Profiling changes in glycosylation during C2C12 myoblast differentiation

Duchenne muscular dystrophy (DMD) affects 1 in 3500 boys. Systemic lack of the protein dystrophin disrupts muscle function by preventing the formation of the transmembrane dystrophin-glycoprotein complex (DGC). The DGC connects the actin cytoskeleton with the ECM, and disruption of this connection causes necrosis, degeneration and ultimately muscle wasting. Under physiologic conditions, a homologous complex, the utrophin glycoprotein complex (UGC) where utrophin substitutes for dystrophin, is restricted to the neuromuscular junction (NMJ). Alpha dystroglycan (αDG), the cell membrane associated glycoprotein that completes the last link between transmembrane βDG and laminin in the extracellular matrix, differs in glycosylation depending upon association with the DGC vs. the UGC. αDG at the DGC binds the lectin wheat germ agglutinin (WGA) while αDG associated with the UGC preferentially binds the lectin Wisteria floribunda agglutinin (WFA). Some current therapies aim to ameliorate DMD by manipulating the complex association-dependent glycosylation of αDG, by increasing WFA reactive DG extrasynaptically and therefore redistributing the utrophin-glycoprotein complex in an effort to substitute for the missing dystrophin. The current work utilized a panel of 12 lectins to perform comprehensive profiling of changes in glycosylation of C2C12 murine myoblasts following differentiation. Significant increases were observed in the binding of PNA and all five GalNAc binding specific lectins following 2 and 7 days of differentiation respectively. Three GalNAc binding lectins (WFA, VVA-B4 and HPA) showed increased binding following only 2 days of differentiation, illustrating differences in time dependency for changes in glycosylation. Lectin binding epitopes were mapped via the use of a pharmacological inhibitor to complex N-glycan creation. Importantly, not all GalNAc binding lectins were dependent upon complex N-glycan creation for reactivity.It is important to note that there is no comprehensive knowledge surrounding ...