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P. Argueso, M. Sumiyoshi, S. Spurr–Michaud, A. Tisdale, I.K. Gipson; Differential O–Glycosylation of Membrane–Associated Mucins in Human Corneal–Limbal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3496.
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Purpose: Membrane–associated mucins are a family of highly O–glycosylated glycoproteins involved in the protection of the ocular surface epithelia. We have hypothesized that differential O–glycosylation of individual mucins modulates their function at the ocular surface. Our aim was to evaluate whether the membrane–associated mucins MUC1 and MUC16 produced by cultures of human corneal epithelial (HCLE) cells are differentially glycosylated. Methods: HCLE cells were grown to confluence in Gibco K–sfm and switched to a medium optimized for their differentiation (DMEM, 10% calf serum) for 7 days. Immunolocalization was carried out using antibodies to MUC1 (HMFG1) and MUC16 (OC125). Carbohydrate epitopes on mucins were detected using the H185 antibody (recognizing an O–acetylated sialic acid) and the lectins PNA and Jacalin (recognizing the T–antigen). Protein extracts from HCLE cells grown for 7 days were applied to a column of equilibrated Sepharose CL–4B (0.7x30 cm) coupled to a chromatography system, eluted in PBS, pH 7.4, and monitored for glycoprotein absorbance at 280 nm. The resulting fractions were concentrated, electrophoresed in 1% agarose gels and transferred to nitrocellulose membranes by vacuum blot. Membrane–associated mucins and carbohydrate epitopes were detected by chemiluminescence. Results: By immunofluorescence microscopy, MUC1 but not MUC16, H185, or the T–antigen, was detected on apical cell surfaces of HCLE cells grown without serum. After serum addition, MUC16, H185 and the T–antigen were detected. By Western blot, both MUC1 and MUC16 extracted from HCLE cells after 7 days in serum had high molecular weights (MW > 250 kDa), with MUC16 having a broad smear and a higher MW than MUC1. Size–exclusion chromatography of HCLE protein extracts rendered three peaks corresponding to high, mid and low MW fractions. MUC16 primarily eluted in the high MW fraction. MUC1 and a minor amount of MUC16 eluted together in the middle peak but were separated in agarose gels. H185 binding co–localized to regions of MUC16 binding in the lower portion of the smear in nitrocellulose membranes. PNA staining was also observed in the lower portion of the MUC16 smear. There was no detectable binding of H185 or PNA to regions of MUC1 binding. Conclusions: These results show that individual mucins are differentially glycosylated in HCLE cells. Membrane–associated mucins, through their individual repertoire of carbohydrate moieties, may have specific roles in the protection of the ocular surface.
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