Human Molecular Genetics, 2000, Vol. 9, No. 2 217-226
© 2000 Oxford University Press
Myoferlin, a candidate gene and potential modifier of muscular dystrophy
Departments of 1Pathology, 2Medicine and 3Human Genetics, University of Chicago, Chicago, IL 60637, USA
Received 30 August 1999; Revised and Accepted 18 November 1999.
DDBJ/EMBL/GenBank accession nos AF182316 and AF182317.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Dysferlin, the gene product of the limb girdle muscular dystrophy (LGMD) 2B locus, encodes a membrane-associated protein with homology to Caenorhabditis elegans fer-1. Humans with mutations in dysferlin (DYSF) develop muscle weakness that affects both proximal and distal muscles. Strikingly, the phenotype in LGMD 2B patients is highly variable, but the type of mutation in DYSF cannot explain this phenotypic variability. Through electronic database searching, we identified a protein highly homologous to dysferlin that we have named myoferlin. Myoferlin mRNA was highly expressed in cardiac muscle and to a lesser degree in skeletal muscle. However, antibodies raised to myoferlin showed abundant expression of myoferlin in both cardiac and skeletal muscle. Within the cell, myoferlin was associated with the plasma membrane but, unlike dysferlin, myoferlin was also associated with the nuclear membrane. Ferlin family members contain C2 domains, and these domains play a role in calcium-mediated membrane fusion events. To investigate this, we studied the expression of myoferlin in the mdx mouse, which lacks dystrophin and whose muscles undergo repeated rounds of degeneration and regeneration. We found upregulation of myoferlin at the membrane in mdx skeletal muscle. Thus, myoferlin (MYOF) is a candidate gene for muscular dystrophy and cardiomyopathy, or possibly a modifier of the muscular dystrophy phenotype.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Mutations in dysferlin, a novel protein of unknown function, were recently identified in patients with limb girdle muscular dystrophy (LGMD) type 2B and in patients with Miyoshi myopathy (MM) (1,2). In LGMD 2B, affected individuals develop muscle weakness in the late second decade and often have normal function during childhood. LGMD 2B patients characteristically have progressive, proximal muscle weakness (3). In contrast, individuals with MM have a much milder course with preferential involvement of the distal musculature (4). Both LGMD 2B and MM patients have markedly elevated serum creatine kinase levels early in life, despite normal muscle function (5,6). Genetic linkage analysis suggested that LGMD 2B and MM were allelic disorders that both mapped to the same small interval at chromosome 2p13 (7,8). The identification of the dysferlin (DYSF) gene confirmed that LGMD 2B and MM arise from mutations in the same gene. Mutation analysis of DYSF revealed nonsense, missense and frameshifting mutations associated with LGMD 2B and MM. Importantly, the type of mutation does not correlate with the phenotypic severity, and the same mutation has been found in patients with both MM and LGMD 2B (9). Therefore, it has been suggested that genetic modifiers may play a role in mediating phenotypic severity.
Dysferlin represents a new class of protein involved in muscular dystrophy. The function of dysferlin is unknown, but dysferlin is highly homologous to fer-1, a Caenorhabditis elegans protein necessary for fusion of the membranous organelle to the sperm plasma membrane (10–12). The primary sequence of dysferlin predicts a protein of 230 kDa with a single transmembrane domain. Dysferlin is thought to be a type II transmembrane protein with a predicted topology that places most of the protein within the cytoplasm anchored by its C-terminal transmembrane domain. Dysferlin contains six C2 domains, independently folding domains found in a number of proteins involved in signal transduction and membrane trafficking (13), including protein kinase C (14,15) and the synaptotagmins (16). C2 domains are known to bind Ca2+, phospholipids and lipid bilayers (17), as well as other proteins in Ca2+-dependent and -independent interactions (18). An antibody to dysferlin was reported recently and showed that dysferlin normally is found at the plasma membrane of skeletal muscle. Patients with dysferlin mutations showed a great reduction of dysferlin on immunoblotting, but did not show a complete absence of full-length dysferlin despite frame- shifting, truncating mutations (9,19).
