 1996 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5, No. 6
801–808
The Emery–Dreifuss muscular dystrophy protein,
emerin, is a nuclear membrane protein
S. Manilal1, Nguyen thi Man1, C. A. Sewry2 and G. E. Morris1,*
1MRIC
Biotechnology Group, N.E. Wales Institute, Deeside, Clwyd CH5 4BR, UK and 2Neuromuscular Unit,
Department of Paediatrics and Neonatal Medicine and Muscle Cell Biology Group, MRC Clinical Sciences
Centre, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, UK
Received February 7, 1996; Revised and Accepted March 21, 1996
A large fragment of emerin cDNA was prepared by PCR
and expressed as a recombinant protein in Escherichia
coli. Using this as immunogen, we prepared a panel of
12 monoclonal antibodies which recognise at least
four different epitopes on emerin in order to ensure
that emerin can be distinguished from non-specific
cross-reacting proteins. All the mAbs recognised a 34
kDa protein in all tissues tested, though minor emerinrelated bands were also detected in some tissues.
Immunofluorescence microscopy showed that emerin
is located at the nuclear rim in all tissues examined. A
muscle biopsy from an Emery–Dreifuss muscular dystrophy (EMDM) patient showed complete absence of
emerin by both Western blotting and immunohistochemistry, suggesting a simple diagnostic antibody
test for EDMD families. Biochemical fractionation of
brain and liver tissues showed that emerin was present
in nuclei purified by centrifugation through 65% sucrose
and was absent from soluble fractions (post-100 000 g).
From these results, together with sequence and structural homologies between emerin, thymopoietins and
the nuclear lamina-associated protein, LAP2, we suggest
that emerin will prove to be one member of a family of
inner nuclear membrane proteins.
in the telomeric region of the Xq28 (2). This region has been
further refined with the aid of physical mapping studies. Of the
eight genes in this region which were highly expressed in brain
and muscle, one in particular, STA, was a strong candidate for
EDMD as mutations in this gene were identified in affected
individuals (3–5).
Although the gene is highly expressed in skeletal muscle and
heart, it has been shown that STA is ubiquitously expressed at high
levels in most tissues. STA encodes a 254 amino acid serine-rich
protein, identified as emerin. The emerin sequence is highly
hydrophilic except for a hydrophobic carboxy-terminal tail. No
homology to other proteins with known functions was apparent,
although two regions of homology with thymopoietins were
observed (3). Thymopoietin (TMPO) was originally isolated as a 5
kDa, 49 amino acid protein from bovine thymus in studies of the
effects of thymic extracts on neuromuscular transmission, though
these effects were later found to be due to a contaminant in the
preparation. Three proteins produced by alternative splicing with
this sequence at their amino-termini were later identified and named
as α (75 kDa), β (51 kDa) and γ (39 kDa) thymopoietins (6), but
there were few clues to their function until the cDNA for a rat
nuclear lamina-associated protein, LAP2, was cloned and found to
be the rat homologue of β-thymopoietin (7,8).
We now describe the PCR cloning and expression of the human
emerin hydrophilic region and its use for the production of
monoclonal antibodies. We show that emerin is a nuclear
membrane protein and suggest that it will prove to be a member
of a family of nuclear lamina-associated proteins (LAPs).
INTRODUCTION
RESULTS
Emery–Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterised by the following features: slowly
progressive skeletal muscle wasting of the shoulder girdle and
distal leg muscles, early contractures of the elbows and Achilles
tendons and cardiomyopathy which often presents as atrial
ventricular block and eventually results in death unless a cardiac
pacemaker is inserted immediately after diagnosis. EDMD is
inherited either as an autosomal dominant form or an X-linked
recessive disorder. The two forms are clinically very similar with
a preponderance of the X-linked form (1).
Linkage analysis of the X-linked disorder has shown this gene
to lie between the loci DXS52/DXS15 and the gene for factor eight
A fragment of STA cDNA encoding the first 188 amino acids of
emerin (out of 254) was amplified from total human skeletal muscle
cDNA and cloned into the expression plasmid, pMW172. The
C-terminal hydrophobic domain of emerin was avoided because it
might have adverse effects on recombinant protein expression (9).
After electroporation into E.coli BL21(DE3), good expression of a
protein of the expected size (21 kDa) was obtained. A panel of 12
mAbs was generated by standard hybridoma methods using partially
purified protein as immunogen.
