The CT carbohydrate, Neu5Ac/Neu5Gcα2,3[GalNAcβ1,4]Galβ1,4GlcNAcβ-, is specifically expressed at the neuromuscular junction in skeletal myofibers of adult vertebrates. When Galgt2, the glycosyltransferase that creates the synaptic β1,4GalNAc portion of this glycan, is overexpressed in extrasynaptic regions of the myofiber membrane, α dystroglycan becomes glycosylated with the CT carbohydrate and this coincides with the ectopic expression of synaptic dystroglycan-binding proteins, including laminin α4, laminin α5, and utrophin. Here we show that both synaptic and extrasynaptic forms of laminin and agrin have increased binding to the CT carbohydrate compared to sialyl-N-acetyllactosamine, its extrasynaptically expressed precursor. Muscle laminins also show increased binding to CT-glycosylated muscle α dystroglycan relative to its non-CT-containing glycoforms. Overexpression of Galgt2 in transgenic mouse skeletal muscle increased the mRNA expression of extracellular matrix (ECM) genes, including agrin and laminin α5, as well as utrophin, integrin α7, and neuregulin. Increased expression of ECM proteins in Galgt2 transgenic skeletal muscles was partially dependent on utrophin, but utrophin was not required for Galgt2-induced changes in muscle growth or neuromuscular development. These experiments demonstrate that overexpression of a synaptic carbohydrate can increase both ECM binding to α dystroglycan and ECM expression in skeletal muscle, and they suggest a mechanism by which Galgt2 overexpression may inhibit muscular dystrophy and affect neuromuscular development.

SummaryAltered glycosylation and/or expression of dystroglycan have been reported in forms of congenital muscular dystrophy as well as in cancers of the breast, colon, and oral epithelium. To date, however, there has been no study of the expression of dystroglycan in pediatric solid tumors. Using a combination of immunostaining on tissue microarrays and immunoblotting of snap-frozen unfixed tissues, we demonstrate a significant reduction in native α dystroglycan expression in pediatric alveolar rhabdomyosarcoma (RMS), embryonal RMS, neuroblastoma (NBL), and medulloblastoma, whereas expression of β dystroglycan, which is cotranslated with α dystroglycan, is largely unchanged. Loss of native α dystroglycan expression was significantly more pronounced in stage 4 NBL than in pooled samples of stage 1 and stage 2 NBL, suggesting that loss of native α dystroglycan expression increases with advancing tumor stage. Neuroblastoma and RMS samples with reduced expression of native α dystroglycan also showed reduced laminin binding in laminin overlay experiments. Expression of natively glycosylated α dystroglycan was not altered in several other pediatric tumor types when compared with appropriate normal tissue controls. These data provide the first evidence that α dystroglycan glycosylation and laminin binding to α dystroglycan are altered in certain pediatric solid tumors and suggest that aberrant dystroglycan glycosylation may contribute to tumor cell biology in patients with RMS, medulloblastoma, and NBL.

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...

Vaccination has become an important therapeutic approach to the treatment of Alzheimer's disease (AD), however, immunization with Aβ amyloid can have unwanted, potentially lethal, side effects. Here we demonstrate an alternative peptide-mimotope vaccine strategy using the SDPM1 peptide. SDPM1 is a 20 amino acid peptide bounded by cysteines that binds tetramer forms of Aβ 1-40 -and Aβ 1-42 -amyloid and blocks subsequent Aβ amyloid aggregation. Immunization of mice with SDPM1 induced peptide mimotope antibodies with the same biological activity as the SDPM1 peptide. When done prior to the onset of amyloid plaque formation, SDPM1 vaccination of APPswePSEN1(A246E) transgenic mice reduced amyloid plaque burden and Aβ 1-40 and Aβ 1-42 levels in the brain, improved cognitive performance in Morris water maze tests, and resulted in no increased T cell responses to immunogenic or Aβ peptides or brain inflammation. When done after plaque burden was already significant, SDPM1 immunization still significantly reduced amyloid plaque burden and Aβ 1-40/1-42 peptide levels in APPswePSEN1(A246E) brain without inducing encephalitogenic T cell responses or brain inflammation, but treatment at this stage did not improve cognitive function. These experiments demonstrate the efficacy of a novel vaccine approach for Alzheimer's disease where immunization with an Aβ 1-40/1-42 amyloidspecific binding and blocking peptide is used to inhibit the development of neuropathology and cognitive dysfunction.