Most recently, another protein with homology to dysferlin and fer-1 was implicated in autosomal recessive, non-syndromic prelingual deafness, DFNB9 (20). This protein, named otoferlin, is smaller than dysferlin, but over its length it has 64% similarity to dysferlin on the amino acid level. Otoferlin also contains three C2 domains and a C-terminal transmembrane domain and is widely expressed by RT–PCR.
Here we describe the identification and characterization of a new member of the ferlin family, which we have called myoferlin, due to its high homology to dysferlin and its expression in cardiac and skeletal muscle. Myoferlin is nearly identical in size to dysferlin. Like dysferlin, myoferlin is predicted to be a type II transmembrane protein with a large cytoplasmic domain containing six C2 domains and a C-terminal membrane-spanning domain. An antibody specific to myoferlin demonstrated that myoferlin was expressed highly in both cardiac and skeletal muscle. Like dysferlin, myoferlin was found at the plasma membrane in muscle, but unlike dysferlin, myoferlin was also found in the nucleus. We also found that myoferlin is upregulated at the plasma membrane in the dystrophin-deficient mdx mouse, suggesting a role in membrane regeneration and, potentially, repair. The gene for myoferlin mapped to chromosome 10q24. Thus, myoferlin (MYOF) is a novel candidate gene for muscular dystrophy or cardiomyopathy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Identification and expression of myoferlin
A translation BLAST search of the expressed sequence tag (EST) database with the dysferlin sequence (GenBank accession no. AF075775) revealed several human ESTs with homology to dysferlin. A number of clones that derived from the same sequence revealed high homology to dysferlin at the amino acid level and lower homology at the nucleotide level. A probe that contained no significant homology to the dysferlin nucleotide sequence was used to detect mRNA expression in multiple human tissues (Fig. 1A). A prominent mRNA, myoferlin, was detected at 7.5 kb, and this mRNA was highly expressed in heart, placenta and lung. On longer exposure, a small amount of myoferlin mRNA was seen in other tissues, including skeletal muscle (Fig. 1A). Actin was used as a loading control and showed that myoferlin mRNA was expressed at levels seven times higher in cardiac versus skeletal muscle. To study further the expression of myoferlin mRNA, in situ hybridization of embryonic day 15 mouse embryos was performed using a probe corresponding to the 3'-untranslated region (3'-UTR) of murine myoferlin (bp 6285–6805). The antisense probe (Fig. 1B) showed abundant expression in skeletal muscle. This was most easily seen in the muscles of the neck (trapezius) and spine (intercostals). Expression was also detected in the heart (Fig. 1D). High level expression of myoferlin in the diaphragm was noted (Fig. 1D, arrow). The expression of myoferlin mRNA was also seen in the gut (Fig. 1F), reflecting its likely expression in smooth muscle.
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The complete myoferlin sequence (GenBank accession no. AF182316) was obtained from a combination of IMAGE clones, cDNA clones isolated from heart and lung cDNA libraries and from RT–PCR (Fig. 2A). The open reading frame of myoferlin was 6183 bp, with 97 bp 5'-UTR and 555 bp 3'-UTR. The open reading frame of myoferlin predicted a protein of 2061 amino acids with an estimated molecular weight of 234 kDa (Fig. 2B). There was a single transmembrane domain (amino acids 2027–2042), and it was preceded by the positively charged residues characteristic of type II transmembrane proteins. Overall, myoferlin was 68% similar to dysferlin at the amino acid level, and this homology was found throughout the myoferlin sequence. Myoferlin was also similar to fer-1 (40% similarity), the C.elegans protein implicated in spermatic membrane fusion, and otoferlin (54% similarity), a protein implicated in non-syndromic deafness.