The mAbs were shown to recognise at least four different
epitopes on emerin as shown in Table 1. Only three mAbs
(MANEM1–3) cross-reacted with rabbit emerin on Western blots
*To whom correspondence should be addressed
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and only one of these (MANEM3) failed to bind when 41 amino
acids were removed from the recombinant emerin by chemical
cleavage at Cys147 using NTCB. This establishes two distinct
epitopes for MANEM1–2 and MANEM3. For seven mAbs
(MANEM4–10) which do not recognise rabbit emerin, Western
blotting of chymotryptic fragments produced two quite distinct
fragmentation patterns (Table 1). This establishes two more
epitopes, making four distinct epitopes in all. We can be confident
that any antigen recognised by mAbs from all four epitope
groups, whether by Western blotting or immunohistochemistry,
will be authentic emerin and not a cross-reacting protein; the latter
is always a possibility when a single antibody or a polyclonal
antiserum is used. None of the 12 mAbs cross-react with mouse
emerin.
Figure 1 shows that the emerin mAbs detect a protein band of
∼34 kDa in normal human muscle. This band is completely absent
from the muscle of an EDMD patient, confirming its identity with
emerin. The muscle membrane dystrophin-associated protein,
43DAG or β-dystroglycan, is used as a control to show that equal
amounts of protein were loaded on the gel [43DAG and
dystrophin itself are unchanged in EDMD (10)]. On SDS–PAGE,
emerin migrates more slowly than expected from its predicted
size (29 kDa). This could be due to post-translational modification, since the emerin sequence has a glycosylation site and
several potential sites for phosphorylation. This might also
explain a faint additional band at ∼36 kDa in Figure 1. The
estimated size for emerin on SDS–PAGE of 34 kDa was
established by careful comparison with several different prestained and unstained marker sets (see Materials and Methods).
Figure 2 shows that emerin is localised almost exclusively at
the periphery of nuclei in frozen sections of human skeletal
muscle. The same result was obtained with all the mAbs, and
double labelling with ethidium bromide and anti-emerin mAbs
(not shown) confirmed the nuclear localisation. The nuclear
membrane staining is seen in normal muscle and in a biopsy from
Figure 1. Western blots of normal muscle and EDMD biopsy muscle extracts.
MANDAG1 mAb to the muscle membrane protein, 43DAG, was used as
control to show that equal amounts of normal and EDMD protein were loaded
onto the gel. A single major band at 34 kDa observed in normal muscle is
completely absent in EDMD muscle following staining with the emerin mAb,
MANEM1.
a patient with an uncharacterised myopathy, but EDMD biopsy
showed no staining with any of the 12 anti-emerin mAbs,
consistent with the Western blot results in Figure 1. EDMD
sections in Figure 2 were also stained with anti-dystrophin mAbs
(confirming that dystrophin is unaffected in EDMD) and
ethidium bromide (to show that nuclei are present in the EDMD
biopsy sections). Careful examination of EDMD sections showed
that all sections tested contained many ethidium bromide-stained
nuclei, but no nuclear staining by anti-emerin mAbs was ever
detected. Staining of major blood vessels in the biopsy sections
(Fig. 3) shows that the presence of emerin in normal nuclei and
its absence from EDMD nuclei is not restricted to myofibre
nuclei. Most of the nuclei are in the smooth muscle layer of the
blood vessels.
Table 1. Characterization of 12 emerin mAbs
mAb
Clone
NTCBa
ChymoTb
Rabbitc
IMFd
Epitope groupe
MANEM1
5D10
1
1
++
++
1
MANEM2
9B2
1
1
++
++
1
MANEM3
6D2
2
2
+
++
2
MANEM4
6C4
1
2
–
++
3
MANEM5
8A1
1
2
–
+
3
MANEM6
8E9
1
2
–
++
3
MANEM7
7A9
1
3
–
++
4
MANEM8
7B9
1
3
–
++
4
MANEM9
9F8
1
3
–
+
4
MANEM10
1H9
1
3
–
+
4
MANEM11
7D9
nd
nd
–
++
nd
MANEM12
5B11
nd
nd
–
weak
nd
aNTCB
(nitrothiocyanobenzoic acid) cleaves the recombinant emerin at Cys-147: group 1 mAbs bind to the large fragment, while the group 2 mAb does not.
