Immunolocalization and characterization of beta-keratins in growing epidermis of chelonians

Beta-keratins constitute most of the corneous material of carapace and plastron of turtles. The production of beta-keratin in the epidermis of a turtle and tortoise (criptodirians) and of a species of pleurodiran turtle was studied after injection of tritiated proline during the growth of carapace, plastron and claws. Growth mainly occurs near hinge regions along the margins of scutes and along most of the claws (growing regions). Proline incorporation occurs mainly in the growing centers, and is more specifically associated with beta-keratin synthesis. Proline-labeled bands of protein at 12-14 kDa and 25-27 kDa, and 37 kDa, in the molecular weight range of beta-keratins, were isolated from the soft epidermis of turtles 3 h after injection of the labeled amino acid. After extraction of epidermal proteins, an antibody directed against a chicken beta-keratin was used for immunoblotting. Bands of beta-keratin at 15-17 kDa, 22-24 kDa, and 36-38 kDa appear in all species. Beta-keratin is present in the growing and compact stratum corneum of the hard (shell) and soft (limbs, neck and tail) epidermis. This was confirmed using a specific antibody against a turtle beta-keratin band of 15-16 kDa. The latter antibody recognized epidermal protein bands in the range of 15-16 kDa and 29-33 kDa, and labels beta-keratin filaments. This result indicates that different forms of beta-keratins are produced from low molecular weight precursors or that larger aggregate form during protein preparation. The present study shows that beta-keratin is abundant in the scaled epidermis of tortoise but also in the soft epidermis of pleurodiran and cryptodiran turtles, indicating that this form of hard keratin is constitutively expressed in the epidermis of chelonians.