May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
Regulation of Muc4 in Cultured Corneal Epithelium Cells by Proteolytic Degradation
Author Affiliations & Notes
  • J. Lomako
    The Retina Group of Florida, Miami, Florida
    Cell Biology&Anatomy-Sch of Med,
  • W. M. Lomako
    The Retina Group of Florida, Miami, Florida
    Cell Biology&Anatomy-Sch of Med,
  • C. A. C. Carraway
    The Retina Group of Florida, Miami, Florida
    Biochemistry and Molecular Biology-Sch of Med,
  • K. L. Carraway, III
    The Retina Group of Florida, Miami, Florida
    Cell Biology&Anatomy-Sch of Med,
  • Footnotes
    Commercial Relationships J. Lomako, None; W.M. Lomako, None; .A.C. Carraway, None; K.L. Carraway, None.
  • Footnotes
    Support NIH Grant EY12343
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2747. doi:
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      J. Lomako, W. M. Lomako, C. A. C. Carraway, K. L. Carraway, III; Regulation of Muc4 in Cultured Corneal Epithelium Cells by Proteolytic Degradation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2747.

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

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Purpose:: Most studies of ocular membrane-spanning mucins have concentrated on cell surface forms. However, these glycoproteins must transit from their site of synthesis in the endoplasmic reticulum to the plasma membrane for their expression. Moreover, we have recently shown that the expression of Muc4 is regulated by intracellular degradation. This study was initiated to determine how Muc4 is distributed among intracellular compartments of cultivated cornea epithelium cells.

Methods:: All experiments were performed on 8 day cultures. Cells were treated with proteasomal inhibitor PSI or with KIF, an inhibitor of mannosidase. The Muc4 level was measured in cultured multilayer epithelial cell sheets by immunoblotting. Biotinylation was performed using PinpointTM Cell Surface Protein Isolation Kit (Pierce). Cellular localization of Muc4 was visualized by double staining confocal fluorescence microscopy (DSCFM) using anti-Muc4 antibodies 4F12 (mAbs) or C-pep (polyclonal) directed respectively against extra- and intra-cellular epitopes in conjunction with antibodies directed against markers of different cellular organelles and vesicles. The chosen markers were as follows: protein disulphate isomerase (PDI} - endoplasmic reticulum (ER); LAMP1 - lysosomes, EEA1 - early endosomes and RAB9 - late endosomes.

Results:: DSCFM revealed that Muc4 was localized in membrane and intracellular compartments of untreated as well as PSI- and KIF-treated cells. Biotinylation analysis showed that in both untreated and treated cells membrane localization was predominant. In DSCFM intracellular Muc4 appeared to be dispersed all over the cells in the form of micro-aggregates or as the constituent of the very small vesicles. In control cells Muc4 did not co-localize with any of chosen subcellular markers, but it was co-localized with endoplasmic reticulum, lysosomes and late endosomes after PSI or KIF treatment. The only exception was the EEA1 marker for early endosomes which did not co-localize with Muc4 under experimental conditions. The lysosomal inhibitior, chloroquine, increased the level of Muc4.

Conclusions:: These studies suggest that in corneal epithelium, under normal conditions, proteosomal degradation is efficient enough to protect against accumulation of insoluble deposits which can be detrimental for vision. However, in unfavorable conditions such as defects in proteasomal degradation or expression of non-mature glycoproteins, an additional protection mechanism may occur by re-routing these glycoproteins to the lysosomes.

Keywords: glycoconjugates/glycoproteins • stress response • proteolysis 

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