| The vertebrate neuromuscular junction (NMJ), the synapse formed between the nerve terminal of the motor neuron and the postsynaptic membrane of the skeletal myofiber, remains the preeminent model synapse to study cellular and molecular aspects of synaptic development. All NMJs studied thusfar, including those in lampreys, axolotols, fish, frogs, chickens, dogs, cats, mice, rats, guinea pigs, hamsters, monkeys and humans, have highly concentrated synaptic localization of terminal β-linked N-acetyl-D-galactosamine (βGalNAc). Such synaptic βGalNAc structures can be made, in part, by Galgt1 and Galgt2, a family of two β1,4GaNAc transferases. While both of these enzymes synthesize synaptic β1,4GalNAc linkages to make Neu5Acα2,3[GalNAcβ1,4]Galβ-R glycans, such as those found at the NMJ, they do so on different classes of substrates, with Galgt1 able to glycosylate ganglioside-type glycolipids while Galgt2 glycosylates certain mucin-like glycoproteins. Here we show that both the Galgt2 and the Galgt1 proteins are concentrated in synaptic regions of skeletal myofibers and can contribute to the synthesis of synaptic βGalNAc glycans but that they do so in different ways, both with regard to expression and function.;Using mice containing genetic deletions of Galgt1, Galgt2, or both genes, we show that Galgt1 controls the expression of pre-synaptic βGalNAc structures on the motor nerve terminal, while Galgt2 primarily controls the expression of postsynaptic ones in the muscle membrane. The Martin Lab has shown previously that overexpression of Galgt2 in skeletal muscle leads to the specific glycosylation of α dystroglycan, a member of the dystrophin-associated glycoprotein complex. Here, we show that loss of dystroglycan at the NMJ leads to loss of βGalNAc in the postsynaptic muscle membrane, suggesting α dystroglycan is required for postsynaptic βGalNAc localization. The Martin lab has also previously shown that Galgt2 overexpression in skeletal muscle induces the overexpression of synaptic dystroglycan-binding proteins and can, via such changes, inhibit the development of muscular dystrophy in multiple forms of the disease. Here, we show that overexpression of Galgt1, by contrast to Galgt2, does not allow for overexpression of extrasynaptic βGalNAc expression in the muscle membrane and instead leads to focal intracellular aggregates of βGalNAc expression that are associated with decreased muscle growth and increased muscle pathology. Thus, Galgt2 is clearly unique as a therapeutic target in muscular dystrophy. Loss of Galgt2 in mice led to an age-dependent disorganization of postsynaptic nicotinic acetylcholine receptors (AChRs), the neurotransmitter receptors responsible for synaptic transmission at the NMJ. AChRs, along with acetylcholinesterase, but not other DAG proteins, were increased within endosomes in Galgt2-deficient muscles, suggesting a role for Galgt2 in AChR membrane stability as muscle age.;Using microarray studies of muscle regeneration data generated, we queried 1800 genes either directly responsible for glycosylation or encoding major glycoproteins or lectins. Of these, Galgt1 was the gene most upregulated in response to muscle damage induced by cardiotoxin. We confirmed an almost 30-fold increase in Galgt1 mRNA a day after cardiotoxin-induced muscle injury by qRT-PCR. FACS cell sorting analysis and double immunostaining shows high increase in Galgt1 expression in satellite cells within 1 day after muscle regeneration begins, while CD31-positive endothelial cells and CD45-positive immune cells, and Sca1-positive mesenchymal stem cells show no increase. These data point to a role for Galgt1 on satellite cells, the stem cells of skeletal muscle. Induction of muscle regeneration was delayed, both in kinetics and extent, in mice lacking Galgt1, in response to cardiotoxin-induced injury. Dystrophin-deficient mdx mice lacking Galgt1 also showed reduced muscle regeneration, as evidenced by myofibers with smaller average diameters and also reduced numbers of myofibers with central nuclei. In addition, in both experiments, large aggregates of mononuclear cells could be seen that were indicative of a potential in vivo migratory defect at sites of injury. These data support a role for Galgt1 in skeletal muscle regeneration. |
