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Unformatted text preview: © 1999 Oxford University Press Human Molecular Genetics, 1999, Vol. 8, No. 5 855–861 Dysferlin is a plasma membrane protein and is
expressed early in human development
Louise V. B. Anderson*, Keith Davison, Jennifer A. Moss, Carol Young,
Michael J. Cullen, John Walsh, Margaret A. Johnson1, Rumaisa Bashir2,
Stephen Britton2, Sharon Keers2, Zohar Argov3, Ibrahim Mahjneh4,
Françoise Fougerousse5, Jacques S. Beckmann5 and Kate M. D. Bushby2
Neurobiology Department and 1Neurology Department, University Medical School, Framlington Place, Newcastle upon
Tyne NE2 4HH, UK, 2School of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne
NE1 7RU, UK, 3Department of Neurology, Hadassah University Hospital, Jerusalem 91120, Israel, 4Division of
Neurology, Kainuun Central Hospital, 87140 Kajaani, Finland and 5Généthon, URA 1922, 1 rue de l’Internationale,
91002 Évry, France
Received December 21, 1998; Revised and Accepted February 3, 1999 Recently, a single gene, DYSF, has been identified which
is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy
(MM). This is of interest because these diseases have
been considered as two distinct clinical conditions since
different muscle groups are the initial targets. Dysferlin,
the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We
have now raised a monoclonal antibody to dysferlin and
report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of ∼230 kDa on western blots of
skeletal muscle, with localization to the muscle fibre
membrane by microscopy at both the light and electron
microscopic level. A specific loss of dysferlin labelling
was observed in patients with mutations in the LGMD2B/
MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of
immunoreactive protein in a manner reminiscent of the
dystrophin expressed in many Duchenne patients.
Analysis of human fetal tissue showed that dysferlin was
expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5–6
weeks). Dysferlin is present, therefore, at a time when the
limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of
muscle involvement that develops later, with the onset of
a muscular dystrophy primarily affecting proximal or distal muscles.
To date, at least eight forms of autosomal recessive muscular
dystrophy have appeared under the general heading of limb- girdle muscular dystrophy (LGMD). These are in two groups:
those with abnormal expression of the dystrophin–glycoprotein
complex (1) and those where labelling of proteins in this complex
is unaffected. Thus, the sarcoglycanopathies (sometimes known
as LGMD types 2C, 2D, 2E and 2F) are caused by defects in the
genes for γ-, α-, β- or δ-sarcoglycan on chromosomes 13q12,
17q12, 4q12 and 5q33, respectively (2–5). Among the dystrophies where expression of the sarcoglycans is normal, the gene
responsible for LGMD2A has been identified as the chromosome
15q15-encoded muscle-specific protease calpain 3 (6), and the
gene for LGMD2B was identified recently as the 2p13-located
DYSF (7,8). Genes for LGMD2G and LGMD2H have been
localized to 17q11–q12 (9) and 9q31–q33 (10), and further
LGMD genes are inferred (9,11). Although clinically heterogeneous in terms of age of onset and rate of progression, the feature
that all these conditions share is weakness that starts with the
proximal limb-girdle muscles. Unexpectedly, a disease characterized by the early involvement of distal calf muscles, Miyoshi
myopathy (MM), was shown to be caused by mutations in the
same gene as LGMD2B (8). Dysferlin, the protein product of the
DYSF gene, is a novel molecule without homology to any known
mammalian protein. However, the gene does have significant
homology, throughout its length, to a nematode spermatogenesis
factor (fer-1), a fact that contributed to the name it was given
(7,8). Here we present the first description of dysferlin protein
Dysferlin antibody generation
A monoclonal antibody was developed that recognizes an epitope
near the C-terminus of dysferlin, within amino acids 1999–2016,
just before the predicted transmembrane domain (7,8). Adsorption with the immunizing peptide removed all immunoreactivity.
Since the intention was to raise a diagnostically useful antibody
that would enable patients with LGMD2B to be identified, the *To whom correspondence should be addressed. Tel: +44 191 222 5728; Fax: +44 191 222 5227; Email: email@example.com 856 Human Molecular Genetics, 1999, Vol. 8, No. 5 Figure 2. Distribution of dysferlin in different rat tissues. Lane 1, stomach;
lane 2, lung; lane 3, kidney; lane 4, uterus; lane 5, skeletal muscle; lane 6,
heart; lane 7, cerebellum; lane 8, brain stem; lane 9, spinal cord; lane 10, sciatic
nerve; lane 11, liver; lane 12, spleen. The blot was labelled with 1/300
anti-dysferlin antibody, NCL-hamlet. Figure 1. Estimation of molecular mass. Four strips from a blot of normal
control muscle labelled with antibodies to dystrophin (lane 1, band at ∼400
kDa), filamin (lane 2, band at ∼280 kDa), dysferlin (lane 3, band estimated to
be at ∼230 kDa by comparison with the others) and myosin heavy chain (lane
4, band at ∼200 kDa). antibody was given the designation NCL-hamlet (as in ‘2B or not
2B?—that is the question’).
