Proteomic Profiling of the Dystrophin Complex and Membrane Fraction
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J Muscle Res Cell Motil (2017) 38:251–268 DOI 10.1007/s10974-017-9478-4 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 Sandra Murphy1 · Heinrich Brinkmeier2 · Mirjam Krautwald2 · Michael Henry3 · Paula Meleady3 · Kay Ohlendieck1 Received: 3 May 2017 / Accepted: 5 August 2017 / Published online: 12 August 2017 © Springer International Publishing AG 2017 Abstract The almost complete loss of the membrane fate of the dystrophin-associated core proteins in muscular cytoskeletal protein dystrophin and concomitant drastic dystrophy. Comparative membrane proteomics was used reduction in dystrophin-associated glycoproteins are the to correlate this novel finding to large-scale changes in underlying mechanisms of the highly progressive neuro- the dystrophic phenotype. A drastic increase in the extra- muscular disorder Duchenne muscular dystrophy. In order cellular stabilizers biglycan and fibronectin was shown by to identify new potential binding partners of dystrophin or both mass spectrometric analysis and immunoblotting. The proteins in close proximity to the sarcolemmal dystrophin reduced expression of desmoglein in dystrophin-deficient complex, proteomic profiling of the isolated dystrophin–gly- skeletal muscles, and simultaneous increase in components coprotein complex was carried out. Subcellular membrane of the extracellular matrix, suggest that muscular dystrophy fractionation and detergent solubilisation, in combination is associated with plasmalemmal disintegration, loss of cel- with ion exchange, lectin chromatography and density gradi- lular linkage and reactive myofibrosis. ent ultracentrifugation, was performed to isolate a dystrophin complex-enriched fraction. Following gradient gel electro- Keywords Biglycan · Desmoglein · Desmoplakin · phoresis and on-membrane digestion, the protein constitu- Dystrophin · Dystrophinopathy · Fibronectin ents of the dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which Introduction act as cytolinker proteins and possibly exist in close prox- imity to the dystrophin complex in the sarcolemma mem- The largest gene in the human genome, the 2.4 Mb Dmd brane. Interestingly, comparative immunoblotting showed a gene, contains seven promoters and encodes a variety of iso- significant reduction in desmoglein in dystrophin-deficient forms of the protein dystrophin ranging in molecular mass mdx skeletal muscles, reminiscent of the pathobiochemical from 71 to 427 kDa (Muntoni et al. 2003). The full-length isoform of dystrophin, Dp427, is a membrane cytoskeletal protein that exists in a membrane-associated supramolecular Electronic supplementary material The online version of this article (doi:10.1007/s10974-017-9478-4) contains complex (Murphy and Ohlendieck 2015). The dystrophin supplementary material, which is available to authorized users. complex mediates membrane stabilization during excita- tion–contraction–relaxation cycles and functions as a scaf- * Kay Ohlendieck fold for various signalling proteins (Le Rumeur et al. 2010; [email protected] Constantin 2014). In Duchenne muscular dystrophy, primary 1 Department of Biology, Maynooth University, National abnormalities in the Dmd gene are the underlying cause for University of Ireland, Maynooth, County Kildare, Ireland the almost complete absence of Dp427 in skeletal muscles 2 Institute of Pathophysiology, University Medicine and the heart, as well as changes in the dystrophin isoform Greifswald, 17495 Karlsburg, Germany expression pattern in the central nervous system (Guiraud 3 National Institute for Cellular Biotechnology, Dublin City et al. 2015). Dystrophin-deficient skeletal muscles are char- University, Dublin 9, Ireland acterized by a drastic reduction in dystrophin-associated Vol.:(0123456789)1 3 252 J Muscle Res Cell Motil (2017) 38:251–268 proteins, such as dystroglycans, sarcoglycans, sarcospan, matrix linker proteins biglycan (Amenta et al. 2011) and dystrobrevin and syntrophins (Ohlendieck 1996). This fibronectin, indicating that sarcolemmal disintegration is results in down-stream abnormalities in cellular signalling, associated with distinct changes in the matrisome (Bowe membrane integrity and energy metabolism (Allen et al. et al. 2000; Holland et al. 2016). Hence, the proteomic and 2016). biochemical profiling of normal versus dystrophic muscles In order to better understand the pathobiochemical com- presented in this report indicates new pathophysiological plexity of dystrophinopathy and the potential interconnec- roles of desmoglein, biglycan and fibronectin in X-linked tivity of damage pathways, mass