Abstract
Abstract: :
Purpose: The ocular surface epithelium produces at least three membrane–associated mucins, MUC 1, 4 and 16, as well as the goblet cell mucin MUC5AC. We sought to develop a sensitive method to detect the mucin protein repertoire in the tear film. Methods: Sixty µl of sterile water was instilled onto the ocular surface of normal individuals and then collected from the inferior fornix. Total protein from tears of a single individual or those pooled from 17 individuals were denatured in non–reducing Laemmli buffer. Proteins were separated under non–reducing conditions in a 1% (w/v) agarose gel in electrophoresis buffer. Proteins were transferred to nitrocellulose membrane and probed with antibodies to MUC1 (HMFG–2), MUC4 (1G8), MUC16 (OC125), a carbohydrate epitope present on MUC16 (H185) and MUC5AC (791). Detection was by chemiluminescence. Results: The membrane–associated mucins MUC1, MUC4, MUC16 and its carbohydrate epitope, H185, as well as the secreted, gel–forming mucin MUC5AC, were all detected in tears of normal individuals. Each of the mucins was detected as a major, polydisperse band migrating slightly above the 250–kDa marker. In addition, comparable bands of higher molecular weight were detected with antibodies recognizing MUC16 and its carbohydrate epitope, H185. MUC5AC detected in human tears is of much lower molecular weight than that isolated from human stomach, gall bladder tissue, or human cervical mucus. Conclusions: The membrane–associated mucins, MUC1, 4 and 16, as well as the gel–forming mucin, MUC5AC, are present in the pre–ocular tear fluid of normal subjects. Separation of tear proteins in agarose gels under non–reducing conditions coupled with immunoblot using the highly sensitive chemiluminescence assay allows detection of differential electrophoretic migration patterns of mucins present in the tear fluid as well as providing an assay to compare their relative amounts in normal and disease states.
Keywords: cornea: surface mucins • cornea: tears/tear film/dry eye • conjunctiva