Abstract
α2-Magcroglobulin (α2M), a major serum protease inhibitor, was localized in mouse skeletal muscle by immunoperoxidase histochemistry. In all muscles examined (mm. soleus, plantaris, and extensor digitorum longus) specific immunoreactivity occurred diffusely in extracellular structures (periendomysium, blood vessel wall) as well as inside about a half of the muscle fibers. This localization pattern did not change substantially by extensively perfusing deeply anesthetized mice with phosphate buffered saline (PBS) to remove serum α2M. In release experiments on fresh (nonfixed) cryostat sections, specific immunoreactivity persisted after an extensive prewash with PBS (up to 5–6h), but a new specific staining appeared inside those fibers that were originally negative. Western blotting experiments were negative on the soluble fraction of muscle homogenate, thus confirming that the perfusion procedure was effective in removing serum α2M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-solubilized extracts (0.3% Triton X-100), indicating the occurrence of tissue-associated α2M. Confocal immunofluorescence microscopy revealed that the intracellular specific staining was associated to a longitudinal network, probably corresponding to the sarcoplasmic reticulum. A multifunctional role of α2M in skeletal muscle was hypothesized.
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Akaaboune M., Ma J., Festoff B. W., Greenberg B. D., and Hantai D. (1994) Neurotrophic regulation of mouse muscle β-amyloid protein precursor and α1-anthichymotrypsin as revealed by axotomy.J. Neurobiol. 25, 503–514.
Askanas V., Engel W. E., and Alvarez R. B. (1992) Strong immunoreactivity of β-amyloid precursor protein sequence, at human neuromuscular junctions.Neurosci. Lett. 143, 96–100.
Bilak M., Askanas V., and Engel W. K. (1993) Strong immunoreactivity of α1-antichymotrypsin colocalizes with β-amyloid protein and ubiquitin in vacuolated muscle fibers of inclusion-body myostis.Acta Neuropathol. 85, 378–382.
Borth W. (1984) α2-Macroglobulin in connective tissue matrix metabolism.Collagen Relat. Res. 4, 83–94.
Bulfield G., Siller W. G., Wight P. A., and Moore K. J. (1984) X chromosome linked muscular dystrophy (mdx) in the mouse.Proc. Natl. Acad. Sci. USA 81, 1189–1192.
Chu C. T. and Pizzo S. V. (1994) α2-Macroglobulin, complement, and biologic defense: antigens, growth factors, microbial proteases, and receptor ligation.Lab. Invest. 71, 792–812.
Festoff B. W., Rao J. S., Rayford A., and Hantai D. (1990) Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpinprotease complex receptors increase during in vitro myogenesis.J. Cell. Physiol. 144, 272–279.
Festoff B. W., Reddy R. B., VanBecelaere M., Smirnova I., and Chao J. (1994) Activation of serpins and their cognate proteases in muscle after crush injury.J. Cell. Physiol. 59, 11–18.
Grounds M. D. and Yablonka-Reuveni Z. (1993) Molecular and cell biology of skeletal muscle regeneration, inMolecular and Cell Biology of Muscular Distrophy (Partridge T., ed.), pp. 210–256, Chapman and Hall, London.
Kim O., Chung S. S., Woo K. M., Ha D. B., and Chung C. H. (1992) Purification and partial characterization of a trypsin inhibitor from chick skeletal muscle.Biochem. Int. 27, 963–972.
Kramer M. D. and Justus C. (1988) The antiproteolytic compound α2-macroglobulin in human skin.Arch. Dermatol. Res. 280, 93–96.
Kuehn L., Dahlmann B., and Reinauer H. (1984) Identification of four distinct serine proteinase inhibitors in rat skeletal muscle.Biochem. Biophys. Res. Commun. 120, 96–102.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685.
LaMarre J., Wollenberg G. K., Gonias S. L., and Hayes M. A. (1991) Cytokine binding and clearance properties of proteinase-activated α2-Macroglobulins.Lab. Invest. 65, 3–14.
Moestrup S. K., Gliemann J., and Pallesen G. (1992) Distribution of the α2-Macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues.Cell. Tissue Res. 269, 375–382.
Moestrup S. K. (1994) The α2macroglobulin receptor and epithelial glycoprotein-330: two giant receptors mediating endocytosis of multiple ligands.Biochem. Biophys. Acta 1197, 197–213.
Nori S. L., Businaro R., Fabrizi C., Moestrup S. K., Starace G., Fumagalli L., and Lauro G. M. (1993) Receptor-mediated endocytosis of α2-macroglobulin by a glioma cell line.Neuroreport 4, 423–426.
Sasaki M., Taniguchi K., Suzuki K., and Imahori K. (1983) Human plasma α2 and α1 thiol proteinase inhibitors strongly inhibit Ca-activated neutral proteinases from muscle.Biochem. Biophys. Res. Commun. 110, 256–261.
Schubert W., Prior R., Weidemann A., Dircksen H., Multhaup G., Masters C. L., and Beyreuther K. (1991) Localization of Alzheimer β-amyloid precursor protein at central and peripheral synaptic sites.Brain Res. 563, 184–194.
Seiler S., Wegener A. D., Whang D. D., Hathaway D. R., and Jones L. R. (1984) High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease.J. Biol. Chem. 259, 8550–8557.
Sohar I. and Katona G. (1992) Regulation of proteinase activation in mammalian tissues.Biol. Chem. Hoppe Seyler 373, 567–572.
Tawa N.E. and Goldberg A. L. (1994) Protein and amino acid metabolism in muscle, inMyology, 2nd ed. (Engel A. G. and Franzini-Armstrong C., eds.), pp. 683–707, McGraw-Hill, New York.
Van Leuven F., Marynen P., Cassiman J. J., and Van den Berghe H. (1987) A mouse monoclonal antibody to human α2M} crossreacts with α2M from mouse. Epitope mapping and characterization of subunit structure of murine α2M.J. Biochem. (Tokyo)101, 1181–1189.
Van Leuven F., Torrekens S., Overbergh, L., Lorent K., De Strooper B., and Van den Berghe H. (1992) The primary sequence and the subunit structure of mouse α2-macroglobuli, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.Eur. J. Biochem. 210, 319–327.
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Fumagalli, L., Businaro, R., Nori, S.L. et al. Evidence for tissue-associated α2-magcroglobulin in mouse skeletal muscle. Molecular and Chemical Neuropathology 27, 211–223 (1996). https://doi.org/10.1007/BF02815105
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DOI: https://doi.org/10.1007/BF02815105