Human Molecular Genetics

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Human Molecular Genetics

Pages 1329-1336

©1999 Oxford University Press

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Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy
Introduction
Results
   Characterization of myotilin cDNA
   Organization of the myotilin gene
   Chromosomal localization of myotilin
   Expression pattern of myotilin
   Subcellular localization of myotilin in skeletal muscle
   Myotilin forms intermolecular interactions and directly binds [alpha]-actinin in vitro
Discussion
Materials And Methods
   cDNA cloning of myotilin and sequence analysis
   Genomic structure of myotilin
   Chromosomal localization of myotilin
   Production of myotilin antibody
   mRNA and protein studies
   Localization of myotilin in myofibrils
   Immunohistochemistry
   Yeast two-hybrid analysis and in vitro binding assay
Acknowledgements
References

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Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy

Paula Salmikangas, Olli-Matti Mykkänen, Mikaela Grönholm, Leena Heiska, Juha Kere¹, Olli Carpén^*

Departments of Pathology and¹Medical Genetics, University of Helsinki, Haartman Institute, Box 21 (Haartmaninkatu 3), FIN-00014 Helsinki, Finland

Received March 9, 1999; Revised and Accepted April 19, 1999

DDBJ/EMBL/GenBank accession no. AF144477

The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I-bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.

INTRODUCTION

Among the various cell types in higher organisms, the striated muscle cells have differentiated to carry out the task of force generation and transduction. To serve this very specialized function, the muscle cells express many gene products or mRNA splice variants that are not found in other cells of the body. Many of the muscle-specific genes encode cytoskeletal proteins by which a highly organized sarcomeric architecture is created (1,2). The major components of thin and thick filaments, actin and myosin, are linked to a variety of molecules regulating the assembly, structural integrity and function of the striated muscle. For instance, the giant protein titin that spans from the M-line of the thick filament to the Z-disk of the thin filament functions as a spring and a ruler of the sarcomere, and [alpha]-actinin, an actin-binding protein, cross-links thin filaments into antiparallel bundles in the Z-disks (2-8). The force generated by cytoskeletal components of the contracting subunits is transduced through the plasma membrane (sarcolemma) to the extracellular matrix via a connecting multi-subunit dystrophin-glycoprotein complex (9,10).

The importance of the individual components of the sarcomeric and sarcolemmal structures is highlighted by recent discoveries that mutations in several different structural proteins result in muscular diseases such as muscular dystrophies and cardiomyopathies (9-12). Many of the identified muscular dystrophy genes encode proteins of the dystrophin-associated sarcolemmal complex, but recently other types of molecule, including regulators of the sarcomeric architecture, have also been indicated to participate in the pathogenesis of certain forms of the disease. Mutations in the [alpha]-tropomyosin gene, TPM3, and the nebulin gene cause an autosomal dominant nemaline myopathy (NEM1) (13) and a recessive form of nemaline myopathy (NEM2) (14), respectively. Moreover, the titin gene is a candidate for autosomal dominant tibial muscular dystrophy (15). In spite of recent advances, several clinically distinquishable forms of muscular dystrophy with unidentified disease genes exist. Two forms of muscular dystrophy, a dominant form of limb-girdle muscular dystrophy (LGMD1A) and a dominant form of distal myopathy with vocal cord and pharyngeal weakness (VCPMD), have been mapped to an overlapping locus in 5q31 (16,17).

Here we describe a novel gene, myotilin, which encodes a structural component of the striated and cardiac muscle cytoskeleton. Myotilin protein contains two C2-type Ig-like domains with considerable homology to certain Ig domains of titin. Myotilin resides both in the sarcomere, where it localizes within the I-bands and is bound to [alpha]-actinin, and along the sarcolemmal membrane. The myotilin gene locates in chromosome 5q31 inside a 2 Mb region, which contains the LGMD1A disease gene (16), and thus is a candidate for LGMD1A.

RESULTS

Characterization of myotilin cDNA

We searched for novel cytoskeletal proteins by a yeast two-hybrid screen using the spectrin-like repeats of [alpha]-actinin as bait. With this approach, we isolated from a skeletal muscle library a partial cDNA encoding a previously unknown polypeptide. Using this cDNA sequence as a probe, we cloned a 2244 bp cDNA from a human skeletal muscle library. The cDNA contains a 1494 bp open reading frame encoding a 498 amino acid polypeptide, which we have termed myotilin (Fig. 1A). The methionine start codon is in partial agreement with the Kozak consensus sequence. The N-terminal sequence is particularly rich in serine residues often arranged in a paired fashion, and contains a 23 amino acid hydrophobic stretch (residues 57-79). Database searches show that the N-terminal sequence is unique and does not contain known structural domains.

