Abstract
PURPOSE: The authors have determined that the corneal and conjunctival epithelia of the rat produce a mucin-like glycoprotein at the apical surface of the epithelium. The purpose of this study was to determine if human ocular surface epithelium produces similar glycoproteins. METHODS: Because our initial attempts at production of monoclonal antibodies yielded blood type A-specific antibodies, corneal epithelia from blood type O donor eyes were used for the production of monoclonal antibodies. Screening of hybridoma products was accomplished by immunofluorescence microscopy of cryostat sections of blood type O donor eyes. The monoclonal antibody produced was used for immunofluorescence microscopy and immunoelectron microscopy to determine tissue and cellular distribution, respectively. Immunoblot analysis of SDS-PAGE-separated proteins from corneal epithelial tissue and from cultured human corneal epithelium was used to determine molecular weight range and epitope binding after periodate oxidation, N-glycanase, and O-glycanase treatment. RESULTS: A monoclonal antibody, designated H185, that binds to apical cell layers of human corneal, conjunctival, laryngeal, and vaginal epithelium was produced. The antibody binds to apical membranes of apical cells, particularly at the tips of microplicae. In subapical cells, the antibody binds to small cytoplasmic vesicles. Cultured human corneal epithelium produces H185 antigen. By immunoblot analysis, H185 binds a high molecular weight protein, > 205 kD, from corneal epithelium and cultured corneal epithelium. The protein band visualized by immunoblot analysis cannot be stained by Coomassie or silver stain on SDS-PAGE, but it does stain with Alcian blue followed by silver stain, indicating that the protein is highly glycosylated. H185 binding to blotted proteins is destroyed by sodium periodate treatment and O-glycanase incubation, suggesting that the epitope of H185 is an O-linked carbohydrate. CONCLUSION: Human corneal and conjunctival epithelia produce a molecule, similar in size, cellular localization, and distribution to the mucin-like glycoprotein of rat ocular surface epithelium. These data suggest that the entire ocular surface epithelium produces mucins for the tear film.