Oral epithelial cells are strategically located at the interface of tissues and environment and are involved in a variety of interactions with saliva, pathogens, adjacent cells, and extracellular matrix proteins. This proposal deals with the distribution and the kind of receptors present on the oral keratinocyte cell surface (e.g. laminin and fibronectin receptors) as well as their biogenesis and a putative keratinocyte secretion. Cells will be grown to confluence in a bicameral culture system to allow separate access to the media on the apical and basal surfaces. Domain-selective biotinylation coupled with precipitation with immobilized lectins will be used to analyze the apical and basal glycoprotein distribution. The biogenesis of epithelial glycoproteins will be studied by combining metabolic radiolabeling and domain-selective biotinylation to monitor the delivery of newly-synthesized glycoproteins to the cell surface. Monolayers will be pulsed with (35)S-methionine and chased at different intervals to permit glycoprotein modifications and arrival at the apical and basal domains. After domain-selective labeling with S-NHS-biotin, cells will be lysed, extracted with TX-114, and the radiolabeled/ biotinylated proteins recovered with immobilized streptavidin. Alternatively, the apical and basal surfaces will be exposed to biotin-hydrazide to directly label glycoproteins and the monolayers processed for semithin frozen sections and immunofluorescence. The biotinylated keratinocyte membrane proteins and the proteins released to the apical and basal compartments will be analyzed by one- and two-dimensional electrophoresis, isofocusing, fluorography, and western-blotting. Use of the proposed in vitro model system for studying oral keratinocyte biology may provide valuable information on: receptors for microorganisms and viruses, receptors for extracellular matrix proteins, cytokines release, transepithelial delivery of secretory IgA, and cell adhesion and migration. The long-term goals of this research are to understand how changes in the oral keratinocyte surface relate to cell-to-cell communication and the etiology of oral mucosa diseases.