digests of the recombinant emerin give three different patterns when different mAbs are used on Western blots.
cOnly three mAbs cross-react with emerin in rabbit muscle extracts.
dmAbs were tested on frozen sections of human muscle by immunofluorescence microscopy.
emAbs were placed in four epitope groups on the basis of a, b and c. All mAbs belong to the IgG1 subclass.
nd, not determined.
bChymotrypsin
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Figure 4a shows that emerin levels are similar in all foetal and
adult human tissues tested. This is consistent with mRNA data (3)
and with a nuclear location. The slightly higher Mr band seen in
Figure 1 is also present in all the human tissues (with a third band
visible in foetal liver). Figure 4b shows that emerin is present at
similar concentrations in all rabbit tissues tested. The main ‘34
kDa’ emerin band migrates slightly faster in the heart, kidney and
skeletal muscle extracts. This is quite reproducible, but its
significance remains to be determined. It is important to examine
carefully any stained protein bands other than the 34 kDa emerin
because of the possible existence of isoforms or modified forms.
The lower Mr bands could be degradation products, but they are
noticeably absent from skeletal muscle and sciatic nerve (see also
the human skeletal muscle extract in Fig. 1) and all the extracts
were freshly prepared. These bands are also present in foetal and
adult human tissues, except that they are much less prominent in
adult skeletal muscle (Fig. 4a).
The nuclear rim localisation was also observed in tissues other
than skeletal muscle, including kidney, heart and spleen (results
not shown). In rabbit sciatic nerve, as in skeletal muscle, emerin
was also found almost exclusively at the nuclear periphery
(Fig. 5). Most of the nuclei are found in the perineurium,
connective tissue which surrounds the fibre, at the bottom of the
figure and appear elongated in this section. The few nuclei visible
in circular section among the axons are likely to be from Schwann
cells or blood vessels. The control section without primary
antibody shows that the faint non-nuclear staining is non-specific.
To confirm the immunohistochemical results, purified nuclei
were prepared from rabbit brain by centrifugation through 65%
sucrose and shown to contain emerin by Western blotting (Fig. 6).
A simple subcellular fractionation by differential centrifugation
was performed as outlined in Figure 7 and the fractions were
tested for emerin by Western blotting (Fig. 7). About half the
emerin was found in the 1000 g pellet containing nuclei and
unbroken cells (lane 1), but none of this was extracted by Triton
X-100 (cf. lanes 2 and 3). This implies that emerin is retained in
nuclei not only via its transmembrane domain but also by
non-membrane-dependent interactions. The nuclear membrane
protein LAP2 is also Triton-insoluble because of its interaction
with nuclear lamins (7). Most of the remaining emerin was found
in the ‘mitochondrial’ pellet (cf. lanes 4 and 5), though this may
also contain some nuclei or nuclear fragments. None of the
emerin remained in the supernatant after high-speed centrifugation (lane 7). Similar results were obtained with rabbit liver
(results not shown). The results cannot be used as evidence for
non-nuclear emerin because we cannot rule out the possibility that
nuclear membrane fragments are responsible for the emerin
content of the ‘mitochondrial’ and ‘microsomal’ pellets obtained
by this very simple fractionation procedure.
DISCUSSION
We have shown that emerin is a nuclear membrane protein. Several
striking sequence and structural homologies with LAP2 (β-thymopoietin) suggest, as a working hypothesis at least, that emerin
belongs to a family of type II integral membrane proteins which are
anchored to the inner nuclear membrane near their carboxy-termini,
with the remainder of the molecule projecting into the nucleoplasm,
as illustrated in Figure 8. A sequence of 39 amino acids at the human
emerin amino-terminus (Asp6–Gln44) shows 41% identity (70%
conserved residues) with a sequence in all three human thymopoie-
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Figure 2. Immunohistochemical localisation of emerin at the nuclear membrane in normal muscle and its absence in EDMD muscle. Frozen sections of
normal muscle, muscle from a patient with an undiagnosed myopathy (control)
and muscle from an EDMD patient were stained with emerin mAb, MANEM1.
