Abstract
Abstract: :
Purpose: Distribution of the monoclonal antibody H185, which recognizes a carbohydrate epitope on the membrane-associated mucin MUC16 in the ocular surface epithelium, is altered in patients with dry eye. We sought to investigate the structural requirements for binding of the H185 antibody to mucin collected from tears. Methods: Tear fluid was collected from the inferior fornix of normal subjects after instillation of 60 µl of sterile water onto the ocular surface. The monoclonal mouse anti-human H185 antibody (isotype IgG1) was obtained from culture supernatants of hybridoma cells. An ELISA protocol was developed to quantify the amount of H185 antigen in mucin secretions. Human tears were enzymatically digested for 1 h with 1, 5 and 25 mU of neuraminidase from S. pneumoniae (α2-3 specific), C. perfringens (α2-3,6 specific), Newcastle disease virus (α2-3,8 specific) and A. ureafaciens (α2-3,6,8,9 specific). De-O-acetylation of sialic acids was carried out in solution by incubation with 0.1 M NaOH at 24°C. Results: The H185 antigen was detected in tears using ELISA. Digestion of tears with increasing concentrations of neuraminidase from A. ureafaciens (broad specificity) resulted in an enzyme concentration-dependent reduction (81-99%) of H185 antibody binding to its antigen as compared to untreated tears. Treatment with neuraminidase from Newcastle disease virus at similar concentrations resulted in a 50-85% loss of reactivity of the H185 antibody. Neuraminidases from S. pneumoniae and C. perfringens, which have restricted specificity, reduced the H185 antibody binding by only 25%. Since O-acetylation in sialic acids confers resistance to hydrolysis by neuraminidases, we sought to determine the effect of de-O-acetylation in H185 antibody binding to tear samples. Removal of O-acetyl groups resulted in a 65% reduction of H185 antibody binding to its epitope. Conclusions: This study demonstrates that O-acetyl sialic acid contributes to the H185 epitope on ocular surface epithelia. Alteration of O-acetylation of sialic acid on mucins may occur as a result of dry eye leading to a change in mucin character.
Keywords: cornea: surface mucins • cornea: tears/tear film/dry eye • protein modifications-post translational