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

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.

Multiomics Analysis of the mdx/mTR Mouse Model of Duchenne Muscular Dystrophy

“…Numerous proteoglycans such as biglycan (Bgn), decorin (Dcn), lumican (Lum), osteopontin (Spp1), tenascin C (Tnc), tenascin X (Tnxb), versican (Vcan), vinculin (Vcl), and vimentin (Vim) were upregulated at the transcript or protein level ( Figure 3D). These observations are consistent with previous findings in 3 month old mdx and 6 month old mdx 4cv animals, which are thought to have a more severe muscle phenotype from the original strain of mdx mice (32,33).…”

Comparative gel‐based proteomic analysis of chemically crosslinked complexes in dystrophic skeletal muscle

“…The following search parameters were used for protein identification: (i) peptide mass tolerance set to 10 ppm, (ii) MS/MS mass tolerance set to 0.02 Da, (iii) an allowance of up to two missed cleavages, (iv) carbamidomethylation set as a fixed modification and (v) methionine oxidation set as a variable modification. Peptides were filtered using a minimum XCorr score of 1.5 for 1, 2.0 for 2, 2.25 for 3 and 2.5 for four charge states, with peptide probability set to high confidence . For the identification of common versus unique protein species, proteins were only considered to be unique if they were detected in all four replicates of one condition and in none of the four replicates in the other condition.…”

Emerging proteomic biomarkers of X-linked muscular dystrophy

“…Both, focused studies on the mass spectrometric identification and biochemical characterization of the dystrophin-glycoprotein complex, as well as systematic cataloguing studies of muscle tissue and cell lines, have been carried out over the last decade. A summary of major proteomic studies on dystrophin and the dystrophin complexome is provided in Table 1 [28][29][30][34][35][36][37][38][39][40][41][42][43][44][45].…”

“…Building on the initial biochemical characterization of the dystrophin complex by density gradient ultracentrifugation and chemical crosslinking analysis [32,46,47], proteomic studies could confirm the close linkage of dystrophin with the integral glycoprotein β-dystroglycan and its associated extracellular laminin-binding protein α-dystroglycan [34,[38][39][40][41][42][43][44][45]. Cortical actin, syntrophins, dystrobrevins, the α,β,γ,δsarcoglycan complex and laminin subunits are routinely identified by mass spectrometric surveys [28][29][30].…”

“…However, the integral protein sarcospan is often only identified by low sequence coverage using peptide mass spectrometry, even in highly enriched subcellular fractions from skeletal muscle [45], which is probably due to its low abundance and highly hydrophobic properties. Components of the wider dystrophin complex include caveolin, the desmoglein/desmoplakin complex, desmin, actinin, synemin, tubulins, vimentin and plectin on the intracellular side, and various collagen isoforms, fibronectin and biglycan on the extracellular side of muscle fibres [38][39][40][41][42][43][44] (Figure 4).…”