A lectin-binding glycoprotein of Mr 135,000 associated with basal keratinocytes in pig epidermis.

Pig epidermis separated by 1 M-CaCl2 treatment was homogenized and separated into three fractions by filtration through nylon mesh and high-speed centrifugation. Lectin-binding glycoproteins were isolated from urea/deoxycholate/mercaptoethanol extracts of the residue fraction that resisted filtration, from deoxycholate extracts of the particulate material in the filtrate and from the soluble fraction. Concanavalin A, Ricinus communis (castor bean) agglutinin 1, peanut (Arachis hypogaea) agglutinin and Ulex europaeus (gorse) agglutinin-binding glycoproteins in the three epidermal fractions were analysed by SDS/polyacrylamide-gel electrophoresis. A major neuraminidase-sensitive glycoprotein component of the particulate fraction of Mr 135,000 was strongly bound by concanavalin A and Ricinus communis agglutinin 1, but only weakly by peanut and Ulex europaeus agglutinins. This glycoprotein was not detected in the residue or soluble fractions of the epidermis, indicating that it had only a limited distribution within the tissue. The 135,000-Mr glycoprotein was one of two major glycoprotein antigens in the particulate fraction. Rabbits immunized with total particulate glycoproteins produced antibodies directed mainly against 135,000- and 110,000-Mr components. Monospecific antibodies were obtained from guinea pigs immunized with the 135,000-Mr glycoprotein band excised from polyacrylamide gels. Indirect immunofluorescence with the use of affinity-purified antibodies showed that the 135,000-Mr glycoprotein was present at the surface of cells in the basal layer of the epidermis as well as at that of other stratified epithelia. It was not present on differentiating cells in the suprabasal layers of the epithelium, suggesting an important role in the attachment or proliferative functions of basal cells in stratified epithelia. Metabolic labelling studies with skin explants cultured in the presence of D-[3H]glucosamine showed that this basal-cell glycoprotein was synthesized by cultured tissue. The major D-[3H]glucosamine-labelled glycoprotein component in the residue and particulate fractions of cultured epidermis had an Mr of 135,000, was immunoprecipitated by rabbit antisera raised against particulate epidermal glycoproteins and was bound by concanavalin A. The labelling of this glycoprotein with D-[3H]glucosamine was sensitive to tunicamycin, indicating that the basal-cell glycoprotein contained N-glycosidically linked oligosaccharides.