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A. Guzman, P. Argueso; Role of T-synthase in Apical Barrier Formation on Ocular Surface Epithelia. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4607.
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We have previously shown that a carbohydrate-dependent association between galectin-3 and highly O-glycosylated mucins contributes to form an apical barrier at the ocular surface epithelia. T-synthase is a branchpoint enzyme responsible for the biosynthesis of the T-antigen, a key carbohydrate precursor for the elongation of mucin-type O-glycans. The purpose of this study was to determine whether T-synthase is involved in the formation of the apical barrier on the ocular surface glycocalyx.
Human corneal-limbal epithelial (HCLE) cells were transfected with a regulatory plasmid encoding a tetracycline repressor, and subsequently infected with retroviruses containing T-synthase shRNAs. T-synthase mRNA expression and protein levels were assessed by conventional PCR and western blot, respectively. Galectin-3 and T-antigen levels on the cell surface were detected using a biotinylation assay. Barrier function was assayed by incubation of cultures with 0.1% rose bengal, and dye uptake quantified using ImageJ.
A stable tetracycline-inducible RNA interfering system to knockdown T-synthase was developed in HCLE cells. Three different shRNAs sequences efficiently reduced T-synthase mRNA expression and protein levels as compared to scramble control. Sequence 1 produced the highest decrease, with 74% mRNA and 70% protein reduction. Sequences 2 and 3 reduced T-synthase mRNA and protein by approximately 50% compared with scramble control. Cell surface T-antigen levels were also suppressed by the three sequences with sequence 1 inducing the highest repression. Concomitantly, a marked decrease of galectin-3 levels on the cell surface glycocalyx was observed in these cells. In functional assays, rose bengal penetrance significantly increased in HCLE cells stably expressing T-synthase shRNAs, especially those transfected with shRNA sequence 1, indicating loss of barrier function.
These results indicate that T-synthase contributes to the formation of an apical barrier that prevents the penetrance of molecules through the epithelial cell surface glycocalyx. Manipulation of the O-glycan barrier could contribute to improving drug delivery into the cornea.
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