The size of dysferlin is expected to be ∼237 kDa, based on the
predicted amino acid sequence (8), but it is notoriously difficult
to estimate the molecular masses of large proteins on blots.
Molecular mass markers are not manufactured in this size range,
so the migration distance of the immunolabelled dysferlin band
was compared with those for dystrophin (∼400 kDa), filamin
(250–280 kDa) and myosin heavy chain (∼200 kDa). From this,
the size of dysferlin was estimated to be ∼230 kDa, in keeping
with the predicted size (Fig. 1). Bands at 230 kDa were also
observed in skeletal muscle from mouse, rat, rabbit, hamster, pig
and dog, but not chicken (data not shown), suggesting that the
amino acid sequence of this region is conserved between
mammals, but differences exist in birds.
Dysferlin expression in normal tissues
Protein expression in different tissues was examined in rat (Fig.
2). On blots, dysferlin appears to have a ubiquitous distribution,
and bands at ∼230 kDa were observed in all the tissues tested:
skeletal muscle, heart and kidney showed the strongest expression; stomach, lung, uterus, liver and spleen also showed clear
labelling, with nervous tissue (cerebellum, brain stem, spinal cord
and sciatic nerve) showing less. Blood vessels are present in all
the tissues, but vascular smooth muscle represents a minor
component of most tissue samples (as judged previously from the
smooth muscle myosin content; data not shown). It is possible
that the band in the peripheral nerve sample represents a slightly
larger protein. In the parts of rat brain available for examination,
no clear evidence was found of a smaller protein that might Figure 3. Dysferlin expression during development. Western blot of fetal limb
tissues at different stages of development. The times indicated are weeks of fetal
age. The blot was labelled with 1/300 anti-dysferlin antibody, NCL-hamlet. correspond to the reported 4 kb transcript (7,8), but faint bands
were seen at ∼60 kDa in brain stem and spinal cord (Fig. 2, lanes
8 and 9), and at 40 kDa in sciatic nerve (lane 10). Few fresh human
tissues were available for examination: dysferlin was detected on
blots of human heart and term placenta, and labelling of skeletal
muscle sections showed weak labelling of peripheral nerve and
the smooth muscle in blood vessels, but no significant labelling
of fibroblasts in the extracellular matrix, or the lymphocytes and
macrophages in inflammatory infiltrates and foci of degeneration.
Dysferlin expression during fetal development
Dysferlin expression was also examined in samples of human
fetal limb tissue of different ages. Western blot analysis demonstrated a clear 230 kDa band from the earliest time point
examined, at Carnegie stage 15 or 16, embryonic age 5–6 weeks
(Fig. 3). No fetal material was available for sectioning at this time.
Localization of dysferlin in skeletal muscle
The localization of dysferlin in skeletal muscle was first
determined by light microscopy. Like dystrophin (Fig. 4A and C),
labelling for dysferlin was observed at the periphery of the muscle 857
Human Molecular Genetics, 1999, Vol.No.No. 5
Nucleic Acids Research, 1994, Vol. 22, 8, 1
fibre (Fig. 4B). The resolution of light microscopy makes it
impossible to determine whether the labelling was at the plasma
membrane or in the basal lamina. Electron microscopic immunogold cytochemistry revealed labelling sites for dysferlin that were
concentrated at the periphery of the myofibres, many being
closely adjacent to the plasma membrane (Fig. 5). The basal
lamina, external to the plasma membrane, was not labelled. No
nuclear membrane labelling was observed, or labelling of the
endoplasmic or sarcoplasmic reticulum. It is not yet possible to
confirm that the N-terminus and bulk of the molecule lie on the
inside of the plasma membrane, but examination of the available
photographs shows labelling of the NCL-hamlet epitope with
78% of the gold particles on the inside (Fig. 5). 857 Dysferlin expression in patients
Immunocytochemical analysis of dysferlin expression was undertaken in LGMD2B patients with homozygous frameshifting
mutations that would cause premature termination of translation
before the amino acids of the antibody-binding site. Some slight
labelling was seen around some fibres (e.g. Fig. 4D), but a single
fibre and parts of adjacent fibres were more strongly labelled in
one patient (Fig. 4E). This pattern of labelling is strikingly similar
to that seen for dystrophin in patients with Duchenne muscular
dystrophy (DMD) where faint labelling may occur and ‘revertant’