A - B

C

Figure 1. Deduced amino acid sequence and schematic structure of myotilin, and sequence comparison with titin. (A) The inferred 498 residue myotilin polypeptide is shown. The regions containing Ig-like domains are boxed. The dashed line indicates the 17 residue peptide used for production of rabbit antiserum. The nucleotide sequence of cDNA for myotilin has been deposited in the GenBank database (accession no. AF144477). (B) A schematic diagram of the protein structure shows the serine-rich region (grey box) containing a hydrophobic stretch (black box) and the two Ig domains (loops). The approximate positions of various regions are shown below. (C) Two paired Ig domains and flanking regions of human myotilin and titin (Ig domains 7 and 8) were aligned using the Clustal W method. Residues belonging to the Ig-like domains are boxed. Black boxes indicate conserved residues and grey boxes indicate conservative substitutions. The asterisks indicate the highly conserved structural residues W and Y of each Ig domain. The GenBank accession no. for titin is I38344.

The C-terminus of the protein is predicted to form two Ig-like domains with conserved key residues (Fig. 1B) (18). Several cytoskeletal proteins involved in organization of the muscle sarcomere recently have been shown to contain such structural units. By sequence comparison, the highest homology is detected between myotilin and the region of human striated muscle titin that contains the Z-disk-associated Ig domains 7 and 8 (Fig. 1C) (residues 1406-1621 of titin) (4). The sequences within the compared regions are 31% identical and 53% conserved, without any introduced gaps. A similarity comparison using the Clustal method indicates a 38.3% similarity between this region of myotilin and titin. The sequence similarity between myotilin and titin is restricted to the Ig domains of myotilin. Other characteristic structural features of titin, the fn(III)-type domains, the specific sequences of the Z-disk, I-band and M-band, or the repeating KSP phosphorylation motif (4), are not present in myotilin. However, the myotilin sequence prediction reveals several other possible sites for phosphorylation, three of them in the serine-rich region.

Organization of the myotilin gene

The organization of myotilin was determined by comparing the myotilin cDNA with the genomic sequence from chromosome 5 PAC clone 9c13 (GenBank accession no. AC006084). All splice junction sequences are in agreement with the GT-AG consensus (Table 1). The exon-intron boundaries were confirmed further by amplification and sequencing of each exon from a commercial P1 clone with intron-specific oligonucleotide primers (data not shown). The gene is composed of 10 exons, and the translation initiation signal is in exon II (Fig. 2). Thus, the small 69 bp first exon is not translated. The size of the entire gene is <20 000 bp without the promoter region. The nucleotides coding for the Ig domains are in exons VI and VII (first Ig domain) and in exons VIII and IX (second Ig domain).

Figure 2. Organization of the myotilin gene. Exons (vertical black boxes), numbered with Roman numerals, and introns are shown to scale. The positions of the translation initiation signal in exon II and the translation stop codon in exon X are indicated. The sizes of introns are in kb.

Table 1. Exon-intron structure and splice junction sites of the human myotilin gene

Exon	Size	Sequence at exon-intron junction
No.	(bp)	5[prime] splice donor	Intron size (kb)	5[prime] splice acceptor
I	69	GGAACTACGGgtaagtccct	2.5	ccttttgaagGAACAATATT
II	567	TGGATTCCAAgtaagtgaat	4.8	ctttttaaagCTATCAACAG
III	175	TGGAAATCAAgtgggcaaga	1.5	ttctctaaagCGTCTAACAT
IV	102	AGACTCGCAGgtaagttaaa	3.2	taatttcaagCAACACAACT
V	50	CACAAGTAAGgtaaaaaatt	1.1	attcttgtagAAGTAGATCA
VI	133	GGACTTCAAAgtaagagaag	1.3	ttctttctagGTGAGTGGAC
VII	208	GATGTCCTTGgtaagcctcc	2.5	taatatatagCAAAAGAACA
VIII	166	ACCGAATAAGgtaggatatg	0.7	tttatttcagCTTATATCAA
IX	134	GACGTTACGGgtatgtcata	0.2	tctatttcagCACGTCCAAA
X	641

