The expression of different keratins in basal cells vs. suprabasal cells of normal human epidermis, and in pathological vs. normal skin, has led to speculation that specific keratins may interact to form filaments of differing structure and function. The primary goal of these studies is to investigate the interaction of keratin polypeptides in keratin filament formation during the course of human epidermal differentiation. Individual human keratin polypeptides extracted from the living layers of normal epidermis pathological epidermis, and cultured epidermal cells will be isolated by column chromatography under reversible denaturing conditions. Different combinations of polypeptides will be mixed, and analyzed for their ability to repolymerize in vitro into filaments. In order to investigate possible differences among keratin filaments, repolymerized filaments of defined composition will be examined under various conditions which reflect possible biologically significant differences within the living layers of the epidermis (e.g. temperature, ion concentration, pH). Individual polypeptides will be identified by their molecular weights and isoelectric points by one- and two-dimensional gel electrophoresis, and by their reactivities with previously defined monoclonal anti-keratin antibodies. Filament formation will be monitored by electron microscopy. These in vitro studies will be correlated with in vivo keratin filament structure by extraction of keratins under specified conditions and by using antibodies to a synthetic 15-mer peptide from the 50kd keratin sequence. Structural analysis of the amino acid sequence of the 50kd keratin (present in both normal and pathological human epidermal cells in vivo and in vitro) suggests that this 15-mer sequence is probably exposed on the filament surface and that it may be a unique antigenic site not shared by other keratin polypeptides. Antibodies to this peptide will be used in immunoelectron microscopy to compare results of reassembled filaments in vitro with filaments in vivo, and to study the fate of the 50kd keratin during epidermal differentiation. This integrated approach of in vitro and in vivo experimentation will lead to greater understanding of keratin filament composition in human epidermis and the functional role of keratin filaments during normal and pathological epidermal differentiation.
