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Pablo Argueso, Nicole McColgan, Marissa Feeley, Ashley M Woodward, Damien Guindolet; Intracellular glycosylation regulates terminal differentiation in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4353.
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The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) plays an important role in orchestrating the transcriptional activity of eukaryotic cells. Here we investigated the contribution of the O-GlcNAc modification to promoting corneal epithelial differentiation and the establishment of barrier function.
Telomerase-immortalized human corneal epithelial cells were grown in conditions that promote cell differentiation and stratification. O-GlcNAc levels were evaluated by using click chemistry and immunoblotting. Cytoplasmic and nuclear protein fractions were extracted using NE-PER extraction reagents. Depletion and quantification of O-GlcNAc transferase was achieved using small interfering RNA and qPCR, respectively. Thiamet G was used as a selective inhibitor of O-GlcNAc hydrolase. Epithelial barrier function was assayed by measuring MUC16 levels, transepithelial electrical resistance and FITC-dextran permeability.
Induction of human corneal epithelial cell differentiation stimulated the global transfer of O-GlcNAc to both nuclear and cytosolic proteins. Inflammatory conditions, on the other hand, were associated with a reduction in O-GlcNAc transferase expression. Loss- and gain-of-function studies using small interfering RNA or Thiamet G, respectively, revealed that the presence of O-GlcNAc was necessary to promote glycocalyx barrier function. Moreover, Thiamet G triggered a correlative increase in both surface expression of MUC16 and apical epithelial cell size, while reducing paracellular permeability.
These results identify intracellular protein O-glycosylation as a novel pathway responsible for promoting terminal differentiation in human corneal epithelial cells.
This is a 2020 ARVO Annual Meeting abstract.
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