Chromosomal localization of myotilin

The chromosomal localization of myotilin was determined by radiation hybrid mapping. The myotilin gene mapped to chromosome 5q31 between the markers AFM350yb1 and D5S500 (Fig. 3). The gene causing LGMD1A has been mapped to chromosome 5q31 between the markers D5S479 and D5S594 (16). The myotilin gene is inside this reported area and, thus, it is a candidate gene for LGMD1A. Furthermore, the gene for an autosomal dominant distal myopathy (VCPMD) resides in an overlapping 12 cM linkage interval between markers D5S458 and D5S1972 (17).

Figure 3. Integrated map of chromosome 5 in the myotilin gene region. Distances between markers are based on combined genetic and physical mapping information (http://www.genome.wi.mit.edu/ ). The myotilin gene is located in chromosome 5q31, 141 cM from the top of the chromosome 5 linkage group (34) and maps within the 2 Mb LGMD1A critical region (16), thus being a positional candidate gene for the disease. IL9, interleukin-9 gene. The orientation of the chromosome is indicated (centromere to the left).

Expression pattern of myotilin

By northern blot analysis, we detected two different transcripts (2.2 and 2.5 kb) strongly expressed in skeletal muscle and weakly in the heart (Fig. 4). The two transcripts in the heart are only seen after a prolonged exposure (data not shown). Smooth muscle and several non-muscular tissues, including brain, placenta, lung, liver, kidney and pancreas, did not contain detectable mRNA. In vitro translation of the full-length cDNA yielded a 57 kDa polypeptide, which is in agreement with the mass of myotilin estimated from the cDNA sequence (Fig. 5A).

Figure 4. Northern blot analysis of myotilin. A commercial multiple tissue mRNA filter was probed with a ³²P-labelled 320 bp fragment of myotilin cDNA. The filter was exposed for 20 h. The probe hybridizes strongly with skeletal muscle RNA and weakly with cardiac muscle RNA, whereas other indicated tissues are negative. The right lane shows a 6 h exposure of the skeletal muscle mRNA, in order to demonstrate two different transcript sizes (2.2 and 2.5 kb).

Figure 5. In vitro translation and western blot analysis of myotilin. (A) The myotilin cDNA was in vitro translated using a coupled reticulocyte lysate kit. A 57 kDa protein band representing the full-size protein is detected. The smaller 45 kDa band in the translation of full-length cDNA is apparently due to an aberrant translational start point in the sequence. Myotilin_215-498 is a deletion construct used in two-hybrid experiments. Mw, molecular weight markers. (B) Lysates from the indicated tissues were used for immunoblotting with affinity-purified myotilin antibody. Western blotting of human skeletal muscle reveals a strong 57 kDa band and a fainter 110 kDa band (arrowheads), whereas the non-muscular tissues show no reactivity. Pre-absorption of the myotilin antibody with a 5-fold molar excess of the antigenic peptide results in loss of immunoreactivity from skeletal muscle lysate (right lane).

We raised an antibody against myotilin by immunizing rabbits with a synthetic branched 17 amino acid peptide encompassing residues 353-369 (Fig. 1A). In western blotting, this antibody revealed a 57 kDa protein band and a fainter band near 110 kDa from skeletal muscle but not from smooth muscle or non-muscular tissues (Fig. 5B). The reactivity could be blocked by incubating the antibody with a 5-fold molar excess of the corresponding peptide (Fig. 5B). Both the mRNA and immunoblotting data thus indicate that myotilin is a muscular protein with a clearly restricted expression pattern. The identity of the 110 kDa band is unclear. As it migrates at a region twice the size of a myotilin monomer and as myotilin is able to form intermolecular interactions (see below), the band possibly represents a myotilin dimer. On treatment of tissues with 1% Triton X-100 or with 1 M KCl, myotilin was retained in the insoluble fraction, suggesting a cytoskeletal association (data not shown).

Subcellular localization of myotilin in skeletal muscle

To characterize the subcellular localization of myotilin, we isolated bundles of striated muscle myofibrils and stained them using the affinity-purified peptide antiserum. The immunostaining pattern was compared with several characterized components of the sarcomere (Fig. 6). Myotilin staining was detected in the I-bands. The staining pattern was reminiscent of [alpha]-actinin, which is known to decorate the Z-disks of the I-bands. Actin, the major component of thin filaments, gave a more diffuse staining pattern along the entire I-bands. A titin monoclonal antibody, which recognizes an epitope at junctions of thin and thick filaments, revealed a staining pattern of a doublet band at each sarcomere. The immunolocalization data demonstrate that myotilin is an integral component of striated muscle sarcomeres.