spectrometry-based prot- muscular dystrophy. eomics has been widely applied to study dystrophic skeletal muscles, cardiac abnormalities, the impaired central nervous system and serum changes, as outlined in recent reviews Experimental section (Fuller et al. 2016; Hathout et al. 2016). The deficiency in dystrophin was shown to be associated with changes in ion Materials homeostasis, excitation–contraction coupling, cellular sig- nalling, the stress response and energy metabolism (Guevel For the gel- and liquid chromatography-based proteomic et al. 2011; Rayavarapu et al. 2013; Roberts et al. 2015; and biochemical profiling of the dystrophin complex and Fröhlich et al. 2016; Lardenois et al. 2016). In addition, membrane fractions from skeletal muscles, analytical grade stabilizing structures of the cytoskeleton and the extracel- reagents and materials were purchased from GE Healthcare lular matrix are drastically altered (Holland et al. 2013). In (Little Chalfont, Buckinghamshire, UK), Sigma Chemical contrast, only a limited number of studies have focused on Company (Dorset, UK), Bio-Rad Laboratories (Hemel- the proteomics of the core dystrophin complex (Murphy Hempstead, Hertfordshire, UK) and National Diagnostics and Ohlendieck 2015). The proteomic characterization of (Atlanta, GA, USA). Proteolytic digestion was carried out the dystrophin–glycoprotein complex has employed mass with sequencing grade modified trypsin and Lys-C enzymes spectrometric surveys of separated protein assemblies using from Promega (Madison, WI, USA). Materials for immunob- one-dimensional gel electrophoresis (Lewis and Ohlendieck lotting included Whatman nitrocellulose transfer membranes 2010), immuno-precipitation (Yoon et al. 2012) and gradient from Invitrogen (Carlsbad, CA, USA), chemiluminescence centrifugation (Turk et al. 2016). substrate from Roche Diagnostics (Mannheim, Germany) Building on these proteomic surveys and previous bio- and the reversible membrane stain MemCode from Thermo chemical purification studies of dystrophin (Ervasti et al. Fisher Scientific (Waltham, MA, USA). Primary antibodies 1990, 1991), we used here a combination of differential were purchased from Abcam, Cambridge, UK (ab124798 to centrifugation, detergent solubilisation, ion exchange chro- desmoglein isoform DSG1, ab2413 to fibronectin, ab58562 matography, lectin affinity chromatography, density gradi- to biglycan, ab52488 to lactate dehydrogenase, ab14739 ent ultracentrifugation, gradient one-dimensional gel elec- to the voltage-dependent anion-selective channel protein trophoresis, on-membrane protein digestion and peptide VDAC-1, and ab16048 to lamin-B1), and Sigma-Aldrich, mass spectrometry to characterize the dystrophin complex- Dorset, UK (L9393 to laminin). Peroxidase-conjugated sec- enriched fraction from skeletal muscle by proteomic means. ondary antibodies were obtained from Chemicon Interna- The mass spectrometric analysis of normal skeletal mus- tional (Temecula, CA, USA). cle resulted in the identification of the cytolinker protein desmoglein (Kljuic and Christiano 2003), in conjunction Skeletal muscle preparations with desmoplakin (Boyer et al. 2010), as an interesting new candidate protein that possibly exists in close relationship The initial proteomic screening for new potential binding to the core sarcolemmal dystrophin–glycoprotein complex. partners of dystrophin was carried out with a membrane Interestingly, comparative immunoblotting revealed a drastic fraction isolated from combined hind limb and back muscle decrease of the desmoglein isoform DSG1 in dystrophin- tissues of normal adult New Zealand white rabbits, which deficient muscles, which resonates the molecular fate of were kept under standard conditions according to Irish legis- other dystrophin-associated proteins (Ervasti et al. 1990; lation on the use of animals in experimental research (Lewis Ohlendieck et al. 1993; Murphy and Ohlendieck 2015). and Ohlendieck 2010). Muscle samples from eight rabbits To relate this novel finding to proteome-wide alterations were obtained as freshly dissected post-mortem specimens in the dystrophic phenotype, a comparative mass spectro- from the Bioresource Facility of the National University of metric analysis of the membrane-enriched fraction from Ireland and transported as quick-frozen tissue specimens dystrophic mdx quadriceps muscle was carried out. The to Maynooth University on dry ice in accordance with the reduction in desmoglein and deficiency in dystrophin was Department of Agriculture animal by-product register num- shown to be related to increased levels of the extracellular ber 2016/16 (Department of Biology, Maynooth University). 1 3 J Muscle Res Cell