Abstract
BackgroundLARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of α-dystroglycan. Overexpression of LARGE induces hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.Methodology/Principal FindingsIn order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with α-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.Conclusions/SignificanceThis work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and α-dystroglycan hyperglycosylation in muscle should be safe.
Highlights
The muscular dystrophies (MDs) are a clinically and genetically heterogeneous group of conditions characterised by progressive muscle degeneration [1]
LARGE is a putative glycosyltransferase mutated in the myodystrophy mouse (LARGE-myd) [27] and in patients affected by MDC1D [10], one of the dystroglycanopathy variants associated with skeletal muscle and structural brain involvement
None of the other enzymes responsible for dystroglycanopathies has a similar effect; we have previously demonstrated that the overexpression of the LARGE paralog GYLTL1B is capable of hyperglycosylating a-DG in cultured cells [29,31,33]; mutations in this gene have not yet been associated with a human pathology
Summary
The muscular dystrophies (MDs) are a clinically and genetically heterogeneous group of conditions characterised by progressive muscle degeneration [1]. LARGE is a putative glycosyltransferase mutated in the myodystrophy mouse (LARGE-myd) [27] and in patients affected by MDC1D [10], one of the dystroglycanopathy variants associated with skeletal muscle and structural brain involvement. The first domain is related to bacterial aglycosyltransferases, while the second is most closely related to human b-1,3-Nacetylglucosaminyltransferase, required for synthesis of the poly-N-acetyllactosamine backbone (Galb1-4GlcNAcb13)n found on N- and O-glycans [28] Neither of these structures is present on a-DG [21], there is strong evidence that LARGE plays a pivotal role in the functional glycosylation of aDG. The forced overexpression of LARGE in mouse skeletal muscle, as well as cultured human and mouse cell lines, results in increased expression of functionally glycosylated a-DG (hyperglycosylation) and a corresponding increase in its binding capacity for laminin and other ligands [30,31]. This was not accompanied by any morphological changes suggesting a mild subclinical defect. a-DG was not hyperglycosylated in brain despite low levels of expression of the transgene, which suggests that higher levels of LARGE are necessary to achieve hyperglycosylation in a tissue, in which high levels of endogenous Large are present
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