Figure 6. Immunolocalization of myotilin in purified myofibrils. Bundles of bovine myofibrils were isolated as described in Materials and Methods and stained with antibodies against myotilin, [alpha]-actinin, actin, titin and a rabbit pre-immune IgG. All analysed proteins localize to I-bands in sarcomeres but the staining patterns differ. Myotilin and [alpha]-actinin decorate the middle of I-bands, whereas actin staining is more diffuse. Titin is detected as a doublet staining the junctions of A- and I-bands. The phase contrast image demonstrates the sarcomeric structure, where the light bands are thin filaments (I-bands) and the dark ones are thick filaments (A-bands). Bar, 5 µm.

To study the localization of myotilin in the striated muscle further, we performed immunohistochemical analyses of frozen tissue sections with the myotilin antibody. In perpendicular sections, where the organization of sarcomeres was visible, we could detect a periodical cross-striated staining of myotilin (Fig. 7A), which was consistent with the pattern in isolated myofibrils. Especially in transverse sections (Fig. 7B), the myotilin staining was also localized at the plasma membrane, indicating that myotilin is also present at the sarcolemma. In addition to these findings, myotilin antibody stained intramuscular nerve fibres (Fig. 7C). The pre-immune serum showed no reactivity (Fig. 7D).

Figure 7. Immunohistochemical staining of myotilin in frozen sections of human skeletal muscle. Frozen sections of skeletal muscle were analysed by the immunoperoxidase technique using an affinity-purified myotilin antibody (A-C) or a control antiserum (D). Myotilin staining is detected in the I-bands of sarcomeres (A), and in transverse sections, also along the sarcolemma of muscle fibres (B). Positive reactivity is also detected in muscular nerves (C). (D) A control staining with pre-immune serum.

Myotilin forms intermolecular interactions and directly binds [alpha]-actinin in vitro

We used the yeast two-hybrid method to study protein interactions of myotilin. Among the tested partners, the strongest interactions were seen between myotilin and [alpha]-actinin (a construct containing spectrin-like repeats R1-R4) and between myotilin molecules (Fig. 8A-C). [alpha]-Actinin is known to form dimers via spectrin-like repeats (19,20) and this could be verified also in our two-hybrid analysis (Fig. 8B). However, quantitation of the [beta]-galactosidase values indicates that the intensity of the reaction was weaker than the interaction between [alpha]-actinin and myotilin and between two myotilin molecules (Fig. 8C). An N-terminal deletion construct containing the Ig domains, myotilin_215-498, bound full-length myotilin, but did not bind [alpha]-actinin (R1-R4). We were unable to express myotilin_215-498 as a bait to test whether the intermolecular interaction of myotilin is mediated by the C-terminal Ig domain-containing region or by N-terminal association with the C-terminal part. The interaction between myotilin and [alpha]-actinin was tested further by an affinity precipitation assay, using in vitro translated ³⁵S-labelled myotilin and GST-[alpha]-actinin constructs bound to glutathione-agarose. Myotilin bound the C-terminal half of [alpha]-actinin (R3/R4/EF-hand), but not the N-terminal half (ABD/R1/R2) or GST alone (Fig. 8D and E). Based on the two-hybrid and affinity precipitation results, residues important for [alpha]-actinin binding reside in the first 215 N-terminal residues of myotilin, and the myotilin-binding site in [alpha]-actinin apparently locates within spectrin-like repeats 3 and 4.