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All of the known ferlin family members shared several conserved features as described below and summarized in Figure 2C. Myoferlin has six C2 domains: C2A at amino acids 1–85, C2B at 200–281, C2C at 359–458, C2D at 1126–1231, C2E at 1538–1638 and C2F at 1790–1903. C2 domains A, C, D and E are directly homologous to those previously identified in dysferlin (2). This search algorithm also identified two additional C2 domains in dysferlin, not previously identified, at amino acids 221–302 (C2B) and 1813–1926 (C2F). Myoferlin also has an SH3 domain at amino acids 676–689 in a region with little or no homology to dysferlin. There were five potential tyrosine phosphorylation sites in myoferlin at amino acids 629, 953, 1593, 1611 and 1835. Three of these tyrosine phosphorylation sites (amino acids 629, 1593 and 1835) were conserved between myoferlin and dysferlin, and one (amino acid 1835) was conserved in otoferlin. There were also three potential cAMP- and cGMP-dependent protein kinase phosphorylation sites at amino acids 35, 183 and 575. The two sites at amino acids 35 and 575 were conserved in dysferlin. Analyses of motifs in ferlin family proteins also identified a saposin type B domain in fer-1 that was not conserved in the other ferlin family members. Saposin-B domains are found in proteins that function in membrane lysis, and may also bind lipid substrates (21,22).
Over 50 ESTs representing myoferlin sequences were found in the database. One EST, N42174, isolated from a normalized melanocyte library, represented a putative splice form of the cDNA. This splice form would generate a protein containing the first three C2 domains in the absence of a transmembrane domain. By northern blotting, the splice form mRNA was not expressed at detectable levels in the representative tissues (data not shown). An alternative 5' end was also identified in several non-identical cDNA clones isolated from a lung cDNA library. In these clones, there was an alternative amino acid sequence that did not contain the N-terminal 88 amino acids and instead contained an alternate 47 amino acids after the proposed alternate start site. This alternate 5' end (GenBank accession no. AF182317) was confirmed by RT–PCR (data not shown). A recent GenBank entry (accession no. AL096713) contained 4845 bp of myoferlin sequence from the 3' end (23). However, this entry was missing a sequence that likely corresponds to a single exon from myoferlin (bp 5360–5450), and contained additional sequences inserted at nucleotide 4415 of myoferlin that were not seen in any other ESTs or RT–PCR sequences in that region.
Fifty-six myoferlin ESTs have been mapped by radiation hybrid mapping and were available in the Unigene database. These ESTs were found between the markers D10S5654 and D10S603, indicating that MYOF mapped to chromosome 10q24. Fifty-two of these ESTs were grouped into a sequence-tagged site (STS) Z40200. Four other ESTs were grouped into a separate site, STS N-22119. These two sites were mapped near each other (physical positions 436.36 and 438.18, respectively). Both were between nearest markers D10S583 and D10S571 according to current marker order. No known muscular dystrophy or cardiomyopathy phenotypes have been associated with these loci.
Myoferlin is highly expressed in skeletal and cardiac muscle
A peptide was synthesized corresponding to myoferlin amino acid residues 1692–1708. These residues showed no significant homology to dysferlin, and the alignment of these regions is shown in Figure 3A. Figure 3B shows that the myoferlin antibody (Myof1) did not cross-react to a glutathione S-transferase (GST) fusion protein of the homologous region of dysferlin. The peptide used for generating Myof1 also showed no significant homology to otoferlin, nor to any other potential protein in the electronic database. We predicted that this peptide would cross react to mouse myoferlin since it was conserved (16 of 17 amino acid residues) in the murine myoferlin sequence (data not shown). The myoferlin peptide was injected into rabbits and polyclonal antiserum (Myof1) was characterized. SDS–PAGE of homogenates prepared from various mouse tissues was blotted, and the membrane was probed with Myof1. A band of 230 kDa, the predicted size of myoferlin, was abundant in skeletal muscle and heart (Fig. 4). The level of protein expression in skeletal muscle seemed equal to or slightly higher than that in heart whereas the expression of myoferlin mRNA on northern blot was higher in heart than in skeletal muscle (Fig. 1A). This may reflect differences between human and murine tissues or may indicate that mRNA expression does not correlate with protein expression, and that post-translational regulation of myoferlin protein may be occurring. Myoferlin was also present in lung, and at very low levels in kidney, placenta and brain.
