Identification of new components of the cornified envelope of human and bovine epidermis

Antibodies raised in rabbits to purified cornified envelopes (CEs) of cultured human keratinocytes reacted in a peripheral fashion with the granular and spinous layers of human, cow, rat, and mouse epidermis. This reaction could not be abolished by absorption of the antibody with purified human involucrin to which the antibody reacted by immunoblot, thus indicating the presence of an additional antigenic determinant(s). Antibodies raised to CEs of human epidermis stained the cytoplasm of epidermal cells and gave a strong reaction to cytokeratins and a weak one to involucrin, indicating that in tissue the keratins are also cross-linked. The antibody prepared to bovine CEs reacted with keratins, but when absorbed with prekeratin it gave a peripheral staining pattern with epidermis and reacted strongly with a 126 kD component of the neutral buffer extract of cow snout epidermis and weakly with 205 kD and 85 kD ones. This antibody reacted with human involucrin by immunoblot while an antibody to involucrin stained 143 kD, 119 kD, 113 kD, and 107 kD polypeptides in the bovine extract. These latter 3 bands were shown to be substrates of transglutaminase. Further, a monoclonal antibody to bovine CEs reacted with the 119 kD and 113 kD bands and gave a peripheral staining pattern in the epidermis. Proof that the 126 kD protein was a precursor of the envelope was obtained by preparing an antibody to it and demonstrating peripheral staining of epidermal cells. These results point out the value of preparing antibodies to CE as an additional approach to studying the composition of CEs and demonstrate previously undescribed components in human and bovine tissue.