N-acetyllactosamine and the CT carbohydrate antigen mediate agrin-dependent activation of MuSK and acetylcholine receptor clustering in skeletal muscle.

Galbeta1,3GalNAc and Galbeta1,4GIcNAc are the subterminal saccharide structures present on the CT carbohydrate antigen GalNAcbeta1,4[NeuAcalpha2,3]-Galbeta1-(3GalNAc or 4GIcNAc)-R, which is localized at the mammalian neuromuscular junction. Here we show that Galbeta1,3GalNAc, Galbeta1,4GIcNAc, and the CT carbohydrate antigen affect postsynaptic assembly in cultured muscle cells. Treatment of C2C12 myotubes with benzyl-O-alpha-GalNAc or neuraminidase increased peanut agglutinin (PNA) expression and AChR clustering. Induction of AChR clustering was blocked by PNA and by muscle agrin. Addition of Galbeta1,4GIcNAc or Galbeta1,3GalNAc increased AChR clustering in myotubes and muscle-specific kinase (MUSK) autophosphorylation in vitro, while NeuAcalpha2,3Galbeta1,4GIcNAc and Galbeta1,4GIc did not. Neural agrin activated MuSK in vitro if the lactosamine-containing mucin domain was present, and this activation was blocked in large part by Galbeta1,3GalNAc and Galbeta1,4GIcNAc. Agrin fragments and MuSK bound to these disaccharides with differing specificities. Overexpression of the CT carbohydrate antigen also increased AChR clustering and MuSK autophosphorylation in the presence of neural agrin. These data suggest a model in which different portions of the CT carbohydrate structure contribute to agrin-dependent signal transduction.