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Myoferlin is found at nuclear and plasma membranes
Myof1 was used on immunocytochemical studies of normal mouse skeletal muscle membrane. Although myoferlin was found at the plasma membrane, it was also noted to be associated abundantly with nuclei (Fig. 5A and B). A second myoferlin antibody (Myof2) was generated to a different, unique peptide of myoferlin that contains no significant homology to dysferlin or otoferlin. Like Myof1, Myof2 shows staining at the plasma membrane and the nucleus. An antibody was also generated to dysferlin using a peptide unique to dysferlin, and this is shown in Figure 5D. To further clarify the intracellular localization of myoferlin, normal skeletal muscle was fractionated into nuclei, light microsomes and heavy microsomes (Fig. 5E). Immunoblotting of these fractions shows that myoferlin was found preferentially in the nuclear fraction (Fig. 5E, lane 3). A protein known to be present at the plasma membrane,
-sarcoglycan, was used to assess the degree of plasma membrane contamination in the nuclear fractions. Although some plasma membrane was present in the nuclear fraction, myoferlin was enriched in the nuclear fraction. A second fractionation protocol was performed to determine whether myoferlin was associated with the nuclear membrane fraction. Figure 5F, lane 3, shows that myoferlin was enriched in the nuclear membranes. Membrane fractions of normal cardiac muscle also demonstrated the presence of myoferlin in the plasma membrane fraction (data not shown).
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Myoferlin expression in muscular dystrophy
We next studied the expression of myoferlin in animal models of muscular dystrophy. Although it was shown previously that components of the dystrophin–glycoprotein complex (DGC) were not altered in chromosome 2p13-linked (DYSF) muscular dystrophy (24), it has not been addressed whether ferlin family members are altered in dystrophin-related muscular dystrophy. To study the potential involvement of myoferlin in muscular dystrophy, we assessed myoferlin expression in two mouse models of muscular dystrophy. The first, the mdx mouse, has a mutation in exon 23 of dystrophin and is a model for Duchenne muscular dystrophy (25). We also studied mice lacking
-sarcoglycan, a dystrophin-associated protein and the gene mutated in LGMD 2C. These mice develop muscular dystrophy and cardiomyopathy (26). We found that myoferlin was upregulated in membrane fractions from mdx muscle (Figure 6). The dihydropyridine receptor (DHPR) was used as a loading control. Normalization to DHPR revealed a 274% increase in myoferlin expression in mdx. This suggests that ferlin family members may play a role in dystrophin-deficient muscular dystrophy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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We report here the isolation and characterization of a novel protein, myoferlin. Myoferlin is a protein of ~230 kDa and is highly homologous to the recently described dysferlin, the protein product of the LGMD 2B gene, over nearly its entire length. Both proteins contain a single transmembrane domain very near to the C-terminus of the protein and appear to be type II transmembrane proteins with the predicted topology of both indicating that the majority of the protein is cytoplasmic. We showed that myoferlin was associated with the plasma membrane fraction of skeletal muscle. Interestingly, myoferlin was also found in nuclei, and enriched in the nuclear membrane fraction. Myoferlin has six putative C2 domains. C2 domains are found in proteins involved in signal transduction or membrane trafficking and, therefore, myoferlin may be placed in one or both of these categories. C2 domains have also been shown to bind phospholipids (17), which suggests a role for myoferlin in membrane fusion or membrane repair in the myocyte. The interaction of C2 domains with phospholipids is often calcium dependent, and elevated levels of intracellular calcium are known to occur in skeletal and cardiac myocytes in response to damage and in Duchenne muscular dystrophy (27–29). C2 domains also are known to bind other proteins in calcium-dependent and -independent interactions (18,30–32), as well as mediate formation of protein–protein dimers (33). Myoferlin, unlike dysferlin, has an SH3 domain that may mediate interactions with other proteins. A number of conserved phosphorylation sites in dysferlin and myoferlin further suggests a potential signaling role for myoferlin.
It has been suggested that genetic modifiers may determine the severity of the phenotype and muscle groups affected when dysferlin is absent (9). Myoferlin seems a likely candidate, in that its high similarity to dysferlin may allow compensation for the loss of dysferlin. The presence of myoferlin may also explain the unexpected presence of a 230 kDa protein in skeletal muscle from patients with mutations in DYSF using the NCL-hamlet antibody (9,19). The monoclonal antibody used to study dysferlin in these reports was raised against a peptide with significant homology to myoferlin across its length (17 of 18 amino acids identical) (9,19). However, the lack of nuclear staining on immunofluorescent staining with the dysferlin monoclonal antibody may indicate specificity of this antibody (9,19).
