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Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

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Abstract

The almost complete loss of the membrane cytoskeletal protein dystrophin and concomitant drastic reduction in dystrophin-associated glycoproteins are the underlying mechanisms of the highly progressive neuromuscular disorder Duchenne muscular dystrophy. In order to identify new potential binding partners of dystrophin or proteins in close proximity to the sarcolemmal dystrophin complex, proteomic profiling of the isolated dystrophin–glycoprotein complex was carried out. Subcellular membrane fractionation and detergent solubilisation, in combination with ion exchange, lectin chromatography and density gradient ultracentrifugation, was performed to isolate a dystrophin complex-enriched fraction. Following gradient gel electrophoresis and on-membrane digestion, the protein constituents of the dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which act as cytolinker proteins and possibly exist in close proximity to the dystrophin complex in the sarcolemma membrane. Interestingly, comparative immunoblotting showed a significant reduction in desmoglein in dystrophin-deficient mdx skeletal muscles, reminiscent of the pathobiochemical fate of the dystrophin-associated core proteins in muscular dystrophy. Comparative membrane proteomics was used to correlate this novel finding to large-scale changes in the dystrophic phenotype. A drastic increase in the extracellular stabilizers biglycan and fibronectin was shown by both mass spectrometric analysis and immunoblotting. The reduced expression of desmoglein in dystrophin-deficient skeletal muscles, and simultaneous increase in components of the extracellular matrix, suggest that muscular dystrophy is associated with plasmalemmal disintegration, loss of cellular linkage and reactive myofibrosis.

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Acknowledgements

Research was supported by project grants from Muscular Dystrophy Ireland and the Irish Health Research Board (HRB/MRCG-2016-20) and a Hume scholarship from Maynooth University. The Q-Exactive quantitative mass spectrometer was funded under the Research Infrastructure Call 2012 by Science Foundation Ireland (SFI-12/RI/2346/3). The authors would like to thank Ms Caroline Batchelor for expert technical support with mass spectrometry.

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  1. Department of Biology, Maynooth University, National University of Ireland, Maynooth, County Kildare, Ireland

    Sandra Murphy & Kay Ohlendieck

  2. Institute of Pathophysiology, University Medicine Greifswald, 17495, Karlsburg, Germany

    Heinrich Brinkmeier & Mirjam Krautwald

  3. National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland

    Michael Henry & Paula Meleady

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  1. Sandra Murphy
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  2. Heinrich Brinkmeier
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Murphy, S., Brinkmeier, H., Krautwald, M. et al. Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin. J Muscle Res Cell Motil 38, 251–268 (2017). https://doi.org/10.1007/s10974-017-9478-4

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