Protein O-fucosyltransferase 1 (Pofut1) transfers fucose to serine or threonine on proteins, including Notch receptors, that contain EGF repeats with a particular consensus sequence. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. Fucosylation of recombinant C45 agrin, both active (neural, z8) and inactive (muscle, z0) splice forms, was eliminated when agrin was overexpressed in Pofut1-deficient cells or by mutation of a consensus site for Pofut1 fucosylation (serine 1726 in the EGF4 domain). Loss of Ofucosylation caused a gain of function for muscle agrin such that it stimulated AChR clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form. Deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increased AChR aggregation. In addition, Pofut1 gene and protein expression and Pofut1 activity of the EGF4 domain of agrin were modulated during neuromuscular development. These data are consistent with a role for Pofut1 in AChR aggregation during synaptogenesis via the regulation of the synaptogenic activity of muscle agrin. Keywords glycosylation; agrin; synapse; neuromuscular junction; fucose; Notch An essential step in the formation of all types of synapses is the concentration of neurotransmitter receptors in the postsynaptic membrane such that they are localized in a manner that allows efficient and robust neurotransmission by the presynaptic nerve terminal. Nowhere is this more dramatically in evidence than at the mammalian neuromuscular junction, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptwhere postsynaptic concentration of the nicotinic acetylcholine receptors (AChRs) approaches a thousand-fold relative to AChR protein expression in the extrasynaptic membrane (Sanes and Lichtman, 2001). While this concentration of AChRs is brought about by multiple cellular and molecular mechanisms, the lateral migration of extrasynaptic AChRs during development to the postsynaptic membrane requires the stimulation of signal transduction pathways in skeletal myofibers by agrin, a motor nerve-derived synaptogenic factor (Kummer et al., 2006).Agrin is a highly glycosylated extracellular matrix protein that is essential for the formation of the mammalian neuromuscular junction (Martin, 2003b;Sanes et al., 1998b). Studies using recombinant forms of agrin have shown that high affinity AChR aggregating activity is contained wi...

Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein Ofucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on ␣ dystroglycan glycosylation.KEYWORDS Notch signaling, glycobiology, muscle aging, sarcopenia S arcopenia, the loss of muscle mass and strength during the course of normal aging, has a profound impact on quality of life for the elderly (1-4). About one third of the muscle mass present in young individuals is lost during the aging process. Increased muscle weakness with age can result in decreased ambulation and in more frequent falls and bone fractures. Sarcopenia encompasses a number of phenotypic changes in skeletal muscle, including muscle atrophy, functional reductions in overall muscle strength, muscle wasting, neuromuscular decay and denervation, alterations in muscle fiber populations and patterning, reduced mitochondrial capacity, and the decreased regenerative capacity of satellite cells. Myriad changes in signaling cascades, endocrine hormones, inflammatory factors, ␤ agonists, muscle factors, antioxidants, and energy metabolites have been suggested as contributing factors to sarcopenia, many of them by altering downstream myogenic regulatory factors (1, 2, 4). Similarly, there are a number of factors that control aging in a more global manner (IgfRs, Klotho, p53, Ku-80,