Protein composition of cornified cell envelopes of epidermal keratinocytes

ABSTRACT Terminally differentiated mammalian epidermal cells are lined with a 15 nm thick layer of proteins cross-linked by isodipeptide and disulfide bonds, called the cornified cell envelope (CE). A number of proteins, including involucrin, loricrin, cystatin A, filaggrin, a cysteine-rich protein (CRP) and the ‘small proline-rich’ proteins (SPRRs) have been reported to be components of this complex, but little infor-mation has been obtained as to their relative abundances because the acute insolubility of the CEs has precluded direct methods of analysis. To address this question, we have determined the amino acid compositions of isolated CEs, and then modelled them in terms of linear combina-tions of the candidate proteins. The results show that stratum corneum CEs have a loricrin content of 65-70% (w/w) in human, and 80-85% in mouse. In human epidermal CEs, the secondary contributors are filaggrin and CRP (each ~10%), with smaller amounts of involucrin, SPRR and cystatin A (2-5% each) also present. Mouse epidermal CEs have about the same amount of filaggrin and somewhat more SPRR, but only trace amounts of the other proteins. In marked contrast, the major constituents of the CEs of cultured keratinocytes induced to terminal differentiation in vitro are cystatin A, involucrin and CRP (each ~30%). No significant amount of loricrin was detected except in sloughed mouse cells, which represent a more advanced state of terminal differentiation than attached cells. These results demonstrate that the CEs of cultured cells are quite different from mature CEs from native epidermis, and further suggest that generation of the CE in vivo is a multi-stage process in which involucrin, SPRR, CRP and cystatin A are initially attached to the cell membrane, and are subsequently overlaid with a heavy deposition of loricrin together with some filaggrin. Our data suggest that cultured CEs approximate only the first stage(s) of this process.