June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Intracellular glycosylation regulates terminal differentiation in human corneal epithelial cells
Author Affiliations & Notes
  • Pablo Argueso
    Schepens Eye Rsrch Inst, MEE, HMS, Boston, Massachusetts, United States
  • Nicole McColgan
    Schepens Eye Rsrch Inst, MEE, HMS, Boston, Massachusetts, United States
  • Marissa Feeley
    Schepens Eye Rsrch Inst, MEE, HMS, Boston, Massachusetts, United States
  • Ashley M Woodward
    Schepens Eye Rsrch Inst, MEE, HMS, Boston, Massachusetts, United States
  • Damien Guindolet
    Schepens Eye Rsrch Inst, MEE, HMS, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Pablo Argueso, None; Nicole McColgan, None; Marissa Feeley, None; Ashley Woodward, None; Damien Guindolet, None
  • Footnotes
    Support  NH Grant R01EY026147
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4353. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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