Figure 8. Homotypic interaction of myotilin and association with [alpha]-actinin. (A) Domain structure of [alpha]-actinin and myotilin and the constructs used in yeast two-hybrid and in vitro binding assays. ABD, actin-binding domain; R, spectrin-like repeat; EF, EF-hand region. The grey box in myotilin indicates the serine-rich region and the loops indicate Ig-like domains. (B) A photomicrograph of the yeast two-hybrid interactions. On the left are the expressed bait fusion proteins and on the top are the prey fusion proteins. EG202, empty bait vector; JG4-5, prey vector. A colour reaction is an indicator of an interaction. (C) Quantitation of [beta]-galactoside values. The bait and prey fusion proteins are as in (B). The [beta]-galactosidase values are categorized as follows: -, <20; +, 21-150; ++, 151-300; +++, 301-450. (D) Affinity precipitation analysis of myotilin-[alpha]-actinin interaction. The N- and C-terminal parts of [alpha]-actinin were expressed as GST fusion proteins, purified and bound to glutathione-agarose beads. ³⁵S-labeled in vitro translated myotilin was allowed to bind to GST-[alpha]-actinin fusion-protein-containing beads. Bound material was separated by SDS-PAGE and autoradiographed. Myotilin binds the R3/R4/EF construct, whereas the ABD/R1/R2 and the GST control do not bind. (E) Coomassie-stained SDS-PAGE demonstrating the constructs used in the binding assay.

DISCUSSION

In this study, we present a detailed description of a novel gene, which encodes a component of the striated muscle sarcomere and sarcolemma. Expression of the gene product, which we have termed myotilin (myofibrillar protein with titin-like Ig domains), is strictly controlled as the transcript is only found in skeletal and cardiac muscle. Several lines of evidence indicate that myotilin is an integral component of striated muscle cytoskeleton. Immunostaining of isolated myofibrils and muscle tissue demonstrates a highly organized pattern along the sarcomere and sarcolemma. On extraction of muscle tissue, myotilin is insoluble in neutral detergent and high salt (data not shown), consistent with the behaviour of several cytoskeletal proteins. Finally, myotilin directly binds [alpha]-actinin, an actin cross-linking protein of the striated muscle Z-disks.

The most striking structural feature of myotilin is the presence of two Ig-like domains in the C-terminus of the molecule. Of several Ig domain-containing cytoskeletal proteins, myotilin shows highest sequence homology to human titin, especially to Ig domains 7 and 8 of skeletal muscle titin. This giant protein is assembled mostly from two types of repetitive subunits, the Ig-like domains and fn(III)-like domains that span throughout the molecule. In the sarcomere, titin molecules extend from the Z-disks to the M-lines, and Ig domains 7 and 8 have been shown to locate on the periphery of the Z-disks (6,21). The homology between these particular Ig domains of titin and myotilin suggests that they share sequence motifs important for subcellular localization.

A common feature among many cytoskeletal components, especially actin cross-linking proteins, is their ability to form non-covalent dimers via modular domains, including spectrin-like repeats in proteins such as [alpha]-actinin and Ig-like domains in proteins such as ABP-120 (22). The dimerization interface provides rigidity for the molecules and it is also thought to act as a ruler separating structural molecules at a proper distance. Our yeast two-hybrid experiments indicate homotypic interaction for myotilin molecules, which possibly occurs by dimer formation through its Ig domains. The intermolecular interaction between myotilin molecules is in line with the possibility that myotilin would serve as a cytoskeleton-organizing protein. The general ability of Ig domains to serve as an interface for protein interactions suggests that myotilin, through this region, may participate in binding to other sarcomeric/sarcolemmal proteins.

In addition to homotypic association, myotilin interacts with [alpha]-actinin, an actin cross-linking protein. Mapping of the binding domains indicated involvement of the first 214 residues of myotilin and the spectrin-like repeats 3-4 of [alpha]-actinin. [alpha]-Actinin is also known to interact with titin (8,23,24). The interaction sites of myotilin and titin, however, appear different. In titin, the Z-repeat between Ig domains 3 and 4 contains the binding site (8). Such a sequence is not contained in myotilin. Other proteins that bind [alpha]-actinin include actin, vinculin, zyxin and two LIM proteins, CRP1 and ALP (25-29). Most of these proteins interact with the globular actin-binding domain of [alpha]-actinin, but the binding sites for titin and ALP reside in the spectrin-like repeats (8,29). It remains to be studied whether the association site of [alpha]-actinin for myotilin and these proteins is overlapping and how the interplay between different binding partners is regulated during Z-disk assembly. The association between myotilin and [alpha]-actinin is in line with their subcellular localization in the I-bands. In the Z-disk, [alpha]-actinin links actin from opposing halves of sarcomeres into antiparallel bundles. It is possible that myotilin also has a regulatory role in the organization of actin filaments in the muscle. Further studies to identify additional binding partners of myotilin and in vitro experiments with purified molecules are required to understand the functional characteristics of myotilin.

