Abstract
Abstract: :
Purpose: Mucins are highly O-glycosylated proteins that play important roles in the protection and maintenance of the ocular surface. The first step in mucin-type O-glycosylation is the enzymatic addition of N-acetyl galactosamine (GalNAc) to serine and threonine residues by a large family of polypeptide GalNAc-transferases (GalNAc-Ts). We sought to determine the repertoire and cellular origin of GalNAc-T isoforms in the human ocular surface and to evaluate their expression in non-keratinized and keratinized epithelium of ocular cicatricial pemphigoid (OCP) patients. Methods: Five conjunctival biopsies and five corneas from normal subjects, and fourteen conjunctival biopsies from OCP patients were obtained in accordance with human study guidelines. Conjunctival biopsies in OCP patients were divided into two groups, non-keratinized (6 patients) and keratinized (8 patients), based on the histologic appearance. Immunofluorescence microscopy with monoclonal antibodies was used to determine the subcellular localization of the GalNAc-T1, T2, T3, T4 and T6 isoenzymes. Coverslips were applied using Vectashield Mounting Medium with propidium iodide to visualize the nuclei of the cells. Results: GalNAc-T2, -T3 and -T4 isoforms were present in Golgi-associated compartments of the stratified epithelium in the normal cornea and conjunctiva. T4 localized primarily in apical cells, whereas T2 was found in the supranuclear region of the basal cell layer. T3 was found throughout the epithelium and T1 was present only in scattered cells of the conjunctiva. T6 localized only in conjunctival goblet cells. By comparison, in non-keratinized epithelium of OCP patients T6 was localized in the apical cells of the stratified epithelium and T1 localized throughout the entire conjunctival epithelium. In all eight keratinized OCP biopsies T6 was absent, and 7 of the 8 biopsies had no detectable T1 antibody binding. Conclusion: The presence of GalNAc-transferase isoforms in human corneal and conjunctival epithelia is cell-layer and cell-type specific and may be associated with the state of epithelial differentiation. The altered apical distribution of T6 and T1 in conjunctiva of OCP patients prior to keratinization may represent a compensatory attempt to glycosylate the apical surface to maintain a wet surface phenotype.
Keywords: 372 cornea: epithelium • 365 conjunctiva • 421 glycoconjugates/glycoproteins