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The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics. [ABSTRACT FROM AUTHOR]

Aims: Variable degrees of inflammation, necrosis, regeneration and fibrofatty replacement are part of the pathological spectrum of the dystrophic process in alpha dystroglycanopathy LGMDR9 (FKRP‐related, OMIM #607155), one of the most prevailing types of LGMDs worldwide. Inflammatory processes and their complex interplay with vascular, myogenic and mesenchymal cells may have a major impact on disease development. The purpose of our study is to describe the specific immune morphological features in muscle tissue of patients with LGMDR9 to enable a better understanding of the phenotype of muscle damage leading to disease progression. Methods: We have analysed skeletal muscle biopsies of 17 patients genetically confirmed as having LGMDR9 by histopathological and molecular techniques. Results: We identified CD206+ MHC class II+ and STAT6+ immune‐repressed macrophages dominating the endomysial infiltrate in areas of myofibre regeneration and fibrosis. Additionally, PDGFRβ+ pericytes were located around MHC class II+ activated capillaries residing in close proximity to areas of fibrosis and regenerating fibres. Expression of VEGF was found on many regenerating neonatal myosin+ fibres, myofibres and CD206+ macrophages also co‐expressed VEGF. Conclusion: Our results show characteristic immune inflammatory features in LGMDR9 and more specifically shed light on the predominant role of macrophages and their function in vascular organisation, fibrosis and myogenesis. Understanding disease‐specific immune phenomena potentially inform about possibilities for anti‐fibrotic and anti‐inflammatory therapeutic strategies, which may complement Ribitol replacement and gene therapies for LGMDR9 that may be available in the future. [ABSTRACT FROM AUTHOR]

Impaired glycosylation of cellular receptor Alpha Dystroglycan (α-DG) leads to dystroglycanopathy. Glycoprotein α-DG is the receptor protein in the Dystrophin Associated Protein Complex (DAPC), a macromolecular gathering on muscle cell membrane to form a bridge between extracellular matrix (ECM) and cellular actin cytoskeleton. Proper glycosylation of α-DG is mediated by the glycosylating enzyme LARGE. Mutations either in α-DG or in LARGE lead to improper glycosylations of α-DG thereby hampering the formation of final Laminin binding form α-DG resulting in dystroglycanopathy. In our current work, we explored the structural changes associated with the presence of mutations in α-DG as well as in the enzyme LARGE. We further extended our research to understand the effect of the mutations onto protein-enzyme interactions. Moreover, since LARGE transfers the sugar moiety (glucuronic acid; GlcA) onto α-DG, we tried to analyze what effect the mutation in LARGE confers on this enzyme ligand interaction. This work for the first time addressed the molecular changes occurring in the structures α-DG, LARGE and their interactions and shed lights on the as yet poorly understood mechanism behind the dystroglycanopathy onset. [ABSTRACT FROM AUTHOR]