EDMD sections from the same biopsy were also stained with anti-dystrophin
mAb, MANDYS1, to show the sarcolemma and with ethidium bromide to show
that nuclei are present in the sections. The different nuclear shapes in different
sections are due to differences in orientation (see Figure 3, where various
intermediates between transverse and longitudinal nuclei are visible in the same
blood vessel).
tins (Glu114–Gln152). The last 35 amino acids of emerin show the
same degree of identity with a sequence very close to the
carboxy-terminus of β- and γ-thymopoietins, including a hydrophobic transmembrane domain of ∼20 amino acids (Fig. 8).
α-Thymopoietin lacks this transmembrane domain and localises to
the nucleoplasm rather than the nuclear membrane (8). In between
the two regions of homology at each end of the molecule, emerin is
about the same length as γ-thymopoietin, but there is little obvious
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Figure 4. Western blots of emerin in adult and foetal human tissues and in rabbit
tissues. (a) Adult and foetal muscle, brain and lung extracts from post-mortem
tissues were screened with the emerin mAb, MANEM1, after SDS–PAGE on
12% polyacrylamide gels. The emerin band is present in all the extracts,
together with a fainter second band at slightly higher Mr of 36 kDa (foetal lung
has a third band). Lower Mr bands may be degradation products, though the
most prominent of them corresponds in size (17 kDa) to the labelled rabbit band
in (b). (b) Rabbit tissue samples were resolved by SDS–PAGE on a 3–12.5%
gradient polyacrylamide gel. The 34 kDa emerin band is present in all tissue
extracts examined, but the lower Mr band indicated is absent from muscle (MU)
and sciatic nerve (SN). Other lanes are Mr: prestained molecular weight
markers, LI: liver, HT: heart, KI: kidney, BR: brain and SP: spleen.
Figure 3. Immunofluorescence of blood vessels in frozen muscle sections stained
with emerin mAb, MANEM1. Staining of nuclear membranes is observed in
normal blood vessel sections but was absent in EDMD biopsy sections. Most of
the nuclei are in the smooth muscle layer of the vessel and can be seen both in
circular section (transverse; right) and in elongated form (top left). The
non-nuclear, fibrous staining in both normal and EDMD sections is due mainly to
autofluorescence from elastic fibres on each side of the smooth muscle layer. This
autofluorescence is red and easily distinguished from the apple-green anti-emerin
staining. In the upper panel, the empty lumen of the blood vessel is clearly visible
in the lower right hand quadrant; in the lower panel, the blood vessel has broken
open in the upper left hand quadrant during sectioning.
sequence homology, apart from the presence of numerous potential
phosphorylation sites.
We have not yet confirmed the orientation of emerin in the
nuclear membrane by electron microscopy, partly because the
mAb epitopes are destroyed by most common fixation procedures, making EM technically difficult. The arrangement of
positively and negatively charged amino acids on each side of the
transmembrane sequence argues strongly against the aminoterminal region being in the lumen between inner and outer
membranes (11). A cytoplasmic orientation in the outer nuclear
membrane is unlikely because it is not clear what could target
emerin to nuclei rather than to the rest of endoplasmic reticulum
(ER), of which the outer nuclear membrane forms a part. In
contrast, LAP2 and other inner nuclear membrane proteins
appear to be inserted first into ER membranes where they diffuse
laterally throughout the ER system until they are fixed in the inner
nuclear membrane only by specific interactions with the nuclear
lamina (12).
LAP2 is known to interact with lamin B polymers below the
inner nuclear membrane, a property shared with another type II
integral membrane protein, LBR (lamin B receptor or p58). The
interaction between LAP2 and lamin B is regulated during
mitosis by phosphorylation of LAP2 which prevents lamin B
binding in vitro (12). This involves cdc2 kinase and emerin does
not appear to have any potential cdc2 kinase sites, suggesting that
emerin does not interact with lamin B in the same way as LAP2,
if at all. The amino-terminal sequence of emerin which is shared
with LAP2 does have three predicted phosphorylation sites which
are also conserved in thymopoietins, and it most likely has a
common function in all four proteins. Neither this sequence nor
the transmembrane sequence appear to be essential for nuclear
membrane localisation, however, since deletion mutants lacking
either or both will still target to the nuclear rim in a Triton-stable
manner in transfected HeLa cells (7). A 73 amino acid sequence
in the middle of LAP2 was identified as particularly important for
targetting to the nuclear rim and the nucleoplasmic α-thymopoietin lacks this, as well as the transmembrane domain (8). Emerin
also lacks this sequence, so proteins other than lamin B could be
responsible for Triton-stable targetting of emerin to the nuclear
rim. Indeed, it is not yet known whether interaction with lamin B
is solely responsible for LAP2 targetting, since the deletion
mutant results suggested that multiple interactions are involved.