The elevated creatine kinase levels in patients with LGMD 2B and MM is suggestive of membrane damage. This damage may be due to the lack of adequate repair normally performed by the dysferlin-like proteins. Membrane damage in muscular dystrophy may lead to additional recruitment of myoferlin to the plasma membrane from the nuclear membrane. This could explain the upregulation of myoferlin in the sarcolemmal fraction of mdx mice. Mice lacking -sarcoglycan display severe muscular dystrophy but showed a lesser degree of myoferlin upregulation. Recently, it has been shown that -sarcoglycan deficiency does not lead to mechanical defects of the plasma membrane such as those seen in the absence of dystrophin (34). Thus, mechanical defects may lead to greater myoferlin upregulation.
The expression of myoferlin at the nuclear membrane suggests a function unique to that of dysferlin, and may explain the need for two highly homologous proteins in the same tissues. Two other proteins associated with the nuclear membrane have been implicated in muscular dystrophy. The first of these, emerin, is the protein product of the Emery–Dreifuss muscular dystrophy gene. Emerin is a lamin-like protein that is found at the nuclear membrane (35,36). By subcellular fractionation, emerin also has been shown to be expressed in plasma membrane fractions (37). A second nuclear membrane protein, lamin A/C, was recently shown to be mutated in patients with autosomal dominant muscular dystrophy and cardiomyopathy with cardiac conduction system defects (38). It remains unclear how absence of nuclear membrane proteins leads to a phenotype of muscular dystrophy. But, given its homology and intracellular location, MYOF is a candidate gene for both myopathic and arrhythmic phenotypes.
The molecular mechanism of membrane maintenance and repair in the myocyte is poorly understood, but this new family of proteins seems likely to play an important role. The broad tissue distribution of these proteins also suggests that this may be a ubiquitous membrane repair mechanism, found in many different cell types. Due to its homology to dysferlin and its expression pattern, it seems likely that myoferlin may be associated with similar clinical phenotypes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Northern blot analysis and in situ hybridization
A probe corresponding to nucleotides 4758–4985 was generated from the EST AA115643 (clone 511703) in the presence of [32P]dCTP. This probe was hybridized to a multiple tissue northern blot containing 2 µg of poly(A)+ mRNA (human MTN1; Clontech, Palo Alto, CA). Northern blots were exposed to Kodak BioMax MS film (Kodak, Rochester, NY) for 1–7 days at –80°C and were also exposed to phosphorimager cassettes and digitally imaged on a STORM 840 (Molecular Dynamics, Sunnyvale, CA) and analyzed with ImageQuant Software (Molecular Dynamics). In situ hybridization was performed on day 15.5 mouse embryos. The 3'-UTR region of mouse myoferlin (bp 6285–6805) was subcloned into pBluescript SK– (Stratagene, La Jolla, CA) and pCR2.1 (Invitrogen, Carlsbad, CA) and was in vitro transcribed using T7 polymerase in the presence of [35S]UTP to generate sense and antisense riboprobes. In situ hybridization was performed as described (39).
Isolation of myoferlin cDNAs
cDNA clones were obtained from Genome Systems (St Louis, MO) and sequenced to confirm the sequence of myoferlin (AA115643, AA664697, R62159, N42174, AI335266 and AI040116). Two libraries were also used to obtain cDNA clones for myoferlin; a heart library (40) and lung library (Clontech) were screened using probes to myoferlin. cDNA clones were subcloned into pBluescript (Stratagene) and fully sequenced on both strands to determine their nucleotide sequences. Cycle sequencing was performed and sequences were analyzed using Sequencher (Gene Codes, Ann Arbor, MI) and MacVector (Oxford Molecular, Campbell, CA). One region of myoferlin (1144–4658) was generated by RT–PCR using heart RNA as the template for reverse transcription. RT–PCR was performed using primer 810F, 5'-CCCTGCTGGCATTGCCCTCCGGTG-3', and primer 561R, 5'-GTCGGATGAAAATGAAGATCCTTCTG-3', and the amplified sequences were directly sequenced. Alignment of myoferlin amino acid sequence with dysferlin, otoferlin and fer-1 was performed with MacVector software. The transmembrane domain and membrane topology was predicted by PSORT II (http://psort.nibb.ac.jp:8800/ ).
