EXPRESSION OF CARBOHYDRATE RESIDUES IN PLASMA-MEMBRANE GLYCOPROTEINS DURING THE DIFFERENTIATION OF AMPHIBIAN EPIDERMAL-CELLS

Expression of various sugar residues on the plasma membrane of frog (R ana per perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectin s. In paraffin-embedded tissue, mannose residues (stained by Concanava lin A) were detected at the keratinocyte cell surface in all epidermal strata. However, Lens culinaris is agglutinin (LCA), also specific fo r mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), lab elled with wheat germ agglutinin (WGA), was maximum at the cell surfac e of basal cells and progressively decreased through the stratum spino sum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, lab elled with Griffonia simplicifolia I (GS I) and Glycine max (SBA) aggl utinins, respectively, were expressed according to the degree of diffe rentiation in amphibian epidermal cells. Sialic acid-containing glycop roteins, labelled with Limax flavus agglutinin (LFA), were found in th e outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding pat terns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane gly coproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and gly coproteins containing terminal Gal residues, that are expressed accord ing to the degree of differentiation in frog epidermis, have been part ially purified by affinity chromatography on a GSI-Sepharose 4 B colum n. The purified fraction was composed by four acidic glycoproteins wit h isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five ma jor protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to th e alpha and beta subunits of amphibian epidermal Na+,K+-ATPase as demo nstrated by specific staining with a polyclonal antibody against the c atalytic subunit of pig kidney proton pump and staining with lectins G SI, GSII, and WGA. Possible relationships between higher molecular wei ght proteins and the constituents of intramembranous particles from th e outermost plasma membranes of the replacement cell layer and the str atum corneum are also discussed.