In a somewhat similar way, both the amino-terminal domain (13)
and the carboxy-terminal transmembrane domain (14) of the
lamin B receptor can independently target proteins to the nuclear
envelope. We are currently investigating possible interactions
between our recombinant emerin and other proteins.
Since EDMD is a variable, but generally rather mild, genetic
disease, the complete absence of emerin in all the nuclei of at least
some patients suggests that emerin function must be redundant in
most situations. This is consistent with emerin being a member of
a family of related proteins, with other members capable of
performing some of its functions. The tissue-specific effects of
EDMD could therefore reflect the tissue distribution of emerin-
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Figure 5. Localisation of emerin in a transverse section of rabbit sciatic nerve.
Nuclear membranes stained with emerin mAb, MANEM1, are visible in
circular section, sparsely distributed amongst the packed myelinated and
unmyelinated axons (these few nuclei are probably derived from Schwann cells
or blood vessels) and, more abundantly, in elongated form in the connective
tissue which surrounds the nerve fibre (perineurium). Faint staining of the axons
is non-specific, since it is also seen in the control section without primary Ab.
related proteins rather than emerin itself. Alternatively, tissuespecific alternative splicing or modification might generate
isoforms of emerin with different functions or localisations [cf.
LAP2 phosphorylation and deletion mutants (7,12)]. Bands other
than 34 kDa emerin are detected by anti-emerin mAbs on Western
blots in many tissues, but they are weak or absent in adult skeletal
muscle and sciatic nerve (Fig. 4). The possibility cannot yet be
excluded that some of these protein bands are different forms of
emerin, generated by post-translational modification, alternative
splicing or proteolysis in vivo, although degradation during
experimental manipulation has not been ruled out as an explanation for the lower Mr bands. The gene for the autosomal form of
EDMD has not yet been identified, but it may well shed further
light on emerin function. Although the severity of EDMD can
vary, there is concordance of severity in affected members within
a family. The clinical variability between families might therefore
relate to the effect of different gene mutations on protein function,
since it is not yet known whether emerin is completely absent in
all EDMD cases.
The discovery that emerin is primarily a nuclear protein was
unexpected. Many other genetic muscle-wasting diseases are
caused by defects in genes encoding sarcolemmal proteins:
Duchenne and Becker MDs by defects in the dystrophin gene (15)
and three of the five limb-girdle MDs by defects in genes for each
of the three sarcoglycan components of the transmembrane
complex which attaches dystrophin to the muscle membrane
(16–18). A calcium-activated protease, calpain, is defective in a
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Figure 6. Isolation of rabbit brain nuclei and emerin analysis by Western blot.
Rabbit brain (0.5 g) was homogenised in 10 ml of RSB buffer (10 mM NaCl; 1.5
mM MgCl2; 10 mM Tris–HCl, pH 7.5) using a Dounce hand homogeniser. The
homogenate was centrifuged at 1000 g for 10 min at 4C. The pellet was
resuspended in 10 ml of RSB solution, homogenised and sedimented as described
above. The crude nuclear pellet was resuspended in 65% sucrose/5 mM MgCl2
and centrifuged at 100 000 g for 80 min in a 3×5 ml swing-out rotor. The nuclear
pellet was washed with RSB to remove residual sucrose and resuspended in RSB.
Phase-contrast microscopy (upper panel) shows that the nuclear preparation is
reasonably pure, though some have ‘cytoplasmic tags’ (cytoskeletal material)
attached. The Western blot (12% polyacrylamide gel) with MANEM1 mAb (lower
panel) shows that emerin is present in the purified nuclei (Mr: prestained markers,
lane 1: rabbit muscle extract, lane 2: purified nuclei).
fourth form of limb-girdle MD (19). Myotonic dystrophy is
associated with changes in a protein kinase gene (20,21), which
may also show some homology with thymopoietins (22). The
homology is at the amino-terminus of both proteins and is
different from the homology shown by emerin towards these
proteins. It does not target the kinase to the nucleus, since the
expressed protein is cytoplasmic in transfected COS-1 cells (22).
