The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt−/− retina, and Notch target gene expression was decreased in Eogt−/−endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.DOI: http://dx.doi.org/10.7554/eLife.24419.001

Notch signalling regulates a plethora of developmental processes and is also essential for the maintenance of tissue homeostasis in adults. Therefore, fine-tuning of Notch signalling strength needs to be tightly regulated. Of key importance for the regulation of Notch signalling are O-fucose, O-GlcNAc and O-glucose glycans attached to the extracellular domain of Notch receptors. The EGF repeats of the Notch receptor extracellular domain harbour consensus sites for addition of the different types of O-glycan to Ser or Thr, which takes place in the endoplasmic reticulum. Studies from Drosophila to mammals have demonstrated the multifaceted roles of O-glycosylation in regulating Notch signalling. O-glycosylation modulates different aspects of Notch signalling including recognition by Notch ligands, the strength of ligand binding, Notch receptor trafficking, stability and activation at the cell surface. Defects in O-glycosylation of Notch receptors give rise to pathologies in humans. This Review summarizes the nature of the O-glycans on Notch receptors and their differential effects on Notch signalling.

Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. We also explored the domains which are responsible for cellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, coupled with homology modeling of SMS1 and SMS2, will be useful for drug development targeting SMS.

Background: To explore underlying molecular mechanisms in the pathogenesis of symptomatic sporadic primary hyperparathyroidism (PHPT). Materials and methods: Forty-one parathyroid adenomas from patients with symptomatic PHPT and ten normal parathyroid glands either from patients with PHPT (nZ3) or from euthyroid patients without PHPT during thyroid surgery (nZ7) were analyzed for vitamin D receptor (VDR), calcium-sensing receptor (CASR), cyclin D1 (CD1), and parathyroid hormone (PTH) expressions. The protein expressions were assessed semiquantitatively by immunohistochemistry, based on percentage of positive cells and staining intensity, and confirmed by quantitative real-time PCR. Results: Immunohistochemistry revealed significant reductions in VDR (both nuclear and cytoplasmic) and CASR expressions and significant increases in CD1 and PTH expressions in adenomatous compared with normal parathyroid tissue. Consistent with immunohistochemistry findings, both VDR and CASR mRNAs were reduced by 0.36-and 0.45-fold change (P!0.001) and CD1 and PTH mRNAs were increased by 9.4-and 17.4-fold change respectively (P!0.001) in adenomatous parathyroid tissue. PTH mRNA correlated with plasma PTH (rZ0.864; P!0.001), but not with adenoma weight, while CD1 mRNA correlated with adenoma weight (rZ0.715; P!0.001). There were no correlations between VDR and CASR mRNA levels and serum Ca, plasma intact PTH, or 25-hydroxyvitamin D levels. In addition, there was no relationship between the decreases in VDR and CASR mRNA expressions and the increases in PTH and CD1 mRNA expressions. Conclusions: The expression of both VDR and CASR are reduced in symptomatic PHPT in Asian Indians. In addition, CD1 expression was greatly increased and correlated with adenoma weight, implying a potential role for CD1 in adenoma growth and differential clinical expression of PHPT.

Methylation-mediated silencing of VDR and CASR promoter does not appear to be associated with reduced expression, indicating the involvement of other factors in specific suppression of VDR and CASR in parathyroid adenomas.