Defects in several genes cause muscular dystrophies, a spectrum of hereditary disorders characterized by muscle weakness and derangement in muscular fine structure. The characterized disease genes encode proteins regulating cytoskeletal or sarcolemmal integrity or cell adhesion (9,10). Mutations in several sarcomeric proteins, such as myosin, troponin T, [alpha]-tropomyosin or myosin-binding protein C, cause hypertrophic cardiomyopathies. However, the phenotypes in muscular dystrophies and cardiomyopathy are often overlapping (12). The gene defects in several muscular dystrophy subtypes are still unknown. We mapped the myotilin gene in chromosome 5q31 to a region overlapping with the segment that contains an unidentified gene causing the autosomal-dominant LGMD1A (16). Recently, VCPMD was also mapped to the same region in 5q31 (17). To our knowledge, the only annotated muscle-specific expressed sequence tag sequences from the critical region are those belonging to myotilin. The current findings, demonstrating myotilin as a component of the muscle sarcomere and sarcolemma and as a positional candidate gene for LGMD1A, should encourage mutational analyses to clarify the role of myotilin in muscular dystrophy.

MATERIALS AND METHODS

cDNA cloning of myotilin and sequence analysis

A partial cDNA was used for screening of the full-length myotilin cDNA from a skeletal muscle library (Stratagene, La Jolla, CA). Positive clones were sequenced with an ABI 310 Genetic Analyzer (Perkin-Elmer, Foster City, CA). Protein database searches were done with the BLAST program (http://www.ncbi.nlm.nih.gov ). Sequence alignments between Ig domains of myotilin and other cytoskeletal proteins were performed with the MegAlign software (DNASTAR). The domain predictions were obtained from Pfam server (http://genome.wustl.edu/Pfam/ ). Protein motif predictions were done with Protein Family alignment Pfam2.1 (http://pfam.wustl.edu/ ) and with Motif (http://www.motif.genome.ad.jp/ ).

Genomic structure of myotilin

The organization of the myotilin gene was determined by comparing the myotilin cDNA with the genomic sequence from chromosome 5 PAC clone 9c13 (GenBank accession no. AC006084). Furthermore, a commercial genomic clone was identified from a P1 library (Genome Systems, St Louis, MO). The clone was used as a template for PCR amplification and sequencing of exon-intron boundaries using gene-specific oligonucleotide primers.

Chromosomal localization of myotilin

The chromosomal localization of myotilin was determined by radiation hybrid mapping using the Genebridge II panel. PCR primers, designed from the 3[prime]-untranslated region of myotilin cDNA, were M1 (5[prime]-ACA GGA AAT CTG GGT ATA TG) and M2 (5[prime]-TGT GGA ACA CTC AGA TAA TC). These primers amplify a 230 bp product from human genomic DNA. PCR assays were performed as duplicates and the resulting data vector was analysed using the Whitehead Genome Center server (http://www-genome.wi.mit.edu ).

Production of myotilin antibody

A polyclonal antibody was raised in rabbits using a synthetic branched, lysine-cored 17 amino acid peptide of myotilin (marked in Fig. 1 with a dashed line) as the antigen. After five immunizations, rabbits were bled. The specific antibody was purified on an affinity column using a corresponding single chain peptide coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) as the ligand. The specificity of the rabbit antibody was verified by reactivity with appropriate GST fusion protein constructs in western blot analysis (data not shown) and by cross-blocking experiments, in which a 5-fold molar excess of the specific peptide but not an irrelevant myotilin peptide (residues 199-217) absorbed the reactivity.

mRNA and protein studies

Northern blot analysis was performed with a multiple tissue mRNA filter (Clontech, Palo Alto, CA) using a ³²P-labelled 320 bp myotilin cDNA fragment, which encodes amino acids 369-471, as a probe. In vitro translations were performed with a coupled reticulocyte lysate kit (Promega, Madison, WI) using ³⁵S-labelled methionine for detection. The templates were full-length myotilin and a construct containing amino acids 215-498 (myotilin_215-498) in Bluescript plasmid vector (Stratagene). For western blotting, fresh tissues were homogenized in reducing Laemmli buffer. Equal amounts of protein, as estimated by Coomassie blue staining, were separated in 8% SDS-PAGE and transferred to nitrocellulose filters (Schleicher & Schuell, Dassel, Germany). The filters were probed with the myotilin antibody or with a control pre-immune serum, followed by peroxidase-conjugated goat anti-rabbit IgG (Dako, Copenhagen, Denmark) and ECL detection (Pierce, Rockford, IL).