dystrophin-positive fibres are a common feature (12–14). Immunolabelling of serial sections showed that these fibres were not Figure 4. Immunocytochemical labelling of skeletal muscle sections from: (A) a normal control labelled with 1/10 Dy8/6C5 to dystrophin (C-terminus); (B) a normal
section labelled with NCL-hamlet to dysferlin; (C) the Libyan Jew with LGMD2B (homozygous frameshifting mutation), labelled with Dy4/6D3 to dystrophin (rod);
(D) the same patient, labelled with NCL-hamlet to dysferlin; (E) the Palestinian Arab with LGMD2B (homozygous frameshifting mutation) labelled with NCL-hamlet;
(F) the normal control and is labelled with the tissue culture medium containing 20% fetal calf serum that was used to grow the NCL-hamlet hybridoma cells (negative
control). 858 Human Molecular Genetics, 1999, Vol. 8, No. 5 Figure 5. Ultrastructural immunocytochemistry. Dysferlin is located at the
periphery of the myofibre. Gold particles (10 nm; arrowed) are seen close to the
plasma membrane (PM). BL, basal lamina. Magnification, ×75 000. labelled with an antibody to developmental myosin heavy chain,
indicating that they were not in the process of regenerating.
Western blot analysis of the patient biopsies demonstrated very
faint bands at 230 kDa, representing an abundance of <5% of
normal (Fig. 6, lanes 2–4). Patients with non-2p13-linked forms
of muscle disease (e.g. dystrophinopathy, sarcoglycanopathy,
calpainopathy, inflammatory myopathy, metabolic myopathy)
showed dysferlin labelling on sections and blots that was
indistinguishable from normal (e.g. Fig. 6, lane 5). Dysferlin
immunoanalysis was also undertaken in patients with a common
DYSF mutation but different clinical phenotypes (LGMD2B
versus MM) (15).
Immunoreactivity to fragments on blots and sections
In control skeletal muscle (normal or non-2p13-linked patient),
multiple bands of smaller size were observed in addition to the
intensely labelled 230 kDa band (Fig. 6, lanes 1 and 5). These
lower bands were missing in patients with mutations in the DYSF
gene (lanes 3 and 4), indicating that these represent fragments of
dysferlin protein, as opposed to protein products of other genes.
A comparable range of lower molecular mass bands below the
full-size protein band is also seen with antibodies to dystrophin
(e.g. Fig. 6, lane 6). Large muscle proteins take a long time to be
synthesized (16) and to be broken down (17), and it is likely that
these bands represent metabolic fragments of the full-size protein.
In this context, it is interesting that the Dy8/6C5 antibody, to an
epitope at the C-terminus of dystrophin, recognizes a single band
on blots and provides very clean labelling on sections (Fig. 4A).
In contrast, the rod antibody Dy4/6D3, which recognizes multiple
fragments of dystrophin on blots (shown in Fig. 6), demonstrates
a slight cytoplasmic labelling (Fig. 4C), suggesting that the
smaller fragments are not able to locate correctly. Some faint
dysferlin labelling was also observed within the muscle cytoplasm (Fig. 4B), and this may also be attributable to unattached
fragments of the full-size protein.
This report describes the production of a monoclonal antibody to
dysferlin and its use in the examination of protein expression in
normal and pathological situations. Dysferlin is a ubiquitously
expressed 230 kDa molecule that is localized to the periphery of
muscle fibres. It therefore appears that the putative transmem- Figure 6. Western blot of human skeletal muscle biopsies. Lanes 1 and 6,
normal control subjects (amputated leg muscles); lane 2, deltoid sample from
a Palestinian LGMD2B patient linked to chromosome 2p13 (unknown
mutation); lane 3, quadriceps sample from a Palestinian Arab LGMD2B patient
(homozygous frameshifting mutation); lane 4, deltoid sample from a Libyan
Jewish patient with LGMD2B (homozygous frameshifting mutation); lane 5,
quadriceps sample from a patient with genetically confirmed LGMD2A. Lanes
1–5 were labelled with 1/300 anti-dysferlin antibody, NCL-hamlet. Lane 6 was
labelled with 1/50 Dy4/6D3 to an epitope in the rod domain of dystrophin. The
lower panel shows the corresponding myosin heavy chain (MHC) bands from
the Coomassie blue-stained gel, and the outline of these bands can be seen at
200 kDa in lanes 1, 5 and 6. The MHC bands on the post-blotted gel are used
to indicate how much muscle protein, as opposed to fat and fibrous connective
tissue, is loaded in each lane. Densitometric estimates of dysferlin/myosin
abundance were expressed as a percentage of the normal sample in lane 1: lane
2, 1.5%; lane 3, 1.8%; lane 4, 4.7%; lane 5, 100.8%. brane domain identified in the dysferlin sequence (7,8) does exist,
with the plasma membrane being the target in skeletal muscle.
