ARTICLE |
Correspondence to: Elena Pompili, University “La Sapienza,” Dept. of Cardiovascular Sciences, Via A. Borelli 50, 00161 Rome, Italy. E-mail: elena.pompili@uniroma1.it
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Summary |
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We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac ß-myosin heavy chain (ß-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac ß-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac ß-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family. (J Histochem Cytochem 51:471478, 2003)
Key Words: non-muscle myosin heavy, chain, immunohistochemistry, neuromuscular junction
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Introduction |
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Myosins are molecular motors that, on interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force. In addition to the well-known sarcomeric muscle myosins, a large number of novel isoforms have been identified in muscle and non-muscle cells (
Although the precise function of the non-sarcomeric myosins (generally called non-muscle myosins) remains to be determined, they appear to play a role in specialized cell functions such as membrane trafficking, cell movements, and signal transduction (
The third non-muscle myosin that has been extensively characterized is myosin-Va (
Here we discuss the identification of a 220-kD protein at the neuromuscular junction, showing biochemical and immunohistochemical features of a novel non-muscle myosin heavy chain (NMHC).
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Materials and Methods |
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Protein Identification
Specimens of bovine skeletal muscle were taken from adult animals at a local slaughterhouse. Samples were immediately snap-frozen in liquid nitrogen and stored at -80C until used. In a typical experiment, approximately 80100 g of muscle was homogenized in 50 mM Tris, pH 7.5, containing 0.15 M NaCl (1:5 w:v), using an UltraTurrax T25 homogenizer (IKA; Staufen, Germany). The homogenate was ultracentrifuged at 105,000 x g for 1 hr at 4C and soluble proteins were partially precipitated with 50% ammonium sulfate. The resulting pellet was resuspended in 50 mM Tris (pH 7.5) containing 0.15 M NaCl and 0.005% Brij-35, and was applied at 4C to a small column (2 ml) of agarose (Sepharose 4B; Pharmacia Biotech, Uppsala, Sweden). After sample application, the column was washed (10 ml/hr) with 10 volumes of equilibrium buffer and finally eluted with 0.05% trifluoroacetic acid (TFA), pH 2.5, containing 0.15 M NaCl. To neutralize the acid pH of the eluent, the eluted material (0.5 ml/tube) was immediately mixed with aliquots of 1 M Tris, pH 7.5 (0.04 ml/tube).
Protein concentration was determined by the method of
Mass Spectrometry and Sequence Analysis
The mixture of proteins eluted from the Sepharose 4B column was electrophoresed on a 7% polyacrylamide gel containing 1% SDS by using a vertical apparatus (Hoefer Pharmacia Biotech; San Francisco, CA) (1.5 mm gel thickness). The gel was stained with Coomassie Blue R250 for 10 min to visualize the bands and then was de-stained by soaking for 3 hr to remove the excess stain. The band of interest (220 kD) was excised from the gel and subjected to trypsin digestion according to
A small aliquot of the gel digested with trypsin was subjected to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) (Keck Facility; Yale University, New Haven, CT). MALDI-MS was carried out on a Micromass TofSpec SE mass spectrometer equipped with delayed extraction and a reflection. To attain the high level of accuracy needed for peptide mass searching, 100 fml bradykinin with a protonated, monoisotopic mass of 1060.57, and an ACTH clip, with a protonated, monoisotopic mass of 2465.2, were added as external calibrants.
The resulting peptide masses were then subjected to peptide mass identification using PeptIdent (at the EBI) and ProFound (at Rockefeller University). Peptide masses were compared with the theoretical masses derived from the sequence contained in SWISS-PROT/TrEMBL databases (release 40.16 and 20.4). The search parameters used were as follows: cysteines unmodified, maximum allowed peptide error 0.1 Dalton.
For amino acid sequence, the tryptic mixture was separated using a Labservice Analytica chromatographer (model LabFlow 4000) on a C18 column (250 mm x 2.1 mm; 5-µm Vydac) in a linear gradient of acetonitrile (170%) in 0.05% TFA. Amino acid sequence was carried out using a sequencer equipped with an on-line HPLC system (Keck Facility, Yale University).
Antibody Preparation
For antigen preparation, the protein mixture eluted from the Sepharose 4B column was subjected to semipreparative SDS-PAGE on 12.5% gels by using a vertical apparatus (Hoefer Pharmacia Biotech; San Francisco, CA) (1.5 mm gel thickness). After electrophoretic separation, the gels were stained faintly with Coomassie Blue and the 220-kD band was excised and used to immunize two New Zealand White rabbits. For each injection two or three slices (containing approximately 200 µg antigen) were pooled, homogenized in 10 mM Tris, pH 7.5 (1 ml), and emulsioned with an equivalent amount of incomplete Freund's adjuvant (Sigma; St Louis, MO). For the first injection, complete Freund's adjuvant (Sigma) was used. Samples were injected into a rabbit's back by multiple intradermal injections (2030 injections). Four antigen administrations were performed at 2830-day intervals.
