June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Mucin N-Glycans Contribute to the Ocular Surface Glycocalyx Barrier
Author Affiliations & Notes
  • Pablo Argueso
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Takazumi Taniguchi
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Ashley Woodward
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Paula Magnelli
    New England Biolabs, Ipswich, Massachusetts, United States
  • Sarah M. Jacopo
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Jerome Mauris
    Schepens Eye Research Institute and Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Pablo Argueso, None; Takazumi Taniguchi, None; Ashley Woodward, None; Paula Magnelli, None; Sarah Jacopo, None; Jerome Mauris, None
  • Footnotes
    Support  NIH R01EY014847, NIH R01EY026147, NIH P30EY003790
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2630. doi:
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      Pablo Argueso, Takazumi Taniguchi, Ashley Woodward, Paula Magnelli, Sarah M. Jacopo, Jerome Mauris; Mucin N-Glycans Contribute to the Ocular Surface Glycocalyx Barrier. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2630.

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

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Abstract

Purpose : Terminally differentiated stratified epithelial cells maintain barrier function at the ocular surface through a specialized protective structure composed of highly O-glycosylated transmembrane mucins. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remains poorly characterized. The purpose of this study was to define the contribution of mucin N-glycans to the ocular surface glycocalyxl barrier.

Methods : Transmembrane mucins were purified from stratified corneal epithelial cell cultures using size exclusion chromatography and isopycnic density centrifugation. N-glycans released from mucin isolates were analyzed by liquid chromatography and electrospray ionization mass spectroscopy. N-glycosylation was inhibited using chemical, enzymatic and siRNA (MGAT1) approaches. Plasma membrane proteins were labeled by biotinylation. Lectin blot, immunoblot, and qPCR were used to determine levels of carbohydrate, protein, and mRNA, respectively. MUC16 affinity was analyzed using a slot blot galectin-3 binding assay. Glycocalyx barrier function was determined using the rose bengal dye penetrance assay.

Results : Structural analysis of the N-glycans in human corneal mucins by mass spectrometry revealed the presence of complex-type structures carrying N-acetyllactosamine residues, a preferred galectin ligand. In N-glycosylation inhibition experiments, we found that treatment with tunicamycin and MGAT1 siRNA induced partial loss of total and cell surface MUC16 mucin, and a concomitant reduction in glycocalyx barrier function. These results correlated with the transcriptional upregulation of the transcription factor CHOP and the molecular chaperone BiP, indicative of cells undergoing endoplasmic reticulum stress and the activation of the unfolded protein response. Finally, we found that N-glycans promoted binding affinity of MUC16 towards galectin-3 and caused retention of the lectin on the epithelial cell surface.

Conclusions : Taken together, these studies define a novel role for N-glycans in supporting the stability and protective function of transmembrane mucins at the ocular surface.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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