Thus, dysferlin joins the group of sarcolemmal proteins located
at the plasma membrane or within the basal lamina, whose
reduced expression cause inherited degenerative myopathies.
Proteins in this group include dystrophin (18), α- (3), β- (19), γ(2) and δ-sarcoglycan (5), the laminin α2 chain of merosin (20),
the integrin α7 chain (21) and caveolin 3 (22). The function of
some or all of these proteins may be structural, whereby the loss
of protein leads to muscle fibre degeneration. Dystrophin and the
associated proteins (dystroglycans and sarcoglycans) are unaffected in LGMD2B/MM (7), so dysferlin is unlikely to be a close
member of that complex. Given the homology of dysferlin to a
nematode spermatogenesis factor that is required for successful
membrane fusion (7,8), it is also conceivable that the lack of
dysferlin may cause faulty myotube fusion and thereby impair
Dysferlin molecules of ∼230 kDa were expressed in all the
tissues tested, with less apparent variation in abundance (per mg
of sample) than might be expected from the distribution of RNA.
Dysferlin RNA appears to be expressed predominantly in skeletal
muscle, although it is also present in heart and placenta, and
weakly expressed in liver, lung, kidney and pancreas (7,8). There 859
Human Molecular Genetics, 1999, Vol.No.No. 5
Nucleic Acids Research, 1994, Vol. 22, 8, 1
have been no reports of tissues other than skeletal muscle being
affected in either LGMD2B or MM. It should be noted that
β-sarcoglycan also has a widespread tissue distribution although
the clinical symptoms of β-sarcoglycanopathy (or LGMD2E) are
restricted to skeletal muscle (4,23). The distribution (and
functions) of dysferlin in non-muscle tissues will also need
Patients with 2p13-linked MD were differentiated from those
with other conditions on the basis of reduced dysferlin expression
on sections and blots. The generation of a diagnostically useful
antibody is important since the dysferlin gene has >50 exons, and
mutation analysis is extremely laborious. It has been estimated
that LGMD2B may be a very common form of milder limb-girdle
dystrophy (24). The widespread tissue distribution may mean that
it is possible to undertake dysferlin immunoanalysis in a sample
other than a muscle biopsy.
In this preliminary study, we found very low levels of
immunolabelling in sections and blots of muscle from two
patients with homozygous frameshifting mutations in the DYSF
gene. The amount of labelling, >5% of normal, is similar to that
of dystrophin seen in cases of DMD with terminating mutations
(12,13). It seems likely that, by analogy, the reading frame has
been restored by exon skipping (25–29) in a small proportion of
dysferlin transcripts. There are many small exons in the dysferlin
gene, which may predispose this gene to this activity. An
alternative explanation for the low level of immunoreactive
protein detected is the existence of a homologous protein of
identical size and location. However, the observation of occasional more brightly labelled ‘revertant’ fibres argues against this
possibility. The lack of correlation with a marker of regeneration
(developmental myosin heavy chain) indicates that the immunolabelling in these few fibres is unlikely to represent re-expression
of a fetal protein.
Muscle weakness in LGMD2B starts in the proximal muscles
of the pelvic girdle and presents as difficulty with running and
climbing stairs. In contrast, initial muscle weakness in MM
characteristically is restricted to the gastrocnemius muscle, as
indicated by an inability to stand on the toes (30–34). The finding
that both these conditions are caused by mutations in the same
gene is interesting because each type of muscular dystrophy is
defined by the muscles involved and, although the clinical
phenotype of LGMD2B and MM may overlap to varying degrees
(30), the possibility of such different patterns of muscle
involvement is unique among the known dystrophies. For
example, Duchenne and Becker muscular dystrophy have
different clinical profiles yet the actual muscle involvement is
identical in both cases (35). This certainly raises the possibility
that dysferlin may interact with a modifying protein that is the
product of another gene (31). It is also possible that dysferlin
levels vary in different muscle groups, or that it plays a role in
development of the distal and proximal musculature in the fetus.
