The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."
A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes.
