Isolation and characterization of a lower molecular weight protein doublet of horny cell outer leaflet: a possible novel epidermal differentiation marker.

The water-soluble fraction of stratum corneum contains numerous unidentifiable proteins and polypeptides when examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Intensely stained polypeptide bands of low molecular weights (LMW) [8 and 10 kDa] are also present, and the abundance and invariable presence of this LMW protein doublet in the soluble proteins of stratum corneum prompted us to isolate and characterize them. Extracts of stratum corneum, using 8 M urea, 0.05 M Tris, pH 9.0, 0.025 M 2-mercaptoethanol, were dialysed against water. Water-insoluble proteins were removed by repeated dialysis and centrifugation. From the remaining soluble proteins, a LMW protein doublet was isolated by Sephadex G50 column chromatography and reversed-phase high-performance liquid chromatography (HPLC). The amino acid compositions of the 8 and 10 kDa proteins were similar to, but different from, those of known structural proteins in the stratum corneum. Rabbit antibody was raised against the 10-kDa protein band and cross-reacted with the 8-kDa protein band by Western immunoblot technique. The antibody recognized only this protein doublet amongst the proteins extracted from cultured keratinocytes. The 8- and 10-kDa proteins therefore appear to be closely related to, but different from, known structural proteins or degradation products. Immunofluorescence and immunoelectron microscopy demonstrated that this protein doublet was located in the cytoplasm of the cells of the malpighian layer, and in the outer layer of the marginal band of the horny layer. Its distribution was shown to be limited to stratified squamous epithelia by organ specificity studies. These results suggest that this protein doublet appears to be a novel soluble protein and a new epidermal differentiation marker of stratified epithelium.