The diseases themselves have in common progressive wasting
and/or weakness of skeletal muscles, but also have important
distinguishing features. In EDMD, for example, a striking feature
is a failure of impulse conduction in the heart, which can be
corrected with a cardiac pacemaker. There is little that is common
to all the gene products, other than a possible association with cell
signalling and signal transduction. Thus, although it is appealing
to look for links between the functions of the various gene
products, such links may prove to be obscure and rather remote.
While this paper was under revision, a paper by Nagano et al.
(27) appeared in which a nuclear membrane localisation for
emerin was demonstrated using two different polyclonal antisera
raised against synthetic peptides derived from the C-terminal half
of the emerin sequence. We concur with this conclusion and have
taken it further by drawing attention to the important relationship
between emerin and LAP2. Most of the specificity problems
encountered by Nagano et al. [non-specific staining of the plasma
membrane and non-specific protein bands on Western blots (27)]
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Figure 8. Schematic representation of predicted organisation of emerin and
thymopoietin in the nuclear membrane. Based on the model for LAP2 (7).
Figure 7. Flow diagram depicting the subcellular fractionation of rabbit brain
by differential centrifugation and Western blot analysis of the subcellular
fractions. The crude nuclear pellet (1) will also contain unbroken cells. The
6600 g pellet (4) will contain mainly mitochondria, ‘heavy’ membranes and
some nuclei. The 100 000 g pellet (6) is the microsomal fraction containing
mainly ER and ‘light’ membranes. Aliquots of each fraction were removed and
analysed on 12% SDS–polyacrylamide gels for Western blotting with
MANEM1 mAb. Lanes 1–7 correspond to the fractions in the flow diagram
above. **Non-specific staining by second antibodies.
are avoided using our panel of 12 mAbs which recognise the
N-terminal region of the emerin molecule. We did not observe in
rabbit tissues the tissue-specific differences in human emerin
distribution reported by Nagano et al. We found emerin in nuclear
membranes in all tissues studied, although this does not rule out
the possibility that the proportion of emerin-positive nuclei, or the
intensity of nuclear rim staining, varies between tissues. More
detailed studies of emerin distribution in both rabbit and human
tissues and with as wide a range of anti-emerin antibodies as
possible are clearly required.
MATERIALS AND METHODS
PCR amplification of STA cDNA
Human skeletal muscle cDNA (10 ng; Clontech Laboratories) was
subjected to PCR amplification using forward (5′-gcggatccgacaactacgcagatctttcg) and reverse (5′-gcaggcctaagaggtggaggaggaagtagg)
primers designed to the cDNA of the STA gene. Restriction sites
were engineered into the 5′ ends of these primers to facilitate cloning
of the amplified products into BamHI and StuI sites of the pMW172
expression vector. PCR was carried out in sterile microfuge tubes in
a final reaction volume of 50 µl consisting of 1.5 mM MgCl2,
50 mM KCl, 10 mM Tris–HCl (pH 8.4), 200 µM dNTP (Pharmacia)
and 1 µM forward and reverse primers. Following a 5 min
denaturation step at 94C, 0.5 U of Taq polymerase was added to
the reaction tube. PCR was performed for 25 cycles (56C,
30 s/72C, 30 s/95C, 30 s) followed by a final 5 min extension step
at 72C. The PCR product was purified from a 1% agarose gel using
Qiaex resin (Diagen, Dusseldorf, Germany).
Cloning of cDNA into pMW172 and expression of
fusion protein
The PCR product was subcloned into the Novagen pT7Blue
vector (50 ng; Novagen) containing compatible single T-nucleotide overhangs. Ligation was carried out in a final reaction
volume of 10 µl in the presence of 100 mM dithiothreitol (DTT),
10 mM ATP and 2 U of T4 DNA ligase (Boehringer Mannheim).
