The synaptic CT carbohydrate modulates binding and expression of extracellular matrix proteins in skeletal muscle: Partial dependence on utrophin

Abstract

The CT carbohydrate, Neu5Ac/Neu5Gcα2,3[GalNAcβ1,4]Galβ1,4GlcNAcβ-, is specifically expressed at the neuromuscular junction in skeletal myofibers of adult vertebrates. When Galgt2, the glycosyltransferase that creates the synaptic β1,4GalNAc portion of this glycan, is overexpressed in extrasynaptic regions of the myofiber membrane, α dystroglycan becomes glycosylated with the CT carbohydrate and this coincides with the ectopic expression of synaptic dystroglycan-binding proteins, including laminin α4, laminin α5, and utrophin. Here we show that both synaptic and extrasynaptic forms of laminin and agrin have increased binding to the CT carbohydrate compared to sialyl-N-acetyllactosamine, its extrasynaptically expressed precursor. Muscle laminins also show increased binding to CT-glycosylated muscle α dystroglycan relative to its non-CT-containing glycoforms. Overexpression of Galgt2 in transgenic mouse skeletal muscle increased the mRNA expression of extracellular matrix (ECM) genes, including agrin and laminin α5, as well as utrophin, integrin α7, and neuregulin. Increased expression of ECM proteins in Galgt2 transgenic skeletal muscles was partially dependent on utrophin, but utrophin was not required for Galgt2-induced changes in muscle growth or neuromuscular development. These experiments demonstrate that overexpression of a synaptic carbohydrate can increase both ECM binding to α dystroglycan and ECM expression in skeletal muscle, and they suggest a mechanism by which Galgt2 overexpression may inhibit muscular dystrophy and affect neuromuscular development.

B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I

“…Thus, native glycosylation of a dystroglycan may be required for effective glycosylation by GALGT2, and this is consistent with previous studies in Chinese hamster ovary cells showing that GALGT2 glycosylation only occurred when like-acetylglucosaminyltransferase was also overexpressed. 42 Thus, improper glycosylation of a dystroglycan resulting from FKRP deficiency negatively affected a number of expected GALGT2-induced molecular changes in skeletal muscle.…”

“…36,37,39,43 In addition, other molecules known to be therapeutic in various forms of MD, including integrin a7, can also be induced by GALGT2 overexpression. 42 Such inductions can occur at both the protein and mRNA level in most instances. We therefore next determined changed protein and mRNA expression for these surrogates using Western blot analysis and quantitative RT-PCR (Figure 9).…”

N-terminal α Dystroglycan (αDG-N): A Potential Serum Biomarker for Duchenne Muscular Dystrophy

“…The plasmid was transfected into HEK293T cells, and αDG-N was purified from the supernatant via anti-FLAG (M2) affinity chromatography as previously described (52). …”

Immunization with the SDPM1 peptide lowers amyloid plaque burden and improves cognitive function in the APPswePSEN1(A246E) transgenic mouse model of Alzheimer's disease

“…Purified antibody was dialyzed in TBS and concentrated, as needed, using a 10,000 MW spin column. Protein concentration was assessed using a modified Bradford assay (Pierce BCA Protein assay kit; Fisher Scientific 23227) coupled with conformation of relative purity by SDS-PAGE with silver staining (Pierce;24597), as before(Yoon et al, 2009). A fraction of these experiments was also analyzed by SDS-PAGE with subsequent immunoblotting for Aβ 1–40/42 as above.…”