Antiadhesive character of mucin O-glycans at the apical surface of corneal epithelial cells.
OBJECTIVE
Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells.


METHODS
Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber.


RESULTS
Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface.


CONCLUSIONS
These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).
Sumiyoshi
Mika
M
Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Ricciuto
Jessica
J
Tisdale
Ann
A
Gipson
Ilene K
IK
Mantelli
Flavio
F
Argüeso
Pablo
P
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D001593:Benzyl Compounds / Q000494:pharmacology; D015153:Blotting, Western; D018394:CA-125 Antigen / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005293:Ferritins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009077:Mucins / Q000502:physiology*; D011134:Polysaccharides / Q000502:physiology*
D001593:Benzyl Compounds; D018394:CA-125 Antigen; C451759:MUC16 protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009077:Mucins; D011134:Polysaccharides; C010297:polycationic ferritin; C080739:benzyl-alpha-N-acetylgalactosamine; D005293:Ferritins; D000116:Acetylgalactosamine