Following incubation at 16C for 5 h, 1 µl of the ligation mixture
was directly transformed into chemically competent pT7 NovaBlue cells (20 µl; available with the kit). Colony PCR was
performed on recombinant clones to confirm the presence of the
cloned PCR product using pT7 forward (5′-taatacgactcactataggg)
and reverse primers (5′-ggttttcccagtcacgacgt). The pT7 construct
(100 ng) was digested with 1 U of BamHI and StuI restriction
enzymes (Boehringer Mannheim) for 1 h at 37C in a final
reaction volume of 20 µl. The digested fragment was purified
using the Qiaex DNA purification kit, cloned into BamHI–StuIdigested, alkaline phosphatase-treated, pMW172 vector and
electrotransformed into 20 µl of electrocompetent BL21(DE3)
cells in ice-cold electroporation cuvettes (0.2 cm). Recombinant
clones were subjected to colony PCR to confirm the presence of
the insert using pMW172 forward (5′-gactcactatagggagaccacaac)
and reverse (5′-gttattgctcagcggtggcagcag) primers.
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Luria Bertani media (100 ml) was inoculated with 1 ml of
overnight culture of pMW172 recombinant clone. The media was
cultured by shaking at 37C until the O.D.600nm was 0.8.
Following induction (0.8 mM IPTG) at 37C for 3 h, the culture
was centrifuged at 6600 g for 10 min at 4C. The pellet was
resuspended in 30 ml of ice-cold TNE buffer, pH 8.0 (50 mM
NaCl, 1.27 mM EDTA, 50 mM Tris–HCl) and sedimented as
described above. This procedure was repeated twice. The
resulting pellet was extracted twice by sonication in 2 M urea.
Sequencing of cloned inserts
DNA sequencing of the pT7 and pMW172 constructs containing
the EDMD cDNA was performed by the dideoxy chain
termination protocol (23). DNA was sequenced in both orientations using vector-specific oligonucleotides and the Sequenase kit
version 2.0 (Amersham Life Science).
Production of monoclonal antibodies
mAbs were produced by immunisation of BALB/c mice and
fusion of spleen cells with Sp2/0 myeloma cells as described
elsewhere (24). Hybridoma culture supernatants were tested by
ELISA (24), and 12 cell lines were successfully established after
two rounds of cloning by limiting dilution.
Clinical details
A needle biopsy from the quadriceps was taken from a boy aged
16 years with features of EDMD. He had contractures of the
elbows and Achilles tendon, weakness of the tibialis anterior, a
stiff spine, progressive muscle weakness and junctional bradiacardia. There was a strong suggestion of X-linked inheritance
with an affected maternal cousin with progressive muscle
weakness, contractures and an arrhythmogenic cardiomyopathy
requiring a pacemaker. This sample was compared with four
controls with no evidence of X-linked inheritance.
Immunofluorescence microscopy
Unfixed, frozen sections of human and rabbit tissues (6 mm) were
mounted on glass slides and stored at –70C. FITC-labelled
anti-mouse IgG (DAKO) was used to detect bound antibody as
described previously (25).
SDS–PAGE and Western blotting
SDS–PAGE and Western blotting were carried out essentially as
described elsewhere (26). Antibody reacting bands were visualised following development with a biotin–avidin detection
system for mouse immunoglobulin (Vectastain kit; Vector
Laboratories) and diaminobenzidine (Sigma, Dorset, UK).
In order to get an accurate estimate of molecular weight for the
emerin protein, a number of different sets of prestained markers
(Novex, CA) were checked and re-calibrated against unstained
Sigma markers (Sigma, SDS-7).
Subcellular fractionation
Rabbit liver and brain tissues (0.5 g) were homogenised in
ice-cold RSB buffer (10 mM NaCl/1.5 mM MgCl2/10 mM
Tris–HCl, pH 7.5) using a Dounce hand homogeniser. All steps
were carried at 4C. The homogenate was centrifuged at 1000 g
807
for 10 min. The pellet was extracted with 1% Triton
X-100/10 mM Tris–HCl, pH 7.5 and centrifuged at 100 000 g for
30 min whilst the supernatant fraction was centrifuged at 6600 g
for 10 min. The supernatant resulting from this fraction was
further subjected to a high-speed spin (100 000 g) for 30 min.
ACKNOWLEDGEMENTS
We thank Francesco Muntoni (RPMS) for valuable comments on
the manuscript and Fiona Wilkinson and Abosede Salako
(NEWI) for technical assistance. This work was supported by a
research development award (DevR) from the Higher Education
Funding Council (Wales).
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