Identification of domains in the amino acid sequence utilized PROSITE-ProfileScan (http://expasy.chuge.ch/sprot/prosite.html ) and MOTIF (http://www.motif.genome.ad.jp ). A BLAST search for other known proteins with homology to myoferlin was performed with Advanced BLAST (http://www.ncbi.nlm.nih.gov/blast/ ). Mapping was performed by searching the Unigene database (http://www.ncbi.nlm.nih.gov/UniGene/index.html ). Myoferlin sequences have been deposited into GenBank with the accession nos AF182316 (myoferlin) and AF182317 (myoferlin alternate 5' end).
Generation of antibodies (Myof1, Myof2, Dysf1)
Peptides for antibody production were selected using MacVector. The peptide SEDGSRIRYGGRDYSLD (amino acids 1692–1709) was synthesized, coupled to KLH and injected into rabbits (Zymed, South San Francisco, CA) to generate Myof1. Crude antiserum was used at 1:1000 on immunoblotting. For immunofluorescence, affinity-purified myoferlin antibody was prepared from a GST fusion protein expressing a portion of myoferlin. A segment of myoferlin (from nucleotide 4916 to 5361) that contained the peptide used for polyclonal antibody production was amplified with the following primers: 4209F-EcoRI, 5'-TCGAATTCCCCGAACTGAGCTGCTACTTACCTCAAG-3'; and 4632R-NotI, 5'-GCACAGCACCTTCCAGCCCAACATTGAGCGGCCGCAT-3'. A segment of dysferlin (from nucleotide 5222 to 5866) that correlates to the region homologous to the peptide used for myoferlin antibody production was also amplified with the following primers: 5222F-EcoRI, 5'-TCGAAATTCCCCAACTACATCCCCTGCACGCTGGAGC-3'; and 5866R-NotI, 5'-GATCCTGGATGACCTGAGCCTCACGGAGCGGCCGCAT-3'. Both products were cloned into the EcoRI/NotI sites of pGex4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ). The myoferlin segment added 14 kDa whereas the dysferlin segment added 21 kDa to the 27 kDa of GST. These fusion proteins were used in SDS–PAGE analysis, and the myoferlin protein was used for affinity purification of the myoferlin antibody as described previously (41). A second myoferlin-specific antibody was generated to the peptide TEFTDEVYQNESRYPGGD (amino acids 928–945) as described above. This region contains no significant homology to dysferlin or otoferlin. Crude antiserum was used at 1:300 for immunofluorescence. An antibody was raised to dysferlin (Dysf1) using the peptide NYTDVNGEKVLPKDDIE; this peptide corresponds to dysferlin amino acids 966–987 and shows no significant homology to myoferlin or otoferlin. Crude antiserum was used for immunofluorescence at 1:300.
Tissues for immunoblot analysis
Tissues from normal mice were processed as follows. Tissues were harvested, ground in liquid nitrogen and homogenized in 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM NaF, 10 mM sodium pyrophosphate, 2 µg/ml aprotinin, 5 ng/ml leupeptin, 1 ng/ml pepstatin and 0.5 mM PMSF. The samples were centrifuged at 16 000 g at 4°C for 15 min. The protein concentration of the supernatant was determined with Bio-Rad (Hercules, CA) protein assay. Total protein (100 mg) was added to an equal volume of 2x SDS loading buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 2% ß-mercaptoethanol and 0.2% bromophenol blue), boiled for 5 min and loaded onto SDS–PAGE. Gels were transferred to a PVDF Immobilon-P membrane (Millipore, Bedford, MA). Transfer was performed overnight at 20 V and 4°C. The membranes were blocked in 5% milk in 1x TBS plus 0.05% Tween-20 (TBS-T) for 1 h. Anti-myoferlin antibody (Myof1) was used at 1:1000 dilution. Anti-DHPR antibody was obtained from Upstate Biotechnology (Lake Placid, NY) and used at 1:3000. Secondary antibody (goat anti-rabbit horseradish peroxidase; Jackson Immunochemicals, West Grove, PA) was used at 1:5000. ECL-Plus chemiluminescence substrate was used for detection (Amersham Pharmacia Biotech). Chemiluminescence was visualized on Kodak Biomax MS film.
