid binding requires
electrostatic interactions.
strand (Fig. 3, asterisk). We introduced the V67D mutation into
dysferlin C2A and found abnormal phospholipid binding through a range
of calcium concentrations (Fig. 6A, dashed line).
A similar mutation was engineered into myoferlin C2A (I67D) and
similarly demonstrated abnormal calcium-dependent
phospholipid binding (data not shown). We determined that the
expression of dysferlin C2A and C2A-V67D was similar and that an
identical amount of protein was assayed for calcium sensitive
phospholipid binding (Fig. 6B). Thus, the abnormal binding
properties of dysferlin C2A-V67D arise from the mutation responsible
for the LGMD2B and MM phenotypes.
View larger version (18K):
[in a new window]
Fig. 6.
V67D mutation in dysferlin C2A abolishes
phospholipid binding. A point mutation in dysferlin (V67D) was
described as being associated with muscular dystrophy (31). Dysferlin
C2A was expressed as a GST fusion protein and purified from E. coli. A, dysferlin C2A binds 50% PS in a
calcium-dependent manner (solid line). A
missense amino acid substitution (V67D) that causes muscular dystrophy
was engineered and tested for its ability to bind phospholipids through
a range of calcium concentrations (dashed line). Partial
phospholipid binding was detected at lower calcium concentrations and
was diminished at higher calcium concentrations (dashed
line). Abnormal calcium-dependent phospholipid binding
combined with an abnormal accumulation of submembraneous vesicles in
dysferlin mutant muscle (5, 6) suggests that membrane fusion is
abnormal in dysferlin-mediated muscular dystrophy. B, the
expression and purification of the normal and V67D dysferlin C2A domain
are shown in lanes 1 and 2, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strands and inserts a novel charge residue.
Molecular modeling suggests that this residue is important to the core
structure of the C2 domain (data not shown). Dysferlin C2A-V67D showed
partial phospholipid binding at lower calcium concentrations but
minimal phospholipid binding at higher calcium concentrations. The peak
intracellular calcium concentration within a myofiber can be as high as
14 µM (45), thus it is highly probable that this mutation
alters phospholipid binding of dysferlin in vivo.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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