Abstract
LARGE is a glycosyltransferase involved in glycosylation of α-dystroglycan (α-DG). Absence of this protein in the LARGEmyd mouse results in α-DG hypoglycosylation, and is associated with central nervous system abnormalities and progressive muscular dystrophy. Up-regulation of LARGE has previously been proposed as a therapy for the secondary dystroglycanopathies: overexpression in cells compensates for defects in multiple dystroglycanopathy genes. Counterintuitively, LARGE overexpression in an FKRP-deficient mouse exacerbates pathology, suggesting that modulation of α-DG glycosylation requires further investigation. Here we demonstrate that transgenic expression of human LARGE (LARGE-LV5) in the LARGEmyd mouse restores α-DG glycosylation (with marked hyperglycosylation in muscle) and that this corrects both the muscle pathology and brain architecture. By quantitative analyses of LARGE transcripts we also here show that levels of transgenic and endogenous LARGE in the brains of transgenic animals are comparable, but that the transgene is markedly overexpressed in heart and particularly skeletal muscle (20–100 fold over endogenous). Our data suggest LARGE overexpression may only be deleterious under a forced regenerative context, such as that resulting from a reduction in FKRP: in the absence of such a defect we show that systemic expression of LARGE can indeed act therapeutically, and that even dramatic LARGE overexpression is well-tolerated in heart and skeletal muscle. Moreover, correction of LARGEmyd brain pathology with only moderate, near-physiological LARGE expression suggests a generous therapeutic window.
Highlights
Dystroglycan was originally identified as the central component of the dystrophin associated glycoprotein complex (DAGC) in skeletal muscle, but has since been shown to be one of the main receptors linking basement membranes to the cell surface in a wide variety of tissues, via association with components such as laminin [1], perlecan, agrin [2] in muscle, neurexin in the brain [3], pikachurin in the eye [4] and most recently Slit [5]
We show here that transgenic expression of human LARGE on the LARGEmyd background restores a normal phenotype and wholly corrects both the muscle pathology and the abnormalities seen in the brain, despite dramatic tissue-specific differences in transgene expression level and extent of α-DG hyperglycosylation
Wild type mice carrying the LARGE-LV5 transgene (WT-LV5) are indistinguishable from non-transgenic littermates [41], while homozygous LARGEmyd mice are readily identified by their smaller size, pronounced kyphosis and distinctive gait abnormalities [12]
Summary
Dystroglycan was originally identified as the central component of the dystrophin associated glycoprotein complex (DAGC) in skeletal muscle, but has since been shown to be one of the main receptors linking basement membranes to the cell surface in a wide variety of tissues, via association with components such as laminin [1], perlecan, agrin [2] in muscle, neurexin in the brain [3], pikachurin in the eye [4] and most recently Slit [5]. Whilst β-dystroglycan is recognised as a transmembrane adhesion protein with many interacting partners [16, 17], α-DG is a heavily-glycosylated peripheral membrane protein that consists of two globular domains connected by an elongated central domain [18]. This central domain is extensively Omannosylated on Ser/Thr residues, and glycoepitopes in this region contribute to the stability of the protein and govern linkage to laminin-G-like (LG) domain-containing extracellular matrix proteins [2, 19], with binding strength and specificity mediated both by the nature and extent of glycosylation
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