Membrane preparations
Three mice of each genotype (C57BL/6, gsg–/–, mdx) were killed by cervical dislocation. Approximately 1 g of tissue was excised from forelimb and hindlimb or heart. The tissues from all three mice of a single genotype were combined. Tissue was homogenized with a Tissue Tearor (Biospec Products, Bartlesville, OK) in 5 ml buffer A. The samples were then homogenized in a dounce homogenizer for 20 strokes with probe A and 20 strokes with probe B. This homogenate was centrifuged at 14 000 g for 15 min at 4°C in a SW50.1 rotor. The pellet was resuspended in 5 ml buffer A and rehomogenized with the dounce for 20 strokes with each probe. This homogenate was centrifuged as above. The pellet was resuspended in 2 ml of 2x SDS–PAGE loading buffer. The supernatants were combined and centrifuged at 14 000 g for 10 min. The supernatant was centrifuged at 30 000 g for 30 min at 4°C in an SW50.1 rotor (Beckman, Fullerton, CA). A KCl buffer was added to the supernatant from this step and incubated at 4°C for 30 min. The light microsomes were then centrifuged at 142 000 g for 30 min at 4°C. This pellet was resuspended in 200 µl of buffer B, and used for analysis in SDS–PAGE. The pellet from the 30 000 g centrifugation (heavy microsomes) was resuspended in 5 ml KCl wash buffer and incubated at 4°C for 30 min and then centrifuged at 142 000 g for 30 min at 4°C. This pellet was resuspended in 500 µl of buffer B and analyzed with SDS–PAGE. This protocol was adapted from the work of Ohlendieck et al. (42). An equal representative volume, 1/50 of total volume of each fraction, was loaded on SDS–PAGE for analysis.
Nuclear preparations
Skeletal muscle from a normal mouse was homogenized in 1x PBS with a Tissue Tearor. Cells were pelleted at 1500 g in a microcentrifuge. The cells were washed in 1x PBS twice at 4°C. Pelleted cells were resuspended in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, plus protease inhibitors listed above), incubated on ice for 15 min and nuclei were pelleted at 14 000 g for 10 s in a microcentrifuge at 4°C. The pellet was resuspended in high-salt buffer (20 mM HEPES pH 7.9), 25% glycerol, 0.42 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, plus protease inhibitors), incubated on ice for 15 min, and nuclear membranes were pelleted at 14 000 g for 5 min in a microcentrifuge at 4°C. Equal volumes of each fraction (15 µl) were loaded.
Immunofluorescence
Tissues were excised from mice killed as described above. The tissues were mounted with OCT, fixed with isopentane and frozen in liquid nitrogen. Sections (10 mm) were cut with a cryostat, fixed in methanol for 2 min and rehydrated in 1x PBS. Sections were blocked in PBS + 5% FBS and incubated with affinity purified antibody against myoferlin without dilution. Secondary antibody (goat anti-rabbit Cy3; Jackson Immunochemicals) was used at a dilution of 1:5000 in PBS + 5% FBS. Sections were mounted with a coverslip and Vectashield (Vector Laboratories, Burlingame, CA). The slides were visualized with Zeiss Axiophot microscope and images were captured on Kodak TMax 400 film.
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ACKNOWLEDGEMENTS |
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We would like to thank Donna Fackenthal for sequencing, Brian Keith for help with in situ hybridization, and David M. Adelman and Ahlke Heydemann for critical reading of the manuscript. E.M.M. is a Charles Culpeper Medical Fellow and is supported by the Muscular Dystrophy Association, the American Heart Association and the NIH. D.B.D. is supported by the MSTP and the American Diabetes Association.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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While this paper was in production, FER1L3 was officially assigned as the approved symbol for myoferlin.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Section of Cardiology, Department of Medicine, 5841 South Maryland, MC 6088, Chicago, IL 60637 USA. Tel: +1 773 702 2672; Fax: +1 773 702 2681; Email: emcnally@medicine.bsd.uchicago.edu
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REFERENCES |
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