Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase ζ (RPTPζ)/Phosphacan with Cell-specific O-Mannosyl Glycans in the Developing Brain

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.     

N-methyl-D-aspartate receptor subunits are non-myosin targets of myosin regulatory light chain

Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the "GXXXR" portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.     

Expression pattern of the homeodomain transcription factor Pitx2 during muscle development

Late-stage Pitx2(+/LacZ) mouse embryos stained with x-gal appeared to have blue muscles, suggesting that Pitx2 expression specifically marks some phase of the myogenic progression or muscle anlagen formation. Detailed temporal and spatial analyses were undertaken to determine the extent and onset of Pitx2 expression in muscle. Pitx2 was specifically expressed in the vast majority of muscles of the head and trunk in late embryos and adults. Early Pitx2 expression in the cephalic mesoderm, first branchial arch and somatopleure preceded specification of head muscle. In contrast, Pitx2 expression appeared to follow muscle specification events in the trunk. However, Pitx2 expression was rapidly upregulated in these myogenic structures by E10.5. Upregulation correlated tightly with the apposition of a non-myogenic, Pitx2-expressing, cell cluster lateral to the dermomyotome. This cluster first appeared at the forelimb level at E10.25, gradually elongated in the posterior direction, appeared to aggregate from delaminated cells emanating from the ventrally located somatopleure, and was named the dorsal somatopleure. Immunohistochemistry on appendicular sections after E10.5 demonstrated that Pitx2 neatly marked the areas of muscle anlagen, that Pax3, Lbx1, and the muscle regulatory factors (MRFs) stained only subsets of Pitx2(+) cells within these areas, and that virtually all Pitx2(+) cells in these areas express at least one of these known myogenic markers. Taken together, the results demonstrate that, within muscle anlagen, Pitx2 marks the muscle lineage more completely that any of the known markers, and are consistent with a role for Pitx2 in muscle anlagen formation or maintenance.     

Selenoprotein W during development and oxidative stress

Selenium is involved in prevention of cancer, heart and muscle diseases, is implicated in immune function, fertility and in delaying the aging process. Selenium deficiency is harmful to brain, heart and skeletal muscles. Selenoprotein W, a member of the selenoprotein family was expressed in developing nervous system, skeletal muscles and heart in mice. Selenoprotein W was highly expressed in proliferating myoblasts and less or not in differentiated myotubes. Selenoprotein W exhibited an immediate response to oxidative stress in proliferating myoblasts, after exposure to hydrogen peroxide, similar to gluteraldehyde-3-phosphate dehydrogenase. We suggest that Selenoprotein W is involved in muscle growth and differentiation by protecting the developing myoblasts from oxidative stress.