Dysferlin was clearly detected at Carnegie stage 15 or 16
(embryonic age 5–6 weeks) and is therefore present at a stage of
development when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the
pattern of muscle involvement that develops later, with the onset
of a muscular dystrophy primarily affecting proximal or distal
muscles. 859 MATERIALS AND METHODS
To generate the new antibodies to dysferlin, the 2080 amino acid
sequence (GenBank accession no. AF075575) was assessed with
Omiga v1.1 analytical software from Oxford Molecular, and a
peptide (C-ERPAGQGRDEPNMNPKLE) corresponding to
amino acids 1999–2016 was chosen for synthesis and conjugation
to keyhole limpet haemocyanin. Following immunizations and
test tail bleeds, a CD1 mouse that had responded well was chosen,
given a final boost of intravenous immunogen in saline, and
killed. The harvested splenocytes were fused with X63.Ag8.653
myeloma cells using polyethylene glycol solutions (36). The
resultant hybridoma wells were screened for specific antibody
activity on sections and blots, before being cloned four times at
0.5 cells/well to ensure monoclonality. The antibody was used
undiluted on tissue sections and at 1/300 dilution on blots.
Antibodies to two epitopes on dystrophin were also used:
Dy4/6D3 (rod domain) and Dy8/6C5 (C-terminus) (13). The
antibody to filamin is a commercially available one (Novocastra,
Newcastle upon Tyne, UK), and antibodies to myosin heavy
chain were generated by immunization with myosin purified
from rat muscle.
Muscle samples from five normal control subjects were obtained,
with permission, from amputated leg tissue. Muscle biopsies
from patients with various muscle diseases were taken as part of
the routine diagnostic protocol, and stored in a liquid nitrogen
archive. Samples from >30 patients with non-2p13-linked
diseases were examined. Six patients from five different families
with 2p13-linked LGMD2B/MM were studied. One Palestinian
Arab patient with classical LGMD2B (30) has a homozygous
frameshifting mutation where a 23 bp insertion produced a stop
codon at position 1633 (7). A further Palestinian patient with the
LGMD2B phenotype was from a family linked to 2p13, in whom
the mutation has not yet been identified. A Libyan Jewish patient
from a large kindred with LGMD2B (33) was identified as having
a homozygous single base deletion producing a stop codon at
position 1729 (7). Other patients with defined mutations have
been examined (15).
All the biopsies showed the classical signs of a muscular
dystrophy including an increase in fibre size variation and foci of
necrosis and regeneration. However, features of note included the
presence of large inflammatory infiltrates or other evidence of
inflammatory processes in half the patients.
Immunoelectrophoresis and western blotting
Polyacrylamide gel electrophoresis, western blotting and densitometric analysis were performed as described in detail previously
(37). All tissue samples were weighed frozen and homogenized
in 19 vol electrophoresis treatment buffer (e.g. 20 mg + 380 µl
buffer), giving a loading concentration of ∼200 µg in 30 µl (17). 860 Human Molecular Genetics, 1999, Vol. 8, No. 5 Labelling of sections
Immunocytochemistry at the light level was performed as
described previously (38) with 6 µm unfixed frozen sections,
monoclonal primary antibodies and 1/100 Dako R260 rhodamine-conjugated secondary antibody (Dako, Cambridge, UK)
diluted in phosphate-buffered saline (PBS) containing 3% bovine
serum albumin (BSA) and 0.1 M lysine. The primary antibodies
were used undiluted, or at the dilutions specified in each figure
legend. Immunogold labelling was also carried out as described
previously (39,40). Briefly, the control muscle sample (from a
case with no evidence of any form of muscular dystrophy) was
fixed, cryoprotected and plunge-frozen in liquid nitrogen.
Sections were cut at a thickness of 80 nm at a block temperature
of –90_C and a knife temperature of –110_C using a Reichert
Ultracut E microtome fitted with an FC4D cryomicrotomy
attachment. The primary antibody, NCL-hamlet, was applied
undiluted. The secondary antibody, British BioCell EM.GAM10
goat anti-mouse immunoglobulins, conjugated with 10 nm gold
particles was diluted 1/20 in PBS containing 0.5% BSA. Sections
processed without a primary antibody, or with an antibody to
dystrophin (Dy8/6C5) were used as negative and positive
BL, basal lamina; BSA, bovine serum albumin; DMD, Duchenne
muscular dystrophy; LGMD, limb-girdle muscular dystrophy;
MHC, myosin heavy chain; MM, Miyoshi myopathy; PBS,
phosphate buffered saline; PM, plasma membrane.
Grants for our work have been awarded by the Muscular
Dystrophy Group of Great Britain and Northern Ireland, the
Medical Research Council of Great Britain, the Association
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