Western Blotting
Immunoblotting experiments were performed essentially according to
Immunohistochemistry
For immunohistochemical experiments, small blocks of skeletal and cardiac muscle were embedded in gum tragacanth (Merck; Darmstadt, Germany) and snap-frozen in isopentane precooled with liquid nitrogen. Serial sections (810 µm thick) were cut in a cryostat at -20C and mounted on gelatin-coated glass slides. Specimens were fixed for 4 min at RT with 4% paraformaldehyde (Merck), quenched with 0.1 M glycine-HCl, pH 7.4, and treated with 3% H2O2 in methanol to inhibit endogenous peroxidase. After extensive washes in PBS, sections were preincubated for 1 hr with 6% nonfat dry milk and incubated with the appropriate antibody up to 72 hr at 4C.
Anti-220-kD protein Igs were isolated on a DEAEAffigel blue column and used at a final concentration of 110 µg/ml. A commercial monoclonal antibody to human cardiac MHC, type /ß (Biocytex Biotechnol; Marseille, France) was also used at a final dilution of 1:100. Immunohistochemical distribution was revealed according to a standard avidinbiotinperoxidase method. Briefly, after washes with PBS0.3% Triton and PBS alone, slides were incubated (1 hr at RT) with a biotinylated secondary antibody (Vector), washed again, and incubated (30 min at RT) with an avidinbiotinperoxidase complex (Vectastain Elite ABC kit; Vector). Finally, sections were washed and treated with 0.05% 3-3' diaminobenzidine and 0.015% H2O2.
Negative controls were done by omission of the first or the secondary antibody or by replacing the primary antibody with an equivalent amount of Igs purified from the preimmune sera. In additional experiments, specific anti-220-kD protein Igs were preabsorbed with saturating amounts of purified antigen (antigen:antibody ratio=5).
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Results |
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Protein Identification and Sequence Analysis
Samples of bovine skeletal muscle were homogenized and precipitated with 50% ammonium sulfate. The resulting pellet was then resuspended in the equilibrium buffer and applied to an agarose (Sepharose 4B) column that is known to selectively bind carbohydrate recognition molecules (
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For protein identification, a sample of the 220-kD band was excised and digested with trypsin. MALDI-MS was carried out on approximately 5% of the tryptic digest. MALDI-MS detected a clear break in the number of peptides matched, with a sufficient number of masses for database searching. The primary program we used for searching was PeptIdent, which relies on SWISS-PROT/TrEMBL databases. This search revealed that our protein had a top score to a myosin heavy chain of various sources, mainly cardiac isoforms. Furthermore, automated Edman degradation of a purified tryptic peptide gave the following sequence: STHPHFVR. This sequence is identical to that present in human cardiac ß-MHC in position 664671. This sequence is conserved in all the other cardiac ß-myosins from various sources whose complete sequences are known, but is not conserved in the primary structure of skeletal myosins.
Immunochemistry and Immunohistochemistry
To study our protein further, we generated a polyclonal antibody in rabbits using the entire protein as antigen (for details see Materials and Methods). The resulting Igs purified from rabbit sera specifically stained a single band of 220 kD in Western blots of muscle homogenate (Fig 2). Furthermore, by using the same Igs we studied the immunohistochemical distribution of the 220-kD protein on cryostatic sections of skeletal and cardiac muscles. In bovine skeletal muscle anti-220-kD protein Igs stained specifically scattered structures near the skeletal muscle fibers, the sarcoplasm of all muscle fibers being completely devoid of immunoreactivity (Fig 3A). In adjacent sections, a classical cholinesterase staining (
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In bovine heart, anti-220-kD protein Igs selectively labeled the intercalated disks of all myocardial cells (Fig 4A). Control experiments were completely negative (Fig 4B). A very different pattern of immunostaining was obtained by using a monoclonal antibody specific for cardiac MHC. In bovine skeletal muscle, anti-cardiac MHC Igs strongly and specifically stained the cytoplasm of only few scattered muscle fibers (Fig 5A). By contrast, as expected, in bovine heart all cardiac fibers were specifically labeled (Fig 5B).
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Discussion |
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The first molecular motor to be discovered was conventional, two-headed, and filament forming myosin II. Because of its important role in muscular contraction, myosin II has been extensively studied at both the molecular and the cellular level.
Myosin II has also been detected in non-muscle cells. In particular, vertebrates have two NMHC genes, encoding for NMHC II-A and NMHC II-B (
Here we have described the identification, the initial characterization, and the immunohistochemical localization of a 220-kD protein that is probably an additional member of this large superfamily. This protein has been isolated from bovine skeletal muscle homogenate by affinity chromatography on an agarose column. This approach has also been used by other workers to purify other NMHCs from mammalian retinal pigment epithelial and endothelial cells (
Several studies have reported the presence of NMHCs Va and VI in neurons and synapses of the CNS (
In the myocardium, our anti-220-kD protein antibody stains exclusively the intercalated disks, where NMHC II-B has been also detected (
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Acknowledgments |
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Supported by the Italian Ministry of University and Technology (Grants University La Sapienza, Facoltà and Ateneo to L. Fumagalli).
Received for publication July 15, 2002; accepted October 2, 2002.
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