Localization of myotilin in myofibrils

Bundles of bovine and human myofibrils were isolated as described (30), cytocentrifuged onto objective slides, fixed in -20°C methanol, and reacted with monoclonal antibody against actin (AC 40; Sigma), titin (T11; Sigma), [alpha]-actinin (67CB11) (31) and a control monoclonal antibody X63 (ATCC, Rockville, MD), or with affinity-purified anti-myotilin antibody or the corresponding pre-immune IgG. Secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel Research Products, Durham, NC) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit F(ab)₂ fragment (Jackson ImmunoResearch Laboratories, West Grove, PA). Staining of bovine and human myofibrils yielded identical results.

Immunohistochemistry

Frozen 2 µm sections of human skeletal muscle were immobilized on poly-L-lysine-coated glass slides, fixed with cold acetone and immediately air-dried. For immunohistochemical staining, the sections were reacted with a 1:100 dilution of affinity-purified myotilin antibody or rabbit pre-immune IgG at a similar concentration. The antibody was detected with Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. The slides were counterstained briefly with haematoxylin and eosin.

Yeast two-hybrid analysis and in vitro binding assay

Full-length myotilin, myotilin_215-498 and spectrin-like repeats R1-R4 (residues 267-749) of chicken smooth muscle [alpha]-hactinin (kindly provided by Dr D. Critchley, University of Leicester, UK) were subcloned into EG202 and JG4-5 plasmids for two-hybrid analysis (32). The C-terminal construct of myotilin was subcloned from a partial cDNA sequence obtained from the skeletal muscle library screen. The authenticity of the constructs was verified by sequencing. The genotype of the Saccharomyces cerevisiae strain BOY1, kindly provided by P. Ljungdahl (Ludwig Institute for Cancer Research, Stockholm, Sweden), is MAT [alpha]his3 trp1 leu2::6LexAop-LEU2 URA3::8LexAop-Gal1-LacZ. BOY1 mating type a was made using the YCpHO CUT4 plasmid (33). Yeast strains were grown at 30°C in rich medium or in synthetic minimal medium with appropriate amino acid supplements. Bait and prey constructs were transformed into BOY1 yeast of both the a and [alpha] mating type using the TRAFO protocol (www.manitoba.ca/faculties/medicine/human-genetics/gietz/trafo.html ) and plated on selection plates. Clones were grown to late logarithmic phase in selective medium. For analysis of fusion protein expression, yeast cells from 1 ml of overnight culture were lysed in reducing Laemmli sample buffer, and the samples were boiled and analysed by SDS-PAGE and immunoblotting. Baits and prey were grown on selection plates, replica plated together on rich media plates for mating overnight and replica plated for double (tryptophan and histidine) or triple (tryptophan, histidine and leucine) selection with or without 5-bromo-4-chloro-3-indolyl-[beta]-D-galactopyranoside (X-gal) (Boehringer Mannheim, Mannheim, Germany) for selection of interactions.

For the in vitro binding assay, GST-[alpha]-actinin fusion proteins, ABD/R1/R2, R3/R4/EF (19) or GST alone were produced in Escherichia coli and purified with glutathione-agarose beads (Pharmacia). A 2 µg aliquot of fusion proteins on glutathione beads was reacted with 20 µl of in vitro translated, ³⁵S-labelled myotilin in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 130 mM KCl, 0.05% Tween-20. After washes with the same buffer, bound material was eluted by boiling in Laemmli buffer, subjected to SDS-PAGE and detected by autoradiography.

ACKNOWLEDGEMENTS

We thank Dr D. Critchley for [alpha]-actinin constructs, Dr I. Virtanen for [alpha]-actinin antibodies and Dr P. Ljungdahl for yeast cells. Mrs Tuula Halmesvaara and Mrs Maija-Liisa Mäntylä are warmly acknowledged for their excellent technical expertise. This study was supported by the Academy of Finland, the Helsinki University Central Hospital, the Sigrid Juselius Foundation and the Finnish Muscular Disease Research Foundation.

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*To whom correspondence should be addressed. Tel: +358 9 1912 6413; Fax: +358 9 1912 6700; Email: olli.carpen{at}helsinki.fi

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