Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle.

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy.

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin- and utrophin-glycoprotein complexes and α7ß1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein-replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein-protein interactions amongst the adhesion complexes and is an important potential therapeutic target. Here, we review current protein-replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic.