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Abstract

In differentiating mammalian keratinocytes proteins are linked to the plasma membrane by epidermal transglutaminases through N-ϵ-(γ-glutamyl)-lysine isopeptide bonds to form the cornified cell envelope. The presence of transglutaminases and their protein substrates in the epidermis of nonmammalian vertebrates is not known. The present study analyses the presence and localization of the above proteins in the epidermis using immuno-cross reactivity across different classes of amniotes. After immunoblotting, some protein bands appear labelled for loricrin, sciellin, and transglutaminase in most species. These proteins are scarce to absent in the epidermis of aquatic species (goldfish and newt) where a stratum corneum is absent or very thin. The molecular weight of transglutaminase immunoreactive bands generally varies between 40 to 62 kDa, with the most represented bands at 52–57 kDa in most species. The more intense loricrin- and sciellin-immunoreactive bands are seen at 50–55–62 kDa, but are weak or absent in aquatic vertebrates. Loricrine-like immunoreactivity is present in the epidermis where α-(soft)-keratinization occurs. Isopeptide bonds are mainly associated to bands in the range of 50–62 kDa. In vertebrates where hard-keratin is expressed (the beta-keratin corneous layer of sauropsids and in feathers) or in hair cortex of mammals, no loricrin-like, transglutaminase-, and isopeptide-bond-immunoreactivities are seen. Immunoblotting however shows loricrin-, sciellin-, and trasnsglutaminase-positive bands in the corneous layers containing β-keratin. Histologically, the epidermis of most amniotes shows variable transglutaminase immunoreactivity, but isopeptide-bond and sciellin immunoreactivities are weak or undetactable in most species. The limitations of immunohistochemical methods are discussed and compared with results from immunoblotting. In reptilian epidermis transglutaminase is mainly localized in 0.15–0.3 µm dense granules or diffuse in transitional α-keratogenic cells. In β-keratogenic cells few small dense granules show a weak immunolabeling. Transglutaminase is present in nuclei of terminal differentiating α- and β-keratinocytes, as in those of mature inner and outer root sheath. The present study suggests that keratinization based on loricrin, sciellin and transglutaminase was probably present in the stratum corneoum of basic amniotes in the Carboniferous. These proteins were mainly maintained in α-keratogenic layers of amniotes but decreased in β-keratogenic layers of sauropsids (reptiles and birds). The study suggests that similar proteins for the formation of the cornified cell envelope are present in α-keratinocytes across vertebrates but not in β-keratinocytes. J. Exp. Zool. (Mol. Dev. Evol.) 000: 000–000, 2004. © 2004 Wiley-Liss, Inc.